WO2021159661A1 - 一种凡纳滨对虾的耐低温关联的snp分子标记、检测引物及其应用 - Google Patents

一种凡纳滨对虾的耐低温关联的snp分子标记、检测引物及其应用 Download PDF

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WO2021159661A1
WO2021159661A1 PCT/CN2020/100608 CN2020100608W WO2021159661A1 WO 2021159661 A1 WO2021159661 A1 WO 2021159661A1 CN 2020100608 W CN2020100608 W CN 2020100608W WO 2021159661 A1 WO2021159661 A1 WO 2021159661A1
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genotype
individuals
low temperature
molecular marker
litopenaeus vannamei
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French (fr)
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黄文�
王国霞
孙慧明
陈晓瑛
舒琥
郑春田
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广东省农业科学院动物科学研究所
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the invention belongs to the field of molecular marker-assisted breeding of aquatic animals, and specifically relates to a SNP molecular marker, detection primer, molecular function and application of microchromosome maintenance gene 2 (MCM2) associated with low temperature tolerance traits of Litopenaeus vannamei.
  • MCM2 microchromosome maintenance gene 2
  • Litopenaeus vannamei (commonly known as Penaeus vannamei) is the most important species of prawn in China.
  • Penaeus vannamei is the most important species of prawn in China.
  • the annual production of Litopenaeus vannamei in China exceeded 1.76 million tons, and the annual demand for seedlings exceeded 1,000 billion.
  • Litopenaeus vannamei aquaculture is of great significance.
  • Low temperature tolerance is an important resistance trait of Litopenaeus vannamei.
  • this species of prawn has been promoted to the waters of northern China for breeding, including the sea and freshwater waters of northern provinces such as Shanxi, northern Shaanxi, and Shandong.
  • Low temperature tolerance has become an important resistance trait pursued by the Litopenaeus vannamei aquaculture industry in northern China.
  • the market price of live Litopenaeus vannamei is more than three times that of the normal season. Farming farmers often risk the time to market to the winter, and often the large-scale death of farmed prawns due to the sudden drop in water temperature caused by the winter cold wave.
  • the breeding of low-temperature-tolerant species is also an urgent need for the Litopenaeus vannamei aquaculture industry in southern China.
  • Molecular breeding that is, molecular marker-assisted selection breeding, refers to the technology of using DNA molecular markers to select breeding materials.
  • the molecular breeding method uses effective molecular markers to select parental candidates, which can more quickly improve the economic traits of offspring.
  • SNP Single Nucleotide Polymorphism
  • SNP Single Nucleotide Polymorphism
  • High-quality DNA molecular markers are the basis for molecular breeding. At present, although many molecular markers for shrimp have been developed for different traits, the molecular functions of molecular markers have not been clarified.
  • Minichromosome Maintenance Gene 2 English name: Minichromosome Maintenance Complex Component 2 (MCM2), is a member of the minichromosome maintenance gene family involved in initiating eukaryotic genome replication. There has never been a report of the MCM2 gene in Litopenaeus vannamei, and no studies have found that this gene is associated with low temperature traits.
  • MCM2 Minichromosome Maintenance Complex Component 2
  • the present invention aims to provide a SNP molecular marker, detection primer, molecular function and application of the microchromosome maintenance gene 2 (MCM2) associated with low temperature tolerance of Litopenaeus vannamei.
  • SNP molecular markers, detection primers, molecular functions and application methods thereof are unpredictable by conventional knowledge or methods currently known.
  • the SNP molecular markers of the present invention can be used to detect low temperature tolerance traits of Litopenaeus vannamei parents, and are provided according to the present invention
  • the method can select the reserve parents with stronger low-temperature tolerance, which is beneficial to quickly improve the low-temperature tolerance traits of the offspring of Litopenaeus vannamei and shorten the breeding cycle of low-temperature-tolerant shrimp lines.
  • the SNP molecular marker associated with the low temperature tolerance traits of Litopenaeus vannamei microchromosome maintenance gene 2 (MCM2) of the present invention is shown in SEQ ID NO.1.
  • the SNP molecular marker is: the G/C genotype or G/G genotype at the 1,925 base position of the sequence shown in SEQ ID NO.1; or the 3,414, 3,475 and 3,537 bases are fully linked, which is the A/A gene Type or A/C genotype; or T/T genotype or T/G genotype at base 3,776; or A/C genotype or A/A genotype at base 6,679.
  • the average survival time of individuals with G/C genotype at base 1,925 under low temperature and pressure conditions is significantly higher than that of individuals with G/G genotype; bases 3,414, 3,475, and 3,537 are fully linked, and their A/A gene
  • the average survival time of individuals with low temperature and pressure is significantly higher than that of A/C genotype individuals; the average survival time of individuals with T/T genotype at base 3,776 under low temperature and pressure is significantly higher than that of T/G Genotype individuals; the average survival time of individuals with A/C genotype at base 6,679 under low temperature and pressure conditions is significantly higher than that of individuals with A/A genotype.
  • the second objective of the present invention is to provide detection primers labeled with the above SNP molecules, as shown in any of the following detection primer pairs
  • g.MCM2-3,414-F 5’-CTGAGGTGACTGGTGTGTACAC-3’;
  • g.MCM2-3,776-F 5’-TGAGAGGATTGTGGCCTCAATT-3’;
  • the third object of the present invention is to provide a detection kit, which includes a detection primer pair labeled with the above-mentioned SNP molecule.
  • the fourth objective of the present invention is to provide the application of the above-mentioned SNP molecular markers, SNP molecular marker detection primers and detection kits associated with low temperature tolerance traits in the breeding of low temperature tolerance and stress tolerance of Litopenaeus vannamei.
  • the fifth object of the present invention is to provide a method for detecting low temperature tolerance of Litopenaeus vannamei, which includes: extracting the genomic DNA of Litopenaeus vannamei to be tested, amplifying the detection primer pair with the above-mentioned SNP molecular marker, and sequencing The sequencing result is obtained, the genotype of the SNP molecular marker is determined based on the sequencing result, and then the low temperature tolerance of the test Litopenaeus vannamei is judged.
  • the average survival time of individuals with G/C genotype at base 1,925 under low temperature and pressure conditions is significantly higher than that of individuals with G/G genotype; bases 3,414, 3,475 and 3,537 are completely linked, and their A/A
  • the average survival time of genotype individuals under low temperature and stress conditions is significantly higher than that of A/C genotype individuals; the average survival time of individuals with T/T genotype at base 3,776 under low temperature and stress conditions is significantly higher than that of T/C genotype individuals.
  • G genotype individuals; the average survival time of individuals with A/C genotype at base 6,679 under low temperature and pressure conditions is significantly higher than that of individuals with A/A genotype.
  • Low temperature tolerance is an important stress resistance trait of Litopenaeus vannamei.
  • the SNP molecular markers and methods of the present invention can be used to screen the reserve parent populations, so as to eliminate low-temperature sensitive species early.
  • the individual parents of Litopenaeus vannamei will help to quickly enhance the low temperature tolerance of the offspring of Litopenaeus vannamei.
  • it is beneficial to quickly improve the low-temperature tolerance traits of the offspring of Litopenaeus vannamei, shorten the breeding cycle of low-temperature-tolerant shrimp strains, and has greater potential application value.
  • Figure 1 is: A: SNP polymorphism site map of Shrimp MCM2 gene, the number represents the base position of the polymorphism site; B: Linkage analysis diagram of SNP polymorphism site of Shrimp MCM2 gene, black represents complete linkage; C: Statistical results of individual genotypes, "***" indicates significance test P ⁇ 0.001; D: indicates the sequence peak map of mutation site; E: correlation analysis of SNP site amino acid and low temperature traits, the size of the ordinate indicates The size of the correlation with low temperature traits.
  • Figure 2 is: A: Under the control temperature condition, after the MCM2 gene is interfered with expression, most of the individual shrimps can still survive normally; B: At 15°C, after the MCM2 gene is interfered with, the shrimp survival rate drops to 50% About; C: Under the condition of lower 13°C, after the MCM2 gene is interfered with, the survival rate of the shrimp drops to 30%.
  • the red line represents the survival curve of the RNAi interference sequence injected with the MCM2 gene, and the blue represents the injection of nonsense RNAi interference.
  • the survival curve of the sequence, black represents the survival curve of DEPC water injection.
  • PCR detection of the MCM2 gene of individual shrimps in the low-temperature sensitive group and low-temperature tolerance group is carried out.
  • the reaction procedure is as follows : Pre-denaturation at 95°C for 4 minutes, (denaturation at 95°C for 30 seconds, annealing at 52-57°C for 20 seconds, and extension at 72°C for 1 minute) ⁇ 35 cycles, and finally extension at 72°C for 10 minutes.
  • the PCR product was sequenced.
  • the sequence of the MCM2 gene is shown in SEQ ID NO. 1
  • the cDNA sequence is shown in SEQ ID NO. 2. According to the statistics of individual genotypes, it is found that:
  • the average survival time of individuals with G/C genotype at base 1,925 under low temperature and pressure conditions is significantly higher than that of individuals with G/G genotype; bases 3,414, 3,475 and 3,537 are completely linked, and its A/
  • the average survival time of individuals with genotype A under low temperature and pressure conditions is significantly higher than that of individuals with genotype A/C; the average survival time of individuals with genotype T/T at base 3,776 under low temperature and pressure conditions is significantly higher than that of T /G genotype individuals; the average survival time of A/C genotype individuals at base 6,679 under low temperature and pressure conditions is significantly higher than that of A/A genotype individuals ( Figure 1-C, Table 1);
  • the G>C missense mutation at the 1,925 base position causes the amino acid encoded by the gene to be mutated from aspartic acid (Asp, D) to glutamic acid (Glu, E); the 3,414 base position A>C A missense mutation causes the amino acid encoded by the gene to be mutated from glutamine (Gln, Q) to lysine (Lys, K); a missense mutation at the 3,776 base position T>G causes the amino acid encoded by the gene to be methionine (Met, M) is mutated to isoleucine (Ile, I); A>C missense mutation at base 6,679 causes the amino acid encoded by this gene to be mutated from histidine (His, H) to proline ( Pro, P); The 3,475 and 3,537 bases are located in the intron of the MCM2 gene, which does not cause changes in the encoded amino acid ( Figure 1-A, D, E);
  • RNAi sequence of the MCM2 gene of the present invention siRNA sense: 5'-AGGCTTCCCTGTGTTCGCGACTGTT-3', siRNA antisense: 5'-CAGTCGCGAACACAGGGAAGCCTTT-3'
  • prawns at 25°C, 15°C, and 13°C were injected respectively, and compared
  • the group was injected with DEPC water and nonsense RNA strands, specifically: siRNA solution and nonsense RNA strands (dissolved in DEPC water) were injected at a dose of RNA/body weight of 3 ⁇ g/g, and the control group was injected with the same volume of DEPC aqueous solution, the total volume of injection It should be less than 50 ⁇ l, and the injection site is the abdominal muscle of the shrimp.
  • RNAi interference sequence disclosed in the present invention can significantly reduce the survival rate of shrimp at low temperature, indicating that the RNAi interference sequence disclosed in the present invention can interfere with the shrimp MCM2 gene. Normal expression indicates that the gene has important molecular functions under low temperature and pressure conditions ( Figure 2).

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Abstract

一种凡纳滨对虾的耐低温关联的SNP分子标记、检测引物及其应用。所述的SNP分子标记的序列如SEQ ID NO.1所示，所述的凡纳滨对虾的SNP分子标记是：SEQ ID NO.1所示序列的第1,925碱基位置的G/C基因型或G/G基因型；或第3,414、3,475和3,537碱基完全连锁，其为A/A基因型或A/C基因型；或第3,776碱基位置的T/T基因型或T/G基因型；或第6,679碱基位置的A/C基因型或A/A基因型。所述分子标记、检测引物及其应用，有利于快速提升凡纳滨对虾子代的耐低温性状，缩短耐低温对虾品系的选育周期，具有较大的潜在应用价值。

Description

一种凡纳滨对虾的耐低温关联的SNP分子标记、检测引物及其应用 技术领域：
本发明属于水产动物分子标记辅助育种领域，具体涉及一种凡纳滨对虾低温耐受性状关联的微小染色体维持基因2(MCM2)的SNP分子标记、检测引物、分子功能及其应用。
背景技术：
凡纳滨对虾(Litopenaeus vannamei，俗称南美白对虾)是中国最重要的对虾养殖品种，2018年中国凡纳滨对虾养殖年产量超过176万吨，种苗年需求量超过10000亿尾，良种繁育对凡纳滨对虾养殖业具有重要意义。耐低温是凡纳滨对虾的重要抗逆性状。一方面，由于凡纳滨对虾具有较高的市场需求、环境适应性广，该种对虾已被推广到中国北方水域进行养殖，其中包括山西、陕北、山东等北方省份的海、淡水水域，耐低温成为中国北方凡纳滨对虾养殖业追求的重要抗逆性状；另一方面，在中国的春节时期，鲜活凡纳滨对虾的市场价格是平常季节的3倍以上，广东等地的对虾养殖农户经常冒险的将上市时间拖到冬季，又往往因冬季寒潮造成水温骤降引起养殖对虾大规模死亡，耐低温品种的选育也是中国南方凡纳滨对虾养殖业的迫切需求。
传统的选育方法依赖于表型选择，具有周期长、不稳定等不利因素。分子育种，即分子标记辅助选择育种，是指利用DNA分子标记对育种材料进行选择的技术，分子育种方法根据有效的分子标记进行后备亲本选择，能更快速地提升子代的经济性状。SNP(Single Nucleotide Polymorphism)是指基因组上单核苷酸的多态性，是目前应用最广、最新的分子标记。高质量的DNA分子标记是开展分子育种的基础。目前，虽开发出了众多针对不同性状的对虾分子标记，但多未阐明分子标记的分子功能。
微小染色体维持基因2，英文名：Minichromosome Maintenance Complex Component 2(MCM2)，是参与启动真核基因组复制的小染色体维持基因家族的成员。在凡纳滨对虾中从未有MCM2基因的报道，也从未有研究发现该基因与低温性状相关。
发明内容：
本发明旨在提供一种凡纳滨对虾低温耐受性状关联的微小染色体维持基因2(MCM2)的SNP分子标记、检测引物、分子功能及其应用，本发明公开的凡纳滨对虾MCM2基因的SNP分子标记、检测引物、分子功能及其应用方法，是目前已知的常规知识或方法无法预测的，本发明的SNP分子标记可用于检测凡纳滨对虾亲本的耐低温性状，根据本发明提供的方法，可筛选耐低温能力更强的后备亲本，有利于快速提升凡纳滨对虾子代的耐低温性状，缩短耐低温对虾品系的选育周期。
本发明的凡纳滨对虾微小染色体维持基因2(MCM2)的耐低温性状关联的SNP分子标记，所述的SNP分子标记的序列如SEQ ID NO.1所示，所述的凡纳滨对虾的SNP分子标记是：SEQ ID NO.1所示序列的第1,925碱基位置的G/C基因型或G/G基因型；或第3,414、3,475和3,537碱基完全连锁，其为A/A基因型或A/C基因型；或第3,776碱基位置的T/T基因型或T/G基因型；或第6,679碱基位置的A/C基因型或A/A基因型。
优选，第1,925碱基位置的G/C基因型个体在低温压力条件下的平均存活时间显著的高于G/G基因型个体；第3,414、3,475和3,537碱基完全连锁，其A/A基因型个体在低温压力条件下的平均存活时间显著的高于A/C基因型个体；第3,776碱基位置的T/T基因型个体在低温压力条件下的平均存活时间显著的高于T/G基因型个体；第6,679碱基位置的A/C基因型个体在低温压力条件下的平均存活时间显著的高于A/A基因型个体。
本发明的第二个目的是提供上述SNP分子标记的检测引物，如下任一检测引物对所示
(1)第1,925位置检测引物对
g.MCM2-1,925-F：5’-GTCTGAGTTATCGGTAGTATTGG-3’；
g.MCM2-1,925-R：5’-CATAGGAACACTTGTTGCAGTC-3’；
(2)第3,414位置检测引物对
g.MCM2-3,414-F：5’-CTGAGGTGACTGGTGTGTACAC-3’；
g.MCM2-3,414-R：5’-GAGTACCTGTGGTGAAGACAGC-3’；
(3)第3,776位置检测引物对
g.MCM2-3,776-F：5’-TGAGAGGATTGTGGCCTCAATT-3’；
g.MCM2-3,776-R：5’-CACCTCATACGAGGTCATATTGTC-3’；
(4)第6,679位置检测引物对
g.MCM2-6,679-F：5’-CTCTATGAGTTGACATTGGATGGG-3’；
g.MCM2-6,679-R：5’-CTCTTGCCGTCATGTGAGAAGTT-3’。
本发明的第三个目的是提供一种检测试剂盒，其包括上述SNP分子标记的检测引物对。
本发明的第四个目的是提供上述耐低温性状关联的SNP分子标记、SNP分子标记的检测引物和检测试剂盒在凡纳滨对虾耐低温抗逆育种中的应用。
本发明的第五个目的是提供一种检测凡纳滨对虾耐低温性能的方法，包括：提取待测凡纳滨对虾的基因组DNA，利用上述SNP分子标记的检测引物对进行扩增，并测序获得测序结果，基于测序结果确定SNP分子标记的基因型，再判断该待测凡纳滨对虾的耐低温性能。
具体是：第1,925碱基位置的G/C基因型个体在低温压力条件下的平均存活时间显著的高于G/G基因型个体；第3,414、3,475和3,537碱基完全连锁，其A/A基因型个体在低温压力条件下的平均存活时间显著的高于A/C基因型个体；第3,776碱基位置的T/T基因型个体在低温压力条件下的平均存活时间显著的高于T/G基因型个体；第6,679碱基位置的A/C基因型个体在低温压力条件下的平均存活时间显著的高于A/A基因型个体。
耐低温是凡纳滨对虾的重要抗逆性状，在凡纳滨对虾耐低温抗逆育种工作中，可利用本发明的SNP分子标记和方法对后备亲本群体进行筛选，及早排除对低温敏感的凡纳滨对虾亲本个体，将有助于快速增强凡纳滨对虾子代的耐低温能力。根据本发明提供的方法，有利于快速提升凡纳滨对虾子代的耐低温性状，缩短耐低温对虾品系的选育周期，具有较大的潜在应用价值。
附图说明：
图1是：A:对虾MCM2基因SNP多态性位点位置图，数字代表多态性位点的碱基位置；B:对虾MCM2基因SNP多态性位点连锁分析图,黑色代表完全连锁；C:个体基因型的统计结果,“***”表示显著性检验P<0.001；D:表示突变位点测序峰图；E:SNP位点氨基酸与低温性状的相关性分析,纵坐标大小表示与低温性状的相关性大小。
图2是：A:对照温度条件下，MCM2基因被干扰表达后，大部分对虾个体仍能正常存活；B:在15℃条件下，MCM2基因被干扰表达后，对虾的存活率降到50％左右；C:在更低13℃条件下，MCM2基因被干扰表达后，对虾的存活率降到30％.红色线条代表注射MCM2基因的RNAi干扰序列的生存曲线，蓝色代表注射无义RNAi干扰序列的生存曲线，黑色代表注射DEPC水的生存曲线.
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。
实施例1：
一、
以13℃±0.5为应激条件，对150头凡纳滨对虾“普瑞莫”与“迈阿密”群体杂交来源的后代进行低温耐受性实验，统计个体的耐受时间，将首先死亡的30头虾作为低温敏感组，将最后仍然存活的30头虾作为低温耐受组。在实验过程中，及时将死亡个体置于95％酒精中保存。低温敏感组和低温耐受组对虾基因组DNA提取方法按照天根海洋动物组织基因组提取试剂盒进行(TIANGEN，天根生化科技有限公司)。
利用本发明公开的检测引物(F：5‘-CGCTACCCTCAACACACTACA-3’，R：5‘-CAACAGCCTTACAACACTAC-3’)，对低温敏感组和低温耐受组对虾个体的MCM2基因进行PCR检测，反应程序为：95℃预变性4分钟，(95℃变性30秒、52-57℃退火20秒、72℃延伸1分钟)×35个循环，最后72℃延伸10分钟。对PCR产物进行测序，MCM2基因的序列如SEQ ID NO.1所示，其cDNA序列如SEQ ID NO.2所示，经个体基因型的统计，发现：
(1)、第1,925碱基位置的G/C基因型个体在低温压力条件下的平均存活时间显著的高于G/G基因型个体；第3,414、3,475和3,537碱基完全连锁，其A/A基因型个体在低温压力条件下的平均存活时间显著的高于A/C基因型个体；第3,776碱基位置的T/T基因型个体在低温压力条件下的平均存活时间显著的高于T/G基因型个体；第6,679碱基位置的A/C基因型个体在低温压力条件下的平均存活时间显著的高于A/A基因型个体(图1-C，表1)；
(2)、第1,925碱基位置G>C错义突变导致该基因编码氨基酸由天冬氨酸(Asp，D)突变成谷氨酸(Glu，E)；第3,414碱基位置A>C错义突变导致该基因编码氨基酸由谷氨酰胺(Gln，Q)突变成赖氨酸(Lys，K)；第3,776碱基位置T>G错义突变导致该基因编码氨基酸由甲硫氨酸(Met，M)突变成异亮氨酸(Ile，I)；第6,679碱基位置A>C错义突变导致该基因编码氨基酸由组氨酸(His，H)突变成脯氨酸(Pro，P)；第3,475和3,537碱基处于MCM2基因的内含子上，不导致编码氨基酸改变(图1-A,D,E)；
(3)、对低温性状的影响重要性进行分析，由高到低为：第1,925碱基位置G>C错义突变；第6,679碱基位置A>C错义突变；第3,414碱基位置A>C错义突变；第3,776碱基位置T>G错义突变(图1-E)；
表1对虾MCM2基因SNP位点与低温性状的关联分析
Figure PCTCN2020100608-appb-000001
二、
采用本发明的MCM2基因的RNAi序列(siRNA sense：5’-AGGCTTCCCTGTGTTCGCGACTGTT-3’，siRNA antisense：5’-CAGTCGCGAACACAGGGAAGCCTTT-3’)，分别对25℃、15℃、13℃的对虾进行注射处理，对照组注射DEPC水及无义RNA链，具体是：按照RNA/ 体重为3μg/g的剂量注射siRNA溶液及无义RNA链(溶解于DEPC水中)，对照组注射相同体积的DEPC水溶液，注射总体积应小于50μl，注射部位为虾的腹部肌肉。人工合成的RNA链干粉及溶液应保存于干冰中，冰上解冻后在半小时内注射完毕。结果如图2所示，从图2可以看出，本发明公开的人工合成的RNAi干扰序列能够显著的降低对虾在低温中的成活率，说明本发明公开的RNAi干扰序列能够干扰对虾MCM2基因的正常表达，说明该基因在低温压力条件下具有重要的分子功能(图2)。
Figure PCTCN2020100608-appb-000002
Figure PCTCN2020100608-appb-000003
Figure PCTCN2020100608-appb-000004
Figure PCTCN2020100608-appb-000005
Figure PCTCN2020100608-appb-000006
Figure PCTCN2020100608-appb-000007
Figure PCTCN2020100608-appb-000008

Claims (7)

  1. 凡纳滨对虾的耐低温性状关联的SNP分子标记，其特征在于，所述的SNP分子标记的序列如SEQ ID NO.1所示，所述的凡纳滨对虾的SNP分子标记是：SEQ ID NO.1所示序列的第1,925碱基位置的G/C基因型或G/G基因型；或第3,414、3,475和3,537碱基完全连锁，其为A/A基因型或A/C基因型；或第3,776碱基位置的T/T基因型或T/G基因型；或第6,679碱基位置的A/C基因型或A/A基因型。
  2. 根据权利要求1所示的SNP分子标记，其特征在于，第1,925碱基位置的G/C基因型个体在低温压力条件下的平均存活时间显著的高于G/G基因型个体；第3,414、3,475和3,537碱基完全连锁，其A/A基因型个体在低温压力条件下的平均存活时间显著的高于A/C基因型个体；第3,776碱基位置的T/T基因型个体在低温压力条件下的平均存活时间显著的高于T/G基因型个体；第6,679碱基位置的A/C基因型个体在低温压力条件下的平均存活时间显著的高于A/A基因型个体。
  3. 一种权利要求1所述的SNP分子标记的检测引物，其特征在于，如下任一检测引物对所示
    (1)第1,925位置检测引物对
    g.MCM2-1,925-F：5’-GTCTGAGTTATCGGTAGTATTGG-3’；
    g.MCM2-1,925-R：5’-CATAGGAACACTTGTTGCAGTC-3’；
    (2)第3,414位置检测引物对
    g.MCM2-3,414-F：5’-CTGAGGTGACTGGTGTGTACAC-3’；
    g.MCM2-3,414-R：5’-GAGTACCTGTGGTGAAGACAGC-3’；
    (3)第3,776位置检测引物对
    g.MCM2-3,776-F：5’-TGAGAGGATTGTGGCCTCAATT-3’；
    g.MCM2-3,776-R：5’-CACCTCATACGAGGTCATATTGTC-3’；
    (4)第6,679位置检测引物对
    g.MCM2-6,679-F：5’-CTCTATGAGTTGACATTGGATGGG-3’；
    g.MCM2-6,679-R：5’-CTCTTGCCGTCATGTGAGAAGTT-3’。
  4. 一种检测试剂盒，其特征在于，包括权利要求3中的任一SNP分子标记的检测引物对。
  5. 权利要求1所述的耐低温性状关联的SNP分子标记、权利要求3所述的SNP分子标记的检测引物和权利要求4所述的检测试剂盒在凡纳滨对虾耐低温抗逆育种中的应用。
  6. 一种检测凡纳滨对虾耐低温性能的方法，其特征在于，包括：提取待测凡纳滨对虾的基因组DNA，利用权利要求3所述的SNP分子标记的检测引物对进行扩增，并测序获得测序结果，基于测序结果确定SNP分子标记的基因型，再判断该待测凡纳滨对虾的耐低温性能。
  7. 根据权利要求6所述的方法，其特征在于，所述的基于测序结果确定SNP分子标记的基因型，再判断该待测凡纳滨对虾的耐低温性能是：第1,925碱基位置的G/C基因型个体在低温压力条件下的平均存活时间显著的高于G/G基因型个体；第3,414、3,475和3,537碱基完全连锁，其A/A基因型个体在低温压力条件下的平均存活时间显著的高于A/C基因型个体；第3,776碱基位置的T/T基因型个体在低温压力条件下的平均存活时间显著的高于T/G基因型个体；第6,679碱基位置的A/C基因型个体在低温压力条件下的平均存活时间显著的高于A/A基因型个体。
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