Responses Of Genomic DNA Methylation To Chromosome Doubling In Maize And Wide Hybridization Between Maizes And Z. Perennis

In this study, diploid maize, autotetraploid maize, the allotriploids and allotetraploids which are hybrids of diploid maize and autotetraploid maize reciprocal cross with Z. perennis (9475) respectively were used as the experimental materials. We used AFLP (amplified fragment length polymorphism) and MSAP (methylation sensitive amplification polymorphism) to analyze the inheritance and alteration of genomic DNA methylation during the chromosome doubling in maize and wide hybridization between maizes and Z. perennis in order to reveal the DNA methylation strategy as a genetic buffer against genomic shock in Zea mays and investigate the potential relationship between phenotypic variation and DNA methylation, which would provide a theoretical foundation for screening of epigenetic mark, explaining the epigenetic mechanisms during genome duplications in plants and wide hybridization, as well as wide hybridization and polyploidy breeding. The main results are as follows:1. The analysis results of biological characteristics indicated autotetraploid maize (Twf9) displayed the ploidy effects on plant height, stalk circumference, leaf length and tassel branch number compared with diploid maize (wf9), but the growth period was postponed and the pollen fertility was poorer. The biological characteristics were compared between the allotriploids (DW9and9DW, which are hybrids of wf9reciprocal cross with9475) and allotetraploids (AW9and9AW, which are hybrids of Twf9reciprocal cross with9475). The results showed that allotriploids were perennial, better vegetative growth such that tillering was earlier and more exuberant, but postponed reproductive growth stage and poorer pollen fertility; Allotetraploids were annual, poorer tillering ability, but better reproductive growth such as tassel branch number, tassel length and pollen fertility were significantly higher than those of allotriploids, earlier seed mature, as well as more row number and grain number per ear. No significant difference was observed on morphology between reciprocal hybrids.2. AFLP was used to identify the DNA sequence polymorphism. There was no different fragment between wf9and Twf9, DW9and AW9,9DW and9AW, DW9and9DW, AW9and9AW, and the genetic similarity coefficients respectively were0.934,0.936,0.918,0.975,0.950. It suggested that the DNA primary structure has no significant change during genome duplication in wf9, and there was no different DNA sequence among allopolyploids.3. We investigated the DNA methylation of leaves and roots during withering period by MSAP. The results showed that the proportions of polymorphic methylated fragments respectively were12.84%～14.08%(11.68%～11.78%),11.68%～12.42%(12.76%～12.86%) and12.76%(12.26%) in9475, maizes and allopolyploids leaves (roots). Internal cytosine methylation of double-stranded DNA was the most often of all types, but in roots, the proportions of internal cytosine methylation were higher whereas the proportion of external cytosine methylation were lower than those in leaves.4. Analyzing DNA methylation level of leaves and roots during chromosome doubling in maize and wide hybridization between maizes and Z. perennis, we found that chromosome doubling in maize reduced the level of DNA methylation slightly, but wide hybridization between maizes and Z. perennis increased the level of DNA methylation of allopolyploid hybrids significantly. Methylation level of leaf was significantly higher than that in root. Methylation level of polymorphic methylated fragments was significantly higher than that of the same methylated fragments.5. The model for inheritance and alteration of DNA methylation of the same methylated fragments in the leaves and roots during chromosome doubling in maize and wide hybridization between maizes and Z. perennis indicated that the heritability of DNA methylation in root was higher than that in leaf, the heritability of internal cytosine methylation of double-stranded DNA was highest in leaf, while the heritability of external cytosine methylation of single-stranded DNA was highest in root. The main alterations of DNA methylation were that diagnostic fragments with unmethylated CCGG sites and common parental fragments with different methylation types were methylated during wide hybridization, DNA methylation variation rate as a male parent was slightly higher than that as female parent. Genome duplication caused not only methylation of unmethylated sites but also demethylation of methylated sites.6. Alterations of DNA methylation were directed or random during chromosome doubling in maize and wide hybridization between maizes and Z. perennis. Sequencing results of directed alteration fragments showed that CCGG sites located in intergenic region or intragenic region, and the intragenic CCGG sites mostly located in the exon region. CCGG sites of directed alteration in maize were distributed in the whole genome, and mostly located on chromosome5. Demethylation of CCGG sites which located inside, upstream or downstream of auxin and photosynthesis related genes were caused by chromosome doubling of wf9and might related to ploidy effects of Twf9. Methylation patterns in upstream CCGG sites of ethylene-response and fatty acid synthesis related genes, intragenic CCGG sites of programmed cell death and cell-cycle regulation related genes were different between allotriploids and allotetraploids, and these different methylation patterns might relate to different traits between allotriploids and allotetraploids, such as growth period, seed maturation and perennation.