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1 HEREDITAS (Beijing) , 31(1): ISSN 研究报告 DOI: /SP.J 古茶园台地茶园遗传多样性的 AFLP 分析 1, 2, 季鹏章 3, 蒋会兵 2 1,, 黄兴奇 3, 张俊 2, 梁名志 2, 王平盛 1., ; 2., ; 3., 采用 AFLP- 毛细管电泳法对云南省西双版纳地区 4 个有代表性的古茶园和 2 个台地茶园 ( 阿萨姆茶 Camellia sinensis var. assamica) 进行遗传多样性分析研究表明 : 阿萨姆茶变种水平的遗传多样性为 : P = 92.31%, 期望杂合度 He = , Shannon 多样性指数 Ho = ; 古茶园居群水平是 45.55%, 勐腊居群最高 P = 59.11%, 勐宋居群变异度最低 P = 36.44%; 而台地茶中, 有性系勐海大叶群体种 P = 35.02%, 无性系云抗 10 号则非常低 P = 13.77%, 台地茶居群水平是 24.2%; 古茶园和台地茶遗传多样性相差很大, 依次是古茶园 > 有性系台地茶 > 无性系台地茶研究还发现古茶园与台地茶园之间, 南糯山居群勐腊易武居群与其他居群间存在多条特异谱带, 可作为南糯山居群和勐腊易武居群的分子指纹图谱, 应用于这两个居群所产晒青毛茶的鉴别 Camellia sinensis var. assamica; AFLP; 遗传多样性 ; 古茶园 ; 台地茶园 Genetic diversity of ancient tea gardens and tableland tea gardens from Yunnan Province as revealed by AFLP marker JI Peng-Zhang 1, 2,3, JIANG Hui-Bing 2, HUANG Xing-Qi 1,3, ZHANG Jun 2, LIANG Min-Zhi 2, WANG Ping-Sheng 2 1. College of Life Sciences, Yunnan University, Kunming , China; 2. Tea Research Institute, Yunnan Academy of Agricultural Sciences, Menghai , China; 3. Biotechnology and Genetic Germplasm Institute, Yunnan Academy of Agricultural Sciences, Kunming , China Abstract: This study was conducted to evaluate the genetic diversity within and among the plants of four ancient tea gardens and two tableland tea gardens form Yunnan Province, China by AFLP technique. The percentage of polymorphic loci (P) of the plants from six tea gardens was 92.31%. The genetic diversity within the six gardens demonstrated by Nei s genetic diversity (He) was estimated to be , while Shannon indices (Ho) were The percentage of polymorphic loci of the four ancient tea populations was 45.55% on average, with a range of 36.44% (Mengsong) to 59.11% (Mengla). But the percentages of polymorphic loci of the plants from two tableland gardens were 13.77% (Yunkang 10) and 24.2% (Menghai Daye), respectively. There was a great genetic difference between ancient tea gardens and tableland tea gardens. The genetic diversity among the plants of the ancient tea garden was higher than those of the sexual tableland tea garden and the clone tableland tea garden based on P valve. The four ancient tea gardens and two tableland gardens could be differentiated with AFLP markers. The results show that AFLP marker is an effective tool in the discrimination of tea germ- 收稿日期 : ; 修回日期 : 基金项目 : ( : 2006FY110700) ( : 2006C0012Z) 作者简介 : (1975 ),,,, : pengzhji@126.com 通讯作者 : (1954 ),,,, : Tel: ; xingqih@hotmail.com

2 102 HEREDITAS (Beijing) plasm, as well as sundried green tea. Keywords: Camellia sinensis var. assamica; AFLP; genetic diversity; ancient tea garden; tableland tea garden [1, 2], [3] (Tea plant (Camellia sinensis var. assamica (Masters) Kitamura)) 4 hm 2, ;, 20 hm 2,,,,,,,,,, 4 ( ) 2, AFLP(Amplified fragment length polymorphism)-,,, (C. sinensis var. assamica), 4 ; 2 10, 1 3~5,, 样本 DNA 的提取和 AFLP-PCR 的扩增 6 C. sinensis var. assamica 1.0 g, CTAB [4] DNA DNA RNA A, 260 nm 280 nm, DNA EcoR Mse ; EcoR Mse T4 ; EcoR Mse, : 94 2 min; s, s, 72 2 min, 20 ; min 20, : 94 2 min; s, 66~56 30 s, 72 2 min, 10, 66, 1 ; s, s, 72 2 min, 20 ; min MJ Research (Waltham, MA, USA) PTC200-well thermal cycler AFLP- 毛细管电泳检测表 1 用于 AFLP 分析的阿萨姆茶 C. sinensis var. assamica 的 6 个居群 (N) (E) MS,, ML,, J,, NNS,, YK,, MD,,

3 1 : AFLP 103 (Beckman Coulter) CEQ8000,,, 0 1, 数据处理和分析, POPGENE version 1.32 [6] ; (P), Nei s [7] ( )(He), Shannon (Ho) (Coefficient of gene differentiation, Gst) AMOVA AMOVA(Analysis of molecular variance), NTSYSpc2.0 UPGMA ;, AFLP 8 2 AFLP, 494, , % : P=92.31%, He=0.1366, Shannon Ho =0.2323, Ht = ; (P) 45.55%, (He) , Shannon's (Ho) 0.166, (P =59.11%, He =0.1192; Ho=0.2009); (P =36.44%, He=0.081; Ho= );, P=35.02%, He=0.0986, Ho=0.1531; 10, P=13.77%, He=0.0437, Ho=0.0666; P=24.4%, He=0.0712, Ho= POPGENE : Gst , 35.03%, 35.03% AMOVA : 6, 37.86%, 62.14% (P<0.001)( 4) 表 2 所用引物组合及其检测效率 (5 3 ) (%) E-ACC/M-CTC GACTGCGTACCAATTCAAC GATGAGTCCTGAGTAACTC E-ACC/M-CTA GACTGCGTACCAATTCAAC GATGAGTCCTGAGTAACTA 表 3 阿萨姆茶 6 个居群的遗传多样性分析 N P He (s.d.) Ho (s.d.) MS (0.1437) (0.2118) ML (0.1398) (0.2090) J (0.1521) (0.2231) NNS (0.1599) (0.2339) PLA (0.1489) (0.2195) YK (0.1216) (0.1791) MD (0.1625) (0.2383) PLT (0.1421) (0.2087) (0.1484) (0.2081) : 1; N: ; P: ; He: ; Ho: Shannon ; ; PLA: ; PLT: ; Variety level:

104 HEREDITAS (Beijing) 2009 31 6 Nei's 5 10, 0.1674,, 0.0122;, 0.9879, 10, 0.

4 104 HEREDITAS (Beijing) Nei's 5 10, ,, ;, , 10, Jaccard UPGMA ( 1), 6 表 4 阿萨姆茶居群间和居群内的遗传变异 P (%) < < 图 1 6 个居群的相似性系数 UPGMA 聚类图

5 1 : AFLP 105 Jaccard : Jaccard 0.18, 6, 10,, 6 Nei s UPGMA ( 2) AFLP ( PCA) AMOVA ( 4), 6 图 2 6 个居群的 Nei s 遗传距离 UPGMA 聚类图 2.4 AFLP 4 AFLP- : 4 2 AFLP,, 6 6 : E-ACC/M-CTC, 2 67 bp 150 bp 383 bp E-ACC/M-CTC, bp 132 bp 337 bp, 4, E-ACC/M-CTC, (NNS) : 160 bp 276 bp 337 bp; (ML) : 67 bp 123 bp 383 bp; (MS) (JH) : 150 bp 205 bp 383 bp E-ACC/M-CTA, (NNS) : 137 bp 150 bp 167 bp 205 bp 383 bp; (ML) : 132 bp 332 bp; (MS) (JH) : 132 bp 337 bp, [8] [9] (94.17%~100%); 图 3 基于 AFLP 表型特征的主成分分析 (PCA)3D 图

6 106 HEREDITAS (Beijing) 表 5 6 个古茶园间的 Nei s 遗传距离和遗传一致度 MS ML JH NNS YK MD MS **** ML **** J **** NNS **** YK **** MD **** : 1,, 图 4 勐海勐宋和勐海大叶种 E-ACC/M-CTC AFLP 选择性扩增电泳图谱,

7 1 : AFLP 107 表 6 E-ACC/M-CTC 和 E-ACC/M-CTA 居群主特征峰带 E-ACC/M-CTC (bp) E-ACC/M-CTA (bp) MS JH ML NNS YK MD : 1; +: ; :, P=92.31%, 45.55%;, P=35.02%; 10 P=13.77%;, > > [10], 0.58~0.84,, 10, ;, [11] [11] ISSR 10, He H 0 P,, ISSR PCR 20, RFLP SSR RAPD [12,13] ISSR DNA,, AMOVA : 37.86%, [11] ISSR 10 AMOVA (39.7% ), 3.2 [14] 3,, : (1), ; (2),,,, [15] 陈亮等曾报道 RAPD 标记在鉴别茶树品种和资源非常有效, 有 3 种独立的方法可以用于鉴别茶树资源 : (1) 特殊的标记 ; (2) 特异的谱带类型 ; (3) 不同引物提供的谱带类型组合我们采用 AFLP- 毛细管电泳技术对 4 个古茶园进行分析, 发现在引物 E-ACC/M-CTC 的 110 bp 和 137 bp 处, 引物 E-ACC/M-CTA 的 276 bp 282 bp 和 337 bp 处, 南糯山居群与其他居群间存在特异带, 综合 2 对引物 5 ;, 2007, 200, 50%,,, 参考文献 (References): [1] Wight W. Nomenclature and classification of tea plant. Nature, 1959, 183(4677): [DOI] [2].., 1986, 6 (1): 1 8. [3].. :, 2004, [4] Doyle J. DNA protocols for plants-ctab total DNA isolation. In: Hewitt GM, Johnston A, eds. Molecular tech-

8 108 HEREDITAS (Beijing) niques in taxonomy. Berlin: Springer, 1991, , 2007, 33(4): [5] GIBCOBRL. Instruction manual AFLPTM analysis system, AFLP start primer kit. Version B, ( ) ISSR., [11],,,,,,. [6] Yeh FC, Yang RC, Boyle T. POPGENE. Microsoft Windows-based freeware for population genetic analysis. Re- [12],,. RAPD ISSR 2007, 27(4): lease Edmonton: University of Alberta, , 2000, 42(7): [7] Nei M. Analysis of gene diversity in subdivided populations. [13] Ge XJ, Sun M. Reproductive biology and genetic diversity of a Proc Natl Acad Sci USA, 1973, 70(12): [DOI] cryptoviviparous mangrove aegiceras corniculatum (Myrsinaceae) using allozyme and intersimple sequence repeat (ISSR) [8] Shao Wan-Fang, Pang Rui-Hua, Wang Pin-Sheng. RAPD analysis of tea relationship in Yunnan. Sci Agri Sin, 2003, analysis. Mol Ecol, 1999, 8(12): [DOI] 36(12): [14],,,,,, [9],,,,,,,... RAPD., 2004, 19 (3): , 2006, 21(4): [10],,,,. [15],,. DNA ISSR.., 2002, 35(10): ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇ 2009 年遗传杂志征稿简则, 1. 征稿范围 : ; ; ; ; ; 2. 稿件要求 :, ; ; ; ;,,, 3. 送审标准 :, PCR, PCR 4. 数据库收录 : 20 ( ) ;, 5. 作者确认 :,, ; ; ; ;,,,,, 4. 投稿方式 : 3, ( yczz@genetics.ac.cn), 5. 审稿流程 :,,,,, 3 6. 稿件费用 :, 200, 800 5, 30,

DNA 2 RAPD 3-5 AFLP 6 GISH RAPD 7 RAPD SCAR 32 8 RAPD SSR DNA CTAB 引物合成 8 RAPD 8 6 SC

DNA 2 RAPD 3-5 AFLP 6 GISH RAPD 7 RAPD SCAR 32 8 RAPD SSR DNA CTAB 引物合成 8 RAPD 8 6 SC 2013 35 2 307-312 http / /xuebao. jxau. edu. cn Acta Agriculturae Universitatis Jiangxiensis E - mail ndxb7775@ sina. com SSR SCAR 310024 RAPD EST - SSR SCAR 26 24 22 18 81. 8% 7 23 S668. 4 Q943. 2 A 1000-2286