CN117551785A - 凡纳滨对虾耐亚硝酸氮性状相关的snp分子标记及其应用 - Google Patents
凡纳滨对虾耐亚硝酸氮性状相关的snp分子标记及其应用 Download PDFInfo
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Abstract
本发明公开了凡纳滨对虾耐亚硝酸氮性状相关SNP分子标记及其应用,在Cpb基因序列编码区共发现3个与凡纳滨对虾亚硝酸氮耐受性状显著相关的SNP分子标记,分别位于SEQ ID NO.1所示序列的694 bp、696 bp、702 bp位点处,3个SNP分子标记位点完全连锁,其基因型分别为AG、CT和CT是凡纳滨对虾亚硝酸氮耐受性状的有利标记,可用于凡纳滨对虾耐亚硝酸氮性状育种的辅助选择。
Description
技术领域
本发明属于水产动物分子标记技术领域,具体涉及凡纳滨对虾耐亚硝酸氮性状相关SNP分子标记及其应用。
背景技术
凡纳滨对虾(Litopenaeus vannamei)也称南美白对虾,因其生长速度快、蛋白质含量高、营养价值丰富、抗逆能力强等优点,成为我国主要的对虾养殖品种之一[1]。在集约化养殖过程中,高放养密度造成残留饵料及粪便的积累,导致环境污染物质的增加,加剧了水体环境恶化,从而影响对虾的健康和产量[2]。而亚硝酸氮是水产养殖系统中常见的环境污染物质之一[3],大量研究表明,水体中过高的亚硝酸氮浓度会导致对虾生长异常,能量代谢、血氧结合、免疫反应和离子调节等多种生理功能紊乱,对水生动物病原体易感性增强[4-7];此外,长时间暴露在高浓度的亚硝酸氮中,还会导致对虾肝胰腺和肠道等组织结构严重受损[8-9],最终造成高死亡率,对养殖生产造成巨大损失。然而在养殖过程中,由于缺乏高效水质调控技术以及生态养殖模式,将养殖水体中的亚硝酸氮浓度维持在较低水平十分困难,因此,培育出凡纳滨对虾耐亚硝酸氮新品种尤为重要。
目前凡纳滨对虾选育多采用群体和家系选育方式,然而这种选育方式具有选育周期长、成本高、不稳定等缺点,分子标记辅助育种则可以克服传统育种方式的弊端,能够准确、高效的实现经济性状的快速改良。单核苷酸多态性标记(single nuclearpolymorphism,SNP)作为第三代分子标记,是指由于单个碱基的变化所引起DNA序列的改变,包括颠换、转换、插入和缺失等形式,多发生在非编码区,少数SNP位于编码区[10],具有位点数量多、多态性丰富、分布广泛、能够稳定遗传等特点,并且检测方法简便、高效[11],在凡纳滨对虾育种中已得到广泛应用。Janpoom等[12]在凡纳滨对虾血蓝蛋白基因(LvHc)中发现1个和高生长、耐氨性状相关的SNP。Yu等[13]对位于多个凡纳滨对虾免疫相关基因中的SNPs进行关联分析,鉴定出5个SNPs与抗白斑综合征病毒(WSSV)性状相关。陈晓敏等[14]在凡纳滨对虾过氧化氢酶(CAT)基因中筛选到1个SNP与低氧耐受性状关联。但是有关凡纳滨对虾耐亚硝酸氮性状的SNP分子标记筛选鲜有报道。
羧肽酶B(Carboxypeptidase B-like,Cpb)是一种可水解蛋白质或肽的羧基端(C端)末端肽键的蛋白酶[15],广泛存在于人类、动物和植物体内,参与机体的各种生化反应。然而Cpb基因在甲壳动物中发挥的功能研究较少。本发明对该基因的编码区序列进行SNPs筛选,最终检测到3个与凡纳滨对虾耐亚硝酸氮性状显著关联的SNP位点,可为选育具有耐亚硝酸氮性状的凡纳滨对虾品种提供技术支撑。
发明内容
本发明的目的在于提供凡纳滨对虾耐亚硝酸氮性状相关的SNP分子标记,用于选育耐亚硝酸氮性状的凡纳滨对虾品种。
为了实现上述目的,本发明采用以下技术方案:
本发明首先随机挑取亚硝酸氮胁迫96h后死亡和存活的凡纳滨对虾cDNA为模板,进行PCR扩增,初步筛选候选基因的SNPs位点,结果在Cpb基因中检测到了SNPs位点。然后以亚硝酸氮胁迫96h后死亡的和存活个体的凡纳滨对虾基因组DNA为模板,通过引物设计、PCR扩增、产物测序来检测凡纳滨对虾Cpb基因上SNPs位点的基因型,利用SPSS软件进行数据处理,卡方检验分析基因型与亚硝酸氮耐受性状的关联,结果发现3个与凡纳滨对虾耐亚硝酸氮性状显著相关的SNP分子标记,包括SNP分子标记c.694G>A、c.696T>C、c.702T>C:
所述SNP分子标记c.694G>A位于SEQ ID NO.1所示序列的694bp位点处,该碱基为G或A,突变类型为G/G纯合型、G/A杂合型和A/A纯合型;
所述SNP分子标记c.696T>C位于SEQ ID NO.1所示序列的696bp位点处,该碱基为C或T,突变类型为C/C纯合型、C/T杂合型和T/T纯合型;
所述SNP分子标记c.702T>C位于SEQ ID NO.1所示序列的702bp位点处,该碱基为C或T,突变类型为C/C纯合型、C/T杂合型和T/T纯合型;
所述3个SNP分子标记位点完全连锁。
上述SNP分子标记在辅助选育耐亚硝酸氮性状的凡纳滨对虾品种中的应用,具体方法为:扩增含有所述SNP位点的片段,通过直接测序法对SNP位点的基因型进行分型,SNP分子标记c.694G>A的GA基因型、SNP分子标记c.696T>C CT基因型、SNP分子标记c.702T>C的CT基因型为凡纳滨对虾亚硝酸氮耐受性状的有利标记。
进一步地,用于扩增所述3个SNP分子标记的引物序列如SEQ ID NO.4和SEQ IDNO.5所示。
附图说明
图1是为凡纳滨对虾Cpb基因3个SNP位点的不同基因型的测序峰图。
图2是凡纳滨对虾Cpb基因3个SNP位点的连锁分析图。
具体实施方式
1.1试验动物
所用的凡纳滨对虾来自广西壮族自治区水产科学研究院国家南美白对虾育种中心,平均体重为32g,规格均匀、体质健壮、附肢完整、健康有活力,运至实验室暂养7d以适应环境。水温27±0.5℃、pH=7.9±0.1、盐度30‰以及溶解氧高于6mg/L。通过添加NaNO2使水体保持在116mg/L水平进行亚硝酸氮协迫实验,每6h统计凡纳滨对虾死亡数,直至胁迫96h,死亡虾归入亚硝酸氮应激敏感群体,存活虾归入亚硝酸氮应激耐受群体。
1.2试验方法
1.2.1凡纳滨对虾基因组DNA和RNA的提取
采取醋酸铵/异丙醇方法提取凡纳滨对虾肌肉组织总DNA,Trizol法提取各组织样品总RNA,用超微量紫外分光光度计与琼脂糖凝胶电泳检测质量及完整性,按照HiScriptⅡReverse Transcriptase反转录试剂盒说明书操作将抽提的Total RNA反转录为cDNA。
1.2.2引物设计
基于本发明的转录组及NCBI中的基因组(Gene ID:113802097)数据,获得的cDNA和基因组序列,用Premier 5.0及NCBI在线软件设计特异性引物,引物序列如表1所示。
表1本发明设计的引物序列及PCR条件
1.2.3PCR扩增
以每组取5尾凡纳滨对虾的cDNA为模板,按照表1中的引物(Cpb-F1和Cpb-R1)及其扩增条件扩增,PCR扩增反应体系为10×PCR Buffer 1μL,上、下游引物(10μmol/L)各0.25μL,Es taq DNA Polymerase 0.1μL,基因组DNA/cDNA模板1μL,补双蒸水至总体积为10μL。扩增程序:95℃5min;95℃30s,58℃30s,72℃1min,30个循环;72℃10min;4℃保存。将产物于1%的琼脂糖凝胶中电泳,成像保存。
1.2.4产物测序及SNP的筛选
PCR产物经过1%琼脂糖凝胶电泳检测后,由武汉天一辉远有限公司纯化测序,测序结果由DNAstar软件进行比对分析,包括核苷酸序列比对及峰图分析,筛选出可能的SNPs位点。
1.2.5直接测序法进行基因分型
对于初步筛选的SNP位点,在敏感和耐受群体中进行PCR扩增,产物直接测序后根据测序峰图判断每个个体的基因型。引物采用表1中的引物对Cpb-F2和Cpb-R2(即SEQ IDNO.4和SEQ ID NO.5),扩增体系见1.2.3。
1.2.6结果分析
凡纳滨对虾Cpb基因基因型峰图见图1。根据分型结果统计c.694G>A、c.696T>C、c.702T>C 3个SNPs位点在凡纳滨对虾亚硝酸氮耐受群体和敏感群体中的基因型,并计算基因型频率及等位基因频率,进行性状关联分析(表2),并对3个SNP位点进行连锁不平衡分析(图2)。
结果表明c.694G>A、c.696T>C、c.702T>C 3位点均与亚硝酸氮耐受性状显著相关(P<0.01),在耐受群体中占优势地位的基因型分别为AG、CT和CT型,且3位点SNP位点完全连锁。因此,在选育及养殖过程中,优先选择位点c.694G>A AG型、c.696T>C CT型、c.702T>C CT型个体。
表2 SNPs位点与凡纳滨对虾亚硝酸氮耐受性状的关联分析
Claims (3)
1.SNP标记在凡纳滨对虾耐亚硝酸氮性状选择中的的应用,其特征在于,所述SNP标记为如下组合:
所述SNP分子标记c.694 G>A位于SEQ ID NO.1所示序列的694bp位点处,多态性位点为G或A;
所述SNP分子标记c.696 T>C位于SEQ ID NO.1所示序列的696bp位点处,多态性位点为C或T;
所述SNP分子标记c.702 T>C位于SEQ ID NO.1所示序列的702bp位点处,多态性位点为C或T。
2.根据权利要求1所述的应用,其特征在于,所述SNP分子标记c.694 G>A的基因型为AG、SNP分子标记c.696 T>C的基因型为CT、c.702 T>C的基因型为CT是凡纳滨对虾耐亚硝酸氮性状的有利标记。
3.用于凡纳滨对虾耐亚硝酸氮性状检测的引物,其特征在于,所述引物的序列如SEQID NO.4和5所示。
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