欧拉羊角性状相关<i>RXFP2</i>基因SNPs检测及分析

doi:10.7668/hbnxb.20194458

华北农学报 ›› 2024, Vol. 39 ›› Issue (2): 200-208. doi: 10.7668/hbnxb.20194458

所属专题： 畜牧；

• 畜牧・水产・兽医 • 上一篇下一篇

欧拉羊角性状相关RXFP2基因SNPs检测及分析

张强龙¹^,²^,³, 吴森¹^,²^,³, 多杰才让⁴, 唐燕花⁵, 马杰⁶

¹ 青海大学畜牧兽医科学院,青海西宁 810016
² 青海省高原家畜遗传资源保护与创新利用重点实验室,青海西宁 810016
³ 农业农村部青藏高原畜禽遗传育种重点实验室,青海西宁 810016
⁴ 河南蒙古族自治县有机产业科技园区管理委员会,青海黄南 811300
⁵ 河南蒙古族自治县生态有机畜牧业技术服务中心,青海黄南 811300
⁶ 河南蒙古族自治县畜牧兽医站,青海黄南 811300

收稿日期:2023-12-01 出版日期:2024-04-28
通讯作者:
吴森(1987—),男,陕西咸阳人,副研究员,博士,硕士生导师,主要从事生物技术育种与健康养殖、动物生长发育调控研究。
作者简介:
张强龙(1990—),男,甘肃定西人,助理研究员,硕士,主要从事绵羊遗传育种与繁殖、高原动物生态健康养殖研究。
基金资助:
青海省重大科技专项(2021-NK-A5); 青海省牧科院基本科研业务费自主选题项目(MKY-2019-04)

Detection and Analysis of RXFP2 Gene SNPs Related to Horn Trait of Oula Sheep

ZHANG Qianglong¹^,²^,³, WU Sen¹^,²^,³, DUOJIE Cairang⁴, TANG Yanhua⁵, MA Jie⁶

¹ Academy of Animal Science and Veterinary,Qinghai University,Xining 810016,China
² Key Laboratory of Plateau Animal Genetic Resources Conservation and Innovative Utilization of Qinghai Province,Xining 810016,China
³ Key Laboratory of Livestock and Poultry Genetics and Breeding on the Qinghai-Tibet Plateau,Ministry of Agriculture and Rural Affairs,Xining 810016,China
⁴ The Henan Mongol Autonomous County Organic Industry Science Park Board,Huangnan 811300,China
⁵ The Technical Service Centre for Henan Mongol Autonomous County,Ecological and Organic Animal Husbandry,Huangnan 811300,China
⁶ The Henan Mongol Autonomous County, Livestock,Veterinary Station,Huangnan 811300,China

Received:2023-12-01 Published:2024-04-28

摘要/Abstract

摘要：

旨在分析有角和无角欧拉羊群体RXFP2基因对犄角表型的调控作用及相关SNP分子标记,以期为欧拉羊的遗传改良、品种培育提供技术支持。通过采集青海省河南县健康成年欧拉羊母羊血液样品100份,其中有角、无角各50份,提取DNA,随机选取有角、无角样品各15份开展全基因组测序,随后采用多重PCR扩增全部血样验证RXFP2基因SNP。全基因组检测结果表明,包含RXFP2基因在内的欧拉羊10号染色体存在强选择信号,最强选择信号出现在RXFP2基因区段;多重PCR检测验证了全基因组重测序筛查到的4个编码区SNP的准确性,并检测到7个欧拉羊RXFP2基因内含子区域高频SNPs(29508704G>A、29509428A>C、29509766G>A、29512170G>A、29512176G>A、29514968T>A、29521377C>A);经统计检验,该7个SNP位点的基因型在犄角有无表型上表现出分离,纯合子基因型均100%表现为无角或有角,杂合子基因型89.66%表现为无角,10.34%表现为有角;突变等位基因频率小于野生等位基因频率,群体表现为哈代温伯格不平衡状态,受到人工选择强度大,但多态信息含量中等,选育改良潜力仍较大。综上,确认了RXFP2基因在欧拉羊群体中对犄角有无的强调控作用,并筛选出了欧拉羊RXFP2基因的7个犄角有无表型的分子标记,相关结果可作为欧拉羊无角性状群体的遗传改良的分子标记。

关键词: 欧拉羊, RXFP2基因, SNPs多态性位点, 犄角性状, 分子标记

Abstract:

The aim of this study was to analyze the regulatory effect of RXFP2 gene on horned phenotype and related SNP markers in horned and hornless Oula sheep populations,in order to provide technical support for genetic improvement and breed breeding of Oula sheep.DNA was extracted from 100 ewe blood samples of healthy adult Oula sheep in Henan County,Qinghai Province,including 50 blood samples with horns and 50 blood samples without horns,respectively,15 samples with and without horns were randomly selected for whole genome sequencing,and then all blood samples were amplified by multiple PCR to verify the SNPs of RXFP2 gene.Genome-wide detection results showed that there were strong selection signals in chromosome 10 of Oula sheep,including RXFP2 gene,and the strongest selection signals appeared in the RXFP2 gene segment.Multiple PCR tests verified the accuracy of the 4 coding SNPs screened by whole genome resequencing.Seven high frequency SNPs in the intron region of RXFP2 gene were detected in Oula sheep(29508704G>A,29509428A>C,29509766G>A,29512170G>A,2951276G>A,29514968T>A,29521377C>A).The genotypes of the 7 SNP loci were separated in hornless phenotypes.100% of homozygous genotypes appeared horned or hornless trait,89.66% of heterozygous genotypes were hornless trait and 10.34% were horned trait.The frequency of mutant alleles was less than that of wild alleles,and the population showed an unbalanced state of Hardy-Weinberg,which meant subject to high artificial selection intensity,but the content of polymorphic information was moderate,and the potential of breeding and improvement was still large.In conclusion,the present study confirmed the strong regulatory effect of RXFP2 gene on horn trait in Oula sheep,and filtered out 7 molecular markers of RXFP2 gene on horn trait in Oula sheep population.The relevant results can be used as molecular markers for genetic improvement of the hornless population of Oula sheep.

Key words: Oula sheep, RXFP2 gene, SNPs polymorphic site, Horn trait, Molecular marker

张强龙, 吴森, 多杰才让, 唐燕花, 马杰. 欧拉羊角性状相关RXFP2基因SNPs检测及分析[J]. 华北农学报, 2024, 39(2): 200-208. doi: 10.7668/hbnxb.20194458.

ZHANG Qianglong, WU Sen, DUOJIE Cairang, TANG Yanhua, MA Jie. Detection and Analysis of RXFP2 Gene SNPs Related to Horn Trait of Oula Sheep[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 200-208. doi: 10.7668/hbnxb.20194458.

http://www.hbnxb.net/CN/Y2024/V39/I2/200

图/表 8

参考文献 26

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位点 Location	引物序列(5'—3') Primer sequences(5'—3')	长度/bp Length	退火温度/℃ Tm
Exon1	F:GGGACTGCAGCTAGTTCACTTT	751	60.3
	R:GGCACTTCTATCCATGGCTCTAT
Exon2	F:CATGCACAAGTAGGACAATCACT	743	64.5
	R:CAGAAGGAAGTGGTCATCGTTTA
Exon3	F:GCTTTTCCTTCCTTACCATTGTC	597	58.0
	R:TCTAGTGGAGCCATGTGAGGTAT
Exon4	F:GCCTTTCTGTGCCATTCAATTA	697	64.5
	R:AACCTGGACAATCCAACATACAG
Exon5	F:AAATCACCGTAGATGTTCCAGTC	667	60.3
	R:ACCACTCTTCCAGGTTAAAAAGC
Exon6	F:ACTTTGGTGGGTTCAAGTCAGAG	659	58.0
	R:TTGCAGCAGGATTCTTTACTACTG
Exon7	F:CCCCAGTGCATTATTTCTGG	738	64.5
	R:TGGGGTAGAGACCACAAAGG
Exon8	F:GAGCCCATAATCAGCCAATAAA	842	58.0
	R:CTAGAATTTTCAGTGACTCTTGCTG
Exon9	F:GGCTGTATGATGCTGGTAATGA	811	64.5
	R:GATAAGAATCCTCCAGCCAATG
Exon10	F:CCATGTCTAAGTGTCAGGCAGA	871	64.5
	R:GCAGCAACACTAACCACTTTCA
Exon11	F:CTTTGCTCTTTCTGGCCTTCT	804	60.1
	R:CATTCAGCAAACTTCAACTGAG
Exon12-13	F:CCTAAAACAGAGGAAACGAATG	835	60.1
	R:TTGTGAGAACCGTAAGATGAGA
Exon14	F:TCAAGCTACAGAGCCAATTTCA	765	62.0
	R:CATCTTTCTTCCTAAGGGCTCA
Exon15	F:TATAACACCCCCAGAGAACCAC	825	65.8
	R:TAATTTTCTGGGCTTGAGGAAC
Exon16	F:TGTGGGTGTTTTCATTAGTTCG	851	64.5
	R:AACTTGGAAGGGTCACAGGAC
Exon17	F:TATCTTTTTGATCTGGCGAACC	842	65.4
	R:ACATGGGTTTGATTTCTGGTCT
Exon18	F:TGACTCCAAACCAGAAGATTCC	962	64.5
	R:TGCTGCAGAGATAGAGATCCAC

位点 Location	引物序列(5'—3') Primer sequences(5'—3')	长度/bp Length	退火温度/℃ Tm
Exon1	F:GGGACTGCAGCTAGTTCACTTT	751	60.3
	R:GGCACTTCTATCCATGGCTCTAT
Exon2	F:CATGCACAAGTAGGACAATCACT	743	64.5
	R:CAGAAGGAAGTGGTCATCGTTTA
Exon3	F:GCTTTTCCTTCCTTACCATTGTC	597	58.0
	R:TCTAGTGGAGCCATGTGAGGTAT
Exon4	F:GCCTTTCTGTGCCATTCAATTA	697	64.5
	R:AACCTGGACAATCCAACATACAG
Exon5	F:AAATCACCGTAGATGTTCCAGTC	667	60.3
	R:ACCACTCTTCCAGGTTAAAAAGC
Exon6	F:ACTTTGGTGGGTTCAAGTCAGAG	659	58.0
	R:TTGCAGCAGGATTCTTTACTACTG
Exon7	F:CCCCAGTGCATTATTTCTGG	738	64.5
	R:TGGGGTAGAGACCACAAAGG
Exon8	F:GAGCCCATAATCAGCCAATAAA	842	58.0
	R:CTAGAATTTTCAGTGACTCTTGCTG
Exon9	F:GGCTGTATGATGCTGGTAATGA	811	64.5
	R:GATAAGAATCCTCCAGCCAATG
Exon10	F:CCATGTCTAAGTGTCAGGCAGA	871	64.5
	R:GCAGCAACACTAACCACTTTCA
Exon11	F:CTTTGCTCTTTCTGGCCTTCT	804	60.1
	R:CATTCAGCAAACTTCAACTGAG
Exon12-13	F:CCTAAAACAGAGGAAACGAATG	835	60.1
	R:TTGTGAGAACCGTAAGATGAGA
Exon14	F:TCAAGCTACAGAGCCAATTTCA	765	62.0
	R:CATCTTTCTTCCTAAGGGCTCA
Exon15	F:TATAACACCCCCAGAGAACCAC	825	65.8
	R:TAATTTTCTGGGCTTGAGGAAC
Exon16	F:TGTGGGTGTTTTCATTAGTTCG	851	64.5
	R:AACTTGGAAGGGTCACAGGAC
Exon17	F:TATCTTTTTGATCTGGCGAACC	842	65.4
	R:ACATGGGTTTGATTTCTGGTCT
Exon18	F:TGACTCCAAACCAGAAGATTCC	962	64.5
	R:TGCTGCAGAGATAGAGATCCAC

群体 Group	染色体 Chromosome	起始位点 Start site	SNP数量 SNP number	Tajima's D检验 Tajima's D test
无角型欧拉羊	NC_019477.2	50280000	160	3.960 54
The hornless Oula sheep	NC_019484.2	66800000	226	3.957 48
	NC_019463.2	90200000	111	3.932 68
	NC_019467.2	42100000	110	3.929 81
	NC_019460.2	130080000	194	3.920 43
	NC_019484.2	66940000	111	3.905 65
	NC_019484.2	66960000	103	3.903 45
	NC_019484.2	67060000	62	3.896 94
	NC_019471.2	14220000	126	3.887 90
	NC_019474.2	58000000	242	3.874 20
有角型欧拉羊	NC_019467.2	32440000	142	3.952 12
The horn Oula sheep	NC_019458.2	109560000	152	3.934 47
	NC_019484.2	76280000	98	3.928 91
	NC_019484.2	84160000	167	3.920 42
	NC_019467.2	41860000	82	3.919 64
	NC_019458.2	199560000	187	3.905 77
	NC_019467.2	41840000	97	3.896 65
	NC_019458.2	109580000	183	3.894 97
	NC_019467.2	41700000	171	3.887 07

群体 Group	染色体 Chromosome	起始位点 Start site	SNP数量 SNP number	Tajima's D检验 Tajima's D test
无角型欧拉羊	NC_019477.2	50280000	160	3.960 54
The hornless Oula sheep	NC_019484.2	66800000	226	3.957 48
	NC_019463.2	90200000	111	3.932 68
	NC_019467.2	42100000	110	3.929 81
	NC_019460.2	130080000	194	3.920 43
	NC_019484.2	66940000	111	3.905 65
	NC_019484.2	66960000	103	3.903 45
	NC_019484.2	67060000	62	3.896 94
	NC_019471.2	14220000	126	3.887 90
	NC_019474.2	58000000	242	3.874 20
有角型欧拉羊	NC_019467.2	32440000	142	3.952 12
The horn Oula sheep	NC_019458.2	109560000	152	3.934 47
	NC_019484.2	76280000	98	3.928 91
	NC_019484.2	84160000	167	3.920 42
	NC_019467.2	41860000	82	3.919 64
	NC_019458.2	199560000	187	3.905 77
	NC_019467.2	41840000	97	3.896 65
	NC_019458.2	109580000	183	3.894 97
	NC_019467.2	41700000	171	3.887 07

群体 Group	染色体 Chromosome	起始位点 Start site	终止位点 End site	SNP数量 SNP number	π值 π value
无角型欧拉羊	NC_019464.2	22640001	22680000	1 281	0.002 218 53
The hornless Oula sheep	NC_019475.2	57520001	57560000	1 006	0.002 367 82
	NC_019464.2	22620001	22660000	972	0.002 187 53
	NC_019464.2	22660001	22700000	989	0.001 798 51
	NC_019460.2	205440001	205480000	927	0.001 593 17
	NC_019460.2	206780001	206820000	900	0.001 169 72
	NC_019458.2	96920001	96960000	836	0.001 220 93
	NC_019470.2	36880001	36920000	749	0.001 442 71
	NC_019464.2	22560001	22600000	867	0.001 033 39
	NC_019460.2	205420001	205460000	837	0.001 196 61
有角型欧拉羊	NC_019464.2	22640001	22680000	1 286	0.011 804 10
The horn Oula sheep	NC_019475.2	57520001	57560000	1 003	0.009 558 05
	NC_019464.2	22660001	22700000	1 000	0.008 712 36
	NC_019464.2	22620001	22660000	966	0.008 669 54
	NC_019460.2	205440001	205480000	929	0.008 469 74
	NC_019458.2	96920001	96960000	828	0.007 802 66
	NC_019460.2	205420001	205460000	835	0.007 780 87
	NC_019460.2	206780001	206820000	896	0.007 728 59
	NC_019472.2	45640001	45680000	898	0.007 637 26
	NC_019470.2	36880001	36920000	726	0.007 569 19

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欧拉羊角性状相关RXFP2基因SNPs检测及分析

Detection and Analysis of RXFP2 Gene SNPs Related to Horn Trait of Oula Sheep

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染色体 Chromosome	起始位点 Start site	终止位点 End site	SNP数量 SNP number	Fst权重值 Fst weighted	Fst 均值 Fst Mean
NC_019467.2	29420001	29460000	185	0.555 574	0.450 275
NC_019467.2	29440001	29480000	184	0.543 529	0.377 100
NC_019467.2	29340001	29380000	346	0.534 190	0.328 112
NC_019467.2	29400001	29440000	238	0.532 660	0.401 596
NC_019467.2	29360001	29400000	270	0.469 524	0.283 048
NC_019467.2	29380001	29420000	299	0.466 929	0.272 309
NC_019467.2	29320001	29360000	324	0.458 475	0.255 278
NC_019467.2	29460001	29500000	152	0.380 597	0.244 133
NC_019467.2	25440001	25480000	236	0.356 358	0.227 202
NC_019469.2	300001	340000	303	0.336 557	0.209 993

基因 Gene	内含子位置 Intron	突变位置 Variation site	突变碱基 Alternate	突变数量 Mutation number	无角(46) Hornless number	有角(48) Horned number
RXFP2	Intro17	29501199	C>T	1	0	1
RXFP2	Intro17	29501256	G>A	1	1	0
RXFP2	Intro17	29501270	A>T	1	0	1
RXFP2	Intro16	29508704	G>A	74	26	48
RXFP2	Intro16	29509428	A>C	74	26	48
RXFP2	Intro14	29509766	G>A	74	26	48
RXFP2	Intro13	29512170	G>A	74	26	48
RXFP2	Intro13	29512176	G>A	74	26	48
RXFP2	Intro12	29514968	T>A	74	26	48
RXFP2	Intro10	29521377	C>A	74	26	48

突变位点 Mutation site	个体数 Number	基因型(频率/%) Genotype (frequency)	表型(频率/%) Phenotype(frequency)	等位基因(频率/%) Alleles (frequency)
29508704G>A	20	GG(21.28)	无角(100)	G(36.70)
	29	GA(30.85)	无/有角(89.66/10.34)	A(63.30)
	45	AA(47.87)	有角(100)
29509428A>C	20	AA(21.28)	无角(100)	A(36.70)
	29	AC(30.85)	无/有角(89.66/10.34)	G(63.30)
	45	CC(47.87)	有角(100)
29509766G>A	20	GG(21.28)	无角(100)	G(36.70)
	29	GA(30.85)	无/有角(89.66/10.34)	A(63.30)
	45	AA(47.87)	有角(100)
29512170G>A	20	GG(21.28)	无角(100)	G(36.70)
	29	GA(30.85)	无/有角(89.66/10.34)	A(63.30)
	45	AA(47.87)	有角(100)
29512176G>A	20	GG(21.28)	无角(100)	G(36.70)
	29	GA(30.85)	无/有角(89.66/10.34)	A(63.30)
	45	AA(47.87)	有角(100)
29514968T>A	20	TT(21.28)	无角(100)	T(36.70)
	29	TA(30.85)	无/有角(89.66/10.34)	A(63.30)
	45	AA(47.87)	有角(100)
29521377C>A	20	CC(21.28)	无角(100)	G(36.70)
	29	CA(30.85)	无/有角(89.66/10.34)	A(63.30)
	45	AA(47.87)	有角(100)

突变位点 Mutation site	个体数 Number	基因型 Genotype	理论个体数 Theoretical number	卡方值 Chi-square value	P值 P value	多态信息含量PIC	观测杂合度Ho	期望杂合度He	有效等位基因数Ne
29508704G>A	20	GG	12.66	10.61	0.00	0.36	0.31	0.46	1.87
	29	GA	43.68
	45	AA	37.66
29509428A>C	20	AA	12.66	10.61	0.00	0.36	0.31	0.46	1.87
	29	AC	43.68
	45	CC	37.66
29509766G>A	20	GG	12.66	10.61	0.00	0.36	0.31	0.46	1.87
	29	GA	43.68
	45	AA	37.66
29512170G>A	20	GG	12.66	10.61	0.00	0.36	0.31	0.46	1.87
	29	GA	43.68
	45	AA	37.66
29512176G>A	20	GG	12.66	10.61	0.00	0.36	0.31	0.46	1.87
	29	GA	43.68
	45	AA	37.66
29514968T>A	20	TT	12.66	10.61	0.00	0.36	0.31	0.46	1.87
	29	TA	43.68
	45	AA	37.66
29521377C>A	20	CC	12.66	10.61	0.00	0.36	0.31	0.46	1.87
	29	CA	43.68
	45	AA	37.66

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欧拉羊角性状相关RXFP2基因SNPs检测及分析

Detection and Analysis of RXFP2 Gene SNPs Related to Horn Trait of Oula Sheep

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