Upland cotton (Gossypium hirsutum L., 2n=4x=52, 2[AD]1) is a major fiber and oilseed crop that contributes significantly to the livelihoods of millions of people, as well as the economies of the USA and many other countries. Genetic improvements of cotton are needed to enhance sustainability, address evolving pest and pathogen pressures, and meet industrial requirements. However, genetic diversity is low among elite Upland cotton cultivars and breeding germplasm, which constrains the effectiveness of breeding efforts, and renders the crop more vulnerable to emerging biotic and abiotic threats. Genetic diversification efforts involving interspecific hybridization, introgression, and genetic dissection would benefit greatly by the availability of the inexpensive assays for targeted genotyping. Single-nucleotide polymorphisms (SNPs) are the most abundant type of marker and most amenable to high-throughput technologies and can be used for marker-assisted selection (MAS) and efficient manipulation of important genes and genome regions, e.g., for genes that enhance yield, pest resistance, and drought tolerance. In this study, we have endeavored to develop a genome-wide panel of map-spaced (cM) simplex SNP marker assays. Using the Illumina(TM) CottonSNP63K array, an associated interspecific linkage map, and new AD genome sequence assemblies, we identified candidate SNPs that were [1] low- or single-copy and polymorphic between Upland cotton and donor species, [2] positioned well (spaced) on the linkage map, and [3] possessed a sequence milieu that seemed amenable to PCR-based KASP/PACE assay development, based on in silico analysis. We identified SNPs from 18,000 mapped interspecific SNPs on CottonSNP63K array that are biallelic and can distinguish Upland cotton, Gossypium hirsutum L., from the three germplasm donor species, Gossypium mustelinum, Gossypium tomentosum and Gossypium barbadense, and their corresponding F1s. About 550 SNP assays, either "Kompetitive Allele Specific PCR" (KASP) or "PCR Allelic Competitive Extension" (PACE), were progressively identified according to their linkage map positions, contribution to genome coverage relative to neighboring SNP markers and their functionality in Phase-I evaluation of the assays for genotyping an Upland cotton inbred line 'TM-1', three donor species and F1 hybrids. For each of the 26 linkage groups, we chose 10 or more spaced simplex assays that should collectively suffice for many interspecific breeding needs. While likely useful for much more, this SNP genotyping assay will be especially useful for efficient introgression of wild species germplasm into an Upland cotton genetic background, genetic dissection, analysis, and breeding manipulations. A set of 453 KASP/PACE assays were subjected to Phase-II evaluations using 88 interspecific F2 hybrids (~30 per donor species) with Fluidigm 96.96 Dynamic Arrays(TM) on the BioMark(TM) platform. The results indicated about 70% of the assays enable genotyping of self-generations, e.g., for recovery of true-breeding BC5S1 chromosome segment substitution lines. A pipeline for in silico assembly-based targeted SNP assay development was developed and should facilitate future completion of genome-wide coverage with map-spaced interspecific AD-genome SNP assays.
Upland cotton (Gossypium hirsutum L., 2n=4x=52, 2[AD]?) is a major fiber and oilseed crop that contributes significantly to the livelihoods of millions of people, as well as the economies of the USA and many other countries. Genetic improvements of cotton are needed to enhance sustainability, address evolving pest and pathogen pressures, and meet industrial requirements. However, genetic diversity is low among elite Upland cotton cultivars and breeding germplasm, which constrains the effectiveness of breeding efforts, and renders the crop more vulnerable to emerging biotic and abiotic threats. Genetic diversification efforts involving interspecific hybridization, introgression, and genetic dissection would benefit greatly by the availability of the inexpensive assays for targeted genotyping. Single-nucleotide polymorphisms (SNPs) are the most abundant type of marker and most amenable to high-throughput technologies and can be used for marker-assisted selection (MAS) and efficient manipulation of important genes and genome regions, e.g., for genes that enhance yield, pest resistance, and drought tolerance. In this study, we have endeavored to develop a genome-wide panel of map-spaced (cM) simplex SNP marker assays. Using the Illumina(TM) CottonSNP63K array, an associated interspecific linkage map, and new AD genome sequence assemblies, we identified candidate SNPs that were [1] low- or single-copy and polymorphic between Upland cotton and donor species, [2] positioned well (spaced) on the linkage map, and [3] possessed a sequence milieu that seemed amenable to PCR-based KASP/PACE assay development, based on in silico analysis. We identified SNPs from 18,000 mapped interspecific SNPs on CottonSNP63K array that are biallelic and can distinguish Upland cotton, Gossypium hirsutum L., from the three germplasm donor species, Gossypium mustelinum, Gossypium tomentosum and Gossypium barbadense, and their corresponding F?s. About 550 SNP assays, either "Kompetitive Allele Specific PCR" (KASP) or "PCR Allelic Competitive Extension" (PACE), were progressively identified according to their linkage map positions, contribution to genome coverage relative to neighboring SNP markers and their functionality in Phase-I evaluation of the assays for genotyping an Upland cotton inbred line 'TM-1', three donor species and F? hybrids. For each of the 26 linkage groups, we chose 10 or more spaced simplex assays that should collectively suffice for many interspecific breeding needs. While likely useful for much more, this SNP genotyping assay will be especially useful for efficient introgression of wild species germplasm into an Upland cotton genetic background, genetic dissection, analysis, and breeding manipulations. A set of 453 KASP/PACE assays were subjected to Phase-II evaluations using 88 interspecific F? hybrids (~30 per donor species) with Fluidigm 96.96 Dynamic Arrays(TM) on the BioMark(TM) platform. The results indicated about 70% of the assays enable genotyping of self-generations, e.g., for recovery of true-breeding BC?S? chromosome segment substitution lines. A pipeline for in silico assembly-based targeted SNP assay development was developed and should facilitate future completion of genome-wide coverage with map-spaced interspecific AD-genome SNP assays.
