DNA Fingerprinting of Individual Microspores from Upland Cotton Chromosome Substitution Lines
Cotton is an important dual use plant for fiber and nutritive seed production, thus innovative genetic improvement procedures are needed for this important crop. Single gamete, including microspores, based genotyping can be used for genetic studies in cotton. The use of single gametes is very useful for identification of genetic makeup of plants as well as true to type identification. Such approach is very useful for enhancing breeding efficiencies, since being cost effective and time saving. The microspore developmental stages, that would allow DNA yield, needs to be identified and correlated to flower bud lengths for harvesting of appropriate samples. Microspores held as tetrads as well as just released at early uninucleate stage, have less developed cell wall and thus can be used to release their DNAs. Early uninucleate microspores stages, just after being released from tetrad, were identified and correlated to flower bud sizes for the three cotton lines. The appropriate microspore stages were found for 3-79, CS-B17 and TM-1 in flower buds of about 9.43mm, 7.05mm and 6.88mm sizes, respectively. The requisite microspores from these three cotton lines were isolated from samples prepared in 27% D-sorbitol and placed individually in separate PCR tubes using a microinjector mounted on a micromanipulator. The microspores genome was then amplified by using multiple displacement method, which was followed by simple sequence repeats (SSR) based analyses of individual gametes. The near isogenic nature of CS-B 17 was confirmed by observing lack of segregation of the parental SSR markers in its microspores. ^
Shreya Singh Hamal,
"DNA Fingerprinting of Individual Microspores from Upland Cotton Chromosome Substitution Lines"
ETD Collection for Tennessee State University.