【作者】 李建华；

【导师】 齐桂年； 马炳田；

【作者基本信息】 四川农业大学 ， 茶学， 2009， 硕士

【摘要】 我国西南部是茶树（Camellia sinensis（L.）O.Kuntze）的起源中心,孕育了丰富的茶树种质资源,为我们进行茶树品种选育和生物技术研究提供了坚实的物质基础。近年来发展起来的SSR（简单重复序列）和ISSR（简单重复序列间隔区）技术能够直接从DNA水平反映材料间的遗传差异,方法简便快捷,是研究生物遗传多样性的有力工具。本文采用SSR和ISSR技术对原产四川及从福建、台湾、浙江、贵州、湖南、广州、安徽、海南引进四川的34份茶树栽培种质资源和3份野生茶树资源的遗传多样性和亲缘关系进行了分析,结果表明:1.SSR最佳反应体系为:25ul反应体系中含15ng模板DNA,12.5ul 2×Master Mix,上、下游引物各0.5umol/L,9ul ddH₂O;最佳扩增程序为:94℃预变性5min,94℃变性1min,52℃或60℃退火30s,72℃延伸90s,35个循环,最后72℃延伸7min。ISSR反应的最佳反应体系为:25ul反应体系中含20ng模板DNA,0.6umol/L引物,0.24mmol/L dNTPs,2.0mmol/LMgCl₂,1.75U Taq聚合酶,1×PCR缓冲液;最佳扩增程序为:94℃预变性5min,94℃变性1min,52℃或60℃退火1min,72℃延伸1min,41个循环,最后72℃延伸7min。在各自的反应体系下,均可获得清晰条带。2.从21对SSR引物中筛选出8对SSR引物对37份供试材料扩增,共扩增出93条多态性条带,占总条带的93%。SSR引物扩增的平均多态性条带数、平均多态性百分比（%）、PIC分别为11.63、92.3、0.91,平均特异谱带22.38个;每个品种扩增出多态性条带数26～45,平均为:36.41,平均多态性百分比（%）为83.68;基于SSR标记的供试品种间的遗传相似系数介于0.490～0.880,平均为0.699。从DNA分子水平显示出供试材料间存在着丰富的遗传多样性。从89条ISSR引物中筛选出14条ISSR引物在37份供试材料共扩增出146条多态性条带,占总条带的94.81%。ISSR引物扩增的平均多态性条带数、平均多态性百分比（%）、PIC分别为10.43、95.19、0.89,平均特异谱带13.93个;每个品种扩增出多态性条带数37～76,平均为:57.97,平均多态性百分比（%）为87.63;基于ISSR标记的供试品种间的遗传相似系数介于0.519～0.851,平均为0.689,显示出供试材料间存在着丰富的遗传多样性。3.基于SSR标记,在遗传相似系数为0.676时,供试品种中的37份材料聚成2个大类群和3个独立组。SSR聚类结果与茶树种质资源的来源和适制性有关,适制性相同的来自福建和台湾的大部分乌龙茶聚为了一类,来源地不同的一些红绿兼制、红茶和绿茶品种也分别聚在了一起。基于ISSR标记,在相似系数为0.658时,供试的37份材料聚成2个大类群和1个独立组。ISSR与SSR分析得出的结果相一致。4.引物SSR8和ISSR12扩增的DNA指纹图谱,可以鉴定所有的供试材料。表明SSR和ISSR技术进行茶树品种分子鉴别的可行性。更多还原

【Abstract】 Southwest of China is the origin of tea plant（Camellia sinensis（L.）O.Kuntze）,which has rich of tea germplasms.It provides rich resources for the application of new tea cultivars.SSR（Simple Sequence Repeat） and ISSR（Inter-Simple Sequence Repeat）,as newly developed molecular marker techniques,are powerful methods for bio-diversity research.They have been popularized for their rapidity,simplicity and capacity of revealing the genetic diversity in DNA level.Genetic diversity and relationship of 34 tea cultivated species and 3 tea wild species Cultivars including those origins in Sichuan and introduced from Fujian,Taiwan,Zhejiang,Guizhou,Hunan,Guangdong,Anhui and Hainan into Sichuan tea areas,as materials,were revealed by SSR and ISSR Markers.The result as follows:1.The proper SSR-PCR reaction system was determined as follows:25μL reaction system containing 15ng template DNA,12.5ul 2×Master Mix,0.5μmol/L Upstream or downstream primer,9ul ddH₂O.The proper amplification was after 1 cycle initial denaturation at 94℃for 5min,followed by 41 cycles of 1min at 94℃,30s annealing at 52℃or 60℃,and extension of 90s at 72℃,and a final 7min extension at 72℃.The proper ISSR-PCR reaction system was determined as follows:25μL reaction system containing 1×PCR buffer,2.0mmol/L MgCl₂,0.24mmol/L dNTPs,0.6μmol/L primer,1.75U Taq DNA polymerase,20ng template DNA.The proper amplification was after 1 cycle initial denaturation at 94℃for 5min,followed by 41 cycles of 1min at 94℃, 1 min annealing at 52℃or 60℃,and extension of 1min at 72℃,and a final 7min extension at 72℃.2.8 SSR primers selected from 21 SSR primers were used to amplify 93 DNA bands（93%）were polymorphic.The average number of polymorphic DNA bands、the percentage of polymorphic bands（%）、PIC amplified by SSR primer were 11.63、92.30、0.91,the average specific bands were 22.38.Each tea cultivar could be amplified 26 to 45 polymorphic DNA bands,with an average of 36.41,the average percentage of polymorphic bands（%） was 83.68.The genetic similarity coefficient between the cultivars varied from 0.490 to 0.880,with an average of 0.699.The SSR results indicated that there was abundant genetic diversity among the tested materials.14 ISSR primers selected from 89 ISSR primers were used to amplify 146 DNA bands（94.81%）were polymorphic.The average number of polymorphic DNA bands、the percentage of polymorphic bands（%）、PIC amplified by ISSR primer were 10.43、95.19、0.89,the average specific bands were 13.93.Each tea cultivar could be amplified 37 to76 polymorphic DNA bands,with an average of 57.97,the average percentage of polymorphic bands（%） was 87.63.The genetic similarity coefficient between the cultivars varied from 0.519 to 0.851,with an average of 0.689.The ISSR results indicated that there was abundant genetic diversity among the tested materials. 3.Based on SSR,at similarity coefficient of 0.676,all the tested materials could be clustered into 2 complex groups and 3 simple groups.It was found that there was a certain correlation between the results and the derivation、productive characters of tea cultivars.Most oolong tea cultivars were clustered into a group,some black、green tea were clustered into a group separately too.Based on ISSR,at similarity coefficient of 0.658, all the tested materials could be clustered into 2 complex groups and 1 simple groups.The ISSR results were similar with SSR.4.By the amplication products from genetic DNA of SSR8 and ISSR12 obtained in the study,all the tested materials could be identified.The results indicated that SSR and ISSR were useful tools for molecular identification of tea identification.更多还原