Soybean fusarium oxysporum root rot resistant molecular marker DNKF765 and primers thereof

Abstract

The invention discloses a soybean fusarium oxysporum root rot resistant molecular marker DNKF765 and primers thereof, and belongs to the technical field of plant disease resistance detection. The invention provides a method for identifying soybean fusarium oxysporum root rot resistance. The invention provides a soybean fusarium oxysporum root rot related molecular marker, the chromosome of the molecular marker is on the No.7 chromosome of soybean, and the locus of the nucleotide at 19078765bp is G or T. The marker can be used for performing genotyping on soybean materials and accurately identifying a sample carrying a fusarium oxysporum root rot resistant gene. After the screened soybean material is inoculated with fusarium oxysporum in the seedling stage, the disease index is obviously lower than that of the soybean material without resistance. In actual production, the marker can be used for effectively screening soybean breeding materials with fusarium oxysporum root rot resistant genotypes.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease resistance detection, and specifically relates to a soybean Fusarium oxysporum root rot resistance molecular marker DNKF765 and a primer thereof. Background Art

[0002] Root rot is a soil-borne disease in which multiple fungi infect plants, threatening global crop yields. Similarly, the occurrence of soybean root rot also affects my country's food security. Fusarium oxysporum is one of the main pathogens of soybean root rot. It has a wide range of pathogenicity and strong pathogenicity. It is also listed as one of the top ten plant pathogens, posing a great threat to crop growth. Breeding new soybean varieties that are resistant to Fusarium oxysporum root rot and reducing the occurrence of soybean root rot are potential ways to increase total soybean production. In order to improve the efficiency of soybean disease-resistant breeding and accelerate the breeding process of excellent disease-resistant varieties, a rapid identification method is urgently needed.

[0003] Disease resistance is one of the main goals of soybean breeding. Fusarium oxysporum is the main pathogen of root rot and has a serious impact on soybean yield. The existing technology for identifying soybean Fusarium oxysporum root rot resistance generally uses artificial inoculation identification, which has a long identification cycle and high labor costs. In addition, the residues left after inoculation and identification of pathogens will seriously affect the surrounding environment and even cause secondary infection to a variety of plants. A safe and rapid identification method is urgently needed. Summary of the invention

[0004] The purpose of the present invention is to provide a method for identifying resistance to soybean fusarium root rot.

[0005] The invention provides a molecular marker related to soybean fusarium root rot. The chromosome of the molecular marker is chromosome 7 of soybean, and the site of the nucleotide at 19078765 bp is G or T.

[0006] The present invention provides a primer combination for amplifying the above-mentioned molecular marker, wherein the primer combination comprises an upstream primer as shown in SEQ ID NO.3 or SEQ ID NO.4; and a downstream primer as shown in SEQ ID NO.5.

[0007] The invention provides a kit for detecting resistance to soybean fusarium root rot, and the kit comprises the primer combination.

[0008] Further defined, the kit also includes FLu-Arms 2XPCR Mix reagent.

[0009] The present invention provides the use of the above molecular marker or the above primer combination in preparing a kit for detecting resistance to soybean Fusarium oxysporum root rot.

[0010] The present invention provides the use of the above molecular marker or the above primer combination in screening soybean breeding for resistance to Fusarium oxysporum root rot.

[0011] The present invention provides a method for identifying soybean resistance to Fusarium oxysporum root rot, and the specific steps of the method are as follows:

[0012] Step 1: Extract the DNA of the soybean to be tested;

[0013] Step 2: Perform a PCR reaction using the primer combination described in claim 2 to detect the genotype of the soybean variety to be detected.

[0014] It is further defined that the PCR reaction conditions in step 2 are pre-denaturation at 95°C for 10 min; amplification cycle 1: denaturation at 95°C for 15 s, 61°C-55°C, -0.6°C / cycles, annealing for 60 s, 10 cycles; amplification cycle 2: denaturation at 95°C for 15 s, annealing at 55°C for 60 s, 32 cycles.

[0015] It is further defined that in step 3, if the genotype of the detected soybean is GG, the soybean is resistant to Fusarium oxysporum root rot, and if the genotype of the detected soybean is TT, the soybean is not resistant to Fusarium oxysporum root rot.

[0016] It is further defined that the TT-type SNP molecular marker is T at position 19078765 on chromosome 7 of soybean, and the GG-type SNP molecular marker is G at position 19078765 on chromosome 7 of soybean.

[0017] Beneficial effects: The present invention relates to a new marker DNKF765 for soybean resistance to Fusarium oxysporum root rot (the marker chromosome is chromosome 7 of soybean, and the site of the nucleotide at 19078765bp is G or T) and its dedicated primers. The marker is a KASP marker related to soybean resistance to Fusarium oxysporum root rot. By inoculating Fusarium oxysporum on the natural soybean population at the seedling stage, identifying the disease index, combining resequencing data, performing genome-wide association analysis (GWAS), mining significantly associated SNP sites, and screening candidate genes from genes linked to the site. Further, extremely resistant materials are selected from the natural population, and association analysis is performed on candidate genes based on their disease index combined with genome resequencing data, mining soybean Fusarium oxysporum root rot resistance-related genes (Glyma.07G155300) and functional SNP sites on the genes, and a KASP marker developed for the site, namely DNKF765. The marker can perform genotyping on soybean materials and accurately identify samples carrying genes for resistance to Fusarium oxysporum root rot. The disease index of the selected soybean materials after inoculation with Fusarium oxysporum at the seedling stage was significantly lower than that of the soybean materials without resistance. In actual production, the marker can be used to effectively screen soybean breeding materials with Fusarium oxysporum root rot resistance genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the frequency distribution result of disease index of 356 varieties;

[0019] Figure 2 This is the result of GWAS analysis of soybean Fusarium oxysporum resistance;

[0020] Figure 3 This is the result of sequence alignment of soybean varieties;

[0021] Figure 4 This is the significance analysis result of Chr.07:19078765KASP typing result;

[0022] Figure 5 These are the typing results of KASP marker DNKF765; A: typing results of the first group of materials; B: typing results of the second group of materials; C: typing results of the third group of materials; D: typing results of the fourth group of materials. DETAILED DESCRIPTION

[0023] Example 1. Screening of molecular markers

[0024] 1. Identification of resistance to Fusarium oxysporum root rot and statistics of disease index

[0025] Take the sorghum particles covered with mycelium and crush them to 1 / 3-1 / 5 of the original volume with a crusher, mix them thoroughly with sterile vermiculite at a ratio of 1:50 (V:V), and plant 4 soybean seeds in a culture pot filled with the inoculum and vermiculite mixture. Five pots were used for each variety. After the soybean seedlings emerged, the seedlings were reduced to 3 plants / pot. The experiment was conducted using a randomized complete block design. The greenhouse environment was maintained at 25°C for 12 hours of light / 18°C for 12 hours of darkness, and the disease index was identified after 15 days. The disease grading standards are shown in Table 1.

[0026] Disease index

[0027] Table 1 Disease classification standards

[0028]

[0029] Results: The disease of the natural population of soybean germplasm (356 germplasms in total) was graded 15 days after inoculation with Fusarium oxysporum, and the disease index of the population members was calculated. The smaller the disease index value of soybean, the stronger the resistance to Fusarium oxysporum root rot. Figure 1 As shown, after the population was inoculated with Fusarium oxysporum, the disease index ranged from 5.35 to 73.62, with an average of 26.60. At the same time, the disease index was distributed in a continuous single peak shape, which was consistent with the quantitative trait characteristics.

[0030] 2. Mining of functional loci and candidate genes related to Fusarium oxysporum root rot

[0031] The natural population of soybean germplasm resources (hereinafter referred to as the population) was resequenced to obtain the whole genome SNP data. The resequencing data of the population and the disease index of the population members after inoculation were used to analyze the disease index of the population members using R 4.4.1 GWAS analysis was performed using the mixed linear model (MLM) in the software and Rstudio software GAPIT 3.0 package. Genes within 50 kb upstream and downstream of the obtained SNP physical position were annotated according to the gene annotation in SoyBase (http: / / www.soybase.org). At the same time, 30 extremely resistant and susceptible materials were selected from the population, and the general linear model (GLM) of TASSEL 5.0 software was used to perform candidate gene-based association analysis to further screen candidate genes. The excellent allele variants that were significantly associated with Fusarium root rot (P value greater than 0.05, i.e., considered significantly associated) were screened, and it was found that SNP Chr.07:19078765 on the Glyma.07G155300 gene was significantly associated with disease resistance and was used for KASP markers.

[0032] Results: First, a genome-wide association analysis was performed based on the population resequencing results (including 1,355,930 SNPs) combined with the disease index obtained after population vaccination. Figure 2 ), mining SNPs significantly associated with soybean Fusarium root rot (-log 10 P>4), and finally the one located on chromosome 7, Chr.07:19078765, was selected.

[0033] Subsequently, the 50kb range upstream and downstream of the significant SNP site was used as the confidence interval. A total of 240 candidate genes in the target interval may be related to Fusarium root rot. Candidate gene association analysis was performed on the predicted candidate genes based on the resequencing data, and it was found that Glyma.07G155300 had 7 significantly (P<0.05) related SNPs (Table 2), among which Chr.07:19078765 was extremely significantly correlated with soybean Fusarium root rot resistance (P<0.01) and was located in the promoter region, so this position was selected for the next step of molecular marker development. At the same time, upstream and downstream primers were designed for this site, and the site could be successfully PCR amplified using the primers. The PCR product length was 379bp, which was verified by first-generation sequencing to confirm that the site really existed ( Figure 3 , Figure 4 ).

[0034] Table 2 SNPs in candidate genes significantly associated with Fusarium root rot

[0035]

[0036] 3. Development and validation of KASP markers at Chr.07:19078765

[0037] The target gene sequence was downloaded using the Wm82.a2.v1 reference genome in the Phytozome (https: / / phytozome-next.jgi.doe.gov) database, and a primer containing the sequence of the target site was designed using Premier5.0 software for the Chr.07:19078765 site on Glyma.07G155300 (Table 3).

[0038] Table 3 Primer information

[0039]

[0040] 30 pairs of extremely resistant materials were used as test samples, DNA of the test samples was extracted, and the concentration and quality of the DNA samples were tested. The reference standard was that the value range of OD 260 / OD 280 was 1.6-2.2, which was a qualified sample.

[0041] PCR amplification was performed using the designed primers and the DNA of the sample to be tested. The amplification procedure was as follows: 98°C pre-denaturation for 3 min; amplification cycle: 98°C denaturation for 10 s, 55.0°C annealing for 5 s, 68°C extension for 5 s, for a total of 35 cycles; 68°C extension for 10 min; 4°C incubation. The PCR amplification product was sequenced to determine the authenticity of the superior allele variation and its association with disease resistance ( Figure 3 , Figure 4 ).

[0042] Table 4 PCR reaction system

[0043]

[0044] The significant SNP site Chr.07:19078765 was used as the target site. The primers for KASP marker (DNKF765) were designed using Primer Premier 5.0 software, including 2 specific primers and 1 universal primer. The terminal bases of the 2 specific primers included the 2 allelic variations of the site, and different fluorescent linkers were added to the 5′ end of the primers (Table 5). The reaction system is shown in Table 6. The reaction procedure is as follows: Phase 1 observation, set the temperature: 30.0℃, keep for 30s. Phase 2 PRC reaction, step 1: set the temperature: 95.0℃, keep for 15s. Step 2: set the temperature: 61.0℃, drop to 55℃. Cycle 10 times, the temperature drops by 0.6℃ each cycle. Phase 3 amplification, step 1: set the temperature: 95.0℃, keep for 15s. Step 2: set the temperature: 55.0℃, keep for 60s. 28 to 35 cycles. Read the plate in stage 4, set the temperature to 30.0°C and hold for 30 seconds.

[0045] Table 5 KASP marker primer sequences

[0046]

[0047] Table 6 KASP reaction system

[0048]

[0049]

[0050] The KASP marker was used to genotype the population and analyze the resistance of different types of germplasm to Fusarium root rot to determine the effectiveness of the KASP marker. Figure 5 ).

[0051] Results: A KASP marker was developed based on the Chr.07:19078765 locus of Glyma.07G155300 and named DNKF765. Figure 5 As shown in the figure, the marker was used to genotype the soybean materials (356 samples in total, divided into 4 groups) used in the whole genome association analysis. The marker can clearly identify the G / T allele variation at the Chr.07:19078765 site, accurately and clearly cluster the test materials with the same genotype. The blue dots close to the Y axis represent the homozygous genotype germplasm resources with the genotype result of TT, and the red dots close to the X axis represent the homozygous genotype germplasm resources with the genotype result of GG. Among all the test materials, 5.9% of the soybean materials carried the homozygous genotype of GG, with an average disease index of 26.1; 94.1% of the soybean materials carried the homozygous genotype of TT, with an average disease index of 35.4. The disease index of the germplasm resources carrying the genotype GG was significantly lower than that of the germplasm resources with the genotype of TT.

[0052] Example 2. Kit for identifying resistance to soybean Fusarium oxysporum root rot

[0053] 1. The primer combination is an upstream primer as shown in SEQ ID NO.3 or SEQ ID NO.4; and a downstream primer as shown in SEQ ID NO.5.

[0054] 2. Step 1: Extract the DNA of the soybean root system at the seedling stage of the soybean to be tested (can be achieved at any stage);

[0055] Step 2: Use the primer combination described in step 1 to perform PCR reaction to detect the genotype of the soybean variety to be tested.

[0056] The reaction procedure is as follows: Phase 1 observation, set temperature: 30.0℃, hold for 30s. Phase 2 PRC reaction, step 1: set temperature: 95.0℃, hold for 15s. Step 2: set temperature: 61.0℃, drop to 55℃. Cycle 10 times, temperature decrease 0.6℃ per cycle. Phase 3 amplification, step 1: set temperature: 95.0℃, hold for 15s. Step 2: set temperature: 55.0℃, hold for 60s. 28-35 cycles. Phase 4 plate reading, set temperature: 30.0℃, hold for 30s.

[0057] If the genotype of the tested soybean is GG, it is a soybean resistant to Fusarium root rot. If the genotype of the tested soybean is TT, it is a soybean not resistant to Fusarium root rot.

[0058] To detect unknown soybean samples resistant to Fusarium oxysporum root rot, the primers marked with GG type indicate soybeans resistant to Fusarium oxysporum root rot. If the tested soybean genotype is TT type, it is not resistant to Fusarium oxysporum root rot. The identified varieties were evaluated for Fusarium oxysporum root rot resistance, and the test results showed that the Fusarium oxysporum root rot resistance of soybeans was consistent with the results of the genotype.

Claims

1. A molecular marker related to soybean Fusarium oxysporum root rot, characterized in that: The chromosome of the molecular marker is chromosome 7 of soybean, and the nucleotide position at 19078765 bp is G or T.

2. A primer combination for amplifying the molecular marker according to claim 1, characterized in that: The primer combination is an upstream primer as shown in SEQ ID NO.3 or SEQ ID NO.4; and a downstream primer as shown in SEQ ID NO.

5.

3. A kit for detecting resistance to soybean Fusarium oxysporum root rot, characterized in that: The kit comprises the primer combination according to claim 2.

4. The kit according to claim 3, characterized in that The kit shown also includes FLu-Arms 2XPCR Mix reagent.

5. Use of the molecular marker according to claim 1 or the primer combination according to claim 2 in preparing a kit for detecting resistance to soybean Fusarium oxysporum root rot.

6. Use of the molecular marker according to claim 1 or the primer combination according to claim 2 in screening soybean breeding for resistance to Fusarium oxysporum root rot.

7. A method for identifying soybean resistance to Fusarium oxysporum root rot, characterized in that: The specific steps of the method are as follows: Step 1: Extract the DNA of the soybean to be tested; Step 2: Perform a PCR reaction using the primer combination described in claim 2 to detect the genotype of the soybean variety to be detected.

8. The method according to claim 7, characterized in that The PCR reaction conditions in step 2 are pre-denaturation at 95°C for 10 min; amplification cycle 1: denaturation at 95°C for 15 s, 61°C-55°C, -0.6°C / cycles, annealing for 60 s, 10 cycles; amplification cycle 2: denaturation at 95°C for 15 s, annealing at 55°C for 60 s, 32 cycles.

9. The method according to claim 7, characterized in that: In step 3, if the genotype of the detected soybean is GG, it is soybean resistant to Fusarium oxysporum root rot; if the genotype of the detected soybean is TT, it is soybean not resistant to Fusarium oxysporum root rot.

10. The method according to claim 9, characterized in that The TT-type SNP molecular marker is T at position 19078765 on chromosome 7 of soybean, and the GG-type SNP molecular marker is G at position 19078765 on chromosome 7 of soybean.

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