CN113337615A - 一种番红砗磲、长砗磲及其杂交子一代的鉴定引物、试剂盒和鉴定方法 - Google Patents
一种番红砗磲、长砗磲及其杂交子一代的鉴定引物、试剂盒和鉴定方法 Download PDFInfo
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Abstract
本发明公开了一种番红砗磲、长砗磲及其杂交子一代的鉴定引物、试剂盒和鉴定方法。所述的鉴定引物为F:CCATTTTGGTGCGTAGTA;R:CTTGAGGGCACCTTTTAG。本发明从150对番红砗磲的转录组微卫星引物中筛选出一对能够在番红砗磲、长砗磲中通用并且可以根据扩增条带大小区分两种砗磲,同时带型清晰、重复性好、可靠性强的微卫星引物:TC111,并在两者间的杂交子一代个体鉴定中具有重要应用价值。
Description
技术领域:
本发明属于水产生物技术中的贝类及其杂交种鉴定领域,具体涉及一种番红砗磲、长砗磲及其杂交子一代的鉴定引物、试剂盒和鉴定方法。
背景技术:
砗磲是一类热带大型海洋珊瑚礁底栖贝类,分布在中国南海的砗磲种类主要有砗磲属的库氏砗磲、无鳞砗磲、鳞砗磲、长砗磲、番红砗磲、诺亚砗磲以及砗蚝属的砗蚝。砗磲属于高经济价值物种,是热带海洋生物和渔业经济的物质基础,提供水族观赏、工艺材料、水产食品等资源。另外砗磲也是热带岛礁生态系统的主要组成部分,为众多海洋生物提供了栖息地、保护地、繁殖和索饵场所,具有极其重要的生态价值。但由于其固着生活在热带浅层透明海域的习性使其极易被捕捞,尤其是自20世纪六七十年代始,随着潜水设备和机动船的普及,砗磲资源受到严重破坏,甚至在许多海域有些品种已经绝迹。1983年《濒危野生动植物种国际贸易公约》(CITES)将砗磲所有种类列为世界稀有海洋生物加以保护,对其国际贸易加以管制。
番红砗磲是个体较小的砗磲种类,外形呈卵圆状,足丝孔较宽。长砗磲的个体也较小,外形成呈长卵圆形,是砗磲中分布最广的种类。番红砗磲和长砗磲都是水族馆中优先选择的观赏种类且存在造礁、护礁功能,具有重要的经济价值和生态价值。近年来随着我国砗磲野生资源的迅速减少和市场需求的不断扩大,番红砗磲和长砗磲将成为重要的养殖经济种类。
发明内容
本发明的第一个目的是提供一种番红砗磲、长砗磲及其杂交子一代的鉴定引物。
经过摸索和研究,我们已经熟练掌握了番红砗磲和长砗磲的大规模人工繁育和养殖技术。为了砗磲新品种培育的需要,我们成功进行了番红砗磲和长砗磲的杂交试验,得到了一批杂交子代。在成功获得番红砗磲和长砗磲杂交子一代的基础上,我们发明了一种利用遗传标记对杂交子一代进行分子鉴定的方法,可以简单高效地对番红砗磲×长砗磲杂交子一代进行大规模物种鉴定。
本发明从150对番红砗磲EST-SSR引物中筛选出1对微卫星(SSR)引物对番红砗磲、长砗磲及其杂交子一代各40个个体的基因组DNA进行PCR扩增,通过扩增图谱可以简单高效的对真正杂交子一代进行分子鉴定。
本发明的番红砗磲、长砗磲及其杂交子一代的鉴定引物(TC111),包括:
F:CCATTTTGGTGCGTAGTA,核苷酸序列如SEQ ID NO.1所示;
R:CTTGAGGGCACCTTTTAG,核苷酸序列如SEQ ID NO.2所示。
本发明的第二个目的是提供一种番红砗磲、长砗磲及其杂交子一代的鉴定试剂盒,其包括PCR检测试剂和鉴定引物,所述的鉴定引物为:
F:CCATTTTGGTGCGTAGTA;
R:CTTGAGGGCACCTTTTAG。
本发明的第三个目的是提供一种番红砗磲、长砗磲及其杂交子一代的鉴定方法,包括以下步骤:提取待鉴定样品的基因组DNA,然后用上述鉴定引物对其扩增,得到扩增产物,对扩增产物进行电泳,若仅在240bp~260bp之间出现一条带,则为番红砗磲,若仅在200bp~230bp之间出现条带,则为长砗磲,若同时在240bp~260bp区间和200bp~230bp区间内各出现1条带,即为番红砗磲与长砗磲杂交子一代。缺少其中的任意一个条带均为假杂种。
所述的扩增,其PCR反应体系为:PCR反应体系的总体积为25μL,其中各种成分及终浓度分别为:DNA模板50ng,Buffer 10mM,引物各0.2mM,dNTP 0.2mM,Mg2+2.0mM,Taq DNA聚合酶1U,用灭菌水补足25μL。
所述的扩增,其PCR反应程序为:94℃预变性5min,94℃变性30s,50℃退火30s,72℃延伸1min,变性至延伸三个步骤重复30次,72℃延伸10min。
所述的对扩增产物进行电泳是PCR扩增产物经8%非变性聚丙烯酰胺凝胶电泳,220V稳压电泳2.5小时,然后用0.1%AgNO3溶液染色10min,银染后用2%NaOH、0.4%甲醛、0.2%NaS2O3、dH2O 1000mL显色,至条带清晰为止,置于UMAX扫描仪下扫描。
由于微卫星引物TC111产生的番红砗磲特异条带在240bp~260bp之间,长砗磲特异性条带范围在200bp~230bp之间,两者之间没有等位基因重叠区,可以明显区分两种砗磲的个体。该标记在番红砗磲×长砗磲杂交子一代鉴定中的应用方法为:样品只有同时在240bp~260bp区间和200bp~230bp区间内各出现1条带,即同时拥有亲本的1条特异性条带的个体才为真正的番红砗磲与长砗磲杂交子一代,缺少其中的任意一个条带均为假杂种。
本发明从150对番红砗磲的转录组微卫星引物中筛选出一对能够在番红砗磲、长砗磲中通用并且可以根据扩增条带大小区分两种砗磲,同时带型清晰、重复性好、可靠性强的微卫星引物:TC111,并在两者间的杂交子一代个体鉴定中具有重要应用价值。
由于幼体阶段的番红砗磲和长砗磲根据形态特征难以区分,杂交种更加难以鉴别。而利用本发明方法,可以快速、准确地鉴定出番红砗磲×长砗磲杂交子一代,具有重要的实际应用价值,同时本发明具有方法简单、结果直观、准确有效、不受环境及发育时期影响、成本低廉等优点。
附图说明
图1是引物TC111在番红砗磲、长砗磲及其杂交子一代部分个体间的电泳染色对比图谱,1~5是番红砗磲个体,6~10是长砗磲个体,11/13/15/16:真正杂交子一代;12/14:非真正杂交子一代。
具体实施方式
下面通过具体实施例和具体实验对本发明所述应用做进一步说明。但不应将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明内容所实现的技术均属于本发明的范围。
实施例1:
从番红砗磲、长砗磲及其杂交种足丝孔中剪取少量外套膜组织,采用酚-氯仿抽提法提取番红砗磲、长砗磲及其杂交子一代各40个个体的基因组DNA。具体是将外套膜组织充分剪碎,用干净的滤纸吸除水分后放入1.5mL离心管中,再加入400μl裂解液和10μl蛋白酶K(10mg/ml),在振荡器上混匀,50℃水浴消化3~5h,直到裂解液澄清为止。加入等体积饱和酚(200μL)、氯仿/异戊醇(24:1)(200μL)混合液抽提三次。用1mL的无水乙醇沉淀DNA,经70%酒精洗涤,室温晾干后溶于100μL的超纯水中。紫外分光光度计检测DNA浓度和纯度,并用1%的琼脂糖凝胶电泳检测DNA的完整性。以提取的三种砗磲基因组DNA为模板,进行PCR扩增反应:PCR反应体系的总体积为25μL,其中各种成分及终浓度分别为:DNA模板50ng,Buffer 10mM,引物各0.2mM,dNTP 0.2mM,Mg2+2.0mM,Taq DNA聚合酶1U,用灭菌水补足25μL。其中引物序列为:
F:CCATTTTGGTGCGTAGTA;
R:CTTGAGGGCACCTTTTAG;
反应程序为:94℃预变性5min,94℃变性30s,50℃退火30s,72℃延伸1min,变性至延伸三个步骤重复30次,72℃延伸10min。
PCR反应结束后,取0.5μL扩增产物在8%非变性聚丙烯酰胺凝胶电泳,22V稳压电泳2.5小时,电泳结束后将凝胶从玻璃板上取下放入托盘中,用去离子水清洗两次,然后加入0.1%AgNO3溶液(AgNO3 1g,dH2O 1000mL)染色10min。染色后加入去离子水再次清洗一次,加入显色液(2%NaOH、0.4%甲醛、0.2%NaS2O3、dH2O 1000mL)显色,直至条带清晰为止,于UMAX扫描仪下扫描后,电泳结果如图1所示,微卫星引物TC111产生的番红砗磲特异条带在240bp~260bp之间,长砗磲特异性条带范围在200bp~230bp之间,若在240bp~260bp区间和200bp~230bp区间内各出现1条带,则该个体为真正的番红砗磲与长砗磲杂交子一代,缺少其中的任意一个条带均为假杂种。
序列表
<110> 中国科学院南海海洋研究所
<120> 一种番红砗磲、长砗磲及其杂交子一代的鉴定引物、试剂盒和鉴定方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccattttggt gcgtagta 18
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cttgagggca ccttttag 18
Claims (6)
1.一种番红砗磲、长砗磲及其杂交子一代的鉴定引物,其特征在于,包括:
F:CCATTTTGGTGCGTAGTA;
R:CTTGAGGGCACCTTTTAG。
2.一种番红砗磲、长砗磲及其杂交子一代的鉴定试剂盒,其特征在于,包括PCR检测试剂和鉴定引物,所述的鉴定引物为:
F:CCATTTTGGTGCGTAGTA;
R:CTTGAGGGCACCTTTTAG。
3.一种番红砗磲、长砗磲及其杂交子一代的鉴定方法,其特征在于,包括以下步骤:提取待鉴定样品的基因组DNA,然后用权利要求1所述的鉴定引物对其扩增,得到扩增产物,对扩增产物进行电泳,若仅在240bp~260bp之间出现一条带,则为番红砗磲,若仅在200bp~230bp之间出现条带,则为长砗磲,若同时在240bp~260bp区间和200bp~230bp区间内各出现1条带,即为番红砗磲与长砗磲杂交子一代。
4.根据权利要求3所述的鉴定方法,其特征在于,所述的扩增,其PCR反应体系为:PCR反应体系的总体积为25μL,其中各种成分及终浓度分别为:DNA模板50ng,Buffer 10mM,引物各0.2mM,dNTP 0.2mM,Mg2+2.0mM,Taq DNA聚合酶1U,用灭菌水补足25μL。
5.根据权利要求3所述的鉴定方法,其特征在于,所述的扩增,其PCR反应程序为:94℃预变性5min,94℃变性30s,50℃退火30s,72℃延伸1min,变性至延伸三个步骤重复30次,72℃延伸10min。
6.根据权利要求3所述的鉴定方法,其特征在于,所述的对扩增产物进行电泳是PCR扩增产物经8%非变性聚丙烯酰胺凝胶电泳,220V稳压电泳2.5小时,然后用0.1%AgNO3溶液染色10min,银染后用2%NaOH、0.4%甲醛、0.2%NaS2O3、dH2O 1000mL显色,至条带清晰为止,置于UMAX扫描仪下扫描。
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