Development and mapping of SNP assays in allotetraploid cotton |
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Authors: | Robert L Byers David B Harker Scott M Yourstone Peter J Maughan Joshua A Udall |
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Institution: | (1) Department of Plant and Wildlife Sciences, Brigham Young University, Provo, UT 84602, USA; |
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Abstract: | A narrow germplasm base and a complex allotetraploid genome have made the discovery of single nucleotide polymorphism (SNP)
markers difficult in cotton (Gossypium
hirsutum). To generate sequence for SNP discovery, we conducted a genome reduction experiment (EcoRI, BafI double digest, followed by adapter ligation, biotin–streptavidin purification, and agarose gel separation) on two accessions
of G. hirsutum and two accessions of G. barbadense. From the genome reduction experiment, a total of 2.04 million genomic sequence reads were assembled into contigs with an
N50 of 508 bp and analyzed for SNPs. A previously generated assembly of expressed sequence tags (ESTs) provided an additional
source for SNP discovery. Using highly conservative parameters (minimum coverage of 8× at each SNP and 20% minor allele frequency),
a total of 11,834 and 1,679 non-genic SNPs were identified between accessions of G. hirsutum and G. barbadense in genome reduction assemblies, respectively. An additional 4,327 genic SNPs were also identified between accessions of G. hirsutum in the EST assembly. KBioscience KASPar assays were designed for a portion of the intra-specific G. hirsutum SNPs. From 704 non-genic and 348 genic markers developed, a total of 367 (267 non-genic, 100 genic) mapped in a segregating
F2 population (Acala Maxxa × TX2094) using the Fluidigm EP1 system. A G. hirsutum genetic linkage map of 1,688 cM was constructed based entirely on these new SNP markers. Of the genic-based SNPs, we were
able to identify within which genome (‘A’ or ‘D’) each SNP resided using diploid species sequence data. Genetic maps generated
by these newly identified markers are being used to locate quantitative, economically important regions within the cotton
genome. |
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