CN114480671B - 用于鉴定细鳞鲑遗传性别的微卫星标记引物对及性别鉴定方法 - Google Patents

用于鉴定细鳞鲑遗传性别的微卫星标记引物对及性别鉴定方法 Download PDF

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CN114480671B
CN114480671B CN202210140776.8A CN202210140776A CN114480671B CN 114480671 B CN114480671 B CN 114480671B CN 202210140776 A CN202210140776 A CN 202210140776A CN 114480671 B CN114480671 B CN 114480671B
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brachymystax lenok
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佟广香
刘冰
宋建臣
刘福祥
韩世成
马凯
匡友谊
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Inner Mongolia Yinchuo Jiliao Water Supply Co ltd
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Abstract

用于鉴定细鳞鲑遗传性别的微卫星标记引物对及性别鉴定方法,涉及一种鉴定鱼类性别的微卫星标记引物对及性别鉴定方法。本发明微卫星标记引物对中上游引物F的序列为5’‑CACCCACACACGTACCAGTC‑3’,该引物对中下游引物R的序列为5’‑TGTGGAGTTCATGTGGGATG‑3’。性别鉴定方法:一、提取待鉴定细鳞鲑的DNA;二、PCR反应;三、琼脂糖凝胶电泳检测。本发明方法通过PCR扩增产物的琼脂糖凝胶电泳条带判断雌、雄,仅需提取DNA样本,可以采集鳍条或鳞片提取DNA,对鱼体伤害小,无需杀鱼,能够用于早期性别鉴定。

Description

用于鉴定细鳞鲑遗传性别的微卫星标记引物对及性别鉴定 方法
技术领域
本发明涉及一种鉴定鱼类性别的微卫星标记引物对及性别鉴定方法。
背景技术
细鳞鲑Brachymystax lenok(Pallas),属鲑形目(Salmoniformes)、鲑科(Salmonidae),细鳞鱼属(Brachymystax)是我国珍稀、名贵的冷水性鱼类,1998年被列入中国濒危动物红皮书,濒危等级为濒危,2021年野外种群被列为国家二级保护动物。
细鳞鲑3~5龄性成熟,性成熟时无第二性征,繁殖季节难以通过外形鉴定性别。细鳞鲑属于珍稀名贵冷水性鱼类,野生资源匮乏,以往鉴定性别需要取性腺,做石蜡切片,过程繁琐,需要杀鱼,而且不能鉴定性腺未发育的小鱼。鉴于以往鉴定性别方法需要杀鱼,但在野生调查过程中,不能将所有的鱼均取性腺,鉴定性别,尤其是细鳞鲑为国家二级保护动物,因此不能全面了解野生鱼性比情况。养殖过程中虽然细鳞鲑雌、雄生长差异较小,但细鳞鲑的卵粒大,与大西洋鲑相似,并且鱼卵营养丰富,可以用来做鱼子酱,商品价值远远超过雄鱼,且性成熟的细鳞鲑雄鱼精液多,人工繁殖时1尾雄鱼的精液能与3~5尾细鳞鲑雌鱼配组,因此商品鱼养殖及后备亲鱼培育时需要优化雌、雄比例,避免饲养过多的雄鱼,造成不必要的浪费。
发明内容
为更有效的在早期鉴别细鳞鲑遗传性别,本发明提供了一种用于鉴定细鳞鲑遗传性别的微卫星标记引物对及性别鉴定方法。
本发明用于鉴定细鳞鲑遗传性别的微卫星标记引物对,其中该引物对中上游引物F的序列为5’-CACCCACACACGTACCAGTC-3’,该引物对中下游引物R的序列为5’-TGTGGAGTTCATGTGGGATG-3’。
细鳞鲑遗传性别的鉴定方法:
一、提取待鉴定细鳞鲑的DNA;
二、PCR反应;PCR反应体系中扩增引物为上述微卫星标记引物对;上游引物F的序列为5’-CACCCACACACGTACCAGTC-3’,下游引物R的序列为5’-TGTGGAGTTCATGTGGGATG-3’;
三、琼脂糖凝胶电泳检测,扩增结果为0条带的是雌鱼,扩增结果为1条带的是雄鱼。
其中,雄鱼所扩增的条带为278bp。
本发明能够简单、快速的鉴定细鳞鲑遗传性别,仅需琼脂糖电泳,无需测序,省时、省力。
本发明方法通过PCR扩增产物的琼脂糖凝胶电泳条带判断雌、雄,仅需采集鳍条或鳞片样本提取DNA,对鱼体伤害小,无需杀鱼,能够用于早期性别鉴定。
附图说明
图1是实施例1中雌、雄细鳞鲑样本鉴定结果图,图中M泳道为DNA marker。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例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。
具体实施方式一:本实施方式用于鉴定细鳞鲑遗传性别的微卫星标记引物对,其中该引物对中上游引物F的序列为5’-CACCCACACACGTACCAGTC-3’,该引物对中下游引物R的序列为5’-TGTGGAGTTCATGTGGGATG-3’。
具体实施方式二:本实施方式细鳞鲑遗传性别的鉴定方法:
一、提取待鉴定细鳞鲑的DNA;
二、PCR反应;PCR反应体系中扩增引物为具体实施方式一所述微卫星标记引物对;上游引物F的序列为5’-CACCCACACACGTACCAGTC-3’,下游引物R的序列为5’-TGTGGAGTTCATGTGGGATG-3’;
三、琼脂糖凝胶检测,扩增结果为0条带的是雌鱼,扩增结果为1条带的是雄鱼;雄鱼所扩增的条带为278bp。
具体实施方式三:本实施方式与具体实施方式二的不同点在于:步骤二PCR反应体系中加入细鳞鲑18s核糖体RNA参照引物作为参照;
细鳞鲑18s核糖体RNA参照引物对中的上游引物18SF为5’-GGTCCGAAGCGTTTACTTTG-3’,下游引物18SR为5’-ACCTCTAGCGGCACAATACG-3’;
步骤三、琼脂糖凝胶检测,扩增结果为1条带的是雌鱼,扩增结果为2条带的是雄鱼。其它与具体实施方式二相同。
本实施方式以细鳞鲑18s核糖体RNA基因为参照,通过参照基因的PCR扩增结果有无,可以判断电泳条带缺失是否由于加样失误、扩增失败及DNA降解等原因造成的,能够提高鉴定的准确率,避免误判。
本实施方式中,雌鱼所扩增的条带为175bp;雄鱼所扩增的条带为175bp和278bp。
具体实施方式四:本实施方式与具体实施方式三的不同点在于:步骤二PCR反应体系为20μl,由10μl 2×PCR Dream Taq master mix、1μl DNA、10μM微卫星标记引物对上游引物F和下游引物R各1μl、1μM的细鳞鲑18s核糖体RNA参照引物对上下游引物各1μl及余量的无酶无菌水组成。步骤二PCR反应条件:95℃预变性3min;95℃变性30s、64℃退火30s、72℃延伸30s,30个循环;最后72℃延伸5min。其它与具体实施方式三相同。
实施例1
细鳞鲑每年4~5月份繁殖,在繁殖季节采集确定遗传性别的细鳞鲑雌、雄鱼样本各50尾,并采剪取鳍条样本放在75%乙醇中保存,24h后换1次75%乙醇,然后保存在-20℃冰箱备用。
用DNA提取试剂盒提取细鳞鲑DNA,将DNA浓度稀释至50ng/μl备用。
PCR反应:PCR反应体系为20μl,由10μl 2×PCR Dream Taq master mix、1μl DNA、10μM微卫星标记引物对上游引物F和下游引物R各1μl、1μM的细鳞鲑18s核糖体RNA参照引物对上下游引物各1μl及余量的无酶无菌水组成。PCR反应条件:95℃预变性3min;95℃变性30s、64℃退火30s、72℃延伸30s,30个循环;最后72℃延伸5min。微卫星标记引物对中上游引物F的序列为5’-CACCCACACACGTACCAGTC-3’,微卫星标记引物对中下游引物R的序列为5’-TGTGGAGTTCATGTGGGATG-3’。细鳞鲑18s核糖体RNA参照引物对中的上游引物18SF为5’-GGTCCGAAGCGTTTACTTTG-3’,下游引物18SR为5’-ACCTCTAGCGGCACAATACG-3’。
琼脂糖凝胶检测:PCR扩增产物用1.8%的琼脂糖凝胶电泳检测,点样5μl,凝胶成像仪成像,记录扩增结果。扩增结果为1条175bp条带的是雌鱼,扩增结果为2条175bp和278bp条带的是雄鱼,扩增结果如图1所示。
50尾细鳞鲑雌鱼样本均扩增出条1条带,50尾细鳞鲑雄鱼样本均扩增出条2条带,与取样结果完全一致。
由于本发明所用的微卫星标记引物对和细鳞鲑18s核糖体RNA参照引物对的扩增效率存在差异,因此,本发明对引物浓度进行了设计,从而保证了本发明所用的微卫星标记引物对和细鳞鲑18s核糖体RNA参照引物对在同一PCR反应体系中能够同时进行有效的扩增,得到较为清晰的条带。细鳞鲑18s核糖体RNA参照引物对的有效扩增,说明PCR扩增不存在加样失误、扩增失败、DNA降解等原因造成的电泳条带缺失,避免了误判。
本实施例的实验结果证实本发明方法能够简单、快速、准确的鉴定细鳞鲑遗传性别。

Claims (5)

1.用于鉴定细鳞鲑遗传性别的微卫星标记引物对,其特征在于该引物对中上游引物F的序列为5’-CACCCACACACGTACCAGTC-3’,该引物对中下游引物R的序列为5’-TGTGGAGTTCATGTGGGATG-3’。
2.细鳞鲑遗传性别的鉴定方法,其特征在于该鉴定方法步骤如下:
一、提取待鉴定细鳞鲑的DNA;
二、PCR反应;PCR反应体系中扩增引物为权利要求1所述微卫星标记引物对;上游引物F的序列为5’-CACCCACACACGTACCAGTC-3’,下游引物R的序列为5’-TGTGGAGTTCATGTGGGATG-3’;
三、琼脂糖凝胶电泳检测,扩增结果为0条带的是雌鱼,扩增结果为1条带的是雄鱼。
3.根据权利要求2所述的细鳞鲑遗传性别的鉴定方法,其特征在于步骤二PCR反应体系中还加入细鳞鲑18s核糖体RNA参照引物对;
细鳞鲑18s核糖体RNA参照引物对中的上游引物18SF为5’-GGTCCGAAGCGTTTACTTTG-3’,下游引物18SR为5’-ACCTCTAGCGGCACAATACG-3’;
步骤三、琼脂糖凝胶检测,扩增结果为1条带的是雌鱼,扩增结果为2条带的是雄鱼。
4.根据权利要求3所述的细鳞鲑遗传性别的鉴定方法,其特征在于步骤二PCR反应体系为20μl,由10μl 2×PCR Dream Taq master mix、1μl DNA、10μM微卫星标记引物对上游引物F和下游引物R各1μl、1μM的细鳞鲑18s核糖体RNA参照引物对上下游引物各1μl及余量的无酶无菌水组成。
5.根据权利要求4所述的细鳞鲑遗传性别的鉴定方法,其特征在于步骤二PCR反应条件:95℃预变性3min;95℃变性30s、64℃退火30s、72℃延伸30s,30个循环;最后72℃延伸5min。
CN202210140776.8A 2022-02-16 2022-02-16 用于鉴定细鳞鲑遗传性别的微卫星标记引物对及性别鉴定方法 Active CN114480671B (zh)

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