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The Japanese crested ibis Nipponia nippon is a critically threatened bird. For the conservation of this species, it is important to maintain genetic diversity in the population. The population size in Japan has now increased to about 150 individuals because of great conservation efforts for captive-breeding programs. However, the current Japanese crested ibis population in Japan is extremely unique because they were originated from the only five founder individuals (2 males and 3 females) donated by China. The understanding of the genetic diversity between the 5 founder individuals was critical for the effective management of captive breeding toward a national project for a tentative release of the Japanese crested ibis. To discover genome-wide single nucleotide polymorphism (SNP) and short tandem repeat (STR) markers and to simultaneously obtain genotype data on their markers in each of 5 founders, five reduced representation libraries (RRLs) were independently prepared from each of the 5 founder genomes and sequenced by using Illumina Hiseq2000 sequencer. 306 million 101bp reads (more than 30 Gb of sequence information) were generated. Consensus sequences were created by clustering sequence reads and then each of sequence reads were mapped to the consensus sequences, resulting in the detection of about 52000 putative SNPs. Moreover 162 putative STRs were found. For the discovery of genome wide SNP and STR markers in Japanese crested ibis, we generated sequences from the Japanese crested ibis RRLs using the next generation sequencer Hiseq2000 (Illumina). Also, we simultaneously obtained the genotype data on each of markers by sequencing the 5 RRLs independently prepared from the 5 founder genomes. At the time of this study, the HiSeq2000 produced sequences of 150-200 million DNA fragments with 100bp read length in one sequencing lane. That allowed us to analyze DNA fragments from 0.5-1 million loci per genome, when the number of DNA sample was five and sequencing depth was about 30 times (5 sample ×1 million loci × 30 depth = 150 million). Pair-end sequencing with 100bp read length of 0.5-1 million fragments resulted in 100-200 Mb, which was estimated to represent 8-13% of the Japanese crested ibis genome, supposing that their genome size was 1.5 Gb based on several entries in the Eukaryotic genome size databases. To prepare RRL including the desired number of fragments, we digested genome DNA with HaeIII or MboI and isolated fragments in the 250-350 bp size range from agarose gel. In a preliminary experiment, the number of size selected DNA fragments by digestion with HaeIII and that by MboI were estimated to be 0.34 million and 0.44 million, respectively from the yield of isolated DNA fragments. Also, additional reason that we chose the DNA fragments in the 250-350 bp size range, was to prevent decreasing sequence data by overlapping of forward and reverse 100 bp sequence reads from a restriction fragment. The restriction fragments by digestion with HaeIII and those by MboI were combined and processed as a single RRL for sequencing. The five RRLs were independently prepared from the 5 founder genomes. Each of RRLs was distinguished by adding the sequence adaptors with different index sequence (barcode sequence). After then, the 5 RRLs were pooled and sequenced by a single sequencing lane on Hiseq2000 for 101 cycles with pair-end mode. |