Genetic Polymorphism of Microsatellite DNA in Two Popula- Tions of Northern Sheatfish (Silurus Soldatovi)

遗传学报 Acta Genetica Sinica, October 2006, 33 (10)：908–916 ISSN 0379-4172 Genetic Polymorphism of Microsatellite DNA in Two Popula- tions of Northern Sheatfish (Silurus soldatovi) QUAN Ying-Chun1,2, SUN Xiao-Wen1,①, LIANG Li-Qun1 1. Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China; 2. College of Aqua-life Science and Technology, Shanghai Fisheries University, Shanghai 200090, China Abstract: In this article, population variations and genetic structures of two populations of northern sheatfish (Silurus soldatovi) were analyzed using 24 microsatellite loci enriched from southern catfish (S. meriaionalis Chen) by magnetic beads. Gene frequency (P), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information contents (PIC), and number of effective alleles (Ne) were determined. One population was wild, ripe individuals collected from Heilongjiang River (HNS); the other was cultured fry collected from Songhuajiang River (SNS). The Hardy-Weinberg equilibrium (HWE) was tested by the genetic departure index (d). The coefficient of gene differentiation GST and ФST by AMOVA (Analysis of Molecular Variety) was im- puted using Arlequin software in this study. In addition, a phylogenetic tree was constructed by UPGMA method based on the pair- wise Nei’s standard distances using PHYLIP. A total of 1 357 fragments with sizes ranging between 102 bp and 385 bp were ac- quired by PCR amplifications. The average number of alleles of the two populations was 8.875. Results indicated that these microsatellite loci were highly polymorphic and could be used as genetic markers. The mean values of the parameters P, Ho, He, PIC, and Ne were 0.165, 0.435, 0.758, 0.742, and 5.019 for HNS and 0.147, 0.299, 0.847, 0.764, and 5.944 for SNS, respectively. Al- though there were differences, there were no significant differentiations except for the locus HLJcf37. These populations to a cer- tain extent deviated from HWE, such as excessive and deficient heterozygote numbers. The value of GST was 0.078 and above 98% of the variation were differences among individuals within the population, so the variation between populations was insignifi- cant. Cluster analysis also showed that the relationships among individuals were very close. In conclusion, the microsatellite markers that were developed through this study are useful for genetic analysis and the genetic culture that was proposed in this study has no significant impact on S. soldatovi. Key words: Silurus soldatovi; microsatellite DNA; genetic polymorphism Microsatellites, also known as simple sequence tionary biology, QTL, conservation biology, and ge- repeats (SSRs), simple sequence length polymo- netic mapping [1-3]. phisms (SSLPs), or short tandem repeats (STR), are Northern sheatfish (Silurus soldatovi Nikolsky et regions of DNA that exhibit short repetitive sequence Soin), also called Suo’s/Su’s sheatfish or Huai Tou motifs. These motifs are often composed of 1-6 bp sheatfish, belongs to the Vertebrata, Osteichthyes, repeat sequences, such as CA, AGA, ATA, and the Siluriformes, Siluridae, and Silurus. Few years ago, it like. Their repeat numbers are variable, which makes was distributed widely in areas along the middle and microsatellites polymorphic. Characterized as lower reaches of the Heilongjiang River and the Liao codominant, highly polymorphic and amenable to River. It is well received with suitable size of the genotyping by polymerase chain reaction (PCR), mi- adult individual, since it is delicious, nutritious, dis- crosatellites have become instrumental as genetic ease-resistant, and easy to capture. It also grows markers in areas such as population genetics, evolu- quickly and can survive over wide temperature ranges. Received: 2005-11-20; Accepted: 2006-02-03 This work is supported by Key Project of Chinese National Programs for Fundamental Research and Development (No. 2004CB117405). ① Corresponding author. E-mail: [email protected] QUAN Ying-Chun et al.: Genetic Polymorphism of Microsatellite DNA in Two Populations of Northern Sheatfish (Silurus soldatovi) 909 The features mentioned above have made northern 1. 2 Methods sheatfish an excellent freshwater fish for the culturists. 1. 2. 1 Extraction of genomic DNA Since most of the captured species are wild, All the fins of the samples were kept in 70% over-fishing and habitat degradation threaten its living ethanol, from which genomic DNAs were extracted condition. Consequently, in recent times it has become following the method of Geng et al [17]. [4] one of the endangered freshwater fishes in China . To 1. 2. 2 Development and screening of microsatellite protect and develop the breed resource, studies on its DNAs biology and genetics are necessary. Although there are Cloning and characterization of southern catfish many reports of channel catfish (Ictalurus punctatus) microsatellite sequences were performed following [2,5,6] [7, 8] , European catfish (S. glanis) , Asian catfish the microsatellite hybrid capture technique of Carle- (Pangasius krempfi, P. bocourti, P. conchophilus, P. ton et al [18]. In brief, genomic DNA was extracted [9] pleurotaenis, and Helicophagus waandersii . S. aso- from the fin of a southern catfish (Wuhan, the middle [10-12] tus Linnaeus and S. meriaionalis Chen ), and so reach of the Yangtze River) and digested with the [13, 14] [15, 16] on, reports about the biology and cytology restriction enzyme Sau3AⅠ. Then short (20 bp) of northern sheatfish are few. Application of microsa- Brown linkers [19] were attached to the DNA. DNA tellite variations in this species has been introduced for fragments (400-900 bp) were hybridized with bio- the first time in this study. tin-labeled oligonucleotide (CA)12 probe and enriched In the earlier research by the authors of this by the magnetic beads coated with streptavidin to cap- study, microsatellite DNAs were isolated from a ture the fragments containing microsatellites. A partial CA/GT enriched library of southern catfish (S. genomic library was then established in a pMD18-T meriaionalis) by combining biotin capture method vector. These clones were screened using the oligonu- 32 and radioactive-labeling hybridization. Southern cat- cleotide (CA)15 radiolabeled with [γ P]-ATP at the 5′ fish and northern sheatfish belong to the same taxo- end. Finally, positive clones harboring strong hybridiza- nomic species; so 40 microsatellite loci were synthe- tion signals would be sequenced. Primer pairs would be sized and screened. Twenty-four microsatellite loci designed according to the flanking sequences of mi- were polymorphic and were used to analyze the crosatellite loci using Primer Premier 5.0 software. population structures and genetic diversities of north- Forty pairs of microsatellite primers were synthe- ern sheatfish. sized and screened by PCR amplification using the mixed genomic DNAs of northern sheatfish. Twenty-four pairs 1 Materials and Methods successfully yielded reproducible and stable amplifications. They were registered in GenBank and were used to 1. 1 Samples, primers, and reagents identify the genetic diversities of northern sheatfish. All the samples of northern sheatfish were col- 1. 2. 3 Conditions and processes of PCR lected from Heilongjiang province on June 2005, in- Amplifications were performed with a reaction cluding 22 wild individuals (9♂, 13♀) gathered from volume [17] of 25 μL using 50-100 ng of template the Heilongjiang River (HNS) and 30 cultured fry (sex DNA, 10 pmoles of each primer, 0.01 mol/L Tris-Cl unknown) rooted in the Songhuajiang River (SNS). (pH 8.3), 0.05 mol/L KCl, 0.0015 mol/L MgCl2, Microsatellite primers were synthesized by 0.01% Gelatin, 0.1% Tween 20, 0.1% NP-40, 0.0002 Shanghai Sangon Biological Engineering & Tech- mol/L dNTP, and 1 U Taq DNA polymerase. The nology and Service Co. Ltd., Shanghai, China. En- profile of thermal cycling (Perkin-Elmer 9700, USA) zymes and vectors were bought from Promega, USA, was as follows: 3 min at 94℃ , followed by 38 cycles and other biochemical reagents were from TaKaRa, of 30 s at 94℃ , 30 s at the selected higher annealing Dalian, China. temperature, 30 s at 72℃ , and 5 min at 72℃ . 910 遗传学报 Acta Genetica Sinica Vol.33 No.10 2006 1. 2. 4 Electrophoresis and visualization of PCR crosatellite sequences. Of all the microsatellites se- products quenced, 90.60% repeated more than 10 times and PCR products were separated by electrophoresis in 75.98% had perfect repeat motifs. There were also CT, 2% agarose gel (0.5 × TBE buffer at 5 V/cm) for 2 h GA, and ATG motifs besides the CA/GT motif. Ac- with 0.02× GoldView (SBS, Beijing China) nucleic acid cording to the flanking regions of these microsatellite stain and visualized under UV light on a GDS 8000 loci, 120 pairs of microsatellite primers were designed. (UVP, USA). Microsatellite alleles were identified by Forty pairs were synthesized and examined in the two their size in base pairs using Gel works software pack- populations of northern sheatfish, and 24 were poly- age (Version 3.0; UVP, USA). Sizes of amplified frag- morphic in at least one population using the 0.95-allele ments were determined by reference to a standard frequency criterion. The primer information and Gen- base-pair ladder, DL2000 (TaKaRa, Dalian, China). Bank accession numbers are shown in Table 1. 1. 2. 5 Statistical analysis 2. 2 PCR amplification The observed number of alleles (A), effective number of alleles (Ne), allele frequency (P), observed Out of the 24 polymorphic microsatellite loci, only heterozygosity (Ho), and expected heterozygosity (He) eight amplified in one of the two populations. The locus were computed by the software of POPGENE (Ver- HLJcf19 amplified no fragment in HNS, neither did loci sion 3.2). Polymorphism information content (PIC) HLJcf9, HLJcf12, HLJcf15, HLJcf17, HLJcf25, HLJcf31, was computed according to the following formula[20]. and HLJcf40 in SNS. Amplifications of locus HLJcf20 nnn−1 were shown as representative examples in Figs.

Load more