SNP discovery from next-generation transcriptome sequencing data and their validation using KASP assay in wheat (Triticum aestivum L.)
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Single nucleotide polymorphisms (SNPs) are becoming the most amenable form of DNA-based molecular markers for genetic analysis. In hexaploid bread wheat (Triticum aestivum L.), it is difficult to discern true polymorphic SNPs due to homoeologous and paralogous genes. Two serial analysis of gene expression (SAGE) libraries were developed utilizing leaves from resistant plants carrying leaf rust resistance gene Lr28; one library was derived from leaves that were mock inoculated and the other was derived from leaves inoculated with the urediniospores of the leaf rust pathogen Puccinia triticina. Next-generation sequencing reads, after quality trimming and removal of fungal sequences, were mapped to wheat reference sequences at Ensembl Plants. CLC Genomics Workbench and Freebayes softwares were employed for SNP calling. A total of 611 SNPs were predicted to be common by both softwares, of which 207 varietal SNPs were identified by ConservedPrimer software. A subset of 100 SNPs was used for validation across 47 wheat genotypes using Kompetitive Allele Specific PCR (KASP) assay; 83 SNPs could be successfully validated. These SNPs were positioned on wheat subgenomes and chromosome arms. When functionally annotated, many sequences harboring SNPs showed homology to resistance and resistance-like genes listed in Plant Resistance Gene database (PRGdb) as well as pathogenesis-related (PR) and stress-responsive genes. The results of the present study involving discovery of SNPs associated with resistance to leaf rust, a major threat to wheat production worldwide, will be valuable for molecular breeding for rust resistance.
KeywordsSingle nucleotide polymorphisms Wheat Transcriptome KASP assay Leaf rust
The authors are thankful to Biotechnology Information System Network Distributed Information Sub Centre (BT/BI/04/065/04) for providing facilities for bioinformatics analyses. This work was supported by Department of Biotechnology, Government of India (Grant No. BT/PR6037/AGR/02/308/05 and BT/PR3925/BID/7/384/2011) and Centre of Excellence, Technical Education Quality Improvement Program-II (Grant No. NPIU/TEQIP II/FIN/31/158).
KM, MK, and PKG designed the experiment and wrote the manuscript; SC, DS, and SK performed SNP genotyping; JP and RP supported the bioinformatics analysis; HSB and KVP revised the manuscript. All the authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
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