Real time PCR mediated determination of the spontaneous occurrence of Sorghum bicolor alleles in wild sorghum populations

Abstract

The study evaluates the utility of Real Time PCR (RT-PCR) in quantitative and qualitative analysis of alleles in sorghum populations and the spontaneous occurrence of Sorghum bicolor alleles in wild populations of sorghum. Leaf and seed material from wild sorghum accesions were sampled in Homabay, Siaya and Busia counties to represent Western Kenya sorghum producing regions. A second sampling was done on S₂ populations of S. bicolor, Sorghum halepense and Sorghum sudanense maintained in the greenhouse. Crop loci were evaluated in all materials using a LightCycler® 2.0 system. Real Time PCR was effective in qualitative and quantitative determination of crop alleles in both crop and weedy backgrounds of S. sudanense, S. halepense and S. verticilliflorum. Crossing point values ranged between 19.7 from 30 ng template to 35.9 from 0.015 pg of template on locus SB1764. Melting peaks analysis ranged between 83.29 to 88°C on locus SB1764 and between 86.01 to 80.88°C on locus SB3420 effectively differentiating the 4 species. RealTime-PCR was successful in quantitative and qualitative analysis of specific crop alleles from loci SB1764 and SB3420 from seed and leaf DNA. Spontaneous occurrence of crop and rare alleles in wild sorghum populations growing in sympatry with crop cultivars showed the presence of crop and rare alleles in wild sorghum populations. Means of wild populations from lower midland₁ (LM₁), LM₂, LM₃ and LM₄ AEZs were not significantly different. It is therefore vital to test S. bicolor seeds and other plant materials in transit, at entry points and populations of growing plants for foreign genes including transgenes using RT-PCR. 

Keywords: Real time polymerase chain reaction (PCR), Sorghum bicolor, Sorghum halepense, Sorghum sudanense.