IDENTIFICATION OF SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) IN BRASSICA JUNCEA
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Date
2017
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Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu
Abstract
Generation of massive sequence data from next-generation sequencing platforms coupled with suitable bioinformatics tools has led to the identification of single nucleotide polymorphisms (SNPs) in various organisms. However, narrow genetic base, complex allotetraploid genome and highly repetitive regions have led the discovery of single nucleotide polymorphism (SNP) in Brassica juncea at a slower pace. To counter this problem, a genome reduction technique known as dd-RAD (double digest- Restriction-site Associated DNA) was followed on three accessions each from Indian (Pusa Tarak, RSPR-01 and Urvashi) and exotic (Donskaja-IV, Zem1 and Cutlas) germplasm. In this method, the genomic DNA of these accessions was digested with two restriction enzymes- MseI and SacI; followed by adapter ligation and sequencing. After sequencing, a total of 25.5 million paired-ends reads were obtained, of which nearly 89 percent reads had a Q score >30 and only 4% (approx.) reads had a Q score of < 10 which depicted a good quality raw data. The sequence data from all the samples were then processed bioinformatically for the identification of SNPs using CLC Genomics Workbench. Three different mapping approaches for the detection of SNP were employed. The mapped reads were then used for the SNP mining by Probabilistic variant detection method using stringent parameters. A total of 10,399 SNPs were identified between different accessions of B. juncea while considering read depth at 10. An in-silico analysis confirmed that out of total SNPs, about 93% of SNPs were found in gene coding region. A subset of 62 genic SNPs were selected and subjected to MassARRAY® platform for validation using diverse germplasm of Brassica juncea. Validation analysis on a set of 98 diverse B. juncea lines showed that 61 markers were functional, polymorphic and of good quality for genotyping. The newly identified SNPs in this study provide an important genomic resource for genetic mapping, association analysis and marker-assisted selection/breeding for B. juncea.
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