ERX1189413 E-MTAB-4014:Sample 1 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951694 SAMEA3644545 E-MTAB-4014:Sample 1 Sample 1 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: shoot organism part ERX1189414 E-MTAB-4014:Sample 10 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951695 SAMEA3644546 E-MTAB-4014:Sample 10 Sample 10 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: shoot organism part ERX1189415 E-MTAB-4014:Sample 11 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951696 SAMEA3644547 E-MTAB-4014:Sample 11 Sample 11 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: shoot organism part ERX1189416 E-MTAB-4014:Sample 12 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951697 SAMEA3644548 E-MTAB-4014:Sample 12 Sample 12 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: shoot organism part ERX1189417 E-MTAB-4014:Sample 13 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951698 SAMEA3644549 E-MTAB-4014:Sample 13 Sample 13 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: shoot organism part ERX1189418 E-MTAB-4014:Sample 14 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951699 SAMEA3644550 E-MTAB-4014:Sample 14 Sample 14 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: shoot organism part ERX1189419 E-MTAB-4014:Sample 15 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951700 SAMEA3644551 E-MTAB-4014:Sample 15 Sample 15 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: shoot organism part ERX1189420 E-MTAB-4014:Sample 16 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951701 SAMEA3644552 E-MTAB-4014:Sample 16 Sample 16 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: shoot organism part ERX1189421 E-MTAB-4014:Sample 17 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951702 SAMEA3644553 E-MTAB-4014:Sample 17 Sample 17 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: shoot organism part ERX1189422 E-MTAB-4014:Sample 18 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951703 SAMEA3644554 E-MTAB-4014:Sample 18 Sample 18 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: shoot organism part ERX1189423 E-MTAB-4014:Sample 19 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951704 SAMEA3644555 E-MTAB-4014:Sample 19 Sample 19 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: shoot organism part ERX1189424 E-MTAB-4014:Sample 2 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951705 SAMEA3644556 E-MTAB-4014:Sample 2 Sample 2 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: shoot organism part ERX1189425 E-MTAB-4014:Sample 20 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951706 SAMEA3644557 E-MTAB-4014:Sample 20 Sample 20 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: root organism part ERX1189426 E-MTAB-4014:Sample 21 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951707 SAMEA3644558 E-MTAB-4014:Sample 21 Sample 21 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: root organism part ERX1189427 E-MTAB-4014:Sample 22 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951708 SAMEA3644559 E-MTAB-4014:Sample 22 Sample 22 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: root organism part ERX1189428 E-MTAB-4014:Sample 23 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951709 SAMEA3644560 E-MTAB-4014:Sample 23 Sample 23 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: root organism part ERX1189429 E-MTAB-4014:Sample 24 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951710 SAMEA3644561 E-MTAB-4014:Sample 24 Sample 24 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: root organism part ERX1189430 E-MTAB-4014:Sample 25 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951711 SAMEA3644562 E-MTAB-4014:Sample 25 Sample 25 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: root organism part ERX1189431 E-MTAB-4014:Sample 26 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951712 SAMEA3644563 E-MTAB-4014:Sample 26 Sample 26 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: root organism part ERX1189432 E-MTAB-4014:Sample 27 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951713 SAMEA3644564 E-MTAB-4014:Sample 27 Sample 27 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: root organism part ERX1189433 E-MTAB-4014:Sample 28 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951714 SAMEA3644565 E-MTAB-4014:Sample 28 Sample 28 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: root organism part ERX1189434 E-MTAB-4014:Sample 29 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951715 SAMEA3644566 E-MTAB-4014:Sample 29 Sample 29 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: root organism part ERX1189435 E-MTAB-4014:Sample 3 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951716 SAMEA3644567 E-MTAB-4014:Sample 3 Sample 3 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: shoot organism part ERX1189436 E-MTAB-4014:Sample 30 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951717 SAMEA3644568 E-MTAB-4014:Sample 30 Sample 30 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: root organism part ERX1189437 E-MTAB-4014:Sample 31 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951718 SAMEA3644569 E-MTAB-4014:Sample 31 Sample 31 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: root organism part ERX1189438 E-MTAB-4014:Sample 32 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951719 SAMEA3644570 E-MTAB-4014:Sample 32 Sample 32 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: root organism part ERX1189439 E-MTAB-4014:Sample 33 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951720 SAMEA3644571 E-MTAB-4014:Sample 33 Sample 33 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: root organism part ERX1189440 E-MTAB-4014:Sample 34 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951721 SAMEA3644572 E-MTAB-4014:Sample 34 Sample 34 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: root organism part ERX1189441 E-MTAB-4014:Sample 35 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951722 SAMEA3644573 E-MTAB-4014:Sample 35 Sample 35 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: root organism part ERX1189442 E-MTAB-4014:Sample 36 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951723 SAMEA3644574 E-MTAB-4014:Sample 36 Sample 36 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto limestone quarry soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: lime quarry soil growth condition Experimental Factor: root organism part ERX1189443 E-MTAB-4014:Sample 37 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951724 SAMEA3644575 E-MTAB-4014:Sample 37 Sample 37 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Plants grown in hydroponics for additional root RNA samples for meta-transcriptome assembly: To obtain some pure plant root RNA for generation of the meta-transcriptome assembly, relevant to acid stress, tillers of one individual from each of the acid and lime ecotype were transferred to 0.5mM CaCl2 solution to equilibrate for 10 days. Tillers were then transferred to 50M AlCl3 solution for 48 hours, followed by harvesting of roots for RNAseq. Roots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: hydroponics plus Al growth condition Experimental Factor: root organism part ERX1189444 E-MTAB-4014:Sample 38 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951725 SAMEA3644576 E-MTAB-4014:Sample 38 Sample 38 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Plants grown in hydroponics for additional root RNA samples for meta-transcriptome assembly: To obtain some pure plant root RNA for generation of the meta-transcriptome assembly, relevant to acid stress, tillers of one individual from each of the acid and lime ecotype were transferred to 0.5mM CaCl2 solution to equilibrate for 10 days. Tillers were then transferred to 50M AlCl3 solution for 48 hours, followed by harvesting of roots for RNAseq. Roots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: hydroponics plus Al growth condition Experimental Factor: root organism part ERX1189445 E-MTAB-4014:Sample 39 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951726 SAMEA3644577 E-MTAB-4014:Sample 39 Sample 39 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Plants grown in hydroponics for additional root RNA samples for meta-transcriptome assembly: To obtain some pure plant root RNA for generation of the meta-transcriptome assembly, tillers of one individual from each of the intermediate ecotype were transferred to 0.5mM CaCl2 solution to equilibrate for 10 days. A tiller from one individual of the intermediate ecotype was transfered to fresh 5mM CaCl2 solution for 48 hours, followed by harvesting of roots for RNAseq. Roots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. " RNA Extraction, QA and RNAseq: RNA extraction of frozen root tissue: Root samples were partly pre-ground, whilst cooling with liquid nitrogen, by hand in 1.5ml microcentrifuge tubes with 106m acid washed glass beads (Sigma-Aldrich) using disposable pestles (Sigma Aldrich), before processing in FastPrep tubes containing Lysing Matrix A on the Precellys 24-Dual using the same conditions as for shoots. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 21/23 root samples passed the QA and were submitted for RNAseq (all three hydroponics root samples passed, but 3 roots samples from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 4 replicates for 3 treatments and 5 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: intermediate ecotype Experimental Factor: hydroponics growth condition Experimental Factor: root organism part ERX1189446 E-MTAB-4014:Sample 4 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951727 SAMEA3644578 E-MTAB-4014:Sample 4 Sample 4 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: shoot organism part ERX1189447 E-MTAB-4014:Sample 5 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951728 SAMEA3644579 E-MTAB-4014:Sample 5 Sample 5 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: acid ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: shoot organism part ERX1189448 E-MTAB-4014:Sample 6 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951729 SAMEA3644580 E-MTAB-4014:Sample 6 Sample 6 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: shoot organism part ERX1189449 E-MTAB-4014:Sample 7 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951730 SAMEA3644581 E-MTAB-4014:Sample 7 Sample 7 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: shoot organism part ERX1189450 E-MTAB-4014:Sample 8 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951731 SAMEA3644582 E-MTAB-4014:Sample 8 Sample 8 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: shoot organism part ERX1189451 E-MTAB-4014:Sample 9 ERP013055 E-MTAB-4014 RNAseq study (root and shoot) of Holcus lanatus ecotypes selected from two widely contrasting habitats, acid bog (pH 3.5) or limestone quarry spoil (pH 7.5) ERS951732 SAMEA3644583 E-MTAB-4014:Sample 9 Sample 9 RNA-Seq TRANSCRIPTOMIC RANDOM Plant and soil collection: H. lanatus plants and topsoil were collected from three locations: 6 plants (6 lime genotypes) were selected from a disused limestone quarry soil (pH 7.5) in County Antrim, NI, hereafter referred to as lime, 6 plants (6 acid genotypes) from an acidic peat bog (pH 3.5) in County Tyrone, NI, hereafter referred to as acid, and 6 plants (6 intermediate genotypes) from an acid mineral rough grassland (pH 5.5) (County Down, NI), hereafter refereed to as intermediate. In each case plants were carefully selected within each habitat, with all plants at least 5m apart, so that each of the 6 plants represents a clone for a unique genotype of that particular habitat. Tillers from each plant were trans-planted into John Innes no. 2 compost to nutritionally neutralize all plants. Tillers from compost grown lime and acid ecotypes (5 genotypes each) were subsequently used for set up of a full factorial soil transplant experiment. Tillers from one genotype of each the acid, lime and intermediate population were subsequently grown in hydroponics for additional root acid stress relevant transcriptome sequencing data. Full factorial reciprocal soil transplant experiment: Tillers from 5 genotypes of each the acid and lime ecotype were transplanted from compost onto acid bog soil (total 10 pots) and grown in the growth chamber for 7 weeks. Replication was at the level of ecotype by using 5 tillers from 5 different clonal plants from each ecotype (5 genotypes per ecotype) rather than using multiple tillers from one plant, to ensure that natural variation within the two H. lanatus ecotypes could be investigated. At harvest at 7 weeks, roots and shoots were washed thoroughly with deionized water, separated, frozen in liquid nitrogen and stored at -80C until RNA extraction. RNA Extraction, QA and RNAseq: RNA extraction of frozen shoot tissue: Shoot samples were homogenized in FastPrep Lysing tubes containing Lysing Matrix D (MP Biomedicals, USA) using two 20-second Precellys 24-Dual (Bertin Technologies, France) runs at 5500rpm to produce a fine powder. Inbetween runs the tubes were submerged into liquid nitrogen to prevent degradation of RNA. RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN), according to protocol and with inclusion of a DNA digestion step (DNase Set, QIAGEN) with the following amendment: 450l Buffer RLT (containing 4.5l -Mercaptoethanol) was added to the Precellys lysing tube containing the ground tissue and homogenised again using the Precellys 24-Dual for 5 seconds at 5500rpm to ensure that the powdered sample was suspended as quickly as possible to prevent RNA degradation. The lysate was transferred to a QIAshredder spin column and centrifuged at 14,000rpm for 2 minutes, followed by RNA extraction according to the standard protocoll. RNA was then eluted twice using the same eluate to further concentrate the RNA and stored at -80 C. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Scientific) and Agilent 2200 Tapestation (Agilent Technologies) to assess quality (purity and integrity) of the RNA. 19/20 shoot samples passed the QA and were submitted for RNAseq. 1 shoots sample from the soil experiment failed the QC and could therefore not be processed for library preparation for the RNAseq study, hence there are 5 replicates for 3 treatments and 4 replicates for 1 treatment. Transcriptome sequencing (Illumina, RNAseq) was conducted at the Genome Analysis Centre (TGAC), Norwich, UK. Barcoded 125bp paired end TruSeq RNA libraries (polyA selected) were generated for each RNA sample and sequenced on the Illumina HiSeq 2500. 252 0 F Application Read Forward 1 1 R Application Read Reverse 127 Illumina HiSeq 2500 Experimental Factor: lime ecotype Experimental Factor: acid bog soil growth condition Experimental Factor: shoot organism part