| Polyploidy has played an important role in the evolution of higher plants,which can evoke new phenotypes to better adapt environment just like drought-resistance,diseases and insects resistance,blooming date,organ size,and so on.DNA methylation is an important epigenetic modification that play an important role incontrolling gene expression and maintaining genomic stability in higher plants.On one hand,we used Amplified fragment length polymorphism(AFLP) molecular marker to analyse the variations of genome in polyploid of Citrus grandis Osb.cv.Shatianyou and Citrus sinensis Osb.cv.Hongjiang;On the other hand,we used Methylation sensitive amplified polymorphism(MSAP) to analyse their DNA methylation level and heredity patterns,and expected to approach the mechanism of inheritance and envolution mode in polyploid citrus,which is singnificance for research the hereditary property,heterosis and ploidy breeding of polyploid fruit trees.The main results were as follow:AFLP analysis of artificial and natural selection of polyploid genome of C.grandis Osb.cv. Shatianyou showed that:912 loci were amplified by 18 pairs of primers,of which polymorphic loci were 382,and the rate was 41.89%.Based on the diploid,the number of discrepancy loci from large to small was X14(266),X18(252),X8(249),X5(243),X1(242),X7(241),X6(240), X4(239),X17(213),X3(213),X15(207),X12(198),X16(195),and the variation rate was between 21%and 29%.That indicated large variation was happened during polyploidy in polyploid genome of C.grandis Osb.cv.Shatianyou,and it was maybe one of the main reason that evoke C.grandis Osb.cv.Shatianyou polyploid phenotypes variation.The result show that, artificial polyploid has a higher aberration rate than natural polyploid,and the strains which have similar trend variation that have the similar origin mode,these suggested that polyploid genome variation is not random. AFLP analysis of polyploid genome of natural C.sinensis Osb.cv.Hongjiang showed that 18 pairs of primers have totally amplified 2107 bands,and each pair elicited 117 bands on average. Among 2107 bands,the disparity bands were 180,and the rate was 8.21%.There were 43 new appearance loci,and the rate was 23.89%,while 137 loci were missing,accounting for 76.11%.In general,disparity bands in the tetraploid strain rough equivalency,X19 for 61(8.74%),X20 for 57(8.14%),X2 for the 62(8.74%).Compared with the diploid plants,8 loci have appeared new bands and 33 loci have deleted at the same time,which suggested that polyploid genome were Similarity.In general,the percentage of difference bands was low,so we presume that Hongjiangcheng tetraploid was formed by doubling the nucellar embryos.The outcome of DNA methylation analysis about 13 natural and artificial polyploid and 1 diploid checking of C.grandis Osb.cv.Shatianyou show that 18 pairs of primers have totally amplified 9688 bands,in which methylated bands were 2368,including 1369 full methylated bands and 999 hemimethylated bands,and the polymorphism was 24.4%.the total methylation ratio of all tetraploid strains was between 21.6%～30.2%,and 23.8%on average;Full methylation ratio was between 11.4%～15.8%,13.9%on average.The total methylation rate of Natural triploid X12 was 26.0%and the full methylation rate was 15.1%.Compared with the diploid plant total methylation rate of 26.0%and full methylation rate of 16.9%,there was little difference.However,methylation patterns have changed a lot from the diploid parents,including A-type,monomorphisml413(36.2%);B-type,demethylation 954(24.5%);C-type, hypermethylation 1057(27.1%);D-type,hypomethylation 437(12.3%).The main variation methylation pattern was hypermethylation,followed with demethylation.The result of methylation analysis in polyploid genome DNA of citrus sinensis Osb.cv. Hongjiang shows that 18 pairs of primers have amplified 2245 bands,of which contain 644 methylated bands,including 328 full methylation bands and 316 hemimethylated bands.The total methylation rates of tetraploid plants were X 19(30.64%),X20(31.01%) and X2(28.24%),higher than the diploid plant(25.04%);the full methylation rates were X19(15.23%),X20(15.05%),X2 (14.21%),a little higher than the diploid female parent(13.99%).The methylation patterns of the tetraploid plants have changed a lot,in which the main type was hypermethylation(C-type), representing 33.29%,and then by demethylation(B-type),accounting for 13.45%; hypomethylation(D-type) occupied the least,accounting for 11.98%.The change of methylation patterns directly impacted the polyploidy genome expression.To sum up,the genome structure have variation great in polyploid C.grandis Osb.cv. Shatianyou and C.sinensis Osb.cv.Hongjiang,we presumed that the reason was homologous genetic recombination and exogenous gene introduction.The DNA methylation pattern has adjustments great which to revoke gene expression and regulation variation in polyploid,that make the phenotype,resistance to change. |
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