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乌龟十个新微卫星位点的分离鉴定及在其 它曲颈龟类中的跨物种扩增# 刘罗1,2，聂刘旺1,2，卜兴江1,2，夏行权1,2，黄振峰 1,2，景万星1,2，蒋远 1,2，汪玲 1,2** 5 （1. 安徽师范大学生命科学学院，安徽 芜湖 241000； 2. 安徽重要生物资源保护与利用研究省级重点实验室，安徽 芜湖 241000） 摘要：Ten polymorphic microsatellite loci were developed for the Chinese Pond ( reevesii) from (AC)n and (GATA)n microsatellite-enriched genomic library. We screened 32 wild individuals from the middle and lower reaches of Changjiang River and detected high levels of 10 polymorphism for all ten loci. The number of alleles/locus ranging from 3 to 18 with the values of expected and observed heterozygosities ranging from 0.585 to 0.907 and 0.469 to 1.000, respectively. Cross- amplification of these microsatellite loci was also tested in additional seven freshwater turtle/half freshwater turtle species (Mauremys megalocephala, Cuora galbinifrons, Cuora flavomarginata, Mauremys mutica, Mauremys sinensis, cantorii, 15 swinhoei) of . All 10 loci were amplified specific products in the five species of , while in the P. cantorii and the R. swinhoei of only one locus and two loci worked, respectively. Therefore, these highly variable loci may serve as a valuable tool for population genetics analyses, gene flow and provide information on the evolutionary history of the species. 20 关键词：Mauremys reevesii; Microsatellites; Genetic diversity; Conservation; Cross-amplificatio 中图分类号：Q959.6; Q951.3

Isolation and characterization of ten novel polymorphic microsatellites loci in the Chinese pond turtle (Mauremys 25 reevesii) and cross-species amplification in other Cryptodira species Liu Luo, Nie Liuwang, Bu Xinjiang, Xia Xingquan, Huang Zhenfeng, Jing Wanxing, Jiang Yuan, Wang Ling (Life Science College, Anhui Normal University, The Provincial Key Lab of the Conservation and 30 Exploitation Research of Biological Resources in Anhui, Wuhu, Anhui, 241000, China;) Abstract: Ten polymorphic microsatellite loci were developed for the Chinese Pond turtle (Mauremys reevesii) from (AC)n and (GATA)n microsatellite-enriched genomic library. We screened 32 wild individuals from the middle and lower reaches of Changjiang River and detected high levels of polymorphism for all ten loci. The number of alleles/locus ranging from 3 to 18 35 with the values of expected and observed heterozygosities ranging from 0.585 to 0.907 and 0.469 to 1.000, respectively. Cross-species amplification of these microsatellite loci was also tested in additional seven freshwater turtle/half freshwater turtle species (Mauremys megalocephala, Cuora galbinifrons, Cuora flavomarginata, Mauremys mutica, Mauremys sinensis, Pelochelys cantorii, Rafetus swinhoei) of Cryptodira. All 10 loci were amplified specific products in the five species of 40 Geoemydidae, while in the P. cantorii and the R. swinhoei of Trionychidae only one locus and two loci worked, respectively. Therefore, these highly variable loci may serve as a valuable tool for population genetics analyses, gene flow and provide information on the evolutionary history of the species. Keywords:Mauremys reevesii;Microsatellites;Genetic diversity;Conservation;Cross-amplificatio

基金项目：This research was supported by the Specialized Research Fund for the Doctoral Program of Higher Education(20080370001) and the National Natural Science Foundation of China (NSFC, #30970351) 作者简介：刘罗,男,硕士研究生 ,主要研究方向为分子生态学 通信联系人：聂刘旺 ,1962 年生,男,博士,教授,博士生导师 . E-mail: [email protected]

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45 The pond turtle Mauremys reevesii, which belongs to Mauremys of Geoemydidae (Testudines), occurs in China, as well as Japan (Honshu and Kyushu) and Korea (Bonin et al., 2006; Rhodin et al., 2010). Because the pond tutle are sold not only for meat, medicinal purposes and tonic products, but also in demand as pets, the large demand for pond have depleted once abundant populations in China. In the past the Chinese pond turtle was especially 50 abundant in lakes, ponds, and rivers in China, but and overhunting causes dramatical decline of the wild populations of this species (Altherr and Freyer, 2000; Zhou and Li, 2007). Consequently, the Chinese pond turtle was listed as an in the Chinese Red List of Threatened species (Wang and Xie, 2004), it was also listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix III in 2005 55 by the Chinese government. In to take effective conservation measures of this species, it is important to characterize their genetic diversity and population structure. Ye et al. developed eight polymorphic microsatellite loci(Ye et al., 2009). In this study, we isolated another ten novel microsatellite loci for M.reevesii with the propose of providing more credible and powerful data to investigate the genetic diversity of this species. 60 All of the microsatellite primers were derived from clones isolated from a highly enriched CA- and GATA-repeat genomic library following the protocol presented by Fischer et al.(1998) and Bloor et al. (2001) with some modifications. Genomic DNA for marker development and subsequent surveys was extracted using TIANamp Genomic DNA Kit (TIANGEN). Three micrograms of genomic DNA from the Chinese pond turtle digested with Sau3A I restriction 65 enzyme (TaKaRa). The restriction fragments between 400bp to 1000bp were size-selected and

ligated to linkers SauL-F (5’- GGC CAG AGA CCC CAA GCT TCG -3’) and SauL-R ( 5’- PO4 - GAT CCG AAG CTT GGG GTC TCT GGC C 3’). The ligated fragments were amplified by polymerase chain reaction (PCR) using SauL-F as forward and reverse primers in a 50 µL reaction system composed of 5 µL template DNA (30–50 ng/µL), 2 U exTaq DNA

70 polymerase (TaKaRa), 5 µL of 10× PCR buffer (TaKaRa), 4 µL of 25 mM MgCl2, 4µL of 25 mM dNTPs, 2 µL of 25 mM primer (SauL-F). PCR conditions were as follows: 95 ℃ for 5 min, then 25 cycles of 94 ℃ for 50 s, 60 ℃ for 45 s, 72 ℃ for 60 s, and a final period at 72 ℃ for 10

min. The purified PCR products were hybridized to biotinylated (CA)12 and (GATA)6 oligonucleotide probes and isolated using streptavidin-coated magnetic beads (Roche). The 75 enriched DNA was amplified again by PCR. PCR products were purified using AxyPrep DNA Gel Extraction kit (AxyGEN) and then ligated into pMD18-T vector (TaKaRa) at 16 ℃ 5 hours, followed transformation into Escherichia coil DH5a competent cells. The colonies were plated on a selective medium and recombinants were identified using blue/white screening on Luria-Bertani agar plates containing ampicillin, X-gal and IPTG. Recombinant clones were amplified using

80 SauL-F and corresponding (CA)12 or (GATA)6 probe sequence as forward and reverse primers, respectively. We screened 256 recombinant colonies and sequenced by ABI-PRISM 3730 Sequencer, 59 primer pairs were designed in the flanking regions using PRIMER PREMIER 5.0 (http://www.premierbiosoft.com/). 17 primer pairs can amplified consistent product, each forward primer of them were labeled with a fluorescent dye (FAM, HEX or TAMRA) at its 5′ end. To 85 characterize these microsatellite loci, each of them was tested with 32 wild individuals of M. reevesii from the middle and lower reaches of Changjiang River PCR was performed in 25 µL total volume reactions containing 1 µL template DNA (30–50 ng/µL), 1U exTaq DNA polymerase

(TaKaRa), 2.5 µL of 10× PCR buffer (TaKaRa), 2µL of 25 mM MgCl2, 2 µL of 25 mM dNTPs, 0.5 µL of each 25 mM primer. PCR conditions were as follows: 95 ℃ for 5 min, then 35 cycles

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90 of 94 ℃ for 50 s, at the annealing temperature (Table 1) for 45 s, 72 ℃ for 60 s, and a final period at 72 ℃ for 10 min. PCR products were analyzed on an ABIPRISM 3730 Genetic Analyzer using ROX 350 or LIZ 500 (Applied Biosystems). The size standard and analyzed using software Genemarker (Applied Biosystems). We used MICRO-CHECKER version 2.2.3 to identify genotyping errors (Van Oosterhout et 95 al., 2004). HO (Observed Heterozygosity), HE (Expected Heterozygosity), LD (Linkage Disequilibrium ) and HWE (Hardy–Weinberg Exact) were conducted using web-based GENEPOP 4.0 (Raymond and Rousset, 1995). CERVUS 3.0 (Kalinowski et al., 2007) was used to calculate frequency of null alleles. The PCR products of 10 in 17 primer pairs showed polymorphism across 32 wild 100 individuals. Locus designations, repeat motifs, primer sequences, range of allele sizes in base pairs, number of alleles per locus and estimates of heterozygosity for ten loci examined are listed in Table 1. The number of alleles/locu ranging from 3 to 18. The values of expected and observed heterozygosities ranged from 0.585 to 0.907 and 0.469 to 1.000, respectively. One of the 10 loci (Mclw06) showed significant deviation from HWE (P<0.01). Deviations from HWE expectations 105 as seen in this study are probably due to the sample size or the presence of null alleles. None of the loci showed significant linkage disequilibrium (P>0.05). Therefore, these highly variable loci will be useful for investigating the genetic diversity, population structure, gene flow and provide information on the evolutionary history of the species. Owing to the high applicability of microsatellite markers in closely related species, 110 cross-species amplification of 10 loci was carried out in other five species (Mauremys megalocephala, Cuora galbinifrons, Cuora flavomarginata, Mauremys mutica, Mauremys sinensis) of Geoemydidae and one specie (Pelochelys cantorii) of Trionychidae. The same PCR conditions were used as described above. All 10 loci were amplified specific products in the five species of Trionychidae, while in the P. cantorii of Trionychidae only one locu worked(Table 2). 115 The number of loci successfully amplified in the five species of Geoemydidae is probably correlated to their phylogenetic affinities with M. reevesii.

Table 1. Characteristics of ten polymorphic microsatellite loci isolated from M. reevesii. Locus, Repeat motif, Primer sequences, Annealing temperature (Ta), Size of amplified product (bp), Number of alleles(NA), Average 120 observed (HO) and Expected (HE) heterozygosities. GenBank accession No. Allele GenBank Repeat Ta Locus Primer sequence (5′-3′) size NA HO HE accession motif (°C) (bp) No. TCCTATACCCAGTGGGACATG M01 (GT) 52.9 165-185 10 0.719 0.818 JF712880 10 AAGTTTCACCCATCCATCAGC ACTGTGCCGGGTCGTGATGGG M02 (CA) 58 255-265 3 1.000 0.589 JF712881 13 GTGGTGTCTGAGTGTTCTTGC GTGGTGGCACAGAGGTAGTTG M03 (TG) 57.5 216-290 18 0.813 0.907 JF712882 11 CTCACATTTTCAGTTTGGTTA GAAAGGCTGCTGCTCACCACG M04 (TG) 57.5 194-228 9 0.656 0.733 JF712883 11 ACTGACCCAACCCTCCCTGCT AGCAAGGCCAGGTAAAGG M05 (TG) (AG) 60 302-346 14 0.875 0.803 JF712884 15 8 CATCTGCGCTGAGGGTTA GGGCTATTTCATTGCTGT M06 (GT) 57.5 284-322 8 0.469 0.810 JF712885 17 CGTCCTTCAAATGCCACC GAAGAGCCGACCGTCAACCT M07 (TG) (AG) 57.5 214-298 10 0.750 0.657 JF712886 9 18 CCCGTAGCCTTTCAAACCAC AGCTCCTCCAGGAACTAAAA M08 (TG) 57.5 239-303 10 0.813 0.828 JF712887 13 AAACCAAAGTCTTTCCAACC GAGCCACCACCATCCCATTT M09 (AC) 51.2 152-184 4 0.844 0.585 JF712888 17 AAGGCATCCAGGCAGCGT CTGAGAAATCCTGGACAC M10 (GATA) 48 151-277 9 0.906 0.728 JF712889 10 CTATGGACTTTGGAAACC

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Table 2. Cross-species amplification of ten microsatellite loci . ( ＋, amplification; －, no amplification for all tested individuals) Locus Species M01 M02 M03 M04 M05 M06 M07 M08 M09 M10 M. megalocephala ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ C. galbinifrons ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ C. flavomarginata ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ M. mutica ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ M. sinensis ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ P. cantorii － － － － － － ＋ － － －

ACKNOWLEDGMENTS 125 This research was supported by the Specialized Research Fund for the Doctoral Program of Higher Education(20080370001) and the National Natural Science Foundation of China (NSFC, #30970351) and the Key Laboratory of Biotic Environment and Ecological Safety of Anhui province.

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