The Study Of DNA Methylation Polymorphism And Genetic Diversity And Copia-like Retrotransposon Reverse Transcriptase Of Annual Wild Soybean (Glycine Soja) And Cultivated Soybean (G.Max)

Soybean belongs to the Glycine of subfamily of Papiliomatae, family Leguminosae. Annual wild soybean (G. soja) and cultivated soybean (G. max) are the two species of Glycine. Compelling evidence has established that cultivated soybean was domesticated from annual wild soybean (G. soja) in China. A good many of wild soybean variations are formed under natural selections. Rare genes in these wild soybean variations may be pivotal for soybean breeding. So, wild soybean is the precious resource to soybean improvement and biotechnology study. Cultivated soybean has been domesticated and cultivated for more than 5000 years in China, and similar to wild soybean, distributes throughout China. In the long domestication history, cultivated soybean also forms variations associated with environment. To study DNA methylation polymorphism and genetic diversity in soybean, 20 and 27 lines respectively of annual wild and cultivated soybeans were selected from Jilin Academy of Agricultudral sciences, and subjected to MSAP and AFLP analysis. Thirteen primer pairs of MSAP generated 984 CG/CNG methylation sites across the 47 lines, and the MSAP data were dissected into methylation-sensitive and -insensitive polymorphisms, MSP and MISP. The calculated MSP vs. MISP for wild and cultivated soybeans are respectively 34.65%vs. 34.76% and 47.05% vs. 47.15%, indicating higher levels of MSP and MISP in cultivated than in wild soybeans, a result incongruent with AFLP analysis of the same soybean lines. These results provide a testable empirical evidence for the hypothesis that diverse epigenotypes may result from"bottlenecked"genotypes due to occurrence of stress-induced or stochastically occurred epiallels that are heritable and visible to selection. All three markers, MSP, MISP and AFLP enabled clustering of the lines into two distinct groups each predominantly containing wild and cultivated ones. The clear clustering of wild and cultivated soybean lines into two distinct groups based on both MSP and MISP suggested that there exist species-specific DNA methylation polymorphism along with genetic polymorphism (e.g., the AFLP results) in soybean, which once occurred are heritable and likely being selected for, and contribute to genetic and epigenetic differentiation of the two soybean species. But because of introgression between wild soybean and cultivated soybean, 2 to 4 cultivated soybean cluster into the wild group in MSAP analysis. Homology search indicated that 12 out of 24 sequenced MSPs had significant similarities to known-function or predicated genes. No significant association between MSP and MISP or MSP and AFLP was detected, suggesting that the mechanisms of generation and maintenance of these two polymorphisms are largely independent. Relevance of DNA methylation polymorphism in soybean evolution and domestication is discussed.copia-like retrotransposons are class I transposable element with long terminal repeat, which transposes via RNA intermediate. This type of retrotransposons is the most abundantly occurred in plant genomes. copia-like sequences were amplified from two annual wild soybean (G. max) genomic DNA, by PCR with degenerate oligonucleotide primers corresponding to highly conserved domains in the copia-like retrotransposons. PCR fragments of roughly 246bp were isolated and cloned, and 131 clones were obtained. According to alignment and phylogenetic analysis, the sequences were divided into nineteen groups. Among these groups, seventeen each contained multiple sequences in the phylogenetic trees and two had only one sequence in each respectively. These 131 sequences from the two wild soybeans are so similar that 12/19 groups composed of mixed sequences. The nucleotide sequence similarity shown by computer analysis between inter- and intra-group was in a wide range, which indicates high level of sequence heterogeneity among these clones and groups. Among these sequences, 63 clones had unique nucleotide sequences (intact sequences), and the others had a frameshift, a stop codon, or both. These results suggest that every group evolved from an ancestor sequence. And the"intact"sequences suggest these sequences can be activated potentially. These sequences are all located in non-centromere regions. Southern blot analysis revealed distribution of these sequences in another 20 wild soybean (G. soja) lines and 30 cultivated soybean lines (G. max), which proved a close relationship between these two species of soybean. The reason of these results maybe the sequences are young in evolutionary history or the reduction rate of LTR retrotransposons by homologous recombination in soybean is slower than other plants. Methylation analysis showed there is no difference in levels of methylation in wild soybean and cultivated soybean. They both have high methylation members/sites and low level methylation members/sites. These results prove that one of DNA methylation functions is maintenance the genome stabilization by repressing the transposable element activity again.