KASP marker primer set and identification method for wheat fusarium head blight resistance candidate gene TaRAR1

Abstract

The present invention provides a KASP marker primer set for wheat fusarium head blight resistance candidate gene TaRAR1, comprising a forward primer FAM, a reverse primer VIC and a common reverse primer, and the nucleotide sequence is shown in SEQ ID NO: 1-3. A method for identifying a wheat fusarium head blight resistance candidate gene is also provided, wherein the KASP marker primer set is used to perform PCR amplification on a test genomic DNA, and a Bio-rad CFX-96 instrument real-time PCR instrument is used to perform genotype identification of the KASP marker on the PCR product. The marker involved in the present invention is closely linked to the major effect QTL of wheat fusarium head blight resistance-QFhb-2DL, and can be used as a diagnostic marker of the resistance QTL site for fusarium head blight resistance assisted selection breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of identification of wheat fusarium resistant candidate genes, and particularly relates to a KASP marker primer set for wheat fusarium resistant candidate gene TaRAR1 and an identification method thereof. Background Art

[0002] Wheat (Triticum aestivum) is the second largest grain crop in my country. The sustainable development of wheat production is of great significance to ensuring national food security. Wheat head blight (Fusarium head blight, FHB) is a fungal disease caused by Fusarium graminearum, which seriously affects wheat yield and quality and is an important disease threatening wheat production worldwide. The occurrence and spread of wheat head blight not only seriously affects wheat yield and quality, but also wheat grains infected by pathogens can produce a variety of mycotoxins that can cause poisoning in humans and livestock, such as deoxynivalenol (DON) and zearalenone, which seriously affect food safety. Therefore, my country, the European Union (European Union), the US FDA (Food and Drag Administration) and many other countries and organizations have set strict DON maximum content standards for imported and exported wheat and related foods.

[0003] Compared with other leaf diseases, Fusarium head blight mainly invades wheat through the ear when it blooms, with a short invasion period and a fast onset. Since there is no specific medicine to effectively prevent and control wheat Fusarium head blight, the effective chemical prevention and control window period of wheat Fusarium head blight is short and is affected by adverse natural conditions such as rainy fields when the medicine is used, making chemical control of the disease difficult and the prevention and control effect is not ideal. At present, the most effective way to reduce the harm of wheat Fusarium head blight is to use host resistance and cultivate and plant disease-resistant varieties. Wheat Fusarium head blight resistance belongs to the quantitative trait type, which is greatly affected by the environment. It takes many years and multiple points to accurately identify the resistance phenotype. Therefore, in the breeding process, it is difficult and inefficient to identify wheat Fusarium head blight resistance based solely on phenotype. Marker-assisted selection is not affected by the environment, which can improve the selection accuracy in the breeding separation era and greatly improve the efficiency of Fusarium head blight resistance breeding.

[0004] Wheat fusarium head blight resistance belongs to the quantitative trait resistance type, which is controlled by quantitative trait loci (QTL). Although the number of QTLs related to wheat fusarium head blight has exceeded 250, which are distributed on 21 chromosomes of wheat, most of the located QTLs have small genetic effects, have not been verified, and the identified markers are far from the target QTL genetic distance, which cannot meet the needs of molecular breeding. At the same time, due to the cumbersome marker typing technology developed, low throughput and high cost, most of the research results have not been effectively applied to wheat fusarium head blight resistance breeding. Therefore, it is of great significance to discover the major effect QTL of wheat fusarium head blight resistance, verify the genetic effect of the target QTL using different genetic populations or near-isogenic line populations, and develop molecular markers closely linked to it on the basis of fine positioning and candidate gene identification for wheat fusarium head blight resistance marker-assisted selection breeding. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a KASP marker primer set for a wheat fusarium spp. resistance candidate gene and an identification method thereof in view of the deficiencies of the above-mentioned prior art. The invention has high diagnosticity, is closely linked to the fusarium spp. resistance at the QFhb-2DL site, and can be used for auxiliary selection of disease-resistant breeding markers at the site.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: a KASP marker primer set for a wheat fusarium rust resistance candidate gene, the KASP marker primer set comprising a forward primer FAM, a reverse primer VIC and a common reverse primer, the nucleotide sequence of the forward primer FAM is shown in SEQ ID NO:1, the nucleotide sequence of the reverse primer VIC is shown in SEQ ID NO:2, and the nucleotide sequence of the common reverse primer is shown in SEQ ID NO:3.

[0007] The present invention also provides a method for identifying a wheat fusarium resistant candidate gene using the above-mentioned KASP marker primer set for wheat fusarium resistant candidate genes, the method comprising:

[0008] S1, extracting genomic DNA from wheat seedling leaves to obtain genomic DNA to be tested;

[0009] S2, using the KASP labeled primer set to perform PCR amplification on the genomic DNA to be tested obtained in S1 to obtain a PCR product;

[0010] The reaction system of the PCR amplification is: 2×KASP Master Mix 4 μL, KASP labeled primer set 0.01 μL, 80 ng of genomic DNA to be tested, and ddH2O supplemented to 8.0 μL;

[0011] The preparation method of the KASP labeling primer set is as follows: 120 μL of 100 μmol / mL forward primer FAM, 120 μL of 100 μmol / mL reverse primer VIC, 300 μL of 100 μmol / mL common reverse primer, and ddH2O to make up to 1000 μL;

[0012] The reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 20 seconds, gradient PCR from 61°C to 55°C, annealing for 40 seconds per cycle, decreasing by -0.6°C per cycle, denaturation at 95°C for 20 seconds, annealing at 55°C for 40 seconds, a total of 28 to 34 cycles; storage at 12°C;

[0013] S3. The PCR product obtained in S2 was subjected to genotyping of the KASP marker using a Bio-rad CFX-96 instrument real-time PCR instrument; the disease-resistant marker type is allel1, and the software typing shows orange, and the disease-susceptible marker type is allel2, and the software typing shows blue.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] The KASP marker primer set for wheat fusarium scabiei resistance candidate gene TaRAR1 of the present invention has high diagnosticity, is closely linked to the fusarium scabiei resistance of the site of QFhb-2DL, and can be used for auxiliary selection of disease resistance breeding markers at the site.

[0016] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a comparison of the resistance of QFhb-2DLNILs to Fusarium head blight in Example 1 of the present invention.

[0018] Figure 2 This is the result of genotyping wheat germplasm resources from different wheat regions in my country using the KASP marker primer set of Example 1 of the present invention. DETAILED DESCRIPTION

[0019] Example 1

[0020] Fusarium head blight resistance identification: Fusarium head blight resistance identification was performed under greenhouse conditions using the single flower instillation method. During the flowering period of wheat, the middle flowering spikelets were selected, and each spikelet was inoculated with 10 μL of bacterial solution (~1000 spores), and covered with a plastic bag to keep moist for 48 hours. The disease situation was investigated 14-18 days after inoculation, and the percentage of diseased spikelets in each spikelet (PSS) was used as the disease resistance evaluation index.

[0021] Development and verification of KASP markers: Based on the resequencing results and the Fusarium resistance of the identified materials, a simple, high-throughput KASP (Kompetitive allele specific PCR, http: / / www.lgcgroup.com / kasp) marker for the TaRAR1 candidate gene (QFhb.cau-2DL) was developed. At the same time, the working efficiency of the developed marker was verified in near-isogenic line populations and natural populations. The specific process and primer sequence information are as follows:

[0022] KASP markers of TaRAR1 gene:

[0023] The KASP marker primer set for the wheat fusarium rust resistance candidate gene of this embodiment comprises a forward primer FAM, a reverse primer VIC and a common reverse primer, the nucleotide sequence of the forward primer FAM is shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer VIC is shown in SEQ ID NO: 2, and the nucleotide sequence of the common reverse primer is shown in SEQ ID NO: 3.

[0024] The present invention also provides a method for identifying a wheat fusarium resistant candidate gene using the above-mentioned KASP marker primer set for wheat fusarium resistant candidate genes, the method comprising:

[0025] S1, extracting genomic DNA from wheat seedling leaves to obtain genomic DNA to be tested;

[0026] S2, using the KASP labeled primer set to perform PCR amplification on the genomic DNA to be tested obtained in S1 to obtain a PCR product;

[0027] The reaction system of the PCR amplification is: 2×KASP Master Mix 4 μL, KASP labeled primer set 0.01 μL, 80 ng of genomic DNA to be tested, and ddH2O supplemented to 8.0 μL;

[0028] The preparation method of the KASP labeling primer set is as follows: 120 μL of 100 μmol / mL forward primer FAM, 120 μL of 100 μmol / mL reverse primer VIC, 300 μL of 100 μmol / mL common reverse primer, and ddH2O to make up to 1000 μL;

[0029] The reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 20 seconds, gradient PCR from 61°C to 55°C, annealing for 40 seconds per cycle, decreasing by -0.6°C per cycle, denaturation at 95°C for 20 seconds, annealing at 55°C for 40 seconds, a total of 28 to 34 cycles; storage at 12°C;

[0030] S3. The PCR product obtained in S2 was subjected to genotyping of the KASP marker using a Bio-rad CFX-96 instrument real-time PCR instrument; the disease-resistant marker type is allel1, and the software typing shows orange, and the disease-susceptible marker type is allel2, and the software typing shows blue.

[0031] Specific operating procedures:

[0032] https: / / biosearch-cdn.azureedge.net / assetsv6 / running-KASP-on-BIO-RAD-CFX.pdf.

[0033] 1. Genetic location and effect verification of major QTLs for resistance to fusarium head blight

[0034] We used the JI5265 / WheatonRIL population and its GBS (Genotyping by sequencing) genotype data to locate a major QTL for Fusarium head blight resistance, QFhb-2DL, on the long arm of chromosome 2D of wheat. Using flanking markers, we identified Line-9, Line-115, and Line-128 in the RILF6 mapping population as heterozygous at the target QTL locus. We selected the remaining heterozygous line Line-9 to derive near-isogenic lines (NILs) of the target gene segment to verify the genetic effect of the target QTL ( Figure 1 ). Figure 1 Figure a shows the resistance performance of NILs 18 days after inoculation with ergot; b shows the comparison of the differences in resistance to ergot in QFhb-2DL NILs in 2020 and 2021. An investigation of the resistance phenotype 18 days after inoculation with ergot found that the rate of expansion of ergot diseased spikelets carrying the same Ji 5265 genotype (NIL-R) was significantly lower than that of the same wheaton genotype (NIL-S). Surveys in two consecutive growing seasons (2020 and 2021) found that the average diseased spikelet rate (Percentage of Symptomatic Spikelets, PSS) of NIL-R averaged 41.8% and 47.3%, which was significantly lower than the proportion of susceptible spikelets of NIL-S (73.9% and 77.9%).

[0035] 2. Prediction and sequence alignment of disease resistance candidate genes within the localization interval

[0036] Based on the physical position information of the flanking markers on the Chinese Spring reference genome sequence, the target QTL was mapped to a 6.8 Mb physical interval on chromosome 2D of wheat. This interval contains a total of 68 annotated genes, among which 13 disease resistance-related genes may be involved in the Fusarium head blight resistance response at this QTL locus (Table 1). These 13 genes were cloned and Sanger sequenced in the near-isogenic lines NIL-R and NIL-S. Sequence alignment of the resistant and susceptible near-isogenic lines found that only two genes had polymorphic differences in the encoded amino acids, namely a gene encoding an RAR1 protein containing cysteine and histidine domains and an ethylene-responsive transcription factor. RAR1 is a eukaryotic zinc-binding protein that participates in various resistance responses as an important disease resistance factor. RAR1 can bind to SGT1 and is involved in the resistance to powdery mildew mediated by Mla12 in barley, and can form a complex with HSP family proteins and participate in rice innate immunity. RAR1 in wheat participates in the resistance to stripe rust by affecting salicylic acid and then affecting the accumulation of reactive oxygen species and hypersensitive response. Therefore, we regarded TaRAR1 as the main candidate gene for QFhb-2DL.

[0037] Table 1. Genes related to disease resistance in the mapped interval

[0038] Serial number Gene number Gene function Genetic differences 1 TraesCS2D02G410300 Zincfingerprotein ZAT1 No difference in test 2 TraesCS2D02G411600 Cysteineandhistidine-richdomain-containingproteinRAR1 Single amino acid changes 3 TraesCS2D02G412200 Ethylene-responsivetranscriptionfactorERF034 No difference in test 4 TraesCS2D02G413600 Disease resistance protein RPM1 No difference in test 5 TraesCS2D02G414000 DiseaseresistanceproteinPiks-2 No difference in test 6 TraesCS2D02G414100 DiseaseresistanceproteinPIK6-NP No difference in test 7 TraesCS2D02G414200 Nudixhydrolase8 No difference in test 8 TraesCS2D02G414300 Ethylene-responsivetranscriptionfactor5 Single amino acid changes 9 TraesCS2D02G414500 Ethylene-responsivetranscriptionfactor5 No difference in test 10 TraesCS2D02G414600 Ethylene-responsivetranscriptionfactor2 No difference in test 11 TraesCS2D02G415600 ProbableLRRreceptor-likeserine / threonine-proteinkinase No difference in test 12 TraesCS2D02G415700 ProbableLRRreceptor-likeserine / threonine-proteinkinase No difference in test 13 TraesCS2D02G415800 ProbableLRRreceptor-likeserine / threonine-proteinkinase No difference in test

[0039] 3. Development and validation of TaRAR1-KASP marker

[0040] According to the SNP differences in the coding region of TaRAR1, it was converted into a KASP marker (Kompetitive allele specific PCR), and this marker was named TaRAR1-KASP. The primer set of the TaRAR1-KASP marker includes a forward primer FAM, a reverse primer VIC, and a common reverse primer. The nucleotide sequence of the forward primer FAM is shown in SEQ ID NO:1, the nucleotide sequence of the reverse primer VIC is shown in SEQ ID NO:2, and the nucleotide sequence of the common reverse primer is shown in SEQ ID NO:3.

[0041] This marker can divide the resistant (Ji 5265) and susceptible (Wheaton) parents into two groups, and can also distinguish the heterozygous single plants in the target segment from the homozygous resistant and susceptible parents, indicating that TaRAR1-KASP is a co-dominant marker type ( Figure 2 ). Genotype analysis of 157 wheat germplasm resources from different wheat-growing regions in China using the KASP marker TaRAR1-KASP showed that the frequency of the resistance allele of QFhb-2DL was only 3.2%, as Figure 2As shown, the disease resistance marker type is allel1, and the software typing shows orange; the disease susceptible marker type is allel2, and the software typing shows blue. This shows that the disease resistance gene has not been selected and utilized in breeding and has great potential utilization value.

[0042] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A KASP marker primer set for wheat fusarium resistance candidate genes, characterized in that: The KASP marker primer set consists of a forward primer FAM, a reverse primer VIC and a common reverse primer, the nucleotide sequence of the forward primer FAM is shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer VIC is shown in SEQ ID NO: 2, and the nucleotide sequence of the common reverse primer is shown in SEQ ID NO:

3.

2. A method for identifying a candidate gene for wheat fusarium resistance using the KASP marker primer set for a candidate gene for wheat fusarium resistance as claimed in claim 1, characterized in that: The method is: S1, extracting genomic DNA from wheat seedling leaves to obtain genomic DNA to be tested; S2, using the KASP labeled primer set to perform PCR amplification on the genomic DNA to be tested obtained in S1 to obtain a PCR product; The reaction system of the PCR amplification is: 2× KASP Master Mix 4 μL, KASP labeled primer set 0.01 μL, genomic DNA to be tested 80 ng, ddH2O supplemented to 8.0 μL; The preparation method of the KASP labeling primer set is as follows: 120 μL of 100 μmol / mL forward primer FAM, 120 μL of 100 μmol / mL reverse primer VIC, 300 μL of 100 μmol / mL common reverse primer, and ddH2O to make up to 1000 μL; The reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 s, gradient PCR from 61°C to 55°C, annealing for 40 s per cycle, decreasing by -0.6°C per cycle, denaturation at 95°C for 20 s, annealing at 55°C for 40 s, a total of 28 to 34 cycles; storage at 12°C; S3. Use Bio-rad CFX-96 instrument real-time PCR instrument to identify the genotype of KASP marker on the PCR product obtained in S2.

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