Study On DNA Methylation Variation Of Cymbidium Hybridium’s Ovary In Different Periods After Pollination

Cymbidium hybridum is one of the main flowers of the international flower market, whichwon the people’s favorite of all over the world. Like other orchids, C.hybridum has specialfeature in its development, which is the ovary development of orchids only starts after successfulpollination and time-consuming. Furthermore, the seeds of orchids are very thin and ateliosis,the endosperm is absent and the germination is difficult in nature. These uniquecharacteristics of orchids make them difficulty in sexual reproduction, which in some wayblocks the basic research of orchids development. In this study, the ovaries of C.hybridiumwere used as research materials, study the ovary unique developmental pattern of C.hybridium inthe level of genetics and epigenetics and understanding the molecular mechanism of ovarydevelopment initiation in orchids after pollination. By the best suitable MSAP markingsystem and methylation sensitive amplification technology, in order to explore the influenceof DNA methylation on C.hybridium’s ovary development. And we strived to discover keygenes that start up the ovary development and the regulation pattern of epigenetics. Theresults are as follows:（1） Comparing with kit method, CTAB method, high-salt CTAB method, optimized CTABmethod and SDS method, we got the optimized CTAB method is the best one for DNA extraction ofC.hybridium’s ovary in the five method mentioned.（2） By theAFLP analysis of DNA sample, the results shows that the DNAprimary structure ofthe ovary genomic did not change during development after pollination, and proved that DNA methylationdoes not change the DNA nucleotide arrangement.（3） MSAP(methylation sensitive amplified polymorphism) analysis was carried out toanalyze differences of the methylation status between ovaries in different periods afterpollination from C. hybridium. The results indicated that DNA methylation events occurredin the whole ovary’s development. After pollination, the DNA total methylation level is42.7%, fullmethylation is31.4%, the level of DNA methylation was curve-like changes. Furthermore, methylationpatterns varied among these ovaries. There are11types of MSAP patterns detected in thestudy belonged to two classes. That distinct patterns of DNA methylation occurred throughdemethylation or de novo methylation may have specialized functions. DNA methylation is animportant regulatory way during the development of C.hybridium’s ovary.（4） After comparison of BLASTN, the two specific sequences have high homology with 16sequences have been reported. These16sequences are derived from the plant genome, andhave relationship with mitochondrial genome. The homology with the mitochondrial genomeof Brassica plants is40%, with Arabidopsis thaliana’s mitochondrial genome is40%, withcucumis sativus cultivar Calypso’s mitochondrial genome is29%.