大银鱼全基因组简单重复序列标记开发及在不同生态型群体中的检验

doi:10.3785/j.issn.1008-9209.2022.04.191

浙江大学学报（农业与生命科学版）

2023, Vol. 49

Issue (3): 413-423 DOI: 10.3785/j.issn.1008-9209.2022.04.191

动物科学与动物医学

大银鱼全基因组简单重复序列标记开发及在不同生态型群体中的检验

唐雪梅¹(

),周彦锋^1,²,方弟安^1,²(

),罗宇婷¹,张敏莹²,蒋书伦²,张希昭²,彭飞¹,尤洋^1,²(

)

^1.南京农业大学无锡渔业学院，江苏无锡 214081
^2.中国水产科学研究院淡水渔业研究中心，农业农村部淡水渔业与种质资源利用重点实验室，江苏无锡 214081

Development of whole-genome simple sequence repeat markers in Proto-salanx chinensis and their test in different ecological populations

Xuemei TANG¹(

),Yanfeng ZHOU^1,²,Di’an FANG^1,²(

),Yuting LUO¹,Minying ZHANG²,Shulun JIANG²,Xizhao ZHANG²,Fei PENG¹,Yang YOU^1,²(

)

^1.Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, Jiangsu, China
^2.Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture and Rural Affairs, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, Jiangsu, China

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摘要：

利用Krait软件对2020年公布的拼接程度更高的大银鱼全基因组中完美型微卫星的分布特征进行分析，并据此开发具有多态性的微卫星DNA（又称简单重复序列）标记。结果显示：在大银鱼全基因组内共获得587 554个完美型微卫星位点，序列总长度为11 803 017 bp，占全基因组长度的2.53%；在6种重复类型的微卫星中，二核苷酸数量最多（401 585个，占比68.35%）。针对微卫星位点设计的99对引物中，有39对具有多态性，选择其中多态性较好的14个微卫星标记分别对大银鱼洄游型群体、陆封型群体及移植型群体中选择的1个具有代表性的群体进行检验，结果表明，多态性较好的14个微卫星标记在3个具有代表性的群体中均能进行有效扩增。对3个群体的遗传多样性和遗传结构进行分析发现，洄游型群体（崇明岛群体）遗传变异丰富（平均期望杂合度为0.614、平均多态信息含量为0.576），与淡水群体［太湖群体（陆封型）和连环湖群体（移植型）］分属于2个不同的遗传群组，两者间具有较大的遗传距离和极高的遗传分化水平（遗传分化指数高于0.25，P＜0.05）；淡水群体（太湖和连环湖群体）的遗传变异相对匮乏，2个群体间的遗传距离较小，虽存在显著的遗传分化，但遗传分化水平较低（遗传分化指数为0.102，P＜0.05）。本研究结果表明，洄游型群体具有潜在的种质资源保护价值，可为后续大银鱼全基因组微卫星标记开发和遗传图谱构建等提供依据，并为后续更大范围的群体种质资源评估与管理提供参考。

关键词： 大银鱼; 基因组简单重复序列; 生态型群体; 遗传分化

Abstract:

Krait software was used to analyze the distribution characteristics of perfect microsatellites in the whole genome of Protosalanx chinensis, which was published in 2020 with a higher degree of splicing, and to develop polymorphic microsatellite DNA (also known as simple sequence repeat) markers. The results showed that a total of 587 554 perfect microsatellite loci were obtained in the whole genome of P. chinensis, with a total sequence length of 11 803 017 bp, accounting for 2.53% of the whole genome length. Among six repeat types of microsatellites, the number of dinucleotide was the largest (401 585, accounting for 68.35%). In the 99 pairs of primers designed for microsatellite loci， 39 were polymorphic. Among them, 14 microsatellite markers with favorable polymorphism were selected to test one representative population selected from each of the migratory, landlocked, and introduced populations. The results indicated that 14 microsatellite markers with favorable polymorphism could achieve effective amplification in the three representative populations. The genetic diversity and genetic structure of the three populations were analyzed, and it was found that the migratory population (Chongming Island population) had abundant genetic variation (the mean expected heterozygosity is 0.614, and the mean polymorphism information content is 0.576), which could be clustered into a genetic group different from the freshwater populations [including Taihu Lake population (landlocked) and Lianhuan Lake population (introduced)], and there were large genetic distance and extremely high level of genetic differentiation level between them [the genetic differentiation index (F_st) is higher than 0.25, P＜0.05]. The genetic variations between the two freshwater populations (Taihu Lake and Lianhuan Lake populations) were relatively scarce and the genetic distance between them was small. Although there was significant genetic differentiation between them, the genetic differentiation level was relatively low (F_st=0.102, P＜0.05). These results indicate that the migratory population has potential conservation value of germplasm resource, which provide basis for the development of microsatellite markers and construction of genetic maps, and furthermore provide references for the subsequent evaluation of large-scale population germplasm resources of P. chinensis.

Key words: Protosalanx chinensis genome simple sequence repeat ecological population genetic differentiation

收稿日期: 2022-04-19 出版日期: 2023-06-25

CLC:

S917.4

基金资助: 国家重点研发计划项目(2020YFD0900500);国家现代农业产业技术体系项目(CARS-46);中国水产科学研究院科技创新团队专项(2020TD61)

通讯作者: 方弟安,尤洋 E-mail: 15729610929@163.com;fangdian@ffrc.cn;youy@ffrc.cn

作者简介: 唐雪梅（https://orcid.org/0000-0002-3223-5985），E-mail：15729610929@163.com

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引用本文:

唐雪梅,周彦锋,方弟安,罗宇婷,张敏莹,蒋书伦,张希昭,彭飞,尤洋. 大银鱼全基因组简单重复序列标记开发及在不同生态型群体中的检验[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(3): 413-423.

Xuemei TANG,Yanfeng ZHOU,Di’an FANG,Yuting LUO,Minying ZHANG,Shulun JIANG,Xizhao ZHANG,Fei PENG,Yang YOU. Development of whole-genome simple sequence repeat markers in Proto-salanx chinensis and their test in different ecological populations. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(3): 413-423.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2022.04.191 或 https://www.zjujournals.com/agr/CN/Y2023/V49/I3/413

表1 大银鱼样品详细信息

微卫星位点 Microsatellite locus	引物序列（5 $′$ →3 $′$ ） Primer sequence (5 $′$ →3 $′$ )	重复基序及数量 Repeat motif and number	产物长度 Product length/bp	退火温度 Annealing temperature/℃
SSR03	F: GGCTATTCTGCTCACTCTG	CA/TG（10）	245	54
SSR03	R: TTGACTAACCTGCTCTACTG	CA/TG（10）	245	54
SSR07	F: GCATCATCATCCTCTTCATCA	AT/AT（11）	237	56
SSR07	R: CACAGAACGCTTGTCCAC	AT/AT（11）	237	56
SSR10	F: GCTCAGACAACTCAGGTT	CG/CG（14）	214	60
SSR10	R: CACAGCCTTAGAACATTCG	CG/CG（14）	214	60
SSR11	F: CCAGCCTCAATAACATTCAA	AAC/GTT（5）	178	56
SSR11	R: TTGCGTGGAACTTCTTGA	AAC/GTT（5）	178	56
SSR22	F: TATAACCTGCTGTGCTTCAT	AAC/GTT（9）	133	56
SSR22	R: GAGTCCTTCTGGAACTAACC	AAC/GTT（9）	133	56
SSR30	F: CCTCCTCCTTCACATCCT	TTC/GAA（5）	129	60
SSR30	R: AGCGTGTTCAACATCATCT	TTC/GAA（5）	129	60
SSR35	F: CATGTCATCAGCAATCAGAG	TCC/GGA（9）	125	60
SSR35	R: CAGAGGAGCCATTAAGAAGA	TCC/GGA（9）	125	60
SSR45	F: GAGGAAGAGCCGTGATTG	TAA/TTA（6）	165	56
SSR45	R: AGCGACTTTGAATACCTTGA	TAA/TTA（6）	165	56
SSR48	F: TTCCTTTGCTTCCCATCG	ATT/AAT（5）	198	60
SSR48	R: GCCACTTGTTCGTATTCATT	ATT/AAT（5）	198	60
SSR74	F: ACTCACCCAATGTTGAAGG	TGC/GCA（5）	280	56
SSR74	R: CAATCCAAGAATACTCAGTACC	TGC/GCA（5）	280	56
SSR84	F: AGGATTAGGTTGCTGTTGAG	GCT/AGC（5）	151	56
SSR84	R: TGAACTGCCTCTGCTGAT	GCT/AGC（5）	151	56
SSR90	F: CTGAGGAATGAGACCAAGG	GATA/TATC（10）	172	56
SSR90	R: GTTAGGAGTTGCATGAAGTG	GATA/TATC（10）	172	56
SSR98	F: CAGGAAGCGAACGATACAA	TTG/CAA（7）	223	60
SSR98	R: GGCTCGGATGTCATAGAAC	TTG/CAA（7）	223	60
SSR99	F: GTCTGATTCTGAAGTGAACTC	TGT/ACA（10）	225	56
SSR99	R: TGACTACACAGCCTCTCC	TGT/ACA（10）	225	56

表2 大银鱼14对多态性微卫星引物信息

表3 大银鱼全基因组完美型微卫星位点分布信息

表4 大银鱼各重复类型微卫星的主要重复基序分布情况

图1 大银鱼全基因组中各重复类型微卫星的拷贝数分布特征

图2 微卫星位点SSR90毛细管电泳基因分型的部分结果ar：峰面积；ht：峰高度；sz：PCR产物条带的大小。

表5 14个具有多态性的微卫星位点在3个大银鱼生态型群体中的遗传多样性检测结果

表6 3个大银鱼生态型群体间的遗传距离和遗传分化指数

图3 最佳亚群数量（ K ）与ΔK 的关系（A）及 K=2时基于Structure软件所做的聚类分析（B）

图4 基于14个微卫星标记的56个大银鱼个体的NJ系统发育树（A）和主坐标分析（B）

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