44 6 Vol.44, No.6 2013 11 OCEANOLOGIA ET LIMNOLOGIA SINICA Nov., 2013 (Pelodiscus sinensis)est-ssr * 1 1 1 1 2 3 4 (1. 215123; 2. 215004; 3. 215006; 4. 221200) EST-SSR, 60 (Pelodiscus sinensis)-,, 15 EST-SSR, 2.76330.5961 0.539177C8387 (P<0.05)(P<0.01), CC AB 4 ; C211 (P<0.01)(P<0.05), C, CC, BB AB ; C5670 (P<0.01)(P<0.05), BB AA ; C1312 C13038 (P<0.01), C1312 BC C13038 BB ; EST-SSR ; ; S941 (Pelodiscus sinensis),,,,,,,, (, 2005),, ( simple sequence repeat, SSR),, SSR(G-SSR) SSR(EST-SSR) EST-SSR,, G-SSR (Gupta et al, 2004; Skaala et al, 2004)Que (2007)Li (2010)Bu (2011) G-SSR,, EST-SSR (Zalapa et al, 2012), EST, QTL SSR (Zhang et al, 2012), (2013) EST, EST *, BE2012357 ;, PJ2012-10,, E-mail: 18862131376@163.com :,, E-mail: suda-shui@163.com : 2012-05-26, : 2012-08-18
6 : (Pelodiscus sinensis)est-ssr 1655 SSR,, EST-SSR EST,, EST-SSR,,, 1 1.1 (Pelodiscus sinensis),,, 60,, 1.2 EST-SSR 15 EST,,, 1 1.3 1.3.1 DNA 60,,, - DNA, (Thermo Fisher) DNA, 100ng/L, 20 1.3.2 15 20L PCR, 2ES TaqMIX 10L, (10mol/L) 1L, DNA 1L, 20L, PCR (TAKARA), : 94 2min, 26 (94 30s, 5060 30s, 72 30s), 72 2min6%, (2009), Gel-Pro Analyzer 4.5 1.3.3 (1) Tab.1 表 1 微卫星位点及引物信息 Information of the microsatellite loci and primers sequence (5 3 ) NR-tophit_description C8433 (AT) 9 C8387 (ATTT) 4 C4663 (GT) 8 C211 (CGG) 5 C5670 (AGG) 5 C5960 (AGC) 4 C0009 (GT) 9 C8513 (GT) 9 C11010 (CCTT) 4 C25692 (GT) 9 C2226 (CT) 10 C4950 (AC) 11 C1312 (CT) 11 C13038 (CCT) 5 C1675 (GT) 11 F: ATGCCAGCGTTGTGCTTTC R: CAAATCTGCCCTACGTCGC F: GCTGTTCCGACCCAGTGTC R: AGCACCTGTAGGAGGACTTG F: AAACAACAGGATTTCCATGCTC R: CCTCCCTCAGACAAGCTCC F: AGTTCCCTGAGCAGTACCC R: ACCTGCTTACAGCTGAGTTTC F: CAACGGATGCACTCTCGC R: AGTGTTAGGGACAGCGCC F: CTGCATCCTTGGCTTCGTG R: CTGCATCCTTGCGTTCGTG F: CATCGCAACCTTGGAGAGAC R: CTGGCTAATTGTGCAGACCG F: CCAATGCGAATGAGTTTGCC R: CTGGCTAATTGTGCAGACCG F: AGCTGGGCAATAGATGGCTG R: ATGAGGCAGACAAGAGCC F: ATCAAACCTCCAGGGCTCC R: GAGATTGTCCCTGGTTGGC F: GGCACAGTTTGATCAGTGGG R: GGGTGAAGACTCTGATGGG F: GCTTGCATAGATCCGGCAC R: TGATACCTAGGACCGCAGG F: AGAGGATTGACCTAAGAAATTG R: AGGGAACTGAGCAAGGGTG F: GATGCAGCGAAGGAACACG R: GGGCTTTGGTGACGACTTG F: CAAGAAGTCCGGCTTTGGC R: CTCCAGAGATATGCCTTCCAG NR-tophit_description: NR E3A[] B [] G 3 [] 2D [] Der1-like 1 [] RNA RPB1 []
1656 44, Gel-Pro Analyzer 4.5 PopGen32 (P)(N a ) (N e )(H o )(H e ), Botstein (1980) PIC (2) SPSS 16.0 Shapiro-Willk 4, (GLM) 15 SSR 4, ANOVA, 4 2 2.1, 60, 4 (, 2011) SPSS 16.0 Kolmogorov-Smirnov, P 0.05, 4 ( 2), 2.2 PCR, 15,,, 1 2 C8433 C0009 表 2 中华鳖的体重 体长 背甲长 裙边宽正态分布检验 Tab.2 The test of normal distribution in body weight, body length, carapace length and side width of P. sinensis P 42.359211.67388 18.44 65.97 0.561 7.98001.30702 5.60 10.20 0.122 6.08000.79814 4.10 7.60 0.631 1.24000.28175 0.70 1.80 0.123 15 27, (N a ), (N e ) (H o )(H e )(PIC) 3.33332.76330.39890.5961 0.539177( 3) 2.3 15 4,, 5, C8387 (P=0.021)(P=0.02) (P=0.032)(P<0.05), (P=0.0087) (P<0.01); C211 (P=0.007) (P=0.009) (P<0.01), (P= 0.028)(P<0.05); C5670 (P=0.000) (P<0.01), (P=0.042) (P<0.05); C1312 C13038 (P=0.009) (P=0.002)(P<0.01) 5, ( 4): C8387, CC 4, AB (P<0.05), CC 4, AB C5670, BB Fig.1 1 C8433 PCR amplification result of C8433 loci in P. sinensis Fig.2 2 C0009 PCR amplification result of C0009 loci in P. sinensis
6 : (Pelodiscus sinensis)est-ssr 1657 表 3 Tab.3 中华鳖群体 15 个微卫星位点的遗传多样性统计 Data of genetic diversity of 15 microsatellite loci for P. sinensis N a N e H o H e PIC C8433 4 3.0756 0.3833 0.6805 0.631108 C4663 3 2.6836 0.4833 0.6326 0.580413 C8387 4 2.7098 0.3333 0.6363 0.608413 C211 3 2.7304 0.3833 0.6391 0.571331 C5670 4 3.5122 0.4667 0.7213 0.686731 C5960 4 3.9024 0.6833 0.7500 0.722843 C0009 7 6.1121 0.6333 0.8434 0.895932 C8513 3 2.8092 0.3667 0.6494 0.609267 C11010 3 1.9667 0.3333 0.4957 0.406474 C25692 3 2.4991 0.3000 0.6049 0.526842 C2226 3 2.0202 0.4000 0.5092 0.393309 C4950 2 2.0000 0.4000 0.5042 0.37500 C1312 3 1.6969 0.2500 0.4141 0.367502 C13038 2 1.8967 0.5333 0.4768 0.36101 C1675 2 1.8349 0.400 0.4588 0.351488 3.3333 2.7633 0.3989 0.5961 0.539177 N a, N e, H o, H e, PIC AA (P<0.05), BB, AA C211 B,, CC AB AA BB (P<0.05), BC AB (P<0.05), ABAABB (P>0.05), C ; CC (P<0.05), 4 (P>0.05); CC, BB (P<0.05), BBBCABAA (P>0.05), CC ; BB AB C1312 Der1-like, 4 表 4 中华鳖 5 个微卫星位点不同基因型体重 体长 背甲长和裙边宽的平均值及多重比较 Tab.4 Means and multiple comparisons of body weight, body length, carapace length and side width of P. sinensis in 5 microsatellite loci (g) (cm) (cm) (cm) C8387 AB 6 28.657510.56721 a 6.47500.7974 a 5.20000.9055 a 1.17920.2653 a AA 36 40.277110.4760 b 7.75831.2703 ab 5.97500.7525 ab 1.00000.2449 ab BC 7 50.89209.6764 b 8.88000.95237 b 6.36000.4159 ab 1.42000.2387 b CC 11 51.23297.1991 b 8.95710.7067 b 6.74290.5533 b 1.45710.2070 b C5670 AA 33 36.25902.0200 a 7.84510.2433 a 5.81310.6142 a 1.09130.0533 a AB 18 47.62312.7360 b 8.76700.3250 a 6.14240.9312 a 1.15130.1022 a BB 9 54.19823.8690 b 8.88300.4591 a 6.93330.5355 b 1.1674+0.0723 a C211 CC 6 24.36326.5334 a 7.27520.7412 a 4.65000.4042 a 0.85110.1915 a BC 5 33.197017.4441 ab 7.73331.2702 a 5.62210.9539 b 0.96670.2309 ab AA 28 43.42407.5511 bc 8.56840.7917 a 6.24740.60311 b 1.11580.1501 ab BB 15 47.063110.9060 bc 8.27341.7340 a 6.29120.5216 b 1.27000.3434 b AB 6 50.412311.971 c 8.32511.4408 a 6.55320.9678 b 1.17510.1707 ab C1312 AB 3 31.695018.7454 a 7.25002.3335 a 5.45000.7778 a 1.00000.4243 a BB 45 40.208710.4746 ab 7.71331.21506 a 5.96330.8037 a 1.19330.2586 a AA 6 50.73009.9586 b 8.85000.6758 a 6.67500.6185 a 1.45000.2646 a BC 6 55.45007.1233 b 9.47500.7805 a 6.67500.3304 a 1.50000.2160 a C13038 AB 33 40.487311.4677 a 7.86361.3040 a 5.81820.8063 a 1.27140.3315 a AA 21 43.341412.9017 a 8.00001.4507 a 6.25710.6802 ab 1.20910.2724 a BB 6 49.21756.0706 a 8.55000.8062 a 6.90000.4243 b 1.30000.1414 a (P<0.05)
1658 44, AB BC AA (P<0.05), 3 (P>0.05) AB,, BC, C13038 RNA RPB1,, BB, AB, BB 3 3.1 15, 2.7633, 3.3333,, (, 2011),, (, 2010)(H o ) 0.3989, (H e )0.5961,,,,,, (PIC), 15, 6 (0.25<PIC<0.5), 9 (PIC>0.5), 15 PIC 0.539177, (PIC>0.5) EST EST-SSR,, 3.2,,,,, 3, -(, 2010) 15 SSR, C8387, ; C211, ; C5670,, C1312 C13038 ; SSR,, (, 2002;, 2011),,, (, 2012), (2013), (P<0.01), C211 C1312 C13038 EST,, ---,,, (, 2009)(, 2010;, 2013) (, 2004;, 2011) (Wang et al, 2013), EST EST-SSR,, EST-SSR QTL, 4 15 EST-SSR,, : (1) 15 (N e )(H o )(H e ) (PIC) 2.76330.39890.5961 0.539177, (2) 15, 5 : C8387 (P<0.01), (P<0.05), C211 (P<0.01), (P<0.05), C5670 (P< 0.01), (P<0.05), C1312 C13038 (P<0.01) (3) C8387 CC 4
6 : (Pelodiscus sinensis)est-ssr 1659, AB ; C5670 BB, AA ; C211 C, CC, BB AB ; C1312 BC C13038 BB,,, 2011. 鲀., 41(5): 9 16,,, 2013.. :, 39(2): 179 182,,, 2013. (Pelodiscus sinensis) (Morganella morganii)., 44(3): 722727, 2009.., 33(5): 923930,,, 2004. RAPD., 28(2): 119126,,, 2010.., 34(2): 169177,,, 2012.., 36(005): 641646,,, 2013.., 17(1): 1518,,, 2010.. :, 7879,,, 2002. QTL., 28(4): 546555,,, 2011.., 35(2): 197 202,,, 2011. F 1, F 2 F 3., 20(2): 161166,,, 2011.,, QTL., 33(12): 13661373,,, 2010.., 17(4): 859868,,, 2009. PAGE., 31(6): 668673,,, 2005.,. :, 18(2): 183 186 Botstein D, White R L, Skolnick M et al, 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics, 32(3): 314 Bu X J, Liu L, Wang L et al, 2011. Isolation and characterization of 21 novel polymorphic microsatellite loci in the Chinese soft-shelled turtle Pelodiscus sinensis. Genetics and Molecular Research, 10(2): 10061010 Gupta P K, Rustgi S, 2004. Molecular markers from the transcribed/expressed region of the genome in higher plants. Functional & Integrative Genomics, 4(3): 139162 Li Z Q, Li J L, Feng X Y et al, 2010. Sixteen polymorphic microsatellites for breeding of Chinese soft-shelled turtles (Pelodiscus sinensis). Animal Genetics, 41(4): 446447 Que Y, Zhu B, Rosenthal H et al, 2007. Isolation and characterization of microsatellites in Chinese soft-shelled turtle, Pelodiscus sinensis. Molecular Ecology Notes, 7(6): 1265 1267 Skaala Ø, Høyim B, Glover K et al, 2004. Microsatellite analysis in domesticated and wild Atlantic salmon (Salmo salar L.): allelic diversity and identification of individuals. Aquaculture, 240(1): 131143 Wang W, Li C, Ge C et al, 2013. De-novo characterization of the soft-shelled turtle Pelodiscus sinensis transcriptome using Illumina RNA-Seq technology. Journal of Zhejiang University Science B, 14(1): 5867 Zalapa J E, Cuevas H, Zhu H et al, 2012. Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences. American Journal of Botany, 99(2): 193208 Zhang H, Wei L, Miao H et al, 2012. Development and validation of genic-ssr markers in sesame by RNA-seq. BMC Genomics, 13(1): 316
1660 44 CORRELATION OF EST-SSR MARKERS WITH GROWTH TRAITS IN CHINESE SOFT-SHELLED TURTLES (PELODISCUS SINENSIS) ZHANG Qun-Ying 1, HUANG He-Zhong 1, YUAN Wen-Cheng 1, LI Wen-Long 1, YONG Fu-He 2, XU Xue-Ying 3, ZHANG Ping 4 (1. School of Medicine and Life Sciences, Medical College of Soochow University, Fisheries Research Institute of Soochow University, Suzhou, 215123; 2. Mashe Special Aquaculture Farms in Xukou Town, Wuzhong District, Suzhou City, Jiangsu Province, Suzhou, 215004; 3. Fisheries Technology Extension Station of Suzhou City, Jiangsu Province, Suzhou, 215006; 4. Fisheries Technology Guide Station of Suining County in Xuzhou City, Jiangsu Province, Xuzhou, 221200) Abstract 15 polymorphic microsatellites were developed from the transcriptome sequencing EST database of Chinese soft-shelled turtles (Pelodiscus sinensis) to analyze the genetic diversity and linkage of 60 individuals. The result indicate that: 15 EST-SSR markers obtained amplified products successfully in a high polymorphism level; the number of mean valid alleles was 2.7633; the value of mean expected heterozygosity was 0.5961; and the mean polymorphism information content (PIC) was 0.539177. The linkage analysis showed the locus C8387 had a significant impact on body weight, body length, carapace length and a extremely significant difference in side width (P<0.01), and CC at C8387 was a favorable genotype for the four growth traits while AB was a disadvantage genotype; locus C211 showed an extremely significant impact on body weight, carapace length (P<0.01) and a significant impact on the side width (P<0.05), among them allele C was negatively associated with the body weight and genotype CC was disadvantage genotype of carapace length and side width, while BB and AB were favorable genotypes of side width and body weight respectively; locus C5670 had an extremely significant impact on body weight (P<0.01) and a significant impact on carapace length (P<0.05), in which BB was the dominant genotype of body weight and carapace length while the AA was the negative genotype; the locus C1312 and C13038 had a extremely significant correlation of body weight, carapace length, respectively (P<0.01), in which BC of locus C1312 and BB of C13038 was dominant genotype of body weight and carapace length, respectively. Key words Pelodiscus sinensis; EST-SSR marker; growth trait; correlation analysis