| Construction Protocol | 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. |
