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1 2012 3, 19(2): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J SSR SRAP 乔慧 1,2, 吴滟 2, 傅洪拓 1,2, 龚永生 2, 蒋速飞 2, 熊贻伟 1., ; 2.,, 摘要 : SSR SRAP (Macrobrachium nipponense) 175 ( 27 SSR 148 SRAP ) 53 2~8, 3 35, 18, 3.3 ; 6.7~91.2 cm, 49.0 cm, 1.4 cm, 13.1 cm cm, cm,, cm, 51.83%,, QTL, : ; ; SSR; SRAP; 中图分类号 : S96 文献标志码 : A 文章编号 : (2012) ,, (marker assisted selection, MAS),, (Cyprinus carpio) (Oncorhynchus mykiss) (Oreochromis spp.) (Crassostrea gigas) (Mytilus edulis) [1 6], [2] [3], QTL, (Penaeus chinensis) [7] (Penaeus japonicus) [8] (Penaeus litopenaeus vanmanici) [9] (Penaeus monodon) [10] (Portunus trituberculatus) [11],, (Macrobrachium nipponense),,,, [12 15], (simple sequence repeat, SSR) (sequence-related amplified polymorphism, SRAP),, 1 1.1,,, 收稿日期 : ; 修订日期 : 基金项目 : (BG ); (2011JBFA02). 作者简介 : (1981 ),,,. qh @126.com 通信作者 :,,. fuht@ffrc.cn

2 2 : SSR SRAP 203, , F 1, 35:29, DNA, Strauss [16] ( ) SRAP Li [17] SRAP ( 1) PCR : 10 PCR 2.5 μl, MgC1 2 (25 mmol/l)1.5 μl, Taq DNA (5 U/μL)0.16 μl, (10 μmol/l) 2.0 μl, dntp(2.5 mmol/l)1.5 μl, DNA 1.0 μl, ddh 2 O 25 μl PCR : 94 5 min; 94 1min, 35 1 min, 72 1min, 5 ; 94 1min, 50 1min, 72 1min, 35 ; min; 4 : 8% SSR SSR : 40 SSR (GenBank GU GU189639), 106 GenBank, GU GU078569, GU GU586097, EU EU130935, GQ GQ primer5, 96, 4, PCR : 10 PCR 2.5 μl, MgC1 2 (25 mmol/l)1.5 μl, Taq DNA (5 U/μL)0.16 μl, (25 μmol/l) 0.1 μl, dntp(2.5 mmol/l)1.5 μl, DNA 1.0 μl, ddh 2 O 25 μl PCR : 94 5 min; s, 30 s, s, 30 ; min; 4 SRAP (P 0.05), SRAP SSR SRAP M(1-10), E(1-10) ( 1) - M7E Me7 Em10, 194 bp (.loc ),, JoinMap3.0(Van Ooijen and Voorrips, SRAP SRAP marker Tab.1 表 1 SRAP 标记上下游引物序列及编号 List of SRAP forward and reverse primer used in this study forward primer SRAP SRAP marker reverse primer Me1 TGAGTCCAAACCGGATA Em1 GACTGCGTACGAATTAAT Me2 TGAGTCCAAACCGGAGC Em2 GACTGCGTACGAATTTGC Me3 TGAGTCCAAACCGGAAT Em3 GACTGCGTACGAATTGAC Me4 TGAGTCCAAACCGGACC Em4 GACTGCGTACGAATTTGA Me5 TGAGTCCAAACCGGAAG Em5 GACTGCGTACGAATTAAC Me6 TGAGTCCAAACCGGACA Em6 GACTGCGTACGAATTGCA Me7 TGAGTCCAAACCGGACG Em7 GACTGCGTACGAATTCAA Me8 TGAGTCCAAACCGGACT Em8 GACTGCGTACGAATTCAC Me9 TGAGTCCAAACCGGAGG Em9 GACTGCGTACGAATTCAG Me10 TGAGTCCAAACCGGAAA Em10 GACTGCGTACGAATTCAT

3 ),, CP(cross-pollinating), (LOD 3.5) 1 1 (P>0.05) Kosambi (Kosambi, 1944) (centimorgan, cm), MapChart version 2.2 (Voorrips, 2002) = - (n); (s)= / ; = -1; = / (1) : (G of ) 4 ; (G oa ) (2) (G e ), G e1 G e2 G el 2, : G e1 =G oa +2 s n; G e2 (m+1)/(m 1), m G e G e1 G e2 (3) C of =G of / G e C oa = G oa / G e SRAP 100 SRAP, 2 724, 88, , 5.4, 18.1% ( ) 344, 69.8%, 12.6% , 132 (38.4%) ; 177(51.4%) (10.2%) % SSR, 6, 52 52, 38, 73.1% 38, ( ), ( ) % SRAP 38, 27 ( 2) 148 SRAP 53, 3 35, 18 2~8, 3.3, 6.7~91.2 cm, 49.0 cm, 1.4 cm, 13.1 cm cm,, cm, G e cm, G e cm,, cm 22.76%, 51.83%( 3) MapChart, ,,,, (, two way pseudo-testcross)

4 2 : SSR SRAP 205 locus Tab. 2 表 2 青虾遗传连锁图谱上的微卫星标记 Microsatellite loci on the linkage map of Macrobrachium nipponense (5 3 ) primers sequence (5 3 ) T m / accession no. WXM01 F:TTCCCCACGCACCTCAAT R:GACATCCATGCAAAGCAACAG 54 GU WXM02 F:GCCATTTTCTCATAAGGGT R: ACGGTGGTATTCAGGGAT 54 GU WXM06 F: TTGGCAAGTCTCGTCTGATG R: CGAGGAAACGCCTGCTAC 54 GU WXM07 F: CGACGAGGCAACAGAATA R: TGATAATGCGAGGGAGTA A 54 GU WXM13 F: ATGAAGGTAAGTGGCTGAGA R: TCACACGTAAAGTTTTTGGA 58 GU WXM15 F: TGACAACGAGACTGCTAA R: TTGACGCTGCTTACATC 60 GU WXM17 F: GAAAGGAACTTCAAGAGGC R: GGACAGTGAGCAAAGCATC 54 GU WXM18 F: TCGATGGCGACTTGCA R: CCTCTGCCCTGACTTGAA 60 GU WXM20 F: TCCCGATGAAAGGCACA R: TAAAGGCGGTGATGAT 60 GU WXM27 F: ACAATTTAGGTGACGCTC R: TGCATGAATGCTGGTAT 58 GU WXM32 F: TTGTGACGCCAGAGTG R: GCTTGTAGAGCGCCTAT 60 GU WXM33 F: TGGTCCTGTTGTCTTTCT R: GTTGCTCGGTCTATGC 54 GU WXM35 F: GTAACAGGGACTAGCAATG R: ACCGCAAGACTCAACC 58 GU WXM41 F: GTGGCCTTTGACTACG R: TGTCAGCAACAACGAGA 54 GU WXM42 F: GTGGGGTTTCACTGGT R: AGCTGCCTCCTGACTAA 54 GU MaN27 F: CATCGAGTGAAGAGGTGT R: GTCATAAATGCTGCTACG 52 GU A28 F: TGCATTCCCAATTTCTC R: CTTAACCAACTTCCATCGT 50 GU B9 F: TGGTGGTAGTTGAATTAGAAGG R: AGCACAGGAAGGGAAGG 52 GU B21 F: GCTTGCTGTAACCGAC R: TTGCTACACCAGGAAAT 50 GU B70 F: CCAACCATCGGCTTTC R: TGACCCGTTTGTAGTGC 50 GU C77 F: GTCCGATAAATCCCACA R: TCCTGCTCTTCCGTTC 50 GU Mni009 F: AGAAAGGGACTGGTGG R: GTCTACAGGAAATGTAAAGC 50 EU Mni055 F: ATCACCCTCAATAGCA R: CCAAGCCTGTAAAATG 50 GQ Mni100 F: GAAATAGAAGACCTCCTGC R: TGTAAACAATCCCACTCAC 50 GQ Mni102 F: ACCTCGGGAATATCTCATC R: AGTGGCATCGTGTCAGC 50 GQ Mni105 F: TGGTCCACCCAAGTCT R: CCTGGCGTCTCATTTA 52 GQ Mni115 F: AACAGTAAACGGAGTAGAAA R: CGAATGGGAATGAAAG 50 GQ 表 3 青虾遗传连锁图谱总结 Tab.3 Summary of the linkage maps of M. nipponense item result linkage groups 53 average no.of markers per group /cm average marker spacing /cm maximum marker spacing /cm maximum length of linkage group /cm maximum length of linkage group /cm observed genome length G of G oa /cm estimated genome length G e1 G e2 G e /% genome coverage C of C oa F 1, F 2 BC1,, F 1, [12 14],,,, 3.2, DNA (random amplified polymorphic DNA, RAPD) AFLP (amplified fragment length polymorphism, AFLP) SSR,

5 206 19

6 2 : SSR SRAP cm. Fig. 1 Linkage map of Macrobrachium nipponense Markers are shown on the right of each group and the adjacent marker spacing is displayed on the left in Kosambi, cm. SSR, GenBank SSR 56,

7 SSR [18] 52 SSR, 54.2% SSR, SSR ( 52.4% [19], 51.8% [10], 33.3% [11] ) 27 20, SRAP,,, [20 25], SRAP 11.3%~35.5%, SRAP 0.7~3.7 [25 27] (Pelteobagrus fulvidraco), SRAP 33.2% [28] 100 SRAP, 88, 88%, SRAP 5.4, AFLP, SRAP, SRAP 18.1%, AFLP [7,11,25,29], SRAP, (Crassostrea gigas) 20.9% SSR [30] 26.9% AFLP [31] ; (Chlamys farreri), 29.96% 30.45% SSR [32] (Ostrea edulis) 32.8% [33], [9] [7] AFLP, 26.9% SSR 30.2% SRAP,, [34] [35] 1 1 (Aa) (aa) 50%,, 1 1, 163, 1:1 154, 85.1%, 3 1 9, 3.3, 104 [36], 52, 53,,,,, 51.83%, [37] (Penaeus japonicus) [8] [9],,, 125 [38],,,, cm [25], 3 584~5 407 cm [9], cm [39], [36] cm cm,

8 2 : SSR SRAP 209,, DNA,,,,,,,, (QTL),, SRAP SSR, SSR SNP,, 参考文献 : [1] Cheng L, Liu L, Yu X, et al. A linkage map of common carp (Cyprinus carpio) based on AFLP and microsatellite markers [J]. Animal Genetics, 2010, 41(2): [2] Nichols K, Young W, Danzmann R, et al. A consolidated linkage map for rainbow trout (Oncorhynchus mykiss) [J]. Animal Genetics, 2003, 34: [3] Lee B Y, Lee W J, Streelman J T, et al. A Second-Generation Genetic Linkage Map of Tilapia (Oreochromis spp.) [J]. Genetics, 2005, 170: [4] Lallias D, Lape'gue S, Hecquet C, et a1. AFLP based genetic linkage maps of the blue mussel (Mytilus edulis) [J]. Anim Gen, 2007, 38: [5] Li L, Guo X M. AFLP-based genetic linkage maps of the pacific oyster Crassostrea gigas Thunberg [J]. Mar Biotechnol, 2004, 6: [6],,,. [J]., 2009, 16(6): [7] 燚,,. [J]., 2008, 53(5): [8] Li Y T, Byme K., Miggiano E., et al. Genetic mapping of the kuruma prawn Penaeus japonicus using AFLP markers [J]. Aquaculture, 2003, 219: [9] Pèrez F, Erazo C, Zhinaula M, et al. A sex specific linkage map of the white shrimp Penaeus(Litopenaeus)vanmanici based on AFLP markers [J]. Aquaculture, 2004, 242: 105-l18. [10] You E M, Liu K F, Huang S W, et al. Construction of integrated genetic linkage maps of the tiger shrimp (Penaeus monodon) using microsatellite and AFLP markers [J]. Anim Gen, 2010,41(4): [11],,,. [J]., 2010, 31(3): [12],,,. 4 RAPD [J]. :, 2006, 3(2): [13],,,. [J]., 2008, 17(5): [14],,,. [J]., 2010, 34 (2): [15],,,. [J]., 2010, 34(5): [16] Strauss W M. Preparation of genomic DNA from mammalian issues [C]. Current Protocol in Molecular Biology. New York: John Wiley and Softs. 1989: [17] 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 [J]. Theoret Appl Gen, 2001, 103: [18] Qiao H, Li F J, Fu H T, et al. Isolation and characterization of 40 microsatellite loci for oriental river prawn (Macrobrachium nipponense) and cross-species utility [J]. Conservation Genetics Resources, 2011, 3: [19] Liu B, Wang Q Y, Li J, et al. A genetic linkage map of marine shrimp Penaeus (Fenneropenaeus) chinensis based on AFLP, SSR, and RAPD markers [J]. Chinese Journal of Oceanology and Limnology, 2010, 28(4): [20],,,. SRAP [J]., 2010, 18(2): [21],,,. SRAP [J]., 2006, 13(4): [22],,,. [J]., 2007, 14(5): [23],,,. SRAP SCAR [J]., 2008, 54(3): [24],,. SRAP [J]., 2009, 33(5): 727-

9 [25],,,. SRAP [J]., 2010, 32(3): [26],,,. SRAP QTL [J]., 2005, 3(6): [27],,,. SRAP ISSR [J]., 2008, 34(11): [28],,,. QTL [J]., 2010, 34(2): [29]. QTL [D]. :, [30] Launey S, Hedgecock D. High genetic load in the Pacific oyster Crassostrea gigas[j]. Genetics, 2001, 159: [31] Li L, Guo X M. AFLP-based genetic linkage maps of the pacific oyster Crassostrea gigas Thunberg [J]. Marine Biology, 2004, 6: [32]. (Chlamys farreri) [D]. :, [33] Lallias D, Beaumont A R, Haley C. et al. A first-generation genetic linkage map of the European flat oyster Ostrea edulis (L.) based on AFLP and microsatellite markers [J]. Animal Genetics, 2007, 38(6): [34] Lyttle T W. Segregation distorters [J]. Annu Rev Genet, 1991, 25: [35] Causse M A, Fulton T M, Cho Y G, et al. Saturated molecular map of the rice genome based on an interspecific backcross population [J].Genetics, 1994, 138: [36],,. [J]., 1994, 25(5): [37] Wilson K, Li Y, Whan V, et al. Genetic mapping of the black tiger shrimp Penaeus mondon with amplified fragment length polymorphism [J]. Aquaculture, 2002, 204: [38],. ( ) [J]., 1991, 1: [39] Moore S S, Whan V, Davis G P, et al. The development and application of genetic markers for the Kuruma prawn Penaeus japonicus[j]. Aquaculture, 1999, 173: Construction of a genetic linkage map for oriental river prawn (Macrobrachium nipponense) using SSR and SRAP markers QIAO Hui 1, 2, WU Yan 2, FU Hongtuo 1, 2, GONG Yongsheng 2, JIANG Sufei 2, XIONG Yiwei 2 1. College of Fisheries, Nanjing Agriculture University, Wuxi , China; 2. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi , China Abstract: The oriental river prawn, Macrobrachium nipponense, is widely distributed throughout China, Japan, Korea, and Burma. This species is of economic importance in China where about t are produced annually by intensive culture. However, the economic value of the species has declined significantly in recent years due to genetic retrogression. To facilitate genetic improvements, we constructed a genetic linkage map for oriental river prawn using SRAP and SSR markers and pseudo-testcross mapping. A total of 175 makers including 27 microsatellite and 148 SRAP makers were mapped in 53 linkage groups. The number of markers per group ranged from 2 to 8 and thirty-five groups included no less than three markers. The average number of markers per group was 3.3. The length of groups ranged from 6.7 cm to 91.2 cm (Kosambi). The maximum space between adjacent markers was 49.0 cm while the minimum space was 1.4 cm, with an average distance between markers of 13.1 cm. The framework map contained 16 groups with a length of cm. The total length of the map was cm. The average estimated genome size for oriental river prawn was cm. Based on the estimated genome lengths, the map accounted for 51.83% of the genome. This is the first genetic linkage map of oriental river prawn. Our data may be used to aid quantitative trait loci (QTL) mapping, gene cloning, and genetic selection. The map also provides a basis for the construction of a higher density genetic linkage map for oriental river prawn. Key words: oriental river prawn; Macrobrachium nipponense; SSR; SRAP; genetic linkage map Corresponding author: FU Hongtuo. fuht@ffrc.cn

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