Abstract
BackgroundSingle nucleotide polymorphisms (SNPs) are an important class of genetic marker for target gene mapping. As of yet, there is no rapid and effective method to identify SNPs linked with agronomic traits in rapeseed and other crop species.Methodology/Principal FindingsWe demonstrate a novel method for identifying SNP markers in rapeseed by deep sequencing a representative library and performing bulk segregant analysis. With this method, SNPs associated with rapeseed pod shatter-resistance were discovered. Firstly, a reduced representation of the rapeseed genome was used. Genomic fragments ranging from 450–550 bp were prepared from the susceptible bulk (ten F2 plants with the silique shattering resistance index, SSRI <0.10) and the resistance bulk (ten F2 plants with SSRI >0.90), and also Solexa sequencing-produced 90 bp reads. Approximately 50 million of these sequence reads were assembled into contigs to a depth of 20-fold coverage. Secondly, 60,396 ‘simple SNPs’ were identified, and the statistical significance was evaluated using Fisher's exact test. There were 70 associated SNPs whose –log10 p value over 16 were selected to be further analyzed. The distribution of these SNPs appeared a tight cluster, which consisted of 14 associated SNPs within a 396 kb region on chromosome A09. Our evidence indicates that this region contains a major quantitative trait locus (QTL). Finally, two associated SNPs from this region were mapped on a major QTL region.Conclusions/Significance70 associated SNPs were discovered and a major QTL for rapeseed pod shatter-resistance was found on chromosome A09 using our novel method. The associated SNP markers were used for mapping of the QTL, and may be useful for improving pod shatter-resistance in rapeseed through marker-assisted selection and map-based cloning. This approach will accelerate the discovery of major QTLs and the cloning of functional genes for important agronomic traits in rapeseed and other crop species.
Highlights
Single nucleotide polymorphisms (SNPs) are the most abundant variations and currently an important class of genetic marker distributed throughout the genome [1]
Ten F2 individuals, which are susceptible to pod shattering (SSRI,0.10), and ten F2 individuals, which are resistance to pod shattering (SSRI .0.90), were pooled to form the susceptible bulk (SK) and the resistance bulk (RK), respectively
After removing 1,102,886 sequences with low-quality scores, 47,786,006 reads (4.3 billion bp) corresponding to 25,048,721 (12,633,495 and 12,415,226 from RK and SK libraries, respectively) unique sequences remained for SNP discovery
Summary
Single nucleotide polymorphisms (SNPs) are the most abundant variations and currently an important class of genetic marker distributed throughout the genome [1]. It is an effective technique in that it detects large effect QTL alleles in a large sample of progenies at a relatively low cost. Because it saves time and cost, it has been widely used for the genetic analysis of qualitative traits [12,13,14]. Single nucleotide polymorphisms (SNPs) are an important class of genetic marker for target gene mapping. There is no rapid and effective method to identify SNPs linked with agronomic traits in rapeseed and other crop species
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