MZRILseq
Bar-coded multiplexed sequencing of a recombinant population from a cross between two rice cultivars Zhenshan 97 and Minghui 63
For genotyping, total genomic DNA was isolated from leaf tissues of RILs derived from a cross between Zhenshan 97 and Minghui 63 (Xing et al. 2002) using the CTAB method. DNA sequencing followed essentially the procedures as previously described (Huang et al. 2009). Briefly, each of the DNA samples was bar-coded and the indexed DNA samples from average 22 RILs were mixed into a pool with an equal molar concentration and then loaded on 2% agarose gels and separated by electrophoresis. The fragments of 200-300 bp were recovered and purified and then enriched by 18 cycles of PCR to obtain a solexa sequencing library. Each library was loaded into one lane of the Illumina GAII for 2*40 bp paired-end sequencing, with the Illumina PhiX sample used as a control. Image analysis and base calling were performed using Illumina GAPipeline-1.4.0. Reads with base quality values in Illumina FASTQ format were converted to Sanger standard FASTQ format using ‘‘sol2sange’’ pipeline in the software MAQ (Li et al. 2008) and sorted for each RIL based on their 5’ bar-codes using a Perl script (Huang et al. 2009). Paired-reads started with different bar-codes were discarded. Thirty-six-mer sequences were obtained after trimming the four-base bar-code for each read.
The bar-coded multiplexed sequencing approach based on the next-generation sequencing technologies provides capacity to sequence a mapping population in a single sequencing run. However, such approach usually generates low-coverage and error-prone sequences for each line in the population. Thus it is a big challenge to genotype the lines in the population for linkage map construction based on such low-coverage sequences without high quality data of the parental genotyping, whereas deep sequencing of the parents is still expensive. In this study, we report a method for constructing ultra-high density linkage map composed of high quality SNPs, based on sequences from very low depth sequencing of a mapping population.