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1 45 2 Vol.45, No OCEANOLOGIA ET LIMNOLOGIA SINICA Mar., 2014 (Pelodiscus sinensis) SNP * 1, 2 1, ( ; ; ) (Pelodiscus sinensis) ( ), SNP,, SNP, bp, SNP 7, 1, 2, 3,, ; ; SNP; S941 doi: /hyhz (Single Nucleotide Polymorphism, SNP), ( ), (Brookes et al, 1998) SNP,, (Polonikov et al, 2008; Wong et al, 2008; Downey et al, 2009) (Brunner et al, 2000; Jeong et al, 2004) (Mabru et al, 2004) SNP (SSCP)(Gonen et al, 1999) - (PCR-RFLP) (Fukuen et al, 2002) (DHPLC) (Wolford et al, 2000) (SBE)(Steemers et al, 2006) (Griffin et al, 2000) (High-resolution melting curve analysis, HRM) (Liew et al, 2004) HRM PCR, PCR, T m (Wittwer, 2009), HRM SNP, (Pelodiscus sinensis),,, RFLP (, 1997;, 2009), RAPD (, 2004) SSR (Zhu et al, 2012),, SNP HRM DNA SNP,, *, 2012C12907,, chao_zhang_nbu@163.com :,,, zmk407@126.com : , :

2 2 : (Pelodiscus sinensis) SNP DNA :, 2, (750 53)g ; :, 2, (475 32)g ; :, 2, (332 37)g ; :, 2, (436 44)g 25, 5, 20, Takara DNA DNA, DNA 100ng/ L, 1.5%, 20 C 1.2 (2005) 5 PCR, Clustal X 2.0 (Larkin et al, 2007), BioEdit SNP Primer5.0 SNP, 19 25bp, bp, C 1.3 HRM SNP 5, PCR ABI 7500 Fast Real-Time System 20 L, 2 HRM MeltDoctor Master Mix 10 L, 10 mol/l 0.4 L, DNA 50ng, ddh 2 O 20 L 95 C 15min; 95 C 15s, 60 C / 30s, C /s 60 C 95 C HRM 2.0 T m SNP 4 20, 22 Primer5.0 7 ( 1) 表 1 引物设计结果 Tab.1 The results of designed primers SNP (5 3 ) (AY687385) 1 F: GCCCCTTCCACCAAACTGTCATAC 486 R: GGGTATCTAATCCCAGTTTGTGTCT 2 F: GGGGCAAGTCGTAACAAGGTAAG 1064 R: GCTCAAAATCAAAATAACCCTGG 3 F: TAAGTAGAGGTGAAAAGCCTAACGA 1559 R: CTGTTTCAATTTGGCTGTACCCTAA 4 F: AAGCATTCTCATCAAAACGAAAAGT 6879 R: AATCCTTCCTTTCTTGTGTTTTGTA 5 F: CACATTCACACCAACTACACAACTCT 8330 R: GTAGTCCTGTGAGTACGGTGGCT 6 F: CGAAGCCACACTAATCCCAACA R: GAAGTAGGTTCCAGCATTTAGTCGT 7 F: TATACTTCAACACCTTAATCCACCG R: CGTATCAAATTAGGTCGGTTAGGTG 2.2 SNP HRM HRM, 1 7 SNP 5 T m, T m ( 2) T m 0.3 C, 表 2 七对引物的扩增变异类型及扩增产物 T m 值 Tab.2 Types of variation and T m values of the products amplified by seven pairs of primers (bp) T m ( C) T/C C/T C/A C/T T/C A/G T/C

3 SNP T m, : 1 ( 1); 2 4 6, 2 2 ; 3 5, 3 3 ; 7 ( 4) SNP Fig.1 SNP typing result in the locus 486 of mitochondrial DNA of P. sinensis using primer HRM, 4 80, 100% SNP, SNP (, 2012),,,, SNP (Sanger ), SNP, PCR,,,, SNP, SNP SNP, SNP, HRM SNP,,,, SNP (Nomoto et al, 2006) (Lochlainn et al, 2011), (Maat et al, 2007) (Aten et al, 2008) SNP PCR, HRM, 1 4, T m, 3.2 DNA, (Cano et al, 1984), (Stoneking et al, 1996), Cyt b 12S rrna (2005, 2006) (Pelodiscus axenaria) (Palea steindachneri) Cyt b 12S rrna, PCR-RFLP

4 2期张超等: 中华鳖(Pelodiscus sinensis)不同品系线粒体 SNP 的分型与鉴定图2 Fig.2 引物 2 中华鳖线粒体 1064 位点的 SNP 分型结果 SNP typing result in the locus 1064 of mitochondrial DNA of P. sinensis using primer 2 图3 引物 3 中华鳖线粒体 1559 位点的 SNP 分型结果 Fig.3 SNP typing result in the locus 1559 of mitochondrial DNA of P. sinensis using primer 3 379

5 380 45,, DNA, DNA,, SNP, SNP, SNP HRM,,,,,,, RAPD., 28(2): SNP,,, Fig.4 SNP typing result in the locus of mitochondrial DNA of P. sinensis using 12S rrna primer 7.,, (2008) 16(3): ,,, Cyt b, mtdna., 4(3): 1 6,,,,, (SNP) ; (2012) 12S rrna,,,., 40(21): ,,, (Pelodiscus sinensis)5 b., 39(3): ,,, (Pelodiscus sinensis) (TYR)., SNP, 40(3): ,,, DNA, SNP (Mabru et al, 2004) b., 30(4): ,,, DNA 12S rrna. SNP, DNA, 29(3): ,,,, 2005., HRM SNP 2, 468, , 21(5): Aten E, White S J, Kalf M E et al, Methods to detect CNVs in the human genome. Cytogenetic and Genome Research,, 13454, 123(1 4): Brookes A, Day I, SNP attack on complex traits. Nature, 20, 100%, Genetics, 20(3): Brunner S, Keller B, Feuillet C, Molecular mapping of the

6 2 : (Pelodiscus sinensis) SNP 381 Rph7.g leaf rust resistance gene in barley (Hordeum vulgare L.). Theoretical and Applied Genetics, 101(5 6): Cano R L, Brown W M, Wilson A C, Polymorphic sites and the mechanism of evolution in human mitochondrial DNA. Genetics, 106: Downey P M, Petro R, Simon S J et al, Identification of single nucleotide polymorphisms of the human metabotropic glutamate receptor 1 gene and pharmacological characterization of a P993S variant Biochemical. Biochemical Pharmacology, 77(7): Fukuen S, Fukuda T, Maune H et al, Novel detection assay by PCR-RFLP and frequency of the CYP3A5 SNPs, CYP3A5*3 and *6, in a Japanese population. Pharmacogenetics, 12(4): Gonen D, Veenstra-Vander Weele J, Yang Z et al, High throughput fluorescent CE-SSCP SNP genotyping. Mol Psychiatry, 4(4): Griffin T J, Smith L M, Single-nucleotide polymorphism analysis by MALDI-TOF mass spectrometry. Trends Biotechnol, 18(2): Jeong S C, Maroof M A S, Detection and genotyping of SNPs tightly linked to two disease resistance loci, Rsv1 and Rsv3, of soybean. Plant Breeding, 123(4): Larkin M A, Blackshields G, Brown N P et al, Clustal W and Clustal X version 2.0. Bioinformatics, 23: Liew M, Pryor R, Palais R et al, Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem, 50(7): Lochlainn S O, Amoah S, Graham N S et al, High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes. Plant Methods, 7(1): 43 Maat W, van der Velden P A, Out-Luiting C et al, Epigenetic inactivation of RASSF1a in uveal melanoma. Investigative Ophthalmology & Visual Science, 48(2): Mabru D, Douet J P, Mouton A et al, PCR-RFLP using a SNP on the mitochondrial Lsu-rDNA as an easy method to differentiate Tuber melanosporum (Perigord truffle) and other truffle species in cans. International Journal of Food Microbiology, 94(1): Nomoto K, Tsuta K, Takano T et al, Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis. American Society for Clinical Pathology, 126(4): Polonikov A V, Ivanov V P, Solodilova M A et al, A common polymorphism G-50T in cytochrome P450 2J2 gene is associated with increased risk of essential hypertension in a Russian population. Disease Markers, 24(2): Steemers F J, Chang W H, Lee G et al, Whole-genome genotyping with the single-base extension assay. Nat Methods, 3(1): Stoneking M, Soodyall H, Human evolution and the mitochondrial genome. Current Opinion in Genetics & Development, 6(6): Wittwer C T, High-resolution DNA melting analysis: advancements and limitations. Hum Mutat, 30(6): Wolford J K, Blunt D, Ballecer C et al, High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC). Hum Genet, 107(5): Wong C, Kanetsky P, Raj D, Genetic polymorphisms of the RAS-cytokine pathway and chronic kidney disease. Pediatr Nephrol, 23(7): Zhu L M, Li Z Q, Li J L et al, Genetic diversity of farmed Chinese soft-shelled turtle (Pelodiscus sinensis) evaluated from microsatellite analysis. Journal of Animal and Veterinary Advances, 11(8): IDENTIFICATION OF DIFFERENT STRAINS OF PELODISCUS SINENSIS BY USING HIGH RESOLUTION MELTING (HRM) ANALYSIS OF SNPS IN MITOCHONDRIAL DNA ZHANG Chao 1, 2, ZHANG Hai-Qi 1, 3, XU Xiao-Jun 1, HE Zhong-Yang 1, WANG Chun-Lin 2, SHAO Jian-Zhong 3 (1. Zhejiang Fisheries Introduction and Breeding Center, Hangzhou , China; 2. School of Marine Science, Ningbo University, Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo University, Ningbo , China; 3. College of Life Science, Zhejiang University, Hangzhou , China) Abstract Partial sequences of mitochondrial DNA from four Pelodiscus sinensis strains (Taihu Lake, Taiwan, Japanese, and Huanghe River strains) were amplified and sequenced to locate strain-specific SNPs. Corresponding primers were designed and high-resolution melting (HRM) technique employed for genotyping these SNPs. Seven SNPs were detected by HRM, whose length of amplified DNA sequence ranged from 80bp to 150bp, of which one was the Taihu Lake strain specific, two specific to the Taiwan strain, one to Japanese strain, and three to the Huanghe River strain. This method is fast and simple, and may offer a technical support to the strain identification and breeding of P. sinensis in practice. Key words Pelodiscus sinensis; mitochondrial; SNP; germplasm identification

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