飘鱼微卫星位点的筛选及珠江流域5个地理群体的遗传多样性分析

阮惠婷; 徐姗楠; 李敏; 戴嘉格; 李振海; 邹柯姝; 刘丽

doi:10.7541/2020.061

飘鱼微卫星位点的筛选及珠江流域5个地理群体的遗传多样性分析

阮惠婷^1,,
徐姗楠²,
李敏³,
戴嘉格¹,
李振海¹,
邹柯姝^1, ,,
刘丽^1, ,

1.
华南农业大学海洋学院, 广州 510642
2.
中国水产科学研究院南海水产研究所, 农业农村部南海渔业资源开发利用重点实验室, 广州 510300
3.
中国水产科学研究院南海水产研究所, 农业农村部外海渔业开发重点实验室, 广州 510300

基金项目: 国家重点研发计划子课题（2018YFD0900802）；农业部南海渔业资源开发利用重点实验室开放课题（FREU2015-09）；中国-东盟海上合作基金（CAMC-2018F）资助

详细信息

作者简介:
阮惠婷(1994—), 女, 硕士研究生; 研究方向为水生生物遗传多样性。E-mail: 1054741781@qq.com

通信作者:
邹柯姝(1987—), 女, 博士, 讲师; 研究方向为水生生物多样性。E-mail: zoukeshu@scau.edu.cn

刘丽(1964—), 女, 博士, 教授; 研究方向为水产种质资源的开发、利用与保护。E-mail: liuli@scau.edu.cn *共同通信作者

中图分类号: Q346⁺.5
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出版历程
- 收稿日期: 2019-05-15
- 修回日期: 2019-10-27
- 网络出版日期: 2020-05-18
- 发布日期: 2020-04-30

MICROSATELLITE PRIMERS SCREENING AND GENETIC DIVERSITY ANALYSIS OF FIVE GEOGRAPHICAL POPULATIONS OF PSEUDOLAUBUCA SINENSIS IN THE PEARL RIVER BASIN

1.
College of Marine Science, South China Agricultural University, Guangzhou 510642, China
2.
Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agricultural and Rural Affairs, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China
3.
Key Laboratory of Open-Sea Fishery Development, Ministry of Agriculture and Rural Affairs; South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China

Funds: Supported by the National Key R&D Program of China (2018YFD0900802) , the Key Laboratory Project of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture and Rural Affairs, P.R. China (FREU2015-09); China-ASEAN Maritime Cooperation Fund (CAMC-2018F)

Corresponding author:

摘要

摘要: 为了解珠江流域飘鱼(Pseudolaubuca sinensis)群体间的遗传多样性及其分化程度, 利用RAD-Seq技术开发出微卫星位点, 并设计了100对微卫星引物, 筛选出66个可稳定扩增出目的条带的位点, 其中16个具有较高的多态性(PIC>0.5), 以2碱基重复居多。利用其中10对多态性位点对珠江流域的郁江(YuJ)、左江(ZJ)、右江(YJ)、融江(RJ)和桂江(GJ)飘鱼群体进行遗传多样性研究, 得到有效等位基因数、观测杂合度、期望杂合度范围分别为5.2028—6.3800、0.6773—0.7667和0.7975—0.8425, 表明珠江流域5个飘鱼群体具有较高的遗传多样性, 以YuJ群体遗传多样性最高, RJ群体遗传多样性为最低。且群体间各个遗传参数相差较小, 说明他们的遗传多样性水平相近。AMOVA分析显示, 遗传变异主要来自于群体内(98.45%), 仅一小部分的变异来自于群体间(1.55%), 总群体的遗传分化系数F_st为0.015, 两两群体间的F_st在–0.0033—0.0295波动, 属于低等程度的分化。群体间基因交流值(N_m)在8.2246—76.0075, 表明群体间基因交流频繁, 对遗传漂变作用的抵制能力较强。研究筛选出了多态性高的微卫星位点, 并利用其对5个飘鱼群体遗传多样性进行评价, 旨在有效地监测飘鱼的种质资源状况, 为其资源的开发利用和保护提供科学指导。
- 飘鱼 /
- 微卫星 /
- 遗传多样性 /
- 珠江流域 /
- 分化
Abstract: To explore the genetic diversiy and diffentation degree of Pseudolaubuca engraulis population in the Pearl River Basin, RAD-Seq technology was used to develop microsatellite loci. A total of 100 pairs of microsatellite primers were designed, 66 of which could stably amplifiy the target band, including 16 polymorphic microsatellite markers (PIC>0.5) which were mostly with 2 base repeats. Ten polymorphic microsatellite loci were used in genetic diversity analysis in five P. sinensis populations of Pearl River Basin (YuJ, ZJ, YJ, RJ, GJ). The effective allele number, observed heterozygosity, and expected heterozygosity varied from 5.2028—6.3800, 0.6773—0.7667, and 0.7975—0.8425, respectively. The results showed that five P. sinensis populations in the Pearl River Basin had a high level of genetic diversity level, the YJ population had the highest genetic diversity while the RJ population had the lowest. The genetic parameters of population differed little, indicating that their genetic diversity level was close. AMOVE analysis showed that the genetic variation mainly come from population (98.45%), and only a small part of the variation came from different population (1.55%). The genetic differentiation coefficient F_st of the total population was 0.015, which was a low degree of differentiation. The gene exchange value (N_m) was between 8.2246 and 64.3526. The results showed that there were frequent gene exchange in different populations and strong resistance to genetic drift. This study screened microsatellite loci with high polymorphism and used the loci to evaluate the genetic diversity of the P. sinensis population, aiming to effectively monitor the germplasm resources of P. sinensis and to provide scientific guidance for its resource development, utilization and protection.
- Pseudolaubuca sinensis /
- Microsatellite /
- Genetic diversity /
- the Peart River Basin /
- Differentiation

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图 1 采样点分布图

Figure 1. The map of sampling

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图 2 YP11在4个飘鱼个体中的毛细血管峰图

Figure 2. Capillary gel electrophoresis of the primer YP11 in four Pseudolaubuca sinensis individuals

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图 3 5个飘鱼群体遗传瓶颈效应分析的模式迁移图

Figure 3. Mode-shift analysis for the test of the genetic bottleneck of five P. sinensis populations

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表 1 样品采集信息

Table 1 Experimental materials

所属水系(简称)River system (abbreviation)	采集地点Collection site	样品数量Sample quantity	样品编号Sample number
郁江YuJ	桂平	25	YuJ 01-25
郁江YuJ	横县	11	YuJ 26-36
左江ZJ	扶绥	23	ZJ 01-23
左江ZJ	龙州	13	ZJ 24-36
融江RJ	融安	16	RJ01-15
右江YJ	田阳	15	YJ01-16
桂江GJ	昭平	22	GJ01-22

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表 2 22个微卫星位点的信息及其遗传参数

Table 2 Information and genetic parameters of 22 microsatellite loci

引物名Primer name	核心序列 Repetitive sequence	两端序列Primer sequence (5′—3′)	产物大小Size (bp)	退火温度T_m (℃)	等位基因数Number of alleles (N_a)	有效等位基因数Effective number of alleles (N_e)	观测杂合度Observed heterozygosity (H_o)	期望杂合度Expected Heterozy-gosity (H_e)	香农指数Shannon’s index (I)	多态信息含量Polymorphic information content (PIC)
YP 04	(AG)₁₀	CACAAAAACAGAGGGAGAGCA TTCCGCCATTTATATCCAGG	261	59	4	1.8228	0.5	0.539	0.8359	0.4041
YP 05	(TA)₁₃	GCCACAATAACGTGGTGTGA TGTCCATCGTAACAGCATGA	110	59	9	5.3581	0.25	0.1693	1.8692	0.7889
YP 08	(AG)₁₁	TTCTGTGGAATTTATTCTGTGTGA TGACAGGGAGAAAGGGAGAA	188	58	8	5.4857	0.3333	0.1649	1.8586	0.7956
YP 10	(TG)₂₄	CCACAATAACCGTGGTGTGA AGCATTGGCGAGCATAAGAT	204	59	21	10.5688	0.4583	0.0754	2.6812	0.8985
YP 11	(ATA)₇	AGGGTTTGGGTTTGTTTGTG GGGACATTAAGTGCTCTCCAA	275	61	9	6.9818	0.2917	0.125	2.0373	0.8402
YP 19	(AATG)₁₂	TTCAGAATGACATGAGTGTGAGAA TTTCTTCAGGCGTACAGTCG	167	58	21	14.961	0.125	0.047	2.8584	0.9292
YP 23	(GT)₁₃	AAATGTCAGGCATTACAGGGTT TGTGAGATGAAAATGTCGCAA	222	59	9	5.6749	0.4167	0.1587	1.9271	0.8025
YP 31	(AAC)₆	TTCTCGTCGAGGAATCAGGT CACAAAGAGGGGAAGGACAA	243	58	7	3.3103	0.2917	0.2872	1.4352	0.6548
YP 32	(ATAG)₆	TGCAAAGACGTAGAGCATCG AAGCACTTTAAAAAGCGTGAGG	251	60	11	6.7368	0.4583	0.1303	2.1257	0.8364
YP37	(GT)₁₁	CAGAATGTCCATGCTGCTTT TGTTGTGCTCAGGGTTTGTC	166	57	9	5.5922	0.125	0.1613	1.9107	0.8002
YP47	(CA)₁₁	TGGAGTGCTAGATGAGGGCT GAATTATAAGTGTAATGTGAGGGGG	193	59	5	2.5946	0.375	0.3723	1.2026	0.5691
YP50	(ATA)6ac aataataata ac(AAT)₅	CCTGTTCGTTTTGTCTTTTTGA AGGTTATTGGACCCCCTGAT	214	59	5	2.7429	0.5	0.3511	1.1497	0.5639
YP58	(TTTAA)₆	TGTGACACCGTCTGTCTTCC GACATGGGGGTGAGTGAAAT	173	57	6	3.8919	0.2917	0.2411	1.5131	0.7027
YP61	(CA)₉	CCCAAAAGGAGGTTTTGTCA GAATGTCCCCCACAATTCAC	183	63	9	3.4491	0.3333	0.2748	1.5733	0.6740
YP65	(GA)₈	CGGGAGAGCGAAGTAAACTG TGACTGCACAAGGCAGATTC	234	56	11	5.8477	0.2083	0.1534	2.0174	0.8105
YP69	(TA)₇	AGAATTGACGCTGTGGGAAC TTTTGGCTGCACTAGGCG	161	56	11	4.4825	0.125	0.2066	1.9007	0.7579
YP70	(AT)₉	GAGTTCCCAGATCGCTTGTC TCCTAAATGGCAACGAGTCC	171	56	5	3.9051	0.9583	0.2402	1.4461	0.6996
YP73	(ATT)₆	CATTTTTGTCTTGATGCTACGC AAAACCAGCAGGGTTTCTGA	255	56	7	2.8029	0.375	0.3431	1.3158	0.5948
YP75	(CT)₁₂	TTGTATGCCTCTGCATCCTG CAACAGCAGCTTCTGCACTC	235	56	5	1.3585	0.75	0.7305	0.6016	0.2543
YP79	(AC)₁₄	TAATCGGCCTGCTGGATTAG AAAGCAGTCTTGAAGCGGAA	275	58	2	1.0868	1	0.9184	0.1732	0.07671
YP88	(TA)₆	GCCCTACAATCAGCAAAGTAAA CGCATTAGCCTCCTTTATGC	187	55	5	2.9091	1	0.3298	1.2566	0.6053
YP91	(AAAT)₆	CGCCAGGGAAATTATTTTGA GCCATTAAAGTCACCAGTTAATCA	278	55	3	1.2374	0.875	0.8041	0.4042	0.1828
Mean					9.2778	4.6728	0.4564	0.3102	1.5497	0.5965

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表 3 五个飘鱼群体的遗传多样性参数

Table 3 Genetic diversity parameters in five populations of P. sinensis from the Pearl River Basin

群体Population	等位基因数Number of alleles (N_a)	有效等位基因Effective number of alleles (N_e)	观测杂合度Observed Heterozygosity (H_o)	期望杂合度Expected heterozygosity (H_e)	香农指数Shannon’s Index (I)	多态信息含量Polymorphic information content (PIC)
GJ	10.1	5.9406	0.6773	0.8165	1.9041	0.7727
RJ	8.6	5.2028	0.7667	0.7975	1.7640	0.7409
YuJ	11.3	6.6279	0.7233	0.8425	2.0255	0.8074
YJ	8.6	5.8498	0.7257	0.8196	1.8383	0.7665
ZJ	10.8	6.3800	0.7167	0.8212	1.9632	0.7856
Mean	9.9	6.0002	0.7219	0.8195	1.8990	0.7746

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表 4 群体间的基因流N_m(对角线左下角)和遗传分化系数F_st及其P值(对角线右上角)

Table 4 Gene flow N_m (below diagonal) and the genetic differentiation coefficient F_st and its P-value (above diagonal) among five populations of P. sinensis

群体Population	GJ	RJ	YuJ	YJ	ZJ
GJ		F_st0.0043 P0.2522	F_s_t0.0126 P0.0360	F_st0.0080 P0.2072	F_st0.0208 P0.0000
RJ	57.8895		F_st0.0154 P0.0180	F_st–0.0033 P0.7477	F_st0.0295 P0.0000
YuJ	19.5913	15.9838		F_st0.0106 P0.0360	F_st 0.0180 P0.0000
YJ	31.0000	76.0075	23.3349		F_st0.0179 P0.0000
ZJ	11.7692	8.2246	13.6389	13.7165

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表 5 五个飘鱼群体间的分子方差分析

Table 5 AMOVA analysis of five populations of P. sinensis

变异来源Source of variation	自由度df	平方和Sum of squares	方差组分Variance component	变异百分比Percentage variation	分化系数F_st	P
群体间 Among populations	4	24.102	0.056	1.55	0.0155	0.000
群体内 Within populations	221	784.668	3.551	98.45
总Total	225	808.770	3.606

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表 6 五个种群在TPM突变模型假设下sign test检验结果

Table 6 Analysis of the possibility of a recent bottleneck using sign tests in the five P. sinensis populations

群体Population	TPM
群体Population	H_E/H_D	P	Mode-Shift
GJ	2/8	0.1777	L型
RJ	3/7	0.3718	L型
YuJ	4/6	0.6301	L型
YJ	2/8	0.1639	L型
ZJ	3/7	0.3855	L型
注: H_E/H_D. 杂合子过剩的个体数量与杂合子缺失的数量之比; P>0.05表示群体没有经历最近的瓶颈Note: H_E/H_D: ratio of the number of individuals with heterozygosity excesses to the number with heterozygosity deficiency; P>0.05 indicates that the population has not experienced a recent bottleneck

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飘鱼微卫星位点的筛选及珠江流域5个地理群体的遗传多样性分析

作者简介: 阮惠婷(1994—), 女, 硕士研究生; 研究方向为水生生物遗传多样性。E-mail: 1054741781@qq.com

通信作者: 邹柯姝(1987—), 女, 博士, 讲师; 研究方向为水生生物多样性。E-mail: zoukeshu@scau.edu.cn 刘丽(1964—), 女, 博士, 教授; 研究方向为水产种质资源的开发、利用与保护。E-mail: liuli@scau.edu.cn *共同通信作者

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MICROSATELLITE PRIMERS SCREENING AND GENETIC DIVERSITY ANALYSIS OF FIVE GEOGRAPHICAL POPULATIONS OF PSEUDOLAUBUCA SINENSIS IN THE PEARL RIVER BASIN

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作者简介:
阮惠婷(1994—), 女, 硕士研究生; 研究方向为水生生物遗传多样性。E-mail: 1054741781@qq.com

通信作者:
邹柯姝(1987—), 女, 博士, 讲师; 研究方向为水生生物多样性。E-mail: zoukeshu@scau.edu.cn

刘丽(1964—), 女, 博士, 教授; 研究方向为水产种质资源的开发、利用与保护。E-mail: liuli@scau.edu.cn *共同通信作者