WO2015192566A1

WO2015192566A1 - Cucumber target leaf spot resistance gene cca as well as linked molecular markers and applications thereof

Info

Publication number: WO2015192566A1
Application number: PCT/CN2014/089045
Authority: WO
Inventors: 温常龙; 许勇; 毛爱军; 于拴仓; 董从娟
Original assignee: 北京市农林科学院
Priority date: 2014-06-16
Filing date: 2014-10-21
Publication date: 2015-12-23

Abstract

Provided are a cucumber target leaf spot resistance gene Cca as well as linked molecular markers and applications thereof in breeding cucumber target leaf spot resistance varieties. The gene has a nucleotide sequence represented by SEQ ID NO:1 in a sequence table. Also provided are specific molecular markers or tightly linked markers thereof generated according to the sequence information of the gene Cca, these markers capable of identifying genotypes of cucumber and descendant plants thereof; and two molecular markers linked to the cucumber target leaf spot resistance gene, the markers capable of identifying the existence of the cucumber target leaf spot resistance gene Cca.

Description

Resistance target gene Cca of cucumber target spot disease and its linkage molecular markers and applications

Technical field

The invention belongs to the field of gene sequences and applications, and particularly relates to a cucumber target spot disease resistance gene Cca and its linked molecular markers and applications.

Background technique

Cucumber (Cucumis Sativus.L) is widely cultivated in the world. China is the country with the largest cucumber cultivation area and the highest total output in the world.

Cucumber leaf spot disease, also known as target spot disease, is a worldwide disease. At present, it has become one of the important diseases that jeopardize the cultivation of cucumbers and protected areas, especially in the protected areas such as wintering greenhouses, winter and spring greenhouses, and spring greenhouses. The main damage to the leaves, the lesions appear pale brown after the brownish green, and the leaves die when severe. When the disease causes the defoliation rate to be less than 5%, the disease spreads slowly, about 2 weeks, and then develops rapidly in the next week, and the defoliation rate can be increased from 5% to 90%. Cucumber planting in the shed indoors is susceptible to epidemics in winter and spring. The incidence rate in the field is generally 10%-25%, and in severe cases it is 60%-70%, resulting in losses of more than 30%.

The pathogen of cucumber target spot disease is Corynespora cassiicola (Berk. Curt.) Wei., which belongs to the fungi. The genus Pseudomonas sp. is branched, translucent, and has a smooth wall and is 2 to 6 μm wide. No child seat. Conidiophores are derived from hyphae, erect, solitary, light brown, with a septum. The top of the conidiophore has 0 to 9 cylindrical layers, and the stalk width is 4 to 11 μm and the length is 110 to 850 μm. Conidia are often solitary, or 2 to 6 strings are born at the top, conidia are erect or slightly curved, cylindrical or inverted stick-shaped, translucent to light olive, 4 to 20 false diaphragms, base umbilical 4 ~ 8 μm wide, spore size is 9-22 μm × 40-220 μm. Pseudomonas aeruginosa is a highly mutated group, which leads to the short-lasting period of disease-resistant varieties and the instability of chemical agents in the prevention and control of the leaf spot leaf spot disease, which not only increases the production input, but also causes environmental safety hazards. Grafting makes technical difficulties and labor costs increase. Therefore, rapid selection of disease-resistant varieties is the best way to solve the target spot hazard.

Cloning genes is one of the most important applications for finding molecular markers linked to a target trait. Map-based cloning is the use of molecular genetic maps to link target traits and molecular markers through genetic linkage mapping, positioning target genes between flanking markers that are closely linked to them; The analytical method (BSA method) or the near-isogenic method obtains a molecular marker linked to the target gene, and then uses the mapping population to position the molecular marker on the map. Then these markers are used to screen large fragment DNA libraries. Identification of the clones associated with the marker, followed by subcloning and Chromosome walking to obtain cloned fragments containing the gene of interest, and finally complemented by transformation and complementation tests (Wang Yongfei, molecular marker in plant genetic breeding) The application principle and status quo, Journal of Northwest A&F University (Natural Science Edition), 2001, 106-113). At present, a large number of plant genes have been isolated using map-based cloning technology, especially genes related to disease resistance (Yan Qitao, map cloning technology for plant gene isolation, molecular plant breeding, 2005, 585-590), but no A report on the successful cloning of a disease resistance gene for cucumber target spot disease. In addition, the cucumber target spot disease resistance gene affects the resistance of cucumber to target spot disease, and its research will promote the process of disease resistance breeding. Marker-assisted selection with molecular markers closely linked to the trait of interest is very effective in cucumber genetic breeding. Molecular markers are the choice of target traits at the DNA level, which are efficient, rapid, and free from environmental conditions. They can be selected at the seedling stage to speed up the breeding process. Therefore, it is important to develop molecular markers for the identification of the cucumber target spot disease resistance gene Cca.

Summary of the invention

In view of the deficiencies of the prior art, one of the objects of the present invention is to provide a cucumber target spot disease resistance gene Cca.

A second object of the present invention is to provide a genetic marker for the above-mentioned cucumber target spot disease resistance gene Cca.

A third object of the present invention is to provide a pair of primers for amplifying the above-mentioned cucumber target spot disease resistance gene Cca.

A fourth object of the present invention is to provide an application of the above-mentioned cucumber target spot disease resistance gene Cca and genetic markers.

The fifth object of the present invention is to provide a linkage molecular marker of a cucumber target spot disease resistance gene and a specific primer and application thereof.

In order to achieve the above object, the present invention adopts the following technical solutions:

The cucumber target spot disease resistance gene Cca, (a) is the nucleotide sequence shown by SEQ ID NO: 1 in the sequence listing, and (b) has 90% or more of the sequence shown by SEQ ID NO: 1 in the sequence listing. A nucleotide sequence which is functional and has the same function as the sequence shown by SEQ ID NO: 1 in the Sequence Listing.

The cucumber target spot disease resistance gene Cca involved in the present invention is obtained by fine-position cloning using an anti-infective genetic population. The invention selects the classic disease-resistant material WI2757 and the important backbone parental susceptible material Xintai Mia, constructs the F _2:3 and F ₂ genetic population, and carries out preliminary localization and fine mapping of the disease resistance gene, and the target target gene is finely positioned. Within the 80 kb range of chromosome 6, there are three genes in the interval, the first gene is the NB-ARC gene, the second gene is the BIM2-like transcription factor-related gene, and the third gene is the 33-like peptide. Repetitive sequence structure protein related genes. It is well known that the conserved domain of the NB-ARC gene is a typical NBS-LRR resistance gene, so it is speculated that the NB-ARC gene is an important candidate gene Cca against target spot disease, and its DNA sequence is through a fine mapping interval. The gene sequence was obtained by sequencing.

The genetic marker of the cucumber target spot disease resistance gene Cca represented by the above SEQ ID NO: 1, the first of the susceptible genes The base T at position 1481 is mutated to base G.

A pair of primers for amplifying the cucumber target spot disease resistance gene Cca shown in the above SEQ ID NO: 1, respectively, having the nucleotide sequences shown by SEQ ID NO: 7 and SEQ ID NO: 8 in the Sequence Listing.

According to the published cucumber genome 9930 and GY14 sequence information, the present invention uses the Softberry online prediction software FGENESH (http://sunl.softberry.com/) to predict the gene structure and design according to the starting and ending coding sequences of the predicted results. A pair of full-length amplification primers of the gene are: the nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 in the sequence table, respectively, and PCR amplification is performed using genomic DNA of the anti-inductive material to obtain an anti-/induction The full-length DNA sequence of the gene Cca in the material is SEQ ID NO: 1 (resistant material) and SEQ ID NO: 2 (sensitive material), the full length is 3255 bp, and the DNA sequences of the two genes in the antagonistic material are compared and analyzed. The DNA sequence of Cca has a SNP site in the anti-sense material, that is, at the 1481th position of the gene, the base of the susceptible material is T, and the base of the disease-resistant material is G, which can be applied to development resistance. Cucumber target spot molecular marker.

The cDNA sequence of the cucumber target spot disease resistance gene Cca represented by the above SEQ ID NO: 1 has the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing.

The cucumber target leaf disease resistance gene Cca cDNA (mRNA) sequence of the present invention is the reverse transcription of the total RNA using the full-length primers SEQ ID NO: 7 and SEQ ID NO: 8 as the upstream and downstream primers. The full-length cDNA (mRNA) sequence of the gene Cca in the anti-inductive material was obtained by PCR amplification of a strand as a template, and SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The cDNA structure was obtained by using Softberry to predict the Cca sequence online. By comparing the sequences of SEQ ID NO: 3 and SEQ ID NO: 4, the cDNA (mRNA) of the cucumber anti-target spot gene Cca was only one in the anti-infective material. The exon has no intron structure, but the base of the disease resistance material at the 981 bp position of the cDNA sequence is G, and the base of the susceptible material is T, which is due to this base in the anti-inductive material. Differences, which result in differences in amino acid coding, result in different functions, which in turn lead to changes in the disease resistance of the NB-ARC gene.

The encoded protein of the cucumber target spot disease resistance gene Cca shown in the above SEQ ID NO: 1 is the amino acid sequence shown by SEQ ID NO: 5 in the Sequence Listing.

The amino acid sequence of the cucumber target spot disease resistance gene involved in the present invention is the cDNA (mRNA) sequence of SEQ ID NO: 3 and SEQ ID NO: 4 using the Cca gene in the anti-inductive material, respectively, and is obtained by the softberry online prediction software translation. The Cca amino acid (protein) sequence of SEQ ID NO: 5 and SEQ ID NO: 6 in the sensing material, and the Cca amino acid sequence in the anti-/sensitive material is aligned, and Cca has one amino acid code in the anti-/pathogenic material. The difference is shown in position 493 (KN). It is speculated that this difference leads to a change in the disease resistance domain, resulting in a change in the disease resistance of the NB-ARC gene.

The above-mentioned cucumber target spot disease resistance gene Cca or the above-mentioned cucumber target spot disease resistance gene Cca cDNA is used for breeding cucumber target spot disease resistant varieties.

The above genetic markers are used in breeding resistant varieties of cucumber target spot disease.

A cucumber target spot disease resistance chain molecular marker, which is one of nucleotide sequences amplified from total cucumber DNA by the following primers: SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing, in the sequence listing SEQ ID NO: 11 and SEQ ID NO: 12.

The primers for obtaining the above-mentioned cucumber target spot disease resistance chain-linked molecular marker are as follows: 1) The primer for obtaining the cucumber target spot disease resistance-linked molecular marker CcaSNP1 is SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence table; 2) obtaining cucumber Primers for the target spot disease resistance-linked molecular marker CcaSNP2 are SEQ ID NO: 11 and SEQ ID NO: 12 in the Sequence Listing.

Using the above primer pair, the above molecular marker can be obtained by a conventional PCR method.

The dCAPs-labeled CcaSNP1 which is co-segregated with cucumber against target spot disease is made by using an anti-/infective genetic group (female: WI2757, anti-target spot disease; male parent: Xintai dense thorn, sensitive target spot disease) Positioned and cloned. The target spot disease resistance gene Cca was initially mapped by using 150 F _2:3 populations, and then the Cca gene was finely mapped into the 80 kb interval using 2000 F ₂ populations. The interval contains three genes, namely NB-ARC resistance gene, BIM2-like transcription factor related gene and 33-like peptide repeat structural protein-related gene. The NB-ARC gene was verified and identified as target plaque. An important candidate gene for the disease resistance gene Cca. By comparing the sequence differences of Cca gene in anti-/susceptive cucumber materials, we found a SNP locus based on http://helix.wustl.edu/dcaps/dcaps.html, online design software, developed and designed The target spot blotch is closely linked to the dCAPs molecular marker CcaSNP1. At the same time, CcaSNP1 marker was used to analyze more than 2000 genetic population materials, and combined with field target spot vaccination identification study, it was found that CcaSNP1 marker was closely linked to anti-infective target spot disease and reached co-segregation.

Another cucumber anti-target spot disease SNP2 key site involved in the present invention results in a change in amino acid coding in the disease resistance gene Cca. The SNP locus was obtained by genomic resequencing of cucumber target leaf disease resistance materials with different genetic backgrounds. By resequencing multiple cucumber resistance materials, BWA-Samtools (v0.1.12a, mpileup according to the default) The software) analyzed the SNP locus of Cca gene in different materials, combined with cucumber genome 9930-V2 and genome annotation, and found that there was a single base difference SNP2 locus (from G to A) at position 210 of Cca gene. Using the SNP2 locus design marker, the CcaSNP2 molecular marker was obtained. By analyzing the genotype of the above cucumber material and identifying the anti-pathogenic phenotype of the material in combination with the field target spot disease, it was found that the genotype of the CcaSNP2 marker analysis was consistent with the anti-infective phenotype. It is proved that CcaSNP2 can be used as a closely linked molecular marker of target spot disease to screen cucumber materials.

The above-mentioned cucumber target spot disease resistance chain molecular marker or the primer for obtaining the above-mentioned cucumber target spot disease resistance chain molecular marker is used for breeding the cucumber target spot disease variety.

In the above application, preferably, the specific method for the application of the molecular marker CcaSNP1 in breeding the cucumber target spot disease species is: using the genomic DNA of the cucumber material to be tested as a template, and using SEQ ID NO: 9 in the sequence listing. The specific primer consisting of the nucleotide set in SEQ ID NO: 10 in the sequence listing is subjected to PCR amplification, and then the PCR product is digested with MaeII endonuclease. If a 165 bp digested product is obtained, the cucumber to be tested is a disease-resistant material; if a 135 bp digested product is obtained, the cucumber to be tested is a susceptible material; more preferably, the cucumber material to be tested is WI2757 or Changchun Mia, or WI2757 and/or Changchun Mia as a parent of the parent;

In the above application, preferably, the specific method for the application of the molecular marker CcaSNP2 in breeding the cucumber target spot disease species is: using the genomic DNA of the cucumber material to be tested as a template, and using SEQ ID NO: 11 in the sequence listing. And the specific primer consisting of the nucleotide sequence shown by SEQ ID NO: 12 in the sequence table is subjected to PCR amplification, and then the PCR product is digested with XbaI endonuclease. If a 164 bp digested product is obtained, the cucumber to be tested is a disease-resistant material; if a 134 bp digested product is obtained, the cucumber to be tested is a susceptible material; more preferably, the cucumber material to be tested is BANNA HUANGGUA, or CUCUMIS HARDWICKII, or DI HUANGGUA, or BANNA HUANGGUA and/or CUCUMIS HARDWICKII is the progeny of the parent, or the progeny obtained by DI HUANGGUA as the parent, or DI HUANGGUA and CUCUMIS HARDWICKII as the progeny of the parent, or the progeny obtained by BANNA HUANGGUA and DI HUANGGUA as the parent.

The invention has the following beneficial effects:

The invention utilizes map cloning and forward genetics method to finely locate and clone the cucumber target spot disease resistance gene Cca, obtain the cucumber target spot disease resistance gene Cca, and the nucleic acid of the gene in the anti-/pathogenic material. The sequence and amino acid sequence were analyzed and aligned to obtain the difference and polymorphism analysis of the cucumber target spot disease resistance gene Cca in the anti/pathogenic material. The invention verifies the function of the cucumber target spot disease resistance gene Cca by sequence analysis test on different anti-inductive materials and comparison with field resistance identification results. The disease resistance gene Cca provided by the invention has important application value, and can generate specific molecular markers or closely linked markers thereof according to the gene sequence information, including but not limited to SNP (single nucleotide polymorphism), InDel (insertion) Missing polymorphism), RFLP (restriction endonuclease polymorphism), CAP (cleavage of amplified fragment polymorphism), these markers can be used to identify genotypes of cucumber and progeny plants for molecular marker-assisted selection breeding, thereby improving Breeding efficiency.

In addition, the dCAPs-labeled CcaSNP1 and CcaSNP2 of the present invention are closely linked to the cucumber target spot disease resistance (co-segregation), and the presence of the target gene cucumber target spot disease resistance gene Cca can be identified, and the indirect selection of the disease-resistant plants can be realized. Because it is not affected by other genetic effects and environmental factors, it can be used for early generation selection, shortening breeding years, improving breeding efficiency, and providing technical support for breeding against cucumber target spot disease.

DRAWINGS

Figure 1 is a map of the linkage map of the disease resistance gene Cca in cucumber target spot. The left picture shows the initial location map of cucumber target spot disease gene. The middle two pictures show the fine mapping of target spot disease resistance genes. The right picture shows the 80kb interval. Gene distribution and gene annotation.

2 is a schematic diagram showing the results of enzyme digestion verification of a CcaSNP1 molecular marker of a Cca gene in a certain population;

Figure 3 is a schematic diagram showing the results of enzyme digestion verification of a CcaSNP2 molecular marker of a Cca gene in a certain population;

Figure 4 is a schematic diagram showing the results of restriction enzyme digestion of the CcaSNP1 molecular marker of Cca gene in another unknown population;

Figure 5 is a schematic diagram showing the results of digestion of the CcaSNP2 molecular marker of Cca gene in another unknown population.

detailed description

The present invention will be described in detail below by way of specific examples, but the invention is not limited thereto.

Example 1: Obtainment of the disease target gene Cca of cucumber target spot disease

I. Preliminary positioning and fine mapping of the disease resistance gene Cca of cucumber target spot disease

The specific positioning method includes the following steps:

A, cucumber target spot disease resistance gene mapping research parent and genetic group:

WI2757 (mother) and Xintai thorn (parent) were selected as anti-infective parents, and 150 F _2:3 populations and over 2000 F ₂ large populations were constructed. Among them, the father Xintai Mia and the female parent WI2757 were purchased from the Beijing Crop Germplasm Resource Bank.

B, cucumber target spot disease resistance gene mapping research disease index survey:

The seeds of the parents and each group were wrapped with gauze, soaked in warm soup, incubated at 28 °C, and then sown in 50-well trays. The seedlings were sterilized in the air-conditioned greenhouse, and the seedling substrate was sterilized perlite or nutrient soil.

Corynespora cassiicola (Berk. Curt.) Wei.] is a study on the population differentiation of Cucumber leaf spot disease in Qingxian County, Hebei Province, North China Agricultural Journal , 2011, 26 (5): 9-15) obtained by the separation method. Using the method of 阚琳娜 et al (in vivo screening of cucumber brown spot control agents, Chinese vegetables, 2007 (4): 22-24), the cucumber target spot bacteria solution was prepared at a concentration of 1×10 ⁵ spores/mL, and the blood cell count plate was determined. Spore concentration. The suspension suspension was sprayed in the cucumber seedling stage, and the prepared suspension liquid was uniformly sprayed on the cucumber leaf with a small hand-held sprayer, and the leaves were dripped with water. After inoculation, it was placed in a 25 ° C light incubator for moisturizing culture. Repeat 3 times and repeat 30 strains each time.

The disease was investigated 7 to 10 days after inoculation as described above. The grading criteria are: level 0: no lesions; level 1: lesion area accounts for less than 5% of the total leaf area; level 2: lesion area accounts for 5% to 25%; level 3: lesion area accounts for 26%~ 50%; Grade 4: 51% to 75% of the lesion area; Level 5: The area of the lesion is more than 75%. Among them, 2 or less are disease-resistant, and 3 or more are susceptible types.

Accurate statistics on the condition of the parents and groups.

C, preliminary location of cucumber target spot disease resistance gene Cca:

Use the high-density genetic map primer information published by Cavagnar et al. (Genome-wide characterization of simple sequence repeats in cucumber. BMC Genomics, 2010, 11:569) and the Indel and SNP primer information developed by the laboratory (see Table 1 for the serial number). For the primer information of 1-8, combined with the accurate statistics of the F _2:3 population disease index of 150 strains, the parental and progeny populations were subjected to molecular marker analysis, and the genotype data of the parents and the individual plants of the population were encoded. The maternal genotype is denoted as A, the paternal genotype is denoted as B, the F ₁ hybrid genotype is denoted as H, and the fuzzy or missing data is denoted as U. The data were subjected to proportional suitability analysis using χ ² test. The linkage map was constructed using JoinMap 4.0 software (Stam 1993; Van Ooijen 2001). The software LOD threshold was set to 4.0 and the Kosambi formula (Kosambi 1944) was selected to target the cucumber target. The disease gene was initially located in the 530 kb (Indel16624801 and Indel17156286) region of chromosome 6, according to Li et al. (RNA-Seq improves annotation of protein-coding genes in the cucumber genome. BMC genomics, 2011, 12: 540). Annotation and prediction by Softberry online software, there is a NBS-LRR tandem resistance gene (NBS-LRR is a conserved gene conserved domain) composed of multiple NBS-LRR resistance genes, which is difficult to predict and select candidate genes. . Therefore, it is necessary to further expand the group for fine positioning research.

D, fine mapping of cucumber target spot disease resistance gene Cca:

After the construction of more than 2000 F ₂ large populations, the target spot disease gene was further targeted for fine mapping. At the same time, the laboratory used the genome re-sequencing of the anti-infective parents, and through the bio-information analysis, developed Indel and SNP among the parents. Molecular markers were selected and 19 pairs of Indel molecular markers (see primers in Table 1 with sequence numbers 9-27) were designed for population analysis within 530 kb of the initial positioning interval. After verification, these 19 pairs of Indel markers were used to counter target spot disease. The gene was subjected to fine mapping studies, in which the synthesis of the primers was completed by Bioengineering Biotechnology (Shanghai) Co., Ltd., and finally finely mapped to 80 kb (Indel 16874230 and Indel 16953846) in the F ₂ large population of 2000 strains, see Figure 1. . There were 4 exchanged single plants in the test material of nearly 2200 strains in the fine positioning interval.

Each PCR reaction system (10 μL) using the above 19 pairs of Indel molecular markers was as follows: 25 ng of template DNA, 0.5 μM upstream primer, 0.5 μM downstream primer, 0.2 mM dNTP mix; 0.5 U Taq DNA polymerase, 1× PCR Buffer (Fermentas) 2 μL, ddH2O was made up to 10 μL.

The PCR reaction procedure was as follows: Stage 1: Pre-denaturation at 95 °C for 3 min; Stage 2: 94 ° C for 30 s, 60 ° C for 1 min, 72 ° C for 1 min, annealing temperature decreased by 1 ° C per cycle for a total of 8 cycles; Stage 3: 94 ° C for 30 s, 53 ° C 30 s, 72 ° C 1 min, a total of 32 cycles; Stage 4: 72 ° C extension 5 min; Stage 5: 4 ° C preservation; wherein the PCR instrument is purchased from Applied Biosystems, Veriti 96well Thermal Cycler.

Table 1 Cca gene localization part molecular marker information

E, anti-target spot disease gene prediction analysis:

In the finely mapped 80 kb interval, there are 3 genes, which are analyzed by NCBI Blast online. The first gene is the NB-ARC gene, the second gene is the BIM2-like transcription factor-related gene, and the third gene is 33-like peptide repeats structural protein-related genes. NB-ARC is a conserved domain commonly found in NBS-LRR resistance genes, so the NB-ARC gene is located as an important candidate gene Cca against target spot disease. The results of fine mapping excluded the interference of the NBS-LRR tandem resistance gene in the initial localization interval. The Cca gene has a SNP between the anti-/inductive materials. At the 1481th position of the gene, the resistance gene base is G, and the susceptible gene base is T. It is speculated that this SNP may lead to a change in disease resistance and can be used to develop a tightly linked molecular marker against target spot disease, and the NB-ARC gene is a candidate gene for disease resistance of target spot disease.

2. Obtaining the full-length DNA sequence of the disease resistance gene Cca of cucumber target spot disease

A. Test materials:

The test material was WI2757, a disease-resistant material, and the sensitive material Xintai Mickey was used as a control.

B. Amplification of the full-length DNA of the cucumber target spot disease resistance gene Cca:

Cucumber genomic DNA was extracted by CTAB method, and the full-length PCR amplification of the upstream and downstream primers SEQ ID NO: 7 and SEQ ID NO: 8 was performed using the Cca gene full-length amplification primers SEQ ID NO: 7 and SEQ ID NO: The full length of the gene of the Cca gene on the anti-sense material was SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the full length was 3255 bp. The full length of the Cca gene of the DNAMAN software was used to analyze the full length of the Cca gene. It was found that there is a SNP at the 1481th position of the Cca gene in the anti-infective material, and the base in the disease-resistant material is G, and the base in the susceptible material For T. The SNP can be used as a molecular marker closely linked to Fusarium wilt, and is applied to the breeding of cucumber target spot disease in the world.

The reaction system of the PCR amplification reaction (20 μL) contains: 25 ng of template DNA, 0.5 μM upstream primer, 0.5 μM downstream primer, 0.2 mM dNTP mix; 0.5 U Taq DNA polymerase, 1×PCR Buffer (Fermentas) 2 μL, ddH ₂ O was made up to 20 μL.

The PCR reaction procedure was: pre-denaturation at 95 ° C for 3 min; 94 ° C for 30 s, 60 ° C for 1 min, 72 ° C for 4 min, for a total of 42 cycles; 72 ° C for 10 min; the PCR instrument was purchased from Applied Biosystems, Veriti 96well Thermal Cycler.

The amplified product was diluted to 80 μL with sterilized deionized water and analyzed by a Caliper nucleic acid automatic analyzer.

The upstream and downstream primers SEQ ID NO: 7 and SEQ ID NO: 8, PCR product sequencing and sequence splicing of the full-length DNA sequence were synthesized and analyzed by Shanghai Biotech Co., Ltd. The sequencing results showed that the full-length DNA sequence of the cucumber target spot disease resistance gene Cca is as shown in SEQ ID NO: 1 in the sequence listing, which is 3255 bp in total.

Example 2: Identification and identification of cDNA and protein sequences of cucumber target spot disease resistance gene Cca

1. Full-length amplification of cDNA (mRNA) of cucumber withered target spot disease gene Cca:

Using the anti-inductive parent material WI2757 and Xintai thorn, the total RNA was extracted from the young leaves, and the first strand (cDNA) of mRNA was synthesized by reverse transcription. The full length of the Cca gene was used as the template. Amplification of the upstream and downstream primers SEQ ID NO: 7 and SEQ ID NO: 8 for PCR amplification, respectively obtaining cDNA (mRNA) sequences in the anti-sense material, SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The full length of the Cca gene of the DNAMAN software was used to compare and analyze the full length of the Cca gene. It was found that the gene has only one exon in the anti-/insensitive material, no intron structure, but resists disease at the position of 981 bp of the cDNA sequence. The base of the material is G, and the base of the susceptible material is T, which is due to the difference in bases in the anti-inductive material, resulting in a difference in amino acid coding, resulting in different functions, which in turn leads to NB- The disease resistance of the ARC gene changes.

The RNA extraction and reverse transcription kits were purchased from Fermentas.

The reaction system of the PCR amplification reaction (50 μL) contains: 25 ng of template DNA, 0.5 μM upstream primer, 0.5 μM downstream primer, 0.2 mM dNTP mix; 0.5 U Taq DNA polymerase, 1×PCR Buffer (Fermentas) 5 μl, ddH ₂ O was made up to 50 μl.

The PCR reaction procedure was: pre-denaturation at 95 ° C for 3 min; 94 ° C for 30 s, 60 ° C for 1 min, 72 ° C for 2 min, for a total of 40 cycles; 72 ° C for 7 min; the PCR instrument was purchased from Applied Biosystems, Veriti 96well Thermal Cycler.

The PCR product sequencing and sequence splicing of the Cca gene cDNA sequence were analyzed by Shanghai Biotech Co., Ltd., wherein the cDNA sequence of the Cca gene in the disease resistant material is shown in SEQ ID NO: 3, and the Cca gene in the susceptible material. The cDNA sequence is SEQ ID NO: 4.

2. Sequence analysis of the encoded protein of the cucumber target spot disease resistance gene Cca:

The amino acid sequence of the cucumber target spot disease resistance gene of the present invention is obtained by translating the cDNA (mRNA) sequences of the Cca gene in the anti-sense material with SEQ ID NO: 3 and SEQ ID NO: 4, respectively, through softberry online prediction software. The Cca amino acid (protein) sequences in the anti-sense material are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and the DNAMAN software amino acid sequence is used for the alignment analysis in the anti-inductive material, and the gene Cca coding is found. There is one amino acid coding difference in the anti-/infective material, which is shown in the 493th (KN). It is speculated that this difference leads to a change in the disease resistance domain, resulting in a change in the disease resistance of the NB-ARC gene.

Example 3: Functional verification of disease resistance gene Cca and its application in breeding resistant varieties of cucumber target spot disease

1. Materials to be tested: WI2757 and GY 14 were used as parents to construct F ₂ genetic population, and 120 individuals were randomly selected for target spot disease identification and sequencing. Among them, the male parent GY 14 and the female parent WI2757 were purchased from the Beijing Crop Germplasm Resource Bank.

2. The seeds of the above-mentioned materials to be tested are wrapped with gauze, soaked in warm water, incubated at 28 °C, and then sown in 50-well trays. The seedlings are sterilized in the air-conditioned greenhouse, and the seedling substrate is sterilized perlite or nutrient soil, and then CTAB method is used. The genomic DNA of each test material was extracted from the seedlings, and the full-length PCR amplification of the Cca gene was carried out by using the genomic DNA as a template, using the upstream and downstream primers SEQ ID NO: 7 and SEQ ID NO: 8, and then the PCR product was sequenced and subjected to The sequence was spliced, and it was finally confirmed that 34 of the above 120 test materials had the Cca gene shown by SEQ ID NO: 1. See the second part of Example 1 for the PCR amplification.

3. Detection of target spot disease resistance in seedlings having the Cca gene represented by SEQ ID NO: 1.

The above 34 strains of the Cca gene having the SEQ ID NO: 1 were subjected to field target spot disease resistance test by the method of the first step B of Example 1.

Through statistical analysis of the disease, it was found that the above-mentioned seedlings having the Cca gene represented by SEQ ID NO: 1 were all below grade 2, of which 12 strains were grade 0, 13 strains were grade 1 and 9 strains were grade 2, indicating that The presence of the Cca gene set forth in SEQ ID NO: 1 is consistent with cucumber resistance to target spot traits.

Example 4: Acquisition and functional verification of dCAPs-labeled CcaSNP1 co-segregating with cucumber target leaf disease resistance gene Cca

First, the acquisition of dCAPs labeled CcaSNP1

It can be seen from Example 1 that the Cca gene has a SNP between the 1481th position of the anti-sense material, the base of the disease-resistant material is G, and the base of the susceptible material is T, and the SNP site is used, according to dCAPs. Primer online design website http://helix.wustl.edu/dcaps/dcaps.html, developed a dCAPs molecular marker that is closely linked to the anti-/Sensor target spot, named CcaSNP1, and its upstream and downstream primers such as SEQ ID in the sequence listing. NO: 9 and SEQ ID NO: 10.

PCR amplification was carried out by using the above primer pairs (SEQ ID NO: 9 and SEQ ID NO: 10 in the Sequence Listing) as genomic DNA of the amphiphilic material (resistant material WI2757 and susceptible material Xintai thorn) as a template, and combined with MaeII The endonuclease digested the PCR product to obtain an anti-pathogenic band, of which the resistance band was 165 bp and the susceptible band was 135 bp.

2. Verification of the dCAPs marker CcaSNP1 co-segregating with the target spot disease resistance gene Cca

Using the above primer pairs (SEQ ID NO: 9 and SEQ ID NO: 10 in the Sequence Listing), 150 strains of F _2:3 population lines constructed in the first step A of Example 1, and 2000 early cucumbers in the F ₂ genetic population were used. The material was subjected to PCR and restriction analysis, that is, genomic DNA of 150 strains of F _2:3 population lines and 2000 early F ₂ genetic populations in the F ₂ genetic population was used as a template, and the above primers were used for PCR reaction, and then the PCR product was subjected to MaeII. Endonuclease digestion showed that the digested product was electrophoresed on a 2% agarose gel. If there is a 165 bp resistant strip in the digested product, the strain is a candidate resistant plant, such as 135 bp in the digested product. The susceptible strips of this strain did not have cucumber target spot resistance, and some electrophoresis results are shown in Fig. 2; and the disease resistance of the 2150 cucumber materials was identified in the field according to the method of the first part of the first part of Example 1, The results showed that the primers paired with the above dCAPs molecular marker CcaSNP1 had an anti-disease rate of 100% in the identification and field identification results, that is, the dCAPs molecular marker in the Cca gene was closely linked to the resistance phenotype of 2150 cucumber materials, and the agreement reached 100. %, achieving total separation Level. It is fully confirmed that the dCAPs molecular markers developed by this SNP site can be applied to target spot disease resistance/susceptibility screening and molecular-assisted breeding research.

The PCR reaction system and the PCR reaction procedure in this example are the same as those in the first part of Example 1.

The digestion system (30 μl) contained: 10 μl of PCR amplification product, 2 μl of 10×Buffer R (purchased from Thermo), 1.0 μl of MaeII (purchased from Thermo), and 30 μl of sterilized double distilled water.

The enzymatic cleavage reaction procedure is: stage 1: 65 ° C incubation for 10 h; stage 2: 80 ° C for 20 min inactivation; stage 3: 4 ° C preservation.

Example 5: Identification of target spot resistance of cucumber material using dCAPs molecular marker CcaSNP1

1. Materials to be tested: WI2757 and Jinyan No. 2 were used as parents to construct the F2 genetic population, and 100 individuals were randomly selected for the following experiments. Among them, the father Jinyan No. 2 and the female parent WI2757 were purchased from the Beijing Crop Germplasm Resource Bank.

2. The seeds of the above-mentioned materials to be tested are wrapped with gauze, soaked in warm water, incubated at 28 °C, and then sown in 50-well trays. The seedlings are sterilized in the air-conditioned greenhouse, and the seedling substrate is sterilized perlite or nutrient soil, and then CTAB method is used. The genomic DNA is extracted from the seedlings of the test material, and the genomic DNA of the above-mentioned test material is used as a template, and the upstream and downstream primers SEQ ID NO: 9 and SEQ ID NO: 10 are used for PCR amplification, and the PCR product is subjected to MaeII incision. Enzyme digestion showed that the digested product was electrophoresed on a 2% agarose gel. If there is a 165 bp resistant strip in the digested product, the strain is a candidate resistant plant, such as a 135 bp in the digested product. In the diseased band, the strain does not have cucumber target spot resistance, and some electrophoresis results are shown in Fig. 4; at the same time, the disease resistance of the cucumber materials to be tested is subjected to field identification according to the method of the first part of the first part of the first embodiment, and the results are found. The primer pair of the above dCAPs molecular marker CcaSNP1 was used to assist the identification and the field identification results were 100%.

The enzyme digestion system and reaction procedure in this example were the same as in Example 4.

Example 6: Acquisition and verification of dCAPs labeled CcaSNP2 co-segregating with cucumber target spot disease resistance gene Cca

The specific obtaining method includes the following steps:

A. Construction of genetic population

BANNA HUANGGUA (mother) and Changchun thorn (parent) were selected as anti-infective parents, and 150 RIL-F ₈ strains and 2000 F ₂ populations were constructed. Among them, the father of Changchun Mia and the female parent BANNA HUANGGUA were purchased from the Beijing Crop Germplasm Resource Bank.

B. Acquisition of dCAPs labeled CcaSNP2 co-segregating with the disease resistance gene Cca

The present invention performs genomic sequence resequencing of the above-mentioned anti-infective parents, and uses the biological information software BWA-Samtools (v0.1.12a, mpileup according to default parameters) and the re-sequencing sequence of the BCFTOOLS antagonistic material to perform the alignment analysis, and the sequence amplification SANGER Sequencing verification confirmed that the full-length sequence of the Cca gene of the anti-inductive material was obtained, and the sequence difference analysis of DNAStar was used to obtain the SNP locus of Cca gene in different materials. Combined with the cucumber genome 9930-V2 and the genome annotation, 210 sites of Cca gene were found. There is a single base difference SNP2 site (from G to A), ie, the base of the susceptible material is G, and the base of the disease resistant material is A. Using this SNP locus, according to the dCAPs primer online design website http://helix.wustl.edu/dcaps/dcaps.html, the dCAPs molecular markers closely linked to the anti-sensory spot disease were developed and the CcaSNP2 molecular marker was obtained. The upstream and downstream primers are shown in SEQ ID NO: 11 and SEQ ID NO: 12 in the Sequence Listing.

PCR amplification was carried out by using the above primer pairs (SEQ ID NO: 11 and SEQ ID NO: 12 in the Sequence Listing) as genomic DNA of the amphiphilic materials (BANNA HUANGGUA and Changchun Mickey) as a template, and combined with XbaI endonuclease The PCR product was digested to obtain an anti-pathogenic band, of which the resistance band was 164 bp and the susceptible band was 134 bp.

C. Verification of dcas-labeled CcaSNP2 co-segregating with the target spot disease resistance gene Cca

PCR and restriction enzyme digestion of 150 RIL-F ₈ strains constructed in this example and 2000 early cucumber materials in the F ₂ genetic population using the above primer pairs (SEQ ID NO: 11 and SEQ ID NO: 12 in the Sequence Listing) The genomic DNA of 150 RIL-F ₈ strains and 2000 early F ₂ genetic populations in the F ₂ genetic population was used as a template to carry out PCR reaction using the above primer pairs, and then the PCR products were identified by XbaI endonuclease digestion. The digested product was electrophoresed on a 2% agarose gel. If there is a 164 bp resistant strip in the digested product, the strain is a candidate resistant plant, such as a 134 bp susceptible strip in the digested product. The strain did not have cucumber target spot resistance, and partial electrophoresis results are shown in Fig. 3; at the same time, the disease resistance of the 2150 cucumber materials was subjected to field identification according to the method of the first part of the first part of Example 1, and it was found that the above dCAPs were utilized. The molecular marker CcaSNP2 primer-pair identification and field identification results showed that the resistance rate reached 100%, that is, the dcas molecular marker in Cca gene was closely linked with 2150 cucumber disease resistance phenotypes, and the agreement reached 100%, achieving co-segregation. Level. It is fully confirmed that the dCAPs molecular markers developed by this SNP site can be applied to target spot disease resistance/susceptibility screening and molecular-assisted breeding research.

The digestion system (20 μl) contained: 2 μl of 10×NEB buffer (purchased from Thermo), 10 μl of PCR amplification product, 0.5 μl of endonuclease XbaI, and 20 μl of sterilized double distilled water.

The digestion reaction procedure is: phase 1: 37 ° C incubation for 10 h; phase 2: 65 ° C for 20 min inactivation; stage 3: 4 ° C preservation.

Example: 7: Identification of target spot resistance of cucumber material using dCAPs molecular marker CcaSNP2

1. Materials to be tested: The DI 2 HUANGGUA and Jinyan 2 were used as parents to construct the F ₂ genetic population, and 100 individuals were randomly selected for the following experiments. Among them, the father Jinyan No. 2 and the female parent DI HUANGGUA were purchased from the Beijing Crop Germplasm Resource Bank.

2. The seeds of the above-mentioned materials to be tested are wrapped with gauze, soaked in warm water, incubated at 28 °C, and then sown in 50-well trays. The seedlings are sterilized in the air-conditioned greenhouse, and the seedling substrate is sterilized perlite or nutrient soil, and then CTAB method is used. The genomic DNA is extracted from the seedlings of the test material, and the genomic DNA of the above-mentioned test material is used as a template, and the upstream and downstream primers SEQ ID NO::11 and SEQ ID NO:12 are used for PCR amplification, and the PCR product is subjected to XbaI. The enzyme was digested and digested with 2% agarose gel. If there is a 164 bp resistant strip in the digested product, the strain is a candidate resistant plant, such as 134 bp in the digested product. In the susceptible strip, the strain does not have cucumber target spot resistance, and partial electrophoresis results are shown in Fig. 5; at the same time, the disease resistance of the cucumber materials to be tested is subjected to field identification according to the method of the first part of the first part of the first embodiment, As a result, it was found that the primer pair of the above-mentioned dCAPs molecular marker CcaSNP2 had an anti-disease coincidence rate of 100% with the help of the field identification.

The enzyme digestion system and reaction procedure in this example were the same as in Example 6.

Claims

The cucumber target spot disease resistance gene Cca, (a) is the nucleotide sequence shown by SEQ ID NO: 1 in the sequence listing, and (b) has 90% or more of the sequence shown by SEQ ID NO: 1 in the sequence listing. A nucleotide sequence which is functional and has the same function as the sequence shown by SEQ ID NO: 1 in the Sequence Listing.
The genetic marker of the cucumber target spot disease resistance gene Cca according to claim 1 (a), wherein the base T of the 1481th gene of the susceptible gene is mutated to the base G.
A pair of primers for amplifying the cucumber target spot disease resistance gene Cca according to claim 1 (a), which are the nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 in the Sequence Listing, respectively.
The cucumber target spot disease resistance gene Cca according to claim 1 or the genetic marker according to claim 2 for use in breeding a cucumber target spot disease resistant variety.
A cucumber target spot disease resistance chain molecular marker, which is one of nucleotide sequences amplified from total cucumber DNA by the following primers: SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing, in the sequence listing SEQ ID NO: 11 and SEQ ID NO: 12.
The primer for obtaining the cucumber target spot disease resistance chain-binding molecular marker of claim 5 is as follows: 1) The primer for obtaining the cucumber target spot disease resistance-linked molecular marker CcaSNP1 is SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing. 2) Primers for obtaining the cucumber target spot disease resistance-linked molecular marker CcaSNP2 are SEQ ID NO: 11 and SEQ ID NO: 12 in the Sequence Listing.
Use of the molecular marker of claim 5 or the primer of claim 6 for breeding an anti-cucumber target spot disease variety.
The application according to claim 7, characterized in that the specific method for the application of the molecular marker CcaSNP1 in breeding the cucumber target spot disease species is as follows: using the genomic DNA of the cucumber material to be tested as a template, in the sequence table Specific primers consisting of the nucleotides shown in SEQ ID NO: 9 and SEQ ID NO: 10 in the Sequence Listing were subjected to PCR amplification, and then the PCR product was digested with MaeII endonuclease, and if a 165 bp digested product was obtained, The cucumber to be tested is a disease-resistant material; if a 135 bp enzyme-cut product is obtained, the cucumber to be tested is a susceptible material.
The application according to claim 8, characterized in that the cucumber material to be tested is WI2757 or Changchun thorn, or the WS2757 and/or Changchun thorn as the progeny of the parent.
The application according to claim 7, characterized in that the specific method for the application of the molecular marker CcaSNP2 in breeding an anti-cucumber target spot disease is: using the genomic DNA of the cucumber material to be tested as a template, in the sequence table Specific primers consisting of the nucleotide composition shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the Sequence Listing were subjected to PCR amplification, and then the PCR product was digested with XbaI endonuclease, and if a 164 bp digested product was obtained, The cucumber to be tested is a disease-resistant material; if a 134 bp digested product is obtained, the cucumber to be tested is a susceptible material.
The application according to claim 10, characterized in that the cucumber material to be tested is BANNA HUANGGUA, or CUCUMIS HARDWICKII, or DI HUANGGUA, or a child obtained by using BANNA HUANGGUA and/or CUCUMIS HARDWICKII as a parent, or in DI HUANGGUA is the progeny of the parent, or DI HUANGGUA and CUCUMIS HARDWICKII as the progeny of the parent, or the progeny obtained by BANNA HUANGGUA and DI HUANGGUA as the parent.