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1 作物学报 ACTA AGRONOMICA SINICA 2008, 34(): ISSN ; CODEN TSHPA9 DOI: /SP.J 烟草 SRAP 和 ISSR 分子遗传连锁图谱构建马红勃陶爱芬祁建民林荔辉,* 李延坤吴建梅梁景霞王涛 2 兰涛陈顺辉 3 ( /, ; 2, ; 3, ) : 7 2, 87 F 2, 68 SRAP ISSR, F 2 PCR, 26 2 (92 SRAP 20 ISSR) cm, 8. cm ~20, 0~29.0 cm 24.%, LG LG4, : ; SRAP; ISSR; Construction of A Molecular Genetic Linkage Map of Tobacco Based on SRAP and ISSR Markers MA Hong-Bo, QI Jian-Min,*, LI Yan-Kun, LIANG Jing-Xia, WANG Tao 2, LAN Tao, CHEN Shun-Hui 3, TAO Ai-Fen, LIN Li-Hui, and WU Jian-Mei ( College of Crop or Life science, Fujian Agriculture and Forestry University, Fuzhou , Fujian; 2 Fujian Shunchang Tobacco Company, Shunchang , Fujian; 3 Fujian Tobacco Company, Fuzhou , Fujian, China) Abstract: High-density genetic linkage maps are useful tools for studying genome, phylogeny and evolution, and for mapping and Cloning genes of interest. In this study, a molecular genetic map was constructed with F 2 population consisting of 87 individuals from a cross of flue-cured tobacco cultivar Taiyan 7 and burley tobacco cultivar Bailei 2 by using a total of 53 SRAP primers and 42 ISSR primers. Approximately 58 out of 53 and 0 out of 42 primers, respectively, showed polymorphisms. Each of this polymorphic primers produced at least one scorable polymorphic DNA band which was visible enough for detection and scoring. The map consisted of 26 linkage groups which included 2 (92 SRAP and 0 ISSR), and covered cm with an average distance of 8. cm. Sixteen were still unlinked. All 26 linkage groups consisted of 2 20 ranging in length from cm. A total of 24.% distorted distributed in the map, mainly concentrated in the LG and LG4 linkage groups, and the others were mapped on diverse linkage groups respectively. This map will provide the basis for gene mapping and marker-assisted selection of important agronomic traits in tobacco. Keywords: Tobacco; SRAP; ISSR; Genetic linkage map DNA, 200, Lin [] RFLP RAPD (N. plumbaginifolia / N. longiflora) 基金项目 : ([2005]) 作者简介 : (983 ),,,, (Corresponding author),,,, Qijm863@63.com; Tel & Fax: Received( ) ; Accepted(

2 : SRAP ISSR F 2, ; Nishi [2] (W6 Michinoku) 25 DH, AFLP ; [3] Speight G-28 NC DH, ISSR RAPD, ; Bindler [4] SSR Hicks Broad Leaf Red Russian 86 F 2, SSR [5],, ( ),, F F 2, SRAP Li Quiros [6],,,,, SRAP ISSR,, ( 7 2), F 2, ( ) 材料与方法. SRAP ISSR [7-9], 7 2, F 87 F 2, F 2:3 7, [7].2 DNA F 2, 5 6, 5 g, CTAB [7] DNA, β- 2.5% CTAB DNA.3 SRAP SRAP-PCR 20 μl, buffer [0 mmol L Tris-HCl, ph 8.0, 50 mmol L KCl, 2.3 mmol L MgCl 2, 0.08% Nonidet P40], dntp ( 200 μmol L ), 33 ng, DNA 20 ng, Taq.5 U (Taq buffer SRAP dntp ) 94 5 min, 5 94 min, 33 min, 72 min; 30 53, 72 5 min, 4 [9] PCR PTC-00 PCR DYY-5, 6% PCR TBE, 300 V, 2.5 h,.4 ISSR ISSR-PCR 25 μl, buffer, (0 mmol L Tris-HCl, ph 8.0, 50 mmol L KCl,.5 mmol L MgCl 2, 0.08% Nonidet P40), DNA 20 ng, Taq.5 U, 0.8 μmol L, dntp 50 μmol L (Taq buffer ISSR dntp ) ISSR 94 5 min; s, 54.5 min, 72.5 min, 4 ; 72 7 min; 4 PCR PTC-00 PCR SRAP.5 +, M9E9-540 SRAP M9E9 540 bp, U ISSR UBC bp, 00 bp DNA ladder marker ( bp) 2 7, F 2 D, B; 2 7, F 2 C, A, (χ 2 ) 3 Mapmaker/Exp Version 3.0b PC 3

3 960 34, Group (LOD=3.0, 37.2 cm); Order Compare Try 3 ; Kosambi, Map, MapDraw V2. [0] ISSR, 42 0, 0 F 2 ISSR, 23, 4, 2.3 UBC 结果与分析 , 87 F 2 SRAP, 53 08, F 2 SRAP, 58 05, 6,.8 M3E7 图 2 引物 UBC835 的部分扩增结果 Fig. 2 Parts of amplifications using primer UBC835 M: marker; P: 2; P2: 7 ; ~9: F 2 ~9 P: Bailei 2; P2: Taiyan 7; 9: F 2 plants 9. Polymorphic fragments are indicated by arrows. M3E7 Fig. Parts of amplifications using primer M3E7 M: marker; P: 2; P2: 7 ; ~9: F 2 ~9 P: Bailei 2; P2: Taiyan 7; 9: F 2 plants 9. Polymorphic fragments are indicated by arrows. 2.2 P=0.05, 28 (23 ISSR, 05 SRAP ), 3 (3 ), ( ), 24.3%, SRAP 25 SRAP 23.8%, ISSR 6 ISSR 26.% 23 (74.2%), 8 ( ) (25.8%) 50 F 2 Molecular Table ( ) primers or primer combinations 表构建遗传图谱所用的分子标记 The molecular used for genetic map construction polymorphic distorted linked unlinked ISSR SRAP Total

4 : SRAP ISSR Mapmaker/Exp Version 3.0b MapDraw V2., , cm, 4.3, 8. cm, 3 ISSR 3 SRAP 26 LG LG2 LG3 LG4, , 2 LG, 29.0 cm, 0 cm, LG0, 48.6 cm, LG23 LG26, 0 cm 3 27, 9, 8, LG2 LG20, LG4 LG, 6 8, 2 3 ISSR SRAP, * Linkage group Length Table 2 表 2 ISSR 和 SRAP 标记在遗传连锁图上的分布 Distribution of ISSR and SRAP on genetic linkage map Average distance distorted Linkage group Length Average distance distorted LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG Total LG amounts 3 讨论 3., [3] 24,,, [5] 9 2 4, Lin [] 0 2~4, [3] 7 2 4, Bindler [4] 2 2~4,,,,, 3.2 SRAP SRAP,,,, [6],,, SRAP, 4 [-4] DNA RFLP RAPD ISSR SSR, RFLP

5 Fig. 3 图 3 烟草 SRAP 和 ISSR 分子遗传连锁图 SRAP and ISSR molecular genetic linkage map of tobacco,,, DNA, ; RAPD, ; SSR, SSR, SSR [] ; SRAP ISSR,,,, SRAP ISSR 3.3, [3] [5] [2] [3] [4] [5-6], Knox Ellis [7] 2, 27 (24.%) 3, [3] DH (4%) F 2,,,,,

6 : SRAP ISSR 963,,, 4 结论,, SRAP ISSR 2 26, cm, 8. cm References [] Lin T Y, Kao Y Y, Lin S, Lin R F, Chen C M, Huang C H, Wang C K, Lin Y Z, Chen C C. A genetic linkage map of Nicotiana plumbaginifolia / Nicotiana longiflora based on RFLP and RAPD. Theor Appl Genet, 200, 03: [2] Nishi T, Tajima T, Noguchi S, Ajisaka H, Negishi H. Identification of DNA of tobacco linked to bacterial wilt resistance. Theor Appl Genet, 2003, 06: [3] Xiao B-G(), Xu Z-L(), Chen X-J(), Shen A-R(), Li Y-P(), Zhu J( ). Genetic linkage map constructed by using a DH population for the flue-cured tobacco. Acta Tab Sin (), 2006, 2(4): 35 40(in Chinese with English abstract) [4] Bindler G, Hoeven R, Gunduz I, Plieske J, Ganal M, Rossi L, Gadani F, Donini P. A microsatellite marker based linkage map of tobacco. Theor Appl Genet, 2007, 4: [5] Yu S-C(), Wang Y-J(), Zheng X-Y(). Construction and analysis of a molecular genetic map of Chinese cabbage. Sci Agric Sin (), 2003, 36(2): 90 95(in Chinese with English abstract) [6] 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, 200, 03: [7] Qi J-M(), Wang T( ), Chen S-H(), Zhou D-X(), Fang P-P(), Liang J-X(), Tao A-F(), Wu W-R(). Genetic diversity and genetic relatives analysis of tobacco germplasm based on inter-simple sequence repeat(issr). Acta Agron Sin ( ), 2006, 32(3): (in Chinese with English abstract) [8] Liang J-X(), Qi J-M(), Fang P-P(), Wang T( ), Chen S-H(), Zhou D-X(), Tao A-F(), Liang K-J(), Wu W-R(). Genetic diversity and genetic relatives analysis of tobacco germplasm based on inter-simple sequence repeat (ISSR). Sci Agric Sin (), 2008, 4(): (in Chinese with English abstract) [9] Liang J-X(), Qi J-M(), Wu W-R(), Zhou D-X(), Chen S-H(), Wang T( ), Tao A-F( ). Studies on DNA extraction and establishment of SRAP reaction system in tobacco. Acta Tab Sin (), 2005, (4): 33 38(in Chinese with English abstract) [0] Liu R-H(), Meng J-L(). MapDraw: A Microsoft Excel macro for drawing genetic linkage maps based on given genetic linkage data. Heraditas ( ), 2003, 25(3): 37 32(in Chinese with English abstract) [] Lin Z-X(), Zhang X-L(), Nie Y-C(), He D-H(), Wu M-Q(). Construction of a SRAP genetic linkage map of cotton. Chin Sci Bull ( ), 2003, 48(5): (in Chinese) [2] Wu X-L(), He C-Y(), Wang Y-J(), Zhang Z-Y(), Dong-Fang Y(), Zhang J-S( ), Chen S-Y(), Gai J-Y(). Construction and analysis of a genetic linkage map of soybean. Acta Genet Sin ( ), 200, 28(): 95 96(in Chinese with English abstract) [3] Lu G-Y(), Yang G-S(), Fu T-D(). Linkage map construction and mapping of a dominant genic male sterility gene (Ms) in Brassica napus. Acta Genet Sin ( ), 2004, 3(): (in Chinese with English abstract) [4] Zhao S-H(), Li Y-Y( 玥 ), Zou J-Q(), Folkertsma R, Hash T C. Construction of a molecular genetic map of sorghum. Rain Fed Crops ( ), 2005, 25(): 3(in Chinese with English abstract) [5] Kinshita T. Report of the committee on gene symbolization nomenclature and linkage group. Rice Genet Newl, 993, 0: 7 39 [6] Coe E H, Polacco M. Gene list and working maps. Maize Genet Coop Newslett, 995, 694: 57 9 [7] Knox M R, Ellis T H N. Excess heterozygosity contributes to genetic map expansion in pea recombinant inbred populations. Genetics, 2002, 62:

7期xin

7期xin 中国农学通报 0,0():- Chinese Agricultural Science Bulletin 花椰菜种质资源遗传多样性 SRAP 标记分析荆赞革, 唐征, 裴徐梨, 刘庆, 张小玲, 罗天宽, 朱世杨 ( 浙南作物育种重点实验室, 温州科技职业学院, 浙江温州 00; 作物遗传