CN114657258B - 一种与猪眼肌深度性状相关的snp分子标记及其应用 - Google Patents
一种与猪眼肌深度性状相关的snp分子标记及其应用 Download PDFInfo
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Abstract
本发明属于猪分子标记技术领域，具体涉及一种与猪眼肌深度性状相关的SNP分子标记及其应用。通过整合NCBI数据库和欧洲生物信息研究所公开的58个品种的469头猪的重测序数据，并将重测序数据比对猪参考基因组序列获得基因分型数据，联合分析GWAS信号与增强子‑启动子三维互作网络，筛选得到一种与猪眼肌深度相关的分子标记，该标记的核苷酸序列如SEQ ID NO：1和2所示，在该序列的第51位碱基处存在一个C/T的等位基因突变，TT基因型有利于猪拥有较高的眼肌深度。
Description
技术领域
本发明属于猪分子标记技术领域，具体涉及与猪眼肌深度性状相关SNP分子标记及其应用。
背景技术
猪是畜牧生产中重要的经济动物。家猪作为人类最早驯化的家养动物之一，起源于东亚野猪(Sus scrofa)，从1万年前开始，猪在欧亚大陆的多个地方被驯化(Groenen MA,Archibald AL,Uenishi H,Tuggle CK,Takeuchi Y,Rothschild MF,et al.Analyses ofpig genomes provide insight into porcine demography andevolution.Nature.2012；491(7424):393-8)。野猪(Sus scrofa)的驯化和随后高强度的人工选择导致了家猪的许多性状的显著表型变化，包括行为、身体组成、繁殖和被毛颜色(Rubin CJ,Megens HJ,Martinez Barrio A,Maqbool K,Sayyab S,Schwochow D,etal.Strong signatures of selection in the domestic pig genome.Proc Natl AcadSci U S A.2012；109(48):19529-36)。由于驯化过程是相互独立的，欧洲猪种和亚洲猪种之间有着区别分明的种质特色，例如欧洲猪种拥有更高的瘦肉率和更大的体型，亚洲猪种则脂肪含量更高，并且拥有高产仔数和早熟的优点。
在猪的生产过程中，猪的眼肌深度是重要的经济性状，在生产活动及市场定价时被用来评定生猪胴体品质(如背膘和胴体瘦肉率)，可以影响消费者的消费选择进而影响经济效益(Pannier L,Gardner GE,Pearce KL,McDonagh M,Ball AJ,Jacob RH,etal.Associations of sire estimated breeding values and objective meat qualitymeasurements with sensory scores in Australian lamb.Meat Sci.2014；96(2Pt B):1076-87.)。因此，探索发现眼肌深度遗传机制的相关分子标记有助于更好的展开猪的育种研究。
大多数遗传变异位于不编码的调控区域，正确识别其靶基因需要对顺式调控元件在生理上相关的细胞或组织类型中的染色质互作进行重构。BL-HiChIP，是一种以蛋白质为中心的染色质构象方法。与ChIA-PET相比，BL-HiChIP将构象信息读取的产量提高了10倍以上，并将输入需求降低了100倍以上(Mumbach MR,Rubin AJ,Flynn RA,Dai C,Khavari PA,Greenleaf WJ,et al.HiChIP:efficient and sensitive analysis of protein-directed genome architecture.Nature Methods.2016；13(11):919-22.)，可用于鉴定与疾病相关的增强子与远端靶基因的作用(Mumbach MR,Satpathy AT,Boyle EA,Dai C,Gowen BG,Cho SW,et al.Enhancer connectome in primary human cells identifiestarget genes of disease-associated DNA elements.Nature Genetics.2017；49(11):1602-12.)，同时将调控元件与靶基因联系起来，揭示疾病中基因表达失调的潜在机制(Song M,Yang X,Ren X,Maliskova L,Li B,Jones IR,et al.Mapping cis-regulatorychromatin contacts in neural cells links neuropsychiatric disorder riskvariants to target genes.Nat Genet.2019；51(8):1252-62.)。本发明通过整合H3K27acBL-HiChIP和顺式调控元件注释，构建了猪全基因组范围内的增强子-启动子三维互作网络，筛选得到一个与猪眼肌深度相关的主效基因及功能突变(SNP)，并在群体内进行了验证，SNP与猪眼肌深度的相关性达到了显著水平，为猪眼肌深度的辅助选择和预测改良提供了新的分子标记，对于猪的辅助筛选具有重要意义。
发明内容
本发明的目的在于提供一种与猪眼肌深度性状相关的分子标记及其应用。
为了实现上述目的，本发明采用以下技术方案：
通过整合NCBI数据库和欧洲生物信息研究所公开的58个品种的469头猪的重测序数据，并与猪参考基因组序列进行比对获得基因分型数据，联合分析GWAS信号与增强子-启动子三维互作网络，筛选得到一种与猪眼肌深度性状相关的SNP分子标记rs321591161，SNP位点位于7号染色体第30341053位碱基，该位点的碱基为T或C，参阅Ensembl得到该SNP位点上下游50bp的核苷酸序列，序列如SEQ ID NO.1或2所示。
上述分子标记在猪眼肌深度性状选择中的应用：当SNP位点为TT基因型时，猪拥有更高的眼肌深度，TT基因型是猪眼肌深度性状的有利标记；当基因型为CC时，猪拥有较低的眼肌深度。
本发明与现有技术相比具有的有益效果：
本发明筛选到与猪眼肌深度显著相关的一个分子标记，其基因型被证明在2795头杜洛克猪群体中与眼肌深度显著相关(Mann-Whitney-U单尾检验，P值：2.0×10-2)，可通过在体外采用基因芯片技术检测该标记的基因型，其结果可作为评价猪的眼肌深度的指标，与目前的PCR-RFLP等方法相比，本发明具有简单、快捷、灵敏度高和特异性好等突出优点。
附图说明
图1：分子标记的开发流程图。
图2：本发明IGV图。研究的是猪眼肌深度性状，rs321591161为本发明筛选的分子标记，该标记位于猪第7号染色体上。
图3：rs321591161分子标记的关联分析结果(P值:2.0×10-2)。**表示P<0.01。
具体实施方式
本发明中的序列和全基因组关联分析结果，是基于猪基因组11.1版本的信息。
实施例1：与猪眼肌深度性状相关的分子标记的筛选
(1)整合下载NCBI数据库(SRA,http://www.ncbi.nlm.nih.gov/sra/)和欧洲生物信息研究所(EMBL-EBI,https://www.ebi.ac.uk/)中公开的包含58个品种的469头猪的重测序数据；
(2)使用SRAToolkit(V2.8.2)将原始数据转换为fastq文件，随后使用Trimmomatic(V0.36)软件对fastq文件进行初步质控；该步骤后通过使用Burrows–WheelerAligner 0.7.17(BWA)软件将剩余的高质量reads与猪参考基因组(版本11.1)进行比对，选择唯一比对序列进行分型；为了获得高质量的变异数据，使用GATK(V4.0.3.0)软件通过‘QUAL<30.0||QD<2.0||FS>60.0||MQ<40.0||SOR>4.0||ReadPosRankSum<-8.0’命令进行质控，获得可用于GWAS分析的基因型数据，对基因型数据进行质检，最终有399个个体和19,487,140个SNP用于GWAS研究；
(3)根据文献的GWAS研究(Yang R,Guo X,Zhu D,Tan C,Bian C,Ren J,etal.Accelerated deciphering of the genetic architecture of agriculturaleconomic traits in pigs using a low-coverage whole-genome sequencingstrategy.Gigascience.2021；10(7).)，得到位于猪7号染色体30,242,768-30,522,596的主效QTL，将该QTL内的SNP位点全部提取出来共1,036个，用于后续表观遗传学筛选；
(4)通过H3K27ac BL-HiChIP构建猪全基因enhancer-promoter三维互作网络，对上述SNP位点进行筛选，仅保留同时符合以下条件的SNP：一、位于BL-HiChIP的loop anchor内；二、位于顺式调控元件内(enhancer或promoter)；三、位于染色质开放域或转录因子足迹内；四、4个商业化瘦肉猪品种(大白、长白、杜洛克和皮特兰)和6个中国地方猪品种(梅山、八眉、金华、通城、荣昌和二花脸猪)的等位基因频率差异大于0.5(此步骤的等位基因频率计算基于步骤2)；五、SNP被motifbreakR软件预测到有影响转录因子结合的强效应；
(5)根据群体的基因型和表型值，对上述过滤后的SNP位点在眼肌深度的效应进行验证，检测不同等位基因的猪群体是否在眼肌深度上存在差异；
(6)最终结果如附图2，在眼肌深度QTL内，通过上述步骤筛选，过滤掉噪音及没有功能的分子标记，最终获得分子标记rs321591161，该标记位于靶基因TAF11上游的顺式调控元件内部的开放染色质区域上，且该顺式调控元件与靶基因经BL-HiChIP鉴定存在染色质互作。
实施例2：rs321591161分子标记与猪眼肌深度性状的关联分析
我们测定了2795头杜洛克猪的眼肌深度(当猪的体重达到100±5公斤时，使用b型超声波扫描设备测量每个个体最后3-4根肋骨的眼肌深度)及其在rs321591161分子标记的基因型，利用R统计环境下MVP软件包中的MLM模型进行全基因组关联分析(GWAS)。
统计方法：具体模型如下：y＝Xβ+Zμ+e，其中，y代表个体的表型值，β代表包括主成分(principle component)在内的固定效应，μ代表服从分布u～N(0,G)的随机效应，该分布中G代表由对应个体的SNP计算的亲缘关系矩阵。X和Z代表β和μ的关联矩阵，e代表残差向量(vector of residual errors)。
由图3可知，对于基因型为CT或TT的个体，其眼肌深度高于CC型个体。在采用Mann-Whitney-U单尾检验下，rs321591161标记达到了全基因组显著水平(P<0.05)，说明该标记不仅与猪的眼肌深度性状显著相关，且当该标记突变为T时，有利于猪拥有更高的眼肌深度。
序列表
<110> 华中农业大学
<120> 一种与猪眼肌深度性状相关的SNP分子标记及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 101
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gagtggagac ccgaaccctg gtcttgttcc aatgctcaca gcttctgcta cggcgggcgg 60
tttgtcctgt ccttcctcgc ttttctgaac cctttactcg t 101
<210> 2
<211> 101
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gagtggagac ccgaaccctg gtcttgttcc aatgctcaca gcttctgcta tggcgggcgg 60
tttgtcctgt ccttcctcgc ttttctgaac cctttactcg t 101
Claims (1)
1.SNP分子标记在猪眼肌深度性状选择中的应用，其特征在于，所述分子标记的核苷酸序列如SEQ ID NO.1和2所示，第51位碱基是T或C，导致多态性，SNP位点的基因型为TT时有利于猪拥有高的眼肌深度，所述的眼肌深度为猪的体重达到100±5公斤时，使用b型超声波扫描设备测量每个个体最后3-4根肋骨的眼肌深度。
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