CN117144017B - 与鸡生长性状相关的分子标记及其应用 - Google Patents
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Abstract
本发明公开了一种与鸡生长性状相关的分子标记及其应用,涉及动物分子生物技术领域,具体的,该分子标记的核苷酸序列如SEQ ID NO.1,其第96位的碱基为T或C。本发明公开的SNP位点可用于对鸡的活体体重和胫长表型性状进行选择,加快生长性能选择育种进程和提高鸡群体型均匀度。
Description
技术领域
本发明涉及动物分子生物技术领域,尤其涉及一种与鸡生长性状相关的分子标记及其应用。
背景技术
鸡作为中国国内畜牧业重要的经济家禽之一,具有风味佳营养价值高的优点,且我国地方品种资源丰富,优质鸡配套系繁多,是较佳的育种素材。但良种繁育体系不够健全,致使鸡群体型均匀度差,市场混乱,而且对生长的遗传规律及机制不深入等因素极大地影响优良品种的资源利用及其商品的经济效益。
一直以来,在我国的大部分地区消费者对优质肉鸡市场需求都很旺盛,其产品销售形式主要有3种,包括活鸡、冰鲜鸡和加工产品。近几年随着禽流感疫情的爆发,保鲜技术不断发展和人们消费观念的改变,冰鲜鸡市场份额显著上升,特别是两广地区。其中,活鸡和冰鲜上市对体重等表观生长性状均匀度的要求随着优质肉鸡的产业化发展越来越高。通过家禽育种技术和标准化饲养等技术手段,可有效提高鸡的生长性状均匀度。家禽育种工作主要集中在提高生长和繁殖性能,鸡的生长性能是动物生产中重要的经济性状,对鸡养殖业的经济效益直接相关。
发明内容
本发明所要解决的技术问题在于,提供一种与鸡生长性状相关的分子标记及其应用,其可为生长性状在种质资源深度鉴评以及育种利用提供参考。
为了解决上述技术问题,本发明提供了一种与鸡生长性状相关的分子标记,其核苷酸序列如SEQ ID NO.1所示,其第96位的碱基为T或C。
相应的,本发明还公开了一种用于检测上述的与鸡生长性状相关的分子标记的引物组合,其包括正向引物和反向引物,所述正向引物的核苷酸序列如SEQ ID NO.2所示,所述反向引物的核苷酸序列如SEQ ID NO.3所示。
相应的,本发明还公开了一种试剂盒或PCR反应体系,其包括上述的引物组合。
相应的,本发明还公开了上述的分子标记在(1)或(2)中的应用:
(1)检测鸡生长性状;
(2)鸡分子育种。
相应的,本发明还公开了上述的引物组合在(1)或(2)中的应用:
(1)检测鸡生长性状;
(2)鸡分子育种。
相应的,本发明还公开了上述的试剂盒或PCR反应体系在(1)或(2)中的应用:
(1)检测鸡生长性状;
(2)鸡分子育种。
相应的,本发明还公开了一种检测鸡生长性状的方法,其采用上述的正向引物、反向引物扩增待测鸡的DNA,检测扩增产物,对其进行SNP筛选、基因分型。
作为上述技术方案的改进,扩增反应条件为:
95℃预变形5min;95℃变性30s,56℃退火30s,72℃延伸40s,共32个循环,最后72℃延伸7min。
相应的,本发明还公开了一种鸡分子育种方法,其包括采用上述的分子标记进行鸡辅助育种的步骤。
实施本发明,具有如下有益效果:
本发明基于对鸡生长性状相关基因pIGR的深度分析,建立了与鸡生长性状相关的分子标记,其可对不同类型鸡的活体体重、胫长表型性状进行早期育种选择,加快生长选择育种进程和提高鸡群体型均匀度。
附图说明
图1为本发明实施例1中鸡pIGR基因编码区中寻找到的SNP序列峰图。
具体实施方式
下面将结合本发明实施例对技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
发明下述实施例中,涉及的设备及试剂包括:
DNA分子标准量Marker购自广州东盛生物科技有限公司;DNA聚合酶购自AG公司;饱和苯酚购自生工生物工程(上海)股份有限公司;DNA提取液二合一、DNA提取液三合一购自Solarbio公司。恒温水浴锅、低温高速离心机、PCR仪、凝胶电泳仪、凝胶成像仪为常规设备。
实施例1鸡生长性状pIGR基因分子检测方法的建立
(一)实验材料
广东省农业科学院动物科学研究所原种保育鸡场的F2资源群体由广东省农业科学院动物科学研究所家禽育种与生产研究室团队建立。利用土鸡专门化品系H(即HuiyangBeared chicken,参Sheng Z,Pettersson M E,Hu X,et al.Genetic dissection ofgrowth traits in a Chinese indigenous×commercial broiler chicken cross[J].Bmc Genomics,2013,14.或Wang Y,Guo F,Qu H,et al.Associations between variantsof bone morphogenetic protein 7gene and growth traits in chickens[J].BrPoult,2018:00071668.2018.1454586.)和快大型专门化品系A(即High Quality ChickenLineA,参Sheng Z,Pettersson M E,Hu X,et al.Genetic dissection of growth traitsin a Chinese indigenous×commercial broiler chicken cross[J].Bmc Genomics,2013,14.或Wang Y,Guo F,Qu H,et al.Associations between variants of bonemorphogenetic protein 7gene and growth traits in chickens[J].Br Poult,2018:00071668.2018.1454586.),按公母比例1:4,进行正反杂交产生F1代,F1代群体内避免半同胞横交,同时产生F2资源群体。随机选取F2同批次群体632羽,每个个体标记唯一的翅号身份标识,在相同饲养条件下饲养。在8、10和12周龄时,分别进行详细的鸡活体重(单位:g,精确到0.5g)、胫长(单位:mm,精确到0.01mm)测定并记录。在12周龄测定后,利用ETDA抗凝真空采血管通过翅下静脉采集全血1mL,保存于-20℃冰箱,用于后续的DNA提取。
(二)DNA提取
所有个体的DNA提取操作均按照饱和苯酚-氯仿传统提取法进行,提取血液样品的基因组DNA后,检测DNA样本浓度和OD值,将DNA浓度大于500ng/μL、OD260/OD280之比值在1.8~2.0之间的样品,保存于-20℃冰箱,用于后续的PCR扩增。
(三)引物
根据NCBI(National Center for Biotechnology Information)官网所提供的鸡(Gallus gallus)pIGR基因编码区的DNA序列(参考序列NCBI登录号为:NC_052557.1)为模板,使用NCBI的Primer BLAST工具进行引物设计一对引物pIGR-4F/pIGR-4R,该引物有效扩增得到如序列SEQ ID No.1所示的DNA序列,该序列全长为484bp。
引物序列由生工生物工程(上海)股份有限公司合成。
表1引物表
(四)PCR扩增
总体积为25μL的PCR反应体系中加入鸡血液DNA模板1μL,2×PCR反应混合物(AG公司)12.5μL,10mM正反引物各1μL,双蒸水9.5μL。
PCR反应条件为:95℃预变性5min;95℃变性30s,56℃退火30s,72℃延伸45s,共32个循环;最后72℃延伸7min,4℃保存。
(五)寻找分子标记:
PCR扩增产物直接测序,运用DNASTAR软件分析序列,筛选SNP位点,寻找到第96位点的一个T-C的碱基突变,利用SPSS23.0软件进行基因型与生长性状进行关联分析。
(六)实验结果
取F2群体DNA为模板,进行PCR扩增,利用琼脂糖凝胶电泳检测片段的完整性,PCR产物直接送生工生物工程(上海)股份有限公司正或反向Sanger测序。利用DNASTAR软件将所获序列峰图(图1)与pIGR基因编码区的DNA序列对比分析,筛选SNP位点结果见表2,寻找到第96位点的一个T/C的碱基突变,对应pIGR基因的CDS区外显子4上发生GTC(缬氨酸V)-GCC(丙氨酸A)的错义突变,基因型分为三个种,分别是TT型,CT型及CC型。
表2鸡pIGR基因SNP位点T/C的基因频率统计表
根据F2群体个体基因分型数据,结合该群体的生长性状表型记录,利用SPSS23.0软件进行基因型与生长性状进行关联分析,结果见表3。该软件利用单因素ANOVA分析检验不同基因型个体间生长性状差异显著性,同列肩标字母相隔表示差异显著(p<0.01),字母相邻表示差异显著(p<0.05),字母相同或无标注者表示差异不显著(p>0.05)。Lamda法检测基因型与性状间关联性,返回的p值表示基因型与性状间关联的显著性,基因型效应λ值的大小表示基因型效应的大小,且值越大,表示对生长性状效应大(即与所对应的生长性状关联性越高);p值≤0.05为相关性显著,p值≤0.01为相关性极显著。
表3不同基因型与生长性状关联分析结果表
注:同列肩标字母相隔表示差异显著(p<0.01),字母相邻表示差异显著(p<0.05),字母相同或无标注者表示差异不显著(p>0.05)。
由表3可知,第96位多态性为T/C的核苷酸序列与体重和胫长性状的相关性达极显著水平(p=0.000),当突变位点的基因型为CC时,鸡群个体表现出最高体重、胫长,当为TT型时,表现出最低体重、胫长。检测pIGR基因的基因型能有效提高鸡群的体型均匀度。
以上所述的仅为本发明一种较佳实施例而已,当然不能以此来限定本发明之权利范围,因此依本发明权利要求所作的等同变化,仍属本发明所涵盖的范围。
Claims (3)
1.一种与鸡生长性状相关的分子标记在检测鸡生长性状或鸡辅助育种的应用,其特征在于,所述检测鸡生长性状或鸡辅助育种的方法为:采用与鸡生长性状相关的分子标记SNP的正向引物、反向引物扩增待测鸡的DNA,检测扩增产物,对其进行SNP筛选、基因分型;
所述正向引物的核苷酸序列如SEQ ID NO.2所示;
所述反向引物的核苷酸序列如SEQ ID NO. 3所示;
所述分子标记的核苷酸序列如SEQ ID NO.1所示,其第96位的碱基为T或C;
当第96位碱基的基因型为CC型时,鸡群个体表现出高体重、胫长;
当第96位碱基的基因型为TT型时,鸡群个体表现出低体重、胫长。
2.根据权利要求1所述的与鸡生长性状相关的分子标记在检测鸡生长性状的应用,其特征在于,扩增反应条件为:
95℃预变形5min;95℃变性30s,56℃退火30s,72℃延伸40s,共32个循环;最后72℃延伸7min。
3.一种权利要求1所述的与鸡生长性状相关的分子标记的引物组合在检测鸡生长性状或鸡辅助育种的应用,所述引物组合为核苷酸序列如SEQ ID NO.2所示的正向引物和核苷酸序列如SEQ ID NO. 3所示的反向引物。
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