作物学报 ACTA AGRONOMICA SINICA 2008, 34(): 958 963 http://www.chinacrops.org/zwxb/ ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn DOI: 0.3724/SP.J.006.2008.0958 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 ( /, 350002; 2, 353200; 3, 350003) : 7 2, 87 F 2, 68 SRAP ISSR, F 2 PCR, 26 2 (92 SRAP 20 ISSR) 560.2 cm, 8. cm 6 26 2~20, 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 350002, Fujian; 2 Fujian Shunchang Tobacco Company, Shunchang 353200, Fujian; 3 Fujian Tobacco Company, Fuzhou 350003, 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 560.2 cm with an average distance of 8. cm. Sixteen were still unlinked. All 26 linkage groups consisted of 2 20 ranging in length from 0 29.0 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),,,, E-mail: Qijm863@63.com; Tel & Fax: 059-87644898 Received( ) 2008-03-23; Accepted( 2008-05-6.
: SRAP ISSR 959 99 F 2, ; Nishi [2] (W6 Michinoku) 25 DH, AFLP ; [3] Speight G-28 NC2326 37 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; 94 45 s, 54.5 min, 72.5 min, 4 ; 72 7 min; 4 PCR PTC-00 PCR SRAP.5 +, M9E9-540 SRAP M9E9 540 bp, U826-860 ISSR UBC826 860 bp, 00 bp DNA ladder marker (00 5 000 bp) 2 7, F 2 D, B; 2 7, F 2 C, A, (χ 2 ) 3 Mapmaker/Exp Version 3.0b PC 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 UBC835 2 2 结果与分析 2. 2 7, 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 0 23 6 20 3 SRAP 58 05 25 92 3 Total 68 28 3 2 6
: SRAP ISSR 96 2.3 Mapmaker/Exp Version 3.0b MapDraw V2., 28 2 26, 560.2 cm, 4.3, 8. cm, 3 ISSR 3 SRAP 26 LG LG2 LG3 LG4, 20 0 2 0, 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 29.0 20 5.3 8 LG5 28.8 2 28.8 0 LG2 209.0 0 23.2 3 LG6 27.7 2 27.7 0 LG3 92.0 2 7.5 2 LG7 23.3 2 23.2 0 LG4 254.0 0 28.2 6 LG8 8.2 2 8.2 0 LG5 74.9 5 8.7 LG9 22.2 2 22.2 0 LG6 73. 7 2.2 2 LG20 6.8 2 6.8 2 LG7 82.0 3 4.0 0 LG2 8.0 2 8.0 0 LG8 25.0 8 3.6 0 LG22 5.0 2 5.0 0 LG9 20.7 2 20.7 0 LG23 0 2 0 0 LG0 48.6 2 48.6 0 LG24 5.0 2 5.0 LG 4.7 2 4.7 0 LG25 2.7 2 2.7 0 LG2 37.0 2 37.0 2 LG26 0 2 0 0 LG3 35.2 2 35.2 0 Total 560.2 2 8. 27 LG4 28.6 3 4.3 0 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
962 34 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,,,,,
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