马口鱼全基因组简单重复序列特征分析与多态性标记开发

doi:10.3969/j.issn.1004-1524.20221569

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (11): 2584-2593.DOI: 10.3969/j.issn.1004-1524.20221569

马口鱼全基因组简单重复序列特征分析与多态性标记开发

葛健辉¹^,²(), 关文志², 任晋东², 牛宝龙², 胡金春⁴, 王伟¹, 翁旭东⁵, 楼宝², 于瑾³^,^*(), 许晓军²^,^*()

1.浙江万里学院生物与环境学院,浙江宁波 315100
2.浙江省农业科学院水生生物研究所,浙江杭州 310021
3.龙游县养殖业发展中心,浙江龙游 324000
4.衢州市水产技术推广中心,浙江衢州 324000
5.浙江渔老大农业科技有限公司,浙江衢州 324000

收稿日期:2022-11-04 出版日期:2023-11-25 发布日期:2023-12-04
作者简介:葛健辉(1999—),男,浙江天台人,硕士研究生,研究方向为水产动物遗传育种。E-mail: 2233167022@qq.com
通讯作者: * 于瑾,E-mail:5653135@qq.com;许晓军,E-mail: xuxj@zaas.ac.cn

Characteristics and polymorphic markers development of whole genome simple sequence repeat of Opsariichthys bidens

GE Jianhui¹^,²(), GUAN Wenzhi², REN Jindong², NIU Baolong², HU Jinchun⁴, WANG Wei¹, WENG Xudong⁵, LOU Bao², YU Jin³^,^*(), XU Xiaojun²^,^*()

1. College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, Zhejiang, China
2. Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. Longyou County Breeding Development Center, Longyou 324000, Zhejiang, China
4. Quzhou Aquatic Technology Promotion Center, Quzhou 324000, Zhejiang, China
5. Zhejiang Yulaoda Agricultural Science and Technology Company, Quzhou 324000, Zhejiang, China

Received:2022-11-04 Online:2023-11-25 Published:2023-12-04

摘要/Abstract

摘要：

为开发马口鱼(Opsariichthys bidens)多态性简单重复序列(simple sequence repeat, SSR)标记,利用微卫星识别工具(microsatellite identification tool, MISA)搜索统计马口鱼全基因组SSR,采用生物信息学方法对其分布特征进行比较分析;选取39个三核苷酸SSR位点设计引物进行多态性检测。结果显示:马口鱼全基因组中SSR总数量为304 870个,占全基因组长度的0.10%;二核苷酸SSR为马口鱼基因组中的主要重复类型。5种重复类型SSR数量为二核苷酸SSR>四核苷酸SSR>三核苷酸SSR>六核苷酸SSR>五核苷酸SSR。内含子区SSR数量最多,为93 380个;5'非翻译区SSR数量最少,为622个。各区域SSR数量分布情况为内含子区>基因间隔区>启动子区>3'非翻译区>编码区>5'非翻译区。编码区数量最多的是三核苷酸SSR,而其他5个区域最多的是二核苷酸SSR。各重复类型拷贝数分布范围为5~350,主要集中在5~30。通过筛选得到15对多态性引物,在野生群体马口鱼中共检测到106个等位基因,观测杂合度为0.125~0.813,期望杂合度为0.359~0.862,多态信息含量(PIC)为0.339~0.830,其中12对引物具有高度多态性(PIC>0.5)。这些微卫星标记为马口鱼的种群遗传多样性分析、亲缘关系鉴定等研究提供了基础数据。

关键词: 马口鱼, 全基因组, 简单重复序列, 多态性

Abstract:

In order to develop polymorphic simple sequence repeat (SSR) markers, the SSR were searched and computed in the genome of Opsariichthys bidens by microsatellite identification tool (MISA). The SSR distribution characteristics were analyzed by bioinformatics methods, and 39 tri-nucleotide loci were selected for primers design and polymorphism detection. The total SSR number detected in the O. bidens genome were 304 870, which accounted for 0.10% of the whole genome length. Di-nucleotide SSR was the dominant repeat type in the genome of O. bidens. The number of 5 SSR repeat types was di->tetra->tri->hexa->penta-nucleotide. The introns had the highest number of SSR, with the number of 93 380, while 5'UTR had the smallest number of SSR, with the number of 622. The order of SSR number of 6 regions was introns>intergenics>promoter>3'UTR>CDS>5'UTR. Tri-nucleotide SSR was the dominant repeat type in coding sequence, but di-nucleotide SSR was the dominant repeat type in the other 5 regions. The repeat number ranged from 5 to 350, mainly distributed in 5 to 30. Fifteen pairs of polymorphic primers were screened, and 106 alleles were detected in a wild population of O. bidens, the observed heterozygosity was 0.125 to 0.813, and the expected heterozygosity was 0.359 to 0.862, the polymorphism information content (PIC) was 0.339 to 0.830, 12 pairs of primers were highly polymorphic (PIC>0.50). These microsatellite markers provided basic data for population genetic diversity and parentage determination of O. bidens.

Key words: Opsariichthys bidens, genome, microsatellite sequence, polymorphism

中图分类号:

S917.4

葛健辉, 关文志, 任晋东, 牛宝龙, 胡金春, 王伟, 翁旭东, 楼宝, 于瑾, 许晓军. 马口鱼全基因组简单重复序列特征分析与多态性标记开发[J]. 浙江农业学报, 2023, 35(11): 2584-2593.

GE Jianhui, GUAN Wenzhi, REN Jindong, NIU Baolong, HU Jinchun, WANG Wei, WENG Xudong, LOU Bao, YU Jin, XU Xiaojun. Characteristics and polymorphic markers development of whole genome simple sequence repeat of Opsariichthys bidens[J]. Acta Agriculturae Zhejiangensis, 2023, 35(11): 2584-2593.

图/表 7

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染色体 Chromosome	染色体大小 Chromosome size/bp	各种重复类型SSR的数量 SSR number of different repeat types					SSR总数 Total SSR number
染色体 Chromosome	染色体大小 Chromosome size/bp	二核苷酸 Di- nucleotide	三核苷酸 Tri- nucleotide	四核苷酸 Tetra- nucleotide	五核苷酸 Penta- nucleotide	六核苷酸 Hexa- nucleotide	SSR总数 Total SSR number
Chr1	42 838 281	7 315	1 179	3 500	227	138	12 359
Chr2	36 373 919	9 019	1 158	2 453	176	311	13 117
Chr3	35 330 835	8 287	1 078	2 517	143	102	12 127
Chr4	34 906 132	8 405	1 005	2 586	171	66	12 233
Chr5	33 884 400	7 248	1 043	2 217	214	45	10 767
Chr6	33 396 297	8 013	1 023	2 972	196	282	12 486
Chr7	30 731 189	7 873	915	1 987	161	30	10 966
Chr8	29 997 807	7 912	797	2 064	138	88	10 999
Chr9	28 946 737	7 701	889	2 365	180	127	11 262
Chr10	26 393 674	5 909	820	1 959	112	71	8 871
Chr11	25 992 790	6 195	795	1 483	196	82	8 751
Chr12	25 378 215	6 727	937	2 550	188	970	11 372
Chr13	25 288 361	5 964	801	2 275	150	69	9 259
Chr14	23 545 662	5 600	751	2 297	125	216	8 989
Chr15	23 207 791	4 237	732	1 747	91	250	7 057
Chr16	22 381 823	5 207	685	2 048	106	69	8 115
Chr17	20 993 647	5 490	747	1 955	165	136	8 493
Chr18	20 921 604	6 099	676	1 467	127	128	8 497
Chr19	20 816 272	3 528	628	1 748	126	62	6 092
Chr20	20 697 575	6 242	716	2 379	127	276	9 740
Chr21	19 715 338	3 954	642	1 191	128	13	5 928
Chr22	19 536 526	5 346	610	1 730	138	37	7 861
Chr23	19 033 442	5 589	650	1 044	126	50	7 459
Chr24	18 926 892	5 034	644	2 661	139	229	8 707
Chr25	18 488 034	5 527	599	1 760	84	19	7 989
Chr26	16 960 603	4 246	564	1 403	105	347	6 665
Chr27	15 245 968	5 377	488	1 706	90	29	7 690
Chr28	13 132 233	3 010	382	879	117	136	4 524
Chr29	12 855 328	3 917	397	650	51	42	5 057
Chr30	12 349 206	4 642	410	1 338	75	38	6 503
Chr31	12 285 430	2 950	313	687	40	92	4 082
Chr32	11 311 424	4 517	493	789	48	39	5 886
Chr33	11 073 039	2 918	309	851	91	5	4 174
Chr34	10 660 901	2 327	274	678	69	52	3 400
Chr35	9 886 416	2 662	312	885	76	75	4 010
Chr36	8 802 955	2 212	278	551	42	181	3 264
Chr37	8 801 959	2 331	243	545	46	29	3 194
Chr38	6 852 437	1 742	236	634	34	11	2 657
Chr39	6 768 732	2 074	153	473	32	2	2 734
总计Total		203 346	25 372	65 024	4 650	4 944	303 336
SSR数量占比		67.04	8.36	21.44	1.53	1.63
Proportion of SSR number/%

染色体 Chromosome	染色体大小 Chromosome size/bp	各种重复类型SSR的数量 SSR number of different repeat types					SSR总数 Total SSR number
染色体 Chromosome	染色体大小 Chromosome size/bp	二核苷酸 Di- nucleotide	三核苷酸 Tri- nucleotide	四核苷酸 Tetra- nucleotide	五核苷酸 Penta- nucleotide	六核苷酸 Hexa- nucleotide	SSR总数 Total SSR number
Chr1	42 838 281	7 315	1 179	3 500	227	138	12 359
Chr2	36 373 919	9 019	1 158	2 453	176	311	13 117
Chr3	35 330 835	8 287	1 078	2 517	143	102	12 127
Chr4	34 906 132	8 405	1 005	2 586	171	66	12 233
Chr5	33 884 400	7 248	1 043	2 217	214	45	10 767
Chr6	33 396 297	8 013	1 023	2 972	196	282	12 486
Chr7	30 731 189	7 873	915	1 987	161	30	10 966
Chr8	29 997 807	7 912	797	2 064	138	88	10 999
Chr9	28 946 737	7 701	889	2 365	180	127	11 262
Chr10	26 393 674	5 909	820	1 959	112	71	8 871
Chr11	25 992 790	6 195	795	1 483	196	82	8 751
Chr12	25 378 215	6 727	937	2 550	188	970	11 372
Chr13	25 288 361	5 964	801	2 275	150	69	9 259
Chr14	23 545 662	5 600	751	2 297	125	216	8 989
Chr15	23 207 791	4 237	732	1 747	91	250	7 057
Chr16	22 381 823	5 207	685	2 048	106	69	8 115
Chr17	20 993 647	5 490	747	1 955	165	136	8 493
Chr18	20 921 604	6 099	676	1 467	127	128	8 497
Chr19	20 816 272	3 528	628	1 748	126	62	6 092
Chr20	20 697 575	6 242	716	2 379	127	276	9 740
Chr21	19 715 338	3 954	642	1 191	128	13	5 928
Chr22	19 536 526	5 346	610	1 730	138	37	7 861
Chr23	19 033 442	5 589	650	1 044	126	50	7 459
Chr24	18 926 892	5 034	644	2 661	139	229	8 707
Chr25	18 488 034	5 527	599	1 760	84	19	7 989
Chr26	16 960 603	4 246	564	1 403	105	347	6 665
Chr27	15 245 968	5 377	488	1 706	90	29	7 690
Chr28	13 132 233	3 010	382	879	117	136	4 524
Chr29	12 855 328	3 917	397	650	51	42	5 057
Chr30	12 349 206	4 642	410	1 338	75	38	6 503
Chr31	12 285 430	2 950	313	687	40	92	4 082
Chr32	11 311 424	4 517	493	789	48	39	5 886
Chr33	11 073 039	2 918	309	851	91	5	4 174
Chr34	10 660 901	2 327	274	678	69	52	3 400
Chr35	9 886 416	2 662	312	885	76	75	4 010
Chr36	8 802 955	2 212	278	551	42	181	3 264
Chr37	8 801 959	2 331	243	545	46	29	3 194
Chr38	6 852 437	1 742	236	634	34	11	2 657
Chr39	6 768 732	2 074	153	473	32	2	2 734
总计Total		203 346	25 372	65 024	4 650	4 944	303 336
SSR数量占比		67.04	8.36	21.44	1.53	1.63
Proportion of SSR number/%

重复类型 Repeat type	SSR总数 Total SSR number	重复单元拷贝数 Copy number of each repeat				数量最多的重复基序 Maximum SSR repeat motif
重复类型 Repeat type	SSR总数 Total SSR number	≤10	11~20	21~30	>30	数量最多的重复基序 Maximum SSR repeat motif
二核苷酸SSR Di-nucleotide SSR	204 353	85 798	93 599	18 034	6 922	AC/GT
三核苷酸SSR Tri-nucleotide SSR	25 431	23 423	1 972	28	8	AAT/ATT
四核苷酸SSR Tetra-nucleotide SSR	65 210	30 829	23 222	9 495	1 664	AGAT/ATCT
五核苷酸SSR Penta-nucleotide SSR	4 653	3 825	606	161	61	AATAT/ATATT
六核苷酸SSR Hexa-nucleotide SSR	5 223	4 844	312	42	25	AACCCT/AGGGTT

重复类型 Repeat type	SSR总数 Total SSR number	重复单元拷贝数 Copy number of each repeat				数量最多的重复基序 Maximum SSR repeat motif
重复类型 Repeat type	SSR总数 Total SSR number	≤10	11~20	21~30	>30	数量最多的重复基序 Maximum SSR repeat motif
二核苷酸SSR Di-nucleotide SSR	204 353	85 798	93 599	18 034	6 922	AC/GT
三核苷酸SSR Tri-nucleotide SSR	25 431	23 423	1 972	28	8	AAT/ATT
四核苷酸SSR Tetra-nucleotide SSR	65 210	30 829	23 222	9 495	1 664	AGAT/ATCT
五核苷酸SSR Penta-nucleotide SSR	4 653	3 825	606	161	61	AATAT/ATATT
六核苷酸SSR Hexa-nucleotide SSR	5 223	4 844	312	42	25	AACCCT/AGGGTT

位点名称 Locus name	重复单元 Repeat motif	正向引物 Forward primer (5'→3')	反向引物 Reverse primer (5'→3')	片段大小 Size/bp	退火温度 Annealing temperature/ ℃	荧光标记物质 Fluorescence label
3Chr1	(TAA)13	GAAATGGAAGAACTCGCTCG	GAGTTGTGTTGAACGTCTGG	241	58	FAM
3Chr3	(TAT)12	GGTTACTCGACATTTCGCTG	CAAACTCACGTGGCTTCATT	237	56	FAM
3Chr7	(AAT)15	GTCAAGAAGAGGGAGCACAT	GCCTTTGCTATTAGTTATGGGA	247	58	HEX
3Chr8	(ATA)12	TTAGGAGCACGTGAGACATC	TGCGATAAACATTGATCTGCA	229	56	HEX
3Chr10	(TGA)11	ACAATCATTTCGTTGCCCAG	AGGAGGAGGTTTCTCAGACT	247	56	FAM
3Chr11	(TAA)10	CAGCAGACCGAAAAGAAAGC	ACACAGGTGGAAACATGTCT	248	56	HEX
3Chr12	(CAA)11	GCATCACATATGCAGGGTTC	TCACTTTTTGCTTGGCAGAC	238	56	FAM
3Chr13	(CAT)11	CTATTGGAGGAGATTGCCGA	AAAGTGTGCATACCTCAGCT	210	56	HEX
3Chr14	(AAT)12	TTAAGGTCGGGTTGTTAGGC	GGGTATATCTGAAGGCCCAG	222	56	FAM
3Chr15	(TAT)13	TCGGCCTAACACCATAGATG	GGGTCCATTCATCTAACCCT	246	56	HEX
3Chr16	(ATT)13	CCATTACCTCTCGCACAAAC	CCCAATAACCGACCCTCTAC	226	56	FAM
3Chr25	(TAT)19	GGCTTGACTTGTTGCATGAC	GCTCATCAATGGTGCTTACC	248	56	HEX
3Chr26	(ATT)15	ATTGGTCTTGGTTCTCAGCT	TCGCCTCCTCACTACAATTC	249	56	FAM
3Chr27	(GGA)11	ATAGCTCCCAAGAACTTCGG	AATTGTGCAGCTGGTAGAGT	214	56	HEX
3Chr30	(ATT)11	CGAAGGTTTGGAAAACCCAC	TCGTCTCCAGTAATAGCCAC	232	56	FAM

马口鱼全基因组简单重复序列特征分析与多态性标记开发

Characteristics and polymorphic markers development of whole genome simple sequence repeat of Opsariichthys bidens

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SSR位点	N_a	N_e	H_o	H_e	PIC	N	HWE	F_null
SSR locus
3Chr1	5	3.0251	0.469	0.680	0.611	32	NS	0.1847
3Chr3	8	5.6110	0.625	0.835	0.798	32	ND	0.1371
3Chr7	7	5.7528	0.688	0.839	0.803	32	ND	0.0953
3Chr8	4	2.6806	0.594	0.637	0.572	32	NS	0.0197
3Chr10	6	1.5468	0.281	0.359	0.339	32	ND	0.1492
3Chr11	7	3.5930	0.531	0.733	0.670	32	ND	0.1591
3Chr12	7	1.6126	0.344	0.386	0.368	32	ND	0.0986
3Chr13	5	2.4586	0.594	0.603	0.553	32	NS	0.0130
3Chr14	9	3.5617	0.656	0.731	0.681	32	NS	0.0459
3Chr15	11	6.3801	0.813	0.857	0.827	32	ND	0.0115
3Chr16	6	2.8132	0.500	0.655	0.580	32	NS	0.1198
3Chr25	9	6.6065	0.719	0.862	0.830	32	ND	0.0862
3Chr26	9	3.6768	0.594	0.740	0.691	32	NS	0.1002
3Chr27	5	2.0771	0.531	0.527	0.491	32	ND	-0.0539
3Chr30	8	2.1810	0.125	0.550	0.517	32	ND	0.6391

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