SNP molecular markers linked to the Ccu gene for identifying resistance to cucumber scab and its application

Abstract

The invention discloses a SNP molecular marker linked to the Ccu gene for identifying cucumber resistance to black scab and its application, belonging to the field of biotechnology-assisted breeding technology, wherein the site of the SNP marker is at the 2,847,451 bp of the physical position of cucumber chromosome 2, the base of the site is C or T, the cucumber material with the base of the site being C is a black scab-resistant cucumber material, and the cucumber material with the base of the site being T is a black scab-susceptible cucumber material. The invention not only lays a foundation for the precise positioning and molecular cloning of the cucumber black scab resistance gene Ccu, but also provides an efficient way for the use of molecular markers to assist in the breeding of new cucumber varieties resistant to cucumber black scab.

Description

Technical Field

[0001] The invention relates to the technical field of biotechnology-assisted breeding, in particular to a SNP molecular marker linked to a Ccu gene for identifying cucumber resistance to black spot disease and an application thereof. Background Art

[0002] Cucumber (Cucumis is a tivus L.) is one of the world's top ten vegetables, especially in facility production. Cucumber cultivation is an important industry for many regions to achieve poverty alleviation and rural revitalization. However, during the cultivation and production of cucumbers, it is susceptible to infection by a variety of diseases, and there are more than 10 common diseases. Cucumber scab is one of the main diseases of cucumber. The pathogen is Cladosporium cucumerinum Ell. et Arthur, which belongs to the Ascomycetes. Cucumber scab is a low-temperature disease that is prone to epidemic disasters in protected areas. It can occur throughout the growth period and can infect any part except the roots. It mainly harms young and tender parts. The rot of the growing point can cause "bald piles". Damage to the melon strips can cause deformed melons and lead to loss of commercial value. In severe cases, the whole plant dies. The disease rate is over 90%, the yield reduction is over 70%, and severe damage can lead to total failure. Cucumber scab has been spreading in China since the 1980s. At present, the prevention and control method of black spot disease in cucumber production is still mainly based on chemical prevention and control, which brings certain hidden dangers to food safety and also pollutes the ecological environment. Breeding and promoting cucumber varieties resistant to black spot disease is the most economical and effective method to prevent and control the disease, and it is also an important guarantee for green and efficient production of cucumbers. Research on the genetic laws and molecular markers of cucumber black spot disease resistance is of great significance for breeding new varieties that meet market demand.

[0003] Bailey and Burgess (1934) first reported that the cucumber scab resistance trait was controlled by a single dominant gene and named it Ccu. There has been no accurate report on the location of genes related to cucumber scab resistance. Previous researchers have conducted a number of studies on linkage markers for cucumber scab resistance. Zhang Guihua et al. (2006) screened AFLP markers linked to cucumber scab and determined that the linkage distance between the specific marker E20M64 and the cucumber scab resistance gene was 4.83 cM; Wang Huizhe et al. (2009) screened the SSR marker CSWCTT02D closely linked to the cucumber scab resistance-related gene, with a distance of 3.1 cM. In terms of gene localization, Zhang et al. (Genetic Mapping of the Scab Resistance Gene in Cucumber, J.AMER.SOC.HORT.SCI.135(1):53–58.2010) located the scab resistance gene in the region of SSR03084-SSR17631, with a physical distance of 751.6 kb on chromosome 2. On this basis, Kang et al. further encrypted the markers and located Ccu in the range of 140 kb by image cloning.

[0004] Cleaved amplified polymorphic sequence (CAPS) marker is a DNA marker for detecting SNP sites 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 polymorphism of the digested fragment, which can be displayed by agarose or polyacrylamide gel electrophoresis and staining.

[0005] So far, there are no reports on SNP markers closely linked to the quality trait gene Ccu developed based on CAPS markers. 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 linked to the Ccu gene for identifying cucumber resistance to black spot disease, wherein the site of the SNP marker is at the physical position 2,847,451bp of cucumber chromosome 2, and the base of the site is C or T. The cucumber material whose base at the site is C is a black spot disease-resistant cucumber material, and the cucumber material whose base at the site is T is a black spot disease-susceptible cucumber material.

[0007] The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.3, wherein the 101st base is C or T.

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

[0009] caps-01-F: 5'-TAGCCAATTTGAAGGCTGCT-3',

[0010] caps-01-R: 5'-AAGAACTTGATATGAAAAAAAAATG-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] Preferably, the kit further 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 further comprises a polyacrylamide gel electrophoresis reagent; preferably, the restriction endonuclease is MseI.

[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 cucumber scab resistant / susceptible materials;

[0015] (2) Identify or assist in identifying the cucumber scab resistance / susceptibility gene Ccu;

[0016] (3) Screening or assisting in the screening of cucumber varieties resistant to black scab;

[0017] (4) Cultivating or assisting in the cultivation of cucumber varieties resistant to black scab;

[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] caps-01-F: 5'-TAGCCAATTTGAAGGCTGCT-3',

[0022] caps-01-R: 5'-AAGAACTTGATATGAAAAAAAAATG-3';

[0023] A 201 bp fragment was obtained by PCR amplification. When sequencing was used for detection, the cucumber material whose 101st base of the PCR amplification fragment was C was a cucumber material resistant to black scab, the cucumber material whose 101st base of the PCR amplification fragment was T was a cucumber material susceptible to black scab, and the cucumber material whose 101st base of the PCR amplification fragment contained both T and C was a heterozygous material resistant to black scab.

[0024] When enzyme digestion and electrophoresis detection is used, the restriction endonuclease is MseI. If a 201bp band is obtained by electrophoresis after enzyme digestion, the detection object is a black spot disease-resistant material; if a 101bp band is obtained by electrophoresis after enzyme digestion, the detection object is a black spot disease-susceptible material; if both 201bp and 101bp bands are obtained by enzyme digestion, the detection object is a hybrid black spot disease-resistant material.

[0025] In the technical solution of the above application, the reaction system of PCR amplification includes DNA template, amplification primers, 2×3GTaq Master Mix for PAGE, and double distilled water;

[0026] The system for MseI digestion 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.

[0027] In the technical solution of the above application, 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;

[0028] The enzyme digestion temperature was 37°C and the enzyme digestion time was 2 h.

[0029] The beneficial effects of the present invention are:

[0030] The present invention not only lays a foundation for the precise positioning and molecular cloning of the cucumber scab resistance gene Ccu, but also provides an efficient approach for the use of molecular markers to assist in the breeding of new cucumber varieties resistant to cucumber scab.

[0031] The present invention provides an application for assisting in screening new cucumber varieties with cucumber scab resistance gene Ccu based on the developed SNP marker. The specific primers caps-01-F / caps-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 MseI enzyme digested for identification. Through the method provided by the present invention, the candidate cucumber materials can be screened for scab resistance at any stage, which has the advantages of high efficiency, few restrictions and accuracy, improves the efficiency of breeding cucumber scab-resistant varieties, and shortens the breeding cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention is a result of electrophoresis detection of cucumber parent materials CsIVF0254 (P1), CsIVF0045 (P2), and F1 generation single plants by using the SNP marker (SNP2847451) of the present invention; lanes 1-3 are the detection results of parent materials CsIVF0254 (P1), CsIVF0045 (P2), and F1 generation single plants, respectively; wherein, P1: CsIVF0254 (resistant to black spot disease) obtained a 201 bp fragment, P2: CsIVF0045 (susceptible to black spot disease) obtained a 101 bp fragment, and both fragments of 201 bp and 101 bp were detected in the F1 generation.

[0033] Figure 2 It is the electrophoresis test result of 64 samples of cucumber materials to verify the SNP marker (SNP2847451). DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0035] 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.

[0036] Materials and Methods

[0037] CsIVF0254(P1): An advanced inbred line of North China spiny cucumber with a European greenhouse genetic background. It has strong growth potential, large leaves, green skin, green flesh, thorny fruits, and white thorns.

[0038] CsIVF0045(P2): It is an advanced inbred line of the North China prickly cucumber type. It has strong growth potential, large leaves, green skin, white thorns, and thorny fruits.

[0039] CSIVF0254 and CSIVF0045 are cucumber materials bred by the Cucumber Research Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. They are preserved in this laboratory and guaranteed to be released to the public for verification experiments within twenty years from the date of application.

[0040] The 64 core germplasm materials for verification are part of the core germplasm containing 130 materials constructed by the Cucumber Research Group Laboratory of the Institute of Vegetable and Floriculture, Chinese Academy of Agricultural Sciences. They were screened out from 3,318 cucumber resources and are preserved in this laboratory, guaranteed to be released to the public for verification experiments within twenty years from the date of application.

[0041] The caps-01 marker primer was designed by our laboratory based on the genome information of the resequencing, using the DCAPS finder 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 "Agenomic 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 MseI 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] Example 1. Obtaining SNP markers linked to the cucumber scab resistance gene Ccu

[0045] In previous studies, we used the methods of preliminary positioning and map-based cloning and fine positioning to locate the cucumber scab resistance gene Ccu within the 85.2kb interval of chromosome chr. 2. Based on the above results, we carried out this study.

[0046] 1. Acquisition of SNP markers

[0047] Combining the data of cucumber reference genome sequence and the resequencing data of parental materials CsIVF0254 and CsIVF0045, bioinformatics was used to combine the genotypes and phenotypes of 23 exchange plants selected from 943 F2 plants (F2 phenotypes were analyzed by F 2:3 Family phenotype determination), at the physical position 2,847,451bp on cucumber chromosome 2, a SNP marker SNP2847451 (T / C) tightly linked to the cucumber black spot resistance gene Ccu was obtained, and it was found that in the genome of material CsIVF0254 (resistant to black spot), the base of this site was C; in the genome of material CsIVF0045 (susceptible to black spot), the base of this site was T.

[0048] 2. Development of CAPS markers based on SNP markers

[0049] Based on the obtained SNP marker SNP2847451 which is closely linked to the black spot disease resistance gene Ccu, a CAPS marker (named as caps-01) linked to the black spot disease resistance gene Ccu was developed. The DNA sequence of the above segment of the cucumber chr.2 chromosome reference genome (the reference genome sequence of the North China cucumber variety "9930", version number Cucumber (Chinese Long) v3 Genome) 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 the dCAPS Finder 2.0 (http: / / helix.wustl.edu / dcaps / dcaps.html) website and primer 3.0 software:

[0050] caps-01-F (SEQ ID NO.1): 5'-TAGCCAATTTGAAGGCTGCT-3',

[0051] caps-01-R (SEQ ID NO. 2): 5'-AAGAACTTGATATGAAAAAAAAATG-3'.

[0052] Due to the relationship of the SNPs obtained above (SNP=T / C), when base T exists, the recognition sequence of the restriction endonuclease MseI is formed (T↓TAA, ↓ is the enzyme cutting site), and the amplified fragment can be cut by the endonuclease MseI; when base C exists, the recognition sequence of the restriction endonuclease MseI cannot be formed, and the amplified fragment cannot be cut by the endonuclease MseI.

[0053] The above primers (caps-01-F / caps-01-R) were used to perform PCR amplification on the parental materials CsIVF0254 and CsIVF0045. In the material CsIVF0254 (resistant to black spot disease), a 201 bp band (nucleotide sequence shown in SEQ ID NO.3) was obtained; in the material CsIVF0045 (susceptible to black spot disease), a 201 bp band (nucleotide sequence shown in SEQ ID NO.4) was also obtained. The amplified fragment was digested with endonuclease MseI to obtain a specific band ( Figure 1 ), in material CsIVF0254 (resistant to black spot disease), a 201 bp band was obtained, and in material CsIVF0045 (susceptible to black spot disease), a 101 bp band was obtained (the nucleotide sequence is shown in SEQ ID NO.5).

[0054] The specific operation method of the detection is as follows:

[0055] Step 1. DNA extraction and PCR amplification

[0056] Young leaves of cucumber plants were taken and the genomic DNA of the parents CsIVF0254 (P1) and CsIVF0045 (P2) as well as F1 and F2 were extracted using a modified CTAB (cetyltrimethylammonium bromide) method.

[0057] The CAPS labeling PCR reaction system is as follows: total reaction system 10 μL, 3 μL DNA (5.0 ng·μL -1 ), forward and reverse primers caps-01-F / caps-01-R (concentration 50ng·μL -1 ) 1 μL each, 5 μL 2×3G Taq Master Mix for PAGE (Red Dye) (product of Vazyme).

[0058] 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.

[0059] Step 2. Complete digestion of PCR product with MseI

[0060] 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.

[0061] Step 3. Result determination

[0062] Method 1: Skip step 2, sequence the PCR product directly without enzyme digestion. The sequence obtained from CsIVF0254 (resistant to black spot) has a T at base 101; the sequence obtained from CsIVF0045 (susceptible to black spot) has a T at base 101; the sequence obtained from F1 has two bases at this position, T and C coexisting.

[0063] Method 2: After complete digestion with endonuclease MseI, 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 1h. After electrophoresis, silver staining was performed and the band types were counted.

[0064] like Figure 1 As shown, CsIVF0254 (resistant to black spot) obtained a 201bp fragment, and the band pattern was recorded as b; CsIVF0045 (susceptible to black spot) obtained a 101bp fragment, and the band pattern was recorded as a; two bands were detected simultaneously in F1, and the band pattern was recorded as h, and its field phenotype was resistant to black spot.

[0065] Example 2. Verification of SNP markers linked to the cucumber scab resistance gene Ccu

[0066] The CAPS marker (caps-01) linked to the Ccu gene obtained in Example 1 was verified using 64 core germplasm materials preserved by our research group to determine the accuracy of the marker for molecular marker-assisted selection. The verification method adopted steps 1 to 3 described in Example 1. The results of field identification and molecular verification are shown in Table 1. The restriction enzyme bands of the PCR products are shown in Table 1. Figure 2 .

[0067] Table 1. Field phenotypes and molecular verification results of 64 natural cucumber population materials susceptible to / resistant to cucumber gray mold

[0068]

[0069]

[0070] *Note: Field phenotypes are expressed using disease indexes. Based on the distribution of disease indexes in the population, disease indexes equal to 45 are used as the boundary. Disease indexes less than 45 are considered to be resistant to black scab, and those greater than 45 are considered to be susceptible to black scab. According to the investigation, the field phenotype data of the 64 materials in Table 1 are: 20 are resistant to black scab, and 44 are susceptible to black scab.

[0071] Compared with the inoculation identification phenotype of the selected materials, the phenotypic data reflected by the banding patterns of all materials in the 64 materials of the SNP marker of the present invention were compared with the inoculation identification survey results, and the accuracy rate of the molecular identification results was 98.4%.

Claims

1. Use of SNP molecular markers for identifying resistance of cucumber to black spot disease in any of the following (1)-(3): (1) Identify or assist in identifying cucumber scab resistant / susceptible materials; (2) Screening or assisting in the screening of cucumber varieties resistant to black scab; (3) Cultivate or assist in the cultivation of cucumber varieties resistant to black scab; The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.3 or SEQ ID NO.4, wherein the 101st base is C or T, and the cucumber material with the base C at this position is a cucumber material resistant to black spot disease, and the cucumber material with the base T at this position is a cucumber material susceptible to black spot disease.

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 for the SNP molecular markers are as follows: caps-01-F: 5'-TAGCCAATTTGAAGGCTGCT-3', caps-01-R: 5'-AAGAACTTGATATGAAAAAAAAATG-3'; A 201 bp fragment was obtained by PCR amplification. When sequencing was used for detection, the cucumber material whose 101st base of the PCR amplification fragment was C was a cucumber material resistant to black scab, the cucumber material whose 101st base of the PCR amplification fragment was T was a cucumber material susceptible to black scab, and the cucumber material whose 101st base of the PCR amplification fragment contained both T and C was a heterozygous material resistant to black scab. When enzyme digestion and electrophoresis detection is used, the restriction endonuclease is MseI. If a 201bp band is obtained by electrophoresis after enzyme digestion, the detection object is a black spot disease-resistant material; if a 101bp band is obtained by electrophoresis after enzyme digestion, the detection object is a black spot disease-susceptible material; if both 201bp and 101bp bands are obtained by enzyme digestion, the detection object is a hybrid black spot disease-resistant material.

4. The use according to claim 3, characterized in that: The reaction system of PCR amplification includes DNA template, amplification primers, 2×3G Taq Master Mix for PAGE, and double distilled water; The system for MseI digestion 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.

5. The use according to claim 4, characterized in that: 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; The enzyme digestion temperature was 37°C and the enzyme digestion time was 2 h.

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