A molecular marker DNKF765 for soybean resistance to Fusarium oxysporum root rot and its primers

Abstract

This invention discloses a molecular marker DNKF765 for soybean resistance to Fusarium oxysporum root rot and its primers, belonging to the field of plant disease resistance detection technology. This invention aims to provide a method for identifying soybean resistance to Fusarium oxysporum root rot. The invention provides a molecular marker related to Fusarium oxysporum root rot in soybean, located on chromosome 7 of soybean, with a G or T nucleotide at a 19,078,765 bp nucleotide position. This marker can perform genotyping of soybean materials, accurately identifying samples carrying the Fusarium oxysporum root rot resistance gene. The selected soybean materials, after inoculation with Fusarium oxysporum at the seedling stage, showed significantly lower disease indices than non-resistant soybean materials. In actual production, this marker can be used to effectively screen soybean breeding materials with Fusarium oxysporum root rot resistance genotypes.

Description

Technical Field

[0001] This invention belongs to the field of plant disease resistance detection technology, specifically involving a soybean molecular marker DNKF765 for resistance to Fusarium oxysporum root rot and its primers. Background Technology

[0002] Root rot is a soil-borne disease caused by a complex infection of multiple fungi, threatening global crop yields. Similarly, soybean root rot also impacts my country's food security. Fusarium oxysporum is one of the main pathogens of soybean root rot, with a wide pathogenic range and high virulence; it is also listed as one of the top ten plant pathogens, posing a significant threat to crop growth. Developing new soybean varieties resistant to Fusarium oxysporum root rot to reduce its occurrence is a potential way to increase total soybean yield. To improve the efficiency of soybean disease resistance breeding and accelerate the development of superior disease-resistant varieties, a rapid identification method is urgently needed.

[0003] Disease resistance is one of the main goals of soybean breeding. Fusarium oxysporum, a major pathogen of root rot, severely impacts soybean yield. Current techniques for identifying soybean resistance to Fusarium oxysporum root rot generally rely on artificial inoculation. This method is time-consuming, costly, and leaves pathogen residues that can severely affect the surrounding environment and even cause secondary infections in various plants. Therefore, a safe and rapid identification method is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for identifying resistance to soybean Fusarium root rot.

[0005] This invention provides a molecular marker associated with soybean Fusarium tumefaciens root rot, wherein the chromosome of the molecular marker is soybean chromosome 7, and the nucleotide position at 19078765 bp is G or T.

[0006] This invention provides a primer combination for amplifying the above-mentioned molecular marker, wherein the primer combination consists of 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] This invention provides a kit for detecting resistance to soybean Fusarium tumefaciens root rot, the kit comprising the primer combination described above.

[0008] To further specify, the kit shown also includes FLu-Arms 2XPCR Mix reagent.

[0009] This invention provides the application of the above-mentioned molecular markers or primer combinations in the preparation of a kit for detecting resistance to soybean Fusarium tumefaciens root rot.

[0010] This invention provides the application of the above-mentioned molecular markers or primer combinations in the breeding of soybeans resistant to Fusarium root rot.

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

[0012] Step 1: Extract DNA from the soybeans to be tested;

[0013] Step 2: Use the primer combination described above to perform a PCR reaction to detect the genotype of the soybean variety to be tested.

[0014] Further specifying the PCR reaction conditions in step 2, the conditions are: 95℃ pre-denaturation for 10 min; amplification cycle 1: 95℃ denaturation for 15 s, 61℃-55℃, -0.6℃ / cycles, annealing for 60 s, 10 cycles; amplification cycle 2: 95℃ denaturation for 15 s, 55℃ annealing for 60 s, 32 cycles.

[0015] To further specify, if the soybean genotype detected in step 3 is GG, then it is a soybean resistant to Fusarium oxysporum root rot; if the soybean genotype detected is TT, then it is a soybean not resistant to Fusarium oxysporum root rot.

[0016] To further specify, the molecular marker for the TT-type SNP is the 19078765th site on soybean chromosome 7, which is T, and the molecular marker for the GG-type SNP is the 19078765th site on soybean chromosome 7, which is G.

[0017] Beneficial Effects: This invention relates to a novel marker for soybean resistance to Fusarium oxysporum root rot, DNKF765 (marked on soybean chromosome 7, with a G or T nucleotide at a 19,078,765 bp nucleotide position) and its specific primers. This marker is a KASP marker associated with soybean resistance to Fusarium oxysporum root rot. By inoculating soybean seedlings with Fusarium oxysporum at the seedling stage, identifying the disease index, and combining resequencing data, genome-wide association analysis (GWAS) was performed to identify significantly associated SNP sites, and candidate genes were screened from those linked to these sites. Furthermore, extremely resistant and susceptible materials were selected from the natural population, and association analysis was performed on candidate genes based on their disease index and genome resequencing data, identifying a gene associated with soybean resistance to Fusarium oxysporum root rot (Glyma.07G155300) and its functional SNP site. The KASP marker DNKF765 was developed targeting this site. This marker can perform genotyping of soybean materials and accurately identify samples carrying the Fusarium oxysporum root rot resistance gene. The selected soybean materials showed significantly lower disease indices after inoculation with Fusarium oxysporum at the seedling stage compared to non-resistant soybean materials. In practical production, this marker can be used to effectively screen soybean breeding materials with Fusarium oxysporum root rot genotypes. Attached Figure Description

[0018] Figure 1 A frequency distribution chart of disease indices for 356 varieties;

[0019] Figure 2 Figure 1 shows the GWAS analysis results of soybean Fusarium oxysporum resistance.

[0020] Figure 3 The results of sequence alignment for soybean varieties;

[0021] Figure 4 The significance analysis results for the KASP typing of Chr.07:19078765 are as follows;

[0022] Figure 5 The following diagrams illustrate the classification results for KASP labeled DNKF765: A: Classification results for the first group of materials; B: Classification results for the second group of materials; C: Classification results for the third group of materials; D: Classification results for the fourth group of materials. Detailed Implementation

[0023] Example 1. Screening for molecular markers

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

[0025] Sorghum granules covered with mycelium were crushed to 1 / 3-1 / 5 of their original volume using a pulverizer and thoroughly mixed with sterile vermiculite at a ratio of 1:50 (V:V). Four soybean seeds were planted in culture pots filled with the inoculum and vermiculite mixture. Five pots were used for each variety. After soybean seedlings emerged, the number of seedlings was reduced to 3 per pot. This experiment was conducted using a randomized complete block design. The greenhouse environment was maintained at 25°C for 12 hours of light and 18°C ​​for 12 hours of darkness. Disease index was identified after 15 days. The disease grading criteria are shown in Table 1.

[0026]

[0027] Table 1 Disease Grading Criteria

[0028]

[0029] Results: Disease severity was graded 15 days after inoculation of a natural population of soybean germplasm (356 accessions), and the disease index of each population member was calculated. A lower disease index value indicated stronger resistance to Fusarium root rot. Based on... Figure 1 As shown, after inoculation with Fusarium oxysporum, the disease index of the population ranged from 5.35 to 73.62, with an average of 26.60. Furthermore, the disease index exhibited a continuous unimodal distribution, consistent with quantitative trait characteristics.

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

[0031] Resequencing was performed on a natural population of soybean germplasm resources (hereinafter referred to as the population), and whole-genome SNP data were obtained. Using the population's resequencing data and disease indices collected after inoculation of population members, R... 4.4.1 GWAS analysis was performed using mixed linear modeling (MLM) in the RStudio GAPIT 3.0 software package. Genes within a 50kb range upstream and downstream of the obtained SNP physical locations were annotated according to gene annotations in SoyBase (http: / / www.soybase.org). Simultaneously, 30 extremely resistant and 30 susceptible accessions were selected from the population, and general linear modeling (GLM) based on candidate genes was used for association analysis to further screen candidate genes. Superior allelic variants significantly associated with Fusarium oxysporum root rot (P-value greater than 0.05 was considered significantly associated) were screened, revealing… Glyma.07G155300 The gene SNPChr.07:19078765 was significantly associated with disease resistance and was used as a KASP marker.

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

[0033] Subsequently, using a 50kb range upstream and downstream of significant SNP sites as a confidence interval, 240 candidate genes were identified within the target interval that might be associated with Fusarium oxysporum root rot. Candidate gene association analysis was performed on the predicted candidate genes based on resequencing data, revealing... Glyma.07G155300 There are 7 significant ( P SNPs related to <0.05 (Table 2) were identified. Among them, Chr.07:19078765 was significantly associated with resistance to soybean tip Fusarium root rot (P<0.01) and was located in the promoter region. Therefore, this position was selected for further molecular marker development. Simultaneously, upstream and downstream primers were designed for this site. PCR amplification of this site was successfully performed using these primers, with a PCR product length of 379 bp. First-generation sequencing was used to verify the existence of this site. Figure 3 , Figure 4 ).

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

[0035]

[0036] 3. Development and validation of the KASP marker for locus Chr.07:19078765

[0037] Using the Wm82.a2.v1 reference genome from the Phytozome database (https: / / phytozome-next.jgi.doe.gov), the target gene sequence was downloaded and targeted. Glyma.07G155300 Using Premier 5.0 software, primers containing a sequence of the target site were designed at the Chr.07:19078765 site (Table 3).

[0038] Table 3 Primer Information

[0039]

[0040] Using 30 pairs of extreme resistant materials as test samples, DNA was extracted from the test samples, 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 considered a qualified sample.

[0041] PCR amplification was performed using designed primers and DNA from the test samples. The amplification program was as follows: 98 ℃ pre-denaturation for 3 min; amplification cycles: 98 ℃ denaturation for 10 s, 55.0 ℃ annealing for 5 s, 68 ℃ extension for 5 s, for a total of 35 cycles; 68 ℃ extension for 10 min; and incubation at 4 ℃. The PCR amplification products were subjected to first-generation sequencing to determine the authenticity of superior allelic variants and their association with disease resistance. Figure 3 , Figure 4 ).

[0042] Table 4 PCR Reaction System

[0043]

[0044] Using the significant SNP site Chr.07:19078765 as the target site, primers for the KASP marker (DNKF765) were designed using Primer Premier 5.0 software, including two specific primers and one universal primer. The two specific primers each contained two allelic variations of the site at their terminal bases, and different fluorescent adapters were added to the 5′ ends of the primers (Table 5). The reaction system is shown in Table 6. The reaction procedure was as follows: Stage 1: Observation, temperature set: 30.0 ℃, hold for 30 s. Stage 2: PCR reaction, Step 1: Temperature set: 95.0 ℃, hold for 15 s. Step 2: Temperature set: 61.0 ℃, decrease to 55 ℃. 10 cycles, with the temperature decreasing by 0.6 ℃ per cycle. Stage 3: Amplification, Step 1: Temperature set: 95.0 ℃, hold for 15 s. Step 2: Temperature set: 55.0 ℃, hold for 60 s. 28–35 cycles. Stage 4: Read the plate, set the temperature to 30.0 ℃, and hold for 30 s.

[0045] Table 5 KASP-labeled primer sequences

[0046]

[0047] Table 6 KASP Reaction System

[0048]

[0049] Genotyping of the population was performed using KASP markers, and resistance to Fusarium root rot was analyzed in different types of germplasm to determine the effectiveness of the KASP markers. Figure 5 ).

[0050] Result: Based on Glyma.07G155300 A KASP marker was developed at the Chr.07:19078765 site and named DNKF765. For example... Figure 5 As shown, this marker was used to genotype soybean materials (356 accessions, divided into 4 groups) used in the genome-wide association analysis. The marker clearly identified the G / T allele variation at the Chr.07:19078765 locus, accurately and clearly clustering experimental materials with the same genotype. Blue dots near the Y-axis represent homozygous genotypes of TT, and red dots near the X-axis represent homozygous genotypes of GG. Among all experimental materials, soybean materials carrying the GG homozygous genotype accounted for 5.9%, with an average disease index of 26.1; soybean materials carrying the TT homozygous genotype accounted for 94.1%, with an average disease index of 35.4. The disease index of germplasm carrying the GG genotype was significantly lower than that of germplasm carrying the TT genotype.

[0051] Example 2. Kit for identifying resistance to soybean Fusarium tumefaciens root rot

[0052] I. The primer combination is as follows: upstream primer as shown in SEQ ID NO.3 or SEQ ID NO.4; downstream primer as shown in SEQ ID NO.5.

[0053] 2. Step 1: Extract DNA from the soybean seedling roots (this can be done at any stage).

[0054] Step 2: Use the primer combination described in Step 1 to perform a PCR reaction to detect the genotype of the soybean variety to be tested.

[0055] The reaction procedure is as follows: Stage 1: Observation, temperature set at 30.0 ℃, hold for 30 s. Stage 2: PCR reaction, step 1: temperature set at 95.0 ℃, hold for 15 s. Step 2: temperature set at 61.0 ℃, then decrease to 55 ℃. Repeat 10 times, decreasing the temperature by 0.6 ℃ per cycle. Stage 3: Amplification, step 1: temperature set at 95.0 ℃, hold for 15 s. Step 2: temperature set at 55.0 ℃, hold for 60 s. Repeat 28-35 times. Stage 4: Plate reading, temperature set at 30.0 ℃, hold for 30 s.

[0056] If the soybean genotype is GG, it is resistant to Fusarium oxysporum root rot; if the soybean genotype is TT, it is not resistant to Fusarium oxysporum root rot.

[0057] To test soybean samples for unknown resistance to Fusarium oxysporum root rot, samples labeled with primers of the GG type were identified as resistant to Fusarium oxysporum root rot, while those with the TT genotype were identified as non-resistant. The identified varieties were then evaluated for resistance to Fusarium oxysporum root rot, and the test results were consistent with the genotype results.

Claims

1. The application of a primer combination for detecting molecular markers of soybean resistance to Fusarium oxysporum root rot in the preparation of a kit for detecting soybean resistance to Fusarium oxysporum root rot, characterized in that, The molecular marker is an SNP site on chromosome 7 of soybean, with the reference genome being Wm82.a2.v1, where the 19078765 bp site is either G or T.

2. The application according to claim 1, characterized in that, The primer combination consists of an upstream primer and a downstream primer, the upstream primers being shown in SEQ ID NO.3 and SEQ ID NO.4; and the downstream primer being shown in SEQ ID NO.

5.

3. The application of the primer combination according to claim 1 or 2 in the breeding of soybeans resistant to Fusarium wilt, characterized in that, If the soybean genotype is GG, it is resistant to Fusarium oxysporum root rot; if the soybean genotype is TT, it is not resistant to Fusarium oxysporum root rot.

4. 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 DNA from the soybeans 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 tested; if the detected soybean genotype is GG, it is a soybean resistant to Fusarium oxysporum root rot; if the detected soybean genotype is TT, it is a soybean not resistant to Fusarium oxysporum root rot.

5. The method according to claim 4, characterized in that, The PCR reaction conditions in step 2 are: 95℃ pre-denaturation for 10 min; amplification cycle 1: 95℃ denaturation for 15 s, 61℃-55℃, -0.6℃ / cycles, annealing for 60 s, 10 cycles; amplification cycle 2: 95℃ denaturation for 15 s, 55℃ annealing for 60 s, 32 cycles.

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