鸡Wnt3a的SNPs筛选及其与皮肤毛囊密度性状关联分析

doi:10.3864/j.issn.0578-1752.2022.23.016

摘要/Abstract

摘要： 目的 Wnt信号通路在动物皮肤毛囊的发生发育中具有重要作用, 前期研究结果表明Wnt3a可能是影响鸡皮肤毛囊密度性状的重要候选基因。为进一步验证Wnt3a在皮肤毛囊生长发育中的作用, 研究以金陵花鸡为研究对象, 筛选Wnt3a SNPs, 并分析其与毛囊密度性状的相关性, 以期找到对皮肤毛囊密度有显著影响的分子标记, 为培育屠体美观的屠宰型肉鸡的“分子编写育种”提供参考。方法采用PCR扩增直接测序法对Wnt3a 的SNPs位点进行筛查, 分析单个SNP标记与皮肤毛囊密度性状的相关性。采用Haploview软件分析这些SNP位点的连锁不平衡（LD）程度, 并分析不同单倍型组合与毛囊密度性状的相关性。结果共发现14个SNP位点, 在第2外显子发现1个SNP位点（SNP1）, 为同义突变；第2内含子发现4个突变位点（ SNP2—SNP5）, 在第3内含子发现9个SNP位点（SNP6—SNP14）。卡方检验表明, 第2外显子的1个突变位点（SNP1）和第2内含子的3个突变位点（SNP3—SNP5）的3个位点均处于哈代-温伯格平衡状态（P>0.05）, 第3内含子的9个SNPs（SNP6-SNP14）偏离哈代-温伯格平衡（P<0.05）。SNP1—SNP5的He均小于0.50, PIC均小于0.25, 这5个SNP位点遗传多样性较低。第3内含子的9个突变位点, SNP6、SNP7、SNP9位点PIC<0.25, 其他6个突变位点 0.25< PIC<0.5, 为中度多态。单标记关联分析表明, 公、母鸡SNP2位点的AG基因型的毛囊密度显著高于GG基因型（P< 0.05）；母鸡SNP8位点的AA与GG基因型的毛囊密度显著高于AG基因型（P < 0.05）, 在公鸡中3种基因型的毛囊密度差异不显著。14个SNPs连锁不平衡分析表明, SNP3、SNP4和SNP5的强连锁产生了2种单倍型, H1（GAT）频率为0.882和H2（TCC）频率为0.118；SNP6—SNP13之间的强连锁产生了5种单倍型, 其中H1（ACATTATC）和H2（ACGTCCCA）, H3（ACGTTCCA）, H4（GTGCTATA）, H5（ACGTCCCC）, 单倍型频率分别为0.469、0.275、0.123、0.113、0.014。SNP3、SNP4和SNP5连锁产生的2 种单倍型组合后产生了3种单倍型组合, 关联分析发现在公、母鸡中3种单倍型组合的毛囊密度均差异不显著；将SNP6—SNP13连锁产生的5种主要单倍型组合后, 公鸡有7种组合单倍型组合, 毛孔数量差异不显著；母鸡有8种主要单倍型组合, 其中H1H1（AACCAATTTTAATTCC）毛囊密度最大。SNP2和SNP8位点与皮肤毛囊密度显著相关, 单倍型组合H1H1（AGAA）、H1H2（AGAG）为母鸡优势单倍型。结论筛选到Wnt3a的14个SNPs位点, 其中, SNP2（rs2587721 G >A）和SNP8（rs2555967G>A）位点与毛囊密度显著相关, 8个SNPs（SNP6—SNP13）位点处于强连锁不平衡, 母鸡H1H1单倍型毛囊密度最大。Wnt3a的SNP2和SNP8位点的单倍型组合H1H1（AGAA）、H1H2（AGAG）和SNP6—SNP13连锁产生的H1H1（AACCAATTTTAATTCC）单倍型组合与母鸡毛囊密度显著相关, 可以为鸡毛囊密度“分子编写育种”提供重要遗传信息。

关键词: 鸡, Wnt3a, 皮肤毛囊, SNP位点, 连锁不平衡

Abstract:

【Objective】 The Wnt signaling pathway plays an important role in the development of animal skin feather follicles. The results of previous studies indicated that the Wnt3a might be an important candidate gene that had effects on the chicken feather follicle density. In order to further verify the role of Wnt3a in the growth and development of skin feather follicle density, Wnt3a SNPs were screened and their association with feather follicle density would be analyzed in Jinling Hua chicken. The study could provide a reference for “breeding by molecular writing” of slaughter-type broilers with beautiful carcasses.【Method】 The SNPs of Wnt3a gene were screened by PCR amplification and direct sequencing, and the correlation between a single SNP marker and skin feather follicle density traits was analyzed. Haploview software was used to analyze the degree of linkage disequilibrium (LD) of these SNP loci, and the correlation between different haplotype combinations and feather follicle density traits was also analyzed..【Result】 A total of 14 SNP sites were found, and one SNP site (SNP1) was found in the second exon, which was a synonymous mutation. Four mutation sites (SNP2-SNP5) were found in the second intron, and 9 SNP sites (SNP6-SNP14) were found in the third intron. The chi-square test showed that one mutation site (SNP1) in the second exon and three mutation sites (SNP3-SNP5) in the second intron were all in the Hardy-Weinberg equilibrium (P>0.05), 9 mutation sites (SNP6-SNP14) of the third intron deviated from Hardy-Weinberg equilibrium (P<0.05). The expected heterozygosity (He) of SNP1-SNP5 was less than 0.50, and the polymorphic information content (PIC) was less than 0.25. The genetic polymorphisms of these 5 SNP loci were low. The third intron had 9 mutation sites, PIC of SNP6, SNP7, SNP9 sites was less than 0.25, and the other 6 mutation sites were 0.25<PIC<0.5, which were moderately polymorphic. Single-marker association analysis showed that the number of skin feather follicle with SNP2 locus of AG genotype in males and females was significantly higher than that of GG genotype (P<0.05). The number of skin feather follicles with SNP8 locus of AA and GG genotypes in females was significantly higher than that of the AG genotype (P<0.05). The skin feather follicles density in the three genotypes in males was not significantly different. The linkage disequilibrium analysis of 14 SNPs showed that SNP6-SNP13 and SNP3-SNP5 had a strong linkage disequilibrium, respectively. SNP3, SNP4, and SNP5 produced three haplotype combinations after the combination of the two haplotypes linked by SNP3- SNP5. Association analysis found that the skin feather follicle density of the three haplotype combinations in males and females were not significantly different. After the combination of 5 main haplotypes at SNP6-SNP13 locus, the males had 7 haplotype combinations, and the skin feather follicles density was not significant. Females had 8 haplotype combinations, of which H1H1 (AACCAATTTTAATTCC) had the highest skin feather follicles density. SNP2 and SNP8 were significantly correlated with skin feather follicle density, and the haplotype combination H1H1(AGAA) and H1H2 (AGAG) were the dominant haplotypes in hens.【Conclusion】 Fourteen SNPs of Wnt3a were screened. Among them, individuals with different genotypes at rs2587721 G>A (SNP2) and rs2555967G>A (SNP8) locus had significant differences in feather follicle density. Eight SNPs (SNP6-SNP13) loci were in strong linkage disequilibrium, and the combination of H1H1 had the highest feather follicles density in females. The haplotype combination of SNP2 and SNP8 of Wnt3a H1H (AGAA), H1H2 (AGAG) and SNP6-SNP13 linked to produce the H1H1 (AACCAATTTTAATTCC) haplotype combination were significantly correlated with feather follicle density in females, which provided important genetic information for “breeding by molecular writing” on chicken skin feather follicle density.

Key words: chicken, Wnt3a, skin feather follicle, SNP sites, linkage disequilibrium

屠云洁,姬改革,章明,刘一帆,巨晓军,单艳菊,邹剑敏,李华,陈智武,束婧婷. 鸡Wnt3a的SNPs筛选及其与皮肤毛囊密度性状关联分析[J]. 中国农业科学, 2022, 55(23): 4769-4780.

TU YunJie,JI GaiGe,ZHANG Ming,LIU YiFan,JU XiaoJun,SHAN YanJu,ZOU JianMin,LI Hua,CHEN ZhiWu,SHU JingTing. Screening of Wnt3a SNPs and Its Association Analysis with Skin Feather Follicle Density Traits in Chicken[J]. Scientia Agricultura Sinica, 2022, 55(23): 4769-4780.

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https://www.chinaagrisci.com/CN/Y2022/V55/I23/4769

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性别 Sex	数量（只） Number (individuals)	4 cm²中毛囊个数 Number of feather follicle in 4 cm²
公Male	162	14.25b
母 Female	220	15.71a

SNP位点 SNP locus	基因型 Gene type	基因型频率 Frequency of gene type	等位基因 Allele	等位基因频率 Frequency of alle	期望杂合度（He） Expected heterozygosity	观察杂合度（Ho） Observe heterozygosity	多态信息含量（PIC） Polymorphic information content	哈温平衡 (P值) HWE (P-value)
SNP1 rs2587569	GG	0.80	G	0.90	0.19	0.19	0.17	1.0
	AA	0.01	A	0.10
	AG	0.19
SNP2 rs2587721	GG	0.79	G	0.89	0.19	0.21	0.17	0.0173
SNP2 rs2587721	AG	0.21	A	0.11	0.19	0.21	0.17	0.0173
SNP3 rs2587832	GG	0.78	G	0.88	0.21	0.20	0.19	0.5168
	TT	0.02	T	0.12
	TG	0.20
SNP4 rs2587845	AA	0.78	A	0.88	0.21	0.20	0.19	0.5168
	CC	0.02	C	0.12
	CA	0.20
SNP5 rs2587849	TT	0.78	T	0.88	0.21	0.20	0.19	0.5168
	CC	0.02	C	0.12
	CT	0.20
SNP6 rs2555891	GG	0.07	G	0.11	0.20	0.09	0.18	1.9837E-16
	AA	0.84	A	0.89
	GA	0.09
SNP7 rs2555961	TT	0.07	T	0.11	0.20	0.09	0.18	1.9837E-16
	CC	0.84	C	0.89
	TC	0.09
SNP8 rs2555967	GG	0.42	G	0.53	0.50	0.23	0.37	8.9336E-28
	AA	0.36	A	0.47
	AG	0.23
SNP9 rs2556041	CC	0.07	C	0.11	0.20	0.09	0.18	1.9837E-16
	TT	0.84	T	0.89
	CT	0.09
SNP10 rs2556078	TT	0.62	C	0.29	0.41	0.18	0.33	4.1697E-26
	CC	0.20	T	0.71
	CT	0.18
SNP11 rs2556088	AA	0.49	A	0.59	0.49	0.19	0.37	1.3379E-33
	CC	0.32	C	0.41
	AC	0.19
ASNP12 rs2556093	TT	0.49	C	0.41	0.49	0.19	0.37	1.3379E-33
	CC	0.32	T	0.59
	TC	0.19
SNP13 rs2556109	AA	0.42	A	0.52	0.50	0.20	0.37	3.0303E-34
	CC	0.38	C	0.48
	AC	0.20
SNP14 rs2556178	GG	0.19	G	0.24	0.37	0.11	0.30	8.4255E-39
	TT	0.70	T	0.76
	TG	0.11

性别 Sex	单倍型组合 Haplotype combination	基因型 Gene type	毛囊密度 Skin feather follicle density (individual/4 cm²)	数量 Number
公 Male	H1H1	GGAATT	14.23±0.20	122
	H1H2	TGCACT	14.37±0.38	35
	H2H2	TTCCCC	14.00±1.00	5
母 Female	H1H1	GGAATT	15.69±0.29	177
母 Female	H1H2	TGCACT	15.81±0.60	41

性别 Sex	单倍型组合 Haplotype combination	基因型 Gene type	毛囊密度（个/4cm²） Skin feather follicle density (individual/4 cm²)	数量 Number
公 Male	H1H1	AACCAATTTTAATTCC	14.20±0.30	55
	H1H2	AACCAGTTCTCACTCA	14.50±0.47	22
	H2H2	AACCGGTTCCCCCCAA	14.28±0.37	36
	H3H3	AACCGGTTTTCCCCAA	14.47±0.51	19
	H1H4	AGCTAGCTTTAATTCA	14.00±0.74	6
	H3H4	AGCTGGCTCTCACTAA	13.17±0.91	5
	H4H4	GGTTGGCCTTAATTAA	14.00±0.74	9
母Female	H1H1	AACCAATTTTAATTCC	16.57±0.42a	81
	H1H2	AACCAGTTCTCACTCA	15.83±0.77ab	24
	H1H5	AACCAGTTCTCACTCC	12.45±1.14c	11
	H2H2	AACCGGTTCCCCCCAA	15.08±0.60b	40
	H3H3	AACCGGTTTTCCCCAA	16.25±0.77ab	24
	H1H4	AGCTAGCTTTAATTCA	14.71±0.91bc	17
	H3H4	AGCTGGCTCTCACTAA	15.75±1.88abc	4
	H4H4	GGTTGGCCTTAATTAA	14.81±0.94bc	16

性别 Sex	SNP2，SNP8单倍型组合 Haplotype combination of SNP2 and SNP8 locus	基因型 Gene type	毛囊密度（个/4 cm²） Skin feather follicle density (individual/4 cm²)	数量 Number
公 Male	H1H1	AGAA	14.40±0.70	10
	H1H2	AGAG	15.00±0.78	8
	H1H3	AGGG	15.14±0.47	22
	H2H1	GGAA	14.16±0.33	45
	H2H2	GGAG	14.08±0.44	25
	H2H3	GGGG	13.90±0.31	52
母 Female	H1H1	AGAA	18.57± 0.78a	21
	H1H2	AGAG	19.00±1.27a	8
	H1H3	AGGG	17.83± 1.04ab	12
	H2H1	GGAA	15.87± 0.46b	60
	H2H2	GGAG	14.04± 0.53c	45
	H2H3	GGGG	15.10± 0.42bc	74