Polymorphism of grain sorghum from VIR world collection for the characters associated with the CMS-RF genetic system

Seven different types of cytoplasmic male sterility (CMS) are known for the grain sorghum ( Sorghum bicolor L. Moench), however, only A1 (milo) is used in heterotic hybrid breeding. The genetic control of pollen fertility restoration of CMS A1 is complex and determined by two or three Rf ( Restoration of Fertility ) genes, and also by a number of modifiers. It is very little known about molecular mechanisms of CMS A1 and fertility restoration. Only one gene, Rf1, is identified at the molecular level (R.R. Klein et al., 2005). In the present paper we have demonstrated for the first time the nucleotide polymorphism in the coding regions of the recessive and dominant alleles of Rf2 gene and also of the candidate RFL-PPR gene homologous to the rice Rf1 gene.. Here, we studied polymorphism of the CMS- Rf genetic system related traits in sorghum accessions from the VIR collection, including the fertility restorers k-928 and k-929; a half-restorer k-1362, the sterile lines A-10598 and A-83 (CMS A1) and their fertile analogs, the F8-F12 BC1-BC2 sister lines resistant to Schizaphis graminum Rond. which have been isolated among the hybrids derived from crosses between the sterile (CMS A1) line N-81 and lines k-929 and k-928, and also hybrids between the sister lines. For investigating variability of candidate genes associated with the CMS- Rf genetic system the reference sequences were selected from the bioinformatic database (http://www.ncbi.nlm.hih.gov), the eighth specific primers were designed, and the fragments amplified on the DNA of genotypes differing by the ability to suppression of the CMS phenotype were sequenced. In the CMS lines and fertility restorers a significant polymorphism (18 polymorphic sites) was revealed in the 825 bp fragment of the Rf2 coding region (reference fragment XM_002459403.1, chromosome SDI02) and also in RFL-PPR candidate gene located in the chromosome 3 (reference fragment XM_002458104.1). The sequenced regions of the structural nuclear gene ALDH2b encoding mitochondrial aldehyde dehydrogenase, the maize Rf2 gene homolog, and also of the mitochondrial F0F1 ATPase alpha subunit were identical in the CMS and fertility restorer lines. Variability of pollen fertility indices was studied using acetocarmine stained cytological preparations. The lines resistant to S. graminum, and their hybrids differed in the percentage of stained (fertile) pollen grains, the presence of anomalous large pollen grains (54-70 mm in diameter), giant pollen grains (up to 84 mm in diameter) and deformed pollen grains. In the fertile F8-F12 BC1-BC2 lines which derived from the hybrids produced in crossings with fertility restorers, the frequency of stained pollen grains was relatively high and reached 72.2-83.8 % for k-929, and 57.4 and 63.4 % in two lines, respectively, for k-928; large pollen grains occurred at different frequency in five lines, and the giant ones were observed in two lines. The variability in pollen fertility could be due to the differences in the alleles derived from the recurrent parent.