CN113736866B - Combination of SNP loci for detection of resistance to yellow leaf curl virus disease in tomato and its application - Google Patents
Combination of SNP loci for detection of resistance to yellow leaf curl virus disease in tomato and its application Download PDFInfo
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Abstract
Description
技术领域technical field
本发明涉及植物生物技术领域，具体涉及一种用于检测番茄黄化曲叶病毒病抗性的SNP位点组合及其应用。The invention relates to the field of plant biotechnology, in particular to a combination of SNP sites for detecting the resistance of tomato yellow leaf curl virus disease and its application.
背景技术Background technique
番茄是世界重要的蔬菜经济作物，具有重要的生产应用和基础研究价值。伴随着番茄在全世界范围内的种植面积逐步扩大，番茄病虫害的影响与日俱增。其中番茄黄化曲叶病毒病(tomato yellow leaf curl virus，TYLCV)是一种危害区域广泛分布于热带和亚热带地区的茄科作物病毒病。主要由烟粉虱携带传播，番茄被侵染后，植株表现明显矮化，叶缘黄化，叶片变小并卷曲，严重危害生长、开花和坐果，果实不能正常转色，更甚至导致毁灭性绝产。番茄黄化曲叶病毒病抗性主要由Ty家族的主效基因控制，其中Ty-1和Ty-3是被研究和利用最广泛的一个基因(位于在Chr.06长臂上)，两者互为等位基因，目前已经成功开发并商业化运用了TG97、P6-25等与Ty-1或Ty-3基因紧密连锁的分子标记；Ty-2也是抗番茄黄化曲叶病毒病的一个重要基因(位于Chr.11长臂上)，已经在番茄育种中被广泛利用，目前该基因已经被克隆(Solyc11g069660)。Tomato is an important vegetable economic crop in the world, with important production application and basic research value. With the gradual expansion of tomato planting area around the world, the impact of tomato pests and diseases is increasing day by day. Among them, tomato yellow leaf curl virus (TYLCV) is a virus disease of Solanaceae crops widely distributed in tropical and subtropical regions. It is mainly carried and spread by Bemisia tabaci. After the tomato is infected, the plants show obvious dwarfing, the leaf margins turn yellow, the leaves become smaller and curled, which seriously damages the growth, flowering and fruit setting. Exterminated. Tomato yellow leaf curl virus disease resistance is mainly controlled by the major genes of the Ty family, of which Ty-1 and Ty-3 are the most widely studied and utilized genes (located on the long arm of Chr. Mutual alleles, TG97, P6-25 and other molecular markers closely linked to Ty-1 or Ty-3 genes have been successfully developed and commercialized; Ty-2 is also a resistance to tomato yellow leaf curl virus disease. An important gene (located on the long arm of Chr.11), which has been widely used in tomato breeding, has been cloned (Solyc11g069660).
目前番茄分子标记辅助育种应用研究中常用类型主要包括微卫星位点SSR标记等第一代分子标记，目标位点序列特异性STS标记、插入缺失InDel标记、限制性酶切多态性CAPS标记等第二代分子标记，以及等位基因特异性PCR(AS-PCR)、高分辨率溶解曲线(HRM)和竞争性等位基因特异PCR(KASP)等第三代SNP分子标记。其中KASP(Kompetitive AlleleSpecific PCR)标记作为当今世界主流的SNP分型方法，具备高特异性、准确率高、快速高效、灵活性强、单数据点成本低、易实现自动化高通量操作等优点，非常适合大量样本的少数标记位点的高通量自动化检测，已经在动植物商业化育种以及基因分型相关基础研究中广泛成熟运用。At present, the commonly used types of molecular marker-assisted breeding applications in tomato mainly include first-generation molecular markers such as microsatellite SSR markers, target locus sequence-specific STS markers, indel markers, and restriction enzyme polymorphism CAPS markers, etc. Second-generation molecular markers, as well as third-generation SNP molecular markers such as allele-specific PCR (AS-PCR), high-resolution melting curve (HRM), and competitive allele-specific PCR (KASP). Among them, KASP (Kompetitive AlleleSpecific PCR) marker, as the mainstream SNP typing method in the world today, has the advantages of high specificity, high accuracy, fast efficiency, strong flexibility, low cost per data point, easy to realize automatic high-throughput operation, etc. It is very suitable for high-throughput automated detection of a few marker loci in a large number of samples, and has been widely used in commercial animal and plant breeding and basic research related to genotyping.
目前针对番茄黄化曲叶病毒病抗性基因Ty-1和Ty-3也有几个KASP标记检测技术方案，但这些方案中目标SNP的选择大多基于单一抗感双亲序列差异，或者不是目标基因直接相关的功能分子标记，在不同遗传背景番茄资源及商业化育种中的有效性、通用性和广适性均不能得到保证，可能导致选择失败，或者连锁累赘带来其他不利基因片段，限制了番茄黄化曲叶病毒病抗性检测效率以及抗病新品种的选育效率。并且目前尚没有针对Ty-2基因的商业化KASP标记检测技术方案。At present, there are also several KASP marker detection technology schemes for tomato yellow leaf curl virus disease resistance genes Ty-1 and Ty-3, but the selection of target SNPs in these schemes is mostly based on the sequence difference of a single resistance-susceptible parent, or not directly from the target gene. The relevant functional molecular markers cannot be guaranteed for their effectiveness, versatility and broad applicability in tomato resources with different genetic backgrounds and commercial breeding, which may lead to selection failure, or other unfavorable gene fragments brought by linkage burden, which limit tomato. Detection efficiency of yellow leaf curl virus disease resistance and breeding efficiency of new disease-resistant varieties. And there is no commercialized KASP marker detection technical solution for Ty-2 gene at present.
因此，开发一种高效、有代表性、通用的，能对番茄黄化曲叶病毒病抗性进行快速有效检测的SNP位点组合及高通量检测应用技术方案，对番茄商业化育种应用和遗传学研究具有十分重要的实践和理论意义。Therefore, to develop an efficient, representative and universal SNP site combination and high-throughput detection application technical scheme that can quickly and effectively detect the resistance of tomato yellow leaf curl virus disease, which can be used for tomato commercial breeding applications and applications. Genetic research has very important practical and theoretical significance.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于克服现有技术的不足，提供一种用于检测番茄黄化曲叶病毒病抗性的SNP位点组合及其应用。通过直接在已知Ty-1/Ty-3和Ty-2基因上下游临近区域和基因内部的大量变异组数据分析鉴别得到了能对番茄黄化曲叶病毒病抗性进行快速有效检测的多个SNP位点，可实现目标基因区域精准鉴定和选择，从而解决现有技术中SNP位点通用性不足，或者不是目标性状/基因直接相关的功能分子标记(仅为连锁标记)，导致目标基因选择无效，或者连锁累赘带来其他非目标不利基因片段等问题。The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a combination of SNP sites for detecting the resistance of tomato yellow leaf curl virus disease and its application. Through the analysis of a large number of variant group data directly in the upstream and downstream adjacent regions of the known Ty-1/Ty-3 and Ty-2 genes and within the genes, a number of variants that can rapidly and effectively detect resistance to tomato yellow leaf curl virus disease were identified. A single SNP site can realize accurate identification and selection of the target gene region, thereby solving the problem of insufficient versatility of SNP sites in the existing technology, or functional molecular markers that are not directly related to the target trait/gene (only linkage markers), resulting in the target gene Selection is ineffective, or linkage drag brings problems such as other non-target unfavorable gene segments.
为实现上述目的，本发明第一方面提供了一种用于检测番茄黄化曲叶病毒病抗性的SNP位点组合，所述SNP位点组合包括位于番茄黄化曲叶病毒病抗性基因Ty-1内部及两侧的第一SNP位点组合和位于番茄黄化曲叶病毒病抗性基因Ty-2内部及两侧的第二SNP位点组合，所述第一SNP位点组合包括以下Ty1-SNP01位点～Ty1-SNP08位点中的一种或多种，所述第二SNP位点组合包括以下Ty2-SNP01位点～Ty2-SNP08位点中的一种或多种：In order to achieve the above object, a first aspect of the present invention provides a combination of SNP sites for detecting resistance to tomato yellow leaf curl virus disease, the SNP site combination comprising a resistance gene located in a tomato yellow leaf curl virus disease. The first SNP site combination inside and on both sides of Ty-1 and the second SNP site combination located inside and on both sides of the tomato yellow leaf curl virus disease resistance gene Ty-2, the first SNP site combination includes One or more of the following Ty1-SNP01 sites to Ty1-SNP08 sites, and the second SNP site combination includes one or more of the following Ty2-SNP01 sites to Ty2-SNP08 sites:
表中，基因序列及SNP物理位置信息对应于番茄(Heinz 1706)参考基因组SL2.50版本。In the table, the gene sequence and SNP physical location information correspond to the tomato (Heinz 1706) reference genome version SL2.50.
本发明第一方面所述SNP位点组合对应的番茄黄化曲叶病毒病抗性基因Ty-1/Ty-3(Solyc06g051170，Solyc06g051180，Solyc06g051190)的基因组信息来源于数据库https://solgenomics.net/locus/26402,26403,26404/view，Ty-2(Solyc11g069660)的基因组信息来源于数据库https://solgenomics.net/locus/40343/view。The genome information of the tomato yellow leaf curl virus disease resistance gene Ty-1/Ty-3 (Solyc06g051170, Solyc06g051180, Solyc06g051190) corresponding to the SNP site combination described in the first aspect of the present invention comes from the database https://solgenomics.net /locus/26402, 26403, 26404/view, the genomic information of Ty-2 (Solyc11g069660) comes from the database https://solgenomics.net/locus/40343/view .
基于本发明第一方面所述SNP位点组合，可实现对番茄黄化曲叶病毒病抗性的高通量SNP分型检测，结果准确性高，一致性好，通用性强，可实现目标基因区域精准鉴定和选择。Based on the SNP site combination described in the first aspect of the present invention, high-throughput SNP typing detection of resistance to tomato yellow leaf curl virus disease can be realized. Accurate identification and selection of gene regions.
在本发明的一个实施方式中，所述Ty1-SNP01位点～Ty1-SNP08位点、Ty2-SNP01位点～Ty2-SNP08位点及其各自的侧翼序列分别如SEQ ID No：1～16所示；In one embodiment of the present invention, the Ty1-SNP01 site to Ty1-SNP08 site, the Ty2-SNP01 site to Ty2-SNP08 site and their respective flanking sequences are as shown in SEQ ID Nos: 1 to 16, respectively. Show;
所述Ty1-SNP01位点～Ty1-SNP08位点、Ty2-SNP01位点～Ty2-SNP08位点分别位于所述SEQ ID No：1～16中的第102位。The Ty1-SNP01 site to Ty1-SNP08 site and the Ty2-SNP01 site to Ty2-SNP08 site are located at positions 102 in the SEQ ID Nos: 1-16, respectively.
在本发明的一个实施方式中，所述第一SNP位点组合包括所述Ty1-SNP02位点、所述Ty1-SNP04位点、所述Ty1-SNP05位点、所述Ty1-SNP06位点以及所述Ty1-SNP08位点中的一个或者多个，所述第二SNP位点组合包括所述Ty2-SNP04位点、所述Ty2-SNP05位点、所述Ty2-SNP07位点以及所述Ty2-SNP08位点中的一个或者多个。In one embodiment of the present invention, the first combination of SNP sites includes the Ty1-SNP02 site, the Ty1-SNP04 site, the Ty1-SNP05 site, the Ty1-SNP06 site and One or more of the Ty1-SNP08 sites, the second SNP site combination includes the Ty2-SNP04 site, the Ty2-SNP05 site, the Ty2-SNP07 site and the Ty2 - one or more of the SNP08 sites.
本发明第二方面提供了一种用于扩增上述SNP位点组合的引物组合，所述引物组合包括第一引物组合和第二引物组合，所述第一引物组合包括以下引物组1-01～引物组1-05中的一组或多组，所述第二引物组合包括以下引物组2-01～引物组2-04中的一组或多组：A second aspect of the present invention provides a primer combination for amplifying the above SNP site combination, the primer combination includes a first primer combination and a second primer combination, and the first primer combination includes the following primer set 1-01 ～one or more sets of primer set 1-05, the second primer set includes one or more sets of the following primer set 2-01 to primer set 2-04:
引物组1-01：SEQ ID No：17～19序列，用于扩增所述Ty1-SNP02位点的引物；Primer set 1-01: SEQ ID Nos: 17-19 sequences, primers for amplifying the Ty1-SNP02 site;
引物组1-02：SEQ ID No：20～22序列，用于扩增所述Ty1-SNP04位点的引物；Primer set 1-02: SEQ ID Nos: 20-22 sequences, primers for amplifying the Ty1-SNP04 site;
引物组1-03：SEQ ID No：23～25序列，用于扩增所述Ty1-SNP05位点的引物；Primer set 1-03: SEQ ID Nos: 23-25 sequences, primers for amplifying the Ty1-SNP05 site;
引物组1-04：SEQ ID No：26～28序列，用于扩增所述Ty1-SNP06位点的引物；Primer set 1-04: SEQ ID Nos: 26-28 sequences, primers for amplifying the Ty1-SNP06 site;
引物组1-05：SEQ ID No：29～31序列，用于扩增所述Ty1-SNP08位点的引物；Primer set 1-05: SEQ ID Nos: 29-31 sequences, primers for amplifying the Ty1-SNP08 site;
引物组2-01：SEQ ID No：32～34序列，用于扩增所述Ty2-SNP04位点的引物；Primer set 2-01: SEQ ID Nos: 32-34 sequences, primers for amplifying the Ty2-SNP04 site;
引物组2-02：SEQ ID No：35～37序列，用于扩增所述Ty2-SNP05位点的引物；Primer set 2-02: SEQ ID Nos: 35-37 sequences, primers for amplifying the Ty2-SNP05 site;
引物组2-03：SEQ ID No：38～40序列，用于扩增所述Ty2-SNP07位点的引物；Primer set 2-03: SEQ ID Nos: 38-40 sequences, primers for amplifying the Ty2-SNP07 site;
引物组2-04：SEQ ID No：41～43序列，用于扩增所述Ty2-SNP08位点的引物。Primer set 2-04: SEQ ID Nos: 41-43 sequences, primers for amplifying the Ty2-SNP08 site.
本发明第三方面提供了一种用于检测番茄黄化曲叶病毒病抗性的试剂盒，其包括粉末状的或液态的本发明第二方面所述引物组合中的所述第一引物组合和所述第二引物组合中的一组或者多组引物组。The third aspect of the present invention provides a kit for detecting resistance to yellow leaf curl virus disease of tomato, which comprises the first primer combination in the primer combination of the second aspect of the present invention in powder or liquid form and one or more sets of primer sets in the second primer combination.
在本发明的一个实施方式中，本发明第三方面所述的试剂盒还包括PCR预混液，所述PCR预混液包括荧光探针、淬灭探针、ROX内参染料、KlearTaq DNA聚合酶、dNTP和MgCl2。In one embodiment of the present invention, the kit according to the third aspect of the present invention further includes a PCR premix solution, the PCR premix solution includes fluorescent probes, quenching probes, ROX internal reference dye, KlearTaq DNA polymerase, dNTPs and MgCl 2 .
优选的，所述荧光探针包括荧光探针A和荧光探针B，所述淬灭探针包括淬灭探针A和淬灭探针B；Preferably, the fluorescent probe includes fluorescent probe A and fluorescent probe B, and the quenching probe includes quenching probe A and quenching probe B;
所述荧光探针A的核苷酸序列如SEQ ID No：44所示，其5’端连接荧光基团FAM；The nucleotide sequence of the fluorescent probe A is shown in SEQ ID No: 44, and its 5' end is connected to a fluorescent group FAM;
所述荧光探针B的核苷酸序列如SEQ ID No：45所示，其3’端连接荧光基团VIC或HEX；The nucleotide sequence of the fluorescent probe B is shown in SEQ ID No: 45, and its 3' end is connected to a fluorescent group VIC or HEX;
所述淬灭探针A的核苷酸序列如SEQ ID No：46所示，其3’端连接淬灭基团BHQ；The nucleotide sequence of the quenching probe A is shown in SEQ ID No: 46, and its 3' end is connected to a quenching group BHQ;
所述淬灭探针B的核苷酸序列如SEQ ID No：47所示，其3’端连接淬灭基团BHQ。The nucleotide sequence of the quenching probe B is shown in SEQ ID No: 47, and its 3' end is connected to a quenching group BHQ.
本发明第四方面提供了本发明第一方面所述的SNP位点组合，或本发明第二方面所述的引物组合，或本发明第三方面所述的试剂盒的如下任意一种应用：The fourth aspect of the present invention provides the SNP site combination described in the first aspect of the present invention, or the primer combination described in the second aspect of the present invention, or any one of the following applications of the kit described in the third aspect of the present invention:
(1)在检测或辅助检测番茄黄化曲叶病毒病抗性中的应用；(1) Application in detection or auxiliary detection of resistance to yellow leaf curl virus disease of tomato;
(2)在制备检测或辅助检测番茄黄化曲叶病毒病抗性的产品中的应用；(2) application in the preparation of products for detection or auxiliary detection of tomato yellow leaf curl virus disease resistance;
(3)在番茄黄化曲叶病毒病抗性育种中的应用；(3) Application in tomato yellow leaf curl virus disease resistance breeding;
(4)在番茄种质资源及新品种鉴定和保护中的应用；(4) Application in the identification and protection of tomato germplasm resources and new varieties;
(5)在番茄种质资源改良和创新中的应用。(5) Application in the improvement and innovation of tomato germplasm resources.
优选的，本发明第四方面提供的应用采用以下技术手段进行：Preferably, the application provided by the fourth aspect of the present invention adopts the following technical means to carry out:
检测本发明第一方面提供的SNP位点组合中的一个或多个SNP位点的多态性或基因型，检测方法包括飞行质谱、液相色谱、重测序、靶向测序和多重PCR测序中的一种或多种。Detect the polymorphism or genotype of one or more SNP loci in the SNP locus combination provided by the first aspect of the present invention, and the detection method includes flight mass spectrometry, liquid chromatography, resequencing, targeted sequencing and multiplex PCR sequencing. one or more of.
优选的，本发明第四方面提供的应用采用以下技术手段进行：Preferably, the application provided by the fourth aspect of the present invention adopts the following technical means to carry out:
利用本发明第一方面提供的SNP位点组合中的一个或多个SNP位点的序列信息开发PCR标记和/或基因芯片，所述PCR标记包括PCR-RFLP标记、TaqMan标记、KASP标记、AS-PCR标记和HRM标记中的一种或多种。Using the sequence information of one or more SNP sites in the SNP site combination provided in the first aspect of the present invention to develop PCR markers and/or gene chips, the PCR markers include PCR-RFLP markers, TaqMan markers, KASP markers, AS markers - one or more of PCR markers and HRM markers.
优选的，本发明第四方面提供的应用采用以下技术手段进行：Preferably, the application provided by the fourth aspect of the present invention adopts the following technical means to carry out:
利用本发明第一方面提供的SNP位点组合中的一个或多个SNP位点开展分子操作实现番茄黄化曲叶病毒病抗性的分子育种改良和种质资源创新，所述分子操作包括基因编辑或遗传转化。Using one or more SNP loci in the SNP locus combination provided in the first aspect of the present invention to carry out molecular manipulation to achieve molecular breeding improvement and germplasm resource innovation of tomato yellow leaf curl virus disease resistance, the molecular manipulation includes gene Editing or genetic transformation.
上述应用具体可根据不同项目要求及目的实施优化调整或替代。The above applications can be optimized, adjusted or replaced according to different project requirements and purposes.
根据需要，可以选择本发明第一方面所述的第一SNP位点组合和第二SNP位点组合中的1个或几个或全部进行SNP位点多态性或基因型检测。在一些实施例中，通过检测其中1个SNP位点来鉴定待测番茄品种中是否含有Ty-1/Ty-3和Ty-2基因和/或鉴定待测番茄品种中Ty-1/Ty-3(互为等位基因)和Ty-2基因区段单倍型(Ty-1/Ty-1/Ty-2/Ty-2，Ty-1/ty-1/Ty-2/Ty-2，ty-1/ty-1/Ty-2/Ty-2；Ty-1/Ty-1/Ty-2/ty-2，Ty-1/ty-1/Ty-2/ty-2，ty-1/ty-1/Ty-2/ty-2；Ty-1/Ty-1/ty-2/ty-2，Ty-1/ty-1/ty-2/ty-2或ty-1/ty-1/ty-2/ty-2)。在另一些实施例中，通过检测其中2个或2个以上或全部的SNP位点来鉴定待测番茄品种中是否含有Ty-1/Ty-3和Ty-2基因和/或鉴定待测番茄品种中Ty-1/Ty-3和Ty-2基因区段单倍型。优选的，通过检测第一SNP位点组合中的Ty1-SNP02位点、Ty1-SNP04位点、Ty1-SNP05位点、Ty1-SNP06位点以及Ty1-SNP08位点中的1个或多个来鉴定待测番茄品种中是否含有Ty-1/Ty-3基因和/或鉴定待测番茄品种中Ty-1/Ty-3基因区段单倍型；通过检测第二SNP位点组合中的Ty2-SNP04位点、Ty2-SNP05位点、Ty2-SNP07位点以及Ty2-SNP08位点中的1个或多个来鉴定待测番茄品种中是否含有Ty-2基因和/或鉴定待测番茄品种中Ty-2基因区段单倍型。According to needs, one or several or all of the first SNP site combination and the second SNP site combination described in the first aspect of the present invention may be selected for SNP site polymorphism or genotype detection. In some embodiments, by detecting one of the SNP loci to identify whether the tested tomato variety contains Ty-1/Ty-3 and Ty-2 genes and/or to identify Ty-1/Ty- in the tested tomato variety 3 (mutual alleles) and Ty-2 gene segment haplotypes (Ty-1/Ty-1/Ty-2/Ty-2, Ty-1/ty-1/Ty-2/Ty-2 , ty-1/ty-1/Ty-2/Ty-2; Ty-1/Ty-1/Ty-2/ty-2, Ty-1/ty-1/Ty-2/ty-2, ty -1/ty-1/Ty-2/ty-2; Ty-1/Ty-1/ty-2/ty-2, Ty-1/ty-1/ty-2/ty-2 or ty-1 /ty-1/ty-2/ty-2). In other embodiments, by detecting 2 or more than 2 or all of the SNP sites to identify whether the tomato variety to be tested contains Ty-1/Ty-3 and Ty-2 genes and/or to identify the tomato to be tested Ty-1/Ty-3 and Ty-2 gene segment haplotypes in the cultivar. Preferably, by detecting one or more of the Ty1-SNP02 site, the Ty1-SNP04 site, the Ty1-SNP05 site, the Ty1-SNP06 site and the Ty1-SNP08 site in the first SNP site combination Identify whether the tomato variety to be tested contains the Ty-1/Ty-3 gene and/or identify the haplotype of the Ty-1/Ty-3 gene segment in the tomato variety to be tested; by detecting Ty2 in the second SNP site combination -One or more of the SNP04 site, Ty2-SNP05 site, Ty2-SNP07 site and Ty2-SNP08 site to identify whether the tomato variety to be tested contains the Ty-2 gene and/or to identify the tomato variety to be tested Ty-2 gene segment haplotypes.
本发明第五方面提供了一种检测番茄黄化曲叶病毒病抗性的方法，其特征在于，对待测番茄品种进行SNP分型检测，包括以下步骤：A fifth aspect of the present invention provides a method for detecting resistance to yellow leaf curl virus disease of tomato, characterized in that, performing SNP typing detection on the tomato variety to be tested, comprising the following steps:
(1)提取所述待测番茄品种的DNA；(1) extracting the DNA of the tomato variety to be tested;
(2)以本发明第二方面所述的引物组合分别对所述DNA进行PCR扩增；(2) PCR-amplifying the DNA with the primer combination according to the second aspect of the present invention;
(3)检查扩增结果，确定所述待测番茄品种在每一引物组对应的SNP位点的基因型。(3) Check the amplification results to determine the genotype of the tomato variety to be tested at the SNP site corresponding to each primer set.
在具体实施方案中，所述的待测番茄品种的DNA可取自番茄植株的叶片、根、茎、花、果实和种子中的任一种。In a specific embodiment, the DNA of the tomato variety to be tested can be obtained from any of the leaves, roots, stems, flowers, fruits and seeds of tomato plants.
在本发明的一个实施方式中，所述对待测番茄品种进行SNP分型检测采用KASP检测法，所述KASP检测法包括：In one embodiment of the present invention, the SNP typing detection of the tomato variety to be tested adopts the KASP detection method, and the KASP detection method includes:
(1)向所述待测番茄品种的叶片DNA中加入引物混合液和PCR预混液，进行KASP扩增；(1) adding primer mixture and PCR premix to the leaf DNA of the tomato variety to be tested, and carrying out KASP amplification;
(2)采用荧光定量设备检测PCR产物，确定所述待测番茄品种在每一引物组对应的SNP位点的基因型；(2) using fluorescence quantitative equipment to detect the PCR product, to determine the genotype of the tomato variety to be tested at the SNP site corresponding to each primer set;
所述引物混合液由本发明第二方面所述引物组合中同一引物组的引物序列组成。The primer mixture is composed of primer sequences of the same primer set in the primer combination of the second aspect of the present invention.
优选的，所述PCR预混液包括荧光探针、淬灭探针、ROX内参染料、KlearTaq DNA聚合酶、dNTP和MgCl2。Preferably, the PCR master mix includes fluorescent probes, quenching probes, ROX internal reference dye, KlearTaq DNA polymerase, dNTPs and MgCl 2 .
优选的，所述荧光探针包括荧光探针A和荧光探针B，所述淬灭探针包括淬灭探针A和淬灭探针B；Preferably, the fluorescent probe includes fluorescent probe A and fluorescent probe B, and the quenching probe includes quenching probe A and quenching probe B;
所述荧光探针A的核苷酸序列如SEQ ID No：44所示，其5’端连接荧光基团FAM；The nucleotide sequence of the fluorescent probe A is shown in SEQ ID No: 44, and its 5' end is connected to a fluorescent group FAM;
所述荧光探针B的核苷酸序列如SEQ ID No：45所示，其3’端连接荧光基团VIC或HEX；The nucleotide sequence of the fluorescent probe B is shown in SEQ ID No: 45, and its 3' end is connected to a fluorescent group VIC or HEX;
所述淬灭探针A的核苷酸序列如SEQ ID No：46所示，其3’端连接淬灭基团BHQ；The nucleotide sequence of the quenching probe A is shown in SEQ ID No: 46, and its 3' end is connected to a quenching group BHQ;
所述淬灭探针B的核苷酸序列如SEQ ID No：47所示，其3’端连接淬灭基团BHQ。The nucleotide sequence of the quenching probe B is shown in SEQ ID No: 47, and its 3' end is connected to a quenching group BHQ.
优选的，所述荧光定量设备包括各品牌荧光定量PCR仪、酶标仪以及IntelliQube、GeneMatrix等高通量基因分型系统(自动化工作站)。Preferably, the fluorescence quantitative equipment includes fluorescence quantitative PCR instruments of various brands, microplate readers, and high-throughput genotyping systems (automated workstations) such as IntelliQube and GeneMatrix.
本发明通过基于本发明第一方面提供的SNP位点组合成功开发能够快速直观有效鉴定区分目标SNP位点基因型状态的引物组合，并采用KASP检测法进行SNP分型检测，从而判定被检测番茄材料中Ty-1/Ty-3和Ty-2基因区段单倍型(Ty-1/Ty-1/Ty-2/Ty-2，Ty-1/ty-1/Ty-2/Ty-2，ty-1/ty-1/Ty-2/Ty-2；Ty-1/Ty-1/Ty-2/ty-2，Ty-1/ty-1/Ty-2/ty-2，ty-1/ty-1/Ty-2/ty-2；Ty-1/Ty-1/ty-2/ty-2，Ty-1/ty-1/ty-2/ty-2或ty-1/ty-1/ty-2/ty-2)，进而辅助开展番茄抗黄化曲叶病毒病基因快速精准转育应用。Based on the SNP site combination provided in the first aspect of the present invention, the present invention successfully develops a primer combination that can quickly and intuitively identify and distinguish the genotype state of the target SNP site, and uses the KASP detection method for SNP typing detection, thereby determining the tomato to be detected. Ty-1/Ty-3 and Ty-2 gene segment haplotypes in the material (Ty-1/Ty-1/Ty-2/Ty-2, Ty-1/ty-1/Ty-2/Ty- 2, ty-1/ty-1/Ty-2/Ty-2; Ty-1/Ty-1/Ty-2/ty-2, Ty-1/ty-1/Ty-2/ty-2, ty-1/ty-1/Ty-2/ty-2; Ty-1/Ty-1/ty-2/ty-2, Ty-1/ty-1/ty-2/ty-2 or ty- 1/ty-1/ty-2/ty-2), and then assist in the rapid and precise application of tomato anti-yellowing leaf curl virus disease gene transfer.
在本发明的一个实施方式中，根据本发明第五方面所述方法，所述PCR的反应体系为：10～20ng/μL模板DNA 0.8μL；PCR预混液0.8μL；引物混合液0.03μL，其中各引物的终浓度均为100pmol/L；In one embodiment of the present invention, according to the method of the fifth aspect of the present invention, the PCR reaction system is: 10-20ng/μL template DNA 0.8 μL; PCR premix 0.8 μL; primer mixture 0.03 μL, wherein The final concentration of each primer was 100 pmol/L;
所述PCR的反应条件为：95℃预变性10min；95℃变性20s，61℃退火延伸60s，每个循环的退火温度降低0.6℃，共10个循环，最终退火温度降到55℃；94℃变性20s，55℃退火延伸60s，共28～32个循环。The PCR reaction conditions were: pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 20 s, annealing at 61 °C for 60 s, the annealing temperature for each cycle was reduced by 0.6 °C, a total of 10 cycles, and the final annealing temperature was reduced to 55 °C; 94 °C Denaturation for 20 s, annealing and extension at 55 °C for 60 s, a total of 28 to 32 cycles.
本发明第五方面提供的方法操作简单，只需将引物混合液和PCR预混液加入到含有DNA样本的PCR微孔反应板中，进行PCR扩增，然后采用荧光定量设备检测分析PCR产物并进行数据分析即可。The method provided by the fifth aspect of the present invention is simple to operate. It only needs to add the primer mixture and the PCR premix into the PCR microwell reaction plate containing the DNA samples, carry out PCR amplification, and then use a fluorescence quantitative equipment to detect and analyze the PCR products and carry out Data analysis can be done.
区别于现有技术，本发明具有以下有益效果：Different from the prior art, the present invention has the following beneficial effects:
(1)本发明通过直接在已知Ty-1/Ty-3和Ty-2基因上下游临近区域和基因内部大量变异组数据分析鉴别得到了高效、有代表性、通用的SNP位点组合，通过不同资源材料广泛验证，实现了目标番茄黄化曲叶病毒病抗性基因的分子标记辅助有效选择，打破了番茄黄化曲叶病毒病抗性基因不利连锁；(1) The present invention obtains efficient, representative and universal SNP site combinations by directly analyzing and identifying a large number of variant group data in the upstream and downstream adjacent regions of the known Ty-1/Ty-3 and Ty-2 genes and within the genes, Through extensive verification of different resource materials, the molecular marker-assisted effective selection of the target tomato yellow leaf curl virus disease resistance gene has been achieved, breaking the unfavorable linkage of tomato yellow leaf curl virus disease resistance genes;
(2)本发明的SNP位点组合(及其各自的侧翼序列信息)可以为靶向测序、基因芯片、探针、PCR标记、基因克隆及功能研究等其他技术拓展或研究提供有力支撑帮助；(2) The combination of SNP sites of the present invention (and their respective flanking sequence information) can provide strong support for the development or research of other technologies such as targeted sequencing, gene chips, probes, PCR markers, gene cloning and functional research;
(3)基于本发明的SNP位点组合所开发的检测物质/产品(例如本发明的引物组合、试剂盒等)可实现对番茄黄化曲叶病毒病抗性基因Ty-1/Ty-3和Ty-2区段单倍型的快速、精准和高通量检测，具有操作简单、成本低廉、可自动化、通量效率高、标记稳定、安全无毒无害等优点，可以在番茄苗期快速、准确和高通量的进行番茄黄化曲叶病毒病抗性鉴定，降低人工接种鉴定和田间移栽工作量，提高育种效率、降低育种成本、加速育种进程，非常适合现代商业化育种应用及大规模遗传改良研究。(3) The detection substance/product developed based on the SNP site combination of the present invention (for example, the primer combination, the kit of the present invention, etc.) can realize the resistance gene Ty-1/Ty-3 of tomato yellow leaf curl virus disease The rapid, accurate and high-throughput detection of haplotypes in and Ty-2 segment has the advantages of simple operation, low cost, automation, high throughput efficiency, stable labeling, safety, non-toxicity and harmlessness. It can be used in the tomato seedling stage. Fast, accurate and high-throughput identification of tomato yellow leaf curl virus disease resistance, reducing manual inoculation identification and field transplanting workload, improving breeding efficiency, reducing breeding costs, and accelerating the breeding process, which is very suitable for modern commercial breeding applications and large-scale genetic improvement studies.
附图说明Description of drawings
图1为本发明检测番茄抗黄化曲叶病毒病基因Ty-1/Ty-3和Ty-2的通用SNP位点发掘及KASP引物组合开发及应用的流程图；Fig. 1 is the flow chart that the present invention detects the general SNP site discovery of tomato anti-yellowing leaf curl virus disease genes Ty-1/Ty-3 and Ty-2 and KASP primer combination development and application;
图2示出了番茄抗黄化曲叶病毒病基因Ty-1/Ty-3(Solyc06g051170，Solyc06g051180，Solyc06g051190)上下游及内部区域5个通用SNP位点(Ty1-SNP02、Ty1-SNP04、Ty1-SNP05、Ty1-SNP06和Ty1-SNP08)的变异组及其在番茄基因组(SL2.50版本)上的位置信息；图中，TS-2、TS-3及Reference genome(Heinz 1706)等为感病基因型对照，其余TS-307～TS313为抗病基因型对照；Figure 2 shows five general SNP sites (Ty1-SNP02, Ty1-SNP04, Ty1- SNP05, Ty1-SNP06 and Ty1-SNP08) and their position information on the tomato genome (version SL2.50); in the figure, TS-2, TS-3 and Reference genome (Heinz 1706) are susceptible Genotype control, other TS-307~TS313 are disease resistance genotype control;
图3示出了番茄抗黄化曲叶病毒病基因Ty-2(Solyc11g069660)上下游及内部区域4个通用SNP位点(Ty2-SNP04、Ty2-SNP05、Ty2-SNP07和Ty2-SNP08)的变异组及其在番茄基因组(SL2.50版本)上的位置信息；图中，TS-2、TS-3及Reference genome(Heinz 1706)等为感病基因型对照，其余TS-402～TS408为抗病基因型对照；Figure 3 shows the variation of four universal SNP sites (Ty2-SNP04, Ty2-SNP05, Ty2-SNP07 and Ty2-SNP08) in the upstream, downstream and inner regions of tomato yellow leaf curl virus disease resistance gene Ty-2 (Solyc11g069660) group and its position information on the tomato genome (version SL2.50); in the figure, TS-2, TS-3 and Reference genome (Heinz 1706) are the susceptible genotype controls, and the other TS-402 to TS408 are resistant genotypes. disease genotype control;
图4示出了番茄抗黄化曲叶病毒病基因Ty-1/Ty-3(Solyc06g051170，Solyc06g051180，Solyc06g051190)通用SNPs位点开发的5个KASP引物组合在大群体中的分型情况；图中，A为引物组1-01(Chr06：34385952)，B为引物组1-02(Chr06：34361543)，C为引物组1-03(Chr06：34378550)，D为引物组1-04(Chr06：34385942)，E为引物组1-05(Chr06：34387918)，横坐标代表FAM荧光信号值(I处圆点，代表抗病基因型)，纵坐标代表HEX荧光信号值(III处圆点，代表感病基因型)，中间II处圆点代表杂合抗病基因型，靠近原点IV处圆点代表NTC阴性对照；Figure 4 shows the typing of five KASP primer combinations developed at the universal SNPs site of the tomato yellow leaf curl virus disease resistance gene Ty-1/Ty-3 (Solyc06g051170, Solyc06g051180, Solyc06g051190) in a large population; Figure 4 , A is primer set 1-01 (Chr06: 34385952), B is primer set 1-02 (Chr06: 34361543), C is primer set 1-03 (Chr06: 34378550), D is primer set 1-04 (Chr06: 34385942), E is primer set 1-05 (Chr06: 34387918), the abscissa represents the FAM fluorescence signal value (the circle at I, represents the disease-resistant genotype), and the ordinate represents the HEX fluorescence signal value (the circle at III, represents the Susceptible genotype), the dots in the middle II represent heterozygous disease-resistant genotypes, and the dots near the origin IV represent NTC negative controls;
图5示出了番茄抗黄化曲叶病毒病基因Ty-2(Solyc11g069660)通用SNPs位点开发的4个KASP引物组合在大群体中的分型情况；图中，A为引物组2-01(Chr11：54290582)，B为引物组2-02(Chr11：54288056)，C为引物组2-03(Chr11：54288729)，D为引物组2-04(Chr11：54289564)，横坐标代表FAM荧光信号值(I处圆点，代表抗病基因型)，纵坐标代表HEX荧光信号值(III处圆点，代表感病基因型)，中间II处圆点代表杂合抗病基因型，靠近原点IV处圆点代表NTC阴性对照。Figure 5 shows the typing of four KASP primer combinations developed at the universal SNPs site of tomato yellow leaf curl virus disease resistance gene Ty-2 (Solyc11g069660) in a large population; in the figure, A is primer set 2-01 (Chr11: 54290582), B is primer set 2-02 (Chr11: 54288056), C is primer set 2-03 (Chr11: 54288729), D is primer set 2-04 (Chr11: 54289564), the abscissa represents FAM fluorescence Signal value (dots at I, representing the disease-resistant genotype), the ordinate represents the HEX fluorescence signal value (dots at III, representing the susceptible genotype), and the dots at the middle II represent heterozygous disease-resistant genotypes, close to the origin Dots at IV represent NTC negative controls.
具体实施方式Detailed ways
为详细说明技术方案的技术内容、构造特征、所实现目的及效果，以下结合具体实施例并配合附图详予说明。本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。In order to describe in detail the technical content, structural features, achieved objectives and effects of the technical solution, the following detailed description is given in conjunction with specific embodiments and accompanying drawings. This embodiment is implemented on the premise of the technical solution of the present invention, and provides a detailed implementation manner and a specific operation process, but the protection scope of the present invention is not limited to the following embodiments.
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA的5’末端核苷酸，末位均为相应DNA的3’末端核苷酸。下述实施例中所用部分已知抗性的番茄检测材料，包括TS系列番茄种质资源为国内外公开资源，社会公众可向中国农业科学院农业基因组研究所或者其他科研单位索取以重复下述实验，其余番茄商品种可依据表2所列来源通过正规商业途径获得。The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5'-terminal nucleotide of the corresponding DNA, and the last position is the 3'-terminal nucleotide of the corresponding DNA. Some of the tomato detection materials with known resistance used in the following examples, including the TS series tomato germplasm resources, are public resources at home and abroad, and the public can obtain from the Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences or other scientific research units to repeat the following experiments. , other tomato commodity varieties can be obtained through formal commercial channels according to the sources listed in Table 2.
在具体实施方案中，所述的待测番茄样品(DNA)可取自番茄植株的叶片、根、茎、花、果实和种子中的任一种。在下述实施例中采用番茄植株的叶片提取DNA，但并不用以限制本发明的保护范围。在下述实施例中采用的PCR试剂、反应体系、平台设备及扩增检测程序为本发明的优选方案，其他类似合理的国产或进口试剂、设备平台、反应体系及扩增程序也可以达到相同检测目的，并不用以限制本发明的保护范围。In a specific embodiment, the tomato sample (DNA) to be tested can be obtained from any of the leaves, roots, stems, flowers, fruits and seeds of tomato plants. In the following examples, the leaves of tomato plants are used to extract DNA, but it is not intended to limit the protection scope of the present invention. The PCR reagents, reaction systems, platform equipment and amplification detection procedures used in the following examples are the preferred solutions of the present invention, and other similar and reasonable domestic or imported reagents, equipment platforms, reaction systems and amplification procedures can also achieve the same detection It is not intended to limit the protection scope of the present invention.
图1为本发明检测番茄抗黄化曲叶病毒病基因Ty-1/Ty-3和Ty-2的通用SNP位点发掘及KASP引物组合开发及应用的流程图。如图1所示，在本发明的具体实施例中，采用KASP检测方法进行SNP分型检测，但并不用以限制本发明的保护范围。本领域技术人员可以基于本发明提供的SNP位点，通过质谱、色谱测序、基因芯片以及其他PCR技术等手段进行SNP分型检测。Fig. 1 is a flow chart showing the general SNP locus discovery and KASP primer combination development and application for detecting tomato yellow leaf curl virus disease resistance genes Ty-1/Ty-3 and Ty-2 according to the present invention. As shown in FIG. 1 , in a specific embodiment of the present invention, the KASP detection method is used for SNP typing detection, but it is not intended to limit the protection scope of the present invention. Those skilled in the art can perform SNP typing detection by means of mass spectrometry, chromatographic sequencing, gene chip and other PCR technologies based on the SNP sites provided by the present invention.
实施例1 SNP位点组合的筛选Example 1 Screening of SNP site combinations
1、实验材料1. Experimental materials
选取世界范围内、不同来源类型、有代表性的660份番茄种质资源的变异组数据用于本实施例中SNP位点筛选，该番茄变异组数据来源主要基于本发明人所在项目组前期工作(Lin,T.,Zhu,G.,Zhang,J.et al.Genomic analyses provide insights into thehistory of tomato breeding[J].Nat Genet,2014,46:1220～1226；Tieman D,Zhu G,Resende M F R,et al.A chemical genetic roadmap to improved tomato flavor[J].Science,2017,355(6323):391)。其中，部分番茄种质资源的基因型数据及Ty-1/Ty-3及Ty-2基因的抗性表型数据来源于现有公开数据库(https://solgenomics.net/)。The variation group data of 660 representative tomato germplasm resources worldwide, from different source types, were selected for the SNP locus screening in this example. The source of the tomato variation group data is mainly based on the preliminary work of the project team where the inventor is located. (Lin, T., Zhu, G., Zhang, J. et al. Genomic analyses provide insights into the history of tomato breeding[J]. Nat Genet, 2014, 46: 1220～1226; Tieman D, Zhu G, Resende M F R , et al. A chemical genetic roadmap to improved tomato flavor[J]. Science, 2017, 355(6323):391). Among them, the genotype data of some tomato germplasm resources and the resistance phenotype data of Ty-1/Ty-3 and Ty-2 genes were obtained from the existing public database (https://solgenomics.net/).
2、SNP位点组合的筛选2. Screening of SNP site combinations
利用目标基因Ty-1/Ty-3基因(Solyc06g051170，Solyc06g051180，Solyc06g051190，数据库来源https://solgenomics.net/locus/26402,26403,26404/view)及Ty-2基因(Solyc11g069660，数据库来源https://solgenomics.net/locus/40343/ view)内部及上下游区域2kb范围内筛选通用SNP位点。Using target genes Ty-1/Ty-3 genes (Solyc06g051170, Solyc06g051180, Solyc06g051190, database source https://solgenomics.net/locus/26402, 26403, 26404/view) and Ty-2 gene (Solyc11g069660, database source https: //solgenomics.net/locus/40343/ view ) to screen for universal SNP sites within 2kb of the internal and upstream and downstream regions.
通过大果番茄、樱桃番茄、鲜食番茄、加工番茄等不同类型品种以及其他野生抗病供体种质资源的全基因组变异图谱分析，获得抗、感材料之间Ty-1/Ty-3基因(Solyc06g051170，Solyc06g051180，Solyc06g051190)及Ty-2基因(Solyc11g069660)内部或两侧存在一致差异的SNP位点。以TS-2(Moneymaker)、TS-3(M-82)及Reference genome(Heinz 1706)等作为已知纯合感病基因型(ty-1/ty-1，ty-2/ty-2)对照，以TS-307～TS-313等作为已知纯合抗病基因型(Ty-1/Ty-1)对照，以TS-402～TS-408等作为已知纯合抗病基因型(Ty-2/Ty-2)对照，对照包括大果番茄、樱桃番茄和野生番茄等不同类型和地理来源的番茄品种资源。按照基因上下游及基因内部不同位置选择，并根据分型结果，对每个位点进行筛选分析。最终成功获得16个在已知抗、感基因型对照的变异组中表现高度一致稳定差异的高质量通用SNP位点(即本发明的SNP位点组合：Ty1-SNP01位点～Ty1-SNP08位点和Ty2-SNP01位点～Ty2-SNP08位点)。由于这16个SNP位点在不同类型及来源的抗、感品种之间表现高度一致变异，因此可以预期其对番茄黄化曲叶病毒病抗性的显著关联表征效果。Through genome-wide variation map analysis of different types of large-fruit tomato, cherry tomato, fresh tomato, processed tomato, and other wild disease-resistant donor germplasm resources, the Ty-1/Ty-3 genes between resistant and susceptible materials were obtained. (Solyc06g051170, Solyc06g051180, Solyc06g051190) and Ty-2 gene (Solyc11g069660) with identically different SNP sites within or on both sides. Take TS-2 (Moneymaker), TS-3 (M-82) and Reference genome (Heinz 1706) as known homozygous susceptible genotypes (ty-1/ty-1, ty-2/ty-2) For control, TS-307~TS-313 etc. were used as known homozygous disease resistance genotypes (Ty-1/Ty-1) controls, and TS-402~TS-408 etc. were used as known homozygous disease resistance genotypes ( Ty-2/Ty-2) control, the control includes tomato variety resources of different types and geographical origins such as large-fruited tomato, cherry tomato and wild tomato. According to the different positions of the upstream and downstream of the gene and within the gene, and according to the typing results, screen and analyze each locus. Finally, 16 high-quality universal SNP loci (that is, the SNP locus combination of the present invention: Ty1-SNP01 locus ~ Ty1-SNP08 locus) showing highly consistent and stable differences in the known resistance and susceptibility genotype control variants were successfully obtained. point and Ty2-SNP01 site ~ Ty2-SNP08 site). Since these 16 SNP loci showed highly consistent variation among resistant and susceptible varieties of different types and origins, it can be expected to have a significant correlation characterization effect on resistance to tomato yellow leaf curl virus disease.
考虑到测序、PCR扩增及基因芯片等SNPs不同检测方法的多样性、成功率和基本原理的通用性，以及已克隆基因功能、分子调控网络机制的不确定性，因此本发明进一步提供了上述抗番茄黄化曲叶病毒病基因Ty-1/Ty-3及Ty-2区域的16个SNP位点及其各自的侧翼序列，用于检测番茄黄化曲叶病毒病抗性、制备相关检测或辅助检测产品以及番茄辅助育种和种质资源保护及创新的应用。Considering the diversity of different detection methods of SNPs such as sequencing, PCR amplification and gene chip, the success rate and the generality of the basic principle, as well as the uncertainty of the cloned gene function and molecular regulation network mechanism, the present invention further provides the above-mentioned 16 SNP sites in the regions of Ty-1/Ty-3 and Ty-2 regions of tomato yellow leaf curl virus disease resistance genes and their respective flanking sequences, used for the detection of tomato yellow leaf curl virus disease resistance and preparation of related assays Or auxiliary detection products and tomato auxiliary breeding and germplasm resources protection and innovative applications.
Ty1-SNP01位点～Ty1-SNP08位点、Ty2-SNP01位点～Ty2-SNP08位点及其各自的侧翼序列分别如SEQ ID No：1～16所示。Ty1-SNP01 site to Ty1-SNP08 site, Ty2-SNP01 site to Ty2-SNP08 site and their respective flanking sequences are shown in SEQ ID Nos: 1-16, respectively.
实施例2引物合成与试剂盒制备Example 2 Primer synthesis and kit preparation
1、引物的设计和筛选1. Primer design and screening
根据实施例1提供的Ty1-SNP01位点～Ty1-SNP08位点和Ty2-SNP01位点～Ty2-SNP08位点的侧翼序列，针对每个SNP位点，采用按照KASP标记设计开发原则(目标产物80～150bp，引物在参考基因组上特异匹配目标区域且位于非SNP密集区域，避免A\T或G\C含量高等复杂序列区域等)，利用Primer3.0软件在SNP位点的上游设计两条正向引物，下游设计一条反向引物。According to the flanking sequences of Ty1-SNP01 site～Ty1-SNP08 site and Ty2-SNP01 site～Ty2-SNP08 site provided in Example 1, for each SNP site, adopt the design and development principle according to KASP marker (target product 80-150bp, the primers specifically match the target region on the reference genome and are located in non-SNP-intensive regions, avoiding complex sequence regions with high A\T or G\C content, etc.), using Primer3.0 software to design two upstream of the SNP site Forward primer, downstream design a reverse primer.
待测番茄样品包括部分已知基因型和黄化曲叶病毒病抗性表型的种质资源及育种材料中随机选取的21个样品作为抗、感或杂合对照，最后加3个ddH2O作为NTC空白对照，共计24份。The tomato samples to be tested include some germplasm resources with known genotypes and yellow leaf curl virus disease resistance phenotypes and 21 randomly selected samples from the breeding materials as resistance, susceptible or heterozygous controls, and finally add 3 ddH 2 O was used as NTC blank control, a total of 24 copies.
DNA提取采用常规CTAB法或国产磁珠试剂盒从测番茄样品的叶片中提取基因组DNA，利用Nanodrop 1000进行核酸浓度测定，稀释并控制DNA模板浓度为10～20ng/μL。DNA extraction Using conventional CTAB method or domestic magnetic bead kit to extract genomic DNA from the leaves of the tested tomato samples, using Nanodrop 1000 to measure the nucleic acid concentration, dilute and control the DNA template concentration to 10-20ng/μL.
PCR的反应体系(10μL)为：10～20ng/μL DNA模板5μL；PCR预混液5μL；引物混合液0.14μL，其中各引物的终浓度均为100pmol/L(优选的，引物混合配比为特异分型引物各12μL、公共引物30μL，再加46μL ddH2O；在其他实施例中，使用其他合理的引物混合配比也可以达到相同的检测目的)。其中，引物混合液由本发明引物组合中同一引物组的引物序列组成。PCR预混液包括荧光探针A、荧光探针B、淬灭探针A、淬灭探针B、ROX内参染料、KlearTaqDNA聚合酶、dNTP和MgCl2。其中，荧光探针A的核苷酸序列如SEQ ID No：44所示，其5’端连接荧光基团FAM；荧光探针B的核苷酸序列如SEQ ID No：45所示，其3’端连接荧光基团VIC或HEX；淬灭探针A的核苷酸序列如SEQ ID No：46所示，其3’端连接淬灭基团BHQ；淬灭探针B的核苷酸序列如SEQ ID No：47所示，其3’端连接淬灭基团BHQ。The PCR reaction system (10 μL) is: 10-20 ng/μL DNA template 5 μL; PCR premix 5 μL; primer mixture 0.14 μL, wherein the final concentration of each primer is 100 pmol/L (preferably, the primer mixing ratio is specific The typing primers are each 12 μL, the common primer is 30 μL, and 46 μL ddH 2 O is added; in other embodiments, other reasonable primer mixing ratios can also achieve the same detection purpose). Wherein, the primer mixture is composed of primer sequences of the same primer set in the primer combination of the present invention. PCR master mix includes fluorescent probe A, fluorescent probe B, quenching probe A, quenching probe B, ROX internal reference dye, KlearTaq DNA polymerase, dNTP and MgCl 2 . Among them, the nucleotide sequence of fluorescent probe A is shown in SEQ ID No: 44, and its 5' end is connected to the fluorescent group FAM; the nucleotide sequence of fluorescent probe B is shown in SEQ ID No: 45, and its 3 The 'end is connected to the fluorescent group VIC or HEX; the nucleotide sequence of the quenching probe A is shown in SEQ ID No: 46, and its 3' end is connected to the quenching group BHQ; the nucleotide sequence of the quenching probe B As shown in SEQ ID No: 47, a quenching group BHQ is attached to its 3' end.
按照Thermo Fisher公司的荧光定量PCR仪(Applied Biosystems Quant Studio3，ABI-Q3)的操作手册，编辑样品及引物排布模板，执行运行程序{30℃读取荧光信号1min；94℃变性15min；94℃变性20s，61℃退火60s，重复此步骤10个循环，每个循环设置Touch-Down降温0.6℃，最终退火温度降到55℃；94℃变性20s，55℃退火60s，重复此步骤28～32个循环；30℃读取荧光信号1min}，分析数据结果，最终分别选取5组和4组FAM信号、VIC信号以及杂合荧光信号聚集分型趋势显著的位于目标基因上下游或内部的引物组构成本发明的引物组合(包括第一引物组合：引物组1-01、引物组1-02、引物组1-03、引物组1-04和引物组1-05，以及第二引物组合：引物组2-01、引物组1-02、引物组1-03和引物组1-04，各引物组所对应的SNP信息如图2和3所示，相应扩增目的条带大小及单倍型信息详见表1)，开展后续大群体Ty-1/Ty-3和Ty-2区域基因型或单倍型验证及育种应用。According to the operation manual of Thermo Fisher's fluorescence quantitative PCR instrument (Applied Biosystems Quant Studio3, ABI-Q3), edit the sample and primer layout template, and execute the running program {30 °C to read the fluorescent signal for 1 min; 94 °C for denaturation for 15 min; 94 °C Denaturation for 20 s, annealing at 61 °C for 60 s, repeat this step for 10 cycles, set Touch-Down to cool down by 0.6 °C in each cycle, and reduce the final annealing temperature to 55 °C; 30 ℃ read the fluorescent signal for 1 min}, analyze the data results, and finally select 5 groups and 4 groups of FAM signal, VIC signal and heterozygous fluorescence signal aggregation and typing trend of the primer sets located in the upstream and downstream or inside the target gene with significant trend. The primer combinations constituting the present invention (including the first primer combination: primer set 1-01, primer set 1-02, primer set 1-03, primer set 1-04 and primer set 1-05, and the second primer combination: primer set Set 2-01, primer set 1-02, primer set 1-03 and primer set 1-04, the SNP information corresponding to each primer set is shown in Figures 2 and 3, and the corresponding amplified target band size and haplotype The information is shown in Table 1), and the follow-up large population Ty-1/Ty-3 and Ty-2 region genotype or haplotype verification and breeding applications are carried out.
引物组1-01：SEQ ID No：17～19序列，用于扩增所述Ty1-SNP02位点的引物；Primer set 1-01: SEQ ID Nos: 17-19 sequences, primers for amplifying the Ty1-SNP02 site;
引物组1-02：SEQ ID No：20～22序列，用于扩增所述Ty1-SNP04位点的引物；Primer set 1-02: SEQ ID Nos: 20-22 sequences, primers for amplifying the Ty1-SNP04 site;
引物组1-03：SEQ ID No：23～25序列，用于扩增所述Ty1-SNP05位点的引物；Primer set 1-03: SEQ ID Nos: 23-25 sequences, primers for amplifying the Ty1-SNP05 site;
引物组1-04：SEQ ID No：26～28序列，用于扩增所述Ty1-SNP06位点的引物；Primer set 1-04: SEQ ID Nos: 26-28 sequences, primers for amplifying the Ty1-SNP06 site;
引物组1-05：SEQ ID No：29～31序列，用于扩增所述Ty1-SNP08位点的引物；Primer set 1-05: SEQ ID Nos: 29-31 sequences, primers for amplifying the Ty1-SNP08 site;
引物组2-01：SEQ ID No：32～34序列，用于扩增所述Ty2-SNP04位点的引物；Primer set 2-01: SEQ ID Nos: 32-34 sequences, primers for amplifying the Ty2-SNP04 site;
引物组2-02：SEQ ID No：35～37序列，用于扩增所述Ty2-SNP05位点的引物；Primer set 2-02: SEQ ID Nos: 35-37 sequences, primers for amplifying the Ty2-SNP05 site;
引物组2-03：SEQ ID No：38～40序列，用于扩增所述Ty2-SNP07位点的引物；Primer set 2-03: SEQ ID Nos: 38-40 sequences, primers for amplifying the Ty2-SNP07 site;
引物组2-04：SEQ ID No：41～43序列，用于扩增所述Ty2-SNP08位点的引物。Primer set 2-04: SEQ ID Nos: 41-43 sequences, primers for amplifying the Ty2-SNP08 site.
表1检测番茄Ty-1/Ty-3和Ty-2基因的引物组合及其对应的SNP位点、扩增片段长度和引物信息Table 1 The primer combinations for detecting tomato Ty-1/Ty-3 and Ty-2 genes and their corresponding SNP sites, amplified fragment lengths and primer information
2、试剂盒的制备2. Preparation of the kit
本实施例将上述引物组合应用于制备试剂盒。该试剂盒除了引物组合，还包括PCR预混液组成。引物混合液由本发明引物组合中同一引物组的引物序列组成。PCR预混液包括荧光探针A、荧光探针B、淬灭探针A、淬灭探针B、ROX内参染料、KlearTaq DNA聚合酶、dNTP和MgCl2。其中，荧光探针A的核苷酸序列如SEQ ID No：44所示，其5’端连接荧光基团FAM；荧光探针B的核苷酸序列如SEQ ID No：45所示，其3’端连接荧光基团VIC或HEX；淬灭探针A的核苷酸序列如SEQ ID No：46所示，其3’端连接淬灭基团BHQ；淬灭探针B的核苷酸序列如SEQID No：47所示，其3’端连接淬灭基团BHQ。每个SNP位点的三条引物(每个引物组)独立分装后包装在一起；PCR预混液独立包装。In this example, the above-mentioned primer combination was applied to the preparation kit. In addition to primer combinations, the kit also includes PCR master mixes. The primer mixture is composed of primer sequences of the same primer set in the primer combination of the present invention. PCR master mix includes fluorescent probe A, fluorescent probe B, quenching probe A, quenching probe B, ROX internal reference dye, KlearTaq DNA polymerase, dNTP and MgCl 2 . Among them, the nucleotide sequence of fluorescent probe A is shown in SEQ ID No: 44, and its 5' end is connected to the fluorescent group FAM; the nucleotide sequence of fluorescent probe B is shown in SEQ ID No: 45, and its 3 The 'end is connected to the fluorescent group VIC or HEX; the nucleotide sequence of the quenching probe A is shown in SEQ ID No: 46, and its 3' end is connected to the quenching group BHQ; the nucleotide sequence of the quenching probe B As shown in SEQ ID No: 47, a quenching group BHQ is attached to its 3' end. The three primers for each SNP site (each primer set) are packaged together after being packaged independently; the PCR master mix is packaged independently.
实施例3番茄抗黄化曲叶病毒病基因Ty-1/Ty-3和Ty-2高效KASP标记的验证与育种应用Example 3 Validation and breeding application of highly efficient KASP markers for tomato yellow leaf curl virus disease resistance genes Ty-1/Ty-3 and Ty-2
本实施例基于实施例1提供的SNP位点，采用实施例2中提供的试剂盒(包括引物组合)进行番茄抗黄化曲叶病毒病基因Ty-1/Ty-3和Ty-2高效KASP标记的验证与育种应用工作。Based on the SNP loci provided in Example 1, this example uses the kit (including primer combination) provided in Example 2 to carry out high-efficiency KASP of tomato anti-yellowing leaf curl virus disease genes Ty-1/Ty-3 and Ty-2 Validation of markers works with breeding applications.
选取381份包含主流商品种、国家资源库核心公开种质、育种中间材料、杂交新组合等有代表性的番茄样品，涉及大果番茄、樱桃番茄、串收番茄、鲜食番茄、加工番茄以及农家种(地方品种)等各种不同类型栽培番茄，其中154份通过第三方商业机构利用紧密连锁SCAR标记获得了Ty-1/Ty-3和Ty-2基因型数据(包括41份黄化曲叶病毒病抗性表型已知的番茄种质资源及商品种，详见表2-1和表2-2)。Selected 381 representative tomato samples including mainstream commercial varieties, core public germplasm of the national resource bank, intermediate breeding materials, new hybrid combinations, etc., involving large fruit tomatoes, cherry tomatoes, bunch tomatoes, fresh tomatoes, processed tomatoes and Farming varieties (landraces) and other types of cultivated tomatoes, 154 of which have obtained Ty-1/Ty-3 and Ty-2 genotype data through third-party commercial institutions using closely linked SCAR markers (including 41 Huanghuaqu Tomato germplasm resources and commercial varieties with known leaf virus disease resistance phenotypes are shown in Table 2-1 and Table 2-2 for details).
具体的，本实施例提供了一种检测番茄黄化曲叶病毒病抗性的方法，采用KASP检测法对待测番茄品种进行SNP分型检测，包括以下步骤：Specifically, the present embodiment provides a method for detecting the resistance of tomato yellow leaf curl virus disease, using the KASP detection method to perform SNP typing detection on the tomato variety to be tested, including the following steps:
(1)提取待测番茄品种的叶片DNA；(1) Extracting the leaf DNA of the tomato variety to be tested;
(2)采用实施例2中提供的试剂盒，向待测番茄品种的叶片DNA中加入引物混合液和PCR预混液，进行KASP扩增(以实施例2中提供的引物组合中的每一引物组分别对叶片DNA进行KASP扩增)；(2) Using the kit provided in Example 2, adding primer mixture and PCR premix to the leaf DNA of the tomato variety to be tested, and performing KASP amplification (with each primer in the primer combination provided in Example 2) Groups performed KASP amplification on leaf DNA respectively);
(3)采用荧光定量PCR仪检测PCR产物，确定待测番茄品种在每一引物组对应的SNP位点的基因型。(3) The PCR product was detected by a fluorescence quantitative PCR instrument, and the genotype of the tomato variety to be tested at the SNP site corresponding to each primer set was determined.
待测番茄品种为上述381份包含部分已知基因型和表型的种质资源及育种材料，最后加上3个ddH2O作为NTC空白对照，共计384份。The tomato varieties to be tested were the above 381 germplasm resources and breeding materials containing some known genotypes and phenotypes. Finally, 3 ddH 2 O were added as the NTC blank control, totaling 384 copies.
待测番茄品种的叶片DNA的提取方法及条件与实施例2中的提取方法及条件相同。The extraction method and conditions of the leaf DNA of the tomato variety to be tested are the same as those in Example 2.
PCR扩增反应体系(1.6μL)为：10～20ng/μL模板DNA 0.8μL；PCR预混液0.8μL；引物混合液0.03μL，其中各引物的终浓度均为100pmol/L，引物混合配比为特异分型引物各12μL、公共引物30μL，再加46μL ddH2O。The PCR amplification reaction system (1.6 μL) is: 10-20 ng/μL template DNA 0.8 μL; PCR premix 0.8 μL; primer mixture 0.03 μL, the final concentration of each primer is 100 pmol/L, and the primer mixing ratio is 12 μL of specific typing primers, 30 μL of common primers, plus 46 μL of ddH 2 O.
按照IntelliQube平台操作手册，编辑样品及引物排布模板，执行运行程序{95℃预变性10min；95℃变性20s，61℃退火延伸60s，每个循环的退火温度降低0.6℃，共10个循环，最终退火温度降到55℃；94℃变性20s，55℃退火延伸60s，共28～32个循环}，读取荧光数据并酌情增加PCR循环数，分析并去除部分不确定、荧光值太低或者异常的数据点，最终大群体分型情况如图4和图5所示，并导出Excel结果(如表2-1和表2-2所示)。According to the IntelliQube platform operation manual, edit the sample and primer arrangement template, and execute the running program {95°C pre-denaturation for 10min; 95°C denaturation for 20s, 61°C annealing and extension for 60s, the annealing temperature of each cycle is reduced by 0.6°C, a total of 10 cycles, The final annealing temperature is reduced to 55°C; denaturation at 94°C for 20s, annealing and extension at 55°C for 60s, a total of 28 to 32 cycles}, read the fluorescence data and increase the number of PCR cycles as appropriate, analyze and remove some uncertainties, fluorescence values are too low or For abnormal data points, the final large group typing situation is shown in Figure 4 and Figure 5, and the Excel results are exported (as shown in Table 2-1 and Table 2-2).
结果发现，实施例2提供的9组KASP标记在381份不同类型和来源的番茄育种大群体中获得了高度一致且界限清晰的群体分型效果(如图4和图5所示)，也即通过实验证实了实施例1提供的SNP位点组合在不同遗传背景的番茄品种资源中具备很好的通用性和稳定性。进一步的，在41份黄化曲叶病毒病抗性表型已知的待测样本中，抗性表型、SCAR标记鉴定基因型和本发明提供的KASP标记(即引物组合)鉴定基因数据完全一致，即一致性P＝100％(如表2-1和表2-2所示)。除此之外，154份有连锁SCAR标记结果参照的待测样本中，用于检测Ty-1/Ty-3的5个引物组与Ty-1连锁SCAR标记的基因型鉴定结果之间的一致性P为78.7％，与Ty-3连锁SCAR标记的基因型鉴定结果之间的一致性P为55.0％，Ty-1与Ty-3连锁SCAR标记之间的一致性P为58.4％；用于检测Ty-2的4个引物组与Ty-2基因紧密连锁SCAR标记的基因型鉴定结果之间的一致性P为96.5％。而用于检测Ty-1/Ty-3的5个引物组的检测结果之间的一致性P均超过95％，用于检测Ty-2的4个引物组的检测结果之间的一致性P均超过98％{一致性计算公式为P＝(有效检测样本数-差异样本数)/有效检测样本数*100％}。进一步分析发现，SCAR标记检测结果中出现相同重复材料样本检测结果矛盾情况(例如，编号Ta246和Ta247，Ta235和Ta292均为两组相同重复材料，故意试验设计重复)，而本发明提供的引物组的检测结果则无此现象。上述结果证明本发明提供的KASP标记检测准确率和稳定性均显著高于现有商用SCAR标记。The results found that the 9 groups of KASP markers provided in Example 2 achieved highly consistent and well-defined population typing effects in 381 large tomato breeding populations of different types and sources (as shown in Figures 4 and 5), that is, It is confirmed by experiments that the SNP site combination provided in Example 1 has good versatility and stability in tomato variety resources with different genetic backgrounds. Further, in the 41 samples to be tested for which the resistance phenotype of yellow leaf curl virus disease is known, the resistance phenotype, SCAR marker identification genotype and KASP marker (ie primer combination) identification gene data provided by the present invention are complete. Consistent, that is, consistency P=100% (as shown in Table 2-1 and Table 2-2). In addition, among the 154 samples to be tested with linked SCAR marker results, the 5 primer sets used to detect Ty-1/Ty-3 were consistent with the genotype identification results of Ty-1 linked SCAR marker. The sex P was 78.7%, the concordance P between the genotype identification results of Ty-3-linked SCAR markers was 55.0%, and the concordance P between Ty-1 and Ty-3-linked SCAR markers was 58.4%; The concordance P between the four primer sets for detecting Ty-2 and the genotype identification results of the closely linked SCAR marker of the Ty-2 gene was 96.5%. The consistency P between the detection results of the 5 primer sets used to detect Ty-1/Ty-3 all exceeded 95%, and the consistency P between the detection results of the 4 primer sets used to detect Ty-2 All exceeded 98% {the consistency calculation formula is P=(the number of valid detection samples - the number of difference samples)/the number of valid detection samples*100%}. Further analysis found that in the SCAR marker detection results, there were contradictions in the detection results of the same duplicate material samples (for example, the numbers Ta246 and Ta247, Ta235 and Ta292 were two groups of identical duplicate materials, and the experiment was designed to be repeated), while the primer set provided by the present invention was used. The test results showed no such phenomenon. The above results prove that the detection accuracy and stability of the KASP marker provided by the present invention are significantly higher than the existing commercial SCAR markers.
此外，本实施例还对227份育种中间材料进行了Ty-1/Ty-3和Ty-2位点基因型检测，结果发现待测样本的基因型检测结果基本与系谱关系相吻合，可通过待测样本Ty-1/Ty-3和Ty-2位点基因型检测结果准确预测其子代或父代的番茄黄化曲叶病毒病抗性。In addition, 227 breeding intermediate materials were also tested for Ty-1/Ty-3 and Ty-2 loci genotypes in this example, and it was found that the genotype test results of the samples to be tested were basically consistent with the pedigree relationship, which can be obtained by The genotype detection results of Ty-1/Ty-3 and Ty-2 loci of the samples to be tested can accurately predict the resistance to tomato yellow leaf curl virus disease of their offspring or fathers.
综上所述，本实施例提供的9组KASP标记在不同类型、来源或遗传背景的栽培番茄品种资源中具备很好的通用性和稳定性，标记相互之间的检测结果基本保持一致，且每组标记检测的准确率和稳定性要显著优于现有的商业化SCAR标记，每一组都可以单独应用于番茄黄化曲叶病毒病抗性的分子检测。以此类推，同样符合前述变异组特征的其余7个SNP位点可以通过测序、基因芯片或其他PCR标记验证并取得相同的预期效果。同时，上述KASP标记组合在一起能更进一步提高检测精确度，可以避免假阳性等实验误差以及目标基因区域未被完整选择或部分丢失等遗传变异因素导致的抗性鉴定错误，取得更好的检测判定效果。因此，本发明提供的高效KASP标记可以直接用于番茄抗黄化曲叶病毒病分子育种商业化应用。To sum up, the 9 groups of KASP markers provided in this example have good versatility and stability in cultivated tomato variety resources of different types, sources or genetic backgrounds, and the detection results of the markers are basically consistent with each other, and The accuracy and stability of the detection of each group of markers are significantly better than the existing commercial SCAR markers, and each group can be individually applied to the molecular detection of resistance to tomato yellow leaf curl virus disease. By analogy, the remaining 7 SNP loci that also meet the aforementioned variant group characteristics can be verified by sequencing, gene chip or other PCR markers and achieve the same expected effect. At the same time, the combination of the above KASP markers can further improve the detection accuracy, avoid experimental errors such as false positives and resistance identification errors caused by genetic variation factors such as incomplete selection or partial loss of the target gene region, and achieve better detection. Judgment effect. Therefore, the high-efficiency KASP marker provided by the present invention can be directly used in the commercial application of tomato resistance to yellow leaf curl virus disease molecular breeding.
表2-1 154份番茄种质资源、品种及育种中间材料的黄化曲叶病毒病抗性表型及标记基因型Table 2-1 Yellow leaf curl virus disease resistance phenotypes and marker genotypes of 154 tomato germplasm resources, varieties and intermediate breeding materials
表2-2 154份番茄种质资源、品种及育种中间材料的黄化曲叶病毒病抗性表型及标记基因型Table 2-2 Yellow leaf curl virus disease resistance phenotypes and marker genotypes of 154 tomato germplasm resources, varieties and intermediate breeding materials
注：表2-1和2-2中样品编号为实际检测样品排布顺序编号，为便于结果比较分析和直观判断，按照来源类别调整排序；其中“抗性”栏代表番茄黄化曲叶病毒病抗性表型，“S”代表感病，“R”代表抗病，“IR”代表中抗；其中“SCAR”栏代表第三方商业机构利用Ty-1、Ty-2和Ty-3基因连锁SCAR标记检测结果，“SS”代表纯合感病基因型，“RR”代表纯合抗病基因型，“H”代表杂合基因型；其中“引物组1-01”“引物组1-04”“引物组2-01”“引物组2-03”检测结果中，“T:T”代表纯合感病基因型，“C:C”代表纯合抗病基因型，“T:C”代表杂合基因型；其中“引物组1-02”检测结果中“C:C”代表纯合感病基因型，“T:T”代表纯合抗病基因型，“C:T”代表杂合基因型；其中“引物组1-03”“引物组2-04”检测结果中，“A:A”代表纯合感病基因型，“G:G”代表纯合抗病基因型，“A:G”代表杂合基因型；其中“引物组1-05”检测结果中，“G:G”代表纯合感病基因型，“A:A”代表纯合抗病基因型，“G:A”代表杂合基因型；其中“引物组2-02”检测结果中“C:C”代表纯合感病基因型，“G:G”代表纯合抗病基因型，“C:G”代表杂合基因型；“-”代表数据缺失；“*”标注代表基因连锁SCAR标记与本发明提供的KASP标记(即引物组合)之间或者本发明提供的KASP标记内不同标记之间不一致结果。Note: The sample numbers in Tables 2-1 and 2-2 are the sequence numbers of the actual detection samples. In order to facilitate the comparative analysis and intuitive judgment of the results, the order is adjusted according to the source category; the "resistance" column represents tomato yellow leaf curl virus Disease resistance phenotype, "S" stands for susceptible, "R" stands for resistance, "IR" stands for intermediate resistance; the column "SCAR" represents the use of Ty-1, Ty-2 and Ty-3 genes by third-party commercial organizations Linked SCAR marker detection results, "SS" stands for homozygous susceptible genotype, "RR" stands for homozygous resistant genotype, "H" stands for heterozygous genotype; among which "primer set 1-01" and "primer set 1- 04", "Primer Set 2-01" and "Primer Set 2-03" test results, "T:T" represents the homozygous susceptible genotype, "C:C" represents the homozygous disease resistance genotype, "T:C" "represents heterozygous genotype; in the test result of "primer set 1-02", "C:C" represents homozygous susceptible genotype, "T:T" represents homozygous resistant genotype, and "C:T" represents Heterozygous genotype; in the test results of "primer set 1-03" and "primer set 2-04", "A:A" represents homozygous susceptible genotype, "G:G" represents homozygous disease resistance genotype, "A:G" represents heterozygous genotype; in the test results of "primer set 1-05", "G:G" represents homozygous susceptible genotype, "A:A" represents homozygous disease resistance genotype, " G:A” represents the heterozygous genotype; in the test result of “Primer Set 2-02”, “C:C” represents the homozygous susceptible genotype, “G:G” represents the homozygous resistant genotype, “C: G" stands for heterozygous genotype; "-" stands for missing data; "*" mark stands for gene-linked SCAR marker and KASP marker provided by the present invention (ie primer combination) or between different markers within the KASP marker provided by the present invention Inconsistent results.
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念和相关原理流程，则可对这些实施例做出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。Although the preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may be made by those skilled in the art once the basic inventive concepts and related principle flows are known. Therefore, the appended claims are intended to be construed to include the preferred embodiment and all changes and modifications that fall within the scope of the present invention.
本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。倘若针对本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。Those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention, and can be implemented in a wide range under equivalent parameters, concentrations and conditions without unnecessary experiments. this invention. Provided that these modifications and variations to the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
虽然本发明给出了特殊的实施例，应该理解为可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。While the present invention has been given specific embodiments, it should be understood that the present invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.
序列表 sequence listing
<110> 中国农业科学院农业基因组研究所<110> Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences
中国农业科学院深圳农业基因组研究所 Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences
<120> 用于检测番茄黄化曲叶病毒病抗性的SNP位点组合及其应用<120> A combination of SNP loci for detecting resistance to tomato yellow leaf curl virus disease and its application
<130> WK21-HCP-CN1-0351<130> WK21-HCP-CN1-0351
<141> 2021-09-30<141> 2021-09-30
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<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=G或A<223> n=G or A
<400> 3<400> 3
caacgtttgt tggagacttt cacttctaat aatgtagaat gaaactcaaa cgcaagagat 60caacgtttgt tggagacttt cacttctaat aatgtagaat gaaactcaaa cgcaagagat 60
aatcaatatt ttgatcactt caatatcata ttggcccaaa antctattaa actaaataca 120aatcaatatt ttgatcactt caatatcata ttggcccaaa antctattaa actaaataca 120
atttggctat gaaaaaggtc aaaaaaatta agttccacct tcttcatgtt actaaataag 180atttggctat gaaaaaggtc aaaaaaatta agttccacct tcttcatgtt actaaataag 180
aaaaaaaaat catgtatcct cac 203aaaaaaaaat catgtatcct cac 203
<210> 4<210> 4
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=C或T<223> n=C or T
<400> 4<400> 4
aatctctgaa agttgatatg aaggaaaaac taaccatctt gtacttcttt ataacttcat 60aatctctgaa agttgatatg aaggaaaaac taaccatctt gtacttcttt ataacttcat 60
tgcaagaggt gattcttagt tcacaatcaa agttcatggc cngtgtcata tcctttttgt 120tgcaagaggt gattcttagt tcacaatcaa agttcatggc cngtgtcata tccttttttgt 120
actcttcata tcttcctctc cacaatgtca tgcatctttg agggatttca acttcaaagc 180actcttcata tcttcctctc cacaatgtca tgcatctttg agggatttca acttcaaagc 180
aaggcagttt agagatttct aca 203aaggcagttt agagatttct aca 203
<210> 5<210> 5
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=A或G<223> n=A or G
<400> 5<400> 5
tcctagatca cgtgttactt ctaaggggta gctcaggggc gtgaaatata actttcagtt 60tcctagatca cgtgttactt ctaaggggta gctcaggggc gtgaaatata actttcagtt 60
tcccattttc tttagaagat gctcagggaa gctctttaca cnacattgaa actgtaaatc 120tcccattttc tttagaagat gctcagggaa gctctttaca cnacattgaa actgtaaatc 120
cgtacacttg ccaatttcag ggtttcactt ctatgaataa agaaattatg catacctcat 180cgtacacttg ccaatttcag ggtttcactt ctatgaataa agaaattatg catacctcat 180
aattttcatc ttttatatat tct 203aattttcatc ttttatatat tct 203
<210> 6<210> 6
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=T或C<223> n=T or C
<400> 6<400> 6
gaataatgtc aaaccaagaa ctaggaaata taattcttaa agaatacaag gagacgcaaa 60gaataatgtc aaaccaagaa ctaggaaata taattcttaa agaatacaag gagacgcaaa 60
ttaaaaacta tcatatttta tgccacacag aaatcctaat gncaaaattg atggtcattg 120ttaaaaacta tcatatttta tgccacacag aaatcctaat gncaaaattg atggtcattg 120
aatgtgctaa aatctcttaa aactgaaaac aaataagata aaggtatttt gtcacaagta 180aatgtgctaa aatctcttaa aactgaaaac aaataagata aaggtatttt gtcacaagta 180
aaaataagaa aaatattctt act 203aaaataagaa aaatattctt act 203
<210> 7<210> 7
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=A或T<223> n=A or T
<400> 7<400> 7
atttgtcaat gacaatctct gaaagttgat atgaaggaaa aactaaccat cttgtacttc 60atttgtcaat gacaatctct gaaagttgat atgaaggaaa aactaaccat cttgtacttc 60
tttataactt cattgcaaga ggtgattctt agttcacaat cnaagttcat ggcccgtgtc 120tttataactt cattgcaaga ggtgattctt agttcacaat cnaagttcat ggcccgtgtc 120
atatcctttt tgtactcttc atatcttcct ctccacaatg tcatgcatct ttgagggatt 180atatcctttt tgtactcttc atatcttcct ctccacaatg tcatgcatct ttgagggatt 180
tcaacttcaa agcaaggcag ttt 203tcaacttcaa agcaaggcag ttt 203
<210> 8<210> 8
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=G或A<223> n=G or A
<400> 8<400> 8
tctgagggct tgcacaggcc aataagccca tctcaagctt tgatttagaa gaactttcac 60tctgagggct tgcacaggcc aataagccca tctcaagctt tgatttagaa gaactttcac 60
cttgtaaacg tttaccctct aagcatagaa agtacaaaca tntagtaaga aatatagtaa 120cttgtaaacg tttaccctct aagcatagaa agtacaaaca tntagtaaga aatatagtaa 120
gaagggggtt tggctaaagc ttaaaagctg gtcaaacaga ctttgatagc ttactacgcg 180gaagggggtt tggctaaagc ttaaaagctg gtcaaacaga ctttgatagc ttactacgcg 180
tttggtaaac acccaagcct tca 203tttggtaaac acccaagcct tca 203
<210> 9<210> 9
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=G或A<223> n=G or A
<400> 9<400> 9
aacaccctct tgcttatatg atttagagag taatagtcat tggttgaaag caatgtcctc 60aacaccctct tgcttatatg atttagagag taatagtcat tggttgaaag caatgtcctc 60
agctgctctg attcagagag ttgtttcaat ttctcaaagt gnccatcttc tcctctggaa 120agctgctctg attcagagag ttgtttcaat ttctcaaagt gnccatcttc tcctctggaa 120
taggacatgt gccgactttg ttccaacata tcagcagatc cttcattctc ttccaaccta 180taggacatgt gccgactttg ttccaacata tcagcagatc cttcattctc ttccaaccta 180
atacaatgtt ttgaagatgc agt 203atacaatgtt ttgaagatgc agt 203
<210> 10<210> 10
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=A或T<223> n=A or T
<400> 10<400> 10
cagagagttg tttcaatttc tcaaagtggc catcttctcc tctggaatag gacatgtgcc 60cagagagttg tttcaatttc tcaaagtggc catcttctcc tctggaatag gacatgtgcc 60
gactttgttc caacatatca gcagatcctt cattctcttc cnacctaata caatgttttg 120gactttgttc caacatatca gcagatcctt cattctcttc cnacctaata caatgttttg 120
aagatgcagt ttgggccaaa tcattgataa ggtcgtgcat tacgaatccc tccgatttcc 180aagatgcagt ttgggccaaa tcattgataa ggtcgtgcat tacgaatccc tccgatttcc 180
attctgattc tgggaccctt tca 203attctgattc tgggaccctt tca 203
<210> 11<210> 11
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=C或T<223> n=C or T
<220><220>
<221> misc_feature<221> misc_feature
<222> (119)..(203)<222> (119)..(203)
<223> n(85)=A、G、C或T<223> n(85)=A, G, C or T
<400> 11<400> 11
ttgggatata tcgcacaaaa agcaaaacat cgcttcaaat gtggaggaag atcattgtag 60ttgggatata tcgcacaaaa agcaaaacat cgcttcaaat gtggaggaag atcattgtag 60
ctcaacatta acgctggtaa tataccattc caacaacttt gntgatccca tatttcacnn 120ctcaacatta acgctggtaa tataccattc caacaacttt gntgatccca tatttcacnn 120
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180
nnnnnnnnnn nnnnnnnnnn nnn 203nnnnnnnnnn nnnnnnnnnn nnn 203
<210> 12<210> 12
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=T或C<223> n=T or C
<220><220>
<221> misc_feature<221> misc_feature
<222> (152)..(203)<222> (152)..(203)
<223> n(52)=A、G、C或T<223> n(52)=A, G, C or T
<400> 12<400> 12
taaataactt ggtctttgaa gaattcatga tctttgggat atatcgcaca aaaagcaaaa 60taaataactt ggtctttgaa gaattcatga tctttgggat atatcgcaca aaaagcaaaa 60
catcgcttca aatgtggagg aagatcattg tagctcaaca tnaacgctgg taatatacca 120catcgcttca aatgtggagg aagatcattg tagctcaaca tnaacgctgg taatatacca 120
ttccaacaac tttgctgatc ccatatttca cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180ttccaacaac tttgctgatc ccatatttca cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180
nnnnnnnnnn nnnnnnnnnn nnn 203nnnnnnnnnn nnnnnnnnnn nnn 203
<210> 13<210> 13
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=C或G<223> n=C or G
<400> 13<400> 13
cacatgcatt caccattgat gtataagatg ggaatttgag caatattttg ccagtatttc 60cacatgcatt caccattgat gtataagatg ggaatttgag caatattttg ccagtatttc 60
cctttgtcaa attccagtag tggttcgagc aatgggcatc tnctaatatc tagtttagag 120cctttgtcaa attccagtag tggttcgagc aatgggcatc tnctaatatc tagtttagag 120
agggaagagg gcatctcttt tactggaagg gattggagat tagggcagtc ggagatgttc 180agggaagagg gcatctcttt tactggaagg gattggagat tagggcagtc ggagatgttc 180
agctgagaga gggaggaggg cag 203agctgagaga gggaggaggg cag 203
<210> 14<210> 14
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=T或C<223> n=T or C
<400> 14<400> 14
tgctgattca gagagtgatt ggagattagg gcaatcgaag atgtgtagat gtataagtga 60tgctgattca gagagtgatt ggagattagg gcaatcgaag atgtgtagat gtataagtga 60
agtgaggtgg gaaagatgta gtgaatggag ctcatcatgg tnatgtaaat gtagctctga 120agtgaggtgg gaaagatgta gtgaatggag ctcatcatgg tnatgtaaat gtagctctga 120
aagagaggag ggaagcccac cttgttccac aagtgacggt atattaccct caacatatag 180aagagaggag ggaagcccac cttgttccac aagtgacggt atattaccct caacatatag 180
atattgaaga gaggtgaggc ttt 203atattgaaga gaggtgaggc ttt 203
<210> 15<210> 15
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=T或C<223> n=T or C
<400> 15<400> 15
aacaccactc ctttcggctg ttcactactt tcttgcaatt actgatcata aggagttgta 60aacaccactc ctttcggctg ttcactactt tcttgcaatt actgatcata aggagttgta 60
aattaaagag atgtatttct ggcagacact tgagcttctc angattccga atatacaaat 120aattaaagag atgtatttct ggcagacact tgagcttctc angattccga atatacaaat 120
acgtcaccgc agagtcaact gccaccgaaa gtatttcaac attctcacaa tcctgaatag 180acgtcaccgc agagtcaact gccaccgaaa gtatttcaac attctcacaa tcctgaatag 180
tgagactttc agtggcagta gga 203tgagactttc agtggcagta gga 203
<210> 16<210> 16
<211> 203<211> 203
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<220><220>
<221> misc_feature<221> misc_feature
<222> (102)..(102)<222> (102)..(102)
<223> n=A或G<223> n=A or G
<400> 16<400> 16
caatttcaca agcttaagaa acaaaggatc agcaagccaa tttgaaaatt ttgcccctct 60caatttcaca agcttaagaa acaaaggatc agcaagccaa tttgaaaatt ttgcccctct 60
atatcctgtg atttctaatc gttttatatt tgtgtatggg tntagctcat caagtatctc 120atatcctgtg atttctaatc gttttatatt tgtgtatggg tntagctcat caagtatctc 120
tctttcagtt tgagaatcgt aagcagaact accgtcactc cactgcaaat acaactcctc 180tctttcagtt tgagaatcgt aagcagaact accgtcactc cactgcaaat acaactcctc 180
aacatgctcc ttttccttca tat 203aacatgctcc ttttccttca tat 203
<210> 17<210> 17
<211> 40<211> 40
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 17<400> 17
gaaggtgacc aagttcatgc tttagcacat tcaatgaccg 40gaaggtgacc aagttcatgc tttagcacat tcaatgaccg 40
<210> 18<210> 18
<211> 40<211> 40
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 18<400> 18
gaaggtcgga gtcaacggat tttagcacat tcaatgacca 40gaaggtcgga gtcaacggat tttagcacat tcaatgacca 40
<210> 19<210> 19
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 19<400> 19
ttaaagaata caaggagacg ca 22ttaaagaata caaggagacg ca 22
<210> 20<210> 20
<211> 44<211> 44
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 20<400> 20
gaaggtgacc aagttcatgc tgagtacaaa aaggatatga caca 44gaaggtgacc aagttcatgc tgagtacaaa aaggatatga caca 44
<210> 21<210> 21
<211> 44<211> 44
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 21<400> 21
gaaggtcgga gtcaacggat tgagtacaaa aaggatatga cacg 44gaaggtcgga gtcaacggat tgagtacaaa aaggatatga cacg 44
<210> 22<210> 22
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 22<400> 22
tccacagcac catatagcaa 20tccacagcac catatagcaa 20
<210> 23<210> 23
<211> 41<211> 41
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 23<400> 23
gaaggtgacc aagttcatgc ttcagggaag ctctttacac g 41gaaggtgacc aagttcatgc ttcagggaag ctctttacac g 41
<210> 24<210> 24
<211> 42<211> 42
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 24<400> 24
gaaggtcgga gtcaacggat tctcagggaa gctctttaca ca 42gaaggtcgga gtcaacggat tctcagggaa gctctttaca ca 42
<210> 25<210> 25
<211> 24<211> 24
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 25<400> 25
tcatagaagt gaaaccctga aatt 24tcatagaagt gaaaccctga aatt 24
<210> 26<210> 26
<211> 41<211> 41
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 26<400> 26
gaaggtgacc aagttcatgc tccacacaga aatcctaatg c 41gaaggtgacc aagttcatgc tccacacaga aatcctaatg c 41
<210> 27<210> 27
<211> 41<211> 41
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 27<400> 27
gaaggtcgga gtcaacggat tccacacaga aatcctaatg t 41gaaggtcgga gtcaacggat tccacacaga aatcctaatg t 41
<210> 28<210> 28
<211> 24<211> 24
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 28<400> 28
acctttatct tatttgtttt cagt 24acctttatct tatttgtttt cagt 24
<210> 29<210> 29
<211> 48<211> 48
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 29<400> 29
gaaggtgacc aagttcatgc tcccccttct tactatattt cttactat 48gaaggtgacc aagttcatgc tcccccttct tactatattt cttactat 48
<210> 30<210> 30
<211> 48<211> 48
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 30<400> 30
gaaggtcgga gtcaacggat tcccccttct tactatattt cttactac 48gaaggtcgga gtcaacggat tcccccttct tactatattt cttactac 48
<210> 31<210> 31
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 31<400> 31
agaactttca ccttgtaaac g 21agaactttca ccttgtaaac g 21
<210> 32<210> 32
<211> 43<211> 43
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 32<400> 32
gaaggtgacc aagttcatgc tgaatggtat attaccagcg ttg 43gaaggtgacc aagttcatgc tgaatggtat attaccagcg ttg 43
<210> 33<210> 33
<211> 44<211> 44
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 33<400> 33
gaaggtcgga gtcaacggat tggaatggta tattaccagc gtta 44gaaggtcgga gtcaacggat tggaatggta tattaccagc gtta 44
<210> 34<210> 34
<211> 24<211> 24
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 34<400> 34
attcatgatc tttgggatat atcg 24attcatgatc tttgggat atcg 24
<210> 35<210> 35
<211> 40<211> 40
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 35<400> 35
gaaggtgacc aagttcatgc tttcgagcaa tgggcatctg 40gaaggtgacc aagttcatgc tttcgagcaa tgggcatctg 40
<210> 36<210> 36
<211> 40<211> 40
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 36<400> 36
gaaggtcgga gtcaacggat tttcgagcaa tgggcatctc 40gaaggtcgga gtcaacggat tttcgagcaa tgggcatctc 40
<210> 37<210> 37
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 37<400> 37
ctgccctaat ctccaatccc 20ctgccctaat ctccaatccc 20
<210> 38<210> 38
<211> 43<211> 43
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 38<400> 38
gaaggtgacc aagttcatgc tgtatttctt gcagcttctc aca 43gaaggtgacc aagttcatgc tgtatttctt gcagcttctc aca 43
<210> 39<210> 39
<211> 43<211> 43
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 39<400> 39
gaaggtcgga gtcaacggat tgcagacact tgsgcttctc atg 43gaaggtcgga gtcaacggat tgcagacact tgsgcttctc atg 43
<210> 40<210> 40
<211> 24<211> 24
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 40<400> 40
agaatgttga aatactttcg gtgg 24agaatgttga aatactttcg gtgg 24
<210> 41<210> 41
<211> 46<211> 46
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 41<400> 41
gaaggtgacc aagttcatgc tatygtttta tatttgtgtr tgggtg 46gaaggtgacc aagttcatgc tatygtttta tatttgtgtr tgggtg 46
<210> 42<210> 42
<211> 46<211> 46
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 42<400> 42
gaaggtcgga gtcaacggat tatcgtttta tatttgtgta tgggta 46gaaggtcgga gtcaacggat tatcgtttta tatttgtgta tgggta 46
<210> 43<210> 43
<211> 23<211> 23
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 43<400> 43
ggtagttctg cttacgattc tca 23ggtagttctg cttacgattc tca 23
<210> 44<210> 44
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 44<400> 44
gaaggtgacc aagttcatgc t 21gaaggtgacc aagttcatgc t 21
<210> 45<210> 45
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 45<400> 45
gaaggtcgga gtcaacggat t 21gaaggtcgga gtcaacggat t 21
<210> 46<210> 46
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 46<400> 46
agcatgaact tggtcacctt c 21agcatgaact tggtcacctt c 21
<210> 47<210> 47
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(Artificial)<213> Artificial Sequence (Artificial)
<400> 47<400> 47
aatccgttga ctccgacctt c 21aatccgttga ctccgacctt c 21
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