Genetic diversity analysis of yellow mustard (Sinapis alba L.) germplasm based on genotyping by sequencing

Abstract

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. We used a GBS protocol consisting of Roche 454 pyrosequencing, genomic reduction and advanced bioinformatics tools to analyze genetic diversity of 24 diverse yellow mustard accessions. One and one half 454 pyrosequencing runs generated roughly 1.2 million sequence reads totaling about 392 million nucleotides. Application of the computational pipeline DIAL identified 512 contigs and 828 SNPs. 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 454 scored SNPs were highly imbalanced among assayed samples. Diversity analysis of these SNPs revealed that 26.1 % of the total variation resided among landrace, cultivar and breeding lines and 24.7 % between yellow-and black-seeded germplasm. Cluster analysis showed that the black-seeded accessions were largely clustered together and the breeding lines were grouped with known origin. Computer simulation was performed to assess the impact of 454 SNPs missing and revealed considerable changes in allelic count, bias in detection of genetic structure, and large deviations from the expected genetic-distance matrix. These findings are useful for parental selection consideration in yellow mustard breeding, and our detailed analyses help illustrate the utility of GBS in genetic-diversity analysis of plant germplasm, particularly for genetic-relationship assessment.

Genotyping-By-Sequencing for Plant Genetic Diversity Analysis: A Lab Guide for SNP Genotyping

“…However, Elshire's GBS protocol is not unique, and many similar GBS protocols were also developed following the same idea of genomic reduction [9] and taking advantage of NGS technology [13][14][15][16]. For example, we also developed a similar GBS protocol using the Roche 454 pyrosequencing platform that was capable of generating thousands of genotyped SNP markers specifically for plant genetic diversity analysis [4,10,17].…”

“…First, it combines the processes of marker discovery and genotyping and provides a rapid, high throughput and cost-effective tool for a genome-wide analysis of genetic diversity [5,10]. Second, it requires no prior sequencing of the plant genome and provides direct genotyping of plants with complex genomes without prior SNP discovery, making the approach more accessible to non-model species.…”

“…Third, and most importantly, it generates a large number of genome-wide SNP data, allowing for better genome sampling. Despite its benefits, the GBS approach also displays some limitations: (1) the presence of large amounts of missing data [4,10,[17][18][19] largely due to the use of low coverage sequencing [20]; and (2) uneven genome coverage [21], due to the use of REs and fragment size selection. Figure 1.…”

“…Since 2009, we have explored workable GBS protocols for plant genetic diversity analysis based on the Roche 454 pyrosequencing platform [4,10,17] and Illumina HiSeq and MiSeq sequencing platforms. Our new Illumina-based GBS protocol, named "gd-GBS" to distinguish it from others, yields more SNP genotype data with fewer missing observations than those based on the Roche 454 platform.…”

“…The GBS approach is based on genome reduction with restriction enzymes [9], does not require a reference genome for SNP discovery, is a combined one-step process of marker discovery and genotyping and provides a rapid, high-throughput and cost-effective tool for a genome-wide analysis of genetic diversity for a range of non-model species and germplasm sets [5,10]. These characteristics are advantageous and encouraging for genetic diversity analysis of plants with no informative markers available.…”