绵羊SMAD1基因组织表达及其多态性与产羔数关联分析

doi:10.3864/j.issn.0578-1752.2019.04.015

中国农业科学 ›› 2019, Vol. 52 ›› Issue (4): 755-766.doi: 10.3864/j.issn.0578-1752.2019.04.015

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

绵羊SMAD1基因组织表达及其多态性与产羔数关联分析

田志龙^1,²,汤继顺¹,孙庆¹,王玉琴²(),张效生³,张金龙³,储明星¹()

¹中国农业科学院北京畜牧兽医研究所/农业部动物遗传育种与繁殖重点实验室,北京 100193
²河南科技大学动物科技学院,河南洛阳 471003
³天津市畜牧兽医研究所,天津 300381

收稿日期:2018-09-03 接受日期:2018-12-29 出版日期:2019-02-16 发布日期:2019-02-27
通讯作者: 王玉琴,储明星
作者简介:田志龙,E-mail： tianzl0515@163.com。
基金资助:
国家自然科学基金(31772580);国家肉羊产业技术体系专项(CARS-38);中国农业科学院科技创新工程(ASTIP-IAS13)

Tissue Expression and Polymorphism of Sheep SMAD1 Gene and Their Association with Litter Size

TIAN ZhiLong^1,²,TANG JiShun¹,SUN Qing¹,WANG YuQin²(),ZHANG XiaoSheng³,ZHANG JinLong³,CHU MingXing¹()

¹Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193
²College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, Henan
³Tianjin Institute of Animal Sciences, Tianjin 300381

Received:2018-09-03 Accepted:2018-12-29 Online:2019-02-16 Published:2019-02-27
Contact: YuQin WANG,MingXing CHU

摘要/Abstract

摘要：

【目的】绵羊产羔数是一个极其复杂的性状,受诸多因素影响。绵羊排卵数是最重要的因素之一,而SMAD蛋白在哺乳动物卵泡发育和颗粒细胞生长分化过程中发挥着重要作用。因此本文基于前期绵羊基因组重测序数据,深入研究SMAD家族成员1 (SMAD family member 1, SMAD1)基因在不同繁殖力小尾寒羊组织表达模式,并探讨其多态性与绵羊产羔数的关系,以揭示其影响产羔数的机制,为绵羊分子育种提供科学依据。【方法】首先,采用反转录PCR技术检测SMAD1基因在不同繁殖力小尾寒羊群体大脑,小脑,下丘脑,垂体,子宫,卵巢,输卵管,心脏,肝脏,脾脏,肺脏,肾脏,肾上腺,甲状腺,大肠,小肠,胰腺,瘤胃,肾脂 19种组织的表达模式,然后利用实时荧光定量PCR技术检测SMAD1基因在不同繁殖力小尾寒羊下丘脑,垂体,子宫,卵巢,输卵管5种繁殖组织的相对表达量。随后采用Sequenom MassARRAY ^?SNP技术对SMAD1基因5个单核苷酸多态性(single nucleotide polymorphism, SNP)位点在小尾寒羊（n=380）、湖羊（n=101）、苏尼特羊（n=21）、草原型藏羊（n=161）、策勒黑羊（n=52）、滩羊（n=22）6个绵羊品种中进行基因型检测,计算其群体遗传学指标,利用SPSS19.0软件分析5个SNPs与小尾寒羊产羔数的相关性。【结果】PCR结果显示,SMAD1基因在不同繁殖力小尾寒羊全身性组织均有表达,在小尾寒羊单羔群体卵巢的表达量极显著高于多羔群体（P<0.01）,小尾寒羊单羔群体下丘脑、垂体的表达量显著高于多羔群体（P<0.05）;基因分型结果表明,g.12485895A>G,g.12487558G>A和g.12487190G>T位点在单、多羔绵羊品种中基因型和等位基因频率均差异显著（P<0.05）;而g.12508874T>C和g.12487467A>G位点在单、多羔绵羊品种中基因型和等位基因频率均差异不显著（P>0.05）;群体遗传学分析表明,g.12485895A>G、g.12487558G>A、g.12508874T>C、g.12487467A>G、g.12487190G>T位点在小尾寒羊、湖羊、策勒黑羊、苏尼特羊、草原型藏羊中均为中度多态（0.25<PIC<0.5）,g.12508874T>C位点在滩羊群体表现为低度多态（PIC<0.25）;卡方适合性检验表明,g.12485895A>G和g.12487467A>G位点在小尾寒羊和草原型藏羊中处在Hardy-Weinberg不平衡状态（P<0.05）,g.12487558G>A在小尾寒羊和湖羊群体处于Hardy-Weinberg不平衡状态（P<0.05）,g.12487190G>T位点在草原型藏羊处于Hardy-Weinberg不平衡状态（P<0.05）。其他位点在6个绵羊品种中均处在Hardy-Weinberg平衡状态（P>0.05）;关联分析表明,g.12485895A>G,g.12487558G>A,g.12508874T>C和g.12487467A>G位点与小尾寒羊产羔数无显著关联。g.12487190G>T位点TT基因型母羊前三胎的产羔数均显著高于GT和GG基因型母羊（P<0.05）。将该位点与小尾寒羊FecB（A746G）不同基因型进行组合后发现,AA-GG、AA-GT、AA-TT型母羊产羔数显著低于其他组合基因型母羊（P<0.05）。【结论】SMAD1与小尾寒羊繁殖密切相关,g.12487190G>T可作为小尾寒羊产羔性状选育的潜在分子标记。

关键词: 绵羊, SMAD1基因, 组织表达, SNP分型, 产羔数

Abstract:

【Objective】Sheep litter size is a very complex trait influenced by many factors. Ovulation is one of the most important factors. SMAD protein plays an important role in the mammalian follicular development as well as growth and differentiation process of granulosa cell. To reveal the relationship between the SMAD family member 1 (SMAD1) gene and the litter size of sheep and its mechanism affecting litter size, meanwhile to provide a scientific basis for sheep molecular breeding, we researched the expression pattern of SMAD1 in Small Tail Han sheep with different fertility and analyzed the relationship between polymorphism and the litter size in sheep, based on the previous sheep genome resequencing data. 【Method】First, reverse transcription PCR was used to detect the expression of SMAD1 gene in Small Tail Han sheep (brain, cerebellum, hypothalamus, pituitary, uterus, ovary, oviduct, heart, liver, spleen, lung, kidney, adrenal glands, thyroid, large intestine, small intestine, pancreas, rumen, and kidney fat) tissues with different lambing abilities. Then, Real-time PCR was performed to investigate the expression of SMAD1 gene in Small Tail Han sheep reproduction tissues (hypothalamus, pituitary, uterus, ovary, and oviduct) with different lambing abilities. Multiparous (Small Tail Han sheep (n=380), Hu sheep (n=101), Cele black sheep (n=52)) and uniparous (Sunite (n=21), Tan sheep (n=22), Prairie Tibetan sheep (n=161)) sheep breeds were selected and Sequenom MassARRAY ^?SNP assay was applied to genotype five single nucleotide polymorphism sites (SNPs) of SMAD1 gene. Then the association was analyzed between SMAD1 and litter size in Small Tail Han sheep by using SPSS 19.0. 【Result】The results of RT-PCR revealed that SMAD1 gene expressed in 19 tissues. The qPCR showed that the expression of SMAD1 was higher in ovary (P<0.01), hypothalamus (P<0.05) and pituitary (P<0.05) of uniparous Small Tail Han sheep than that in multiparous sheep. From genotyping, this study found that the genotype frequencies and allele frequencies of the g.12485895A>G, g.12487558G>A and g.12487190G>T of SMAD1 gene were significantly different between uniparous and multiparous sheep (P<0.05). However, g.12508874T>C and g.12487467A>G had no significantly different between uniparous and multiparous sheep (P>0.05). Population genetic analysis indicated that g.12485895A>G, g.12487558G>A, g.12508874T>C, g.12487467A>G, and g.12487190G>T loci were at moderate polymorphism (0.25<PIC<0.5) in Small Tail Han sheep, Hu sheep, Prairie Tibetan sheep, Cele black sheep, Sunite and Tan sheep, while g.12508874T>C was at low polymorphism (PIC<0.25) in Tan sheep. The χ ² test indicated that the g.12485895A>G and g.12487467 A>G were not under Hardy-Weinberg equilibrium (P<0.05) in Small Tail Han sheep and Prairie Tibetan sheep. g.12487558 G>A was not under Hardy-Weinberg equilibrium (P<0.05) in Small Tail Han sheep and Hu sheep. g.12487190 G>T was not under Hardy-Weinberg equilibrium (P<0.05) in Prairie Tibetan sheep. Other loci were under Hardy-Weinberg equilibrium (P>0.05) in six sheep breeds. Association analysis indicated that the polymorphism of g.12487190 G>T had significant correlation with the litter size in Small Tail Han sheep, while the litter size of TT was higher than GT and GG in the first three births (P<0.05). However, the polymorphism of g.12485895A>G, g.12487558G>A, g.12508874T>C and g.12487467A>G had no significant correlation with the litter size in Small Tail Han sheep. The combination of SMAD1 gene g.12487190G>T and FecB (A746G) gene showed that the number of lambs in AA-GG, AA-GT, AA-TT genotypes was significantly low than that of other genotypes (P<0.05), respectively. 【Conclusion】Therefore, it could be concluded that SMAD1 might be the key gene for litter size and the g.12487190G>T locus might provide a basis for litter size trait selection in sheep.

Key words: sheep, SMAD1 gene, tissue expression, SNP genotyping, litter size

田志龙,汤继顺,孙庆,王玉琴,张效生,张金龙,储明星. 绵羊SMAD1基因组织表达及其多态性与产羔数关联分析[J]. 中国农业科学, 2019, 52(4): 755-766.

TIAN ZhiLong,TANG JiShun,SUN Qing,WANG YuQin,ZHANG XiaoSheng,ZHANG JinLong,CHU MingXing. Tissue Expression and Polymorphism of Sheep SMAD1 Gene and Their Association with Litter Size[J]. Scientia Agricultura Sinica, 2019, 52(4): 755-766.

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图/表 9

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SMAD1基因不同位点在不同绵羊品种中的群体遗传学分析"

位点 Locus	品种 Breed	基因型频率 Genotype frequency			基因频率 Allele frequency		多态信息含量（PIC） Polymorphism information content	杂合度（HE） Heterozygosity (HE)	有效等位基因数 Effective number of allele (NE)	卡方值（P） χ² test (P value)
g.12485895A>G		AA	AG	GG	A	G
	小尾寒羊 Small Tail Han sheep	0.33	0.44	0.23	0.55	0.45	0.37	0.49	1.98	0.02
	湖羊 Hu sheep	0.35	0.54	0.11	0.62	0.38	0.36	0.47	1.89	0.12
	草原型藏羊 Prairie Tibetan sheep	0.58	0.32	0.10	0.73	0.27	0.31	0.39	1.64	0.02
	策勒黑羊 Cele black sheep	0.06	0.50	0.44	0.31	0.69	0.34	0.43	1.74	0.21
	苏尼特羊 Sunite sheep	0.29	0.48	0.23	0.52	0.48	0.37	0.50	2.00	0.83
	滩羊 Tan sheep	0.27	0.41	0.32	0.48	0.52	0.37	0.50	2.00	0.40
g.12487558G>A		AA	GA	GG	A	G
	小尾寒羊 Small Tail Han sheep	0.25	0.43	0.32	0.47	0.53	0.37	0.50	1.99	0.01
	湖羊 Hu sheep	0.18	0.34	0.48	0.36	0.64	0.35	0.46	1.85	0.00
	草原型藏羊 Prairie Tibetan sheep	0.10	0.40	0.50	0.30	0.70	0.33	0.42	1.73	0.60
	策勒黑羊 Cele black sheep	0.35	0.52	0.13	0.61	0.39	0.36	0.48	1.91	0.53
位点 Locus	品种 Breed	基因型频率 Genotype frequency			基因频率 Allele frequency		多态信息含量（PIC） Polymorphism information content	杂合度（HE） Heterozygosity (HE)	有效等位基因数 Effective number of allele (NE)	卡方值（P） χ² test (P value)
	苏尼特羊 Sunite sheep	0.33	0.29	0.38	0.48	0.52	0.37	0.50	2.00	0.05
	滩羊 Tan sheep	0.41	0.45	0.14	0.64	0.36	0.36	0.46	1.86	0.93
g.12508874T>C		CC	CT	TT	C	T
	小尾寒羊 Small Tail Han sheep	0.61	0.34	0.05	0.78	0.22	0.29	0.35	1.53	0.64
	湖羊 Hu sheep	0.34	0.50	0.16	0.59	0.41	0.37	0.48	1.94	0.67
	草原型藏羊 Prairie Tibetan sheep	0.56	0.38	0.06	0.75	0.25	0.31	0.38	1.60	0.94
	策勒黑羊 Cele black sheep	0.58	0.38	0.04	0.77	0.23	0.29	0.36	1.55	0.55
	苏尼特羊 Sunite sheep	0.57	0.38	0.05	0.76	0.24	0.30	0.36	1.57	0.82
	滩羊 Tan sheep	0.73	0.27	0.00	0.86	0.14	0.21	0.24	1.31	0.46
g.12487467A>G		AA	GA	GG	A	G
	小尾寒羊 Small Tail Han sheep	0.54	0.30	0.16	0.69	0.31	0.34	0.43	1.75	0.00
	湖羊 Hu sheep	0.62	0.33	0.05	0.78	0.22	0.28	0.34	1.52	0.84
	草原型藏羊 Prairie Tibetan sheep	0.65	0.25	0.10	0.78	0.22	0.28	0.34	1.52	0.00
	策勒黑羊 Cele black sheep	0.22	0.55	0.23	0.49	0.51	0.37	0.50	2.00	0.46
	苏尼特羊Sunite sheep	0.43	0.38	0.19	0.62	0.38	0.36	0.47	1.88	0.42
	滩羊 Tan sheep	0.29	0.38	0.33	0.48	0.52	0.37	0.50	2.00	0.28
g.12487190G>T		GG	GT	TT	G	T
	小尾寒羊 Small Tail Han sheep	0.45	0.41	0.14	0.65	0.35	0.35	0.45	1.83	0.08
	湖羊 Hu sheep	0.61	0.34	0.05	0.78	0.22	0.28	0.34	1.52	0.90
	草原型藏羊 Prairie Tibetan sheep	0.62	0.29	0.09	0.77	0.23	0.29	0.35	1.55	0.00
	策勒黑羊 Cele black sheep	0.19	0.58	0.23	0.48	0.52	0.37	0.50	2.00	0.26
	苏尼特羊 Sunite sheep	0.38	0.48	0.14	0.62	0.38	0.36	0.47	1.89	0.96
	滩羊 Tan sheep	0.27	0.41	0.32	0.48	0.52	0.37	0.50	2.00	0.40

表3

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引物及探针名称 Primer names	引物序列 Primer sequences	退火温度 Annealing temperature (℃)	扩增片段(bp) Product size	用途 Usage
SMAD1	F: TGGTTCCAAGACACAGCGAAT R: GGTGTATCTGCTGGCATCTGA	60	253	半定量RT-PCR semi-quantitative RT-PCR
SMAD1Q	F: CCCGAGTGGGTGTAGTTT R: TCCTGGCGGTGGTATTCT	60	162	荧光定量PCR qPCR
GAPDH	F: TGACGCTCCCATGTTTGTGA R: TCATAAGTCCCTCCACGATGC	60	149	半定量RT-PCR 和荧光定量PCR semi-quantitative RT-PCR and qPCR
FecB-Taqman	F: CCAGCTGGTTCCGAGAGACA R: CTTATACTCACCCAAGATGTTTTCATG	60	73	FecB-Taqman分型 FecB-Taqman genotyping
FecB-Taqman P-G FecB-Taqman P-A	AAATATATCGGACGGTGTT-MGB AAATATATCAGACGGTGTTG-MGB	60	73	FecB-Taqman分型 FecB-Taqman genotyping

位点 Locus	基因型 Genotype	第一胎样本数 Number of the first parity	第一胎产羔数 Litter size of the first parity	第二胎样本数 Number of the second parity	第二胎产羔数 Litter size of the second parity	第三胎样本数 Number of the third parity	第三胎产羔数 Litter size of the third parity
g.12485895A>G	AA	114	2.11±0.08	109	2.33±0.09	45	2.80±0.15
	GG	80	2.14±0.09	74	2.24±0.11	27	2.93±0.20
	GA	144	2.12±0.07	139	2.37±0.08	55	2.70±0.14
g.12487190G>T	GG	157	2.08±0.07a	150	2.28±0.07a	57	2.67±0.14a
	GT	144	2.16±0.07a	138	2.34±0.08a	60	2.72±0.13a
	TT	51	2.39±0.12b	46	2.65±0.13b	16	3.75±0.25b
g.12487467A>G	AA	159	2.11±0.06	152	2.29±0.07	56	2.70±0.13
	GG	48	2.35±0.12	43	2.47±0.14	15	2.80±0.24
	GA	67	2.17±0.09	85	2.31±0.10	36	2.67±0.16
g.12487558G>A	AA	92	2.28±0.09	87	2.43±0.10	35	2.91±0.17
	GG	107	2.06±0.08	99	2.31±0.09	44	2.82±0.15
	GA	142	2.16±0.07	138	2.38±0.08	50	2.64±0.14
g.12508874T>C	CC	204	2.10±0.06	193	2.40±0.07	66	2.82±0.13
	CT	120	2.27±0.08	113	2.35±0.09	54	2.82±0.14
	TT	18	2.11±0.02	19	2.21±0.21	9	2.56±0.35

基因型 Genotype	第一胎样本数 Number of the first parity	第一胎产羔数 Litter size of the first parity	第二胎样本数 Number of the second parity	第二胎产羔数 Litter size of the second parity	第三胎样本数 Number of the third parity	第三胎产羔数 Litter size of the third parity
AA-GG	23	1.00±0.16a	23	1.00±0.18a	23	1.04±0.19a
AA-GT	14	1.36±0.21a	14	1.36±0.22a	14	1.07±0.24a
AA-TT	10	1.10±0.25a	10	1.30±0.27a	10	1.10±0.29a
AG-GG	77	2.04±0.09b	75	2.40±0.10b	27	2.67±0.18b
AG-GT	70	2.07±0.09b	67	2.16±0.10b	34	2.65±0.16b
AG-TT	27	2.14±0.15b	24	2.50±0.17b	10	3.40±0.29b
GG-GG	57	2.33±0.21b	53	2.38±0.12b	22	3.00±0.19b
GG-GT	60	2.40±0.10b	57	2.67±0.11b	18	3.22±0.21b
GG-TT	14	2.29±0.10b	13	2.31±0.23b	6	3.33±0.37b

绵羊SMAD1基因组织表达及其多态性与产羔数关联分析

Tissue Expression and Polymorphism of Sheep SMAD1 Gene and Their Association with Litter Size

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位点 Locus	基因型 Genotype	多羔基因型频率 Genotype frequency in multiparous sheep	单羔基因型频率 Genotype frequency in uniparous sheep	卡方检验 χ² test (P value)	等位基因 Allele	多羔中基因频率 Allele frequency in multiparous sheep	单羔中基因频率 Allele frequency in uniparous sheep	卡方检验 χ² test (P value)
g.12485895A>G	AA	0.30(167)	0.52 (104)	0.00	A	0.54	0.68	0.00
	GA	0.47 (254)	0.34 (70)		G	0.46	0.32
	GG	0.23 (124)	0.14 (29)
g.12487558G>A	AA	0.25 (139)	0.15 (32)	0.00	A	0.48	0.36	0.00
	GA	0.45 (248)	0.40 (81)		G	0.52	0.64
	GG	0.30(161)	0.45 (91)
g.12508874T>C	CC	0.56 (307)	0.58 (118)	0.69	C	0.74	0.76	0.50
	CT	0.37 (203)	0.37 (75)		T	0.26	0.24
	TT	0.07 (39)	0.05(11)
g.12487467A>G	AA	0.51 (249)	0.59 (111)	0.15	A	0.69	0.73	0.11
	GA	0.35 (169)	0.28 (52)		G	0.31	0.27
	GG	0.14 (68)	0.13 (24)
g.12487190G>T	GG	0.44 (250)	0.56 (115)	0.01	G	0.66	0.72	0.01
	GT	0.43 (239)	0.32 (65)		T	0.34	0.28
	TT	0.13 (73)	0.12(24)

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