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.

[Construction of physical map and polymorphism analysis of mtDNA region R from Mo17CMS-J of maize].

Total DNA from twenty-six CMS lines of maize under Mo17, 77 and W23 nuclear background were used for PCR amplification, including N, T, C, S four groups of cytoplasms. The primers was prefabricated according the sequence of R region published by Zebala (1997). Through these amplifications, mitochondrial DNA fragments were obtained from maize total DNA. Generally the results in one group are identical. And they are different from the others. The amplified fragments were sequenced and also give us much more information about the structure of mitochondrial genes that may lead to CMS. In order to isolate and identify the CMS genes, we developed a new platform to construct physical map of chromosome DNA by means of restriction enzyme double-digestion. The elongation of contigs is based on Southern hybridization. Having retrieved DNA from agarose gel after electrophoresis by beta-agarase, we labelled it with 32P-dCTP as a probe. We detected the positive clones in the gene library. Two contigs were revealed. And a restriction map covering 40 kb was constructed, including R region.

[Progress of molecular biology of CMS in maize].

In the paper,we have summarized the molecular biological accomplishment acquired and accepted by most of maize researchers on CMS of maize. A brief review of current molecular biological progress of CMS of maize are displayed in the paper. These progresses concern in the positioning,cloning and maker-assisted selection of nucleic genes associated with fertility,expression and cloning of cytoplasmic genes associated with male sterility. In order to elucidate the molecular mechanism of CMS of maize, the areas about cloning and expression profiling of male sterile nucleic genes,and functional genomics of mitochondria,and interaction cytoplasmic genes with nucleic genes will need to be researched in the future.