SNP molecular markers for identifying resistance to bacterial stem rot in cucumber and its application

Abstract

The invention discloses a SNP molecular marker for identifying resistance of cucumber to bacterial stem rot and its application, belonging to the field of biotechnology-assisted breeding technology. The site of the SNP marker is at the 10,867,465 bp of the physical position of cucumber chromosome 6, the base of the site is A or G, the cucumber material with the base of the site A is a cucumber material resistant to bacterial stem rot, and the cucumber material with the base of the site G is a cucumber material susceptible to bacterial stem rot. The invention not only lays a foundation for the precise positioning and molecular cloning of the cucumber bacterial stem rot resistance gene BSSR, but also provides an efficient way for the use of molecular markers to assist in the breeding of new cucumber varieties resistant to cucumber bacterial stem rot.

Description

Technical Field

[0001] The invention relates to the technical field of biotechnology-assisted breeding, in particular to a SNP molecular marker for identifying resistance of cucumber to bacterial soft stem rot and an application thereof. Background Art

[0002] Cucumber is an important vegetable crop in my country, especially in facility production. It is also a model plant for sex determination, differentiation and vascular biology research (Li et al., 2019). However, in the process of cucumber cultivation and production, it is susceptible to infection by a variety of diseases. In recent years, the previously non-epidemic bacterial disease stem soft rot has gradually become an emerging epidemic disease of cucumber, which will lead to a 20%-30% reduction in yield, quality decline, and serious impact on plant growth. Bacterial stem soft rot (BSSR) of cucumber caused by Pectobacterium carotovorum subsp. brasiliense (Pcb) was first reported in 2017 (Feng Zhiqin, 2017). At present, the prevention and control methods for stem soft rot in cucumber production are mainly prevention and treatment. In production, although it can be prevented and controlled by physical and biochemical methods such as seed disinfection, grafting, and crop rotation, they cannot fundamentally solve the harm to cucumber production. Breeding and promoting cucumber varieties resistant to bacterial stem rot is the most economical and effective way to prevent and control the disease, and is also an important guarantee for green and efficient cucumber production. Breeding and promoting cucumber varieties resistant to bacterial stem rot is the most economical and effective way to prevent and control the disease, and is also an important guarantee for green and efficient cucumber production. Researching the genetic laws and molecular markers of resistance to bacterial stem rot in cucumbers is of great significance for breeding new varieties that meet market demand.

[0003] Genetic studies have shown that plant resistance to Pectobacterium carrotii subspecies carrotii (Pcc) is a quantitative trait controlled by multiple genes (Yamagishi H, 1990). QTL analysis of potatoes found that the genetic control of potato resistance to Pca is very complex, with at least 13 loci controlling potato tuber resistance to Pca (Zimnoch-Guzowska E, 2000). Using the model plant Arabidopsis as the experimental material, the disease-resistant ecotype CS906 and the disease-susceptible ecotype CS20 were screened out, and the F2 population of the hybrid offspring of the two was used for QTL analysis, and a major QTL locus for resistance to soft rot, qAtRECC1, was discovered. The locus is 66 kb in size on the Arabidopsis genome and contains a total of 19 genes (Ye Xueling, 2008).

[0004] The cleaved amplified polymorphic sequence (CAPS) marker is a DNA marker for detecting SNP sites that is generated by combining specific primer PCR with restriction enzyme digestion. The principle is that the SNP mutation site is located on the recognition sequence of a certain endonuclease. After PCR amplification with specific primers, the PCR product is digested with the corresponding endonuclease to produce polymorphisms in the digested fragments, which can be displayed by agarose or polyacrylamide gel electrophoresis and staining. However, it is relatively rare for SNP to be located at a certain restriction enzyme digestion site. Michaels and Amasino (1998) and Neff (1998) developed a derived cleaved amplified polymorphic sequence (dCAPS) marker with artificially introduced mismatched bases based on the CAPS marker, which can convert almost all SNP sites into markers that can be detected by electrophoresis. Both CAPS and dCAPS have the characteristics of co-dominance, site specificity, simple operation, rapid detection, low cost and independence from precision instruments and equipment. They can be used for plant genotyping, positioning, genetic diversity analysis, variety identification and other aspects.

[0005] So far, no SNP markers closely linked to the cucumber bacterial stem rot resistance gene BSSR have been reported. Summary of the invention

[0006] One of the purposes of the present invention is to address the above-mentioned problem and provide a SNP molecular marker for identifying cucumber resistance to bacterial stem rot, the site of the SNP marker is the physical position 10,867,465bp of cucumber chromosome 6, the base of the site is A or G, the cucumber material whose base at the site is A is a cucumber material resistant to bacterial stem rot, and the cucumber material whose base at the site is G is a cucumber material susceptible to bacterial stem rot.

[0007] The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.3, wherein the 27th base is A or G.

[0008] Another object of the present invention is to provide a specific primer pair for detecting the above-mentioned SNP molecular marker, including primers BSSR-SNP-01-F and BSSR-SNP-01-R, and the nucleic acid sequences of the primers are as follows:

[0009] BSSR-SNP-01-F:5'-ACCTTTATAAAAATATTGTGTACTTA-3',

[0010] BSSR-SNP-01-R: 5'-GAGTTTCATCATCTACCATC-3'.

[0011] Another object of the present invention is to provide a kit for detecting the above-mentioned SNP molecular markers, comprising the above-mentioned specific primer pair.

[0012] The kit also comprises a cucumber genomic DNA extraction reagent, a PCR amplification reaction reagent, a PCR amplification product sequencing reagent or a SNP differential site recognition reagent; preferably, the SNP differential site recognition reagent comprises a restriction endonuclease capable of recognizing SNP differential sites, and preferably also comprises a polyacrylamide gel electrophoresis reagent; preferably, the restriction endonuclease is DdeI.

[0013] The final object of the present invention is to provide the use of the above-mentioned SNP molecular marker in any of the following (1)-(5):

[0014] (1) Identify or assist in identifying materials resistant / susceptible to bacterial stem rot of cucumber;

[0015] (2) Identify or assist in identifying the BSSR gene that is resistant / susceptible to bacterial stem rot in cucumber;

[0016] (3) Screening or assisting in the screening of cucumber varieties resistant to bacterial stem rot;

[0017] (4) Cultivating or assisting in the cultivation of cucumber varieties resistant to bacterial stem rot;

[0018] (5) Cucumber breeding.

[0019] In the technical scheme of the above application, the application includes the following steps: extracting genomic DNA of the sample to be tested as a template, using the amplification primers of the above SNP molecular markers to perform PCR amplification, sequencing or enzyme digestion electrophoresis detection of the PCR amplification products, and the enzyme digestion electrophoresis detection refers to using a restriction endonuclease that can recognize the difference site of the SNP molecular marker to digest the PCR amplification products, and then electrophoresing the amplification products, preferably the electrophoresis uses non-denaturing polyacrylamide gel electrophoresis.

[0020] In the technical solution of the above application, the amplification primers of the SNP molecular marker are as follows:

[0021] BSSR-SNP-01-F:5'-ACCTTTATAAAAATATTGTGTACTTA-3',

[0022] BSSR-SNP-01-R: 5'-GAGTTTCATCATCTACCATC-3';

[0023] A 235 bp fragment was obtained by PCR amplification. When sequencing was used for detection, the cucumber material whose 27th base of the PCR amplification fragment was A was a cucumber material resistant to bacterial stem soft rot; the cucumber material whose 27th base of the PCR amplification fragment was G was a cucumber material susceptible to bacterial stem soft rot; the cucumber material whose 27th base of the PCR amplification fragment contained both A and G was a heterozygous material resistant to bacterial stem soft rot;

[0024] When enzyme digestion and electrophoresis detection is used, the restriction endonuclease is DdeI. If a 212bp band is obtained by electrophoresis after enzyme digestion, the detection object is a material susceptible to bacterial stem soft rot; if a 235bp band is obtained by electrophoresis after enzyme digestion, the detection object is a material resistant to bacterial stem soft rot; if both 212bp and 235bp bands are obtained by enzyme digestion, the detection object is a hybrid material resistant to bacterial stem soft rot.

[0025] In the technical solution of the above application,

[0026] The reaction system of PCR amplification includes DNA template, amplification primers, 2×3G Taq Master Mix for PAGE, and double distilled water;

[0027] The DdeI digestion system is: 3 μl of PCR product, 0.2 μl of endonuclease, 1 μl of NEB cutsmart buffer, and 5.8 μl of double distilled water.

[0028] The reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, 35 cycles; insulation at 72°C for 5 min;

[0029] The enzyme digestion temperature was 37°C and the enzyme digestion time was 2 h.

[0030] The beneficial effects of the present invention are:

[0031] The present invention not only lays a foundation for the precise positioning and molecular cloning of the cucumber bacterial stem rot resistance gene BSSR, but also provides an efficient approach for the molecular marker-assisted breeding of new cucumber varieties resistant to cucumber bacterial stem rot.

[0032] The present invention provides an application for assisting in screening new cucumber varieties with a cucumber bacterial stem soft rot resistance gene BSSR based on the developed SNP marker. The specific primers BSSR-SNP-01-F / BSSR-SNP-01-R of the SNP marker are used to amplify the genomic DNA of the material to be tested, and then the amplified product is sequenced or DdeI enzyme digested for identification. Through the method provided by the present invention, the resistance to bacterial stem soft rot of candidate cucumber materials can be screened at the seedling stage, which has the advantages of high efficiency, few restrictions and accuracy, improves the efficiency of breeding cucumber varieties resistant to bacterial stem soft rot, and shortens the breeding cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The SNP marker (SNP10867465) of the present invention is used to detect the cucumber parent material CG104 (P 1 ), CG37(P 2 ), F 1 The electrophoresis test results of the generation of single plants; Lanes 1-3 are the parent material CG104 (P 1 ), CG37(P 2 ), F 1 The test results of the individual plants of different generations; 1 : CG104 (resistant to bacterial stem rot) obtained a 235 bp fragment, P 2 : CG37 (susceptible to bacterial stem rot) obtained a 212 bp fragment, F 1 Two fragments of 235 bp and 212 bp were detected simultaneously.

[0034] Figure 2 This is the result of electrophoresis detection of 64 samples of cucumber materials to verify the SNP marker (SNP10867465). DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0036] In the following examples, all experimental reagents not otherwise specified are conventional reagents in the art, which can be prepared according to conventional methods in the art or purchased commercially; all experimental methods not otherwise specified are conventional methods in the art, and reference may be made to the Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or to the methods described in the manufacturer's instructions.

[0037] Materials and Methods

[0038] CG104(P 1): Strong growth potential, medium branching, long internode length, large leaves, thick stems, the first female flower node is the 4th node, the female flower node rate is about 30%, the melon color is dark green, not shiny, the fruit powder is light, the thorns are white and sparse, the tumors are large, slightly ridged, and there are few lines. It is a material resistant to bacterial stem soft rot.

[0039] CG37(P 2 ): Medium growth potential, medium branching, medium internode length, small leaves, medium stem thickness, more than 10 nodes of the first female flower, female flower node rate is about 17%, dark green melon color, heavy fruit powder, black and dense thorns, no tumors, no edges, no lines, and is susceptible to bacterial stem soft rot.

[0040] CG104, CG37 and 64 core germplasm materials were selected by the cucumber research group of the Institute of Vegetables and Flowers of the Chinese Academy of Agricultural Sciences from 3,318 collected cucumber resources. With 'CG104' as the female parent and 'CG37' as the male parent, and their hybrid progeny, a RIL recombinant inbred line population of 188 strains was constructed through self-pollination for many years by single seed propagation. They are preserved in this laboratory and guaranteed to be released to the public for verification experiments within 20 years from the date of application.

[0041] The BSSR-SNP-01 marker primer was designed by our laboratory based on the genome information of the resequencing, using dCAPS finder2.0 software (http: / / helix.wustl.edu / dcaps / dcaps.html) and primer 3.0 software, and synthesized at Beijing Bioengineering Co., Ltd. The genome information of the resequencing was detailed in the paper "A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity" published by Qi et al. in Nature Genetics in 2013.

[0042] Main reagents

[0043] PCR experiments used 2×3G Taq Master Mix for PAGE (RedDye) from Vazyme; enzyme digestion used restriction endonuclease DdeI from New England Biolabs; gel electrophoresis used 40% non-denaturing polyacrylamide from Beijing Coolbo Technology Co., Ltd., which was diluted to 6% before use. Sequencing was performed at Beijing Bioengineering Co., Ltd.

[0044] In the following examples, all experimental reagents not otherwise specified are conventional reagents in the art, which can be prepared according to conventional methods in the art or purchased commercially; all experimental methods not otherwise specified are conventional methods in the art, and reference may be made to the Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or to the methods described in the manufacturer's instructions.

[0045] Example 1. Acquisition of SNP markers linked to the BSSR gene for resistance to bacterial stem rot in cucumber

[0046] In the previous study, we used the bacterial stem soft rot resistant material 'CG104' as the female parent and the bacterial stem soft rot susceptible material 'CG37' as the male parent, and their hybrid progeny were self-pollinated for many years through single seed transmission to construct a RIL recombinant inbred line population of 188 strains as the experimental material, and carried out two bacterial stem soft rot inoculations and identifications at the cucumber seedling stage in 2021 and 2022. The number and area of ​​lesions on the leaves were used as the grading standards, divided into 0, 1, 3, 5, 7, and 9 levels, and the disease index (DI) was calculated, and the results are shown in Table 1. Genetic analysis based on the disease index showed that the disease index between the parents was significantly different, and the disease index of the RIL population was normally distributed, which was consistent with quantitative trait inheritance.

[0047] Table 1 Disease index of bacterial stem rot of cucumber

[0048]

[0049] Using the disease index of the RIL population combined with the SSR genetic map to conduct QTL positioning research, the bacterial stem rot resistance locus BSSR6.1 was repeatedly detected on cucumber chromosome 6 for the first time. The physical position was between 10,633,896-10,867,192, and the LOD value was 6.29. Based on the above results, we carried out this study.

[0050] 1. Acquisition of SNP markers

[0051] Combined with the genome information resequencing by the research team, based on the polymorphism of the parental SNP sites, it was found that at the physical position 10,867,465bp on chromosome 6 of cucumber, in the genome of material CG104 (resistant to bacterial stem rot), the base of this site was A; in the genome of material CG37 (susceptible to bacterial stem rot), the base of this site was G, and the SNP marker SNP10867465 (A / G) related to resistance and susceptibility to bacterial stem rot of cucumber was obtained.

[0052] 2. Development of dCAPS markers based on SNP markers

[0053] Based on the obtained SNP marker SNP10867465 which is closely linked to the bacterial stem soft rot resistance gene BSSR, a dCAPS marker (named BSSR-SNP-01) linked to the bacterial stem soft rot resistance gene BSSR was developed. The DNA sequence of the above segment of the reference genome of cucumber chromosome 6 (the reference genome sequence of the North China cucumber variety "9930", version number V3) was downloaded from the cucumber genome database website (http: / / cucurbitgenomics.org / ), and a pair of primers were designed according to the position of the SNP using dCAPS finder 2.0 software (http: / / helix.wustl.edu / dcaps / dcaps.html) and primer 3.0 software:

[0054] BSSR-SNP-01-F (SEQ ID NO.1): 5'-ACCTTTATAAAAATATTGTGTACTTA-3',

[0055] BSSR-SNP-01-R (SEQ ID NO. 2): 5'-GAGTTTCATCATCTACCATC-3'.

[0056] Due to the relationship of the SNPs obtained above (SNP=A / G), when base G is present, the recognition sequence of the restriction endonuclease DdeI is formed (C↓TTAG, ↓ is the restriction site), and the amplified fragment can be cut by the endonuclease DdeI; when base A is present, the recognition sequence of the restriction endonuclease DdeI cannot be formed, and the amplified fragment cannot be cut by the endonuclease DdeI.

[0057] PCR amplification was performed on the parental materials CG104 and CG37 using the above primers (BSSR-SNP-01-F / BSSR-SNP-01-R). In material CG104 (resistant to bacterial stem soft rot), a 235 bp band (nucleotide sequence shown in SEQ ID NO.3) was obtained; in material CG37 (susceptible to bacterial stem soft rot), a 235 bp band (nucleotide sequence shown in SEQ ID NO.4) was also obtained. The amplified fragment was digested with endonuclease DdeI to obtain specific bands: in material CG104 (resistant to bacterial stem soft rot), a 235 bp band was obtained, and in material CG37 (susceptible to bacterial stem soft rot), a 212 bp band (nucleotide sequence shown in SEQ ID NO.5) was obtained.

[0058] The specific operation method of the detection is as follows :

[0059] Step 1. DNA extraction and PCR amplification

[0060] The young leaves of cucumber plants were taken and the parent CG104 (P 1 ) and CG37(P 2 ) and F 1 Genomic DNA of individual strains.

[0061] The dCAPS-labeled PCR reaction system is as follows: total reaction system 10 μL, 3 μL DNA (5.0 ng·μL -1 ), forward and reverse primers BSSR-SNP-01-F / BSSR-SNP-01-R (50 ng·μL -1 ) 1 μL each, 5 μL 2×3G Taq Master Mix for PAGE (Red Dye) (product of Vazyme).

[0062] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, 35 cycles; incubation at 72°C for 5 min, and storage at 16°C.

[0063] Step 2. Complete digestion of PCR product with DdeI

[0064] The enzyme digestion system was as follows: PCR product 3 μl, endonuclease 0.2 μl, NEB cutsmart buffer 1 μl, double distilled water 5.8 μl. The digestion temperature was 37°C and the digestion time was 2 h.

[0065] Step 3. Result determination

[0066] Method 1: Skip step 2 and sequence the PCR product directly without enzyme digestion. The sequence obtained for CG104 (resistant to bacterial stem rot) has an A at base 27; the sequence obtained for CG37 (susceptible to bacterial stem rot) has a G at base 27; 1 The resulting sequence has two bases at this position, A and G, present simultaneously.

[0067] Method 2: After complete digestion with endonuclease DdeI, the digestion products were separated by 6% non-denaturing polyacrylamide gel, the electrophoresis buffer was 0.5×TBE, and the electrophoresis was separated at 150V constant power for 1h35min. After electrophoresis, silver staining was performed and the band types were counted.

[0068] like Figure 1 As shown, CG104 (resistant to bacterial stem rot) obtained a 235 bp fragment, and the band pattern was recorded as a; CG37 (susceptible to bacterial stem rot) obtained a 212 bp fragment, and the band pattern was recorded as b; F 1Two bands were detected simultaneously in (resistance to bacterial stem soft rot of cucumber), and the band type was recorded as h. Its field phenotype was resistance to bacterial stem soft rot of cucumber.

[0069] Example 2. Validation of SNP markers linked to the cucumber bacterial stem rot resistance gene BSSR

[0070] The 64 core germplasm materials preserved by our research group were used to verify the SNP marker (BSSR-SNP-01) linked to the BSSR gene obtained in Example 1 to determine the accuracy of the marker for molecular marker-assisted selection. The verification method adopted steps 1 to 3 described in Example 1.

[0071] The results of the test material verification are shown in Table 2. The enzyme cleavage bands of the PCR products are shown in Figure 2 . Compared with the inoculation identification phenotype of the selected materials, the SNP markers of the present invention are: 37 materials were identified as resistant to bacterial stem soft rot in the field phenotype, of which 36 materials were identified as a, and 1 material was identified as b, and the accuracy of molecular identification in the 37 materials was 97.3%; 27 materials were identified as susceptible to bacterial stem soft rot in the field phenotype, of which 3 materials were identified as h, 3 materials were identified as a, and the remaining 21 materials were identified as b, and the accuracy of molecular identification in the 27 materials was 77.78%; in the identification of all 64 materials, the consistency rate of molecular identification and field phenotypic identification was 89%. The molecular identification method of the present invention can be used to identify resistance to bacterial stem soft rot of cucumber for molecular-assisted breeding.

[0072] Table 2 Test material verification results

[0073]

[0074]

[0075] Note: The field investigation of disease resistance at seedling stage was carried out according to the method in Example 1, and the disease index was calculated. Plants with a disease index <18 were materials resistant to bacterial stem soft rot, and the field phenotype was recorded as R in Table 2;

Claims

1. Use of a reagent for detecting SNP molecular markers in any of the following (1)-(3): (1) Identification of cucumber materials resistant / susceptible to bacterial stem rot; (2) Screening of cucumber varieties resistant to bacterial stem rot; (3) Cultivate cucumber varieties resistant to bacterial stem rot; The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.3, wherein the 27th base is A or G, and the cucumber material with the base A at this position is a cucumber material resistant to bacterial stem soft rot, and the cucumber material with the base G at this position is a cucumber material susceptible to bacterial stem soft rot.

2. The use according to claim 1, characterized in that: The application comprises the following steps: extracting genomic DNA of the sample to be tested as a template, performing PCR amplification using the amplification primers of the SNP molecular marker, and sequencing or enzyme-cutting electrophoresis detection on the PCR amplification product. The enzyme-cutting electrophoresis detection refers to using a restriction endonuclease that can recognize the differential site of the SNP molecular marker to cut the PCR amplification product, and then performing electrophoresis on the amplification product.

3. The use according to claim 2, characterized in that: The amplification primers of the SNP molecular markers are as follows: BSSR-SNP-01-F: 5'-ACCTTTATAAAAATATTGTGTACTTA-3', BSSR-SNP-01-R: 5'-GAGTTTCATCATCTACCATC-3'; A 235 bp fragment was obtained by PCR amplification. When sequencing was used for detection, the cucumber material with A at the 27th base of the PCR amplification fragment was resistant to bacterial stem soft rot; the cucumber material with G at the 27th base of the PCR amplification fragment was susceptible to bacterial stem soft rot; the cucumber material with both A and G at the 27th base of the PCR amplification fragment was heterozygous for bacterial stem soft rot resistance. When enzyme digestion and electrophoresis are used for detection, the restriction endonuclease is DdeI. If a 212 bp band is obtained by electrophoresis after enzyme digestion, the detection object is a material susceptible to bacterial stem soft rot; if a 235 bp band is obtained by electrophoresis after enzyme digestion, the detection object is a material resistant to bacterial stem soft rot; if both 212 bp and 235 bp bands are obtained by enzyme digestion, the detection object is a hybrid material resistant to bacterial stem soft rot.

4. The use according to claim 3, characterized in that: The reaction system for PCR amplification included DNA template, amplification primers, 2 × 3G Taq Master Mix for PAGE, and double-distilled water; The DdeI digestion system was as follows: 3 μl of PCR product, 0.2 μl of endonuclease, 1 μl of NEB cutsmart buffer, and 5.8 μl of double distilled water.

5. The use according to claim 4, characterized in that: The reaction procedure of the PCR amplification is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, 35 cycles; insulation at 72°C for 5 min; The digestion temperature was 37°C and the digestion time was 2 h.

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