Mining single-nucleotide polymorphisms from hexaploid wheat ESTs

Abstract

Single-nucleotide polymorphisms (SNPs) represent a new form of functional marker, particularly when they are derived from expressed sequence tags (ESTs). A bioinformatics strategy was developed to discover SNPs within a large wheat EST database and to demonstrate the utility of SNPs in genetic mapping and genetic diversity applications. A collection of > 90000 wheat ESTs was assembled into contiguous sequences (contigs), and 45 random contigs were then visually inspected to identify primer pairs capable of amplifying specific alleles. We estimate that homoeologue sequence variants occurred 1 in 24 bp and the frequency of SNPs between wheat genotypes was 1 SNP/540 bp (theta = 0.0069). Furthermore, we estimate that one diagnostic SNP test can be developed from every contig with 10-60 EST members. Thus, EST databases are an abundant source of SNP markers. Polymorphism information content for SNPs ranged from 0.04 to 0.50 and ESTs could be mapped into a framework of microsatellite markers using segregating populations. The results showed that SNPs in wheat can be discovered in ESTs, validated, and be applied to conventional genetic studies.

EST-SNPs in bread wheat: discovery, validation, genotyping and haplotype structure

“…The HSVs could also be utilized for designing primers in such a manner that each primer pair allows the amplification of only one of the three homoeologous loci. This strategy was successfully utilized by Somers et al (2003) and the present study confirms the utility of this approach for SNP discovery in bread wheat. However, surprisingly, in the present study, 1 HSV occurred every 136.7 bp as against 1 HSV every 24 bp reported by Somers et al (2003).…”

“…Therefore, for SNP discovery in bread wheat, EST contigs are first separated into subcontigs, each representing a locus on an individual chromosome. This is made possible by identifying polymorphisms, described as homoeologue sequence variants (HSVs), which characterize each of the triplicate homoeoloci within an EST-contig (Somers et al 2003). The HSVs could also be utilized for designing primers in such a manner that each primer pair allows the amplification of only one of the three homoeologous loci.…”

“…The EST-contigs were visualized using JalView (M. Clamp; http://www. ebi.ac.uk/~michele/jalview), following Somers et al (2003), to identify homoeologue specific EST clusters (hereafter called EST-subcontigs), which were defined by the presence of homoeologue sequence variants (HSVs) at specific nucleotides. Subsequently in each homoeologue specific ESTsubcontig, SNPs were detected manually.…”

“…The number of HSVs per contig ranged from 2 to 26, giving an average density of 1 HSV every 136.7 bp (for HSV, Somers et al 2003). Utilizing information on HSVs, ESTs in 48 ESTcontigs were grouped into 175 subcontigs, with an average of 3.70 subcontigs per contig; the number of ESTs in individual subcontigs ranged from 2 to 30.…”

“…A number of studies for developing SNP markers in bread wheat have been conducted (Mochida et al 2003;Schwarz et al 2003;Somers et al 2003;Yanagisawa et al 2003;Zhang et al 2003;Blake et al 2004;Caldwell et al 2004;Massa et al 2004;Beales et al 2005;Huang & Rőder 2005;Ravel et al 2006;Chao et al 2009) but in most of these studies, SNPs have been utilized for characterizing polymorphism within specific genes (Schwarz et al 2003;Yanagisawa et al 2003;Zhang et al 2003;Blake et al 2004;Caldwell et al 2004;Massa et al 2004;Beales et al 2005;Huang & Rőder 2005). Only in a few of these studies, SNPs have been developed from ESTs (Mochida et al 2003;Somers et al 2003;Ravel et al 2006;Chao et al 2009), and no study on random genomic SNPs is available. Thus, there is a need for further work on development and use of SNPs in bread wheat, where triplicate loci due to three closely related sub-genomes and paralogues due to further duplication of loci make the development of SNPs rather difficult.…”