Transcriptome and Complexity-Reduced-DNA Based Identification of Intraspecies Single Nucleotide Polymorphisms in the Polyploid Gossypium hirsutum L

Varietal single nucleotide polymorphisms (SNPs) are the differences within one of the two sub-genomes between different tetraploid cotton varieties and have not been practically used in cotton genetics and breeding because they are difficult to identify due to low genetic diversity and very high sequence identity between homoeologous genes in cotton. We have used transcriptome and restriction-site associated DNA (RAD) sequencing to identify varietal SNPs among 18 Gossypium hirsutum varieties based on the rationale that varietal SNPs can be more confidently called when flanked by sub-genome-specific SNPs. Using transcriptome data, we successfully identified 37,413 varietal SNPs and of these, 22,121 did not have an additional varietal SNP within their 20-bp flanking regions so can be used in most SNP genotyping assays. From RAD sequencing data, we identified an additional 3,090 varietal SNPs between two of the varieties. Of the 1,583 successful SNP assays achieved using different genotyping platforms, 1,363 were verified. Many of the SNPs behaved as dominant markers because of co-amplification from homoeologous loci, but the number of SNPs acting as co-dominant markers increased when one or more sub-genome-specific SNP(s) were incorporated in their assay primers, giving them greater utility for breeding applications. A G. hirsutum genetic map with 1,244 SNP markers was constructed covering 5557.42 centiMorgan and used to map qualitative and quantitative traits. This collection of G. hirsutum varietal SNPs complements existing intra-specific SNPs and provides the cotton community with a valuable marker resource applicable to genetic analyses and breeding programs.