[논문]새로 개발한 미세위성체 마커를 이용한 한국 대하의 유전다양성 및 집단구조

신은하; 공희정; 남보혜; 김영옥; 김봉석; 김동균; 안철민; 정형택; 김우진

doi:10.5352/jls.2015.25.12.1347

새로 개발한 미세위성체 마커를 이용한 한국 대하의 유전다양성 및 집단구조
The Study of Genetic Diversity and Population Structure of the Korean Fleshy Shrimp, Fenneropenaeus chinensis, Using Newly Developed Microsatellite Markers 원문보기

생명과학회지 = Journal of life science, v.25 no.12 = no.188, 2015년, pp.1347 - 1353

신은하 (국립수산과학원 생명공학과) , 공희정 (국립수산과학원 생명공학과) , 남보혜 (국립수산과학원 생명공학과) , 김영옥 (국립수산과학원 생명공학과) , 김봉석 (국립수산과학원 미래양식연구센터) , 김동균 (국립수산과학원 생명공학과) , 안철민 (국립수산과학원 생명공학과) , 정형택 (국립수산과학원 생명공학과) , 김우진 (국립수산과학원 생명공학과)

초록
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대하(Fenneropenaeus chinensis)는 우리나라에서 경제적으로 가장 중요한 양식생물 중 하나이다. 그러나 대하의 유전적 특성에 대한 연구는 전무하다. 본 연구에서는 새로 개발된 13개 미세위성체 유전자좌를 이용하여 우리나라에 서식하는 4개 지역 대하의 유전 다양성 및 집단간 관련성을 분석하였다. 평균 대립유전자 richness =16.87, 평균 이형접합률 =0.845를 보여 유전 다양성은 비교적 높은 수준을 보였다. 52개 유전좌중에서 13개 유전자좌가 집단간 분석에서 Hardy–Weinberg 평형에서 유의적인 차이로 벗어났다. Neighbor-joining, principal coordinate 및 molecular variance 분석 결과로 우리나라 대하 집단은 3개 집단(나라도, 천수만, 법성포 및 채석포)으로 구성되어 있으며, 이 결과는 유전적 거리에 근거한 군집 결과와 일치하였다. 본 연구에서 조사된 유전 다양성 및 분화결과는 앞으로 대하의 지속 가능한 자원관리 및 선발 육종을 통한 유전적 개량에 적용될 수 있을 것이다.

Abstract ▼ AI-Helper

The fleshy shrimp, Fenneropenaeus chinensis, is the family of Penaeidae and one of the most economically important marine culture species in Korea. However, its genetic characteristics have never been studied. In this study, a total of 240 wild F. chinensis individuals were collected from four locations as follows: Narodo (NRD, n = 60), Beopseongpo (BSP, n = 60), Chaesukpo (CSP, n = 60), and Cheonsuman (CSM, n = 60). Genetic variability and the relationships among four wild F. chinensis populations were analyzed using 13 newly developed microsatellite loci. Relatively high levels of genetic variability (mean allelic richness = 16.87; mean heterozygosity = 0.845) were found among localities. Among the 52 population loci, 13 showed significant deviation from the Hardy–Weinberg equilibrium. Neighbor-joining, principal coordinate, and molecular variance analyses revealed the presence of three subpopulations (NRD, CSM, BSP and CSP), which was consistent with clustering based on genetic distance. The mean observed heterozygosity values of the NRD, CSM, BSP, and CSP populations were 0.724, 0.821, 0.814, and 0.785 over all loci, respectively. These genetic variability and differentiation results of the four wild populations can be applied for future genetic improvement using selective breeding and to design suitable management guidelines for Korean F. chinensis culture.

주제어

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제안 방법

Detailed information about the primers is presented in Table 2. Each PCR contained three primer sets that were labeled at the 5‘ end of the for- ward primer with 6-FAM, HEX, or NED dyes (Applied Biosystems, Foster City, CA, USA). PCR amplification was carried out in a 10-μL reaction mixture containing 0.
The PCR con- ditions were: 5 min at 95℃, followed by 35 cycles of 30 sec at 95℃, 45 sec at 58℃, and 45 sec at 72℃, with a final ex- tension of 10 min at 72℃. Microsatellite polymorphisms were screened using an ABI PRISM 3130 XL automated DNA sequencer (Applied Biosystems), and alleles were des- ignated according to PCR product size relative to molecular size markers (GENESCAN 400 HD [ROX]; Applied Biosystems).
0. The population structure patterns were further investigated using a model-based Bayesian clustering proce- dure in STRUCTURE (ver. 2.3; [21]), which assigns in- dividuals to K populations based on their multilocus genotype. STRUCTURE was run for K = 2 using a burn-in length of 50, 000 and a run of 50, 000 steps.

대상 데이터

A total of 240 wild F. chinensis individuals were collected from four locations as follows: Narodo (NRD, n=60), Beop- seongpo (BSP, n=60), Chaesukpo (CSP, n=60), and Cheonsu- man (CSM, n=60) (Fig. 1 and Table 1). All muscle tissue sam- ples were stored in 100% ethanol prior to DNA extraction.
1. Sampling sites ( ) for the four wild Fenneropenaeus chi- nensis populations: Narodo (NRD); Beopseongpo (BSP); Chaesukpo (CSP); and Cheonsuman (CSM).

데이터처리

0. Analysis of molecular variance (AMOVA; [7]) was used to test for population structure with Arlequin 3.0. A principal coordinate analysis (PCoA) was performed using GENALEX (ver.
0; [8]). Tests for allelic richness (A_R), number of alleles per locus (Na), and devia- tions from Hardy–Weinberg equilibrium (HWE) were esti- mated using GENEPOP ver. 4.0 (http://kimura.univ- montp2. fr/~rousset/Genepop.htm), and the adjusted P-val- ues for both analyses were obtained using a sequential Bonferroni test for multiple comparisons. MICRO-CHECKER 2.

성능/효과

The NJ tree topology and AMOVA results suggest that the four populations could be assigned to a group that in- cluded the NRD population, a group that included the CSM population, and a group that included the BSP and CSP pop- ulations (Table 5). An evaluation of the evolutionary rela- tionships among the four wild populations showed that BSP and CSP had the highest degree of genetic identity, followed by CSM and BSP, CSM and CSP, NRD and CSM, and NRD and BSP, whereas NRD and CSP were most distantly related. A previous study demonstrated the most likely explanation for this result is that wild populations of Chinese F.
Our results reveal that the newly developed microsatellite markers were a powerful approach to monitoring genetic diversity between the four geographically different wild Korean F. chinensis populations investigated. Heterozygosity is an important measure of population diversity at the genet- ic level.
05 indicates little genetic differentiation [26]. Significant pairwise F_ST values (p<0.05) were observed between the NRD and CSM, NRD and BSP, NRD and CSP, CSM and BSP, and CSM and CSP pop- ulations (Table 4). In our study, pairwise F_ST tests detected low levels of genetic differentiation among the populations, particularly between the CSP and BSP populations, indicating that a geographical barrier was not effectively maintain- ing genetic integrity among the populations in the four areas.
2). The NJ tree constructed based on D_CE, indicated that the four populations were allocated into three major groups (Fig. 3); that is, one group included the CSM population, one group included the BSP and CSP populations, and one group included the NRD population. The PCoA produced a result similar to that of the cluster analysis (Fig.
Heterozygosity is an important measure of population diversity at the genet- ic level. The mean observed heterozygosity values of the NRD, CSM, BSP, and CSP populations were 0.724, 0.821, 0.814, and 0.785 over all loci, respectively. These values were lower than the mean expected heterozygosity for the four populations.
The results of hierarchical AMOVA tests to form putative groups estimated from each NJ tree topology provided addi- tional evidence to support previous findings in F. chinensis populations. The NJ tree topology included a group for the NRD population and another group for the CSM, BSP, and CSP populations.
3582. The smallest estimate for D_CE was between BSP and CSP (0.0380), whereas the highest estimate was between NRD and CSP (0.3582); the genetic distances between NRD and CSM, NRD and BSP, CSM and BSP, and CSM and CSP were 0.1864, 0.3030, 0.0991, and 0.1216, respectively (Table 4). The result of the genetic distances among the F.

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