四川凉山州3个黑绵羊群体<i>Prdm9</i>基因锌指序列的比较研究

doi:10.7668/hbnxb.20193737

摘要/Abstract

摘要：

旨在通过测定Prdm9基因锌指域序列,探索四川3个黑绵羊群体间的亲缘关系。采集黑绵羊群体抗凝全血,提取基因组DNA,用PCR特异扩增Prdm9基因锌指域,进一步通过克隆测序以分析遗传多样性。结果显示,黑绵羊中鉴定出存在9种锌指,锌指间存在1~5个氨基酸差异,共构成5种Prdm9锌指域,分别定义为不同的等位基因(A、B、C、D和E),其中A~D包含8个锌指,这是试验黑绵羊Prdm9锌指域的主要类型,E包含9个锌指且仅发现于盐源黑绵羊中,其等位基因频率仅为0.050。这些等位基因形成6种基因型,包括AA、AC、BB、BD、CC和EE,在布拖黑绵羊和普格黑绵羊中均检测到4种基因型,而盐源黑绵羊中有6种,且BB和EE仅在该群体中观察到。Prdm9的等位基因频率和PIC值在3个黑绵羊群体中存在不同程度差异,有较丰富的遗传多态性,其中盐源黑绵羊相对最丰富。3个黑绵羊群体Prdm9锌指域的等位基因频率分布均极显著偏离哈代-温伯格平衡 (P<0.01),提示它们在育种中受到较大的人工选择影响。综上所述,黑绵羊群体中Prdm9有较丰富的遗传多样性,3个群体间亲缘关系均较近。

关键词: 黑绵羊, Prdm9, 锌指, 克隆测序, 亲缘关系

Abstract:

It aimed to explore the kinship between three black sheep populations in Sichuan by determining the sequence of the zinc finger domain of Prdm9.Anticoagulated whole blood was collected from the black sheep population,genomic DNA was extracted,and the zinc finger domain of the Prdm9 was specifically amplified by PCR,and further sequenced by cloning to analyze genetic diversity.The results showed that nine types of zinc fingers were identified in black sheep,with 1—5 amino acid differences between zinc fingers,constituting five Prdm9 zinc finger domains,defined as different alleles(A,B,C,D and E),of which A to D contained eight zinc fingers,which were the main types of Prdm9 zinc finger domains in the tested black sheep,and E contained nine zinc fingers and was only found in Yanyuan black sheep,with an allele frequency of 0.050.These alleles formed six genotypes,including AA,AC,BB,BD,CC and EE,and four genotypes were detected in both Butuo and Puge black sheep,while six were found in Yanyuan black sheep,and BB and EE were only observed in this population.The Prdm9 allele frequencies and PIC values varied among the three black sheep populations,with richer genetic polymorphisms,of which the Yanyuan black sheep were relatively the most abundant.The allele frequency distribution of the Prdm9 zinc finger domain in all three black sheep populations deviated significantly from Hardy-Weinberg equilibrium(P<0.01),suggesting that they were influenced by artificial selection in breeding.In conclusion,Prdm9 has rich genetic diversity in the black sheep population,and all three populations are closely related to each other.

Key words: Black sheep, Prdm9, Zinc finger, Clonal sequencing, Relationship

张芬芬, 黄卫平, 黄林, 金素钰, 郑玉才. 四川凉山州3个黑绵羊群体Prdm9基因锌指序列的比较研究[J]. 华北农学报, 2023, 38(S1): 408-412. doi: 10.7668/hbnxb.20193737.

ZHANG Fenfen, HUANG Weiping, HUANG Lin, JIN Suyu, ZHENG Yucai. Comparative Study of the Zinc Finger Sequences of Prdm9 Gene in Three Black Sheep Populations in Liangshan Prefecture,Sichuan[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(S1): 408-412. doi: 10.7668/hbnxb.20193737.

http://www.hbnxb.net/CN/Y2023/V38/IS1/408

图/表 7

图1 黑绵羊Prdm9基因片段PCR扩增产物的琼脂糖凝胶电泳 M.DNA Marker; 1—6.6只黑绵羊的PCR产物。

Fig.1 Agarose gel electrophoresis of PCR amplification products of Prdm9 gene fragments in black sheep M.DNA Marker; 1—6.PCR products from 6 black sheep.

图2 黑绵羊中检测到的9种Prdm9锌指 1—9.不同的Prdm9锌指,每个锌指含28个氨基酸;5个氨基酸变异位点在锌指序列的-9、-5、-1、+3、+6处,其中★为正向选择位点。

Fig.2 Nine Prdm9 zinc fingers detected in black sheep 1—9.Different Prdm9 zinc fingers,each containing 28 amino acids; five amino acid variant sites are at -9,-5,-1,+3,+6 of the zinc finger sequence,where ★ is a positive selection site.

图3 黑绵羊和普通绵羊的Prdm9锌指域结构示意图 ZF1~ZF9为Prdm9基因中的锌指顺序。图中包含了8~9个锌指的共5种锌指域结构,其中ZFD为普通绵羊Prdm9锌指域结构(GenBank No.KX109932.1),ZFD1~ZFD5为本研究黑绵羊Prdm9锌指域结构,各锌指的序列和编号见图2。

Fig.3 Schematic structure of the Prdm9 zinc finger domain in black and common sheep ZF1—ZF9 are the zinc finger sequences in Prdm9 gene.A total of five zinc finger domain structures containing eight to nine zinc fingers are shown in the figure,among which ZFD is the common sheep Prdm9 zinc finger domain structure(GenBank No.KX109932.1)and ZFD1—ZFD5 are the Prdm9 zinc finger domain structures of this study black sheep,the sequence and number of each zinc finger are shown in Fig.2.

表1 3个黑绵羊群体Prdm9基因的等位基因频率

Tab.1 Allele frequencies of the Prdm9 gene in three black sheep populations

等位基因 Alleles	锌指数量 Number	布拖黑绵羊 Butuo black sheep (n=20)	普格黑绵羊 Puge black sheep (n=20)	盐源黑绵羊 Yanyuan black sheep(n=20)	平均值 Average (n=60)
A	8	0.375 0	0.300 0	0.275 0	0.317 0
B	8	0.050 0	0.075 0	0.250 0	0.125 0
C	8	0.525 0	0.550 0	0.375 0	0.483 0
D	8	0.050 0	0.075 0	0.050 0	0.058 0
E	9	0.000 0	0.000 0	0.050 0	0.017 0

表2 3个黑绵羊群体Prdm9基因的基因型频率

Tab.2 Genotype frequencies of the Prdm9 gene in three black sheep populations

基因型 Genotype	布拖黑绵羊 Butuo black sheep (n=20)	普格黑绵羊 Puge black sheep (n=20)	盐源黑绵羊 Yanyuan black sheep(n=20)	平均值 Average (n=60)
AA	0.100 0	0.100 0	0.150 0	0.117 0
AC	0.550 0	0.400 0	0.250 0	0.400 0
BB	0.000 0	0.000 0	0.200 0	0.067 0
BD	0.100 0	0.150 0	0.100 0	0.117 0
CC	0.250 0	0.350 0	0.250 0	0.283 0
EE	0.000 0	0.000 0	0.050 0	0.016 0

表3 3个黑绵羊群体Prdm9基因的遗传变异

Tab.3 Genetic variation of the Prdm9 gene in three black sheep populations

参数 Parameter	布拖黑绵羊 Butuo black sheep	普格黑绵羊 Puge black sheep	盐源黑绵羊 Yanyuan black sheep
等位基因数Number of alleles	4	4	5
基因型数Number of genotypes	4	4	6
有效等位基因数Effective number of alleles	2.374	2.477	3.524
观察杂合度Observed heterozygosity	0.650 0	0.550 0	0.350 0
期望杂合度Expected heterozygosity	0.579 0	0.596 0	0.716 0
多态信息含量Polymorphism information contents	0.497 0	0.533 0	0.665 0

图4 3个黑绵羊群体的N-J进化树

Fig.4 N-J evolutionary trees of three black sheep populations

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四川凉山州3个黑绵羊群体Prdm9基因锌指序列的比较研究

Comparative Study of the Zinc Finger Sequences of Prdm9 Gene in Three Black Sheep Populations in Liangshan Prefecture,Sichuan

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四川凉山州3个黑绵羊群体Prdm9基因锌指序列的比较研究

Comparative Study of the Zinc Finger Sequences of Prdm9 Gene in Three Black Sheep Populations in Liangshan Prefecture,Sichuan

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