Whole Genome Amplification and Single Nucleotide Polymorphisms from Sweet Sorghum Microspores

Sweet sorghum (Sorghum bicolor L.) has been growing in Tennessee for over a century and recently been recognized as a potential biofuel crop per its production of syrup which is directly fermentable to ethanol. A total of five varieties (Achi Turi, Dale, Dasht Local, Topper 76-6, and RTx 430) were maintained and monitored in a greenhouse per their flowering characteristics. A combined total of 170 free early microspores were harvested and individually isolated in aqueous solution (6% D-sorbitol) for subsequent whole genome amplifications (WGA). WGA yields were quantified using a spectrophotometer which gave UV absorbance (A230/260) readings of 1.8 for single gametophyte DNAs with 2800 - 3000 (ng/µL) which were also confirmed by gel electrophoresis. Twenty single nucleotides polymorphic (SNP) sequences relevant to sorghum production traits, were selected, from a total of 1351 to provide high level of marker reproducibility along with good separation applicability for homo/heterozygote genotypes. High-quality DNAs were extracted from young leaves tips of 15 to 20 days old healthy parental plants and sent to LGC genomics (Teddington, Middlesex, U.K.) for purchasing SNP primers after validations. After conducting KASP assays on above sorghum plants and microspores; SNP density, markers’ segregation, and genetic variations were recorded. The KASP genotyping success rate was 80% for microspores’ DNAs, while it was 91% for the leaf of 78.5 to 88.4% DNA samples. These results also confirmed previously reported KASP assay genotyping success rate for diploid plants