Genome Variation And DNA Methylation Analysis Of Natural And Artificial Triploid Loquats

The utilization of heterosis included that of diploid,polyploid,and functional gene ascendancy.It is an inevitable result that all living beings evolve from monoploid to polyploid.So the exploitation in heterosis of field crops in the future will be at polyploidy level.New phenotypes often appeared on polyploid plants,like drought-resistance,asexual propagation,disease and insect resistance,blooming date, organ size and biotic variations,and so on,which would made it to fit itself or agricultural production.Triploid heterosis performs especially obviously on plants cultivated for vegetation and propagated by agamic means.Triploid loquat shows growth traits heterosis in growth potential,shoot diameters,size and thickness of leaves,leaf organization structure,floral organ and fruit characters,etc.At present,the study on mechanism to form heterosis carried out in structure of genomic and epigenetic variation.And DNA methylation analysis is being the important and hot pot territory.In this thesis,natural and artificial triploid Ioquats,including 13 individuals and their parent plants were used as materials.On one hand,genomic variations showed up in the process of recombination and triploidization were studied in natural and artificial triploid loquats;On the other hand,their DNA methylation level and heredity patterns were statistically analyzed.The main results were as follow.(1)Artificial triploid loquats were obtained by sexual hybridization between tetraploid "jiefangzhong" and diploid "hunanzaoshu".But none was obtained by crossing of tetraploid as male parent and diploid as maternal plant due to cultural management.All materials were F₁ hybrids obtained by sexual hybridization that tetraploid was maternal plant and diploid was male plant.9 of the 30 F₁ seeds gained were triploids by chromosome counting,and the rate was 30%,other seeds contained 2 diploids,3 tetraploids and 16 aneuploids,the rates of them were 6.7%,10%and 53.3%. The F₁ triploids were carefully cultured,and their shoots thickened,their leaf sizes were increased and leaf colour got dark green.(2)Results of GISH in artificial triploid loquats showed that the source of hybrid somatic chromosomes can be accurately distinguished with genome DNA of one parent as probe,and that of the other parent as blocking DNA for 40 times concentration of probe.The results showed that 17 chromosomes were from male parent,and 34 from maternal parent.No conspicuous chromosome structure variation such as interchange and inversion was found.(3)The results of ISSR and AFLP analysis in 6 natural triploid loquats and their maternal parent indicated that:1540 bands were amplified by 11 ISSR primers,one primer amplified 140 bands on average,and 9 primers amplified 26 diversity bands,difference rate was 1.7%. Polymorphic bands indicated 3 types that included added,lost and brightening bands. The numbers of these three types were 11,13 and 2 respectively,the rate was 42.3%, 50%and 7.7%.Every natural triploid line had difference on types and number of sites with maternal parent,increased 1-3 new bands,1.83 on average;bands lostl-3,2.17 on average;bands brightening were only found one site each in A386 and A322. According to the difference between triploid lines with maternal parent from large to small,that was A368,A322,A35,A348,A369,A313.But in general,the percentage of difference bands percent was lower,only 1.7%.So we could presume that triploid genome may mainly be origin from maternal parent,that is to say,the process of triploid formation was likely to relate to 2n female gametes.12 pairs of AFLP primers amplified 2454 bands,one couple of primers amplified 204.5 bands on average.Contrasted with maternal parent,triploids had 208 difference bands,the rate was 8.5%.There were 2 types,added and lost bands,had 112 bands and 96 bands respectively,the rate was 53.9%and 46.1%.According to the number of the difference sites between the triploid with maternal parent from large to small,that was A369,A348,A35,A368,A322,A313.There were different and same result between AFLP and ISSR analysis.â‘ The same result was that the genome difference between A35 and its maternal parent occupied the middle among the 6 lines,and A313 had the least difference with its maternal parent.Otherwise,the difference between A368 and A322 and their maternal parent were similar,the same with A348 and A369.â‘¡The different result was that ISSR analysis showed large difference between A368 and A322 with their maternal parent,small difference in A348 and A369,but AFLP got just the reverse.The diference in detection efficiency and range of these two methods may be the reason for this discordance.But the deduction on triploid formation by AFLP technique was same as ISSR because of the low polymorphism.(4)The results of ISSR and AFLP analysis in artificial triploid loquats suggested that:11 ISSR primers amplified 1989 bands,one primer amplified 180.8 bands on average.To contrast with parents,9 primers amplified 33 diversity bands,and these diversity bands displayed 4 types that included added,lost,agnate and maternal bands. The numbers of sites for these four types were 1,4,9 and 19 respectively,the rates was 3.0%,12.1%,27.3%and 57.6%.Thus it is clear that the genome of hybrid progenies are much more similar to maternal parent which supplied more chromosomes.But added and lost bands different from both parents proved a few genome variation.In 7 triploid lines,hybrid No.6 and No.3 had more differences with parents,each had 7and 6 difference sites,and some bands which neither parents possessed occurred in both of them.The number of difference sites on the other 5 lines was 4 and no added or lost bands on these lines.The AFLP analysis results of 7 artificial triploid lines showed that 12 pairs of primers were amplified 3122 bands,one couple of primers amplified 260.2 bands on average,and there were 294 different bands,the rate was 9.4%.Different bands turned up 4 types,those were added,lost,agnate and maternal bands.The numbers of the sites for these four types were 82,58,49 and 105 respectively,the diference rates was 27.9%,19.7%,16.7%and 35.7%.Thus it is clear that genome variation occurred in the process of artificial triploid loquats formation,there were a certain proportional bands added and lost.According to the numbers of the polymorphism sites between hybrids and parent plants from large to small,that was hybrid No.8,hybrid No.4, hybrid No.1,hybrid No.6,hybrid No.19,hybrid No.3,hybrid No.2.Contrasted with ISSR,AFLP has higher amplification efficiency.(5)The outcome of DNA methylation analysis in 6 natural triploid loquat lines and their maternal plant showed that 12 primers amplified total 3879 bands together, there were 363 full methylated bands,241 hemimethylated bands and the total bands was 604.Total methylation ratios of 6 triploid lines were 12.9%-18.3%,15.8%on average,full methylation ratios were 7.6%-11.7%,9.7%on average,hemimethylation ratios were 4.7%-6.1%,5.6%on average.The methylation ratios,full methylation ratios and hemimethylation ratios of diploid maternal plant were 14.1%,7.7%and 6.5%respectively.According to DNA total methylation ratios of triploid lines from high to low,the sequence was A348(18.3%),A35(17.8%),A313(16.0%),A369(15.7%),A322(14.3%),A368(12.9%).Except A368 reduced contrasted with maternal plant,that of the other 5 natural triploid lines all raised.The sequence of full methylation ratios from high to low was A348(12.5%),A35(11.7%),A313(10.1%),A369(9.9%),A322(9.6%),A368(7.6%),it is the same with toal methylation ratios that A368 reduced,and the other 5 all raised.Contrasted with maternal parent, hemimethylation ratios of all triploid lines turned to cut down,A35 was the highest, 6.1%,next were A313,A348,and A369,5.8%,A322 was the lowest,4.7%.There were 14 types in DNA methylation of 6 natural triploid lines and their maternal parent,belong to four patterns that was monomorphism(A1andA2), demethylation(B),hypermethylation(C),hypomethylation(D),those number of sites and frequency were 251 and 29.2%,171 and 19.9%,334 and 38.9%,103 and 12.0% respectively,and all of them exisited in all triploid lines.According to the numbers of the different patterns from large to small,the sequence was A348,A35,A322,A313,A368 and A369.The main variations occurred of A313 were demethylation and hypermethylation,those of the other 5 lines were hypermethylation.Generally speaking,in the process of genome recombination and triploidization in natural triploid, there proceeded a great number of hypermethylation,then demethylation, accompanied hypomethylation and maintained methylation hereditary patterns.(6)The outcome of DNA methylation analysis in 7 artificial triploid loquat lines and their tetraploid and diploid parents showed that 12 primers amplified total 5302 bands,there were 838 toal methylation bands,605 full methylation bands,233 hemimethylation bands,polymorphism ratio was 15.8%.Total methylation,full methylation and hemimethylation ratios of maternal plant were 17.6%,11.5%and 6.2%respectively,those of male parent were 16.9%,11.1%and 5.9%.Total methylation ratios of 7 triploid hybrid F₁ were 13.2%～17.8%,15.4%on average,full methylation ratios were 10.5%～12.2%,11.7%on average, hemimethylation rate were 2.2%～5.5%,3.9%on average.Contrasted with parent plants,we can summarize as follow,â‘ toal methylation ratios of artificial triploid loquats transmutation tendency was not obvious,which was possible to raise,reduce or maintain between two parent plants.Hybrid No.19 had raised(17.8),hybrid No.3 had maintained between two parent plants(17.6%),others had cut down,the ratios were 13.2%-16.8%.â‘¡full methylation ratios took on the same law with toal methylation ratios,but most hybrids full methylation ratios had raised,including hybrid No.1(11.7%),hybrid No.3(13.5%)and hybrid No.19(12.3%),then hybrid No.4(10.9%) and hybrid No.6(10.5%)had reduced contrasted with parent plants,and the other two maintained between their parent plants,hybrid No.2(11.5%)and hybrid No.8(11.4%).â‘¢Hemimethylation ratios turned out the same with that of natural triploid,all had cut down,the hemimethylation ratios of artificial triploids were 2.2%-5.5%,lower than that of their parent plants,6.2%and 5.9%.There were 51 types of DNA methylation patterns in 7 artificial triploid lines and their parent plants,including monomorphism(A1andA2),demethylation(B), hypermethylation(C),hypomethylation(D)and intermediate pattern(E),frequencies of those were 24.2%,28.8%,38.5%,6.6%and 1.9%respectively.Overall, hypermethylation occurred mainly in the process of gene recombination and triploidization in artificial triploids,then demethylation,accompanied hypomethylation,and methylation maintain and intermediate pattern that methylation state maintained between parent plants.There were 5 methylation types existed in every artificial triploid loquats lines.According to the numbers of the polymorphic loci from large to small,the sequence was hybrid No.3,hybrid No.1,hybrid No.6,hybrid No.8,hybrid No.19,hybrid 4,hybrid No.2.The main variations occurred to hybrid No.3,No.1,No.8,No.19,No.4 and No.2 were hypermethylation,but a devil of hypermethylation and demethylation variations simultaneously occured on hybrid No.6.