CN112725347B - 许氏平鲉雄性特异基因序列和伪雌、伪雄鉴定引物及方法 - Google Patents

许氏平鲉雄性特异基因序列和伪雌、伪雄鉴定引物及方法 Download PDF

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CN112725347B
CN112725347B CN202110142976.2A CN202110142976A CN112725347B CN 112725347 B CN112725347 B CN 112725347B CN 202110142976 A CN202110142976 A CN 202110142976A CN 112725347 B CN112725347 B CN 112725347B
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齐洁
王成龙
贺艳
张全启
宋伟豪
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Abstract

本发明涉及一段许氏平鲉雄性特异基因序列和伪雌、伪雄鉴定引物及方法；属于分子生物领域，所述雄性特异基因序列如SEQ IP NO.1所示，片段为17307bp，显示出雄性和伪雌特异性扩增。本发明还运用特异基因序列中任何一段序列设计特异性引物、运用PCR扩增技术在雌性个体、雄性个体、伪雄个体和伪雌个体的基因组DNA中进行特异性扩增，从而达到区分许氏平鲉雌、雄、伪雌和伪雄的目的。为降低制种成本、提高许氏平鲉养殖的经济效益、促进许氏平鲉养殖业的可持续发展具有重要意义。

Description

许氏平鲉雄性特异基因序列和伪雌、伪雄鉴定引物及方法
技术领域
本发明属于分子生物领域，具体的说是一种许氏平鲉性别特异基因序列，具体涉及雌雄鱼遗传性别、伪雄鱼和伪雌鱼的性别鉴定及性别控制育种的应用。
背景技术
许氏平鲉，Sebastes schlegelii(Hilgendorf,1880)，属鲉形目平鲉科平鲉属的一种暖温性底层鱼类，分布于西北太平洋近岸的温水性鱼种，是我国重要的经济鱼类之一。其同龄雌雄鱼中存在着雌性个体大于雄性个体的生长二态性，因此在许氏平鲉中进行全雌苗种的选育是提高生产效益非常有效的措施。全雄苗种的培育虽然不及全雌苗种有更直观的经济效益，但对于许氏平鲉性控育种的理论研究也是非常有价值的。在全雌苗种和全雄苗种培育过程中，伪雄鱼和伪雌鱼的鉴定和筛选是该项目实现的关键核心技术。伪雄是指许氏平鲉在表观性状上表现为雄性，但在遗传上为雌性；伪雌是指许氏平鲉在表观性状上表现为雌性，但在遗传上为雄性；要制备遗传上的全雌苗种、全雄苗种，就必须要对亲本个体进行伪雄、伪雌的鉴定。但目前缺少对许氏平鲉伪雄和伪雌的鉴定技术，从而制约了全雌苗种和全雄苗种的培育。
发明内容
本发明要解决的技术问题在于提供一段许氏平鲉雄性特异基因序列和伪雌、伪雄鉴定引物及方法；运用特异基因序列中任何一段序列设计特异性引物、运用PCR扩增技术在雌性个体、雄性个体以及伪雄个体和伪雌个体的基因组DNA中进行特异性扩增，从而达到区分许氏平鲉雌、雄、伪雌和伪雄的目的。
本发明是通过如下技术方案来实现的：
一段许氏平鲉雄性特异基因序列，所述基因的序列为SEQ IP NO.1所示。所述雄性特异的片段为17307bp，显示出雄性和伪雌特异性表达。
基于上述许氏平鲉雄性特异基因序列建立的雌性、雄性、伪雌、伪雄鉴定引物，所述引物为SEQ IP NO.2和SEQ IP NO.3或SEQ IP NO.4和SEQ IP NO.5或SEQ IP NO.6和SEQIP NO.7或SEQ IP NO.8和SEQ IP NO.9。
本发明还提供利用所述引物进行伪雌、伪雄鉴定的PCR方法，利用SEQ IP NO.2和SEQ IP NO.3引物进行PCR反应体系如下：5×PrimeSTAR-GXL-Buffer10μL dNTP(各2.5mM)4.0μL，正向/反向引物(10mM)各1μL，cDNA模板5.0μL，GXL Polymerase 1μL，dd H2O补齐到50μL。
反应条件为：98℃10sec，68℃10min，30cycles。
2kb<目的片段<10kb，利用引物SEQ IP NO.4和SEQ IP NO.5或SEQ IP NO.6和SEQIP NO.7或SEQ IP NO.8和SEQ IP NO.9进行PCR反应体系如下：10×LA buffer 5μL，dNTP(各2.5mM)4.0μL，正向/反向引物(10mM)各1μL，cDNA模板2.0μL，LA Taq DNA聚合酶(5U/μL)1μL，dd H2O补齐到50μL。
PCR反应条件为：94℃,3min；94℃,30s；56℃(视引物退火温度而定),30s；68℃,5min(1kb/min)30cycles；68℃,7min；12℃,∞。
PCR反应结束后，制备1％的琼脂糖凝胶进行电泳检测。
本发明还提供上述许氏平鲉雄性特异基因序列在鉴别雌性、雄性、伪雄鱼和伪雌鱼中的应用。
本发明还提供上述任一组引物在鉴别雌性、雄性、伪雄鱼和伪雌鱼中的应用。
本发明还提供利用所述许氏平鲉雄性特异基因序列制备的鉴定雌性、雄性、伪雄鱼和伪雌鱼的试剂盒。
本发明还提供所述任一对引物制备的鉴定雌性、雄性、伪雄鱼和伪雌鱼的试剂盒。
本发明与现有技术相比的有益效果：
1、结合许氏平鲉的生理性状，能够将表观上的雌鱼和雄鱼区分开，再利用本发明任何雄性特异的片段及其包括的序列中的任一组鉴定引物，都能够将伪雄鱼和伪雌鱼从雄鱼和雌鱼中区分开，本发明方法鉴定方法简单、高效；
2、利用本专利中的雄性特有片段鉴定出的伪雄许氏平鲉(性染色体为XX)与普通雌性许氏平鲉(性染色体为XX)交尾繁殖的后代性染色体均为XX，普通方式养殖即可获得全雌许氏平鲉，1代即可实现全雌许氏平鲉规模化制种。
3、利用本专利中的雄性特有片段鉴定出的伪雌许氏平鲉(性染色体为XY)与普通雄性许氏平鲉(性染色体为XY)交尾繁殖的后代性染色体为1XX：2XY：1YY，可筛选出超雄鱼YY，进而YY超雄鱼与XX普通雌性个体杂交即可获得XY的全雄鱼。
本专利为许氏平鲉的全雌苗种和全雄苗种的繁育奠定坚实的理论基础，为降低制种成本、提高许氏平鲉养殖的经济效益、促进许氏平鲉养殖业的可持续发展具有重要意义。
附图说明
图1为4对引物的扩增产物电泳图谱；
图2为分段扩增序列与雄性特异性基因序列的关系图；
图3为许氏平鲉雄性、伪雌特异染色体特异区域分段验证1；
图4为许氏平鲉雄性、伪雌特异染色体特异区域分段验证2；
图5为许氏平鲉雄性、伪雌特异染色体特异区域分段验证3。
具体实施方式
下面通过实施例来对本发明的技术方案做进一步解释，但本发明的保护范围不受实施例任何形式上的限制。
实施例1
1、DNA的提取
取出保存在-80℃的许氏平鲉肌肉组织放入1.5mL的EP管中，加入TNES溶液和蛋白酶K并在56℃水浴5h，加入平衡酚/氯仿/异戊醇混匀，13000rpm离心20min；吸取上清液，再加入氯仿/异戊醇(24：1)，轻轻晃动20min，13000rpm离心20min。重复一次，去除肌肉DNA中的残留酚。吸取上清，加入-20℃预冷的异丙醇和3M NaAc，置于-20℃3h或过夜，4℃13000rpm/min离心10min。取上清，加入2倍体积的预冷的70％乙醇，4℃13000rpm离心10min。留沉淀，加入预冷的无水乙醇，4℃，13000rpm/min离心10min。去掉上清液，加入无水乙醇洗涤，加入1×TE缓冲液(预先按浓度要求加入RNaseA)溶解DNA沉淀。
2、许氏平鲉DNA质量检测
1)DNA浓度检测
使用Nanodrop对提取的DNA样品进行浓度检测，吸取1μL的TE溶液进行调零测试，再吸取1μL的待测DNA样品进行浓度检测，并读取A260/A280和A260/A230的OD值，OD260/OD280≈1.8>1.9，表明有RNA污染；OD<1.6，表明有蛋白质、酚等污染。
2)电泳检测DNA完整性
1×TAE中溶解琼脂糖凝胶，取30mL的琼脂糖凝胶并加入Gelred染液混匀，倒入预先洗干净的胶槽中，插上梳子，待胶凝固后吸取DNA溶液1μL，加入2μL的loading buffer，3μL的ddH2O，180v电泳20min。
3、许氏平鲉性别特异标记的扩增
以Ss_male-17K-fw和Ss_male-17K-rv为引物，以提取的多个表观上已经进行雌雄鉴别的许氏平鲉DNA为模板，模板终浓度是20ng/μL，用PrimeSTAR GXL DNA Polymerase(Takara公司)对筛选出的雄性特异片段进行扩增；另外分别以Ss_male-5K1-fw和Ss_male-5K1-rv，Ss_male-5K2-fw和Ss_male-5K2-rv，Ss_male-5K3-fw和Ss_male-5K3-rv为引物，以提取的多个个体的许氏平鲉DNA为模板(20ng/μL)，用LA Taq DNA聚合酶(Takara公司)对筛选出的雄性特异片段的中间区段进行扩增，PCR的反应体系和反应条件如下所示。雄性特异区段PCR扩增引物如表1。
以Ss_male-17K-fw和Ss_male-17K-rv为引物，17037bp长度的目的片段PCR反应扩增条件如下：5×PrimeSTAR-GXL-Buffer 10μL dNTP(各2.5mM)4.0μL，正向/反向引物(10mM)各1μL，cDNA模板5.0μL，GXL Polymerase 1μL，dd H2O补齐到50μL。
反应条件为：98℃10sec，68℃10min，30cycles。
分别以Ss_male-5K1-fw和Ss_male-5K1-rv，Ss_male-5K2-fw和Ss_male-5K2-rv，Ss_male-5K3-fw和Ss_male-5K3-rv为引物，2kb<目的片段<10kb，使用LA Taq DNA聚合酶进行扩增，具体反应条件和体系如下：10×LA buffer 5μL，dNTP(各2.5mM)4.0μL，正向/反向引物(10mM)各1μL，cDNA模板2.0μL，LA Taq DNA聚合酶(5U/μL)1μL，dd H2O补齐到50μL。
反应条件为：94℃,3min；94℃,30s；56℃(视引物退火温度而定),30s；68℃,5min(1kb/min),30cycles；68℃,7min；12℃,∞。
表1雄性特异区段PCR扩增中用到的引物。
Figure BDA0002929963820000051
Figure BDA0002929963820000061
PCR反应结束后，制备1％的琼脂糖凝胶，吸取5μL的产物与loading buffer混合均匀后进行电泳检测和测序验证。
以Ss_male-17K-fw和Ss_male-17K-rv为引物，扩增出的目的片段17307bp，显示出雄性和伪雌特异性扩增(图1)。并且对此雄性特异区段进行分段式PCR扩增(分段扩增产物位置如图2所示)，均显示出雄性和伪雌特异性扩增(图3-5)，说明结果的正确性。且Ss_male-5K1-fw和Ss_male-5K1-rv，Ss_male-5K2-fw和Ss_male-5K2-rv，Ss_male-5K3-fw和Ss_male-5K3-rv 3对引物扩增出的序列正好覆盖许氏平鲉雄性特异基因序列。
许氏平鲉雄性特异基因序列片段，总长17307bp，序列如下：
agtccatagttcatccgtccaggagagttactgtagcagccacgctgctgcatgtattgtcccagtaagtctccaccacatcatttagctgcgaggttgtcagctgtttgtctgcctggcgtacttagtgagcacggcgtagctttaccaagtgtccggtcgagagacagagcgtgtgtgtatggcagcggtgtactcttgtcaacctggcgttatgcacataggattaaaaaaatcctacatgctatgtttttaatgttcctacttcttggatctgtcttcttatgtttttttataaagcacattgcatggcatatttttgtatgaaatgtgtaatataaataaagtttaattttgccacaattcactaaatgagtgaaatgaattggagcccttgcatgcgcatcaagagccctgtaggactcacaaattgatctgcaatttggttgcaacaaaacgtgaaactgccaccatttgcagtctgtgcttttgtgaaacaggctccagatgttcagactcgacaactgtggccttgatgacacatctcagctttagttggttaatttgatgtcgcattatcattaaagcaaagtaactgtcatatcaaatttactcgagatgcttaagacaccacagttaaaactaaagcagccttccaccatgtgtgcagtagtaagtgtgtctacatgcatgacaggtgccctttgtttgattgatatcatcatgattgtggcctaaagtcttgtgattgcttcactgatgttattcatgttggaactttgcgatacctcagggattcatgaaccgccgaatgctgctgctgacagactaaaaacatgcagctcagactggaatttacgagcaggggataagcatgtgtactttgtattttttccacttaactccgcctttccgtgaggatctctccatgcttgtgtgtgtgtgggtgtgagatattccagaggtcattgtctctgggtccagacggctccgactcaggttctcagtgtggcttcactaatctgccgtaattatgtttcacagccagctcactcacagtcacacacatacattgactccctgtctctttgactctctctcttttttccctctttttctcaagagaccagagtcgtggtcgcctcaggacggctagcagtgcatgtgtgtgtgaatgtatatgcgtataacacagtgttacccatgcaatggggctatatcctaccctgctgacacacaactcaaaggactcctttcacagcttcagggagaaaaaagagatgccttcgtgacttgccatatagtgaaaggggtttccaatgcaggagtcatccgctgcacgcatcacactcatctccccccgccctgcccctgtgacgcatatcttccttgtgtttcatcaaccgtcgcacctcagggtgtgtggtgacgcaggagatgtgtgggcatcctaaatccagactacctgaccagattcatgtgagttcagttcagggggaagccctcgctataaacaattttattcctttgacacacaaatgggtgtttgttgtaaagatatagacattttaatgagttggaaacactgagggatccaatgatcggttgagcttgtaaaggataattgtggtttaggcaaggcaaggcaaggcagctttatttgtatagcacatttcaacaacagggcaattcaaagtgctttacagaaacattcaagaacattgtgacaaaatgcaaaagaacattaagaaataattaaaacagttataaaaacataaaaacattaaagattagaaaataaaaacaagctaaaaataaaagctaggatggaagctaaaattgcataaaactcaaaagagtaaaagttatagtgcagtgtcagaataaaaggcacccgcaaacaggaaagttttaagctttgttttaaaagaagtgagagttggagcggtcctgcaggtttctgggagcttgttccagatatttggtgcataaaaactgaatgctgcttctgcatgtttagttctgaatctggggacactaagcagacctgatccagatgacctgagaggtctggatggttcataacacagcataagatcagaaatgtattttggcgctaaaccatgtagtgctttgtaaaccagcagcagtattttgaagtcaattctctgagagacaggcagccagtgtagagacttcagaactggactgatgtgatccactttcttggtcttagtgaggactctagcagcagcgttctgaatcagctgcagccgtctgatcgattttttaggaagaccggtaaagacgccgttacagtagtcaagtcggctgaagataaatgcatggactagtttttccaaatcctgctgagacatcagtcctctaattcttcttatattcttcaggtgatagaaggctgtctttgtaattgtcttaatatggctgttaaagcttaggtctgagtccatgactacaccaaggtttctggcttggtctgagctttttaacatcacagattgtaggtgagcactaacttttaatctttcttttttggctccaaaaactacgacctcagttttgtctttgttcagctgaagaaaattctggcacatccaattattgacttgttcaatgcactcactcagtgcttgtattggactgtagtctcctggtgatagagttatgtaaatttgtgtgtcgtctgcatagttatggtaatttattttgttgttctcaaaaatctgacccagtggaagcatgtagatgttaaacaaaagaggccccaagatggagccttggggaactccgcacgtgattttgttcagctcagatttgcaattacctatagacacaaagaagtccctgtcttttaggtaggatttaaaccagctgagtgctatgccagaaataccgacccagttttccagtcggtctagtaatatattatggtcaaccatgtcgaatgcagcactgagatccagtaaaactaaaactgtagttctaccactgtctgtgttcaatcggatgtcattaaagaccttaacaagagcagtctcagtgctgtggttttgtcgaaaacctgactggaagacatcaaaagagttgtttagtgccagaaagctatttaatttttgaaaaactgttttttcaattattttagacaagaatggaagatttgatatcggtctgtaattatttactattgatgagtccagattgttctttttgaggagtggtttgatgacagcagttttcagggcctgtgggaagactcctgaaacaagagatgcattaacaatctgtaaaagctctgagaccatacaatctgatacttttttgaaaaggcctgttggcagaatgtcaaggcagcatgaggaggattttagatgttgtatgatttcctccaggtatgtctgatttattggataaaactgtgtcatggtatttgcgccaggtggacgcaggggaaacaaacatcctgtacctgatatggaggaagtgactgcttgtctgattttgtgaattttttctgtaaaaaaggaagcaaattcattgcaggccctggaggataaaagttcagaggctactgacacaggagggtttgttagcctgtcgacagtagcaaacagggcacgtgcattgttattgtttttggtgatgatgtcagagaagaaggaccgccttgcatttttcagttctgaattataaatgcaaagtctctctttataggtgtcataatgaacttgtagatttgttttccgccactggcgttcagcttttcgacactctcttttttcatttcttaccaacatggtatttctccatggagattttttcttaccagagacaatcttcagttttgggtgcaatggtatcaataacatttgtcattttagaattgaaattatctacaagctcattgacagaggtccaggggagggtgggtgtggaagagaaagcctgaacaaattgatcactggtattttcagtgatatacctttttgtgattacctctgtcttatcatttctgtgcacaggaagagtactctcaaagaatacacatgaatgatcagacagagcaacatccatcactaaaaccttggaaatgttcaggccctttgatatgattaagtccaaagtgtgtcccctattgtgtgtgggctctgtcacatgctgactcattccatagttatcaagaacctgacacagttcattggcccctctgtcctgttggttgtcaacatggatgttaaaatcgccaacaataactacacagtcaaagtcaacacaaattacagacagcagttcagtaaaatcatcaaaaaagtttgcacagtatttaggtggcctgtagatattgagaaacaaagctcgagaagaggagttcagctgaagagcgacatattcaaaagaaccaaaatgtccataagatatctgtttgtattggagagaatcattaaacaatatggcaactccacctcctttcttatgcattctagcttcgctcataaaactaaaattgggaggagttgactcgataagaacagttgcactgttattttggtctaaccaagtttcagttaaaaacataaaatcaagcttgtgctcaataataaaatcattgattaaaaaactttttcctgccaaagacctgacgtttaacagggctagctttaatgtgctagaagaattattattattttttgggacatggtttggctggcaaggaatcaatgctaaatttgataaattagcactatcagtaaaaacatcaacattttgtaaaggaacatccttatcaggctgcggagacagaggctgattccattgtggaggggatggaggtggtgggtgtcgtgcttgtggagatagtgccaaagttgttcgaggtcgacctcgtgggggaatcatgggagagaaaatgggtgcagggcggggagtaagtttagttccagcattgaccagctccttcatgtggtcagtgaactccaagaggggggagtttttcatcaccaaaaacaataacaatgcacgtttattacagcttctgtcttattttggtaggtttggccatctttcctactcaccaagttaagtactgagatctggaaatgcagcaacatcactaaggaggtcaagaaacatgagcagtcatgtgatcatacaggttgcaaacaaatcccctgaactgattttggaaaagaaaacaaagatgacttatttcccaaactgtaaacacccatcacagtgtaaaataaaagggtgtgacgccaaggggcggtatgtgacgagttgggatgagaatgtgttggttggactttttgccatattcccatatctcagcaattactttggttagagcaaagctatcattagtattaggaaaaatggcccgacctatgaaagtgagacttataattaaccagaaccatcctataaagtcagatttataatgctagattaatatgttttcctatgtccttttccttcccccagtttttccactgtgtagcactcattataccattccacattatcctagagtttttgcataatagtaagaaattatcatcacaataaaaagaaagcccatggattcatttgatgctgaatagctgagcctgaaatttccatcctcattgtttccacaaaacactcttgaaccactccctctgcctaagcatgactcgtgaacacataaaatgtttatctttggttttgccaccaaaacaaacagttttcatagtacaataaatctaaaactagaaaatgtagttatactaccacaaggacaaaaacgataattatcaaagtttgcaagtttatatttgtgtggcacttttcacacaattgtaaggcacagaaaacaacagttatgatacaaacacacagtgttgacaccctcaaaggagaacacacaggatagttagagatatcaggtttcattcttgctgtacatcattaaaaaacactttaaagtaaatccaaaccttaatttggttatgaccattttatttagttgttatttaaaggtacagtgtgtaggatttggtggcatctagtggtgtggttgcagattgcaaccaactgaatacccctccgctcactcccccttgtccaatactgcggtaacgtgagctgccgagtgcgaaaccgtggtaacgctgttcaactcgctcagaggccatccttaccatgataacactactttaggagcaatggtacaacggtttagcactatgcagctcattaccacagtttcacaagcgtgtcggagaactacggtggtcttcaggtaacctaaaaacatgaatggctctctctagagccagtgtttggtttgtcggttctgggctactgtagaaacatggcgtagcaacatgctccctatgtagatatgaagggctcattctaagctaatgaaaacacaatgattcttagtttcagaagtgacagaaagaatttaaagtctcctccagaaaataatctctcaaaaatatgtcgccttttggactttattgtcttatggaaatcttttcatggaaattaaaatacgtaaaatacatttcaagtgagttttaaaagacagcagtgagcgtggtaagatcacagaaaaagatatgtggtataaacaaggaaatgaaaagtgatattatatatcatttcatatgacagaatgggagcatgcacataaggaaacaaaacatataatgaacccaaaacaataattaaaaatagttaaaaaataattaacttaatttgatctaactattaattgaccctgactgcgccttctataataacacagctctttaaattgcaggtatcataactataatgaagagttgtggttggcactcatttatattgcagtgatgtatgttgaagtgaactctatttaacccgtagtaatttattcacagggcaggttggccttgcaggacaaacctgtttcttttataaggaactctgtggagcctttggcccgtgtgtcagtttgtgtgtgaaccccactgagctcattaaactgttaatatcgctgtcttcctcttcacaaaacctgaaaccccacaagatattgcatgctcagtagctgaaacacacagcccggcacacacacaaagcctcataaagcttttctgactaaatgcgtgactggctgcttgcttaaacacacacacactcacacacagacgtacacacacacggctgcagcacagcgctcttatttaaactgatgtgagtgctgtcacgacatccgactagtcctatttgttccacagaagccgtggcgtgagagagcagggtcaccccactagctctcatgcaaatccattcagcacacacgtgcgcgcacacacacacacacacacacacacacacacacagacacagacacacgcacacacttaatagtcctgcagataagcaaaaagacagacagatagacaggcaggcaagcagagagagacgagtggacagaaaaaacattgagcagacagaagaacaaaatgaagggcagacagacagacagatagacagacaaacaaacatcaaatcctaattaaagctgcaagcagcgatgaacgggccctcgcccacacaggcacgtctgcagaacgcgcacgtcggggtacccgctgtcgttcattcccgtgaaagtcggaaactgcacgcaacagttagagtgaattttctacttggagcatgtggcgctggaaatgaactgtgagaacatgtggggcatcctttaaagcacttctatgagggtggctggcagtatagaaccatttcctattgtcagtaggtggcgctatgactttcactcataataccacgtgtatgtcttcaggccaagattcttataaaacatgtcaaatttggggaagattggacaatgtatagtcaatttacaacgacttcctggcggagatcaaggatcacagcgtccttatcgcgctgttcctggcagagatcaagtatcacagcgtccttatcgcgctgctcctggcggagatcaaggatcacagcatccttatcgtgctgctcctggcggagatcaaggatcagagcgtgctgttcctggcggagatcaagtatcacagcgtccttatcgcgctgctcctggcggagatcaaggatcacagcgtccttatcgtgctgttcctggcggagatcaaggatcagagcgtactgttcctggcggagatcaaggatcacagcgtccttatcgtgctgctcctggcggagatcaaggatcagagcgtgctgttcctggcggagatcaaggatcacagcgtccttatcatgctgctcctggcggagaccaaggattacagcgtgctgttcttggcagagatcaaggatcacattgtccttatcatgctgctcctggcggagaccaaggattacagcgtccttatcgtgctgcttctggcagagatcaaggatcacagcgtccttatgctgctggggaatgttatcatgttggggaagattggacaatgtataatcaatttataacaatttcctgtttcctgttggaggcgctatgactttcactcaaaatggcacatatatgtcttcaggctgggactattatccagcttgtgaaatttggggcagattggactatgtaaagtcaggttaaaacagcttcctgtgtcattgcgaaacatcaaaattcgccacgccgccccaagaacgccgttgcacgaaaacacaaaagcttcgcaatttagcatcgtgaaggtgttgagattctgctgcccgactttgagatggatcgcttgtatcctctaggaggagtatcgcaaagttccatacctaaaaagagacaaaatggcgtttttgcacatgacgtatgacatcaccgttcgagcgatcaaatatttcttcgcaatttagcatcacagtagtttgatggttgtaccacccaaatttgacgcggatccgataaactctctaggaggagttcgtaaaagtatgcataaaatacatgaaaaacgcacaaaatccaatatggccgacttcagggtgggcggagctaatgaaacccgatgaggaatatgtttcaaataatgagcaggatgtacctacaacctttcatgaatataggatcaactttgaccaaactatggccttccacgcgttagggggcgctaaagagcgggctaaacgcactgaacctaatggctgtacatttacataaagttcacaggtgtcaatcattttgccaagtttcatgagttttcgagtacctcaaggtatgttcaaaacctaaaagacataagggggcgctagagagccaacatgccatgcccaagcaaaatttcaccactaaatcaaagtaattatgagtttggatgtgcatgtaaattttcatgagtttttgtgcatcctaaagtcttcaaacatgtgttcgtaaattttaaaatcatgcaggaaatccaaattggctgacttcctgttggccgaaaaaatctcaaaaatatttaacacgaacttcaagatgtatgcaattacatacttgaatttcgtgaagatcgaagaaactttccctgaaaaactgcctacattagggggcgctatctcgcccgctggcgacgcccaagtcgaatcactacagaactttgaatttcgcgccacttctgacgcatattccacttttggtgagtttttggggaaggaaaaggtcccaaaaacgtgatattccagcagaaaaagaataattaaagctgcaagcagcgatgaacgggccctcgcccttgcgggcacgtttggggaacacgcacgtcggagcacgtacatatctttaggccaagactcttttaaaacatgtcaaatttggggaagattggacaatgtatagtcaatttaaaacaacttgctgtttcctgtaggggcgctatgactatcacgcataataccatgtatatgtcttcaggccggcacccttataaaacatatcaaatttggggaagattggaagaagatt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aaaatgctgctgaacaacacaatagtcaatagttatatttcaatagggaacatgaaagccaagaccaccttatatcagttcctgttcctccagagaaaagcagaagtggaatcatgctgatgttacttcctgtttttctgccaacaaggatatcttttaggttttggtctgcaaagagaggaaatgtgagaagagtgcacacgaaacaagaaattctatcttcattgtcagccctgacctatttggtgccctaggcaagattttagctggtgcccctccacctctctttgcacagcgtgacctgccgggaggttctcagtgttatttttgtgcatacaggatgtgtagcctcttaacctgcttataataaagctaccatattgatcttgattataagaccagaaaaaaagtattaaatctgaccagaatgcaccagtaacagctgatatatatcttttaaatgacattgtcatgtttgaaaaggccaaaaaataaactgcagcccactggtaggcctatttctaacataatttaaccatctaaaagtgaaaaatagaatcataggttatattataataacactctgcctgtctcattgatcaccatgatcataaaattggaattattctcctcctcagttcttggtttactacttttaaccctatgaaaatgaaggtggatcatctgactggcattaacattactgtcttttgaaggctgttgcgggtttcaggtggacatacagttgtatcatatgaatcttgaggttgcgatgaatccgttgatgccaaacatgtcatgctagcttactgggaaagagggtaaatattgctccaaagtagagctgtctttaccgcgaggtccaccacacctcagtggcaaacggcagagttatggccctctggcacttggtgccctaatcacagcatgtgatacatgtgtgcatagcggcagcgctgtatatcttactcgtgtcaaatcttactcatatcaggggttttggtctcaacagaaatcctccatttccagtgaacgtcgccctctgatgcttttcctgttagcattatgtagtgcgtttctcctgagatgcacacagtctagaaaagaccacaacagaaaaacaacagtaatatcagcaatgaaatcatataaatagatttaggatcttttcattcatgcattattcaacacaaggcctttctttacctgtgtgttaggatgcagcgactgactagtgaaaatgacgtctgtgtttcctgccctgagggggctttcaaatgccaagagcagcacaggcttcagcttgagcaaaggtgactgtgggagtgcaaaggtcaacataggtgttcctccatcatcttcctctgtcaagagcactgatggagaaaacaagacatactgtaagataaaaacaaacaatcaaacaataatgtattgcaatcagacagctctgatacctccagttggatgcagaacctccagtccactcctctggtttctgcttgtttccttagcgagctgtgataagactgaggctgatgagctgccagacacggtgcagattccaaactgagtcaaactgcggtttgtgagctcgccgtcgctcccaacagcttcacgcaatgctgcgaatatgtcatccacaaagcacggagcatgttgagggctgtgacgagagactgtggaggctgatgaggtctctgttgatgggaaggtgtgctctgcatttaaaacaaaaacaacaaatttaaataaaatgatttatgtattacataaagagacttgctccttttgctatttgacttgatgatttgtgatgagattaaaaaaaataaaaaaatgtgttgttcctgcatgcctctatggtcaacggataatccaaaaacttgatgaattgaagtaaattcatatcaaaacaattcattttggtaagggaaagcttgaagcaggaaccacatgcaatccaaaagacactcattgacaagataaaaaagagcaaaacaacatcaaataaaggctttgcatatagaattgcatgaatattaatgcatgaaatgttcagaggtttcatttgtgcagagtctgacctgtcactgaggggtcgtgggccgggatcagctgtgagtcctgcagggccacacagatcctggtgtagcagagcattaatgatccgcagcagaagatgtccaaaaacaacatacttcactgcctgtgcgtgtgcctgtgtggtcagacctctctcagtcgcttgttttgaatgggtcaagctcccgaggaaggttgtttctgcgttccctaataatactt
序列表
<110> 中国海洋大学
<120> 许氏平鲉雄性特异基因序列和伪雌、伪雄鉴定引物及方法
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17307
<212> DNA
<213> 许氏平鲉(Sebastes schlegelii)
<400> 1
agtccatagt tcatccgtcc aggagagtta ctgtagcagc cacgctgctg catgtattgt 60
cccagtaagt ctccaccaca tcatttagct gcgaggttgt cagctgtttg tctgcctggc 120
gtacttagtg agcacggcgt agctttacca agtgtccggt cgagagacag agcgtgtgtg 180
tatggcagcg gtgtactctt gtcaacctgg cgttatgcac ataggattaa aaaaatccta 240
catgctatgt ttttaatgtt cctacttctt ggatctgtct tcttatgttt ttttataaag 300
cacattgcat ggcatatttt tgtatgaaat gtgtaatata aataaagttt aattttgcca 360
caattcacta aatgagtgaa atgaattgga gcccttgcat gcgcatcaag agccctgtag 420
gactcacaaa ttgatctgca atttggttgc aacaaaacgt gaaactgcca ccatttgcag 480
tctgtgcttt tgtgaaacag gctccagatg ttcagactcg acaactgtgg ccttgatgac 540
acatctcagc tttagttggt taatttgatg tcgcattatc attaaagcaa agtaactgtc 600
atatcaaatt tactcgagat gcttaagaca ccacagttaa aactaaagca gccttccacc 660
atgtgtgcag tagtaagtgt gtctacatgc atgacaggtg ccctttgttt gattgatatc 720
atcatgattg tggcctaaag tcttgtgatt gcttcactga tgttattcat gttggaactt 780
tgcgatacct cagggattca tgaaccgccg aatgctgctg ctgacagact aaaaacatgc 840
agctcagact ggaatttacg agcaggggat aagcatgtgt actttgtatt ttttccactt 900
aactccgcct ttccgtgagg atctctccat gcttgtgtgt gtgtgggtgt gagatattcc 960
agaggtcatt gtctctgggt ccagacggct ccgactcagg ttctcagtgt ggcttcacta 1020
atctgccgta attatgtttc acagccagct cactcacagt cacacacata cattgactcc 1080
ctgtctcttt gactctctct cttttttccc tctttttctc aagagaccag agtcgtggtc 1140
gcctcaggac ggctagcagt gcatgtgtgt gtgaatgtat atgcgtataa cacagtgtta 1200
cccatgcaat ggggctatat cctaccctgc tgacacacaa ctcaaaggac tcctttcaca 1260
gcttcaggga gaaaaaagag atgccttcgt gacttgccat atagtgaaag gggtttccaa 1320
tgcaggagtc atccgctgca cgcatcacac tcatctcccc ccgccctgcc cctgtgacgc 1380
atatcttcct tgtgtttcat caaccgtcgc acctcagggt gtgtggtgac gcaggagatg 1440
tgtgggcatc ctaaatccag actacctgac cagattcatg tgagttcagt tcagggggaa 1500
gccctcgcta taaacaattt tattcctttg acacacaaat gggtgtttgt tgtaaagata 1560
tagacatttt aatgagttgg aaacactgag ggatccaatg atcggttgag cttgtaaagg 1620
ataattgtgg tttaggcaag gcaaggcaag gcagctttat ttgtatagca catttcaaca 1680
acagggcaat tcaaagtgct ttacagaaac attcaagaac attgtgacaa aatgcaaaag 1740
aacattaaga aataattaaa acagttataa aaacataaaa acattaaaga ttagaaaata 1800
aaaacaagct aaaaataaaa gctaggatgg aagctaaaat tgcataaaac tcaaaagagt 1860
aaaagttata gtgcagtgtc agaataaaag gcacccgcaa acaggaaagt tttaagcttt 1920
gttttaaaag aagtgagagt tggagcggtc ctgcaggttt ctgggagctt gttccagata 1980
tttggtgcat aaaaactgaa tgctgcttct gcatgtttag ttctgaatct ggggacacta 2040
agcagacctg atccagatga cctgagaggt ctggatggtt cataacacag cataagatca 2100
gaaatgtatt ttggcgctaa accatgtagt gctttgtaaa ccagcagcag tattttgaag 2160
tcaattctct gagagacagg cagccagtgt agagacttca gaactggact gatgtgatcc 2220
actttcttgg tcttagtgag gactctagca gcagcgttct gaatcagctg cagccgtctg 2280
atcgattttt taggaagacc ggtaaagacg ccgttacagt agtcaagtcg gctgaagata 2340
aatgcatgga ctagtttttc caaatcctgc tgagacatca gtcctctaat tcttcttata 2400
ttcttcaggt gatagaaggc tgtctttgta attgtcttaa tatggctgtt aaagcttagg 2460
tctgagtcca tgactacacc aaggtttctg gcttggtctg agctttttaa catcacagat 2520
tgtaggtgag cactaacttt taatctttct tttttggctc caaaaactac gacctcagtt 2580
ttgtctttgt tcagctgaag aaaattctgg cacatccaat tattgacttg ttcaatgcac 2640
tcactcagtg cttgtattgg actgtagtct cctggtgata gagttatgta aatttgtgtg 2700
tcgtctgcat agttatggta atttattttg ttgttctcaa aaatctgacc cagtggaagc 2760
atgtagatgt taaacaaaag aggccccaag atggagcctt ggggaactcc gcacgtgatt 2820
ttgttcagct cagatttgca attacctata gacacaaaga agtccctgtc ttttaggtag 2880
gatttaaacc agctgagtgc tatgccagaa ataccgaccc agttttccag tcggtctagt 2940
aatatattat ggtcaaccat gtcgaatgca gcactgagat ccagtaaaac taaaactgta 3000
gttctaccac tgtctgtgtt caatcggatg tcattaaaga ccttaacaag agcagtctca 3060
gtgctgtggt tttgtcgaaa acctgactgg aagacatcaa aagagttgtt tagtgccaga 3120
aagctattta atttttgaaa aactgttttt tcaattattt tagacaagaa tggaagattt 3180
gatatcggtc tgtaattatt tactattgat gagtccagat tgttcttttt gaggagtggt 3240
ttgatgacag cagttttcag ggcctgtggg aagactcctg aaacaagaga tgcattaaca 3300
atctgtaaaa gctctgagac catacaatct gatacttttt tgaaaaggcc tgttggcaga 3360
atgtcaaggc agcatgagga ggattttaga tgttgtatga tttcctccag gtatgtctga 3420
tttattggat aaaactgtgt catggtattt gcgccaggtg gacgcagggg aaacaaacat 3480
cctgtacctg atatggagga agtgactgct tgtctgattt tgtgaatttt ttctgtaaaa 3540
aaggaagcaa attcattgca ggccctggag gataaaagtt cagaggctac tgacacagga 3600
gggtttgtta gcctgtcgac agtagcaaac agggcacgtg cattgttatt gtttttggtg 3660
atgatgtcag agaagaagga ccgccttgca tttttcagtt ctgaattata aatgcaaagt 3720
ctctctttat aggtgtcata atgaacttgt agatttgttt tccgccactg gcgttcagct 3780
tttcgacact ctcttttttc atttcttacc aacatggtat ttctccatgg agattttttc 3840
ttaccagaga caatcttcag ttttgggtgc aatggtatca ataacatttg tcattttaga 3900
attgaaatta tctacaagct cattgacaga ggtccagggg agggtgggtg tggaagagaa 3960
agcctgaaca aattgatcac tggtattttc agtgatatac ctttttgtga ttacctctgt 4020
cttatcattt ctgtgcacag gaagagtact ctcaaagaat acacatgaat gatcagacag 4080
agcaacatcc atcactaaaa ccttggaaat gttcaggccc tttgatatga ttaagtccaa 4140
agtgtgtccc ctattgtgtg tgggctctgt cacatgctga ctcattccat agttatcaag 4200
aacctgacac agttcattgg cccctctgtc ctgttggttg tcaacatgga tgttaaaatc 4260
gccaacaata actacacagt caaagtcaac acaaattaca gacagcagtt cagtaaaatc 4320
atcaaaaaag tttgcacagt atttaggtgg cctgtagata ttgagaaaca aagctcgaga 4380
agaggagttc agctgaagag cgacatattc aaaagaacca aaatgtccat aagatatctg 4440
tttgtattgg agagaatcat taaacaatat ggcaactcca cctcctttct tatgcattct 4500
agcttcgctc ataaaactaa aattgggagg agttgactcg ataagaacag ttgcactgtt 4560
attttggtct aaccaagttt cagttaaaaa cataaaatca agcttgtgct caataataaa 4620
atcattgatt aaaaaacttt ttcctgccaa agacctgacg tttaacaggg ctagctttaa 4680
tgtgctagaa gaattattat tattttttgg gacatggttt ggctggcaag gaatcaatgc 4740
taaatttgat aaattagcac tatcagtaaa aacatcaaca ttttgtaaag gaacatcctt 4800
atcaggctgc ggagacagag gctgattcca ttgtggaggg gatggaggtg gtgggtgtcg 4860
tgcttgtgga gatagtgcca aagttgttcg aggtcgacct cgtgggggaa tcatgggaga 4920
gaaaatgggt gcagggcggg gagtaagttt agttccagca ttgaccagct ccttcatgtg 4980
gtcagtgaac tccaagaggg gggagttttt catcaccaaa aacaataaca atgcacgttt 5040
attacagctt ctgtcttatt ttggtaggtt tggccatctt tcctactcac caagttaagt 5100
actgagatct ggaaatgcag caacatcact aaggaggtca agaaacatga gcagtcatgt 5160
gatcatacag gttgcaaaca aatcccctga actgattttg gaaaagaaaa caaagatgac 5220
ttatttccca aactgtaaac acccatcaca gtgtaaaata aaagggtgtg acgccaaggg 5280
gcggtatgtg acgagttggg atgagaatgt gttggttgga ctttttgcca tattcccata 5340
tctcagcaat tactttggtt agagcaaagc tatcattagt attaggaaaa atggcccgac 5400
ctatgaaagt gagacttata attaaccaga accatcctat aaagtcagat ttataatgct 5460
agattaatat gttttcctat gtccttttcc ttcccccagt ttttccactg tgtagcactc 5520
attataccat tccacattat cctagagttt ttgcataata gtaagaaatt atcatcacaa 5580
taaaaagaaa gcccatggat tcatttgatg ctgaatagct gagcctgaaa tttccatcct 5640
cattgtttcc acaaaacact cttgaaccac tccctctgcc taagcatgac tcgtgaacac 5700
ataaaatgtt tatctttggt tttgccacca aaacaaacag ttttcatagt acaataaatc 5760
taaaactaga aaatgtagtt atactaccac aaggacaaaa acgataatta tcaaagtttg 5820
caagtttata tttgtgtggc acttttcaca caattgtaag gcacagaaaa caacagttat 5880
gatacaaaca cacagtgttg acaccctcaa aggagaacac acaggatagt tagagatatc 5940
aggtttcatt cttgctgtac atcattaaaa aacactttaa agtaaatcca aaccttaatt 6000
tggttatgac cattttattt agttgttatt taaaggtaca gtgtgtagga tttggtggca 6060
tctagtggtg tggttgcaga ttgcaaccaa ctgaataccc ctccgctcac tcccccttgt 6120
ccaatactgc ggtaacgtga gctgccgagt gcgaaaccgt ggtaacgctg ttcaactcgc 6180
tcagaggcca tccttaccat gataacacta ctttaggagc aatggtacaa cggtttagca 6240
ctatgcagct cattaccaca gtttcacaag cgtgtcggag aactacggtg gtcttcaggt 6300
aacctaaaaa catgaatggc tctctctaga gccagtgttt ggtttgtcgg ttctgggcta 6360
ctgtagaaac atggcgtagc aacatgctcc ctatgtagat atgaagggct cattctaagc 6420
taatgaaaac acaatgattc ttagtttcag aagtgacaga aagaatttaa agtctcctcc 6480
agaaaataat ctctcaaaaa tatgtcgcct tttggacttt attgtcttat ggaaatcttt 6540
tcatggaaat taaaatacgt aaaatacatt tcaagtgagt tttaaaagac agcagtgagc 6600
gtggtaagat cacagaaaaa gatatgtggt ataaacaagg aaatgaaaag tgatattata 6660
tatcatttca tatgacagaa tgggagcatg cacataagga aacaaaacat ataatgaacc 6720
caaaacaata attaaaaata gttaaaaaat aattaactta atttgatcta actattaatt 6780
gaccctgact gcgccttcta taataacaca gctctttaaa ttgcaggtat cataactata 6840
atgaagagtt gtggttggca ctcatttata ttgcagtgat gtatgttgaa gtgaactcta 6900
tttaacccgt agtaatttat tcacagggca ggttggcctt gcaggacaaa cctgtttctt 6960
ttataaggaa ctctgtggag cctttggccc gtgtgtcagt ttgtgtgtga accccactga 7020
gctcattaaa ctgttaatat cgctgtcttc ctcttcacaa aacctgaaac cccacaagat 7080
attgcatgct cagtagctga aacacacagc ccggcacaca cacaaagcct cataaagctt 7140
ttctgactaa atgcgtgact ggctgcttgc ttaaacacac acacactcac acacagacgt 7200
acacacacac ggctgcagca cagcgctctt atttaaactg atgtgagtgc tgtcacgaca 7260
tccgactagt cctatttgtt ccacagaagc cgtggcgtga gagagcaggg tcaccccact 7320
agctctcatg caaatccatt cagcacacac gtgcgcgcac acacacacac acacacacac 7380
acacacacac agacacagac acacgcacac acttaatagt cctgcagata agcaaaaaga 7440
cagacagata gacaggcagg caagcagaga gagacgagtg gacagaaaaa acattgagca 7500
gacagaagaa caaaatgaag ggcagacaga cagacagata gacagacaaa caaacatcaa 7560
atcctaatta aagctgcaag cagcgatgaa cgggccctcg cccacacagg cacgtctgca 7620
gaacgcgcac gtcggggtac ccgctgtcgt tcattcccgt gaaagtcgga aactgcacgc 7680
aacagttaga gtgaattttc tacttggagc atgtggcgct ggaaatgaac tgtgagaaca 7740
tgtggggcat cctttaaagc acttctatga gggtggctgg cagtatagaa ccatttccta 7800
ttgtcagtag gtggcgctat gactttcact cataatacca cgtgtatgtc ttcaggccaa 7860
gattcttata aaacatgtca aatttgggga agattggaca atgtatagtc aatttacaac 7920
gacttcctgg cggagatcaa ggatcacagc gtccttatcg cgctgttcct ggcagagatc 7980
aagtatcaca gcgtccttat cgcgctgctc ctggcggaga tcaaggatca cagcatcctt 8040
atcgtgctgc tcctggcgga gatcaaggat cagagcgtgc tgttcctggc ggagatcaag 8100
tatcacagcg tccttatcgc gctgctcctg gcggagatca aggatcacag cgtccttatc 8160
gtgctgttcc tggcggagat caaggatcag agcgtactgt tcctggcgga gatcaaggat 8220
cacagcgtcc ttatcgtgct gctcctggcg gagatcaagg atcagagcgt gctgttcctg 8280
gcggagatca aggatcacag cgtccttatc atgctgctcc tggcggagac caaggattac 8340
agcgtgctgt tcttggcaga gatcaaggat cacattgtcc ttatcatgct gctcctggcg 8400
gagaccaagg attacagcgt ccttatcgtg ctgcttctgg cagagatcaa ggatcacagc 8460
gtccttatgc tgctggggaa tgttatcatg ttggggaaga ttggacaatg tataatcaat 8520
ttataacaat ttcctgtttc ctgttggagg cgctatgact ttcactcaaa atggcacata 8580
tatgtcttca ggctgggact attatccagc ttgtgaaatt tggggcagat tggactatgt 8640
aaagtcaggt taaaacagct tcctgtgtca ttgcgaaaca tcaaaattcg ccacgccgcc 8700
ccaagaacgc cgttgcacga aaacacaaaa gcttcgcaat ttagcatcgt gaaggtgttg 8760
agattctgct gcccgacttt gagatggatc gcttgtatcc tctaggagga gtatcgcaaa 8820
gttccatacc taaaaagaga caaaatggcg tttttgcaca tgacgtatga catcaccgtt 8880
cgagcgatca aatatttctt cgcaatttag catcacagta gtttgatggt tgtaccaccc 8940
aaatttgacg cggatccgat aaactctcta ggaggagttc gtaaaagtat gcataaaata 9000
catgaaaaac gcacaaaatc caatatggcc gacttcaggg tgggcggagc taatgaaacc 9060
cgatgaggaa tatgtttcaa ataatgagca ggatgtacct acaacctttc atgaatatag 9120
gatcaacttt gaccaaacta tggccttcca cgcgttaggg ggcgctaaag agcgggctaa 9180
acgcactgaa cctaatggct gtacatttac ataaagttca caggtgtcaa tcattttgcc 9240
aagtttcatg agttttcgag tacctcaagg tatgttcaaa acctaaaaga cataaggggg 9300
cgctagagag ccaacatgcc atgcccaagc aaaatttcac cactaaatca aagtaattat 9360
gagtttggat gtgcatgtaa attttcatga gtttttgtgc atcctaaagt cttcaaacat 9420
gtgttcgtaa attttaaaat catgcaggaa atccaaattg gctgacttcc tgttggccga 9480
aaaaatctca aaaatattta acacgaactt caagatgtat gcaattacat acttgaattt 9540
cgtgaagatc gaagaaactt tccctgaaaa actgcctaca ttagggggcg ctatctcgcc 9600
cgctggcgac gcccaagtcg aatcactaca gaactttgaa tttcgcgcca cttctgacgc 9660
atattccact tttggtgagt ttttggggaa ggaaaaggtc ccaaaaacgt gatattccag 9720
cagaaaaaga ataattaaag ctgcaagcag cgatgaacgg gccctcgccc ttgcgggcac 9780
gtttggggaa cacgcacgtc ggagcacgta catatcttta ggccaagact cttttaaaac 9840
atgtcaaatt tggggaagat tggacaatgt atagtcaatt taaaacaact tgctgtttcc 9900
tgtaggggcg ctatgactat cacgcataat accatgtata tgtcttcagg ccggcaccct 9960
tataaaacat atcaaatttg gggaagattg gaagaagatt ggtcttcaaa tatgcgttcg 10020
taaattctaa aatgacgcag gaagtccaac atggctgact tcctgtttgc cgcaaaaatc 10080
tgaaaatcat tttttccagg tatgagggcg tgagcaacga catacttgaa tttcgtgaag 10140
atcgaagaaa ctttctcggg aaacctgcgt gcgttagggg gcgctatgga gccctatgga 10200
gacacccgag cccagtcact ccagaacatt gaatttccca ccagttctga cgcattctcc 10260
aattttcgtg agttttgggg atggctaagg tcgtcaaaaa cgcgatcatt cgggcggaaa 10320
aataataata attaaagctg caagcagcga tgaacgggcc ctcgcccctt gcgagtcggg 10380
gacgctggcg ggacgccgac cgacgcggcc gggcaacgtg aagccgaaac tctgacgagc 10440
gccgcgacgc ctgccgcgag gctctacgac aagcggttca cgagttatga aggggggcgt 10500
ggctaatgtg tagggggagg ggataacgac accaacttaa caggcactct ctgctgagcg 10560
atacgacacc tcccgcaaga ctctacgaca aacggttcac gagttatgaa agggggcgtg 10620
gctaatgtgt agggggcggg gataaccttc accaatgaaa caggcactct ctgccgagtg 10680
atatgacacc tcccgcaaga ctctacgaca aacggttcat gagttatgaa agggggcggg 10740
gctaacgtct tggggcgggg ctatgagtat aatttttcat atacatgtca tcagtagtgg 10800
accaccatca tacctgagag atttggggca gatcggacaa tgtacagttg agttacaatg 10860
actgcctgtt tcatggcgaa tggctcaaaa tggccgccac gctacggtcc gatcgttaag 10920
tggacactca tcattttaat aacttttcat cttcaaggtc ttaagatggt cctgaccaaa 10980
tttcaacacg atctgatcaa atctctagga ggagttcgtt aaagtacgcg gcctataaaa 11040
tgctaaaagt gacatgaaat ccaatatggc cgacttccgg gggggcggag ctaatgaaac 11100
ccaatttgca atatgtttca aatgatgaga gggatatgca taccaaattt catgaatatc 11160
ggattaactt tgacaaagtt aaaatttcaa cgcgttaggg ggcgctatag agccggctaa 11220
acacactgag cctaatggct atatatttac ataaagttaa caggtgtcta tcatgttgcc 11280
aaatttcatg agttttcgag cacctaaagg tatgttaaaa atctgaaaga cataaggggg 11340
cgctagagag ccaacatgcc acgcccaagc aaaatttcac cactaaaatt aagtaattac 11400
tagtttggat gtgtgtgtaa agtttcatac atttttgtgc atcctaaagt cttcaaatgt 11460
gcgttcgtaa attctaaatt cacgcaggaa atccaacatg gctgacttcc tgtttgccgc 11520
aaaaatctca aaataatttt ttccaggtat gagggcgtga gcaatgacat acttgaattt 11580
cgtgaagatc gaagaaactt tctcagaaaa actgcatacg ttagggggcg ctatggagcc 11640
ctatggagac acccgagccc agtcactcca gaacattgaa tttcccacca gttctgatgc 11700
atactccaat tttcgtgagt tttggggatg gctaaggtcg tcaaaaacgc gatctcgcag 11760
cggaaaaaga ataataatcc ccagaaaaac aataggttcc ttgcacttcg tgccaggaca 11820
ccgttgggtc ctggcacttt cgtgctcggg ccctaataat aatactgaca aaaacaatag 11880
gttccttgca cttcgtgcca ggacaccgtt gggtcctggc actttcgtgc tcgggcccta 11940
ataataatac tgacaaaaac agtaggttcc ttgcacttcg tgccaggaca ccgttgggtc 12000
ctggcacttt cgtgctcggg ccctaataat aataatactg acaaaaacag taggttcctt 12060
gcacttcgtg ccaggacacc attgggtcct ggcactttcg tgctcgggcc ctaattaaag 12120
ccgaaatcca atatggctga cttccggggg ggcggagcta atgaaaccca atgaggaata 12180
tgtttcaaat gatgagtggg atatgcatac caaatttcat gaatatcgga tcaactttga 12240
caaagttaaa atttcaacgc gttagggggc gctatagagc cggctaaaca cactgagcct 12300
aatggctata cagttacata aagttcacag gtgtctatca ttttgccaaa tttcatgagt 12360
tttcgagtac ctaaaggtat gttaaaaatc tgaaagacat aagggggcgc tagagagcca 12420
acatgccatg cccaagcaaa atttcaccac taaaattaag taattagtag tttggatgtg 12480
tgtgtaaagt ttcataaatt tttgtgcatc ctaaagtctt caaatatgcg ttcgtaaatt 12540
ctaaaatcac gcaggaaatc caacatggct gacttcctgt ttgccgcaaa aatctcaaaa 12600
taattttttc cagttatgag ggtgtgagca acgacatact tgaatttcgt gaagatcgaa 12660
gaaactttct cgggaaaact gcgtacgtta gggggcgcta tggagcctta tggagacacc 12720
cgagcccagt cactccagaa cattgaattt cccaccagtt ctgacgcata ctccaatttt 12780
cgtgagtttt ggggatggct aaggtcgtca aaaacgcgat ctcgcagcgg aaaaagaata 12840
ataataccca gaaaaacaat aggtccgtgc caggacccaa cggtgtcctg gcacgaagtg 12900
ctcgggccct aattaaagct accaaatgta ttagctccgc ccacctggaa gtcggccatc 12960
ttgaattttg agtttttcat gtaatttatg tattttatgc atactgttac gaactcctcc 13020
tagagattta tcccgatcaa cttcaaattt ggtcagtaca ttcttgagac ctttataatg 13080
aaaagttcct caaatcgtga gttttcatga aacggtgttg acgtggcgtg gcggagaatt 13140
tacatgattc gccattaaat gacaaactgt tgtaactcga cggtacttgg tcaaatctgc 13200
ccccaatttg acatacttca taagagtgca ggcttcagga catctaaatg gcaatattgg 13260
ctcatagttg ggtttcatta gctacgccca cccggaagtc ggccatcttg aattctgtgt 13320
ttttcatata atttatgtat tttatgcata cttttacaaa cttctcctac agatttatcc 13380
cgatcaactt caaacttggt cagtacagtc ttgagacctt tatgatgaaa agttcctcaa 13440
atcgtgcgtt ttcatgaaac ggtgttgacg tggcgtggcg gagaatttac atgattcgcc 13500
attaaatgac aaactgttgt aactcgacgg tagttggtca aatctgcccc caaatgtacc 13560
tacttcataa tagtccaggc ctgaagacat atacgtgcca cgacagcggg taccccgacg 13620
tgcgtgttcc cccaacgtgc ccgcgggggc gagggcccgt tcatcgctgc tcgcagcttt 13680
ctgaataaag aacacaaaca gtattcaata tactttgcat ttaataaaat aatgttacat 13740
ttattaatcc atttttgttg ttgtattgta gagaacagga ccttttagta tcactaattt 13800
agaaatgtca ttaaccacca cattgtaaca caatcttata atcagtcatc aagttattaa 13860
caattttaga acaatatttc aatgataact ggaatttgta acacttattt tggaataagt 13920
ttttaacacc taacgtgttt gacacttaaa agtgtcaaat aggctatggt cagttaaata 13980
tgtaaaacca tcttttgtgt cacaactaca cgctaggaag aaaacagctt tagcggcatc 14040
cgcactgcct cgccaccacg tttggtttaa tggagaggta agtcccgtgt tcgttcaagt 14100
ccaccacctc cagggcttcg taggccacag gcacgcaaca tggcgcccgc tcgtccacgc 14160
tctcgctctc aatgtgggag ttgagcagga cggtgtggat gttggcgtta gtcattggga 14220
aagcacaaga gccgtggcag ttgttgatgt tgacagtgtt tgggagcccg aggcgttttt 14280
ctagggacac ggtgaggctc ttcagcccac atatgttgcc ccttaatgag gtgatggggc 14340
cagatctggt ggcccgcagt cctctctgca cctcgtacac gcgggccacc gtctgcaggg 14400
ccttcagcag aagaaacgca cggtactggc tctctcctgt tctagaggta acacaaatat 14460
cagttaaaaa gtgggaatag aaatctaaat ttatgtcaga tgggctgatt tataatgcga 14520
acaattgctg gaggagtttc cttgtacaca tcacctgctg tcggtcccat cgtcggaaac 14580
gcactgagtt ctttgagcct tcccagcctc tccgtggctc tgtgacccac ctcctcctcc 14640
cgtattacct ccattatctg cattccagtc tgctccatcc tctgcttgag ctcctccagc 14700
agtgtaggag acaaagccag ttctccacga tgcacctgaa acatggagaa ctggctaggg 14760
aaggagaaga tggtggggga agaggagttg atgagtcctg ccagcagggt gtcgctggag 14820
gataagccta gtgtgagggg aggaagggac ggtgaggagt ccagctggag tcgaggggac 14880
tcggggcggt cctgaggcag gacatcaccc aggaaccgct tcagctcaca gaggaaggag 14940
ctctgtgtag aggcaggggg tggacctgaa gtgtgtgtat gtctgagaaa gagagtaaat 15000
aaaatgctgc tgaacaacac aatagtcaat agttatattt caatagggaa catgaaagcc 15060
aagaccacct tatatcagtt cctgttcctc cagagaaaag cagaagtgga atcatgctga 15120
tgttacttcc tgtttttctg ccaacaagga tatcttttag gttttggtct gcaaagagag 15180
gaaatgtgag aagagtgcac acgaaacaag aaattctatc ttcattgtca gccctgacct 15240
atttggtgcc ctaggcaaga ttttagctgg tgcccctcca cctctctttg cacagcgtga 15300
cctgccggga ggttctcagt gttatttttg tgcatacagg atgtgtagcc tcttaacctg 15360
cttataataa agctaccata ttgatcttga ttataagacc agaaaaaaag tattaaatct 15420
gaccagaatg caccagtaac agctgatata tatcttttaa atgacattgt catgtttgaa 15480
aaggccaaaa aataaactgc agcccactgg taggcctatt tctaacataa tttaaccatc 15540
taaaagtgaa aaatagaatc ataggttata ttataataac actctgcctg tctcattgat 15600
caccatgatc ataaaattgg aattattctc ctcctcagtt cttggtttac tacttttaac 15660
cctatgaaaa tgaaggtgga tcatctgact ggcattaaca ttactgtctt ttgaaggctg 15720
ttgcgggttt caggtggaca tacagttgta tcatatgaat cttgaggttg cgatgaatcc 15780
gttgatgcca aacatgtcat gctagcttac tgggaaagag ggtaaatatt gctccaaagt 15840
agagctgtct ttaccgcgag gtccaccaca cctcagtggc aaacggcaga gttatggccc 15900
tctggcactt ggtgccctaa tcacagcatg tgatacatgt gtgcatagcg gcagcgctgt 15960
atatcttact cgtgtcaaat cttactcata tcaggggttt tggtctcaac agaaatcctc 16020
catttccagt gaacgtcgcc ctctgatgct tttcctgtta gcattatgta gtgcgtttct 16080
cctgagatgc acacagtcta gaaaagacca caacagaaaa acaacagtaa tatcagcaat 16140
gaaatcatat aaatagattt aggatctttt cattcatgca ttattcaaca caaggccttt 16200
ctttacctgt gtgttaggat gcagcgactg actagtgaaa atgacgtctg tgtttcctgc 16260
cctgaggggg ctttcaaatg ccaagagcag cacaggcttc agcttgagca aaggtgactg 16320
tgggagtgca aaggtcaaca taggtgttcc tccatcatct tcctctgtca agagcactga 16380
tggagaaaac aagacatact gtaagataaa aacaaacaat caaacaataa tgtattgcaa 16440
tcagacagct ctgatacctc cagttggatg cagaacctcc agtccactcc tctggtttct 16500
gcttgtttcc ttagcgagct gtgataagac tgaggctgat gagctgccag acacggtgca 16560
gattccaaac tgagtcaaac tgcggtttgt gagctcgccg tcgctcccaa cagcttcacg 16620
caatgctgcg aatatgtcat ccacaaagca cggagcatgt tgagggctgt gacgagagac 16680
tgtggaggct gatgaggtct ctgttgatgg gaaggtgtgc tctgcattta aaacaaaaac 16740
aacaaattta aataaaatga tttatgtatt acataaagag acttgctcct tttgctattt 16800
gacttgatga tttgtgatga gattaaaaaa aataaaaaaa tgtgttgttc ctgcatgcct 16860
ctatggtcaa cggataatcc aaaaacttga tgaattgaag taaattcata tcaaaacaat 16920
tcattttggt aagggaaagc ttgaagcagg aaccacatgc aatccaaaag acactcattg 16980
acaagataaa aaagagcaaa acaacatcaa ataaaggctt tgcatataga attgcatgaa 17040
tattaatgca tgaaatgttc agaggtttca tttgtgcaga gtctgacctg tcactgaggg 17100
gtcgtgggcc gggatcagct gtgagtcctg cagggccaca cagatcctgg tgtagcagag 17160
cattaatgat ccgcagcaga agatgtccaa aaacaacata cttcactgcc tgtgcgtgtg 17220
cctgtgtggt cagacctctc tcagtcgctt gttttgaatg ggtcaagctc ccgaggaagg 17280
ttgtttctgc gttccctaat aatactt 17307
<210> 2
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
acgaacgcac atttgaagac t 21
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctgcgttccc taataatact t 21
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgaccctgac tgcgccttct at 22
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cctcattggg tttcattagc t 21
<210> 6
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agtccatagt tcatccgtcc a 21
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgtgaagagg aagacagcga 20

Claims (5)

1.许氏平鲉雄性特异基因序列建立的雌性、雄性、伪雌、伪雄鉴定引物对，其特征在于，所述引物对为SEQ ID NO.4和SEQ ID NO.5或SEQ ID NO.6和SEQ ID NO.7。
2.利用权利要求1所述引物对进行许氏平鲉伪雌、伪雄鉴定的PCR方法，其特征在于，利用引物对SEQ ID NO.4和SEQ ID NO.5或SEQ ID NO.6和SEQ ID NO.7进行PCR反应，反应体系如下：10×LA buffer 5 μL，2.5mM dNTP4.0 μL，10mM正向/反向引物各1 μL，cDNA模板2.0 μL，LA Taq DNA聚合酶5U/μL 1 μL，dd H2O 补齐到50 μL。
3.根据权利要求2所述的方法，其特征在于，所述PCR反应条件为：94℃, 3min; 94℃,30s; 56℃, 30s; 68℃, 5min;30cycles; 68℃, 7min; 12℃, ∞。
4.权利要求1所述任一引物对在鉴别许氏平鲉雌性、雄性、伪雄鱼和伪雌鱼中的应用。
5.利用权利要求1所述任一引物对制备的鉴定许氏平鲉雌性、雄性、伪雄鱼和伪雌鱼的试剂盒。
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CN108192954A (zh) * 2017-05-04 2018-06-22 中国水产科学研究院长江水产研究所 一种rad测序筛选中国大鲵雌性特异片段及遗传性别检测方法
CN110172520A (zh) * 2019-06-25 2019-08-27 中国海洋大学 一种平鲉鱼类性别特异分子标记引物、鉴别方法及其应用

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CN108192954A (zh) * 2017-05-04 2018-06-22 中国水产科学研究院长江水产研究所 一种rad测序筛选中国大鲵雌性特异片段及遗传性别检测方法
CN110172520A (zh) * 2019-06-25 2019-08-27 中国海洋大学 一种平鲉鱼类性别特异分子标记引物、鉴别方法及其应用

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