Expression analysis of orf77 and R region in mitochondrial DNA of S-type CMS maize

It is generally recognized that S type of CMS in maize associated with the recombination region R in mitochondria. Complicated DNA recombination and changes of transcripts of R are observed in several subgroups of S cytoplasm. R region includes two open reading frames (orf355 and orf77) and they are all chemeric. Among the sequence of orf77, there are three stretches similar to atp9 of mitochondria. Many different S cytoplasms are found in maize in China and researches on them show that similar R region and sequence are also found in the mitochondrial DNA in Tangxu, Shuang and J (cytoplasm. Northern analysis of Tangxu, J cytoplasm with Probe R and orf77 indicates that orf77 co-transcribes with R region and the nuclear restorer gene Rf3 affects their expression. In all tested S cytoplasm plants with the genotype of s-rf3rf3, both probe orf77 and R can detect six transcripts of 2.8, 1.6, 1.1, 0.9, 0.7 and 0.4 kb. In the presence of the nuclear restorer-of-fertility gene Rf3, previous transcripts of 2.8 kb and 1.6 kb are disappeared, but the other four transcripts are not changed. In T and C cytoplasm of CMS, only four transcripts of 1.1, 0.9, 0.7 and 0.4 kb appear when hybrid to probe R. Further Northern analysis of probe atp9 proved that all the four transcripts of 1.1, 0.9, 0.7 and 0.4 kb were actually transcripts of gene atp9, so only the transcripts of 2.8 kb and 1.6 kb are particular to R region of S. In addition, atp9, atp6 and cox II each has the same transcription pattern in tassel with different genotypes respectively. It can be included that R region and orf77 are the most important candidate gene for S-CMS.