Development of SNP Markers in Tetraploid Cotton Genomes by Haplotype Clustering

Abstract:
Single nucleotide polymorphism (SNP) markers have become markers of choice for marker assisted selection (MAS) in several crop improvement programs because of their higher abundance, amenability for automation and availability of high throughput genotyping platforms. Transcriptome and genome complexity reduction techniques combined with high throughput sequencing technologies enable rapid development of informative SNP markers. However, high genomic complexity, narrow genetic base, allotetraploid nature and lack of reference genome hinder development of candidate SNP markers in cultivated cotton species. To increase the efficiency of SNP detection from homologous sequences, and reduce the risk of high false positive rate due to homeologous genomes in cotton, we have implemented ‘QualitySNP’ program with additional modifications. Sequence contigs generated at high stringency were searched for haplotype information, allelic frequency in respective genotypes along with progenitor sequence information to discriminate paralogous/homeologous contigs from homologous contigs. Additional scripts were incorporated for the classification of identified SNPs into Type I (true SNPs from single locus); Type II (heterologous in one genotype and homologous in other genotype) and Type III (paralogous/homeologous SNPs within each genotype). Type I and II markers can be used for genotyping and mapping purposes. The modified program was automated to parse the contig information and generate SNP markers with flanking sequence information for direct use in assay design with high throughput genotyping platforms. This SNP detection pipeline achieved higher validation rate compared to other approaches due to its efficiency in classifying three different types of SNPs in silico.