WO2018214187A1 - 一种与凡纳滨对虾耐低盐性状相关的snp标记、扩增引物及其应用 - Google Patents
一种与凡纳滨对虾耐低盐性状相关的snp标记、扩增引物及其应用 Download PDFInfo
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- WO2018214187A1 WO2018214187A1 PCT/CN2017/088329 CN2017088329W WO2018214187A1 WO 2018214187 A1 WO2018214187 A1 WO 2018214187A1 CN 2017088329 W CN2017088329 W CN 2017088329W WO 2018214187 A1 WO2018214187 A1 WO 2018214187A1
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- 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
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- C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
- C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the invention relates to the technical field of water production, in particular to a SNP marker, an amplification primer and an application thereof related to low salt tolerance of L. vannamei.
- Litopenaeus vannamei is the world's largest producer of cultured prawns. In recent years, freshwater aquaculture of P. vannamei has developed rapidly, and the demand for low-salt varieties of P. vannamei has gradually increased. Therefore, many scientific research institutions and shrimp companies have begun to carry out the breeding of low-salt varieties of Penaeus vannamei.
- Single Nucleotide Polymorphism is a SNP that refers to DNA sequence polymorphism caused by genomic single nucleotide variation (including base conversion, transversion, single base insertion or deletion, etc.). Sex is the latest third-generation DNA molecular marker. It is widely used in molecular marker-assisted breeding because of its large number, wide distribution, genetic stability, accurate and simple measurement, hidden polymorphism that can not be detected by other technologies, and many advantages related to gene function.
- the Na, K-ATPase gene is an important osmotic regulation gene in crustaceans. Studies have shown that the Na, K-ATPase gene is a functional gene related to the salinity resistance of Litopenaeus vannamei. Therefore, the present invention aims to develop a SNP marker related to low salt tolerance traits of Litopenaeus vannamei for the Na, K-ATPase alpha subunit of Litopenaeus vannamei, in order to assist in the selective breeding of low salt tolerance varieties of Litopenaeus vannamei, thereby accelerating Breeding process of low-salt varieties of Penaeus vannamei.
- the present invention detects the genomic DNA of the low-salt and low-tolerant low-salt P. vannamei as a template, and detects the template in the Na, K-ATPase ⁇ subunit by PCR amplification and sequencing.
- the genotype of each SNP locus contained in the genomic sequence was determined by association analysis method to identify low salt-tolerant SNP markers, and SNP site amplification primers were provided to establish molecular marker-assisted selection of low salt tolerance varieties of Litopenaeus vannamei
- the technical system of breeding lays the foundation for the rapid selection of low-salt varieties of Penaeus vannamei.
- a first object of the present invention is to provide a SNP marker associated with a low salt tolerance trait of S. vannamei, the SNP marker located at base 379 from the 5' end of the sequence set forth in SEQ ID NO.
- the base is C or T.
- the sequence of SEQ ID NO. 2 at Y at 379 bp represents C or T, and the italicized portion represents an intron sequence.
- a second object of the present invention is to provide a SNP-tagged amplification primer associated with low salt tolerance of L. vannamei, including the following primers:
- Lv-R 5'-GAGAAACCACCGAAGAGG-3'.
- a third object of the present invention is to provide the above-mentioned SNP marker associated with low salt tolerance of L. vannamei in molecular marker-assisted breeding of Penaeus vannamei.
- a fourth object of the present invention is to provide a method for breeding a low salt-tolerant species of Penaeus vannamei, comprising the following steps:
- the PCR amplification method preferably has a reaction system of 25 ⁇ L, including: 0.5 ⁇ L of 10 ⁇ PCR buffer without Mg 2+ , 2.0 ⁇ L of 25 mM MgCl 2 , 0.5 ⁇ L of 10 mM dNTP, and 5 ⁇ / ⁇ L.
- HS DNA Polymerase 0.2 ⁇ L, 10 ⁇ M forward primer 0.5 ⁇ L, 10 ⁇ M reverse primer 0.5 ⁇ L, DNA template 12.5 ng, and the rest was supplemented with sterile water to 25 ⁇ L.
- the reaction procedure is preferably: pre-denaturation at 95 ° C for 3 minutes; denaturation at 95 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 1 minute for 35 cycles, and extension at 72 ° C for 10 minutes.
- the present invention designs primers based on the mRNA sequence of the Na, K-ATPase ⁇ subunit of the Penaeus vannamei obtained from NCBI (GenBank: KF765670.1), respectively, and the genome of the shrimp with low salt tolerance and low salt tolerance
- NCBI GenBank: KF765670.1
- the DNA was used as a template for PCR amplification, and a PCR product of 533 bp in length was obtained. It contains one intron of 353 bp in length.
- the sequencing results were compared and analyzed. A total of 11 SNP loci were detected in the sequence. By correlating the genotypes of these templates at each SNP locus, a SNP marker related to low salt tolerance was identified. .
- the invention establishes a technical system for molecular marker-assisted breeding of low salt tolerance varieties of L. vannamei, and lays a foundation for rapidly selecting and breeding low-salt varieties of Penaeus vannamei.
- Figure 1 is a genotype peak map of the Lv-HR08 locus.
- Test shrimp number (bar) 426 Stress 1h deaths (articles) 0 Stress 2h deaths (articles) 186 Stress 4h deaths (articles) 137 Stress 7h deaths (articles) 31 Stress 10h deaths (articles) 19 Stress 10h survival number (bar) 53
- the shrimp seedlings of P. vannamei which survived after 10 hours of stress, were used as low-salt-resistant samples, and shrimps that died after 2 hours of stress were used as low-salt samples.
- Genomic DNA quantified using NanoDrop TM 2000 spectrophotometer completed, quality detected by agarose electrophoresis.
- Primer design requirements are: primer length 18-22bp, GC content 40-60%, Tm value 50-62 ° C, the difference between the Tm values of the upstream and downstream primers is not more than 5, and try to avoid primer dimer, hairpin Structure and mismatch, etc.
- the primer sequences are as follows:
- Lv-R (647-630): 5'-GAGAAACCACCGAAGAGG-3'.
- the number in parentheses represents the position of the nucleotide in the primer in the Na, K-ATPase alpha subunit mRNA sequence.
- a random selection of the genomic DNA of P. vannamei extracted from step 2.1 was used as a template, and the DNA was amplified by PCR using the primer Lv-F/Lv-R designed in step 2.2.
- the reaction system was 25 ⁇ L, including: 10 ⁇ PCR buffer. (without Mg 2+ ) 2.5 ⁇ L, MgCl 2 (25 mM) 2.0 ⁇ L, dNTP (10 mM) 0.5 ⁇ L, LA Taq enzyme (5 U/ ⁇ L) 0.2 ⁇ L, forward primer (10 ⁇ M) 0.5 ⁇ L, reverse primer (10 ⁇ M) 0.5 ⁇ L, DNA template (25 ng/ ⁇ L) 0.5 ⁇ L, and sterile water 18.3 ⁇ L.
- the reaction procedure was: pre-denaturation at 95 ° C for 3 minutes; denaturation at 95 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 2 minutes for a total of 35 cycles; 72 ° C for another 10 minutes.
- the PCR amplification product was detected by 1% agarose electrophoresis, which showed that the primer pair could stably amplify a single band.
- the amplified product was cloned and sequenced. The result showed that the amplified product was 533 bp in length. In addition to the nucleotide sequence of interest, it also contained an intron sequence of 353 bp in length.
- the nucleotide sequence of the sequence was SEQ ID NO. Shown as .1 (designated as SEQ IDPGLv-NK, the italicized portion of the sequence shown in SEQ ID NO. 1 represents an intron sequence).
- the low-salt and low-tolerant low-salt prawn genomic DNA extracted in step 2.1 was used as a template, and PCR amplification was carried out using the primer Lv-F/Lv-R described in step 2.2.
- the reaction system and reaction procedure are basically the same as described in step 2.3, except that the high-fidelity PCR enzyme is used in the PCR system for screening SNP sites.
- HS DNA Polymerase replaced the LA Taq enzyme and the 72 ° C extension was reduced from 2 minutes to 1 minute.
- the PCR amplification products were first detected by 2% agarose electrophoresis and then sequenced using a 3730XL sequencer.
- the SNP marker is resistant to low salt labeling of the prawn, the italic part represents the intron sequence, and the SNP site is the bold mixed base Y, Y represents T or C) has three genotypes of CC, CT and TT (see Figure 1 for peak map), of which TT genotype is only detected in low salt-tolerant L. vannamei (Table 2), suggesting that TT genotype is low tolerance.
- the salt-preferred genotype can be used as a low salt-tolerant molecular marker for Penaeus vannamei and is used for the auxiliary breeding of low-salt varieties of Penaeus vannamei.
- the number in the second column indicates the position of the SNP locus in the SEQ IDPGLv-NK sequence.
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Abstract
Description
试验虾苗数量(条) | 426 |
应激1h死亡数量(条) | 0 |
应激2h死亡数量(条) | 186 |
应激4h死亡数量(条) | 137 |
应激7h死亡数量(条) | 31 |
应激10h死亡数量(条) | 19 |
应激10h存活数量(条) | 53 |
Claims (6)
- 一种与凡纳滨对虾耐低盐性状相关的SNP标记,其特征在于,所述的SNP标记位于SEQ ID NO.2所示序列自5’端起第379位碱基处,碱基为C或T。
- 一种与凡纳滨对虾耐低盐性状相关的SNP标记的扩增引物,其特征在于,包括以下引物:Lv-F:5’-TGATGAGCACAAGGTCCCA-3’;Lv-R:5’-GAGAAACCACCGAAGAGG-3’。
- 权利要求1所述的与凡纳滨对虾耐低盐性状相关的SNP标记在凡纳滨对虾分子标记辅助育种中的应用。
- 一种耐低盐凡纳滨对虾品种的选育方法,其特征在于,包括以下步骤:a、提取待测凡纳滨对虾基因组DNA;b、采用权利要求2所述的扩增引物Lv-F和Lv-R对待测凡纳滨对虾基因组DNA进行PCR扩增;c、对扩增产物进行测序,确定权利要求1所述的SNP标记的基因型,选择TT基因型的个体作为后备亲本进行耐低盐凡纳滨对虾品种育种。
- 根据权利要求4或5所述的选育方法,其特征在于,所述的PCR扩增,其反应程序为:95℃预变性3分钟;95℃变性30秒,55℃退火30秒,72℃延伸1分钟,共35个循环;72℃再延伸10分钟。
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CN114592068A (zh) * | 2022-03-02 | 2022-06-07 | 中国水产科学研究院黄海水产研究所 | 一种凡纳滨对虾高繁殖力的分子标记x1w1及其应用 |
CN114686603A (zh) * | 2022-03-02 | 2022-07-01 | 中国水产科学研究院黄海水产研究所 | 分子标记21w2及其用于筛选具有高繁殖力的凡纳滨对虾群体中的应用 |
CN114686603B (zh) * | 2022-03-02 | 2023-05-30 | 中国水产科学研究院黄海水产研究所 | 分子标记21w2及其用于筛选具有高繁殖力的凡纳滨对虾群体中的应用 |
CN114752685B (zh) * | 2022-03-02 | 2023-05-30 | 中国水产科学研究院黄海水产研究所 | 一种凡尔纳对虾分子标记及其引物和应用 |
CN114752685B9 (zh) * | 2022-03-02 | 2023-08-15 | 中国水产科学研究院黄海水产研究所 | 一种凡纳滨对虾分子标记及其引物和应用 |
CN114574594A (zh) * | 2022-03-03 | 2022-06-03 | 佛山市南海百容水产良种有限公司 | 加州鲈性别特异snp分子标记引物及其应用 |
CN114574594B (zh) * | 2022-03-03 | 2023-12-22 | 广东百容水产良种集团有限公司 | 加州鲈性别特异snp分子标记引物及其应用 |
WO2023245401A1 (zh) * | 2022-06-21 | 2023-12-28 | 中国海洋大学 | 一种基于特征snp标记的凡纳滨对虾养殖品种鉴定方法 |
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