Analysis Of DNA Methylation In Different Ploidy Maizes And Z. Perennis And Their F1 Hybrids

Maize wf9, autotetraploid maize Twf9, Z. perennis 9475, allotriploids MP30 and PM30, allotetraploids MP40 and PM40 were used as the experimental materials in this study. Allotrip-loids were created by reciprocal crossing between wf9 and 9475. Allotetraploids were created by reciprocal crossing between Twf9 and 9475. We used cytogenetic techniques to identify and select 9475, Twf9, MP30, PM30, MP40 and PM40. Genomic in situ hybridization was used to analyze chromosome composition of selected Fl hybrids. On this basis, we used AFLP and MSAP tech-nique to analyze inheritance and variation of DNA sequence and DNA methylation during the chromosome doubling of maize and wide hybridization between maize and Z. perennis. We tried to reveal the strategy and regular pattern of genetic and epigenetic as genomic shock as well as use the epigenetic mechanisms in wide hybridization and polyploidy breeding. The main results are as follows:1. The materials must meet the conditions that 10 Zea mays chromosomes and 20 Zea perennis chromosomes were contained in the reciprocal hybrids of MP30 and PM30,20 Zea mays and 20 Zea perennis chromosomes were contained in the reciprocal hybrids of MP40 and PM40, and no translocation phenomenon occurred between the maize chromosomes and 9475chromosomes in allotriploids and allotetraploids. Screening more than five plants of variety materials that meet the above criteria were cultivated.2. The results of DNA sequence polymorphisms comparative analysis between parental and F1 hybrids showed that no losing fragments in wf9 and an emerging band existed in Twf9 when wf9 were doubled to Twf9. The genetic similarity coefficient is 0.9982 between wf9 and Twf9. DNA sequence variation is very small when maize doubling from wf9 to Twf9. Most of the parent fragments were inherited and a small part of them showed variation when maize reciprocal cross with Z. perennis to create allotriploids and allotetraploids. In all genetic fragments, genetic proba-bility of common parent fragments is bigger than unique parent fragments. Variation of DNA fragments usually reflected in fragments missing of parent, especially in unique parent fragments. When the same material was crossed as male in distant hybridization process, the probability of DNA sequence variation is greater than as female. The genetic similarity coefficient was over 0.9781 between four newly synthesized F1 hybrids.3. The results of DNA methylation status analysis showed that manly CCGG sites revealed no-methylation status. In all methylation status, fully methylated sites (inner Cs) were predomi-nantly higher percentage than other methylation patterns. Analysis of monomorphic fragments and polymorphic fragments showed that most fragments were monomorphic, but also had a small part of polymorphic fragments between the repeat units. Analysis of DNA Methylation level showed that DNA Methylation level of Twf9 was slightly lower than wf9. The DNA Methylation level of four F1 hybrids is significant higher than APV. There was no significant difference in methylation level between four F1 hybrids.4. The inheritance and variation of monomorphic fragments have been investigated in our study. It was found that a great majority of the methylation sites in parents manifested faithful Mendelian inheritance to offspring and a few of them exhibited variation or non-additive. Genome duplica-tion of maize not only caused CCGG sites from methylated to unmethylated, but also caused CCGG sites from unmethylated to methylated. The main alterations of DNA methylation were that diagnostic fragments with unmethylated CCGG sites and common parental fragments with different methylation types during wide hybridization. An amount of methylation mutation of CCGG sites affected by TCM or TCdM pathway in the F1 hybrids.5. Sequencing results of directed alteration fragments showed that CCGG sites located in inter-genic region or intragenic region, and the intragenic CCGG sites mostly located in the exon region. One CCGG site occurred demethylation when the maize chromosome doubling. The site is located gene which may affect the synthesis of cytokinins. This may be related to the ploidy advantages which were emerged in Twf9. There were some CCGG sites which had different methylation pat-terns between allotriploids and allotetraploids. These sites are located or upstream or downstream gene which may affect the synthesis of cytokinins, zinc finger protein activity, serine/threonine kinase synthesis gene activity and ATP6 gene activity.