黄鳍棘鲷SNP标记开发与验证

doi:10.11978/2022108

摘要/Abstract

摘要：

文章以我国东南沿海重要的经济鱼类黄鳍棘鲷(Acanthopagrus latus)为研究对象, 通过对50尾野生黄鳍棘鲷基因组重测序开发单核苷酸多态性(single nucleotide polymorphism, SNP)标记, 并利用MassARRAY^®质谱分析系统对部分SNP标记进行验证。研究结果如下: 1) 50尾野生黄鳍棘鲷重测序共产生约233.48Gb原始数据(raw data), 过滤接头和低质量数据后, 共获得233.43Gb数据, 每个样品平均数据量为4.67Gb, 平均鸟嘌呤胞嘧啶(guanine cytosine, GC)含量为42.85%, Q20在96.56%以上, Q30在91.1%以上, 过滤后数据与参考基因组的比对率为98.06% ~ 99.47%; 2) 通过基因分析工具(genome analysis toolkit, GATK)分析, 从50个个体中共挖掘出13843766个SNP, 经过滤后获得6501个高质量SNP; 3) 从高质量的SNP标记中, 随机挑选30个SNP使用MassARRAY技术进行分型, 其检出率(可以分型的位点)为98%, MassARRAY分型结果与基因组重测序分型结果一致率为64.83%, 这说明两种技术在SNP分型方面存在差异。综上所述, 本研究初步建立了黄鳍棘鲷SNP标记挖掘、过滤与验证的方法, 开发的SNP位点可用于后续黄鳍棘鲷增殖放流效果评估及基因组选择育种研究。

关键词: 黄鳍棘鲷, 重测序, 单核苷酸多态性, 质谱分析

Abstract:

In this experiment, 'Acanthopagrus latus', an important economic fish in the southeast coast of China, was used as the experimental material. The SNPs were discovered by DNA re-sequencing in fifty A. latus, and partial SNPs were genotyped using MassARRAY^® DNA mass spectrometry. The results are presented as followed: 1) the re-sequencing of 50 wild A. latus generated a total of about 233.48 GB raw data. After filtering adapters and low-quality data, 233.43 Gb clear data were obtained. The average data size of each sample was 4.67 GB, and the average GC content was 42.85%, Q20 is above 96.56%, Q30 is above 91.1%, and the comparison rate between the clear data and the reference genome is 98.06% ~ 99.47%; 2) a total of 13843766 SNPs were discovered from 50 individuals by GATK, and 6501 high-quality SNPs were obtained after filtering; 3) thirty SNPs from the high-quality SNP were selected randomly and genotyped using MassARRAY technology. The detection rate (loci that can be genotyped) was reached at 98%. The consistency between the genome re-sequencing and the MassARRAY results was 64.83%, which indicated that the two techniques were different in detecting SNPs. In summary, a method for mining, filtering and validating SNP markers of A. latus bream has been established in this study, and the developed SNP loci can be used in evaluation of proliferation and stocking effect and genome selection breeding of A. latus in the future.

Key words: Acanthopagrus latus, Re-sequencing, SNP, MassARRAY

郑国彬, 赵虹博, 黄良敏, 张静, 刘贤德. 黄鳍棘鲷SNP标记开发与验证[J]. 热带海洋学报, 2023, 42(2): 78-86.

ZHENG Guobin, ZHAO Hongbo, HUANG Liangmin, ZHANG Jing, LIU Xiande. Discovery and verification of SNP in Acanthopagrus latus[J]. Journal of Tropical Oceanography, 2023, 42(2): 78-86.

图/表 7

表1

图1

表2

重测序数据质量统计结果"

样品	原始数据/Gb	过滤数据/Gb	比对率/%	测序深度/×	Q20/%	Q30/%	GC含量/%	样品	原始数据/Gb	过滤数据/Gb	比对率/%	测序深度/×	Q20/%	Q30/%	GC含量/%
Y01	4.71	4.71	95.90	6.15	97.34	92.78	42.69	Y26	4.79	4.79	97.00	6.33	97.33	92.77	42.80
Y02	5.00	4.99	95.49	6.52	97.39	92.88	42.69	Y27	4.59	4.59	96.96	6.09	97.27	92.52	42.93
Y03	4.76	4.76	96.20	6.22	97.43	92.94	42.61	Y28	4.63	4.63	96.76	6.10	96.56	91.10	42.78
Y04	4.55	4.55	96.83	6.03	97.49	93.13	42.73	Y29	4.74	4.74	96.74	6.27	97.34	92.84	42.83
Y05	4.58	4.58	96.38	6.06	97.43	93.03	42.76	Y30	4.48	4.48	96.96	5.96	97.54	93.17	42.66
Y06	4.46	4.46	96.46	5.92	97.44	93.02	42.69	Y31	4.85	4.85	97.2	6.41	97.47	93.06	42.66
Y07	4.71	4.71	97.03	6.25	97.45	93.06	42.76	Y32	4.17	4.17	97.10	5.59	97.26	92.60	42.75
Y08	4.79	4.79	96.69	6.32	97.21	92.52	42.80	Y33	4.98	4.98	97.27	6.55	97.27	92.68	42.99
Y09	4.86	4.86	97.36	6.43	97.34	92.82	43.18	Y34	3.69	3.69	96.8	5.00	97.29	92.64	42.96
Y10	4.78	4.77	96.60	6.30	97.19	92.46	42.87	Y35	4.92	4.92	97.33	6.48	97.12	92.31	43.03
Y11	4.43	4.43	96.56	5.88	96.97	92.00	42.83	Y36	4.86	4.86	97.64	6.41	97.23	92.58	42.95
Y12	4.59	4.59	96.60	6.06	97.09	92.23	42.95	Y37	4.88	4.88	97.32	6.45	97.04	92.13	42.79
Y13	4.63	4.63	96.87	6.13	97.26	92.64	42.78	Y38	4.84	4.84	96.84	6.35	97.25	92.59	42.74
Y14	4.82	4.82	96.55	6.32	97.08	92.24	42.71	Y39	4.59	4.59	96.43	6.07	97.28	92.65	42.85
Y15	4.63	4.63	96.57	6.12	97.22	92.49	42.77	Y40	4.23	4.23	97.54	5.66	97.36	92.82	42.95
Y16	4.70	4.69	96.80	6.20	97.24	92.61	42.97	Y41	4.73	4.73	97.65	6.26	97.49	93.13	43.01
Y17	4.93	4.93	96.95	6.50	97.17	92.43	42.91	Y42	4.37	4.37	96.58	5.77	97.26	92.61	43.08
Y18	4.33	4.33	97.05	5.79	97.10	92.22	42.81	Y43	4.90	4.90	97.08	6.45	97.42	92.99	43.01
Y19	4.85	4.85	96.91	6.40	97.25	92.59	42.95	Y44	4.82	4.81	97.36	6.36	97.49	93.09	42.99
Y20	4.52	4.52	96.45	5.96	97.33	92.76	42.75	Y45	4.95	4.95	97.14	6.52	97.50	93.16	43.02
Y21	4.79	4.79	97.00	6.32	97.20	92.46	42.73	Y46	4.61	4.61	97.09	5.85	97.46	93.06	42.68
Y22	4.84	4.84	97.00	6.36	97.54	93.21	42.54	Y47	4.75	4.75	96.72	6.27	97.42	92.98	42.86
Y23	4.71	4.71	96.41	6.17	96.71	91.43	42.72	Y48	4.50	4.50	96.85	5.96	97.44	93.02	42.90
Y24	4.58	4.58	96.84	6.08	97.25	92.56	42.75	Y49	4.42	4.42	97.43	5.90	97.17	92.46	43.33
Y25	4.75	4.75	97.22	6.27	97.33	92.74	42.80	Y50	4.88	4.88	97.36	6.44	97.38	92.88	43.03

表2

表3

图2

表4

表5

基因组重测序与MassARRAY分型结果比较"

SNP	染色体名称	位置	MassARRAY分型检测				全基因组重测序
SNP	染色体名称	位置	检测结果	检出率/%	MF	MAF	检测结果	检出率/%	MF	MAF
Shq_178_1	NC_051039.1	33527645	CC、CG	85	0.85	0.15	CC、CG	95	0.74	0.26
Shq_178_2	NC_051040.1	32016498	TT	100	1.00	0	TT、TG	100	0.88	0.13
Shq_178_3	NC_051041.1	20806265	TT、TG	100	0.95	0.05	TT、TG	100	0.75	0.25
Shq_178_6	NC_051042.1	30329	AA、AG	90	0.69	0.31	AA、AG	95	0.63	0.37
Shq_178_8	NC_051042.1	7311706	TT、TC	100	0.95	0.05	TT、TC	100	0.95	0.05
Shq_178_19	NC_051048.1	22404460	AA	95	1.00	0	AA、AG	90	0.92	0.08
Shq_178_26	NC_051050.1	34885	CC	95	1.00	0	CC、CA	95	0.92	0.08
Shq_178_39	NC_051050.1	38757	GG	100	1.00	0	GG、GA	90	0.94	0.06
Shq_178_40	NC_051050.1	42037	TT	100	1.00	0	TT、TC	95	0.87	0.13
Shq_178_48	NC_051050.1	59154	TT、TC	85	0.91	0.09	TT、TC	100	0.65	0.35
Shq_178_60	NC_051050.1	74750	GG	100	1.00	0	GG、GA	90	0.94	0.06
Shq_178_63	NC_051050.1	75488	GG	100	1.00	0	GG、GC	95	0.95	0.05
Shq_178_65	NC_051050.1	76081	AA	100	1.00	0	AA、AC	85	0.88	0.12
Shq_178_70	NC_051050.1	87063	GG	100	1.00	0	GG、GA	90	0.89	0.11
Shq_178_72	NC_051050.1	93481	CC、CT	100	0.98	0.03	CC、CT	100	0.95	0.05
Shq_178_73	NC_051050.1	96282	CC	100	1.00	0	CC、CA	95	0.82	0.18
Shq_178_77	NC_051050.1	101791	CC	100	1.00	0	CC、CT	80	0.94	0.06
Shq_178_86	NC_051050.1	106638	GG、GA	100	0.97	0.03	GG、GA	80	0.97	0.03
Shq_178_92	NC_051050.1	128900	AA	100	1.00	0	AA、AG	100	0.93	0.08
Shq_178_96	NC_051050.1	142395	GG	100	1.00	0	GG、GA	90	0.92	0.08
Shq_178_104	NC_051052.1	1588997	AA	100	1.00	0	AA、AC	100	0.75	0.25
Shq_178_112	NC_051053.1	1538	GG	100	1.00	0	AA、AG、GG	95	0.53	0.47
Shq_178_138	NC_051053.1	8936396	AA	100	1.00	0	AA、AG	95	0.76	0.24
Shq_178_149	NC_051056.1	31720087	CC、CT	100	0.95	0.05	CC、CT	90	0.86	0.14
Shq_178_150	NC_051056.1	31723482	TT	100	1.00	0	TT、TC	95	0.89	0.11
Shq_178_157	NC_051058.1	24478462	AA、AG	100	0.95	0.05	AA、AC	100	0.95	0.05
Shq_178_158	NC_051058.1	24478507	CC、CT	95	0.97	0.03	CC、CT	100	0.95	0.05
Shq_178_171	NC_051060.1	26149536	GG	100	1.00	0	GG、GC	100	0.58	0.43
Shq_178_172	NC_051061.1	336649	TT	100	1.00	0	TT、TG	100	0.55	0.45
Shq_178_173	NC_051061.1	22105514	GG	100	1.00	0	GG、GA	100	0.55	0.45

表5

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取样地	数量/尾	体长/cm	体重/g	体厚/cm
厦门近海海区	37	20.52 ± 0.10	190.15 ± 5.92	7.27 ± 0.05
漳州东山岛海区	13	21.23 ± 0.29	212.53 ± 6.19	7.52 ± 0.08

染色体名称	SNP初始数	质控过滤后SNP数	属性过滤后SNP数	高质量过滤后SNP数
NC_051039.1	692976	632996	144274	364
NC_051040.1	646881	595158	134036	221
NC_051041.1	566757	522152	117157	325
NC_051042.1	690338	640940	142536	172
NC_051043.1	606086	565937	129160	157
NC_051044.1	697405	638444	144000	265
NC_051045.1	589449	548080	124463	366
NC_051046.1	615425	574376	130360	228
NC_051047.1	622596	577032	131271	203
NC_051048.1	476295	405575	97778	820
NC_051049.1	629215	586030	132799	263
NC_051050.1	574454	532521	120384	237
NC_051051.1	567923	530390	119320	139
NC_051052.1	462308	429823	94073	109
NC_051053.1	538049	501748	110871	172
NC_051054.1	535520	495987	112649	173
NC_051055.1	613575	567469	128275	221
NC_051056.1	669814	618189	137376	256
NC_051057.1	520747	477109	104765	187
NC_051058.1	509767	475642	106603	97
NC_051059.1	583650	535046	121219	370
NC_051060.1	563226	523705	117774	169
NC_051061.1	518795	479194	110565	299
NC_051062.1	329951	298031	67740	688
未知序列	22564	0	0	0
合计	13843766	12751574	2879448	6501

多态性参数	最大值	最小值	平均值
观测杂合度(H_o)	0.966	0.034	0.499
期望杂合度(H_e)	0.600	0.034	0.376
多态信息含量(PIC)	0.375	0.033	0.279