Genomic DNA Verification in Single Gametes of Cotton Chromosomal Substitution Lines through SSR and AFLP Markers
Cotton (Gossypium) brings enormous humanitarian and economic benefits through fiber, food and feed. Upland cotton (G. hirsutum) which constitutes 90% of production levels in the world requires to be improved upon since favorable alleles have been found to be fixed in the elite breeding gene pool. This required introgressing genes from other tetraploids i.e. G.barbadense, G. tomentosum, and G. mustelinum which have very unique and favorable effects to significantly broaden the genetic diversity of Upland cotton. In this study, two chromosome substitutions (CS) lines of Upland cotton with Pima cotton foreground were used. These two lines which had chromosome pairs 17 (CS-B17) and 25 (CS-B25) replaced by the respective pair from G. barbadense doubled-haploid line 3-79 were used for pollen DNA analyses. The primer extension pre-amplification (PEP) protocol was also successful on the pollen grains. The use of pollen is a very useful strategy to enhance breeding efficiencies due time and space savings. Mature individual pollen grains were collected and isolated from the Upland cotton chromosome substitution lines (CS-B17, CS-B25), germinated in a media and subjected to a PEP protocol to increase the minute quantity of DNAs. DNAs were also extracted from leave tissues of CS-B17, CS-B25 as well as their parents, TM-1 and 3-79. 20 microsatellite (simple sequence repeat) based DNA markers ranging 150-300bp amplified from the leave tissues of the two CS-B lines and their both parents were employed to confirm the near isogenic nature of the CS-B lines through pollen analyses. Seven SSRs markers were detected from the DNAs of CSB-25 pollen samples while only one microsatellite marker was amplifiable from CS-B17 pollen grains. 29 amplified fragment length polymorphism (AFLP) primer pairs labelled IRD-800 and IRD-700 (Li-Cor, Lincoln, NE) fluorescent dye were used to analyze the pollen samples as well as the parental lines. The near isogenic nature of the CS-B lines was confirmed by observing lack of the segregation of both the SSR and AFLP markers in the pollen populations.
Abdul Mujeed Yakubu,
"Genomic DNA Verification in Single Gametes of Cotton Chromosomal Substitution Lines through SSR and AFLP Markers"
ETD Collection for Tennessee State University.