CN113637772B - 一种提高绵羊产羔数的snp标记及方法 - Google Patents
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Abstract
本发明公开一种影响绵羊产羔数的GPR54基因外显子区的SNP标记基因型的方法及其应用。该标记位于绵羊5号染色体的GPR54基因上,该基因第5外显子区域的核苷酸序列为SEQ ID NO:1,其中第514bp处的GG基因型绵羊的产羔数显著多于GT基因型和TT基因型个体,且GG为绵羊的优势基因型。本发明还公开了扩增SEQ ID NO:1序列所用的引物对及扩增方法。本发明以夷陵绵羊为研究对象,利用PCR扩增绵羊GPR54基因第5外显子区域的DNA序列变异,确定该变异位点的基因型并据此选留绵羊个体留种,分析该变异导致的氨基酸变化及GPR54基因特定位点基因型对绵羊产羔数的影响,可用于提高夷陵绵羊产羔数,为绵羊产羔数的标记辅助选择育种提供新的标记资源。
Description
技术领域
本发明属于动物分子育种技术领域,涉及影响绵羊繁殖性状的SNP标记、检测方法及其在绵羊育种中的应用。
背景技术
产羔数是绵羊的一个重要经济性状,显著影响羊场的经济效益。湖羊是我国特有的绵羊品种,源于蒙古羊,经人为驯养选育,具有性成熟早、四季发情、高产羔率等繁殖特征。夷陵绵羊是以东弗利生羊、小尾寒羊和湖羊三品种杂交选育而成的绵羊群体,其突出特点是产多羔,因此研究影响夷陵绵羊产羔数的分子标记对于绵羊多胎资源的开发利用及生产实践有重要意义。
Kiss1/GPR54系统参与调控动物青春期和繁殖功能,在雌性动物的卵巢发育、胚胎附植、妊娠维持和雄性动物睾丸发育、精子发生等过程中发挥功能,Kiss1/GPR54的突变会影响性发育及性腺功能。
绵羊GPR54基因具有多态性。在高繁殖力绵羊品种(小尾寒羊和湖羊)和低繁殖力绵羊品种(中国美利奴羊和考利代羊)GPR54基因上游的5’端调控区-905碱基处检测到1个A/G核苷酸突变位点、-867bp至-863bp区域的一个5bp缺失/插入突变(TTCTT),且该缺失/插入突变与小尾寒羊产羔数有关。湖羊GPR54基因第二外显子有2个多态位点T2360C、A2411C,分别引起氨基酸改变M90T、D107A。在梅赫拉班羊、沙尓羊以及两者与罗曼诺夫羊的杂交品种中,GPR54基因5’UTR区域、第四和第五外显子上存在多态位点,其中第四外显子突变C3431A与梅赫拉班羊产羔数和出生重呈负相关。本发明发现与绵羊产羔数性状相关的GPR54基因SNP标记,为绵羊产羔数性状的标记辅助选择提供新的标记资源。
发明内容
本发明旨在解决现有技术中对绵羊产羔数遗传选育所存在的技术问题之一。
本发明的第一个目的在于提供一种与绵羊产羔数性状相关、且能够有效用于夷陵绵羊产羔数选育的SNP标记及其应用。
根据本发明的实施例,所述SNP标记位于SEQ ID NO:1所示核苷酸序列的下划线标记处。
根据本发明的实施例,该SNP标记的GG基因型个体的产羔数显著高于GT基因型和TT基因型的个体。根据本发明的实施例,通过检测绵羊的上述SNP标记,能够有效地预测其产羔数,从而能够根据该SNP标记的基因型评估绵羊繁殖性能。因此本发明的SNP标记与绵羊的产羔数性状紧密相关,能够有效应用于绵羊的分子标记辅助育种,进而能够根据实际育种需求选择绵羊育种素材,从而能够准确、高效地挑选出产羔数多的优良个体,提高育种选择的效率和准确性。
本发明的第二个目的是提供一种用于检测权利要求1所述SNP标记的引物对。根据本发明的实施例,所述引物为具有SEQ ID NO:2和SEQIDNO:3所示的核苷酸序列,用于检测所述SNP标记。引物对的序列如下所示。
P1:GCCTTCGCTCTCTACAACCT(SEQ 1D NO:2)
P2:AGGGCAAGGCCTTTTCACTAT(SEQ ID NO:3)
根据本发明的实施例,利用本发明的引物对能够有效地扩增待测绵羊的上述与产羔数相关的SNP标记所在DNA片段,通过测序能够有效检测此SNP标记,确定待测绵羊该SNP标记的基因型,进而能够有效预测待测绵羊的产羔数。具体地,该SNP位点的GG基因型个体产羔数显著高于GT基因型和TT基因型个体,表明该SNP位点的GG基因型可以作为判断绵羊产羔数多少的重要标准。在绵羊选育工作中可以通过保留该SNP位点基因型为GG的个体,淘汰该SNP位点基因型为GT或TT的个体,从而逐步提高绵羊群体的产羔数。由此,本发明用于检测前面所述SNP标记的引物对,能够有效用于绵羊的分子标记辅助育种,进而实现低成本、高准确性地选择出产羔数高的绵羊群体。
根据本发明的实施例,利用本发明的引物对及PCR扩增体系、扩增程序,对扩增产物进行测序,能够有效实现对待测绵羊的上述与绵羊产羔数相关的SNP标记的多态性检测,确定待测绵羊该SNP标记位点的基因型,进而能够有效预测待测绵羊的产羔数性状,从而能够有效辅助绵羊选育。
本发明的第三个目的是提供一种预测绵羊产羔数性状并进行遗传改良的方法。根据本发明的实施例,该方法利用上述位于绵羊5号染色体上与产羔数相关的SNP标记筛选产羔数多的绵羊个体的方法,包含如下步骤:检测上述与绵羊产羔数相关的SNP分子标记,确定所述SEQ ID NO:1序列第514位核苷酸是G还是T,淘汰该SNP标记处基因型为TT和GT的个体,选留基因型为GG的个体留种。
本发明提供了一种筛选具有高产羔数绵羊的方法,包含以下步骤:
(1)提取绵羊基因组DNA;
(2)利用两条特异性引物PCR扩增GPR54基因第五外显子区序列,得到884bp扩增产物;所述的两条特异性引物序列为P1:SEQ ID NO:2和P2:SEQ ID NO:2;
(3)对所述PCR扩增产物进行测序,获得测序结果;
(4)根据测序结果确定待测绵羊样品在所述SNP标记的基因型;
(5)选择上述SNP标记位点的GG基因型的绵羊个体留种。
本发明所述的分子标记和引物对可应用于筛选出具有高产羔数的夷陵绵羊个体。
本发明具有以下有益效果:(1)本发明提供的SNP标记与夷陵绵羊的产羔数显著相关,GG基因型绵羊的产羔数显著高于GT基因型和TT基因型母羊。(2)该SNP标记可以用于夷陵绵羊产羔数的辅助选择,筛选具有产多羔潜能的绵羊,对进一步提高绵羊繁殖力和利用特定绵羊为素材进行品种(或品系)选育具有重要的实际应用价值。
附图说明
本发明的上述方面结合附图将更容易理解对实施例的描述,图1展示本发明SNP标记的GG、GT、TT基因型测序峰图。
具体实施例
下面详细描述本发明的实施例,结合实施例对本发明进行进一步详细说明,此处实施例仅用于说明本发明,而不能理解为对本发明的限制。
1.实验样本
选择湖北致清和农牧有限公司149只具有三胎以上产羔数的夷陵绵羊为对象,采集血液5ml,用乙二胺四乙酸二钾(EDTA-2K)抗凝,样品-20℃保存。记录母羊三胎以上的产羔数。
2.基因组DNA提取
采用酚-氯仿法提取血液样本中的基因组DNA,测定样本DNA的OD260和OD280,OD260/OD280值在1.6~1.8之间个体的样本DNA用于后续实验,测定样本DNA浓度并稀释至50μg/μL,4℃保存。
3.引物设计
根据绵羊GPR54基因(Gene Bank登录号:NC_040256.1)序列,用Primer-BLAST设计一对特异性引物(P1:SEQ ID NO:2,P2 SEQ ID NO:3)扩增GPR54基因第5外显子区域的一段DNA序列,扩增产物884bp,该DNA序列为序列表中SEQID NO:1所示。
4.聚合酶链式反应(PCR)扩增样本DNA的目标序列并测序
(1)PCR反应体系:1μL DNA(50μg/μL),引物P1和P2(100μM)各1μL,2×Phanta MaxBuffer 12.5μL,dNTP Mix(10mM each)0.5μL,Phanta Max Super-Fidelity DNAPolymerase 0.5μL,8.5μL蒸馏水。扩增程序:95℃预变性3min,95℃变性15s,60℃退火15s,72℃延伸45s,35个循环,72℃延伸5min。
(2)PCR扩增产物送天一辉远生物科技有限公司测序。用Chromas软件分析测序结果,将扩增产物测序结果与GPR54基因序列进行比对,判定序列表中SEQID NO:1所示核苷酸序列第514bp位点的基因型。
5.夷陵绵羊GPR54基因序列变异
序列表中SEQ ID NO:1第514bp的SNP标记位点的碱基G突变为T使编码半胱氨酸(Cys)的密码子改变为编码苯丙氨酸(Phe)的密码子。
6.夷陵绵羊GPR54基因SNP标记与产羔数性状的关联分析
用SPSS软件中的单因素方差分析进行基因型与产羔数性状之间的关联分析。具体的线性分析模型如下所述:
Yij=μ+Gi+eij
其中:Yij为个体表型记录;μ为群体均值;Gi为各位点的基因型效应;eij为随机误差。
7.夷陵绵羊不同基因型间产羔数差异显著性分析
夷陵绵羊不同基因型产羔数性状分析结果见表1。从表1可以看出,该位点有三种基因型,采用单因素方差分析比较不同基因型间平均产羔数的差异,发现GG基因型绵羊的产羔数显著高于GT基因型和TT基因型个体(P<0.05),表明该SNP位点的GG基因型可以作为判断绵羊高产羔数的重要标准。在绵羊选育工作中可以通过保留该位点基因型为GG的个体,淘汰该位点基因型为GT或TT的个体,直至剔除TT或GT基因型个体,从而逐步改良群体的繁殖性能。
表1 GPR54基因不同基因型与产羔数的相关性
注:不同的肩标字母表示差异显著(p<0.05)。
Claims (2)
1.一种根据与绵羊产羔数相关的分子标记基因型选择绵羊的方法,所述分子标记的核苷酸序列为SEQ ID NO:1,其中该序列第514位点的GG基因型绵羊个体产羔数显著高于GT基因型和TT基因型个体;其方法包括以下步骤:
(1)提取绵羊的基因组DNA;
(2)利用两条特异性引物进行PCR扩增,得到884bp的扩增产物,所述的两条特异性引物序列为P1:SEQ ID NO:2和P2:SEQ ID NO:3;
(3)对所述的PCR扩增产物进行测序,获得测序结果;
(4)根据测序结果确定待测绵羊样品在所述分子标记的基因型;
(5)选择上述分子标记的GG基因型的绵羊个体留种;
(6)所述绵羊为夷陵绵羊。
2.权利要求1所述的分子标记基因型在筛选具有高产羔数夷陵绵羊中的应用,选择所述分子标记的GG基因型的绵羊个体。
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