WO2022068215A1 - 凡纳滨对虾抗弧菌相关est-str标记及其特异性引物和检测方法 - Google Patents

凡纳滨对虾抗弧菌相关est-str标记及其特异性引物和检测方法 Download PDF

Info

Publication number
WO2022068215A1
WO2022068215A1 PCT/CN2021/094239 CN2021094239W WO2022068215A1 WO 2022068215 A1 WO2022068215 A1 WO 2022068215A1 CN 2021094239 W CN2021094239 W CN 2021094239W WO 2022068215 A1 WO2022068215 A1 WO 2022068215A1
Authority
WO
WIPO (PCT)
Prior art keywords
site
litopenaeus vannamei
est
primer
str
Prior art date
Application number
PCT/CN2021/094239
Other languages
English (en)
French (fr)
Inventor
王艳红
胡超群
罗鹏
李卓波
李活
任春华
江晓
陈廷
刘锦上
Original Assignee
中国科学院南海海洋研究所
广东金阳生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院南海海洋研究所, 广东金阳生物技术有限公司 filed Critical 中国科学院南海海洋研究所
Priority to JP2023548987A priority Critical patent/JP2023546627A/ja
Publication of WO2022068215A1 publication Critical patent/WO2022068215A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention belongs to the technical field of biomolecular markers, and in particular relates to an anti-Vibrio-related EST-STR marker of Penaeus vannamei and a specific primer and a detection method thereof.
  • Litopenaeus vannamei also known as White pacific shrimp. Wild populations are distributed in Central and South America, from Mexico to Chile, in the Pacific coastal waters where the natural water temperature is above 20°C all year round. It is a typical tropical prawn. There are seawater and freshwater aquaculture in the coastal areas and many inland areas of my country from south to north. At present, Penaeus vannamei is the world's first farmed shrimp, accounting for about 70% of the world's farmed shrimp production and 80% to 90% of my country's shrimp farming output. At present, the annual aquaculture production of L. vannamei in my country exceeds 1.6 million tons.
  • the purpose of the present invention is to overcome the deficiencies in the prior art, provide a kind of EST-STR marker of Litopenaeus vannamei and its amplification primer, detection method and application, and provide for genetic diversity analysis and molecular marker-assisted breeding of Litopenaeus vannamei. Highly polymorphic molecular markers.
  • the present invention utilizes the Litopenaeus vannamei seedlings constructed by the team, the transcriptome library data constructed under the infection of Vibrio harveyyi, and the constructed sequencing library in Huada Gene Co., Ltd. (BGI, Shenzhen, China). ) on the Illumina HiSeq 4000 platform (Illumina, San Diego, USA).
  • the EST-SSR sequences of Litopenaeus vannamei obtained from the database were used to develop highly polymorphic EST-STR markers of Litopenaeus vannamei, and provide polymorphic primers for the study of genetic diversity of Litopenaeus vannamei and the assisted breeding of excellent varieties. Provides highly polymorphic molecular markers.
  • the present invention selects 50 pairs of primers to carry out PCR amplification on the genomic DNA of Litopenaeus vannamei from the EST-SSR database of Vibrio harvetiensis-infected according to the results of function prediction and core region repeat difference, among which 6 The primers can stably amplify the target band.
  • STR samples were detected by 3730xl equipment, and SSR data were analyzed by Genmapper software, and 6 highly polymorphic EST-STR markers of Litopenaeus vannamei were finally identified.
  • the first object of the present invention is to provide an EST-STR marker of Litopenaeus vannamei, the EST-STR markers are numbered as Lv-Vp001, Lv-Vp011, Lv-Vp012, Lv-Vp023, Lv-Vp029 and Lv -Vp041;
  • the nucleotide sequence of described Lv-Vp001 is shown in SEQ ID NO.1;
  • the nucleotide sequence of described Lv-Vp011 is shown in SEQ ID NO.2;
  • the nucleotide sequence of described Lv-Vp012 is shown in SEQ ID NO.3;
  • the nucleotide sequence of described Lv-Vp029 is as shown in SEQ ID NO.5;
  • the nucleotide sequence of the Lv-Vp041 is shown in SEQ ID NO.6.
  • the second object of the present invention is to provide a kind of amplification primers marked by the above-mentioned EST-STR of Litopenaeus vannamei, and the amplification primers include:
  • Lv-Vp001-F 5'-ACTGGAGGATCCTGAGAGATAGC-3';
  • Lv-Vp001-R 5'-GCTTCGTTCTCTCACTCTTCTCTT-3';
  • Lv-Vp011-F 5'-AAGGATTTCTCTTACACGAACCC-3';
  • Lv-Vp011-R 5'-ACCAAAAAGAAAAACAGAATGGC-3';
  • Lv-Vp012-F 5'-AATCTACAGGACTCGAATGCACA-3';
  • Lv-Vp012-R 5'-CTTTTGTCCTCCACTTTACATGC-3';
  • Lv-Vp23-F 5'-CGAGAAAGAGGGAGAAAAAGA-3';
  • Lv-Vp23-R 5'-TCATGGAGATGATGACTGGATAA-3';
  • Lv-Vp29-F 5'-CACCATATTCTCCAAGAAGCAGT-3';
  • Lv-Vp29-R 5'-TTACTCATCCTGCTTCAACACCT-3';
  • Lv-Vp41-F 5'-CTTTTCGTTTGATGAGTCTTTCCA-3';
  • Lv-Vp41-R 5'-AGGGAGAAAGAGTGGAGTGAGAT-3'.
  • the 5' end of the forward primer of the amplification primer is labeled with a fluorescent group.
  • the fluorescent group is FAM or HEX.
  • the third object of the present invention is to provide a method for detecting the EST-STR marker of Litopenaeus vannamei, comprising the following steps:
  • step (2) Using the genomic DNA extracted in step (1) as a template, use the above-mentioned primer pair Lv-Vp001-F/R for Lv-Vp001 site, and primer pair Lv-Vp011-F for Lv-Vp011 site respectively /R, primer pair Lv-Vp012-F/R for Lv-Vp012 site, primer pair Lv-Vp023-F/R for Lv-Vp023 site, primer pair Lv-Vp029- for Lv-Vp029 site F/R, PCR amplification of Lv-Vp041-F/R with primers for Lv-Vp041 site;
  • the reaction system is 25 ⁇ L, including: 2.5 ⁇ L of 10 ⁇ PCR buffer containing Mg 2+ , 1 ⁇ L of 10 mM dNTPs, 2U of Taq enzyme, 1 ⁇ L of 10 ⁇ M forward primer, 1 ⁇ L of 10 ⁇ M reverse primer, DNA template 10ng, and the rest were made up to 25 ⁇ L with sterile double distilled water.
  • the reaction procedure is: 95°C pre-denaturation for 3 minutes; 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, and 72°C extension for 30 seconds, a total of 10 cycles; 94°C denaturation for 30 seconds, Annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, a total of 35 cycles, and extension at 72°C for 5-8 minutes.
  • the fourth object of the present invention is to provide the above-mentioned EST-STR markers or amplification primers for the genetic diversity research, gene mapping, genetic linkage map construction, germplasm identification, species classification or molecular analysis of the Litopenaeus vannamei population Applications in marker-assisted breeding.
  • the application is an application in an EST-STR marker or amplification primer of Litopenaeus vannamei related to an antibacterial and/or insect resistance trait.
  • the antibacterial and/or anti-insect is anti-Vibrio harvetii and/or anti-C. hepatica.
  • the present invention utilizes the EST sequences of Litopenaeus vannamei obtained from the NCBI database to develop and obtain 6 EST-STR markers of Litopenaeus vannamei with high polymorphism, and the numbers are respectively Lv-Vp001, Lv-Vp011, Lv-Vp012, Lv - Vp023, Lv-Vp029 and Lv-Vp041.
  • the EST-STR marker of Litopenaeus vannamei of the invention can be used for genetic relationship analysis and molecular marker-assisted breeding of Litopenaeus vannamei, laying a foundation for genetic diversity research and assisted breeding of fine varieties of Litopenaeus vannamei.
  • the molecular markers related to the antibacterial properties of Litopenaeus vannamei excavated by the invention have great application value for the screening of seedlings with strong antibacterial ability and the screening of excellent antibacterial germplasm of Litopenaeus vannamei.
  • Fig. 1 is a STR genotyping map of 20 genomic DNAs of Litopenaeus vannamei amplified by primers at Lv-Vp001 locus; S1-S20 represent 20 samples.
  • Fig. 2 is a STR genotyping diagram of 20 genomic DNAs of Litopenaeus vannamei amplified by primers at the Lv-Vp011 site; S1-S20 represent 20 samples.
  • Fig. 3 is a STR phenotyping diagram of 20 genomic DNAs of Litopenaeus vannamei amplified by primers at the Lv-Vp012 site; S1-S20 represent 20 samples.
  • Fig. 4 is a STR genotyping diagram of 20 pieces of Litopenaeus vannamei genomic DNA amplified by primers at Lv-Vp023 site; S1-S20 represent 20 samples.
  • Fig. 5 is a STR genotyping diagram of 20 genomic DNAs of Litopenaeus vannamei amplified by primers at the Lv-Vp029 site; S1-S20 represent 20 samples.
  • Fig. 6 is a STR genotyping diagram of 20 pieces of Litopenaeus vannamei genomic DNA amplified by primers at Lv-Vp041 site; S1-S20 represent 20 samples.
  • the present invention selects 50 pairs of EST-STR primers (as shown in Table 1) from the EST-SSR database of Vibrio harvetii-infected prawns, based on the results of disease-related functional prediction and core region repeat differences Polymorphism screening.
  • the DNA samples were 5 Vibrio-sensitive individuals and 5 non-susceptible individuals; 5 individuals against Enterospora hepatis and 5 controls, totaling 20 samples.
  • Table 2 shows the sample conditions. Twenty tails of Litopenaeus vannamei from 20 farms in Zhanjiang, Xuwen, Maoming and other places were collected from muscle tissue, and the genomic DNA of Litopenaeus vannamei was extracted using a marine animal tissue genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing). , the operation steps are carried out in strict accordance with the instructions. Genomic DNA quantification was performed using a NanoDrop TM 2000 spectrophotometer, and the quality was detected by agarose electrophoresis.
  • the 25 ⁇ L reaction system included: 10 ⁇ Mg 2+ PCR buffer 2.5 ⁇ L, 10mM dNTPs 1 ⁇ L, Taq enzyme 2U, 10 ⁇ M forward primer 1 ⁇ L, 10 ⁇ M reverse primer 1 ⁇ L, DNA template 10ng, the rest were made up to 25 ⁇ L with sterile double-distilled water.
  • Reaction program pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 50°C-60°C for 30 seconds, extension at 72°C for 30 seconds, a total of 10 cycles; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and annealing at 72°C for 30 seconds Extend for 30 seconds for a total of 35 cycles, and extend for 5-8 minutes at 72°C.
  • the PCR amplification products were detected by 2% agarose electrophoresis, and it was shown that 6 pairs of primers could stably amplify the bands at a specific annealing temperature.
  • the amplification products were sequenced respectively, and the results showed that the 6 pairs of primers could amplify the target bands, and the sites were: Lv-Vp001 (as shown in SEQ ID NO. 1), Lv-Vp011 (as shown in SEQ ID NO.
  • Lv-Vp012 (as shown in SEQ ID NO.3)
  • Lv-Vp023 (as shown in SEQ ID NO.4)
  • Lv-Vp029 (as shown in SEQ ID NO.5)
  • Lv- Vp041 (as shown in SEQ ID NO. 6).
  • the selected primers are listed in Table 3.
  • the 5' ends of the upstream primers of the 6 pairs of primers that were preliminarily screened were fluorescently labeled with FAM and HEX, respectively, and PCR amplification was performed using the 20 genomic DNAs of Litopenaeus vannamei extracted above as templates.
  • the reaction system was the same as that in step 2.2, and the reaction procedure was the same as that described in step 2.2 except that the annealing temperature was 60°C.
  • PCR amplification products were typed by 3730XL sequencer, and GeneMapper3.2 software was used to interpret the specific values of allele fragments, and 6 polymorphic EST-STR markers of functional genes of Litopenaeus vannamei were identified (see Figure 1).
  • the sites are: Lv-Vp001 (as shown in SEQ ID NO.1), Lv-Vp011 (as shown in SEQ ID NO.2), Lv-Vp012 (as shown in SEQ ID NO.3) , Lv-Vp023 (as shown in SEQ ID NO. 4), Lv-Vp029 (as shown in SEQ ID NO. 5) and Lv-Vp041 (as shown in SEQ ID NO. 6).
  • the expected heterozygosity, observed heterozygosity and polymorphic information content (PIC) were calculated using Cervus 3.0 software. The results showed that the number of alleles of the above 6 polymorphic microsatellite markers were 5, 2, 14, 6, 9 and 4, respectively; the observed heterozygosity was 0.45, 0.00, 0.35, 0.70, 0.60 and 0.20, respectively; expected The heterozygosity was 0.7654, 0.1846, 0.9013, 0.8231, 0.8500 and 0.6679, respectively; the PIC values were 0.7040, 0.1638, 0.8687, 0.7738, 0.8071 and 0.5834 (Table 4), except for Lv-Vp011, the rest were all greater than 0.5, indicating that these six EST-STR markers are highly polymorphic and can be used for genetic relationship analysis and molecular marker-assisted breeding of Litopenaeus vannamei.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

凡纳滨对虾抗弧菌相关EST-STR标记及其特异性引物和检测方法。利用已经构建的转录组文库，开发获得6个具有高度多态性的凡纳滨对虾EST-STR标记，编号分别为Lv-Vp001、Lv-Vp011、Lv-Vp012、Lv-Vp023、Lv-Vp029和Lv-Vp041，并分别设计了其特异性引物。凡纳滨对虾EST-SSR标记及引物可用于凡纳滨对虾遗传多样性研究及优良品种的辅助选育。

Description

凡纳滨对虾抗弧菌相关EST-STR标记及其特异性引物和检测方法 技术领域
本发明属于生物分子标记技术领域，具体涉及凡纳滨对虾抗弧菌相关EST-STR标记及其特异性引物和检测方法。
背景技术
凡纳滨对虾(Litopenaeus vannamei)，又称太平洋对虾(White pacific shrimp)。野生种群分布于中南美洲，从墨西哥到智利自然水温常年20℃以上的太平洋沿岸海域，是典型的热带性对虾。我国从南到北的沿海和多个内陆地区的海水和淡水均有养殖。目前凡纳滨对虾是世界第一养殖虾类，约占全球养殖对虾产量70％，占我国对虾养殖产量80％-90％。目前我国每年凡纳滨对虾的养殖产量超过160万吨。近年来，对虾白斑综合症病毒(WSSV)病和早期死亡综合症(EMS)细菌病等频繁暴发，养殖水环境异常波动造成养殖对虾大量死亡，另一方面市场对养殖对虾的品质要求也越来越高。市场变化、病害流行和养殖环境变化导致抗病新育种的需求日益迫切。
传统的育种方法周期长、效率低。随着现代生物技术和遗传学技术在水产领域的快速转化，以性状相关功能基因为基础的分子标记技术成为水产遗传育种的关键技术之一。微卫星序列(简单重复序列Simple Sequence repeats，SSR；或称短串联重复序列short tandem repeats,STR)等分子标记技术为基础的分子标记辅助选育技术已成为当前水产育种的关键技术。目前基于抗菌EST(Expressed Sequence Tag)的SSR标记的开发较少。
发明内容
本发明的目的在于克服现有技术中的不足，提供一种凡纳滨对虾EST-STR标记及其扩增引物、检测方法和应用，为凡纳滨对虾遗传多样性分析及分子标记辅助育种提供高度多态性的分子标记。
为实现上述发明目的，本发明利用团队构建的凡纳滨对虾幼苗，在哈氏弧菌Vibrio harveyi感染下构建的转录组文库数据，构建的测序文库在华大基因有限公司(华大基因，深圳)的Illumina HiSeq 4000平台(Illumina,San Diego,USA)进行测序。从数据库获得的凡纳滨对虾EST-SSR序列，开发凡纳滨对虾高度多态性EST-STR标记，并提供多态性引物，为凡纳滨对虾遗传多样性研究及优良品种的辅助选育提供高度多态性的分子标记。
本发明从哈氏弧菌感染的凡纳滨对虾EST-SSR数据库中根据功能预测和核心区重复差异的结果，选择了50对引物对凡纳滨对虾的基因组DNA进行PCR扩增，其中有6对引物能 稳定扩增出目的条带。采用3730xl设备检测STR样本，并用Genemapper软件分析SSR数据，最终确定了6个具有高度多态性的凡纳滨对虾EST-STR标记。
本发明的第一个目的是提供一种凡纳滨对虾EST-STR标记，所述的EST-STR标记编号为Lv-Vp001、Lv-Vp011、Lv-Vp012、Lv-Vp023、Lv-Vp029和Lv-Vp041；
所述的Lv-Vp001的核苷酸序列如SEQ ID NO.1所示；
所述的Lv-Vp011的核苷酸序列如SEQ ID NO.2所示；
所述的Lv-Vp012的核苷酸序列如SEQ ID NO.3所示；
所述的Lv-Vp023的核苷酸序列如SEQ ID NO.4所示；
所述的Lv-Vp029的核苷酸序列如SEQ ID NO.5所示；
所述的Lv-Vp041的核苷酸序列如SEQ ID NO.6所示。
本发明的第二个目的是提供一种上述的凡纳滨对虾EST-STR标记的扩增引物，所述的扩增引物包括：
针对Lv-Vp001位点：
Lv-Vp001-F：5’-ACTGGAGGATCCTGAGAGATAGC-3’；
Lv-Vp001-R：5’-GCTTCGTTCTCTCACTCTTCTCTT-3’；
针对Lv-Vp011位点：
Lv-Vp011-F：5’-AAGGATTTCTCTTACACGAACCC-3’；
Lv-Vp011-R：5’-ACCAAAAAGAAAAACAGAATGGC-3’；
针对Lv-Vp012位点：
Lv-Vp012-F：5’-AATCTACAGGACTCGAATGCACA-3’；
Lv-Vp012-R：5’-CTTTTGTCCTCCACTTTACATGC-3’；
针对Lv-Vp23位点：
Lv-Vp23-F：5’-CGAGAGAAAGAGGGAGAAAAAGA-3’；
Lv-Vp23-R：5’-TCATGGAGATGATGACTGGATAA-3’；
针对Lv-Vp29位点：
Lv-Vp29-F：5’-CACCATATTCTCCAAGAAGCAGT-3’；
Lv-Vp29-R：5’-TTACTCATCCTGCTTCAACACCT-3’；
针对Lv-Vp41位点：
Lv-Vp41-F：5’-CTTTTCGTTGATGAGTCTTTCCA-3’；
Lv-Vp41-R：5’-AGGGAGAAAGAGTGGAGTGAGAT-3’。
优选，所述的扩增引物的正向引物的5’端标记有荧光基团。
优选，所述的荧光基团为FAM或HEX。
本发明的第三个目的是提供一种凡纳滨对虾EST-STR标记的检测方法，包括以下步骤：
(1)提取凡纳滨对虾基因组DNA；
(2)以步骤(1)提取的基因组DNA为模板，分别利用上述的针对Lv-Vp001位点的引物对Lv-Vp001-F/R、针对Lv-Vp011位点的引物对Lv-Vp011-F/R、针对Lv-Vp012位点的引物对Lv-Vp012-F/R、针对Lv-Vp023位点的引物对Lv-Vp023-F/R、针对Lv-Vp029位点的引物对Lv-Vp029-F/R、针对Lv-Vp041位点的引物对Lv-Vp041-F/R进行PCR扩增；
(3)利用测序仪对PCR扩增产物进行分型。
优选，所述的PCR扩增，其反应体系25μL，包括：含Mg 2+的10×PCR buffer 2.5μL、10mM dNTPs 1μL、Taq酶2U、10μM正向引物1μL、10μM反向引物1μL、DNA模板10ng，其余由无菌双蒸水补足至25μL。
优选，所述的PCR扩增，其反应程序为：95℃预变性3分钟；94℃变性30秒，60℃退火30秒，72℃延伸30秒，共10个循环；94℃变性30秒，55℃退火30秒，72℃延伸30秒，共35个循环，72℃再延伸5-8分钟。
本发明的第四个目的是提供上述的凡纳滨对虾EST-STR标记或扩增引物在凡纳滨对虾群体遗传多样性研究、基因定位、遗传连锁图谱构建、种质鉴定、品种分类或分子标记辅助育种中的应用。
优选，所述的应用为在作为抗菌和/或抗虫性状相关的凡纳滨对虾EST-STR标记或扩增引物中的应用。
优选，所述的抗菌和/或抗虫为抗哈氏弧菌和/或抗对虾肝肠孢虫。
本发明利用从NCBI数据库获得的凡纳滨对虾EST序列，开发获得6个具有高度多态性的凡纳滨对虾EST-STR标记，编号分别为Lv-Vp001、Lv-Vp011、Lv-Vp012、Lv-Vp023、Lv-Vp029和Lv-Vp041。本发明的凡纳滨对虾EST-STR标记可用于凡纳滨对虾遗传关系分析及分子标记辅助育种，为凡纳滨对虾遗传多样性研究及优良品种的辅助选育奠定基础。
本发明发掘的凡纳滨对虾抗菌性状相关的分子标记，对凡纳滨对虾抗菌能力强苗种筛选和优良抗菌种质的筛选，均有重大的应用价值。
附图说明
图1是Lv-Vp001位点引物扩增20个凡纳滨对虾基因组DNA的STR分型图；其中S1-S20代表20个样品。
图2是Lv-Vp011位点引物扩增20个凡纳滨对虾基因组DNA的STR分型图；其中S1-S20代表20个样品。
图3是Lv-Vp012位点引物扩增20个凡纳滨对虾基因组DNA的STR分型图；其中S1-S20代表20个样品。
图4是Lv-Vp023位点引物扩增20个凡纳滨对虾基因组DNA的STR分型图；其中S1-S20代表20个样品。
图5是Lv-Vp029位点引物扩增20个凡纳滨对虾基因组DNA的STR分型图；其中S1-S20代表20个样品。
图6是Lv-Vp041位点引物扩增20个凡纳滨对虾基因组DNA的STR分型图；其中S1-S20代表20个样品。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法或者按照试剂盒说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径获得。引物合成和测序由上海生物工程有限公司完成。
实施例1：
1、凡纳滨对虾EST-STR序列的筛选
本发明从哈氏弧菌感染的凡纳滨对虾EST-SSR数据库中，依据和病害相关的功能预测和核心区重复差异的结果，选择了50对EST-STR引物(如表1所示)进行多态性筛选。
表1 凡纳滨对虾EST-STR标记引物特性表
Figure PCTCN2021094239-appb-000001
Figure PCTCN2021094239-appb-000002
Figure PCTCN2021094239-appb-000003
2、凡纳滨对虾EST-STR标记引物的筛选及结果分析
2.1凡纳滨对虾基因组DNA的提取
DNA样本是分别为弧菌敏感个体5个、不敏感个体5个；抗对虾肝肠孢虫个体和对照各5个，共计20个样本，表2为样本情况，为选取来自于广东深圳、珠海、湛江、徐闻和茂名等地20个养殖场的凡纳滨对虾20尾，分别取肌肉组织，采用海洋动物组织基因组DNA提取试剂盒(天根生化科技有限公司，北京)提取凡纳滨对虾基因组DNA，操作步骤严格按照 说明书进行。基因组DNA定量采用NanoDrop TM2000分光光度计完成，质量采用琼脂糖电泳来检测。
表2 样本情况
Figure PCTCN2021094239-appb-000004
2.2引物的初步筛选
从上述提取的凡纳滨对虾基因组DNA中随机抽取5份做模板，分别采用表1中的50对引物对该基因组DNA进行PCR梯度扩增，25μL反应体系，包括：含Mg 2+的10×PCR buffer 2.5μL、10mM dNTPs 1μL、Taq酶2U、10μM正向引物1μL、10μM反向引物1μL、DNA模板10ng，其余由无菌双蒸水补足至25μL。反应程序：95℃预变性3分钟；94℃变性30秒，50℃-60℃退火30秒，72℃延伸30秒，共10个循环；94℃变性30秒，55℃退火30秒，72℃延伸30秒，共35个循环，72℃再延伸5-8分钟。
PCR扩增产物经2％琼脂糖电泳检测，显示有6对引物在特定退火温度下能稳定扩增出条带。分别对扩增产物进行测序，结果显示该6对引物均可扩增出目的条带，位点分别为：Lv-Vp001(如SEQ ID NO.1所示)、Lv-Vp011(如SEQ ID NO.2所示)、Lv-Vp012(如SEQ ID NO.3所示)、Lv-Vp023(如SEQ ID NO.4所示)、Lv-Vp029(如SEQ ID NO.5所示)和Lv-Vp041(如SEQ ID NO.6所示)。筛选出来的引物见表3。
表3 筛选出的EST-SST引物
Figure PCTCN2021094239-appb-000005
Figure PCTCN2021094239-appb-000006
2.3多态性引物的筛选及结果分析
对上述初步筛选出的6对引物的上游引物的5’端分别用FAM和HEX进行荧光标记，以上述提取的20个凡纳滨对虾基因组DNA为模板进行PCR扩增。反应体系同步骤2.2，反应程序除退火温度为60℃外其它均与步骤2.2中所述一致。PCR扩增产物采用3730XL测序仪进行分型，并使用GeneMapper3.2软件判读等位基因片段的具体数值，确定了6个具有多态性的凡纳滨对虾功能基因EST-STR标记(见图1-6)，位点分别为：Lv-Vp001(如SEQ ID NO.1所示)、Lv-Vp011(如SEQ ID NO.2所示)、Lv-Vp012(如SEQ ID NO.3所示)、Lv-Vp023(如SEQ ID NO.4所示)、Lv-Vp029(如SEQ ID NO.5所示)和Lv-Vp041(如SEQ ID NO.6所示)。
采用Cervus 3.0软件计算期望杂合度、观察杂合度及多态性信息含量(polymorphic information content,PIC)。结果表明：上述6个多态性微卫星标记的等位基因数分别为5、2、14、6、9和4个；观测杂合度分别为0.45、0.00、0.35、0.70、0.60和0.20；期望杂合度分别为0.7654、0.1846、0.9013、0.8231、0.8500和0.6679；PIC值分别为0.7040、0.1638、0.8687、0.7738、0.8071和0.5834(表4)，除了Lv-Vp011，其余均大于0.5，说明这6个EST-STR标记均具有高度多态性，可用于凡纳滨对虾遗传关系分析及分子标记辅助育种。
表4 凡纳滨对虾功能基因EST-STR标记特性表
Figure PCTCN2021094239-appb-000007
Figure PCTCN2021094239-appb-000008
Figure PCTCN2021094239-appb-000009
Figure PCTCN2021094239-appb-000010

Claims (10)

  1. 一种凡纳滨对虾EST-STR标记，其特征在于，所述的EST-STR标记编号为Lv-Vp001、Lv-Vp011、Lv-Vp012、Lv-Vp023、Lv-Vp029和Lv-Vp041；
    所述的Lv-Vp001的核苷酸序列如SEQ ID NO.1所示；
    所述的Lv-Vp011的核苷酸序列如SEQ ID NO.2所示；
    所述的Lv-Vp012的核苷酸序列如SEQ ID NO.3所示；
    所述的Lv-Vp023的核苷酸序列如SEQ ID NO.4所示；
    所述的Lv-Vp029的核苷酸序列如SEQ ID NO.5所示；
    所述的Lv-Vp041的核苷酸序列如SEQ ID NO.6所示。
  2. 一种权利要求1所述的凡纳滨对虾EST-STR标记的扩增引物，其特征在于，所述的扩增引物包括：
    针对Lv-Vp001位点：
    Lv-Vp001-F：5’-ACTGGAGGATCCTGAGAGATAGC-3’；
    Lv-Vp001-R：5’-GCTTCGTTCTCTCACTCTTCTCTT-3’；
    针对Lv-Vp011位点：
    Lv-Vp011-F：5’-AAGGATTTCTCTTACACGAACCC-3’；
    Lv-Vp011-R：5’-ACCAAAAAGAAAAACAGAATGGC-3’；
    针对Lv-Vp012位点：
    Lv-Vp012-F：5’-AATCTACAGGACTCGAATGCACA-3’；
    Lv-Vp012-R：5’-CTTTTGTCCTCCACTTTACATGC-3’；
    针对Lv-Vp23位点：
    Lv-Vp23-F：5’-CGAGAGAAAGAGGGAGAAAAAGA-3’；
    Lv-Vp23-R：5’-TCATGGAGATGATGACTGGATAA-3’；
    针对Lv-Vp29位点：
    Lv-Vp29-F：5’-CACCATATTCTCCAAGAAGCAGT-3’；
    Lv-Vp29-R：5’-TTACTCATCCTGCTTCAACACCT-3’；
    针对Lv-Vp41位点：
    Lv-Vp41-F：5’-CTTTTCGTTGATGAGTCTTTCCA-3’；
    Lv-Vp41-R：5’-AGGGAGAAAGAGTGGAGTGAGAT-3’。
  3. 根据权利要求2所述的扩增引物，其特征在于，所述的扩增引物的正向引物的5’端标记有荧光基团。
  4. 根据权利要求3所述的扩增引物，其特征在于，所述的荧光基团为FAM或HEX。
  5. 一种凡纳滨对虾EST-STR标记的检测方法，其特征在于，包括以下步骤：
    (1)提取凡纳滨对虾基因组DNA；
    (2)以步骤(1)提取的基因组DNA为模板，分别利用上述的针对Lv-Vp001位点的引物对Lv-Vp001-F/R、针对Lv-Vp011位点的引物对Lv-Vp011-F/R、针对Lv-Vp012位点的引物对Lv-Vp012-F/R、针对Lv-Vp023位点的引物对Lv-Vp023-F/R、针对Lv-Vp029位点的引物对Lv-Vp029-F/R、针对Lv-Vp041位点的引物对Lv-Vp041-F/R进行PCR扩增；
    (3)利用测序仪对PCR扩增产物进行分型。
  6. 根据权利要求5所述的检测方法，其特征在于，所述的PCR扩增，其反应体系25μL，包括：含Mg 2+的10×PCR buffer 2.5μL、10mM dNTPs 1μL、Taq酶2U、10μM正向引物1μL、10μM反向引物1μL、DNA模板10ng，其余由无菌双蒸水补足至25μL。
  7. 根据权利要求5所述的检测方法，其特征在于，所述的PCR扩增，其反应程序为：95℃预变性3分钟；94℃变性30秒，60℃退火30秒，72℃延伸30秒，共10个循环；94℃变性30秒，55℃退火30秒，72℃延伸30秒，共35个循环，72℃再延伸5-8分钟。
  8. 权利要求1所述的凡纳滨对虾EST-STR标记或权利要求2所述的扩增引物在凡纳滨对虾群体遗传多样性研究、基因定位、遗传连锁图谱构建、种质鉴定、品种分类或分子标记辅助育种中的应用。
  9. 根据权利要求8所述的应用，其特征在于，为在作为抗菌和/或抗虫性状相关的凡纳滨对虾EST-STR标记或扩增引物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述的抗菌和/或抗虫为抗哈氏弧菌和/或抗对虾肝肠孢虫。
PCT/CN2021/094239 2021-04-15 2021-05-18 凡纳滨对虾抗弧菌相关est-str标记及其特异性引物和检测方法 WO2022068215A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023548987A JP2023546627A (ja) 2021-04-15 2021-05-18 バナメイエビのビブリオ抵抗性関連ｅｓｔ‐ｓｔｒマーカーおよびその特異のプライマー並びに検出方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110405484.8A CN113005204A (zh) 2021-04-15 2021-04-15 凡纳滨对虾抗弧菌相关est-str标记及其特异性引物和检测方法
CN202110405484.8 2021-04-15

Publications (1)

Publication Number Publication Date
WO2022068215A1 true WO2022068215A1 (zh) 2022-04-07

Family

ID=76389349

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/094239 WO2022068215A1 (zh) 2021-04-15 2021-05-18 凡纳滨对虾抗弧菌相关est-str标记及其特异性引物和检测方法

Country Status (3)

Country Link
JP (1) JP2023546627A (zh)
CN (1) CN113005204A (zh)
WO (1) WO2022068215A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113862276A (zh) * 2021-09-03 2021-12-31 中国科学院海洋研究所 一组评价对虾抗副溶血弧菌性状的指标基因及其应用
CN114736972A (zh) * 2022-05-12 2022-07-12 岭南师范学院 一种用于评估四指马鲅生长相关性状的试剂

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107287296A (zh) * 2017-06-20 2017-10-24 中国科学院南海海洋研究所 凡纳滨对虾功能基因est‑ssr标记及其特异性引物和检测方法
WO2017215055A1 (zh) * 2016-06-14 2017-12-21 中国科学院南海海洋研究所 凡纳滨对虾渗透压调节相关功能基因est-ssr标记及其特异性引物和检测方法
CN107881246A (zh) * 2017-12-01 2018-04-06 中国科学院南海海洋研究所 凡纳滨对虾est‑str标记及其扩增引物、检测方法和应用
CN108034729A (zh) * 2017-12-01 2018-05-15 中国科学院南海海洋研究所 一种与凡纳滨对虾生长性状相关的EST-STR标记Lv-F36a及其扩增引物和应用
CN108611429A (zh) * 2018-05-10 2018-10-02 中山大学 凡纳滨对虾抗病性相关est-ssr分子标记及其应用
CN110791571A (zh) * 2019-11-15 2020-02-14 中国科学院南海海洋研究所 区分凡纳滨对虾抗哈氏弧菌感染能力的snp标记及其检测方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110452992A (zh) * 2019-06-27 2019-11-15 浙江省海洋水产养殖研究所 一种凡纳滨对虾est微卫星位点引物的标记方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017215055A1 (zh) * 2016-06-14 2017-12-21 中国科学院南海海洋研究所 凡纳滨对虾渗透压调节相关功能基因est-ssr标记及其特异性引物和检测方法
CN107287296A (zh) * 2017-06-20 2017-10-24 中国科学院南海海洋研究所 凡纳滨对虾功能基因est‑ssr标记及其特异性引物和检测方法
CN107881246A (zh) * 2017-12-01 2018-04-06 中国科学院南海海洋研究所 凡纳滨对虾est‑str标记及其扩增引物、检测方法和应用
CN108034729A (zh) * 2017-12-01 2018-05-15 中国科学院南海海洋研究所 一种与凡纳滨对虾生长性状相关的EST-STR标记Lv-F36a及其扩增引物和应用
CN108611429A (zh) * 2018-05-10 2018-10-02 中山大学 凡纳滨对虾抗病性相关est-ssr分子标记及其应用
CN110791571A (zh) * 2019-11-15 2020-02-14 中国科学院南海海洋研究所 区分凡纳滨对虾抗哈氏弧菌感染能力的snp标记及其检测方法和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FRANKLIN PÉREZ ; JUAN ORTIZ ; MARIUXI ZHINAULA ; CESAR GONZABAY ; JORGE CALDERÓN ; FILIP A.M.J. VOLCKAERT: "Development of EST-SSR Markers by Data Mining in Three Species of Shrimp: Litopenaeus vannamei, Litopenaeus stylirostris, and Trachypenaeus birdy", MARINE BIOTECHNOLOGY, vol. 7, no. 5, 1 October 2005 (2005-10-01), Ne , pages 554 - 569, XP019368157, ISSN: 1436-2236 *
SANTOS CAMILLA A, ROSSINI BRUNO C, MARQUES CARLA G, GALETTI PEDRO M, FREITAS PATRÍCIA D: "Characterization and genomic annotation of polymorphic EST-SSR loci in Litopenaeus vannamei shrimp", AQUACULUTRE RESERCH, vol. 43, no. 10, 1 September 2012 (2012-09-01), GB , pages 1567 - 1570, XP055917590, ISSN: 1355-557X, DOI: 10.1111/j.1365-2109.2011.02956.x *
ZHU, QIJIAN ET AL.: "Disease Resistance and Genetic Diversity Analysis in Selected Populations of Macrobrachium Rosenbergii", JOURNAL OF FISHERIES OF CHINA, vol. 37, no. 10, 15 October 2013 (2013-10-15), pages 1468 - 1478, XP055917585 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113862276A (zh) * 2021-09-03 2021-12-31 中国科学院海洋研究所 一组评价对虾抗副溶血弧菌性状的指标基因及其应用
CN113862276B (zh) * 2021-09-03 2023-10-13 中国科学院海洋研究所 一组评价对虾抗副溶血弧菌性状的指标基因及其应用
CN114736972A (zh) * 2022-05-12 2022-07-12 岭南师范学院 一种用于评估四指马鲅生长相关性状的试剂
CN114736972B (zh) * 2022-05-12 2024-01-30 岭南师范学院 一种用于评估四指马鲅生长相关性状的试剂

Also Published As

Publication number Publication date
CN113005204A (zh) 2021-06-22
JP2023546627A (ja) 2023-11-06

Similar Documents

Publication Publication Date Title
WO2017215055A1 (zh) 凡纳滨对虾渗透压调节相关功能基因est-ssr标记及其特异性引物和检测方法
EP2010674B1 (en) A sex-specific marker for shrimps and prawns
CN113913533B (zh) 与草鱼性状相关的snp分子标记及其应用
WO2022068215A1 (zh) 凡纳滨对虾抗弧菌相关est-str标记及其特异性引物和检测方法
CN105506162B (zh) 长牡蛎快速生长相关的snp标记及其鉴定方法和应用
JP6877680B2 (ja) 成長性遺伝形質を有するアカマダラハタの識別方法
CN108559782B (zh) 短体副鳅微卫星位点及其引物和应用
CN107881246B (zh) 凡纳滨对虾est-str标记及其扩增引物、检测方法和应用
Aoki et al. Second generation physical and linkage maps of yellowtail (Seriola quinqueradiata) and comparison of synteny with four model fish
CN110760599B (zh) 大麻哈鱼微卫星分子标记位点及多态性引物和应用
Feng et al. Characterization of 40 single nucleotide polymorphism (SNP) via T m-shift assay in the mud crab (Scylla paramamosain)
CN113637765A (zh) 鉴定大口黑鲈遗传性别的分子标记及应用
CN108588238B (zh) 一种快速鉴定锈斑蟳性别特异分子标记和遗传性别的方法
CN109182546B (zh) 用于穿山甲亲子鉴定的ssr荧光标记引物及应用
CN114891900B (zh) 一种棕点石斑鱼微卫星标记及其引物
CN105603097B (zh) 用于合浦珠母贝微卫星家系鉴定的微卫星标记引物及鉴定方法和应用
Ludanny et al. Polymorphism of microsatellite markers in Russian common carp (Cyprinus carpio L.) breeds
Wang et al. Development of EST-SSRs from the ark shell (Scapharca broughtonii) transcriptome and their application in genetic analysis of four populations
CN110951892A (zh) 用于几种鲟鱼物种鉴定的ssr引物对组、试剂盒、鉴定方法及应用
Wu et al. Analysis on microsatellite loci of Daphnia similoides sinensis and rapid development of primer based on transcriptome sequencing
CN112391481B (zh) 鉴别尖塘鳢鱼种的SNPs及基于SNPs的AS-PCR引物组及其检测方法与应用
KR101437381B1 (ko) 참굴 삼배체 판별용 유전자 키트 및 이를 이용한 판별 방법
CN113215280B (zh) 一种鉴别棘头梅童鱼南北群体通用的遗传性别分子标记、引物及其应用
CN117512135A (zh) 基于转录组测序的凡纳滨对虾pH相关多态性微卫星标记及其扩增引物和应用
KR102074139B1 (ko) 미꾸리과 어종 판별용 유전자 마커 및 판별방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21873878

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023548987

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21873878

Country of ref document: EP

Kind code of ref document: A1