【作者】 张水明；

【导师】 陈昆松； 高中山； 徐昌杰；

【作者基本信息】 浙江大学 ， 果树学， 2009， 博士

【摘要】 杨梅(Myrica rubra Sieb．& Zucc．)原产于中国，是我国南方著名特产果树，其栽培历史悠久，种质资源丰富，目前杨梅产业已进入快速发展期。本研究旨在从DNA分子标记水平上探讨中国杨梅种质资源的遗传多样性和亲缘关系，为有效保护杨梅种质资源和进行种质资源创新利用提供理论依据，主要研究结果如下：1．基于AFLP标记技术的杨梅遗传多样性分析针对杨梅叶片富含多糖和多酚类等次生物质的特点，用CsCl超速离心法对杨梅基因组DNA进行纯化，对AFLP-银染技术体系进行了优化改进，建立了杨梅AFLP-银染技术体系，并筛选出6对选择性引物组合：EAA／M47、EAA／M54、EAC／M56、EAC／M50、EAG／M51和EAT／M61；这6对AFLP引物组合在101个供试杨梅材料中分别产生34，35，45，37，44，41个位点，总计获得236个位点，其中多态性位点177个，每对引物的多态性比率在64．86-86．67％之间，平均多态性比率为75．01％；6对AFLP引物组合的香农指数Ⅰ在0．21-0．36之间，基因多样性指数He在0．1332-0．2464之间，揭示了杨梅种质资源丰富的遗传多样性；对AFLP扩增结果采用UPGMA法进行聚类分析，可以把101个杨梅材料分为5组，第1组有29个材料，包括浙江的水晶种、荸荠、东魁和广东乌西梅、广东早梅、广东黑梅等材料；第2组有63个材料，包括浙江的早荠蜜梅、晚荠蜜梅，湖南的桐子梅、江苏的大浮大叶细蒂大浮、大浮小叶细蒂和乌梅；第3组有7个材料，包括浙江的6个材料和江苏的大浮乌梅；第4组只有1个浙江杭州的大叶黄；第5组为1个美国杨梅(Myrica cerifera L．)。聚类结果与地域来源并不完全一致，同时也与果实色泽及植株性别不一致，反映出在杨梅悠久的栽培历史中存在着普遍的种质资源交流。2．杨梅EST-SSR信息分析与SSR标记开发数量迅速增加的EST为开发新的SSR标记提供了宝贵的资源，本研究从课题组提供的395条杨梅Unigene序列中查找出21条符合筛选条件EST含有SSR，占EST序列总数的5．31％，在21条序列中，以二核苷酸为重复单元的序列最多，为12条，占57．14％，其次是三核苷酸重复类型，有5条，占全部SSR的23．81％；其它重复类型所占比例均不足20％，包括2条四核苷酸重复和2条六核苷酸重复。另外，在相关试验中还发现两条与MYB转录因子有关的序列中含有SSR。通过设计SSR引物，在优选的PCR反应体系下，对SSR位点的多态性进行评价，结果发现有11个位点呈现多态性，其等位基因数(A)为3到12，观察到的杂合度(H_O)从0．1250到0．9667，期望杂合度(H_E)从0．2359到0．8790，多态性信息含量(PIC)从0．2285到0．8516。以上结果说明所开发的11个SSR标记能很好的运用于杨梅遗传多样性分析等相关方面的研究。3．基于SSR标记技术的杨梅遗传多样性分析本研究利用11个SSR标记，选择100个杨梅材料进行遗传多样性分析，每对SSR引物在100个杨梅材料中分别产生6-1 5个多态性位点，总共1 22个多态性位点，平均每对引物扩增出11个多态性位点。将11个SSR标记在100个杨梅材料上的扩增结果，进行UPGMA法聚类分析，结果也可以把100个杨梅材料分为5组，第1组包括晚稻杨梅、水晶种、荸荠、甬选56号、桐子梅等56个材料；第2组包括东魁、晚荠蜜梅等33个材料；第3组包括江苏的桃红、广东早梅、广东黑梅、广东大梅、湖南的墙背梅5个材料；第4组包括4个浙江材料和1个贵州材料；第5组为1个美国杨梅(Myrica cerifera L．)。聚类结果与AFLP并不完全相同，可能原因是AFLP标记为显性标记，而SSR则为共显性标记，分类的原理不同。更多还原

【Abstract】 Chinese bayberry(Myrica rubra Sieb.& Zucc.)is a subtropical fruit tree nativeto China and it is one of the most important fruit tree crops in south China.In Chinabayberry has a long history of cultivation and a lot of varieties and cultivars were developed.This study analyzed the genetic diversity and relationship of 101 commonly cultivatedbayberry accessions based on molecular markers of AFLP and SSR.The results willaid not only the classification and management of bayberry germplasms,but will alsobe useful for innovation and utilization the germplasms.The main results weresummarized as followings:1.Applications of AFLP technology in genetic diversity analysis of ChinesebayberryAs the bayberry leaves are highly rich in impurities including polysaccharides andpolyphenols,we used ultracentrifugation in CsC1 gradient to purify the genome DNAextracted from bayberry leaves.An improved AFLP protocol suitable for bayberrywas provided.Six EcoR I+2 bases/Mse I+3 bases primer combinations(EAA/M47、EAA/M54、EAC/M56、EAC/M50、EAG/M51和EAT/M61)were used.A total of 236bands were detected,of which 177 bands were polymorphic.The average percentageof polymorphic bands was 75.01.Nei’s gene diversity was between 0.1332 and0.2464,Shannon’s information index was between 0.2101 and 0.36000.An unweightedpair-group method of the arithmetic averages(UPGMA)was used to analyze thegenetic relationships.The Dice’s similarity coefficient among the accessions rangedfrom 0.49 to 1.00,and 101 accessions were divided mainly into five groups.Group 1included a total of 29 accessions.Group 2 included a total of 63 accessions.Group 3included a total of 7 accessions.While group 4 included only one accession ofdayehuang and group 5 included one accession of Myrica cerifera L.The accessionsfrom the same origin did not completely belong to one cluster,which suggested thatthe accessions were not completely clustered by their origin,and an extensive geneticexchange existed among the accessions.And the clusters did not match with the ripefruit color,suggesting that the fruit color of bayberry were independent mutations. 2.Development and characterization of microsatellite markers for ChinesebayberryExpressed sequence tags(ESTs)have been increased rapidly in number recently,which are important resources for development of new SSR markers.In this study,395 Unigenes of Chinese bayberry fruit were screened and 21 SSRs were mined out,accounting for 5.31% of ESTs.Dinucleotide and trinucleotide repeats,with highfrequency and accounting for 57.12 % and 23.81% respectively in all SSRs.Whilethe frequency for other repeat type is below 20 %.In addition we found another twoSSRs as we cloning MYB related genes.We also designed primers and named themas MYBSSR1 and MYBSSR2.Under a optimize PCR amplification,11 polymorphicloci were generated.Polymorphism of these 11 loci was assessed in 32 individualsand the number of alleles(A)ranged from 3 to 12,observed heterozygosity(Ho)andexpected heterozygosity(H_E)ranged from 0.1250 to 0.9667 and 0.2359 to 0.8790respectively,polymorphism information content(PIC)ranged from 0.2285 to 0.8516.These microsatellite loci should be useful in the studies of genetic diversity of Myricarubra.3.Genetic diversity analysis of Chinese bayberry based on SSR markersEleven SSR loci(MRU11,MRU155,MYBSSR1,MYBSSR2,my0043,my0186,my0427,my0792,my0793,my0889,my0972)were used to evaluate the genetic diversity of 100bayberry accessions.A total of 122 polymorphic bands were detected and the averagenumber of polymorphic bands was 11 per locus.An unweighted pair-group method ofthe arithmetic averages(UPGMA)was also used to analyze the genetic relationships.The Dice’s similarity coefficient among the accessions ranged from 0.73 to 1.00,and100 accessions were divided into five groups.Group 1 included a total of 56accessions.Group 2 included a total of 33 accessions.Group 3 included 5 accessions.Group 4 included 5 accessions too and group 5 included one accession of Myricacerifera L.The cluster results based on 11 SSR loci were not completely match withthat of AFLP markers.更多还原