Microsoft Word - 21-房祖业_new_.doc

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蝶威穆
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1 49 1 Vol.49, No OCEANOLOGIA ET LIMNOLOGIA SINICA Jan., 2018 (Mastacembelus armatus) * 瑢 ( ; ) (Mastacembelus armatus), 105 ( 6), 50 (47.6%),, 38, 10, 6, (1) 50%, 31.1% 35% (2) 0.649, (0.572), , 93.3%, , 66.7% (3),,,,, ; ; ; Q789; S968 doi: /hyhz (Mastacembelus armatus), (Synbranchiformes) (Mastacembelidae) (Mastacembelus) (Nelson, 2006),, (, 1985;, 1989;, 1991;, 2006),, (, 2006),,,, (, 2005),,, (, 2008),, (Carvalho et al, 1994) (Brooker et al, 1994) (, 2009) QTL (, 2005;, 2011), (2014) (Pelteobagrus fulvidraco) * ( ), ;, A201601A05 ;, 2016A , 2014A ;, @qq.com :,,, shuhu001@126.com : , :

2 1 : (Mastacembelus armatus) , (2008) (Perca fluviatilis), (Sequeira et al, 2005;, 2012) (Ctenopharyngodon idellus) (Hypophthalmichthys molitrix) (Cyprinus carpio) (, 2009;, 2011;, 2011) (2016), (2017) 7,,,, DNA , ( 17.88g, 19.44cm) ( 24.99g, 20.89cm) 30, 90% 25mg, Ezup DNA, 100ng/μL 1.2 DNA,, 105, 5 27, 15 25, 8 11, 5 21, DNA, PCR 24μL, Mix 12.5μL, 50ng/μL DNA 2μL, 50μmol/L 1μL, ddh 2 O 7.5μL 94ºC 5min, 94ºC 40s, 45s( 1), 72ºC 90s, 35, 72ºC 10min PCR 1.5% , bp 8% 50, ( Mg 2+ ) 1.4 5' (FAM HEX TEMED) 30 DNA PCR, 10μL : Mix 5.0μL 50μmol/L F 0.5μL, 50μmol/L R 0.5μL, 50ng/μL DNA 1.0μL ddh 2 O 3.0μL, PCR STR 1.5 PopGene (allele frequency, P) (observed number of alleles, N a ) (effective number of alleles, N e ) (observed heterozygosity, H o ) (expected heterozygosity, H e ) (Yeh et al, 1999) PIC-CALC (polymorphism information content, PIC) : / 100% ( ), 50 38, 10, 10/20 100% = 50%, 14, 14/45 100% = 31.1%, 14/40 100% = 35% ER6 1

176 49 1 ER6 Fig.1 The amplification results of Beijiang M. armatus using microsatellite primer ER6 : M DNA Marker; 1 30 ER6 30 表 1 38 对在大刺鳅中具有多态性的微卫星引物信息 Tab.

3 ER6 Fig.1 The amplification results of Beijiang M. armatus using microsatellite primer ER6 : M DNA Marker; 1 30 ER6 30 表 1 38 对在大刺鳅中具有多态性的微卫星引物信息 Tab.1 Information of 38 pairs of primers with highly polymorphism in M. armatus (5 3 ) (bp) (T m ) ER2 ER5 ER6 ER9 ER10 ER15 ER18 ER22 ER27 ER29 SAN1 SAN3 SAN7 SAN8 SAN9 SAN15 SAN16 SAN17 F:TATTGAATGTAAACGGCGGC R:GCTTTGAATGAGACGCACAC F:AATTCCTACGAGCCACATGC R:AGTTCATTGGCACCCTGAAA F: GGGGATATTGAGAAGGACAAA R: ACCAAAAAGGCAACCCTAGA F:AGTTCTGTTAACCTCTAACCTCCA R:TAGCTGAGCGTGGTGCTTTA F:TCGATAGCCCAATGTTCCTC R:TCCCGATCCATTCAGAAGAC F:TGTTTTAGTGTGCCACAGGG R:TGCACATTTTCCCCTCTTTC F:AAATAAGCAGAGGGGGTTGG R:AGACCTGGAGCAGCTTTCAA F:GTGTTCAGCCCACTCACTCA R:GGAGCACTGTGCAGACAGAA F:ATTCACCATGGGAACACAGG R:GTGCTATCCGTGTGAGGGTT F:CCAGGACATCTACAGGCGAT R:ATGTTTTGCTGCTGTTGCTG F:TTCACTGAAGAATGAACCAGTTG R:GCACATGCAGACATAAACTGC F:TCTGCCAAATGGCTGTGTTA R:CCTGGCAACAGTGGGATAGT F:GCCATAGAGGAGACTGTCAGC R:TCCGTGAAGAGTTTCAGCCT F:TGTATCCAAAATGGGGGAAA R:GTGGGACAAAAGGAAGACCA F:GAAGATGAAAGAACAGCGCC R:TTGCATTATACAGCTGGGGG F:GGCTGTAGTTCCATGCACAG R:ACCACCATGTGGCCATTTAT F:AGATGCAGAAGGATTGGAGG R:TGCCACAGGCTGTTGATAAT F:AGCACAGCAACAGCAACAAC R:CCCATAACACAGCAAAACAAAA (CA) (GT) (AC) (TG) (CA) (AC) (GT) (CA) (AC) (CA) (TTA) (TTA) (AAT) (ATT) (AAT) (TTC) (TAA) (CAG)

4 1 : (Mastacembelus armatus) 177 (5 3 ) (bp) (T m ) SAN19 SAN20 SAN21 SAN23 SAN26 SAN29 SI1 SI2 SI6 SI7 SI8 SI9 SI10 SI11 SI12 SI17 SI18 SI20 SI24 SI25 F:CCGATTATCAGGAAATGGGA R:GGGGTCAGTGTGCTGTTTTT F:CGCTGGTTTCACCACTTTTT R:TGACGACTGCTGCTGCTAAT F:GCAGTGACAATGATCCACGA R:AATGATAGATTCCCACAGGGG F:ATTTGACCCCTCCCAATACC R:AATGATAGATTCCCACAGGGG F:TGGTGTAAATGCACAGCCAT R:TAAACGAATCAGTCCCCGAC F:CAAACCAGCCTCCATCATTT R:AATGATAGATTCCCACAGGGG F:ACTTCGTCCACCAGGATGAG R:CGAAGCACATGAGAACCAGA F:TAAGCTAGTGGCATGGCAAA R:TGAAGGTCGTGACTGATTGC F:GGTGGAGGTACTCAAGCTGC R:CTTTCACCCGAGAGAAGCTG F:GCCTTTGCAATGTTGGTTTT R:CCTGGTTAAGGAATAAGGGGG F:AACTGCACCTTAAAAGCAATTACA R:CGACACAACACACGTCAGTG F:GCCCACAGAAAGGTTGTGTT R:TGGTAACTGATCATTTTTGTATTTTCA F:TTAAAGCCTCCAAATCGCTG R:CCCATGAAGTTTCACATCCA F:ACTTGTCAGATAGACGGGGC R:GGTGTGTGGAGACTACGCCT F:TGGTTACGAAGAACATGGCA R:TGTTGTAGTGTGTGGCCCTG F:CCTGTGACCCTGGTTAAGGA R:GAATGACCATTGTGATGAATCC F:GATGGCACACCAGCAGTAAA R:CCTGTGACCCTGGTTAAGGA F:CCTTTCACCCAAGTGATGCT R:ATTTTTACCAGTGGCACGCT F:TAAGGGACAGGTGGACGGTA R:CAGCGCTGTAGAACTTTGCTT F:TCCTCCCACAGTCCAAAAAC R:TGAATCGTTGTGTGTCAGGAA (TGA) (TAT) (GCT) (AGG) (TAA) (TTA) (AAGA) (ATAG) (TCCA) (ATCC) (ATAG) (TATT) (AAAG) (ATCA) (TTAT) (GATG) (ATCC) (ATGG) (TGAA) (ATGG) , 30,, , SI2 20 ; 3 20, (9.500) (6.000) (8.000), bp; ; (Ho) ; ; PIC ,, (N a ) (N e ) (H o ) (H e ) (PIC) ,

5 : (PIC=0.735) (PIC=0.605) (PIC= 0.606) 30 2 表 2 30 对微卫星引物对北江群体大刺鳅的遗传多样性评估 Tab.2 The 30 pairs of the primers on the assessment of genetic diversity of Beijiang River population (N a ) (N e ) (H o ) (H e ) (PIC) ER ER ER ER ER ER ER ER ER ER SAN SAN SAN SAN SAN SAN SAN SAN SAN SAN SI SI SI SI SI SI SI SI SI SI , 30 PopGene 18,, , 6.722, SAN21 2, SI1 20, , 3.224, (Ho) , 0.822; (He) , PIC CALC, PIC , 0.572, SAN16 PIC, 0.283, SI2 PIC, 0.847,, (PIC=0.627)> (PIC=0.584)> (PIC=0.506) 18 3, 4

6 1 : (Mastacembelus armatus) 179 表 3 18 对微卫星引物对澜沧江群体大刺鳅的遗传多样性评估 Tab.3 The 18 pairs of primers on the assessment of genetic diversity of Lancang River population (N a ) (N e ) (H o ) (H e ) (PIC) ER ER ER ER ER ER SAN SAN SAN SAN SAN SAN SI SI SI SI SI SI Tab.4 表 4 不同类型核心序列的微卫星对两个群体的评估效果比较 Comparison in the result of assessment on genetic structure of the two populations in different repeat motif microsatellites (N a ) (N e ) (H o ) (H e ) (PIC) DNA, 105, PCR,,, 50, 38, 36.2%,,, (2016) (2013) (Acrossocheilus monticola), , 11.04% 30, Ellegren(2002) , 8 9, 5 7 (2009) (Anguilla japonica),

7 PCR 55, DNA,, 3.2,, ;, ;, ,, Brenner (1993), (Crooijmans et al, 1997; Maguire et al, 2000; Tóth et al, 2000; Serapion et al, 2004), Weber(1990), 12, PIC; 16, PIC 0.5, 19, PIC 0.5,, PIC>0.5, ; 0.25<PIC<0.5, ; PIC<0.25, (Botstein et al, 1980), Feresu-Shonhiwa (1998) PIC>0.70, 30 28, 93.3%, 8 (ER5 ER6 ER10 ER22 ER27 SI1 SI2) PIC>0.7, SI2 PIC 0.916; 2 SAN23 SI18 PIC , PIC>0.25,, 6.67%,, , , (N a N e H e H o PIC), (2012),, H o H e,, (2016), (Coilia macrognathos Bleeker) (C. idellus) (Squaliobarbus ourriculus) 鲌 (Erythroculter ilishaeformis) (Monopterus albus) (Odontobutis potamophila),,,, 3.3 N a N e H e H o PIC,,,,,, (Hearne et al, 1992; Diwan et al, 1997;, 2009), (2005),,,, (2013) (A. monticola),, (Schlötterer et al, 1992; Hite et al, 1996) (2009),,, ER5 ER6 ER10 ER22 ER27

8 1 : (Mastacembelus armatus) 181 SI1 SI2,,, (, 2011),,, :, , :,,, DNA., 20(1): 51 58, :, ,,, , 2(2): , :,,, , 32(2): ,,, , 15(4): ,,, , 18(4): ,,, ISSR., 40(1): 63 70,, , 15(4): , , (10): 22 25, : 2016, 101, :, ,,, SSR. ( ), 38(5): ,, , 26(5): ,,, , 1(6): 90 94,,, , 16(1): ,,, 鲉 (Sebastes schlegeli)., 47(1): ,,, ( ), 16(3): 8 14,,, , 17(6): , :, , , (10): 17,,, , 20(3): Botstein D, White R L, Skolnick M et al, Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Society of Human Genetics, 32(3): Brenner S, Elgar G, Sanford R et al, Characterization of the pufferfish (Fugu) genome as a compact model vertebrate genome. Nature, 366(6452): Brooker A L, Cook D, Bentzen P et al, Organization of microsatellites differs between mammals and cold-water teleost fishes. Canadian Journal of Fisheries and Aquatic Sciences, 51(9): Carvalho G R, Hauser L, Molecular genetics and the stock concept in fisheries. Reviews in Fish Biology and Fisheries, 4(3): Crooijmans R P M A, Van der Poel J J, Groenen M A M et al, Microsatellite markers in common carp (Cyprinus carpio L.). Animal Genetics, 28(2): Diwan N, Cregan P B, Automated sizing of fluorescentlabeled simple sequence repeat (SSR) markers to assay genetic variation in soybean. Theoretical and Applied Genetics, 95(5 6): Ellegren H, Microsatellite evolution: a battle between replication slippage and point mutation. Trends in Genetics, 18(2): 70 Feresu-Shonhiwa F, Howard J H, Electrophoretic identification and phylogenetic relationships of indigenous tilapiine species of Zimbabwe. Journal of Fish Biology, 53(6): Hearne C M, Ghosh S, Todd J A, Microsatellites for linkage analysis of genetic traits. Trends in Genetics, 8(8): Hite J M, Eckert K A, Cheng K C, Factors affecting fidelity of DNA synthesis during PCR amplification of d(c-a) n d(c-t) n microsatellite repeats. Nucleic Acids Research, 24(12): Maguire T L, Edwards K J, Saenger P et al, Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae). Theoretical and Applied Genetics, 101(1 2): Nelson J S, Fishes of the World. 4th ed. Hoboken: Wiley and Sons,

9 Schlötterer C, Tautz D, Slippage synthesis of simple sequence DNA. Nucleic Acids Research, 20(2): Serapion J, Kucuktas H, Feng J N et al, Bioinformatic mining of type microsatellites from expressed sequence tags of channel catfish (Ictalurus punctatus). Marine Biotechnology, 6(4): Sequeira F, Rocha S, Ferrand N et al, Isolation and characterization of seven microsatellite loci in Chioglossa lusitanica (Urodela: Salamandridae). Molecular Ecology Notes, 5(2): Tóth G, Gáspári Z, Jurka J, Microsatellites in different eukaryotic genomes: survey and analysis. Genome Research, 10(7): Weber J L, Informativeness of human (dc-da) n (dg-dt) n polymorphisms. Genomics, 7(4): Yeh F C, Yang R C, Boyle T, POPGENE version 1.31: Microsoft window-based freeware for population genetic analysis. Alberta: University of Alberta SCREENING AND CHARACTERISTIC ANALYSIS ON DI-/TRI-/TETRA-NUCLEOTIDE-REPEATED MICROSATELLITES IN MASTACEMBELUS ARMATUS FANG Zu-Ye 1, CHEN Xiao-Dong 1, WU Yong-Shi 1, TAN Jie-Rong 1, ZHANG Wei-En 1, WANG Zheng-Yu 1, LIN Ting-Ting 1, ZHA Guang-Cai 2, SHU Hu 1 (1. College of Life Science, Guangzhou University, Guangzhou , China; 2. School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou , China) Abstract Thirty individuals of two wild fish Mastacembelus armatus populations from Beijiang River (BJP), Guangdong (average body weight 17.88g, and average body length 19.44cm) and from Lancang River (LCP), Yunnan (24.99g, and 20.89cm) were sampled to study the reduced-representation genome sequencing, from which 105 pairs of microsatellite primers were developed and were of di-/tri-/tetra-nucleotide-repeated microsatellite (the number of repeat was six or more, and 50 pairs (47.6%) of them yielded steady amplifiable primers. In addition, we found 38 microsatellite loci who had high polymorphism, from which we randomly selected 10 and 6 pairs each from the three types of repeat from BJP and LCP, respectively, to study the genetic diversity. Result presents that: (1) the efficiency of screening the di-nucleotide-repeated microsatellite primer was 50%, while that of the tri- and tetra-nucleotide-repeated ones was only 31.1% and 35%, respectively. (2) The average polymorphism information content (PIC) was higher in BJP (0.649) than LCP (0.572). We detected 235 alleles from 30 microsatellite loci in BJP and 93.3% of them were in high polymorphism level, while 120 alleles in 18 microsatellite loci in LCP and 66.7% of them were in high polymorphism level. (3) The di-nucleotide-repeated microsatellite markers in the two wild populations presented greater values of five polymorphic indexes than tri- or tetra-nucleotide-repeated ones. The above-mentioned findings indicate that di- and tri-nucleotiderepeated microsatellites are more effective in screening and more polymorphic than those of the tri- and tetra-repeated. BJP showed higher genetic diversity than LCP. The primers screened in this study could provide a reference to analyze genetic diversity among different populations, to establish their genetic linkage, to determine genetic relationships etc., and lay a solid ground for the protection of germplasm resources of M. armatus. Key words Mastacembelus armatus; microsatellite; di-/tri-/tetra-nucleotide repeat; polymorphism

Microsoft Word - 李学军.doc

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