Construction Of Physical Map And Polymorphism Analysis Of MtDNA Region R From Maize Mo17CMS-J

Cytoplasmic male sterility (CMS) is a kind of common phenomena in higher plants and is manifested as the inability of a plant to produce viable pollen; female fertility is unaffected. CMS is a trait of economic importance in the production of hybrid seed as it eliminates the need for emasculation to pollination, thus reducing labor costs. A correlation between CMS and unique arrangements of the mitochondrial genome has been found. Nuclear restorer-of-fertility genes suppress CMS phenomenon to produce normal pollen.Total DNA from twenty-six CMS lines of maize under Mo 17, 77 and W23 nuclear background were used for PCR amplification, including N, T, C, S four groups of cytoplasms. The primers were 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.that is, we could amplify orf77 and R when the templates were DNA from T and S group, no matter what nuclear background they are under. And we could not get anything when the templates were from N or C group. The results can partly confirm Zhang's (2000) conclusion that homologous sequences of R also present in N and T-groups, but not in C group. The amplified fragments were sequenced and also give us much more information about the structure of mitochondrial genes that may lead to CMS.The DNA sequences are almost the same as the sequences in R region of CMS-S. The orf77 in T group was different from that in S group, For T grouphad only part of R region. The heterogeneity of R region suggests that the organization and rearrangement of mtDNA controlled by the nuclear background. R region seems to be involved in the mechanism of S group CMS. Varieties of structures of R, the ratio of three R types, different DNA sequences in R region of different CMS maybe concerned with instability of S group.In order to isolate mitochondrial genes easily, we set up 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 3 -agarase , we label 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 40kb was constructed, including R region.