An InDel marker primer for identifying tomato spotted wilt susceptibility and its application

Abstract

The present invention discloses an InDel marker primer related to the identification of susceptibility to tomato spotted wilt disease and its application, belonging to the technical field of molecular biology. The InDel marker primer of the present invention includes InDc10_221-F with a nucleotide sequence such as SEQ ID NO.1 and InDc10_221-R with a nucleotide sequence such as SEQ ID NO.2, which can achieve highly specific and accurate detection of tomato varieties carrying the tomato spotted wilt disease susceptibility gene Sldnaj<supgt;G< / supgt>. The operation steps are simple and the interpretation method is intuitive, providing a simple, fast, economical and effective molecular tool for screening tomato germplasm resources resistant to tomato spotted wilt disease and screening out tomato varieties susceptible to tomato spotted wilt disease. It can be applied to commercial disease-resistant breeding of tomatoes and applied research on high-quality genetic improvement, providing a new option for the application of tomato spotted wilt disease-resistant breeding.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to an InDel marker primer for identifying tomato spotted wilt susceptibility-related disease and an application thereof. Background Art

[0002] As one of the most widely cultivated and consumed and important vegetable crops in the world, tomato yield and quality are important indicators affecting tomato production and processing. In recent years, large-scale outbreaks of tomato spotted wilt have seriously affected the yield and quality of tomatoes. The highly resistant tomato spotted wilt disease-resistant gene Sw-5 has been cloned and has broad-spectrum resistance, and has been widely used in disease-resistant breeding. However, with the evolutionary mutation and long-term application of Tomato spotted wilt virus (TSWV), Sw-5 resistance has gradually weakened or lost in some tomato varieties. At present, the development of susceptible genes in the process of crop breeding can obtain long-lasting resistance to pathogens by in-depth invention of the loss of susceptible gene function, which can be used as a new idea to replace disease-resistant breeding to create new disease-resistant varieties. The present invention uses susceptible and disease-resistant homozygous varieties to mine and obtain a tomato spotted wilt susceptible gene Sldnaj. G The molecular markers developed based on the differences in the varieties can be used to efficiently and specifically identify the disease-susceptibility gene Sldnaj G The selection and breeding of hybrid progeny provide technical support for creating new varieties of tomato spotted wilt with persistent resistance using susceptible genes.

[0003] Previous studies have conducted in-depth investigations on the resistance traits of tomato spotted wilt resistant variety R6 and susceptible variety M82, and found that the susceptible and resistant traits of tomato spotted wilt are controlled by a pair of alleles (Sldnaj and SldnaJ genes), and completed the genetic positioning and gene cloning analysis of the tomato spotted wilt susceptible gene Sldnaj. Using the differences in resistance genes between homozygous susceptible materials and resistant materials, insertion-deletion (InDel) molecular markers were developed, which can efficiently and specifically detect whether unknown tomato materials carry the tomato spotted wilt susceptible gene Sldnaj. G , providing effective support for commercial disease-resistant breeding of tomatoes, cultivation of new tomato varieties with sustained stability and high resistance to tomato spotted wilt, and research on genetic improvement applications.

[0004] At present, there is no related literature report for identifying InDel molecular markers associated with tomato spotted wilt susceptibility gene Sldnaj. To this end, the present invention develops InDel molecular markers for identifying and detecting tomato spotted wilt susceptibility gene Sldnaj based on insertion and deletion sites. The present invention can effectively identify tomato plants carrying tomato susceptibility gene Sldnaj. GThe identification of homozygous varieties, susceptible varieties and hybrid first generation provides a new option for improving the application of tomato spotted wilt resistance breeding. Summary of the invention

[0005] The purpose of the present invention is to provide an InDel marker primer for identifying tomato spotted wilt susceptibility and its application, so as to solve the problems existing in the above-mentioned prior art. The InDel marker primer can realize the accurate, rapid and efficient detection of the tomato spotted wilt susceptibility gene Sldnaj, has simple operation steps and efficient detection, and provides a new choice for application in tomato spotted wilt resistance breeding.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an InDel marker primer for identifying tomato spotted wilt susceptibility, wherein the InDel marker primer comprises InDc10_221-F and InDc10_221-R;

[0008] The nucleotide sequence of the InDc10_221-F is shown in SEQ ID NO.1, and the nucleotide sequence of the InDc10_221-R is shown in SEQ ID NO.2.

[0009] The present invention also provides the use of the InDel marker primer in preparing a product for detecting susceptibility to tomato spotted wilt.

[0010] Preferably, the product comprises a detection reagent or a detection kit.

[0011] The invention also provides a detection kit for identifying susceptibility of tomato spotted wilt, comprising the InDel labeled primer.

[0012] Preferably, it also includes DNA extraction reagents, PCR reaction reagents and DNA electrophoresis reagents.

[0013] The present invention also provides the use of the InDel marker primer or the detection kit in screening or identifying tomato spotted wilt susceptible varieties.

[0014] The present invention also provides a method for screening or identifying tomato spotted wilt susceptible varieties, comprising the following steps:

[0015] Extracting genomic DNA of the tomato to be tested;

[0016] Performing PCR amplification on the genomic DNA using the InDel labeled primers or the detection kit;

[0017] The PCR amplification products were subjected to electrophoresis, staining, development, and band interpretation.

[0018] Preferably, the reading band is:

[0019] If a single band of 223 bp is amplified, the tomato to be tested is a variety susceptible to tomato spotted wilt;

[0020] If two bands of 223 bp and 284 bp are amplified, the tomato to be tested is carrying the tomato spotted wilt susceptibility gene Sldnaj G tomato varieties;

[0021] If a single band of 284 bp is amplified, the tomato to be tested does not carry the tomato spotted wilt susceptibility gene Sldnaj. G tomato varieties.

[0022] Preferably, the reaction procedure of the PCR amplification is:

[0023] Pre-denaturation at 94°C for 1.5 min; denaturation at 94°C for 20 s, annealing at 52°C for 20 s, extension at 72°C for 30 s, 32 cycles; extension at 72°C for 5 min; insulation at 4°C.

[0024] The present invention also provides the use of the InDel marker primer or the detection kit in tomato molecular marker-assisted breeding.

[0025] The present invention discloses the following technical effects:

[0026] The InDel marker primers designed by the present invention can be used to detect the tomato spotted wilt susceptible gene Sldnaj G It can accurately detect tomato varieties with high specificity, simple operation steps and intuitive interpretation method, providing a simple, fast, economical and effective molecular tool for screening tomato germplasm resources resistant to tomato spotted wilt and screening out tomato spotted wilt susceptible varieties. It can be applied to commercial disease-resistant breeding of tomatoes and high-quality genetic improvement application research, providing a new choice for the application of tomato spotted wilt resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The figure is a graph showing the agarose gel electrophoresis detection result of the InDc10_221 molecular marker in the tomato materials M82, R6, F1 population and F2 population to be detected; wherein, M is D2000 DNA Marker, and - is sterile double distilled water;

[0029] Figure 2 The figure is the agarose gel electrophoresis detection result of InDc10_221 molecular marker in the tomato material to be detected; wherein, M is D2000 DNA Marker, and - is sterile double distilled water. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The tomato disease susceptibility gene Sldnaj used in the present invention GThe homozygous tomato spotted wilt susceptible tomato variety M82, and the homozygous tomato spotted wilt resistant tomato varieties H8, H145, H149 and H19 have been published in the document "Shi Yan, Mo Ning, Qi Shiming, Liang Yan. 2020. Screening of tomato spotted wilt virus-resistant germplasm and optimization of artificial inoculation identification methods. Chinese Vegetables, (06): 39-43.", and the homozygous tomato spotted wilt resistant tomato variety R6 has been published in the document "Qi Shiming. Fine positioning and functional analysis of tomato TSWV resistance gene Sl5R-1[D]. Northwest A&F University, 2022.".

[0036] The homozygous tomato spotted wilt susceptible tomato varieties Moneymaker (MM) and Ailsa Craig (AC) carrying the tomato disease susceptibility gene Sldnaj have been published in the literature "Wang Nan. Functional research and application of tomato wilt resistance gene SlySGT1 [D]. Yangzhou University, 2023" and "Yang Jianyu. Research on the signal transduction and transcriptional regulation functions of exogenous spermidine in enhancing tomato salt-alkali stress tolerance [D]. Northwest A&F University, 2022." respectively.

[0037] Conventional hybrid tomato varieties C134, C248, C267, Black Coral (CHsh), Summer Light (CXrzg), Poppy (CYmr), and Little Red Jade (CXhy) were provided by Zhangye Fuxing Modern Agriculture Co., Ltd.

[0038] The present invention found that there is a 61bp InDel insertion and deletion site at bases 61,443,987 to 61,444,048 of tomato chromosome SL4.0ch10, and the polymorphism is 62 / 1bp. Based on this insertion and deletion site, an InDel marker primer for identifying and detecting the susceptible gene of tomato spotted wilt was developed, which is suitable for screening of tomato spotted wilt resistant and susceptible germplasm resources, germplasm identification and molecular marker-assisted breeding research. The details are as follows:

[0039] Example 1

[0040] 1. Primer Design

[0041] This embodiment provides InDel marker primers for identifying tomato spotted wilt susceptibility genes, including an upstream primer InDc10_221-F and a downstream primer InDc10_221-R, wherein the InDc10_221-F is located at bases 61,443,872-61,443,894 of the tomato SL4.0ch10 chromosome; the InDc10_221-R is located at bases 61,444,134-61,444,156 of the tomato SL4.0ch10 chromosome.

[0042] The sequences of the InDel marker primers are as follows:

[0043] InDc10_221-F: 5′-ATGCTATGTGTTTCACAAAGTGA-3′ (SEQ ID NO. 1);

[0044] InDc10_221-R: 5′-GAGGTGTGGTGGAGTGATAAGTA-3′ (SEQ ID NO. 2).

[0045] 2. Selection and Construction of Tomato Samples

[0046] 2.1 Building a group

[0047] Selecting tomato disease susceptibility gene Sldnaj G The homozygous tomato spotted wilt susceptible tomato variety M82 and the homozygous tomato spotted wilt resistant tomato variety R6 were used to construct F1 population and F2 population (including F2-resistant homozygous population, F2-susceptible homozygous population, and F2-resistant heterozygous population). The population construction method was based on the literature “Qi, S, Shen, Y, Wang, X, Zhang, S, Li, Y, Islam, MM, Wang, J, Zhao, P, Zhan, X, Zhang, F, Liang, Y. 2022. A new NLR gene for resistance to Tomato spotted wilt virus in tomato (Solanum lycopersicum). Theoretical and Applied Genetics, 135(5): 1493-1509.”.

[0048] 2.2 Sample selection

[0049] In addition to the above-mentioned M82, R6, F1 populations and F2 populations, the homozygous tomato spotted wilt susceptible tomato varieties carrying the tomato disease susceptibility gene Sldnaj: Moneymaker (MM) and Ailsa Craig (AC), homozygous tomato spotted wilt resistant tomato varieties: H8, H145, H149 and H19, and conventional hybrid tomato varieties: C134, C248, C267, Black Coral (CHsh), Summer Light (CXrzg), Poppy (CYmr), and Little Red Ruby (CXhy) were also selected for InDel marker identification.

[0050] 3. Identification methods

[0051] The method for identifying InDel molecular markers related to tomato spotted wilt susceptibility genes is as follows:

[0052] S1, extract genomic DNA of tomato samples to be tested

[0053] The tomato samples to be tested include homozygous tomato spotted wilt susceptible tomato varieties: M82, MM, AC; homozygous tomato spotted wilt resistant tomato varieties: R6, H8, H145, H149 and H19; F1 populations and F2 populations constructed with M82 and R6 (including F2-disease-resistant homozygous population, F2-susceptible homozygous population, and F2-disease-resistant heterozygous population); conventional hybrid tomato varieties: C134, C248, C267, CHsh, CXrzg, CYmr, CXhy; and sterile double distilled water (ddH2O) was used as a negative control.

[0054] S2, PCR amplification reaction

[0055] The genomic DNA of the tomato sample to be tested extracted from S1 was subjected to PCR amplification reaction using the InDel-labeled upstream primer InDc10_221-F and the downstream primer InDc10_221-R.

[0056] PCR amplification reaction system: 20 μL system, the content of each component substance is: 10 μL 2×Fast PfuMaster Mix (2×Fast Pfu Master Mix kit, Jinan Protein Technology Co., Ltd.), 2 μL DNA template (50 ng / μL), 0.6 μL upstream primer InDc10_221-F (10 μM), 0.6 μL downstream primer InDc10_221-R (10 μM), 6.8 μL sterile ddH2O, centrifuge and mix.

[0057] PCR amplification program: pre-denaturation at 94°C for 1.5 min; denaturation at 94°C for 20 s, annealing at 52°C for 20 s, extension at 72°C for 30 s, 32 cycles; extension at 72°C for 5 min; insulation at 4°C.

[0058] After PCR amplification, the PCR product was electrophoresed on 3% agarose gel to detect the size of the band.

[0059] S3, test result judgment

[0060] The PCR product band detection result is a single band of 284 bp, which means it is a homozygous disease-resistant tomato variety carrying the tomato spotted wilt resistance gene SlDnaJ;

[0061] The PCR product band detection result is a single band of 223bp, which is the gene carrying the tomato spotted wilt susceptibility gene Sldnaj. G The homozygous tomato variety M82;

[0062] The PCR product band detection results showed two bands of 223bp and 284bp, which was the gene carrying the tomato spotted wilt susceptibility gene Sldnaj. G of heterozygous plants.

[0063] 4. Experimental Results

[0064] like Figure 1 As shown, a band of 223 bp was detected in both the homozygous tomato spotted wilt susceptible tomato material M82 and the F2-susceptible homozygous population; a band of 284 bp was detected in both the homozygous tomato spotted wilt resistant tomato material R6 and the F2-resistant homozygous population; two bands of 223 bp and 284 bp were detected in both the heterozygous tomato spotted wilt resistant tomato material F1 population and the F2-resistant heterozygous population.

[0065] like Figure 2 As shown, a 223 bp band was detected in the homozygous tomato spotted wilt susceptible tomato material M82; a 284 bp band was detected in the homozygous tomato spotted wilt resistant tomato material R6; and a 284 bp band was detected in the homozygous tomato spotted wilt susceptible tomato materials (Moneymaker and AilsaCraig), homozygous tomato spotted wilt resistant tomato materials (H8, H145, H149 and H19) and conventional tomato varieties (C134, C248, C267, CHsh, CXrzg, CYmr and CXhy).

[0066] The results showed that a 223bp band was detected in the homozygous tomato spotted wilt susceptible tomato variety M82, which was 61bp less than that in the homozygous tomato spotted wilt resistant tomato variety R6, indicating that it carried the tomato spotted wilt susceptible gene Sldnaj. G The detection results of the F1 population and the F2-disease-resistant heterozygous population showed two bands, which were the carriers of the tomato spotted wilt susceptible gene Sldnaj G of heterozygous plants.

[0067] No 223 bp band was detected in the homozygous tomato spotted wilt susceptible tomato materials Moneymaker and Ailsa Craig carrying the Sldnaj gene, indicating that the susceptible material M82 showed high specificity; after analysis, the InDel mutation discovered by the present invention was located in the promoter region of the gene, and the tomato spotted wilt susceptible gene Sldnaj G Same coding sequence as Sldnaj.

[0068] The experimental results confirmed that the InDel marker primers provided by the present invention can specifically and accurately identify the tomato spotted wilt susceptibility gene Sldnaj G The tomato varieties provided a simple, fast, economical and effective molecular tool for screening germplasm resources for tomato spotted wilt resistance.

[0069] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of an InDel marker primer for detecting susceptibility to tomato spotted wilt in the preparation of a product for detecting susceptibility to tomato spotted wilt, characterized in that: The InDel marker is based on a 61 bp InDel insertion / deletion site at bases 61,443,987 to 61,444,048 of tomato SL4.0 ch10 chromosome, with a polymorphism of 62 / 1 bp; The InDel marker primers include InDc10_221-F and InDc10_221-R; The nucleotide sequence of InDc10_221-F is shown in SEQ ID NO.1, and the nucleotide sequence of InDc10_221-R is shown in SEQ ID NO.2; The detection of tomato spotted wilt susceptibility comprises: Extracting genomic DNA of the tomato to be tested; Performing PCR amplification on the genomic DNA using the InDel labeled primers; The PCR amplification products were subjected to electrophoresis, staining, development, and band interpretation; The interpretation bands are: If a single band of 223 bp is amplified, the tomato to be tested is a variety susceptible to tomato spotted wilt; If two bands of 223 bp and 284 bp are amplified, the tomato to be tested is carrying the tomato spotted wilt susceptibility gene. Sldnaj G tomato varieties; If a single band of 284 bp is amplified, the tomato to be tested does not carry the tomato spotted wilt susceptibility gene. Sldnaj G tomato varieties.

2. The use according to claim 1, characterized in that The products include detection reagents or detection kits.

3. The use according to claim 2, characterized in that The detection kit also includes a DNA extraction reagent, a PCR reaction reagent and a DNA electrophoresis reagent.

4. Use of the InDel labeled primer as claimed in claim 1 or the detection kit as claimed in claim 2 or 3 in screening or identifying tomato spotted wilt susceptible varieties, characterized in that: The applications include: Extracting genomic DNA of the tomato to be tested; Performing PCR amplification on the genomic DNA using the InDel labeled primers or the detection kit; The PCR amplification products were subjected to electrophoresis, staining, development, and band interpretation; The interpretation bands are: If a single band of 223 bp is amplified, the tomato to be tested is a variety susceptible to tomato spotted wilt; If two bands of 223 bp and 284 bp are amplified, the tomato to be tested is carrying the tomato spotted wilt susceptibility gene. Sldnaj G tomato varieties; If a single band of 284 bp is amplified, the tomato to be tested does not carry the tomato spotted wilt susceptibility gene. Sldnaj G tomato varieties.

5. A method for screening or identifying tomato varieties susceptible to spotted wilt, characterized in that: The following steps are involved: Extracting genomic DNA of the tomato to be tested; Performing PCR amplification on the genomic DNA using the InDel labeled primers described in claim 1 or the detection kit described in claim 2 or 3; The PCR amplification products were subjected to electrophoresis, staining, development, and band interpretation; The interpretation bands are: If a single band of 223 bp is amplified, the tomato to be tested is a variety susceptible to tomato spotted wilt; If two bands of 223 bp and 284 bp are amplified, the tomato to be tested is carrying the tomato spotted wilt susceptibility gene. Sldnaj G tomato varieties; If a single band of 284 bp is amplified, the tomato to be tested does not carry the tomato spotted wilt susceptibility gene. Sldnaj G tomato varieties.

6. The method according to claim 5, characterized in that The reaction procedure of the PCR amplification is: Pre-denaturation at 94°C for 1.5 min; denaturation at 94°C for 20 s, annealing at 52°C for 20 s, extension at 72°C for 30 s, 32 cycles; extension at 72°C for 5 min; insulation at 4°C.

7. Use of the InDel marker primer as claimed in claim 1 or the detection kit as claimed in claim 2 or 3 in tomato molecular marker-assisted breeding, characterized in that: The tomato molecular marker-assisted breeding is to identify the tomato spotted wilt susceptibility gene Sldnaj G tomato varieties, and screen germplasm resources for breeding of tomato spotted wilt resistance; Identification of tomato spotted wilt susceptibility gene Sldnaj G Tomato varieties include: Extracting genomic DNA of the tomato to be tested; Performing PCR amplification on the genomic DNA using the InDel labeled primers or the detection kit; The PCR amplification products were subjected to electrophoresis, staining, development, and band interpretation; The interpretation bands are: If a single band of 223 bp is amplified, the tomato to be tested is a variety susceptible to tomato spotted wilt; If two bands of 223 bp and 284 bp are amplified, the tomato to be tested is carrying the tomato spotted wilt susceptibility gene. Sldnaj G tomato varieties; If a single band of 284 bp is amplified, the tomato to be tested does not carry the tomato spotted wilt susceptibility gene. Sldnaj G tomato varieties.

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