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Discovery of pod shatter-resistant associated SNPs by deep sequencing of a representative library followed by bulk segregant analysis in rapeseed.

ABSTRACT: BACKGROUND: Single 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 FINDINGS: We 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 -log(10)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/SIGNIFICANCE: 70 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.

SUBMITTER: Hu Z

PROVIDER: S-EPMC3328475 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Discovery of pod shatter-resistant associated SNPs by deep sequencing of a representative library followed by bulk segregant analysis in rapeseed.

Hu Zhiyong Z Hua Wei W Huang Shunmou S Yang Hongli H Zhan Gaomiao G Wang Xinfa X Liu Guihua G Wang Hanzhong H

PloS one 20120417 4

<h4>Background</h4>Single 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.<h4>Methodology/principal findings</h4>We 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 ...[more]

PMID: 22529909

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Project description:BackgroundGenome wide association studies (GWAS) are a powerful tool for identifying quantitative trait loci (QTL) and causal single nucleotide polymorphisms (SNPs)/genes associated with various important traits in crop species. Typically, GWAS in crops are performed using a panel of inbred lines, where multiple replicates of the same inbred are measured and the average phenotype is taken as the response variable. Here we describe and evaluate single plant GWAS (sp-GWAS) for performing a GWAS on individual plants, which does not require an association panel of inbreds. Instead sp-GWAS relies on the phenotypes and genotypes from individual plants sampled from a randomly mating population. Importantly, we demonstrate how sp-GWAS can be efficiently combined with a bulk segregant analysis (BSA) experiment to rapidly corroborate evidence for significant SNPs.ResultsIn this study we used the Shoepeg maize landrace, collected as an open pollinating variety from a farm in Southern Missouri in the 1960's, to evaluate whether sp-GWAS coupled with BSA can efficiently and powerfully used to detect significant association of SNPs for plant height (PH). Plant were grown in 8 locations across two years and in total 768 individuals were genotyped and phenotyped for sp-GWAS. A total of 306?k polymorphic markers in 768 individuals evaluated via association analysis detected 25 significant SNPs (P???0.00001) for PH. The results from our single-plant GWAS were further validated by bulk segregant analysis (BSA) for PH. BSA sequencing was performed on the same population by selecting tall and short plants as separate bulks. This approach identified 37 genomic regions for plant height. Of the 25 significant SNPs from GWAS, the three most significant SNPs co-localize with regions identified by BSA.ConclusionOverall, this study demonstrates that sp-GWAS coupled with BSA can be a useful tool for detecting significant SNPs and identifying candidate genes. This result is particularly useful for species/populations where association panels are not readily available.

Dataset Information

Discovery of pod shatter-resistant associated SNPs by deep sequencing of a representative library followed by bulk segregant analysis in rapeseed.

Publications

Discovery of pod shatter-resistant associated SNPs by deep sequencing of a representative library followed by bulk segregant analysis in rapeseed.

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