巴马香猪周身皮肤厚度的测量及其与7号染色体候选SNPs位点的关联分析

【目的】测定中国地方小型猪品种巴马香猪成年时周身9个典型部位的皮肤厚度,揭示巴马香猪不同部位皮肤厚度变化规律,进行9个部位皮肤厚度与候选SNPs位点的关联分析,在巴马香猪群体中验证影响皮肤厚度的7号染色体主效QTL,为进一步在巴马香猪群体中大规模开展皮肤厚度等形态变化的分子遗传控制机理及其相关基因功能研究奠定基础,从而增强人们对猪皮肤的认知。【方法】从一个由319头300日龄巴马香猪组成的成年屠宰群体中,随机选取50头,包括27头母猪和23头阉割公猪,分别取头脸、肩、背、肷、臀、胸、下腹、腋下和管等9个部位的皮肤,利用电子游标卡尺对这些不同部位皮肤厚度进行精确测量,利用R语言基本统计包进行不同部位和不同性别间皮肤厚度的差异分析以及不同部位间皮肤厚度的相关分析。在猪7号染色体34.5-36.2Mb的区域选取46个SNPs位点,利用MassARRAY时间飞行质谱技术进行基因分型,结合上述测定的皮厚表型,利用广义混合线性模型及R语言SNPassoc软件包进行目标候选区域的关联分析。根据关联分析结果和基因的生物学功能确定可能的位置候选基因。【结果】(1)单因素方差分析表明巴马香猪9个部位的皮肤厚度存在极显著差异(P=2.95×10~(-117)),肷部和背部的皮肤最厚,分别为(5.15±0.92)和(4.97±0.85)mm,下腹和腋下的皮肤最薄,分别为(1.77±0.36)和(1.97±0.68)mm。皮肤厚度从厚到薄依次是肷部、背部、肩部、头脸、臀部、管部、胸部、腋下和下腹。(2)阉割公猪腋下皮肤厚度显著小于母猪的(P=0.021),其它部位皮肤厚度在母猪和阉割公猪间差异均不显著。(3)除了下腹皮肤厚度与背部、肩部、头脸部的不相关(P0.05)外,巴马香猪不同部位两两之间皮肤厚度均呈现不同程度地显著或极显著正相关。(4)关联分析结果表明,9个不同部位的皮肤厚度表型均与候选区域的某些SNPs存在极显著的关联,从强到弱依次是肷部、肩部、背部、腋下、臀部、胸部、头脸、管部和下腹。从而,证实了巴马香猪群体存在影响猪皮肤厚度的7号染色体主效QTL。(5)3个与皮肤厚度关联性最强的SNPs值得进一步关注,分别位于7号染色体的34856565、35543837和35573869位置。肷部的皮肤厚度与SNP(chr7:34856565)的关联显著性最强(P_(cor)=5.15×10~(-6)),这个SNP也是肩部皮肤厚度最关联(P_(cor)=5.75×10~(-6))的位点。SNP(chr7:35543837)是腋下(P_(cor)=3.05×10~(-5))、臀部(P_(cor)=0.010)、胸部(P_(cor)=0.013)和头脸(P_(cor)=0.025)皮肤厚度的最关联位点,也是肩部皮肤厚度的次最关联位点。SNP(chr7:35573869)则是背部皮肤厚度的最关联位点(P_(cor)=1.17×10~(-5)),SNPs(chr7:35543837和chr7:34856565)次之。(6)根据最强关联SNPs所在基因及基因生物学功能,初步推测ANKS1A和HMGA1基因可能是影响皮肤厚度的候选因果基因。【结论】较全面地测量了中国小型地方猪品种巴马香猪周身皮肤厚度,揭示了巴马香猪皮肤厚度在不同部位之间的变化规律。在巴马香猪群体中验证了影响皮肤厚度的7号染色体主效QTL位点,肷部、背部和肩部皮肤厚度表型性状与候选SNPs位点关联性更强,可能适合下一步大规模深入分析。ANKS1A和HMGA1基因可能是影响皮肤厚度的候选因果基因,但需要进一步生物学功能试验证明。

Measurement of Skin Thickness Along the Whole Body Sites and Association Study with Candidate SNPs on Sus Scrofa Chromosome 7 in Bamaxiang Pigs

【Objective】Skin thickness at 9 typical sites of the whole pig body were measured in Bamaxiang pig breed, one of the Chinese local miniature pig breeds. There are 46 candidate SNPs located in a major QTL affecting skin thickness on sus scrofa chromosome 7 (SSC7) were genotyped in the Bamaxiang pig population. One aim of this study is to make clear variation rule of skin thickness at different body sites in Bamaxiang pigs. The other aim is to verify the major QTL affecting skin thickness on SSC7 in Bamaxiang pig population by association study in a specific genomic region. Result of this study will contribute to further revealing the genetic mechanism of swine skin thickness in a larger pig population at whole genome level, and increase our understanding about pig skin. 【Method】A totle of 50 Bamaxiang pigs, including 27 females and 23 castrated males, were randomly selected from a Bamaxiang pig population consisting of 319 adult pigs, which were slaughtered at 300 days. The skin with 5 cm² area were cut from 9 body sites of cheek, shoulder, back, loin, rump, chest, belly, armpit and trotter. Skin thickness at these 9 different body sites were accurately measured using digital caliper. The difference of skin thickness between pairwise body sites and the difference between females and castrated males were tested using the basic statistic package of R language. Pairwise Pearson''s correlation coefficients were calculated between skin thickness at different body sites, meanwhile their significant tests were done. A total of 46 SNPs located in the region from 34.5 to 36.2 Mb on SSC7 were selected and designed for genotyping on a SEQUENOM MassARRAY SNP Genotyping platform. Combined with the above phenotypes of skin thickness, association studies on the target candidate region were performed by a general mixed linear model using SNPasscoc package of R language. Based on the association results and biological function of genes, positional candidate genes were determined. 【Result】One-way analysis of variance showed that an extreme significant difference with a 2.95×10^-117 probability existed among skin thickness at 9 body sites in Bamaxiang pigs. The thickest body sites were loin and back with skin thickness of (5.15 ± 0.92) and (4.97 ± 0.85) mm, respectively, while the thinnest sites were belly and armpit with skin thickness of (1.77 ± 0.36) and (1.97 ± 0.68) mm, respectively. The body sites with skin thickness from thick to thin in turn were loin, back, shoulder, cheek, rump, trotter, chest, armpit and belly. Skin thickness of castrated male pig at armpit was significantly thinner than female pig with a 0.021 probability. When compared between other pairwise body sites, skin thickness had no significant differences. There was no significant correlation between skin thickness at belly and back, between belly and shoulder, and between belly and cheek. Other pairwise combinations had a significant positive correlation. Association analysis between skin thickness at 9 different body sites and the above candidate SNPs suggested that the major QTL on SSC7 affecting skin thickness was also identified in Bamaxiang pig population. Three most promising SNPs were worthy of being further focused on. SNP (chr7:34856565) showed most significant association with skin thickness at loin (P_cor = 5.15×10^-6), which was also the most significant site associated with skin thickness at shoulder (P_cor = 5.75×10^-6). SNP (chr7:35543837) was the most significant site associated with skin thickness at armpit (P_cor = 3.05×10^-5), at rump (P_cor = 0.010), at chest (P_cor = 0.013) and cheek (P_cor = 0.025), also was the second most significant site with shoulder. SNP (chr7:35573869) was the top significant site associated with back skin thickness (P_cor = 1.17×10^-5), and followed by SNPs (chr7:35543837 and chr7: 34856565). Based on nearest genes of top SNPs and their biological function, ANKS1A and HMGA1 were preliminarily suggested to be positional candidate genes possibly affecting skin thickness in pigs.【Conclusion】It was the first time to measure skin thickness at 9 different body sites and report the variation rule of skin thickness along the whole body in Bamaxiang pig breed, one of the Chinese local miniature pig breed. Meanwhile the major QTL affecting skin thickness on SSC7 was verified in Bamaxiang pig population, it provided an important clue and base to reveal the genetic mechanism of pig skin thickness in future. ANKS1A and HMGA1 might be positional causal genes affecting skin thickness in pigs, and it needs further verification of biological functional test.