Investigation of the sugar beet (Betavulgaris l. ssp. vulgaris) microsatellite loci structure to develop a technology for genetic analysis of sugar beet lines and hybrids

The quality control in the course of maintenance and reproduction of sugar beet ( Beta vulgaris L . ssp. vulgaris ) hybrid parent lines upon seed production is highly important. The method of microsatellite analysis seems to be very perspective tool to provide genotyping during breeding and seed production. Different research groups reported about microsatellite loci in the sugar beet genome. However, the implementation of this technique into the breeding process requires the development of robust and high-throughput technology of analysis. To develop a technology for obtaining stable DNA profiles, a more detailed study of the sugar beet genome microsatellite loci is required using a large set of verified breeding material. The sequencing a number of sugar beet genome regions containing microsatellite loci to clarify the nature of polymorphism as well as ability for providing the stable DNA profiles has been made in this study. Together with breeders (Pervomayskaya Selection and Experimental Station, Krasnodar Krai), a collection of 146 sugar beet plant samples was selected, including 28 male-sterile (MS) lines, 28 O-type lines, 82 pollinator lines, 6 hybrids of Russian selection (Azimut, Corvette, Pervomaisky, Rubin, Fregate, Uspekh) as well as Dobrava and Dorothea hybrids. Five plants of each sample were analyzed for 12 microsatellite loci, FDSB 502, FBSB 1001, FDSB 1033, Unigene 27833, Unigene 26753, Unigene 16898, Unigene 17623B, Unigene 15915, Unigene 17923, SB 04, SB 09, and SB 15. Allelic variants of each locus were amplified, cloned into the pAL2-T plasmid vector and sequenced. The results of sequencing the microsatellite loci FDSB 1001, FDSB 1033, Unigene 16898, Unigene 17623B, Unigene 26753, Unigene 17923, Unigene 27833, and SB 04 revealed that their length polymorphism is solely due to the different number of tandem repeats in the amplified DNA fragment. The locus Unigene 15915 was excluded from further work because of insertions and deletions in the flanking regions of microsatellite repeats (AC)n in its allelic variants. The polymorphism of allelic variants of the microsatellite loci SB 09, SB 15, and FDSB 502 is due to the complex (composite) repeats. Nevertheless, the SB 09 and SB 15 loci were approved for further study, since they produced stable DNA profiles. The allelic variants of the locus FDSB 502 contained the (TC)n(GAT)n(AAG)n sequence, which in some cases may complicate the analysis. To use this locus for the genetic analysis of sugar beet lines and hybrids, we propose the primers flanking only variable microsatellite repeatsб the (GAT)n and (AAG)n separately. The results we report here are prospective to develop a technology for the genetic analysis of sugar beet lines and hybrids as a reliable tool for both breeding and seed production.