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1 作物学报 ACTA AGRONOMICA SINICA 2009, 35(10): ISSN ; CODEN TSHPA9 DOI: /SP.J 应用 SRAP 标记分析黑芝麻核心种质遗传多样性车卓 1,2 张艳欣 1,** 孙建 1 张秀荣 1,* 尚勋武 2 王化俊 2 1, ; 2 /, : SRAP, , 126, 69.2%, ; 0.469~0.986, 0.726, UPGMA, ,, ; (0.3557) (0.3415) (0.2986),, : ; ; SRAP; Genetic Diversity Analysis of Black Sesame (Sesamum indicum DC) Core Collection of China Using SRAP Markers CHE Zhuo 1,2, ZHANG Yan-Xin 1,**, SUN Jian 1, ZHANG Xiu-Rong 1,*, SHANG Xun-Wu 2, and WANG Hua-Jun 2 1 Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan , China; 2 Gansu Agricultural University / Gansu Key Laboratory of Crop Genetic Improvement and Germplasm Enhancement, Lanzhou , China Abstract: China has a long history and rich germplasm resources for growing black sesame. Black sesame is well used because of its nutrition superior to black rice and black soybean. However, Research on black sesame is relatively less than that on other crops. In the paper, genetic diversity of black sesame core collection of China was analyzed using SRAP (sequence-related amplified polymorphism) markers. The results showed that 13 SRAP primer combinations were employed to evaluate the genetic diversity of 100 black sesame accessions, a total of 182 amplified fragments were detected and 126 of them were polymorphic, the polymorphism percentage was 69.2%, the number of amplified fragments and polymorphic fragments of each primer combination were 14.0 and 9.7, respectively. The 100 accessions were grouped into five clusters at genetic similarity of 0.68, indicating the genetic diversity of these accessions was abundant. As regards geographic regions, Shannon's information index of black sesame accessions in south China (0.3557) was the highest, the followings were that in central regions of China (0.3415) and north China (0.2986). The characteristics of genetic diversity of black sesame core collection of China were fully revealed in this study, which provided the theoretical foundations for further exploring, collecting and introducing black sesame germplasm as well as mining and utilizing excellent black sesame germplasm in the future. Keywords: Black sesame; Core Collection; SRAP; Genetic diversity,,, [3-4],, DNA [5-12] Bhat [5] RAPD 38, ; Kim [6] 14 ISSR 75 ; (2005DKA ), (2006BAD13B05-2), (NB ,35) * (Corresponding author): zhangxr@oilcrops.cn ** : chezhuo1110@163.com Received(): ; Accepted( :

2 10 : SRAP 1937 Laurentin [8] AFLP 32, 8 457, 93% ; [11] RAPD, 14, 19 Li [13] 2001 PCR SRAP (sequence-related amplified polymorphism), RFLP RAPD SSR AFLP, DNA, 1 材料与方法 1.1 ( 453, ) 100 ( 92, 8 ), (, ) [14] 1.2 DNA Doyle [15] CTAB DNA, RNaseA RNA, 0.6% DNA, DNA, DNA, 20 ng μl 1, PCR Li [13] SRAP, 11 ( 1), SRAP-PCR (15 μl) 10 mmol L 1 dntps 0.30 μl, 25 mmol L 1 Mg μl, 50 ng, DNA 80 ng, 10 buffer 1.5 μl, 1 U Taq DNA ( MBI ) [16] Li [13] PCR, 94 3 min; 94 1 min, 35 1 min, 72 1 min, 5 ; 94 1 min, 50 1 min, 72 1 min, 35 ; min, 4 Bassam [17] 6% PCR, DYY-12 DYCZ-20C, 0.5 TBE, 80 W, 30 min, 50 (98%, 10 mmol L 1 EDTA, 0.05%, 0.05%) 8 μl SRAP, 95 5 min,, 3 μl, 6%, 10 cm 1.4 SRAP, 1, 0,, NTSYS-pc2.10, SimQual Nei-Li, SAHN UPGMA ; POPGENE1.32 Shannon 2 结果与分析 2.1 SRAP 1 11, 50 SRAP 6 DNA, 29, 13, , 14.0, 126, %~90.0%, 69.2%( 2) 1 Me09/Em SRAP 100 (GS),, Primer code 表 1 SRAP 分析所用的引物及其序列 Table 1 SRAP primers sequences Forward primer (5 3 ) Reverse primer (5 3 ) Primer sequence Primer code Primer sequence Me01 TGAGTCCAAACCGGATA Em01 GACTGCGTACGAATTAAT Me02 TGAGTCCAAACCGGAGC Em02 GACTGCGTACGAATTTGC Me03 TGAGTCCAAACCGGAAT Em03 GACTGCGTACGAATTGAC Me04 TGAGTCCAAACCGGACC Em04 GACTGCGTACGAATTTGA Me05 TGAGTCCAAACCGGAAG Em05 GACTGCGTACGAATTAAC Me06 TGAGTCCAAACCGGTAA Em06 GACTGCGTACGAATTGCA Me07 TGAGTCCAAACCGGTCC Em07 GACTGCGTACGAATTCAA Me08 TGAGTCCAAACCGGTGC Em08 GACTGCGTACGAATTCTG Me09 TGAGTCCAAACCGGACG Em09 GACTGCGTACGAATTCGA Me10 TGAGTCCAAACCGGACT Em10 GACTGCGTACGAATTCAG Me11 TGAGTCCAAACCGGAGG Em11 GACTGCGTACGAATTCCA

3 Table 2 Primer combination 2 SRAP The primer and the amplified results of SRAP analysis Total locus Polymorphic locus Percentage of polymorphic locus (%) Me01/Em Me01/Em Me02/Em Me02/Em Me03/Em Me06/Em Me07/Em Me08/Em Me08/Em Me09/Em Me09/Em Me09/Em Me10/Em Total Average ~0.986, 0.726, (GS 0.986), (GS ), (UPGMA), ( 2), I, 72, GS 0.820,, ; II, 6, 13 (, ) , 89 92, GS 0.736; III, 9, TKV-306(20)96 662(30)97 100, GS 0.767; IV, 11, , GS 0.734; V, 2, 15 77, GS VI V I IV, 2.3 [18] [19], 92 3, 1 (); 2 (); 3 () 3,, 60.1%, 58.2% 45.6%,, 31.9%; GS (0.7188) (0.7289) (0.7546) (0.7755); (0.3557) (0.3415) (0.2986) (0.2422),, ; GS,, ( 8 ) Fig. 1 图 1 引物组合 Me09/Em06 的扩增结果 SRAP fingerprint of black sesame accessions from primer combination Me09/Em06 1 The code is same as Table 1.

4 10 : SRAP 1939 Fig. 2 图份黑芝麻材料的聚类图 Dendrogram of 100 black sesame accessions 3 讨论 SRAP DNA PCR DNA RFLP DNA, SSR, AFLP, SRAP AFLP,, SRAP

5 Germplasm Table 3 Accession number 表 3 不同地理区域材料遗传多样性比较 Comparison of genetic diversity from different regions Rate of polymorphic bands (%) Minimum genetic similarity Maximum genetic similarity Average genetic similarity Shannon s information index South China Central zone of China North China Oversea [20-24] [23] 49, 11 SRAP 199, 86, 43.7%, [24] 132 SRAP, 36 20, 419 SRAP 100, , 126, 69.2%,, DNA,,, (92% ), 0469~0.986,, Bhat [5] RAPD 21 (22 ) 18 (36 ), 0.19~0.89; Laurentin [8] AFLP 32 ( ), 93%, 0.38~0.85, 0.65; Laurentin [9] 8 AFLP, 91%, 0.31~0.78, 0.52,,, DNA [25] [26] [27],,,,,,,,,, [7-9,12] References [1] Shen M( ). Measurements oil trace elements of black sesame and white sesame. Chin J Health Lab Tech (), 2007, 17(2): (in Chinese with English abstract) [2] Chen H-X( ), Liu F-L( ), Zhao Y-Z( ). Breeding for Zhongzhi 9, a black seed coat sesame cultivar with high quality. Oil Crop Chin (), 1994, 16(4): (in Chinese with English abstract) [3] Xiao T-X( ), Feng X-Y( ), Zhang X-R( ). Analysis of the distribution and main characteristics of black seed coat sesame germplasm in china. Oil Crop Chin (), 1992, 14(2): (in Chinese with English abstract) [4] Le M-W( ), Zhang D-X( ), Xiong Y-P( ), Zhou H-Y( ). Principal component analysis on quantitative characters and its application in black sesame. Oil Crop Chin ( ), 1995, 17(3): (in Chinese with English abstract) [5] Bhat K V, Babrekar P P, Lakhanpaul S. Study of genetic diversity in Indian and exotic sesame (Sesamum indicum L.) germplasm using random amplified polymorphic DNA (RAPD) markers. Euphytica, 1999, 110: [6] Kim D H, Zur G, Danin P Y, Lee S W, Shim K B, Kang C W, Kashi Y. Genetic relationships of sesame germplasm collection as revealed by inter-simple sequence repeats. Plant Breed, 2002, 121: [7] Ercan A G, Taskin M, Turgut K. Analysis of genetic diversity in Turkish sesame (Sesamum indicum L.) populations using RAPD markers. Genet Resour Crop Evol, 2004, 51: [8] Laurentin H E, Karlovsky P. Genetic relationship and diversity in a sesame (Sesamum indicum L.) germplasm collection using amplified fragment length polymorphism (AFLP). BMC Genetics, 2006, 7: 10 [9] Laurentin H E, Karlovsky P. AFLP fingerprinting of sesame (Sesamum indicum L.) cultivars: Identification, genetic relationship and comparison of AFLP informativeness parameters. Genet Resour Crop Evol, 2007, 54: [10] Laurentin H E, Karlovsky P. Relationship between metabolic and genomic diversity in sesame (Sesamum indicum L.). BMC Genomics, 2008, 9: 250 [11] Zhang X-R( ), Chen K-R( ), Peng J( ), Xu

6 10 : SRAP 1941 Z-Y( ). The RAPD analysis and genetic diversity of selected sesame germplasms. Chin J Oil Crop Sci ( ), 2004, 26(4): (in Chinese with English abstract) [12] Zhang P( ), Zhang H-Y( ), Guo W-Z( ), Zheng Y-Z( ), Wei L-B( ), Zhang T-Z( ). Genetic diversity analysis of Sesamum indicum L. germplasms using SRAP and EST-SSR markers. Acta Agron Sin (), 2007, 33(10): (in Chinese with English abstract) [13] Li G, Quiros C F. Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: Its application to mapping and gene tagging in Brassica. Theor Appl Genet, 2001, 103: [14] Zhang X R, Zhao Y Z, Cheng Y, Feng X Y, Guo Q Y, Zhou M D, Hodgkin T. Establishment of sesame germplasm core collection in China. Genet Resour Crop Evol, 2000, 47: [15] Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull, 1987, 19: [16] Che Z( ), Zhang Y-X( ), Sun J( ), Huang B( ), Zhang X-R( ). Establishment and optimization of SRAP reaction system in sesame. Chin Agric Sci Bull ( ), 2008, 24(10): (in Chinese with English abstract) [17] Bassam B, Caetano-Anolles G, Gresshoff P M. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem, 1991, 196: [18] Chen C-Y( ). Sesame Cultivars Resources in China ( ). Beijing: Agriculture Press, pp 5 33 (in Chinese) [19] Oil Crop Research Institute of CAAS ( ). The Catalogue of Varietal Germplasm Resources of sesame in China ( ), Vol. 3. Beijing: China Agricultural Scientech Press, 1997 (in Chinese) [20] Wen Y-C( ), Wang H-Z( ), Shen J-X( ), Liu G-H( ), Zhang X-F( ). Analysis of genetic diversity and genetic basis of Chinese rapeseed cultivars (Brassica napus L.) by sequence-related amplified polymorphism markers. Sci Agric Sin ( ), 2006, 39(2): (in Chinese with English abstract) [21] Budak H, Shearman R C, Parmaksiz I, Gaussoin R E, Riordan T P, Dweikat I. Molecular characterization of buffalo grass germplasm using sequence-related amplified polymorphism markers. Theor Appl Genet, 2004, 108: [22] Ferriol M, Pioó B, Nuez E. Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP markers. Theor Appl Genet, 2003, 107: [23] Wang H-Z( ), Wu Z-D( ), Wang X-W( ), Fang Z-Y( ). Analysis of the genetic diversity in different types of sugar beets by SRAP and SSR markers. Acta Agron Sin (), 2008, 34(1): (in Chinese with English abstract) [24] Li W( ), Ni W( ), Lin Z-X( ), Zhang X-L( ). Genetic diversity analysis of sea-island cotton cultivars using SRAP markers. Acta Agron Sin ( ), 2008, 34(5): (in Chinese with English abstract) [25] Jin W-L( ), Wen Z-X( ), Pu S-J( ), Zhao B( ). Genetic diversity and evolution of adzuki bean (Vigna angularis) germplasm resources based on RAPD markers. Sci Agric Sin ( ), 2005, 38(2): (in Chinese with English abstract) [26] Chen H( ), Fan Y-H( ), Shi X-W( ), Cai Q( ), Zhang M( ), Zhang Y-P( ). Research on genetic diversity and systemic evolution in Saccharum spontaneum L. Acta Agron Sin (), 2001, 27(5): (in Chinese with English abstract) [27] Gai J-Y( ), Xu D-H( ), Gao Z( ), Yoshiya S( ), Jun A( ), Hirofumi F(), Shunji K( ). Studies on the evolutionary relationship among eco-types of G. max and G. soja in China. Acta Agron Sin (), 2000, 26(5): (in Chinese with English abstract)

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L 2012 13 2 201-206 Journal of Plant Genetic Resources / 071001 41 SSR 21 SSR 41 52 2 ~ 4 2. 5 Shannon 0. 21 ~ 1. 40 0. 73 5. 54 3 7 9 13. 16% ~ 186. 49% 5. 48 6 11 SSR Genetic Diversity of Peanut Arachis

Microsoft Word - 车 卓.doc

Microsoft Word - 车卓.doc