Genetic Diversity Analysis of Yellow Mustard Germplasm Using Genotyping-By-Sequencing.

Recent advances in next generation sequencing technologies make genotyping by sequencing (GBS) more feasible for molecular characterization of plant germplasm with complex and unsequenced genomes. Attempt was made here to analyze genetic diversity of 24 diverse yellow mustard accessions using a GBS protocol consisting of Roche 454 pyrosequencing, genomic reduction and advanced bioinformatics tools. One and half 454 pyrosequencing runs generated roughly 1.2 million sequence reads of about 392 Mbp of DNA sequence. Application of the computational pipeline DIAL identified 512 contigs and 828 SNPs. Search with the BLAST algorithm revealed alignments of 214 contigs with the sequences reported in NCBI nr/nt database. Sanger sequencing confirmed 95% of 41 selected contigs and 94% of 240 putative SNPs. The scored 454 SNPs were highly umbalanced for assayed samples. Diversity analysis of the 454 SNP data revealed 26.1% SNP variation resided among landrace, cultivar and breeding lines and 24.7% between yellow and black seed germplasm. Cluster analysis showed that the black seed germplasm was largely grouped together, while the breeding lines were widely spread into different groups. Computer simulation on the impact of 454 SNPs missing revealed considerable changes in allelic count, bias in detection of genetic structure, and large deviation from expected genetic distance matrix. These findings are useful for parental selection for yellow mustard breeding and the detailed analyses help to illustrate the utility of GBS in genetic diversity analysis of plant germplasm, particularly in genetic relationship assessment.