CN116676414A - Application of SNP molecular marker A08-12266524 in identification of cabbage clubroot resistance - Google Patents
Application of SNP molecular marker A08-12266524 in identification of cabbage clubroot resistance Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular marker assisted breeding, and relates to application of SNP molecular marker A08-12266524 in identification of cabbage clubroot resistance and/or cabbage breeding resistant to clubroot. The invention provides an application of SNP molecular markers A08-12266524 in identifying cabbage clubroot resistance and/or clubroot resistance cabbage breeding, wherein the sequence of the SNP molecular markers A08-12266524 is shown as SEQ ID NO.1, the 70 th base from the 5' end of the sequence shown as SEQ ID NO.1 is a SNP locus, and the base is A or G. The SNP molecular marker A08-12266524 can realize identification of the anti-clubroot Chinese cabbage, and is used for screening the breeding of the anti-clubroot Chinese cabbage.
Description
Technical Field
The invention belongs to the technical field of molecular marker assisted breeding, and particularly relates to application of SNP molecular marker A08-12266524 in identification of cabbage clubroot resistance and/or clubroot-resistant cabbage breeding.
Background
Clubroot, also known as "root cancer", is a worldwide soil-borne disease caused by infection with brassica napus (plasmmodiophora brassicae woron), mainly infecting cruciferous plants such as chinese cabbage, radish, broccoli, mustard, canola, etc. After infection, the rhizomatosis can cause abnormal proliferation of root cells and form tumors, thereby affecting the transportation of moisture and nutrient substances and causing the lack of nutrient wilt of the overground parts of plants until the death of the whole plants. The method for cultivating the disease-resistant variety by utilizing the clubroot-resistant gene has the characteristics of safety, high efficiency and economy, is one of important ways for fundamentally solving the trouble of the clubroot, and can greatly improve the disease-resistant breeding efficiency by searching a molecular marker closely linked with the clubroot-resistant gene and utilizing the molecular marker to assist in selective breeding (marker-assisted selection). Therefore, screening more SNP related to cabbage clubroot resistance genes, being applied to the selection of cabbage disease-resistant materials and the breeding of disease-resistant varieties, and having important significance in improving the yield and quality of cabbage and the like.
Disclosure of Invention
The invention aims to provide an application of SNP molecular markers A08-12266524 in identifying the clubroot resistance of Chinese cabbages and/or the breeding of the Chinese cabbages with clubroot resistance. The SNP molecular marker A08-12266524 can realize identification of the anti-clubroot Chinese cabbage, and is used for screening the breeding of the anti-clubroot Chinese cabbage.
The invention provides an application of SNP molecular markers A08-12266524 in identifying cabbage clubroot resistance and/or clubroot resistance cabbage breeding, wherein the sequence of the SNP molecular markers A08-12266524 is shown as SEQ ID NO.1, the 70 th base from the 5' end of the sequence shown as SEQ ID NO.1 is a SNP locus, and the base is A or G.
The invention also provides a reagent for detecting the SNP molecular marker A08-12266524 in the application of the technical scheme.
Preferably, the reagent comprises primer set Crr5-funK3, and the primer set Crr5-funK3 comprises Crr5-funK3Ra, crr5-funK3Rb, and Crr5-funK3F; the nucleotide sequence of Crr5-funK3Ra is shown in SEQ ID NO. 2; the nucleotide sequence of Crr5-funK3Rb is shown in SEQ ID NO. 3; the nucleotide sequence of Crr-funK 3F is shown in SEQ ID NO. 4.
Preferably, the Crr5-funK3Ra and Crr5-funK3Rb are labeled with different colored fluorophores, respectively.
Preferably, the fluorescent groups include FAM and HEX.
The invention also provides a kit for detecting the SNP molecular marker A08-12266524 in the application of the technical scheme, and the kit comprises the reagent and the reaction solution in the technical scheme.
The invention also provides the application of the reagent or the kit in the technical scheme in identifying the clubroot resistance of the Chinese cabbage and/or the breeding of the Chinese cabbage with clubroot resistance.
The invention also provides a method for identifying the clubroot resistance of the celery cabbage, which comprises the following steps:
the reagent or the kit of the technical proposal is used for carrying out competitive allele specific PCR amplification on the genome DNA of the Chinese cabbage, and finally, the KASP amplification product is subjected to end-point method fluorescent signal reading to obtain a genotyping result, when the genotype is AA or AG, the Chinese cabbage is identified as the Chinese cabbage with clubroot resistance, and when the genotype is GG, the Chinese cabbage is identified as the Chinese cabbage with clubroot resistance.
Preferably, the PCR amplification reaction system comprises, in 8. Mu.L, 1.5. Mu.L of genomic DNA, 4. Mu.L of KASP Mastermix (2X), 0.14. Mu.L of primer set Crr-funK 3 and the balance of water.
Preferably, the reaction procedure of the PCR amplification comprises: 94 ℃ for 15min;94℃for 20s and 61℃for 60s, for a total of 10 cycles, each cycle being reduced by 0.6℃from the second cycle; 94 ℃ for 20s and 55 ℃ for 60s, 26 cycles are total; 1min at 37 ℃.
The invention provides an application of SNP molecular markers A08-12266524 in identifying cabbage clubroot resistance and/or cabbage breeding resistant to clubroot. The SNP molecular marker A08-12266524 can realize identification of the anti-clubroot Chinese cabbage, and is used for screening the breeding of the anti-clubroot Chinese cabbage. Specifically, the detection is carried out by using the reagent for detecting the SNP molecular marker A08-12266524, so that the differentiation and identification of the clubroot resistant and clubroot-sensitive materials of the cabbages can be realized, the individuals containing the clubroot resistant gene Crr in the offspring population of the cabbages can be rapidly screened on the molecular level, the accuracy is high, and the disease-resistant breeding efficiency can be greatly improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of the present invention using the marker Crr5-funK3 for F 2 Performing KASP genotyping result graph on the population;
FIG. 2 is a graph showing the results of KASP genotyping of natural populations using the marker Crr5-funK3 provided by the present invention.
Detailed Description
The invention provides an application of SNP molecular markers A08-12266524 in identifying cabbage clubroot resistance and/or clubroot resistance cabbage breeding, wherein the sequence of the SNP molecular markers A08-12266524 is shown as SEQ ID NO.1 (TAACACACATACTGCAAAATGCGAAATGTTTCTTCAGTTTCCATCAACATGAGAGAGCTAAGCTTTCTARAGGACGTAGCCCGCATTCTTTAATCTCCCCGTTTTTTTTTTCGAACCTGAACTCAAAGACTAATTCGGG, R=A/G), the 70 th base from the 5' end of the sequence shown as SEQ ID NO.1 is an SNP locus, and the base is A or G. The SNP molecular marker A08-12266524 is a SNP molecular marker of a cabbage clubroot resistance related gene Crr. The invention discloses SNP molecular markers A08-12266524 related to a Chinese cabbage clubroot resistance trait gene Crr5 for the first time. The detection is carried out by using the reagent for detecting the SNP molecular marker A08-12266524, so that the differentiation and identification of the anti-clubroot and clubroot-sensing materials of the cabbages can be realized, individuals containing the anti-clubroot gene Crr5 in the offspring population of the cabbages can be rapidly screened on the molecular level, the accuracy is high, and the disease-resistant breeding efficiency can be greatly improved.
The invention also provides a reagent for detecting the SNP molecular marker A08-12266524 in the application of the technical scheme. In the present invention, the primer preferably includes a KASP (Kompetitive Allele Specific PCR, competitive allele-specific PCR) primer. In the present invention, the reagent preferably includes a primer set Crr5-funK3, and the primer set Crr5-funK3 includes Crr5-funK3Ra, crr5-funK3Rb, and Crr5-funK3F; the nucleotide sequence of the Crr5-funK3Ra is 5' as shown in SEQ ID NO.2GAAGGTGACCAAGTTCATGCTAGAATGCGGGCTACGTCCTT-3'; the nucleotide sequence of the Crr5-funK3Rb is 5' shown in SEQ ID NO.3GAAGGTCGGAGTCAACGGATTGAATGCGGACTACGTCCTC-3'; the nucleotide sequence of Crr-funK 3F is shown in SEQ ID NO. 4: 5'-CATCAACATGAGAGAGCTAAGC-3'. In the present invention, the Crr5-funK3Ra and Crr5-funK3Rb are preferably labeled with fluorescent groups of different colors, respectively. In the present invention, the fluorescent group preferably includes FAM and HEX. In the present invention, the Crr5-funK3Ra is preferably labeled FAM, and the Crr5-funK3Rb is preferably labeled HEX. The invention discloses a detection primer group Crr-funK 3 of SNP molecular marker A08-12266524 related to Chinese cabbage clubroot resistance character for the first time, which comprises three primers, wherein DNA of F2 generation separation group generated after DH line of disease-resistant and disease-resistant Chinese cabbage and hybridization of the two is amplified by PCR, and detected by KASP gene analysis system, crr-funK 3 can obviously distinguish clubroot resistance and clubroot resistance materials, and Chinese cabbage offspring group can be rapidly screened at molecular level to contain clubroot resistanceIndividuals with disease gene Crr.
The invention also provides a kit for detecting the SNP molecular marker A08-12266524 in the application of the technical scheme, and the kit comprises the reagent and the reaction solution in the technical scheme. In the present invention, the reaction liquid preferably includes KASP Mastermix (2×).
The invention also provides the application of the reagent or the kit in the technical scheme in identifying the clubroot resistance of the Chinese cabbage and/or the breeding of the Chinese cabbage with clubroot resistance.
The invention also provides a method for identifying the clubroot resistance of the celery cabbage, which comprises the following steps:
the reagent or the kit of the technical proposal is used for carrying out competitive allele specific PCR amplification on the genome DNA of the Chinese cabbage, and finally, the KASP amplification product is subjected to end-point method fluorescent signal reading to obtain a genotyping result, when the genotype is AA or AG, the Chinese cabbage is identified as the Chinese cabbage with clubroot resistance, and when the genotype is GG, the Chinese cabbage is identified as the Chinese cabbage with clubroot resistance.
In the present invention, the reaction system for PCR amplification preferably comprises 1.5. Mu.L of genomic DNA, 4. Mu.L of 2 XSP Mastermix, 0.14. Mu.L of primer set Crr-funK 3 and the balance of water in terms of 8. Mu.L. In the present invention, the concentration of each primer in the primer set Crr5-funK3 is preferably 100. Mu. Mol/L. In the present invention, crr-funK 3Ra, crr5-funK3Rb, and Crr5-funK3F and ddH in the primer set Crr5-funK3 2 The mixing volume ratio of O is preferably 12:12:30:46. the method for extracting the genomic DNA of the Chinese cabbage is not particularly limited, and a conventional genomic DNA extraction method well known to a person skilled in the art can be adopted. In the present invention, the reaction procedure of the PCR amplification preferably includes: 94 ℃ for 15min;94℃for 20s and 61℃for 60s, for a total of 10 cycles, each cycle being reduced by 0.6℃from the second cycle; 94 ℃ for 20s and 55 ℃ for 60s, 26 cycles are total; 1min at 37 ℃.
After KASP amplification products are obtained, fluorescent signals are read by an end-point method, and genotyping results are obtained: and when the genotype is AA or AG, identifying the Chinese cabbage as the Chinese cabbage with the clubroot, and when the genotype is GG, identifying the Chinese cabbage as the Chinese cabbage with the clubroot.
In the invention, the fluorescent signal reading by the end-point method is preferably performed by using a Roche fluorescent quantitative P-C R Instrument LightCycler480 Instrument II (LC 480 II). SNP typing results were analyzed using LC480 software v 1.5.1: the signal point of the homozygous disease-resistant material is blue, and the 5' -end is connected with the competitive amplification of the primer of FAM fluorescent tag sequence, and the primer is polymerized near the X axis; the signal point of the homozygous infectious material is green, the 5' end is connected with the primer competitive amplification of HEX fluorescent label sequence, and the primer is polymerized near the Y axis; the signal points of the hybrid disease-resistant material are red and are polymerized near the diagonal.
For further explanation of the present invention, the application of the SNP molecular markers A08-12266524 provided by the present invention in the identification of the clubroot resistance of Chinese cabbage and/or the breeding of the clubroot-resistant Chinese cabbage will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1.1 investigation of test Material and disease resistance Properties
Selecting Chinese cabbage disease-resistant DH series material 1EDHR1 (P) 1 ) And a disease-causing material 1EDHS1 (P) 2 ) Positioning of the cabbage clubroot resistance gene BraA.Pb.8.4 as a parent (Yang Shuangjuan et al; KASP marker development; gardening journal, 2021, 48 (7): 1317-1328. The biological material is available to the public from the applicant and is used only for repeated experiments in connection with the invention and is not used for other purposes. The two materials are subjected to forward and backward intersection to obtain F 1 Seed generation, F 1 Selfing the material to obtain F 2 Seed. P pair P 1 ，P 2 ，F 1 And F 2 Inoculating and identifying in seedling stage, wherein the bacterial source is Henan New wild new bacterium XY-2, the bacterial source is No.4 physiological race according to a Williams system identification system, and is ECD21/31/31 race according to an ECD identification system (Yuxiang, zhao Yanyan, wei Xiaochun, etc. 2017. Henan Chinese cabbage clubroot germ physiological race identification. Henan agricultural science, 46 (7): 71-76). The disease level of each individual was investigated 6 weeks after inoculation and classified into 0, 1, 3, 5 and 7, and specific investigationMethods are described in Zhao Yanyan et al (Zhao Yanyan, jiang Wusheng, yuxiang et al. Chinese cabbage clubroot indoor artificial inoculation methods and condition comparison and identification of resistance to different varieties, gardening journal, 2014, 41 (S1): 2675).
Disease resistance identification results show that the disease resistance parent 1EDHR1 (P 1 ) The 10 strains of (a) all had a disease level of 0, 10 strains of 1EDHS1 (P) 2 ) The disease incidence of the strain is all 7, 10 strain F 1 The onset grade of the material is 0 grade (Table 1), F 2 159 strains in the population are 0 grade, 156 strains are 1 grade, 9 strains are 3 grade, 15 strains are 5 grade, and 96 strains are 7 grade. Classifying level 0 and level 1 into disease resistance type, classifying level 3, level 5 and level 7 into disease susceptibility type, F 2 The disease-resistant single plant in the population has 315 strains, the disease-resistant single plant is 120 strains, and the separation ratio (Table 1) of the disease-resistant single plant accords with 3:1 through the square test, which shows that 1EDHR1 (P) 1 ) The carried gene for resisting clubroot is controlled by 1 pair of dominant nuclear genes, and the disease resistance is dominant to the disease.
TABLE 1 isolation of the parent and its progeny from disease-resistant and susceptible plants of Chinese cabbage
1.2 genomic DNA extraction
Extracting two parents and F by adopting improved CTAB method 1 And F 2 Genomic DNA of each individual strain. The specific steps are as follows.
Fresh leaves were taken in 2.0mL Eppendorf centrifuge tubes, 1 shot of steel balls (5 MM diameter) was placed into each tube, after which 1000. Mu.L of 2% CTAB extraction buffer was added and the cells were broken on a tissue breaker (model: retsch MM400, germany) at a frequency of 30 times/sec for 1min. After cooling, adding 500 mu L of 24:1 chloroform/isoamyl alcohol extract, shaking up and down for 30 times, standing for delamination, centrifuging (12000 r/min) for 10min, sucking 400 mu L of supernatant into a new 1.5mL centrifuge tube, adding 400 mu L of isopropanol for precipitating DNA, centrifuging (12000 r/min) for 5min after mixing up and down, discarding supernatant, adding 70% ethanol for 750 mu L for cleaning DNA precipitation, centrifuging (12000 r/min) for 2min, discarding supernatant, airing DNA at room temperature, adding 100 mu L of ddH2O for dissolving DNA, and placing in a refrigerator at-20 ℃ for standby.
1.3 discovery of SNP marker linked to clubroot Gene Crr5 and development of detection primer set Crr-funK 3
The candidate gene of the cabbage clubroot resistance gene Crr is determined by a gene positioning method, sequencing and sequence comparison are carried out on the candidate gene, and SNP variation A/G is found at the 12266524bp position of an A08 chromosome (Brapa_Chiifu_V3.0 reference gene), wherein the base in disease resistant materials is A, the base in disease resistant materials is G, and the variation is called SNP markers A08-12266524 as shown in the following figure at the 70bp position.
TAACACACATACTGCAAAATGCGAAATGTTTCTTCAGTTTCCATCAACATGAGAGAGCTAAGCTTTCTA[A/G]AGGACGTAGCCCGCATTCTTTAATCTCCCCGTTTTTTTTTTCGAACCTGAACTCAAAGACTAATTCGGG(SEQ ID NO.1)。
The KASP marker Crr5-funK3 was designed for this SNP marker A08-12266524, comprising three primers:
Crr5-funK3Ra：5'-GAAGGTGACCAAGTTCATGCTAGAATGCGGGCTACGTCCTT-3'(SEQ ID NO.2)；
Crr5-funK3Rb：5'-GAAGGTCGGAGTCAACGGATTGAATGCGGACTACGTCCTC-3'(SEQ ID NO.3)；
Crr5-funK3F：5'-CATCAACATGAGAGAGCTAAGC-3'(SEQ ID NO.4)。
crr5-funK3Ra and Crr5-funK3Rb are two allele-specific reverse primers, crr5-funK3Ra is a specific primer of the disease-resistant genotype, FAM fluorescent sequence tag sequences (underlined) are respectively added to the 5 'ends, crr5-funK3Rb is a specific primer of the disease-resistant genotype, and HEX fluorescent sequence tag sequences (underlined) are added to the 5' ends. Crr5-funK3F is a common forward primer.
The KASP-PCR reaction was performed on a 96-well PCR apparatus with 8. Mu.L of the reaction system: 1.5. Mu.L of DNA (80 ng/. Mu.L), 4. Mu.LKASP Mastermix (2X), 0.14. Mu.L of primer mix (prepared from Crr5-funK3Ra, crr5-funK3Rb, crr5-funK3F and ddH at a concentration of 100. Mu. Mol/L 2 O is mixed according to the volume ratio of 12:12:30:46, and the rest is ddH 2 And (3) supplementing O.
The KASP-PCR amplification procedure was: denaturation at 94℃for 15min in the first stage; the second stage of denaturation at 94℃for 20s and annealing at 61℃for 60s, for a total of 10 cycles (0.6℃decrease per cycle starting from the second cycle); the third stage is denaturation at 94 ℃ for 20s and annealing at 55 ℃ for 60s, 26 cycles are carried out; the fourth stage is carried out at 37 ℃ for 1min.
KASP-PCR amplified products were read for endpoint fluorescence signal using a Roche fluorescent quantitative PCR Instrument, lightCycler480 Instrument II (LC 480 II). SNP typing results were analyzed using LC480 software v 1.5.1: the signal point of the homozygous disease-resistant material is blue, the 5' end is connected with the competitive amplification of the primer of FAM fluorescent tag sequence, the primer is polymerized near the X axis, and the genotype is AA; the signal point of the homozygous infectious material is green, the 5' end is connected with the primer competitive amplification of HEX fluorescent tag sequence, and the primer is polymerized near the Y axis, and the genotype is GG; the signal point of the hybrid disease-resistant material is red, the genotype is AG, and the hybrid disease-resistant material is polymerized near the diagonal (FIG. 1, the marker Crr-funK 3 is used for F 2 KASP genotyping results plots were performed for the population). Crr5-funK3 marker can significantly distinguish two homozygous genotypes, and can identify heterozygous genotypes, has the characteristic of co-dominant marker, and the marker development is successful.
Using Crr-funK 3 marker pair F 2 Genotyping 86 individual plants of the population, marking the genotype of homozygous disease resistance as a, the genotype of homozygous disease resistance as b and the heterozygosity as h. As a result, 18 individuals with genotype a had a disease level of 0, 21 individuals with genotype h had a disease level of 0, 47 individuals with genotype b had a disease level of 7 (FIG. 1). The genotypic and phenotypic concordance rate of marker Crr5-funK3 in 86F 2 individuals reached 100%. Crr5-funK3 marker can be used for molecular auxiliary breeding of clubroot resistant varieties of Chinese cabbage.
1.4 application of SNP marker detection primer
The extraction of leaf DNA was performed by CTAB method. The materials are 47 DH-series materials such as Y636-9, Y663-8, Y623-1, Y578-2, etc. The KASP marker Crr5-funK3 was used for verification in 47 DH populations, 1EDHR1 was used as a disease-resistant control, and the result shows that 47 pathogenic DH-series materials are all polymerized near the Y axis and are expressed as pathogenic genotypes, and 1EDHR1 is aggregated near the X axis as a disease-resistant material. The genotypic and phenotypic concordance rate of the marker Crr5-funK3 in 48 DH-series materials reaches 100%. The marker Crr-funK 3 has good universality and accuracy, and can be used for molecular marker assisted selection of the celery cabbage clubroot material.
Conclusion: the invention discloses SNP markers A08-12266524 related to a Chinese cabbage clubroot disease resistance gene Crr5 and discloses a detection primer group Crr5-funK3 for detecting the markers. The SNP marker and the detection primer set Crr5-funK3 thereof can accurately and efficiently detect disease-resistant and disease-sensitive material types. The screening method is not influenced by environmental factors, can greatly reduce the field selection workload, and is helpful for helping to accelerate the disease-resistant breeding of the Chinese cabbages.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The sequence of the SNP molecular marker A08-12266524 is shown as SEQ ID NO.1, the 70 th base from the 5' end of the sequence shown as SEQ ID NO.1 is a SNP locus, and the base is A or G.
2. A reagent for detecting SNP molecular marker A08-12266524 in the use as set forth in claim 1.
3. The reagent of claim 2, wherein the reagent comprises primer set Crr5-funK3, the primer set Crr5-funK3 comprising Crr5-funK3Ra, crr5-funK3Rb and Crr5-funK3F; the nucleotide sequence of Crr5-funK3Ra is shown in SEQ ID NO. 2; the nucleotide sequence of Crr5-funK3Rb is shown in SEQ ID NO. 3; the nucleotide sequence of Crr-funK 3F is shown in SEQ ID NO. 4.
4. A reagent according to claim 3, wherein the Crr5-funK3Ra and Crr5-funK3Rb are labelled with fluorescent groups of different colours respectively.
5. The reagent of claim 4, wherein the fluorescent groups comprise FAM and HEX.
6. A kit for detecting the SNP molecular marker a08-12266524 in the application of claim 1, wherein the kit comprises the reagent according to any one of claims 2 to 5 and a reaction solution.
7. Use of the reagent according to any one of claims 2 to 5 or the kit according to claim 6 for identifying cabbage clubroot resistance and/or cabbage breeding against clubroot.
8. A method for identifying clubroot resistance of chinese cabbage, comprising the steps of:
performing competitive allele-specific PCR amplification on the genomic DNA of the Chinese cabbage by using the reagent according to any one of claims 2 to 5 or the kit according to claim 6, and finally performing end-point fluorescent signal reading on the KASP amplification product to obtain a genotyping result, and identifying the Chinese cabbage as the anti-clubroot Chinese cabbage when the genotype is AA or AG and identifying the Chinese cabbage as the clubroot Chinese cabbage when the genotype is GG.
9. The method according to claim 8, wherein the PCR amplification reaction system comprises 1.5. Mu.L of genomic DNA, 4. Mu.L of 2 XSKASPMastermix, 0.14. Mu.L of primer set Crr-funK 3 and the balance of water in 8. Mu.L.
10. The method of claim 8, wherein the reaction procedure for PCR amplification comprises: 94 ℃ for 15min;94℃for 20s and 61℃for 60s, for a total of 10 cycles, each cycle being reduced by 0.6℃from the second cycle; 94 ℃ for 20s and 55 ℃ for 60s, 26 cycles are total; 1min at 37 ℃.
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