摘要
耳朵形状和大小是猪品种的重要特征,同时,外耳是哺乳动物听觉系统的重要组成部分,人类先天性外耳疾病已多次见诸报道,但其遗传机理尚未阐明。本实验前期利用二花脸×白色杜洛克F:资源群体,通过全基因组扫描,在SSC5标记SWR453到SW1987之间定位到一个显著影响耳面积的QTL,该QTL解释了耳面积表型变异的17.14%,置信区间约为11cM。
本研究在此基础上首先通过在置信区间内增加4个微卫星标记对资源家系进行扫描,将QTL精细定位到SSC5P3到SW2003标记之间的8.7cM(55.6cM-64.3cM)内;随后,根据外耳组织结构特点,在初步精细定位的8.7cM区间内选取3个与软骨细胞发育显著相关的位置功能候选基因:HMGA2,SOX5和PTHLH,分析了3个基因的遗传变异及其与耳面积的关联性。通过对资源家系4头F1公猪PCR产物直接测序,在3个基因中共鉴别到70个多态位点,选择其中多态性较好的7个SNPs采用SNaPShOt法判定其在资源家系中的基因型。标准关联分析和标记辅助关联分析均显示HMGA2g.2836A>G位点与耳面积关联性最强,P值分别为9.19E-46和1.92E-05;F-drop检验显示,在QTL分析中将HMGA2g.2836A>G位点作为固定效应时,F值下降了97.6%;单倍型分析显示,HMGA2所有单倍型解释了QTL全部的表型变异(P=1.31E-31)。然而HMGA2g.2836A>G位点与苏太、苏姜和苏钟3个商业合成品系耳面积的关联性分析结果显示,该位点只在苏太群体中达到了显著水平(P<0.05)。
为了获得更高的QTL定位精度,本研究利用猪全基因组高通量60K SNP芯片对资源家系和苏太猪群体进行扫描。资源家系全基因组关联分析(GWAS)结果显示,在SSC5上共鉴别到21个SNP位点与耳面积表型显著关联,其中关联性最强的三个点为H3GA0016181,ALGA0031524,ASGA0025253(corrected P value=0.0096);苏太猪群体GWAS分析结果显示,H3GA0016181和ASGA0025253两个标记与耳面积关联性最显著(corrected P value=0.00049),分别位于MSRB3和HMGA2基因之间的间隔区和HMGA2基因内含子上;资源家系全基因组连锁和连锁不平衡(LD-LA)以及连锁(LA)分析结果均显示ALGA0031518和ALGA0031519两个位点与耳面积表型关联性最强,P值分别为1.656E-45,7.13E-49,这两个标记均位于MSRB3基因内含子上;利用最显著关联SNP标记的-Loglo(P值)下降2的方法,在LDLA分析中将QTL精细定位到ALGA0031489与ASGA0025275标记之间的2.67Mb区间内,在LA分析中将QTL精细定位到ALGA0031500与ASGA0025287标记之间2.32Mb范围内。进一步利用DualPHASE软件构建了资源家系19头祖代(2头公猪和17头母猪)个体在上述精细定位后的2.32Mb QTL区间的单倍型。结果显示,F0二花脸个体中除3条染色体外,其余均共享一段1.28Mb的单倍型区域(EQ),该单倍型有增加耳面积的效应。对1.28Mb区域内的6个己注释基因进行耳组织内的RT-PCR分析,除WIF1基因外,XPOT、TBC1D30、LEMD3、MSRB3和HMGA2等5个基因在耳组织中均表达。为进一步精细定位QTL区间,在1.28Mb区间内增加191个SNP标记,对屠宰的苏太猪群体和现场表型测定的吴江成年苏太母猪群体进行扫描,进一步将QTL精细定位到925Kb区间。该区间对应于人的同源区包括WIF1, LEMD3, MSRB3和HMGA2等4个已注释的基因,除WIF1基因外,LEMD3, MSRB3和HMGA2等3个基因都是QTL的候选因果基因。
本研究结果为最终鉴别SSC5耳性状QTL的因果基因及其因果突变位点奠定了重要的前期工作基础。
Ear size and erectness are important conformation characteristics of pig breeds. Moreover, ear is an important part of the auditory system. There have been many reports about congenital external ear diseases in human. In our previous study, we identified a significant QTL for ear size within an11.0-cM interval between SWR453and SW1987on SSC5through a genome-wide scan in a large scale White Duroc x Erhualian F2intercross. This QTL explained17.14%of the phenotypic variance.
Here we fine-mapped this major QTL on SSC5to identify the causative gene for ear size. We developed4new microsatellite markers in the QTL interval and genotyped these markers across the White Duroc x Erhualian resource population. The QTL was then refined to an8.7cM interval between markers SSC5P3and SW2003. Subsequently, HMGA2, SOX5and PTHLH located in the8.7cM QTL region was selected as the positional candidate genes. Direct sequencing of4F1boars,70polymorphisms were detected within these genes and seven highly informative SNPs were selected and genotyped in the F2population by SNaPShot assays. Of the seven SNP, HMGA2SNP g.2836A>G showed the strongest association with ear size in the standard association test and marker-assisted association test (P=9.19E-46and1.92E-05, respectively). With the F-drop test, F value decreased by more than97%only when the genotypes of HMGA2g.2836A>G were included as a fixed effect. The haplotype-based association test showed that the phenotypic variance explained by HMGA2was similar to that explained by the QTL. However, the significant association between HMGA2g.2836A>G and ear size was only observed in the synthetic commercial Sutai line, but not in Sujiang and Suzhong pigs.
To further improve the mapping resolution, the White Duroc x Erhualian intercross and a Sutai pig line were genotyped with Illumina porcine60k DNA chips. Genome-wide association (GWAS) results showed that21SNPs on SSC5were significantly associated with ear size in the White Duroc x Erhualian intercross, of which H3GA0016181, ALGA0031524and ASGA0025253were the three most significantly SNPs (corrected P value=0.0096). H3GA0016181and ASGA0025253were the most significant SNPs associated with ear size in the Sutai pig line (corrected P value=0.00049). The two SNPs located within the intergenic region between MSRB3and HMGA2and the intron region of HMGA2, respectively. The results from both Linkage disequilibrium and Linkage analysis(LDLA) and Linkage analysis (LA) showed that ALGA0031518and ALGA0031519were the most significantly associated SNP (P=1.656E-45and7.13E-49, respectively). Both SNPs were mapped to the intronic region of MSRB3. The most likely QTL positions mapped to a2.67Mb (ALGA0031489-ASGA0025275) or2.32Mb (ALGA0031500-ASGA0025287) region through LOD score dropoff of2of the most significant SNPs. We reconstructed the haplotypes of all19founder animals (2sires and17dams) in the2.32Mb refined QTL region with DualPHASE software. A haplotype of-1.28 Mb within the refined2.32Mb interval was shared by31Erhualian Q-bearing chromosomes. This shared haplotype is associated with an increase in ear size. Taken together, we refined the QTL region to the1.28Mb chromosome segment. This region encompasses6annotated genes included XPOT, TBC1D30, WIF1, LEMD3, MSRB3and HMGA2. RT-PCR was performed to detect expression levels of these genes in ear tissues. The results showed that except WIF1, all genes were expressed in the ear tissue. To further narrow the confidence interval of SSC5QTL,191SNPs in the1.28Mb critical interval were selected and genotyped in the slaughtered Sutai pig line and another well phenotyped Wujiang adult Sutai pig line. The association results showed that the most likely QTL position (LOD dropoff of2) located in a-925Kb region. This region encompasses4annotated genes included WIF1, LEMD3, MSRB3and HMGA2. Because WIF1was not expressed in the ear tissue, LEMD3, MSRB3and HMGA2is hence the most likely responsible gene for the major QTL.
This study contributed to identify the causative gene and mutation underlying this major QTL.
引文
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