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

doi:10.3785/j.issn.1008-9209.2022.04.191

Journal of Zhejiang University (Agriculture and Life Sciences)

2023, Vol. 49

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

Animal sciences & veterinary medicines

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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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

Received: 19 April 2022 Published: 25 June 2023

CLC:

S917.4

Corresponding Authors: Di’an FANG,Yang YOU E-mail: 15729610929@163.com;fangdian@ffrc.cn;youy@ffrc.cn

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	Xuemei TANG
	Yanfeng ZHOU
	Di’an FANG
	Yuting LUO
	Minying ZHANG
	Shulun JIANG
	Xizhao ZHANG
	Fei PENG
	Yang YOU

Cite this article:

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.

URL:

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

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

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

Table 1 Details for the samples of P. chinensis

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

Table 2 Information of 14 pairs of polymorphic microsatellite primers from P. chinensis

Table 3 Distribution information of perfect microsatellite loci in the whole genome of P. chinensis

Table 4 Distribution of the main repeat motifs in microsatellite of each repeat type in P. chinensis

Fig. 1 Copy number distribution characteristics of microsatellite of each repeat type in the whole genome of P. chinensis

Fig. 2 Partial results of capillary electrophoresis genotyping of microsatellite locus SSR90ar: Area of the peak; ht: Height of the peak; sz: Size of PCR product strip.

Table 5 Genetic diversity detection results of 14 polymorphic microsatellite loci in three ecological populations of P. chinensis

Table 6 Genetic distances and genetic differentiation indexes of three ecologicalpopulations of P. chinensis

Fig. 3 Relationship between the optimal number of subpopu-lation (K) and the ΔK (A) and cluster analysis at K=2 by Structure software (B)

Fig. 4 NJ phylogenetic tree (A) and principal coordinate analysis (PCoA) (B) of 56 individuals of P. chinensis based on 14 microsatellite markers


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