Genetic Diversity Analysis Of Prunus Mume Sieb. Et Zucc. Based On REMAP And IRAP Molecular Markers

Mei(Prunus mume Sieb. et Zucc.), native to China, is famous for viewing and processing of special healthy food species. The cultivation has a long history and rich germplasm resource. Retrotransposons transpose through an RNA intermediate by a’copy and paste’mechanism and widely distribute and copy in the genome with high heterogeneous populations. It shows high sequence difference and rich insert polymorphism in one or multiple different species which exhibits stable mutation. Compared with normal molecular marker technique, retrotransposons polymorphism molecular marker developed according to above-mentioned characteristics shows the benefits of genome-wide coverage and rich polymorphism. This study aims to explore the genetic diversity, kinship and to provide theoretical basis for effective development and innovation of germplasm resources from the DNA molecule level of germplasm. The results are as follows:1. The genetic diversity of mei analysis based on the mathematic classification of important morphologic characters84 mei varieties (including 43 fruiting mei and 41 flowering mei) located in National mei germplasm pool of Nanjing Agricultural University were taken as test materials. Q clustering, principal component and factor analysis to 31 important morphologic characters such as leaves, flower and fruit with SPSS 11.5,84 mei varieties were divided into 5 groups, including 13 fruiting mei for the first group such as’Changnong17’ ,30 fruiting mei for the second group such as’Hongmei’ ,15 flowering mei for the third group such as ’Chongyemei’,9 flowering mei for the fourth group such as’Yinhongzhusha’,17 flowering mei for the fifth group such as’Xiaolve’. Fruiting mei and flowering mei clustered respectively. The’Touguhong’of fruiting mei gaultheria preference flowering mei.10 morphologic characters among 31 morphologic characters, like L-fruit, b-fruit, fruit across diameter, weight of per fruit, fruit side diameter, stone weight, petal numbers, petal layers, a-fruit and fruit high play an important role to mei mathematic classification.2. The genetic diversity analysis of mei based on REMAP markerThrough studying on DNA purification methods, five factors L16 (45) 4 levels completely random orthogonal test, the various PAGE concentrations, base of primer 3’, and so on, the final optimal system of REMAP marker technique was established. REMAP system was totally in 25μl, composed of template DNA 40 ng,2.5 mmol/L MgCl2 1.5μL, 2.5 mmol/L dNTPs 2.0μL,10×Buffer 2.5μL, Taq DNA polymerase 1.5 U,10μmol/L LTR primer 1.0μL,10μmol/L SSR primer 1.0μL. Once cleavage was the best of the four treatments in quality of template DNA. The selective base at 3’-end of SSR primer influenced on REMAP notably. The best was one base on 3’-end of SSR primer.5% PAGE was the best concentration of non-denaturing polyacrylamide gel, showing the most and clearest electrophoresis line and could present genetic diversity and had the characteristic of convenience and no crash.5 couples of LTR-SSR primers are screened out for REMAP genetic diversity analysis to 84 mei varieties and produced a total of 122 polymorphisms. For 5 couples of primers, the Shannon’s information index was from 0.3897 to 0.6364, Nei’s gene diversity from 0.2395 to 0.4455. UPGMA clustering to REMAP amplification results,84 mei varieties can be divided into 18 groups at the similarity coefficient of 0.766, fruiting mei and flowering mei were clustered in different group. ’Xiaoyezhugan’ was not in the same cluster with ’Dayezhugan’ because of distinct genetic differentiation. ’Xiaoyezhugan’ had spesical bands of 198bp and 600bp bands under REMAP, which may related to the fact that retrotransposon insertion causing the differentiation. It was proved by PopGen32 that Japanese fruiting mei ware introduced from Zhejiang province and Japanese flowering mei from Jiangsu province of China. Three layers of petal was the jumping-off point for genetic variation,3-5 layers genetic stability. One layer of petal in fruiting mei and 5 layers in flowering mei had obvious genetic diversity. Among 84 mei varieties, the least of genetic distance was 0.059 between’Shuangfencuizhi’and ’Fenpigongfen’and the farmost one was 0.63 between’Yunnanhongmei’and’Dali’. Among fruiting mei, the least one was 0.104 between’Huangxiaoda’and’Fujianbaifen’and the farmost one was 0.487 between’Siyuemei’and’Koumeshinano’. Among flowering mei, the least one was 0.059 between’Shuangfencuizhi’and’Fenpigongfen’and the farmost one was 0.556 between’Zhongyugongfen’and’Yunnanfenghou’. The genetic flower strength group transfer number for varieties group of mei was the largest, which illustrated varieties group of mei had genetic diversity in abundance.3. The genetic diversity of mei analysis based on IRAP markerThrough five factors four levels L16 (45) completely random orthogonal test, IRAP molecular marker system was established for mei. IRAP system was total in 25μl, composed of template DNA 30 ng,2.5 mmol/L MgCl2 2.0μL,2.5 mmol/L dNTPs 2.5μL, 10×Buffer 2.5μL, Taq DNA polymerase 1.0 U,10μmol/L LTR primer 1.0μL.6% and 8% PAGE were the better concentration of non-denaturing polyacrylamide gel and could present genetic diversity. Six LTR primers were screened out for IRAP genetic diversity analysis to 84 mei and produced a total of 99 polymorphisms, and the average polymorphism ratio was 83.20%. For 6 primers, the Shannon’s information index was in the range from 0.3359 to 0.5807, Nei’s gene diversity in the range from 0.1967 to 0.3976. UPGMA clustering to IRAP amplification results,84 mei could be divided into 41 groups at the similarity coefficient of 0.766.’Changnong17’of fruiting mei was clustered together with’Fenpigongfen’,’Xiangxuegongfen’,’Yunnanhongmei’and’Xiaoyugongfen’, Pink double form of flowering mei and’Meirenmei’of flowering mei was clustered together with’Koumeshinano’. The rest fruiting mei and flowering mei were clustered in different group, respectively. Among Japanese fruiting mei,’Gyokouei’,’Shirokaga’,’Gessekai’, ’Gojirou’,’Setsuda’and’Hanakami’were clustered together.’Dayezhugan’was not clustered in the same group with’Xiaoyezhugan’. By PopGen32, Japanese mei was the most similar to Zhejiang mei and Jiangsu mei. It could be confered Japanese fruiting mei was introduced from Zhejiang province in China and Japanese flowering mei was introduced from Jiangsu province in China. Three layers of petal was the jumping-off point for genetic variation,3-5 layers genetic stability. Within 84 mei varieties, the least of genetic distance was 0.107 between’Shirokaga’and’Gessekai’and the farmost one was 0.683 between’Koume’and’Benishidale’. Within fruiting mei, the least one was 0.107 between’Shirokaga’and’Gessekai’and the farmost one was 0.588 between ’Dongshanlimei’and’Henghe’. Within flowering mei, the least one was 0.118 between ’Xiangxuegongfen’and’Yunnanhongmei’and the farmost one was 0.663 between ’Yunnanfenghou’and’Benishidale’.4. The genetic diversity of analysis based on the REMAP and IRAP molecular markersThe genetic diversity analysis combined REMAP with IRAP technique showed that the observed number of alleles was 1.9955 in mei populations, effective number of alleles was 1.4887, Nei’s gene diversity was 0.2910, Shannon’s information index was 0.4465. UPGMA cluster, at the similarity coefficient of 0.766,84 mei varieties could be divided into 30 groups, and 20 groups at the similarity coefficient of 0.752. Fruiting mei and flowering mei were clustered respectively showed less genes penetration occur between fruiting mei and flowering mei. Among Japanese fruiting mei,’Shirokaga’,’Gessekai’ and’Gojirou’,’Yourou3’ and ’Gyokouei’,’Koume’,’Gyokouei’ and ’Hanakami’ were similar. Green mei gather together as groups, but part of red mei were assembled into green mei, white mei was similar to green mei, but ’Dayezhugan’ is not similar to group with ’Xiaoyezhugan’ . Within flowering groups, cinnabar purple form was tightly as the same group, pendant form was loosely as one group, albo-Plena Form was (except for ’Longquanyudie’ ) one group, after-spring form and green calyx form as one group separately, pink double form assembles as three groups which showed the complexity of inheritance and evolution extent was different. PopGen32 analysis showed Japanese fruiting mei were introduced from Zhejiang province of China and Japanese flowering mei from Jiangsu province of China. Three layers mei club was obvious change for multilayer ones, gene of 1 layer and 2 layer had big different from 3-5 layer ones. Within 84 mei varieties, the least of genetic distance was 0.136 between ’Shirokaga’ and ’Gessekai’ and the farmost one was 0.602 between ’Fenghou’ and ’Henghe’ . Within fruiting mei, the least one was 0.136 between ’Shirokaga’ and ’Gessekai’ and the farmost one was 0.464 between ’Dongshanlimei’ and ’Henghe’ . Within flowering mei, the least one was 0.141 between ’Xiangxuegongfen’ and ’Fenpigongfen’ & ’Yinhongzhusha’ and ’Nanjinghongxu’ and the farmost one was 0.508 between ’Benishidale’ and ’Yunnanfenghou’ .