WO2024027254A1 - 一种凡纳滨对虾肝肠胞虫抗性性状的组合分子标记、kasp检测引物及应用 - Google Patents

一种凡纳滨对虾肝肠胞虫抗性性状的组合分子标记、kasp检测引物及应用 Download PDF

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WO2024027254A1
WO2024027254A1 PCT/CN2023/092906 CN2023092906W WO2024027254A1 WO 2024027254 A1 WO2024027254 A1 WO 2024027254A1 CN 2023092906 W CN2023092906 W CN 2023092906W WO 2024027254 A1 WO2024027254 A1 WO 2024027254A1
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site
genotype
litopenaeus vannamei
primer set
hepatica
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PCT/CN2023/092906
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French (fr)
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罗鹏
吕颖
张鑫
何熙平
胡超群
王艳红
任春华
陈廷
江晓
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中国科学院南海海洋研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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  • the invention belongs to the technical field of aquatic animal breeding, and specifically relates to a combined molecular marker, a KASP detection primer and an application for the hepatica resistance traits of Litopenaeus vannamei.
  • the seed industry is the "chip" of agriculture, and the Litopenaeus vannamei seed industry is crucial to supporting the development of the entire shrimp farming industry.
  • Litopenaeus vannamei also known as Penaeus vannamei
  • Penaeus vannamei is the largest cultured species of shrimp in my country and the world.
  • my country's shrimp farming society and ecological environment have undergone significant changes.
  • disease outbreaks have occurred frequently, and the shrimp farming industry has suffered a great impact.
  • farmers' demand for shrimp culture varieties has shifted from a single pursuit of growth speed to a demand for disease resistance, growth and other excellent traits.
  • 14 new varieties of Litopenaeus vannamei have been cultivated (as of December 2022).
  • the existing new varieties of Litopenaeus vannamei have outstanding traits in a single category, and most of them are There are no reports on growth, stress resistance or antiviral traits, but there are no reports on resistance breeding or new resistant varieties targeting other pathogenic categories.
  • Enterohepaticus shrimp also known as "intestinal epithelial cell sporidia” was first discovered in 2003. It was isolated and named from the slow-growing Penaeus monodon in 2009. It mainly infects Penaeus monodon and Litopenaeus vannamei. , has started to spread in Southeast Asia and China since 2012, and has gradually become a worldwide shrimp farming disease.
  • Shrimp Enterocystis belonging to the kingdom Fungi, is an intracellular parasitic microsporidian that mainly lives in shrimp hepatopancreas tubule epithelial cells and intestinal epithelial cells. Mature spores are oval and covered with a spore wall. , has strong resistance to harsh environments.
  • KASP Allele-specific PCR
  • the purpose of the present invention is to provide a combination of molecular markers related to the resistance to Enterocystis hepatica in Litopenaeus vannamei, a KASP primer combination for detecting the genotype of the molecular markers, and their application.
  • a combination of molecular markers related to the resistance to Enterocystis hepatica in Litopenaeus vannamei located at the P 358 site (base 549245 of the genome Scaffold fragment NW_020870611.1) and P 339 upstream of the uncharacterized gene LOC113820209 in the genome of Litopenaeus vannamei point (base 549264 of genome Scaffold fragment NW_020870611.1), of which the P 358 site is a C/T-type SNP marker and the P 339 site is a T/C-type SNP marker.
  • the genotype combination of P 358 site and P 339 site is T/T or C/C
  • Litopenaeus vannamei is more resistant to Hepatica cystis.
  • the genotype combination of P 358 site and P 339 site When combined with other types, Litopenaeus vannamei is susceptible to Hepatica cystis.
  • the present invention also provides a primer set for amplifying the above-mentioned molecular marker combination.
  • the primer set is composed of a primer set for detecting the genotype of the P 358 site and a primer set for detecting the genotype of the P 339 site;
  • the primer set for detecting the genotype of the P 358 site is as follows:
  • the primer set for detecting the genotype of the P 339 site is as follows:
  • 549264F1 GAAGGTGACCAAGTTCATGCT GGGACCGTGTCTTATACAGTCTTGT;
  • 549264F2 GAAGGTCGGAGTCAACGGATT GGGACCGTGTCTTATACAGTCTTGC;
  • 549264R CCATTTTCTGTTTTCTCGGTAACTCCGTGC.
  • the SNP site corresponding to the 3' end of the primer set 549245F1/549245F2 is the P 358 site upstream of the uncharacterized gene LOC113820209 of Litopenaeus vannamei (corresponding to the 101st nucleotide of SEQ ID NO.1 in the sequence list), where
  • the underlined part of the 549245F1 primer is the FAM fluorescent specific tag sequence, and the underlined part of the 549245F2 primer is the HEX fluorescent specific tag sequence.
  • the SNP site corresponding to the 3' end of the primer set 549264F1/549264F2 is the P 339 site upstream of the uncharacterized gene LOC113820209 of Litopenaeus vannamei (corresponding to the 120th nucleotide of SEQ ID NO.1 in the sequence list), where
  • the underlined part of the 549264F1 primer is the FAM fluorescent specific tag sequence, and the underlined part of the 549264F2 primer is the HEX fluorescent specific tag sequence.
  • the present invention also provides the application of the above primer set in preparing a kit for detecting the resistance traits of Hepatica cyst in Litopenaeus vannamei.
  • the present invention also provides a kit comprising the above primer set.
  • the present invention also provides the application of the above-mentioned molecular marker combination, primer set or kit in the molecular detection of hepatica resistance traits in Litopenaeus vannamei.
  • the present invention also provides the application of the above-mentioned molecular marker combination, primer set or kit in molecular genetic breeding of Litopenaeus vannamei.
  • it is used in the breeding of hepatica resistance in Litopenaeus vannamei.
  • the invention also provides a method for detecting the resistance traits of Hepatica cyst in Litopenaeus vannamei, which includes the following steps:
  • genotype fluorescence detection is performed on the P 358 site:
  • the P 358 site is the C/C homozygous genotype
  • the P 358 site is the T/T homozygous genotype
  • the P 358 site is a C/T heterozygote.
  • genotype fluorescence detection is performed on the P 339 site:
  • the P 339 site is the T/T homozygous genotype
  • the P 339 site is the C/C homozygous genotype
  • the P 339 site is a T/C heterozygote.
  • the invention provides a combined molecular marker, a KASP detection primer and a combination of resistance traits to Enterocystis hepatica in Litopenaeus vannamei and its application.
  • the combined molecular marker is located at the P 358 and P 339 sites upstream of the uncharacterized gene LOC113820209 in the genome of Litopenaeus vannamei.
  • the P 358 site is a C/T type SNP marker and the P 339 site is a T/C type SNP marker.
  • the combined molecular marker is detected using KASP technology and includes three primer combinations for detecting the genotype of the P 358 site and three primer combinations for detecting the genotype of the P 339 site.
  • Litopenaeus vannamei is more resistant to Hepatica cystis.
  • the genotype combination of P 358 site and P 339 site When combined with other types, they are susceptible to Enterocystis hepatica.
  • This combination of molecular markers and KASP detection primers can be used to detect the genotypes of the P 358 site and the P 339 site, and can be used to screen breeding materials for Litopenaeus vannamei, which is of great significance for promoting the breeding of shrimps resistant to Hepatica cystis.
  • Figure 1 shows the fluorescence detection results of the P 358 locus genotype of 96 shrimps.
  • Square box individuals detected as T/T genotype
  • round box individuals detected as C/C genotype
  • triangle box individuals detected as T/C genotype.
  • Figure 2 shows the fluorescence detection results of the P 339 locus genotype of 96 shrimps.
  • Square box individuals detected as C/C genotype
  • round box individuals detected as T/T genotype
  • triangle box individuals detected as C/T genotype.
  • EH infection levels of E. hepatica are obtained ( EH) graded samples total 75.
  • a similar method was used to collect 75 Vibrio infection degree (AB) graded samples, 75 growth traits (GW) graded samples, and 75 hepatopancreatic index (EI) graded samples, for a total of 300 samples.
  • AB Vibrio infection degree
  • GW growth traits
  • EI hepatopancreatic index
  • DNA was extracted from 300 samples. After DNA extraction, conventional second-generation sequencing library construction and sequencing (Beijing Biomic Biotechnology Co., Ltd.) were performed. The sequencing results were compared with the Litopenaeus vannamei reference genome using GATK (V3.4.6). The software performs SNP and InDel searches, filters SNP sites based on minor allele frequency and site integrity, and retains highly consistent and accurate SNP sites. Combined with EMMAX software, a mixed linear model is used for correlation analysis and trait correlation analysis. Candidate sites were corrected using Bonrroni (Beijing Biomark Biotechnology Co., Ltd.).
  • SEQ ID NO.1 The sequence shown in SEQ ID NO.1 is as follows:
  • the KASP primer set was designed based on the upstream and downstream sequences of the P 358 and P 339 sites of the uncharacterized gene LOC113820209 in Litopenaeus vannamei.
  • the sequence of the primer combination for detecting the genotype of the P 358 site is shown in SEQ ID NO. 2-4, specifically as follows:
  • the SNP site corresponding to the 3' end of primers 549245F1/549245F2 is the P 358 site upstream of the uncharacterized gene LOC113820209 in Litopenaeus vannamei.
  • the underlined part of primer 549245F1 is the FAM fluorescence specific tag sequence, and the underlined part of primer 549245F2 is HEX fluorescence. Specific tag sequence.
  • the sequence of the primer combination for detecting the genotype of the P 339 site is shown in SEQ ID NO. 5-7, specifically as follows:
  • 549264F1 GAAGGTGACCAAGTTCATGCT GGGACCGTGTCTTATACAGTCTTGT;
  • 549264F2 GAAGGTCGGAGTCAACGGATT GGGACCGTGTCTTATACAGTCTTGC;
  • 549264R CCATTTTCTGTTTTCTCGGTAACTCCGTGC.
  • the SNP site corresponding to the 3' end of primers 549264F1/549264F2 is the P 339 site upstream of the uncharacterized gene LOC113820209 in Litopenaeus vannamei.
  • the underlined part of primer 549264F1 is the FAM fluorescence specific tag sequence, and the underlined part of primer 549264F2 is HEX fluorescence. Specific tag sequence.
  • the method for detecting the combined molecular marker KASP using the primer set designed in Example 2 above includes the following steps:
  • the KASP amplification system for detecting the P 358 and P 339 sites upstream of the uncharacterized gene LOC113820209 in Litopenaeus vannamei is shown in Table 1.
  • the Flu-Arms 2 ⁇ PCR mix was purchased from Guangzhou Gude Biotechnology Co., Ltd., and the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.
  • Table 1 KASP amplification system for detecting SNP sites in shrimp genome
  • Amplification was performed using a fluorescence quantitative PCR instrument (Tianlong TL988). Amplification conditions: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 15 seconds, annealing and extension at 61-55°C for 60 seconds, for 10 cycles, with each cycle lowering by 0.6°C; denaturation at 95°C for 15 seconds, annealing and extension at 55°C for 60 seconds, for 35 cycle; end amplification at 30°C.
  • the FAM excitation wavelength is 485nm and the emission wavelength is 520nm.
  • the HEX excitation wavelength is 528nm and the emission wavelength is 560nm.
  • the system reference fluorescence ROX excitation wavelength is 575nm.
  • the emission wavelength is 610nm.
  • FAM fluorescence is detected in the amplification product of the P 358 site molecular marker, it is recorded as C/C genotype; if the amplification product only has FAM fluorescence If HEX fluorescence is detected, it is recorded as T/T genotype; if the amplification product detects both FAM and HEX signals, it is recorded as C/T heterozygote.
  • FAM fluorescence was detected in the amplification product of the P 339 site molecular marker, it is recorded as T/T genotype; if only HEX fluorescence is detected in the amplification product, it is recorded as C/C genotype; if both the amplification product and HEX fluorescence are detected, it is recorded as C/C genotype.
  • FAM and HEX signals were recorded as heterozygous T/C heterozygotes.
  • the P 339 site upstream of the uncharacterized gene LOC113820209 and the genotype combination of the P 339 site are T/T and C/C shrimp individuals, which are judged to have strong resistance to Hepatica infection.
  • the P 339 site and Other genotype combinations at the P 339 locus were determined to be susceptible to E. hepatica .
  • Example 2 Different batches of shrimps were sampled, 50 in each batch, and the method in Example 1 was used to classify the degree of resistance to Hepatocystis infection. The proportion of non-infected shrimps in each batch was ⁇ 5%. A total of 4 batches of shrimps met the requirements, and the total number of samples was 96 Litopenaeus vannamei, body length 9-11cm. The results of the detection of Hepatocystis infection in 96 shrimps are shown in Table 2. A total of 4 shrimps out of the 96 shrimps were detected to be not infected with Hepaticocystis.
  • the primer combination in Example 2 and the KASP detection method in Example 3 were used to detect the P 339 site and the P 339 site genotype on 96 shrimps.
  • the fluorescence detection results of the P 358 locus genotype are shown in Figure 1, and the fluorescence detection results of the P 339 locus genotype are shown in Figure 2.
  • the genotype detection results of P 358 and P 339 loci of 96 shrimps are shown in Table 2.
  • a total of 3 shrimps had a genotype combination of the above two loci of T/T and C/C. None of the 3 shrimps were infected with liver intestine. Cytozoa.

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Abstract

本发明公开了一种凡纳滨对虾肝肠胞虫抗性性状的组合分子标记、KASP检测引物及应用。该组合分子标记位于凡纳滨对虾基因组未特征化基因LOC113820209上游P 358位点和P 339位点，其中P 358位点为C/T型SNP标记，P 339位点为T/C型SNP标记。该组合分子标记采用KASP技术进行检测，当P 358位点和P 339位点的基因型组合为T/T、C/C时，对肝肠胞虫抗性较强，当P 358位点和P 339位点的基因型组合为其它类型时，对肝肠胞虫敏感。

Description

一种凡纳滨对虾肝肠胞虫抗性性状的组合分子标记、KASP检测引物及应用 技术领域：
本发明属于水产动物育种技术领域，具体涉及一种凡纳滨对虾肝肠胞虫抗性性状的组合分子标记、KASP检测引物及应用。
背景技术：
种业是农业的“芯片”，凡纳滨对虾种业对于支撑整个对虾养殖产业的发展至关重要。凡纳滨对虾，又称南美白对虾，是我国及世界对虾第一大养殖品种。近十余年，我国对虾养殖社会和生态环境发生了明显变化，同时病害爆发频繁，对虾养殖产业遭受到较大冲击。养殖户对于对虾养殖品种的需求已经从单一的追求生长速度转化为抗病兼具生长等多优性状的需求。近十余年，经过我国科学家的持续努力，已经培育出14个南美白对虾新品种(截止2022年12月)，但现有的凡纳滨对虾新品种均为单一类别性状突出，并且多为生长、抗逆或抗病毒性状，对于针对其它病原类别的抗性育种或抗性新品种未见报道。
虾肝肠胞虫(EHP)，又名“肠道上皮细胞孢子虫”，于2003年被首次发现，2009年从生长缓慢的斑节对虾中分离并命名，主要感染斑节对虾、凡纳滨对虾，2012年以来开始在东南亚、中国爆发流行，并逐步成为世界性的对虾养殖病害。虾肝肠胞虫，属真菌界，是一种细胞内寄生的微孢子虫，主要寄生于对虾肝胰腺小管上皮细胞及肠道上皮细胞内，成熟孢子为卵圆形，孢子外被一层孢壁，对恶劣环境具有很强的抵抗力，这一特征导致肝肠胞虫广泛分布于对虾养殖环境，对虾一旦感染，极难从养殖系统内清除。凡纳滨对虾感染肝肠胞虫并不会导致急性死亡，患病对虾的病程往往较长，严重影响养殖的经济效益，当前我国养殖的凡纳滨对虾普遍存在肝肠胞虫感染现象，从感染率来讲，肝肠胞虫已经成 为危害凡纳滨对虾养殖最为严重的病原种类。
综合上述，选育具有肝肠胞虫较强抗性的对虾具有十分重要的经济意义，当前国内外均未见针对抗肝肠胞虫感染性状对虾选育的报道。分子标记辅助育种是指通过QTL定位或者基因组关联分析的方法，挖掘出与性状直接相关的分子标记，实现对目标性状的直接选择，可以极大地提高对虾选育效率和准确率，目前未见在对虾中挖掘与抗肝肠胞虫感染相关的分子标记的报道。竞争性等位基因特异性PCR(CompetitiveAllele-specific PCR，KASP)是一种基于常规PCR和荧光检测的基因型分型技术，KASP技术具有成本低、准确、快速等优点，能够在普通实验室实现高通量基因分型的需求，因此在分子标记的发现以及大规模的目标基因型检测方面具有很好的应用前景。至今，在对虾分子标记的检测方法，未见采用KASP检测方法的报道。
发明内容：
本发明的目的是提供一种与凡纳滨对虾抗肝肠胞虫性状相关的组合分子标记、检测分子标记基因型的KASP引物组合及其应用。
为实现上述目的，本发明采用技术方案如下：
一种与凡纳滨对虾抗肝肠胞虫性状相关的分子标记组合，位于凡纳滨对虾基因组未特征化基因LOC113820209上游P358位点(基因组Scaffold片段NW_020870611.1第549245碱基)和P339位点(基因组Scaffold片段NW_020870611.1第549264碱基)，其中P358位点为C/T型SNP标记，P339位点为T/C型SNP标记。当P358位点和P339位点的基因型组合为T/T、C/C时，凡纳滨对虾对肝肠胞虫抗性较强，当P358位点和P339位点的基因型组合为其它类型时，凡纳滨对虾对肝肠胞虫敏感。
本发明还提供一种用于扩增上述分子标记组合的引物组，所述的引物组由检测P358位点基因型的引物组和检测P339位点基因型的引物组组成；
所述检测P358位点基因型的引物组如下：
549245F1：GAAGGTGACCAAGTTCATGCTCGGTACAAGGAAAGGAACAGGGACC；
549245F2：GAAGGTCGGAGTCAACGGATTCGGTACAAGGAAAGGAACAGGGACT；
549245R：GGTAACTCCGTGCATCAACAAGACTGTAT；
所述检测P339位点基因型的引物组如下：
549264F1：GAAGGTGACCAAGTTCATGCTGGGACCGTGTCTTATACAGTCTTGT；
549264F2：GAAGGTCGGAGTCAACGGATTGGGACCGTGTCTTATACAGTCTTGC；
549264R：CCATTTTCTGTTTCTCGGTAACTCCGTGC。
所述引物组549245F1/549245F2的3’末端对应的SNP位点为凡纳滨对虾未特征化基因LOC113820209上游P358位点(对应序列表中SEQ ID NO.1第101位核苷酸)，其中549245F1引物划线部分为FAM荧光特异标签序列，549245F2引物划线部分为HEX荧光特异标签序列。
所述引物组549264F1/549264F2的3’末端对应的SNP位点为凡纳滨对虾未特征化基因LOC113820209上游P339位点(对应序列表中SEQ ID NO.1第120位核苷酸)，其中549264F1引物划线部分为FAM荧光特异标签序列，549264F2引物划线部分为HEX荧光特异标签序列。
本发明还提供上述的引物组在制备凡纳滨对虾肝肠胞虫抗性性状检测的试剂盒中的应用。
本发明还提供一种试剂盒，其包含上述的引物组。
本发明还提供上述的分子标记组合、引物组或试剂盒在凡纳滨对虾肝肠胞虫抗性性状分子检测中的应用。
本发明还提供上述的分子标记组合、引物组或试剂盒在凡纳滨对虾分子遗传育种中的应用。
优选的，是在凡纳滨对虾肝肠胞虫抗性育种中的应用。
本发明还提供一种检测凡纳滨对虾肝肠胞虫抗性性状的方法，包括以下步骤：
(1)获得待测样品基因组DNA；
(2)以基因组DNA为模板，利用上述的引物组进行PCR扩增；
(3)收集每个反应孔产生的荧光信号，根据荧光信号的类型判断分子标记的基因型，进而确定凡纳滨对虾对肝肠胞虫的抗性。
优选的，对P358位点进行基因型的荧光检测：
若仅检测到FAM荧光，则P358位点为C/C纯合基因型；
若仅检测到HEX荧光，则P358位点为T/T纯合基因型；
若同时检测到FAM和HEX荧光，则P358位点为C/T杂合子。
优选的，对P339位点进行基因型的荧光检测：
若仅检测到FAM荧光，则P339位点为T/T纯合基因型；
若仅检测到HEX荧光，则P339位点为C/C纯合基因型；
若同时检测到FAM和HEX荧光，则P339位点为T/C杂合子。
本发明提供了一种凡纳滨对虾肝肠胞虫抗性性状的组合分子标记、KASP检测引物及其 应用。该组合分子标记位于凡纳滨对虾基因组未特征化基因LOC113820209上游P358位点和P339位点，其中P358位点为C/T型SNP标记，P339位点为T/C型SNP标记。该组合分子标记采用KASP技术进行检测，包括了用于检测P358位点基因型的三条引物组合和用于检测P339位点基因型的三条引物组合。当P358位点和P339位点的基因型组合为T/T、C/C时，凡纳滨对虾对肝肠胞虫抗性较强，当P358位点和P339位点的基因型组合为其它类型时，对肝肠胞虫敏感。该组合分子标记、KASP检测引物可用于检测P358位点和P339位点基因型，并用于凡纳滨对虾育种材料的筛选，对于促进抗肝肠胞虫对虾育种具有重要意义。
附图说明
图1为96尾对虾P358位点基因型荧光检测结果。方框：检测为T/T基因型的个体；圆框：检测为C/C基因型的个体；三角框：检测为T/C基因型的个体。
图2为96尾对虾P339位点基因型荧光检测结果。方框：检测为C/C基因型的个体；圆框：检测为T/T基因型的个体；三角框：检测为C/T基因型的个体。
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
凡纳滨对虾抗肝肠胞虫相关分子标记的获得。
(1)凡纳滨对虾样品采集及性状分级
采集不同养殖场及市场来源的对虾样品，每一批对虾50尾，取肝胰腺进行肝胰腺肝肠胞虫携带量半定量PCR检测，以携带量区分对虾的肝肠胞虫感染程度，其中同一批次虾中，不感染的对虾(PCR阴性)被认定为对肝肠胞虫具有较强抗性，其余按PCR检测的条带亮 度分为轻度感染、中度感染、重度感染，其中不感染的对虾在同一批次对虾中占比≤5％，不满足此条件的取样批次淘汰，共获得肝肠胞虫不同感染程度(EH)分级样品总计75个。采用类似方法取弧菌感染程度(AB)分级样品75个，生长性状(GW)分级样品75个，肝胰腺指数(EI)分级样品75个，总计300个样品。
(2)全基因关联分析挖掘肝肠胞虫性状相关SNP
对300个样品进行DNA提取，提取DNA后进行常规二代测序文库构建和测序(北京百迈客生物科技有限公司)，测序结果与凡纳滨对虾参考基因组进行比较，采用GATK(V3.4.6)软件进行SNP和InDel查找，基于次要等位基因频率和位点完整度过滤SNP位点，保留高一致性、确性SNP位点，结合EMMAX软件，采用混合线性模型进行关联分析，性状关联分析候选位点使用Bonrroni校正(北京百迈客生物科技有限公司)。
(3)抗肝肠胞虫相关分子标记挖掘
根据GWAS分析的性状关联的SNP位点的P值大小进行排列，筛选与抗肝肠胞虫性状显著相关的独特SNP位点，从中再筛选位于基因启动子区的SNP和基因内部引起氨基酸编码突变的SNP，结果发现未特征化基因LOC113820209上游358位点(P358)和上游339位点(P339)与肝肠胞虫抗性显著相关(P<0.05)，P358和P339相差仅19个碱基，均位于未特征化基因LOC113820209上游启动子区。P358和P339位点及上下游序列如SEQ ID NO.1所示。
SEQ ID NO.1所示的序列如下所示：

GWAS分析结果显示，对肝肠胞虫抗性较强(不感染)的对虾个体P358和P339位点基因型与T/T、C/C基因型组合显著相关，对肝肠胞虫敏感(轻度感染、中度感染、重度感染)的对虾个体基因型组合为其它类型的基因型组合。
实施例2
凡纳滨对虾肝肠胞虫抗性性状KASP分子标记位点检测引物设计。
根据凡纳滨对虾未特征化基因LOC113820209上游P358和P339位点上游及下游序列，设计KASP引物组。
检测P358位点基因型的引物组合的序列如SEQ ID NO.2-4所示，具体如下：
549245F1：GAAGGTGACCAAGTTCATGCTCGGTACAAGGAAAGGAACAGGGACC；
549245F2：GAAGGTCGGAGTCAACGGATTCGGTACAAGGAAAGGAACAGGGACT；
549245R：GGTAACTCCGTGCATCAACAAGACTGTAT；
引物549245F1/549245F2的3’末端对应的SNP位点为凡纳滨对虾未特征化基因LOC113820209上游P358位点，其中549245F1引物划线部分为FAM荧光特异标签序列，549245F2引物划线部分为HEX荧光特异标签序列。
检测P339位点基因型的引物组合的序列如SEQ ID NO.5-7所示，具体如下：
549264F1：GAAGGTGACCAAGTTCATGCTGGGACCGTGTCTTATACAGTCTTGT；
549264F2：GAAGGTCGGAGTCAACGGATTGGGACCGTGTCTTATACAGTCTTGC；
549264R：CCATTTTCTGTTTCTCGGTAACTCCGTGC。
引物549264F1/549264F2的3’末端对应的SNP位点为凡纳滨对虾未特征化基因LOC113820209上游P339位点，其中549264F1引物划线部分为FAM荧光特异标签序列，549264F2引物划线部分为HEX荧光特异标签序列。
实施例3
凡纳滨对虾肝肠胞虫抗性性状KASP分子标记位点的KASP检测。
利用上述实施例2设计的引物组进行组合分子标记KASP检测的方法，包含以下步骤：
(1)对虾基因组提取
剪取对虾附肢末端或取粪样，采用海洋动物基因组提取试剂盒(天根生化，中国)进行对虾基因组DNA提取。
(2)KASP扩增体系配制
凡纳滨对虾未特征化基因LOC113820209上游P358和P339位点检测的KASP扩增体系如表1所示。其中Flu-Arms 2×PCR mix购自广州固得生物技术有限公司，引物均由上海生工生物工程有限公司合成。
表1对虾基因组SNP位点检测KASP扩增体系
(3)KASP扩增程序
采用荧光定量PCR仪(天隆TL988)进行扩增。扩增条件：95℃预变性10min；95℃变性15sec，61-55℃退火和延伸60sec，进行10个循环，每个循环降低0.6℃；95℃变性15sec，55℃退火和延伸60sec，进行35个循环；30℃结束扩增。
(4)读板检测
扩增结束后，直接对PCR板上的PCR扩增产物进行扫描，FAM激发波长为485nm，发射波长为520nm，HEX激发波长为528nm，发射波长为560nm，系统参比荧光ROX激发波长为575nm，发射波长为610nm。
P358位点分子标记的扩增产物若仅检测到FAM荧光，记为C/C基因型；扩增产物若仅 检测到HEX荧光，记为T/T基因型；扩增产物若同时检测到FAM和HEX信号，记为C/T杂合子。
P339位点分子标记的扩增产物若仅检测到FAM荧光，记为T/T基因型；扩增产物若仅检测到HEX荧光，记为C/C基因型；扩增产物若同时检测到FAM和HEX信号，记为杂合T/C杂合子。
未特征化基因LOC113820209上游P339位点和P339位点的基因型组合为T/T、C/C的对虾个体，判定为对肝肠胞虫感染具有较强的抗性，P339位点和P339位点的其它基因型组合判定为对肝肠胞虫易感。
实施例4
凡纳滨对虾肝肠胞虫抗性性状KASP分子标记验证。
采样不同批次对虾，每批50尾，采用实施例1中的方法进行抗肝肠胞虫感染程度分级，每批中不感染对虾占比≤5％，共有4个批次对虾达到要求，共计采样96尾凡纳滨对虾，体长9-11cm。96尾对虾的肝肠胞虫感染检测结果如表2所示，96尾对虾中共检测到4尾对虾未感染肝肠胞虫。
采用实施例2中的引物组合及实施例3中的KASP检测方法，对96尾对虾进行P339位点和P339位点基因型检测。P358位点基因型荧光检测结果如图1所示，P339位点基因型荧光检测结果如图2所示。96尾对虾的P358和P339位点基因型检测结果如表2所示，共有3尾对虾的上述二个位点基因型组合为T/T、C/C，3尾对虾均未感染肝肠胞虫。
表2 96尾对虾P358和P339位点基因型检测及感染情况检测

以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种与凡纳滨对虾抗肝肠胞虫性状相关的分子标记组合，其特征在于，位于凡纳滨对虾基因组未特征化基因LOC113820209上游P358位点和P339位点，所述的P358位点位于凡纳滨对虾基因组Scaffold片段NW_020870611.1第549245碱基，所述碱基为C/T；所述的P339位点位于凡纳滨对虾基因组Scaffold片段NW_020870611.1第549264碱基，所述碱基为T/C；当P358位点和P339位点的基因型组合为T/T、C/C时，凡纳滨对虾对肝肠胞虫抗性较强，当P358位点和P339位点的基因型组合为其它类型时，凡纳滨对虾对肝肠胞虫敏感。
  2. 用于扩增权利要求1所述分子标记组合的引物组，其特征在于，所述的引物组由检测P358位点基因型的引物组和检测P339位点基因型的引物组组成；
    所述检测P358位点基因型的引物组如下：
    549245F1：GAAGGTGACCAAGTTCATGCTCGGTACAAGGAAAGGAACAGGGACC；
    549245F2：GAAGGTCGGAGTCAACGGATTCGGTACAAGGAAAGGAACAGGGACT；
    549245R：GGTAACTCCGTGCATCAACAAGACTGTAT；
    所述检测P339位点基因型的引物组如下：
    549264F1：GAAGGTGACCAAGTTCATGCTGGGACCGTGTCTTATACAGTCTTGT；
    549264F2：GAAGGTCGGAGTCAACGGATTGGGACCGTGTCTTATACAGTCTTGC；
    549264R：CCATTTTCTGTTTCTCGGTAACTCCGTGC。
  3. 权利要求2所述的引物组在制备凡纳滨对虾肝肠胞虫抗性性状检测的试剂盒中的应用。
  4. 一种试剂盒，其特征在于，包含权利要求2所述的引物组。
  5. 权利要求1所述的分子标记组合、权利要求2所述的引物组或权利要求4所述的试剂盒 在凡纳滨对虾肝肠胞虫抗性性状分子检测中的应用。
  6. 权利要求1所述的分子标记组合、权利要求2所述的引物组或权利要求4所述的试剂盒在凡纳滨对虾分子遗传育种中的应用。
  7. 根据权利要求6所述的应用，其特征在于，是在凡纳滨对虾肝肠胞虫抗性育种中的应用。
  8. 一种检测凡纳滨对虾肝肠胞虫抗性性状的方法，其特征在于，包括以下步骤：
    (1)获得待测样品基因组DNA；
    (2)以基因组DNA为模板，利用权利要求2所述的引物组进行PCR扩增；
    (3)收集每个反应孔产生的荧光信号，根据荧光信号的类型判断分子标记的基因型，进而确定凡纳滨对虾对肝肠胞虫的抗性。
  9. 根据权利要求8所述的方法，其特征在于，对P358位点进行基因型的荧光检测：
    若仅检测到FAM荧光，则P358位点为C/C纯合基因型；
    若仅检测到HEX荧光，则P358位点为T/T纯合基因型；
    若同时检测到FAM和HEX荧光，则P358位点为C/T杂合子。
  10. 根据权利要求8所述的方法，其特征在于，对P339位点进行基因型的荧光检测：
    若仅检测到FAM荧光，则P339位点为T/T纯合基因型；
    若仅检测到HEX荧光，则P339位点为C/C纯合基因型；
    若同时检测到FAM和HEX荧光，则P339位点为T/C杂合子。
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