CN112143823A - KASP marker of Chinese cabbage clubroot resistance gene Crr5 and application thereof - Google Patents
KASP marker of Chinese cabbage clubroot resistance gene Crr5 and application thereof Download PDFInfo
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Abstract
The invention relates to a KASP marker of a Chinese cabbage clubroot resistance gene Crr5 and application thereof, in particular to application of Crr5-1 in identification or auxiliary identification of clubroot resistance of Chinese cabbage; crr5-1 is a SNP locus of the genome of the Chinese cabbage, is the 101 th nucleotide of SEQ ID No.1, and is C or T. The invention provides an SNP locus Crr5-1 (T/C SNP variation occurs on 101 site of a sequence shown as a sequence 1 on a Chinese cabbage genome) closely linked with a clubroot resistance gene Crr5, and develops KASP Crr5-KASP1, wherein the marker can detect Crr5 genotype at high flux, can be applied to molecular marker assisted breeding of a new variety with clubroot resistance of Chinese cabbage at high efficiency and low cost, and has very important significance.
Description
Technical Field
The invention relates to the technical field of crop molecular marker assisted breeding, in particular to a high-throughput detection marker of a Chinese cabbage clubroot disease resistance gene Crr5 and application thereof in breeding.
Background
Clubroot, also known as "root cancer", is a worldwide soil-borne disease caused by infection with Plasmodiophora brasiliensis, and mainly infects brassicaceous plants such as chinese cabbage, pakchoi, cabbage, radish, cauliflower, mustard, rape, etc. After the plasmodiophora root is infected, root cells are abnormally proliferated to form tumors, so that the transportation of water and nutrient substances is influenced, and the overground parts of plants lack nutrients and wither until the whole plants die. The method for breeding disease-resistant varieties by using the clubroot-resistant genes has the characteristics of safety, high efficiency and economy, is one of important ways for fundamentally solving the puzzlement of clubroot, and can greatly improve the disease-resistant breeding efficiency by searching molecular markers closely linked with the clubroot-resistant genes and using molecular marker-assisted selection breeding (marker-assisted selection).
At present, more than 10 clubroot resistance gene loci, namely Crr1a, Crr1b, Crr2, Crr3, Crr4, CRa, CRb, CRc, CRk, PbBa3.1, PbBa3.3 and PbBa8.1, are detected at home and abroad, and some disease-resistant varieties are also cultivated. However, the cultivated anti-clubroot variety has the phenomenon of resistance decline or loss in the cultivation process, which is related to the physiological microspecies variation of clubroot on one hand and is caused by the fact that the disease-resistant gene in the material is single on the other hand. Therefore, the development of new clubroot resistance genes, the development of molecular markers closely linked with the new clubroot resistance genes and the breeding of disease-resistant varieties carrying a plurality of disease-resistant genes are necessary ways for solving the damage of clubroot. KASP (competitive allele specific PCR) can accurately judge SNPs or InDels widely existing in genome DNA, and is a high-throughput, economical and effective SNP typing technology.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a new clubroot resistance gene Crr5, and develop a KASP marker tightly linked to the gene so as to lay a foundation for molecular marker screening and aggregate breeding of Chinese cabbage clubroot resistance breeding.
The invention claims Crr5-1 for identifying or assisting in identifying clubroot resistance of Chinese cabbage; crr5-1 is an SNP locus on the genome of the Chinese cabbage, Crr5-1 is an SNP locus closely linked with the clubroot resistance gene Crr5 of the Chinese cabbage, is the 101 th nucleotide of SEQ ID No.1, and is C or T.
The application of the substance for detecting Crr5-1 polymorphism or genotype in the Chinese cabbage genome in identifying or assisting in identifying the clubroot resistance of the Chinese cabbage also belongs to the protection scope of the invention; crr5-1 is a SNP locus on the genome of Chinese cabbage, Crr5-1 is located at the 101 st nucleotide of SEQ ID No.1 on the genome Crr5 of Chinese cabbage, and the nucleotide is C or T.
The substance for detecting the polymorphism or the genotype of Crr5-1 in the genome of the Chinese cabbage contains a primer for amplifying the DNA fragment of the genome of the Chinese cabbage including Crr 5-1.
The PCR primers may or may not be labeled with a label.
The label refers to any atom or molecule that can be used to provide a detectable effect and that can be attached to a nucleic acid. Labels include, but are not limited to, dyes; radiolabels, e.g.32P; binding moieties such as biotin (biotin); haptens such as Digoxin (DIG); a luminescent, phosphorescent, or fluorescent moiety; and a fluorescent dye alone or in combination with a portion of the emission spectrum that can be suppressed or shifted by Fluorescence Resonance Energy Transfer (FRET). Labels can provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Labels can be charged moieties (positive or negative) or alternatively, can be charge neutral. The label may comprise or be combined with a nucleic acid or protein sequence, provided that the sequence comprising the label is detectable. In some embodiments, the coreThe acid is detected directly without the label (e.g., direct sequence read). The PCR primer can be a primer group consisting of single-stranded DNA with the nucleotide sequence of 22 th to 41 th positions of SEQ ID No.2 in the sequence table, single-stranded DNA with the nucleotide sequence of 22 nd to 41 th positions of SEQ ID No.2 in the sequence table and single-stranded DNA with the nucleotide sequence of SEQ ID No.4 in the sequence table, and the PCR primer can also be a primer group consisting of single-stranded DNA shown by SEQ ID No.2 in the sequence table, single-stranded DNA shown by SEQ ID No.3 in the sequence table and single-stranded DNA shown by SEQ ID No.4 in the sequence table. SEQ ID No.2 in the sequence table consists of 41 nucleotides, the 1 st to 21 st nucleotides are FAM sequences (as markers), and the 22 nd to 41 th nucleotides are specific sequences; SEQ ID No.3 in the sequence table is composed of 41 nucleotides, the 1 st to 21 st nucleotides are FAM sequences (as markers), and the 22 nd to 41 th nucleotides are specific sequences.
The primer is a primer composition consisting of single-stranded DNA shown in SEQ ID No.2, single-stranded DNA shown in SEQ ID No.3 and single-stranded DNA shown in SEQ ID No. 4.
The method for identifying or assisting in identifying the clubroot resistance of the Chinese cabbage comprises the steps of detecting the genotype of Crr5-1 of the Chinese cabbage to be detected, and identifying or assisting in identifying the clubroot resistance of the Chinese cabbage to be detected according to the genotype of the Chinese cabbage to be detected.
The application of Crr5-1 in Chinese cabbage breeding is also within the protection scope of the invention.
The Chinese cabbage breeding is to select and breed the clubroot-resistant Chinese cabbage.
The application of the method for identifying or assisting in identifying the clubroot resistance of the Chinese cabbage in breeding the Chinese cabbage also belongs to the protection scope of the invention.
The Chinese cabbage breeding is to select and breed the clubroot-resistant Chinese cabbage.
The clubroot disease resisting Chinese cabbage breeding process includes the following steps: detecting the Crr5-1 genotype in the Chinese cabbage genome, and selecting the Chinese cabbage with the Crr5-1 genotype of CC in the Chinese cabbage genome as a parent breeding.
The above primers should also be within the scope of the present invention:
1) a primer pair for amplifying a DNA fragment containing the 101 st nucleotide of SEQ ID No.1 on the Chinese cabbage genome Crr 5-1;
2) a primer composition consisting of the single-stranded DNA shown in SEQ ID No.2, the single-stranded DNA shown in SEQ ID No.3 and the single-stranded DNA shown in SEQ ID No. 4.
The invention provides an SNP locus Crr5-1 (T/C SNP variation occurs on 101 site of a sequence shown as a sequence 1 on a Chinese cabbage genome) closely linked with a clubroot resistance gene Crr5, and develops KASP Crr5-KASP1, wherein the marker can detect Crr5 genotype at high flux, can be applied to molecular marker assisted breeding of a new variety with clubroot resistance of Chinese cabbage at high efficiency and low cost, and has very important significance.
Drawings
FIG. 1 shows the BSA-Seq mapping results for the anti-clubroot gene of population 1e519F 2.
FIG. 2 shows KASP genotyping of 150 individuals in the 1e519F2 population using marker Crr5-KASP 1.
FIG. 3 is an analysis of variance between genotype and susceptibility grade of 150 individuals of the 1e519F2 population.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples
1 materials and methods
The Chinese cabbage Y510-9 is clubroot disease-sensitive material (Yang Shuan Juan et al, 2018, optimization and establishment of the KASP reaction system of the Chinese cabbage, Horticulture proceedings, 45(12):2442 and 2452) and the biological material can be obtained by the applicant, is only used for repeating the relevant experiments of the invention and cannot be used for other purposes. The turnip ECD01 is an anti-clubroot material (Yuxiang, Zhaoyuan, Weixiaochun, etc.. 2017. identification of physiological races of clubroot germs of Chinese cabbage in Henan province. Henan agricultural science 46 (7): 71-76) and can be obtained from the applicant by the public, and the biological material is only used for repeating relevant experiments of the invention and can not be used for other purposes. The Chinese cabbage clubroot bacterium XY-2 is characterized in that the bacterium source is a physiological race of Chinese cabbage clubroot bacterium No.4 according to a Williams system identification system, and is an ECD21/31/31 race (original jade incense, Zhaoyyan, Wei Xiaochun, and the like. 2017. identification of the physiological race of Chinese cabbage clubroot bacterium in Henan province. Henan agricultural science, 46 (7): 71-76)
The invention uses Chinese cabbage susceptible material Chinese cabbage Y510-9 (P)1As female parent) and the disease-resistant material European turnip ECD01 (P)2As the male parent), the two materials are hybridized to obtain F1Generation of seed, F1Selfing the material to obtain F2Generation of seed, F2The population is named as 1e519F2. For 10 strains P1、P2And F1And 150 strains F2And (4) performing inoculation identification in the seedling stage, wherein the bacterial source is celery cabbage plasmodiophora XY-2. The disease level of each individual was investigated 6 weeks after inoculation, and the disease level was classified into 0, 1, 3, 5 and 7. The specific grading standard is as follows: level 0: the root has no tumor; level 1: small tumors were found in lateral roots; and 3, level: the main root is swollen, and the diameter of the main root is less than 2 times of the base of the stem; and 5, stage: the main root is swollen, and the diameter of the main root is 2 to 3 times of the base of the stem; grade 7, swelling of the main root, the diameter of which is more than 3 times of the base of the stem. The specific investigation method refers to the original jade incense and the like (original jade incense, Zhaoyuan, Weixiaochun and the like, 2017, identification of physiological races of clubroot germs of Chinese cabbage in Henan province, Henan agricultural science, 46(7), 71-76).
The disease resistance identification result shows that the disease incidence grades of 10 strains of the susceptible parent Y510-9 are all 7 grades, and 10 strains of ECD01 (P)2) All the disease levels of (A) were 0, 10 strains F1The disease rating of the material was 0 (Table 1), F2Population 1e519F2The middle 73 plants were rated 0, 32 plants were rated 1, 9 plants were rated 3, 8 plants were rated 5, and 28 plants were rated 7. Grade 0 and grade 1 are classified as disease-resistant type, grade 3, grade 5 and grade 7 are classified as disease-susceptible type, F2Population 1e519F2The resistant single plants comprise 105 plantsThe infected individuals are 45 individuals, and the separation ratio of the infected individuals is 3: 1 (Table 1) according to the Caliper test, which shows that the ECD01 (P)2) The carried gene for resisting the clubroot is controlled by 1 pair of dominant nuclear genes, and the disease resistance is dominant to the infection.
TABLE 1 Chinese cabbage parent and its progeny for disease-resistant and disease-susceptible plant isolation
2. Discovery of SNP (single nucleotide polymorphism) locus related to clubroot resistance of Chinese cabbage
2.1 genomic DNA extraction
Extracting two parents F by improved CTAB method1And F2Genomic DNA of each individual. The method comprises the following specific steps.
Fresh leaves were taken in a 2.0mL Eppendorf centrifuge tube, 1 bead (5 MM in diameter) was placed in each tube, 1000. mu.L of 2% CTAB extraction buffer was added, and the mixture was disrupted on a tissue disruptor (model: Retsch MM400, Germany) for 1min at a frequency of 30 cycles/sec. Bathing at 65 ℃ for 1h, cooling, adding 500 mu L of 24:1 chloroform/isoamylol extraction liquid, shaking up and down for 30 times, standing for layering, centrifuging (12000r/min) for 10min, sucking 400 mu L of supernatant into a new 1.5mL centrifuge tube, adding 400uL of isopropanol to precipitate DNA, mixing up and down, centrifuging (12000r/min) for 5min, removing supernatant, adding 750 mu L of 70% ethanol to clean DNA precipitate, centrifuging (12000r/min) for 2min, removing supernatant, drying DNA at room temperature, adding 100 mu L of ddH2O to dissolve DNA, and placing in a refrigerator at-20 ℃ for later use.
2.2 location of the novel clubroot-resistant Gene Crr5
With the above-mentioned F2Separating groups as test material, selecting disease grade of 0 grade and 7 grade F2And (3) constructing two extreme gene mixed pools for disease resistance and disease susceptibility of each 25 single plants, and performing whole genome re-sequencing on the two mixed pools by using a BSA-Seq technology. Using Chinese cabbage Chiifu-401-42 genome sequence (V1.5) as a reference sequence, respectively aligning clean reads of parents and two pools to the reference genome by BWA (v0.7.17) software, and performing SNP calling by using Samtools (V1.9) and Bcftools (V1.9) to obtain SNP loci of each sample. And calculating the SNP-index of the disease-resistant mixed pool and the SNP-index of the disease-sensitive mixed pool respectively, subtracting the SNP-index of the disease-sensitive pool from the SNP-index of the disease-resistant pool to obtain a delta SNP-index between the two mixed pools, and performing sliding window analysis by taking 1Mb as a window and 50kb as a step length to determine a candidate interval of the disease-resistant gene. By significance analysis, the disease resistance gene was located in the interval of 7.40-8.85Mb of chromosome A08 (as shown in FIG. 1), with the interval length of 1.45 Mb. The Mapping interval of the gene is the same as Crr1(9.6-11.7Mb, survey et al, 2006.Simple sequence repeat-based comparative genes between the viral rapa and the Arabidopsis thaliana: the genetic origin of the closed resistance), Rcr9 (peak at 10.27Mb, Yu et al, 2017. genetic-by-genetic-Mapping genetic sample QTL for closed resistance to diseases of the fungal strain, and CRs (10.75-11.5Mb, Laila et al, 2019.Mapping of non-viral strain library branched QTL, Crr 5. the gene has no temporary resistance to diseases.
The relation between the SNP locus and clubroot resistance of the Chinese cabbage is analyzed, and the T/C SNP locus at the position of 7,953,439bp in the positioning interval (7.40-8.85Mb) of the novel gene Crr5, namely the SNP locus corresponding to the 101 st locus of the sequence 1 is found to be related to the clubroot resistance, and is named as Crr 5-1. Wherein Y at position 101 in SEQ ID No.1 indicates that the corresponding nucleotide may be T or C.
3. Detection of Chinese cabbage clubroot disease resistance by using SNP locus Crr5-1
3.1 KASP marker design at SNP site Crr5-1
A KASP marker Crr5-KASP1 is designed according to the SNP site Crr5-1 and consists of the following three primers: crr5-KASP1 Fa: 5' -GAAGGTGACCAAGTTCATGCTCGTGGTGGAAAGAAATGGTT-3' (SEQ ID NO: 2); crr5-KASP1 Fb: 5' -GAAGGTCGGAGTCAACGGATTCGTGGTGGAAAGAAATGGTC-3' (SEQ ID NO: 3); crr5-KASP 1R: 5'-CGTCCTCCGAGAAGCACACACC-3' (SEQ ID NO: 4). Crr5-KASP1Fa and Crr5-KASP1Fb are two allele-specific forward primers,
crr5-KASP1Fa is a specific primer with SNP position Crr5-1 as T, and FAM fluorescent sequence tag sequence (underlined) is added to 5 'end, Crr5-KASP1Fb is a specific primer with SNP position Crr5-1 as C, and HEX fluorescent sequence tag sequence (underlined) is added to 5' end. Crr5-KASP1R is a common reverse primer.
Amplifying a fragment with the SNP site Crr5-1 as T by the single-stranded DNA molecules shown in the sequence 2 and the sequence 4, and reading a fluorescent signal of a fluorescent group combined with the FAM sequence in the KASP master mix by using a microplate reader or a fluorescent quantitative PCR (polymerase chain reaction) instrument;
the single-stranded DNA molecules shown in the sequence 3 and the sequence 4 amplify the fragment with the SNP site Crr5-1 as C, and the fluorescence signal of the fluorescent group combined with the HEX sequence in the KASP master mix can be read by a microplate reader or a fluorescent quantitative PCR instrument.
Extract 1e519F2The genomic DNA of 150 individuals in the population was subjected to KASP-PCR using KASP marker Crr5-KASP 1. The KASP-PCR reaction was performed on a 384-well PCR instrument (Eppendorf Mastercycler pro) in a reaction system comprising per 4. mu.L reaction system: mu.L of DNA (60 ng. mu.L-1), 2. mu.L of KASP Master mix (2X), 0.07. mu.L of primer mix (from a concentration of 100. mu. mol. L)-1Crr5-KASP1Fa, Crr5-KASP1Fb, Crr5-KASP1R and ddH2O is mixed according to the volume ratio of 12:12:30: 46), and the rest is ddH2And (4) supplementing and finishing.
The KASP-PCR amplification program was: first stage denaturation at 94 deg.C for 15 min; the second stage is denaturation at 94 ℃ for 20s and annealing at 61 ℃ for 60s for 10 cycles (from the second cycle, each cycle is reduced by 0.6 ℃); in the third stage, denaturation at 94 ℃ is carried out for 20s, annealing at 55 ℃ is carried out for 60s, and 26 cycles are carried out; fourth stage 37 deg.C for 1 min.
The KASP-PCR amplification product was read by end-point method using a Roche fluorescent quantitative PCR Instrument LightCycler 480Instrument II (LC480 II). If the fluorescent signal of only the fluorescent group combined with the FAM sequence is displayed (the signal point is blue), the genotype of the SNP site Crr5-1 of the Chinese cabbage is TT (namely the SNP site Crr5-1 in the genome of the Chinese cabbage is homozygote of T, and is called genotype TT for short); if the fluorescent signal of only the fluorescent group combined with the HEX sequence is displayed (the signal point is green), the genotype of the SNP site Crr5-1 of the Chinese cabbage is CC (namely, the SNP site Crr5-1 in the genome of the Chinese cabbage is homozygote of C, and is hereinafter referred to as genotype CC); if the fluorescent signal of the fluorophore combined with the FAM sequence and the fluorescent signal of the fluorophore combined with the HEX sequence are displayed (the signal point is red), the genotype of the SNP site Crr5-1 of the Chinese cabbage is TC (namely, the SNP site Crr5-1 in the genome of the Chinese cabbage is the homozygous type of T and C, and is hereinafter referred to as the genotype TC).
The SNP typing results were analyzed using LC480 software v1.5.1:
1e519F2the genotype detection results of 150 individuals of the population are shown in figure 2, the signal point of the SNP site Crr5-1 of the Chinese cabbage with the genotype of TT is blue, the 5' end is connected with a primer of a FAM fluorescent label sequence for competitive amplification, and the polymerization is near an X axis; the signal point of the Chinese cabbage SNP locus Crr5-1 with the genotype of CC is green, the 5' end is connected with the primer of the HEX fluorescent label sequence for competitive amplification, and the polymerization is near the Y axis; the signal point of the Chinese cabbage SNP site Crr5-1 with the genotype TC is red and is aggregated near the diagonal (FIG. 2). Crr5-KASP1 mark can obviously distinguish two homozygous genotypes, can identify the heterozygous genotype, has the characteristic of codominant mark, and is successfully developed.
3.2 detection of F by the KASP molecular marker of the novel clubroot-resistant Gene Crr52Clubroot resistance of individual plants in a population
Using the marker Crr5-KASP1, in accordance with the method described above, for F2Carrying out genotyping on 150 individuals of the population, and naming the genotype of homozygous disease-resistant as a, wherein the genotype of the corresponding Crr5-1 locus is CC; the genotype of the homozygous susceptible is named as b, and the genotype of the corresponding Crr5-1 site is TT; the heterozygous type was designated as h, and the genotype at the corresponding Crr5-1 site was TC. As a result, 28 individuals having genotype a had an average disease level of 1.18, 91 individuals having genotype h had an average disease level of 1.09, and 31 individuals having genotype b had an average disease level of 5.29 (FIG. 3). Analysis of variance shows that the disease grade between the genotype a and the disease grade of the disease-resistant material is not different, the disease grade between the genotype b of the disease-sensitive material and the genotype (a and h) of the disease-resistant material is very obvious, and the Crr5-KASP1 marker genotype is highly related to the disease-resistant grade, so that the disease-sensitive material and the disease-resistant material can be definitely identified, and the molecular assisted breeding method can be used for molecular assisted breeding of clubroot resistant varieties of Chinese cabbages.
TABLE 2F2Genotyping and disease-level of 150 individuals of a population
3.3 detecting the anti-clubroot performance of F1 generation Chinese cabbage by KASP molecular marker of clubroot resistance new gene Crr5
Using KASP marker Crr5-KASP1 at 4 BC2F2Population (1e177 BC)2F2-5，1e177BC2F2-24，1e177BC2F2-25，1e177BC2F226) in total 156 individuals, and the genotypes of the 156 individuals were determined by KASP marker Crr5-KASP1 as described above, and the genotypes were compared with the disease levels of the 156 individuals after inoculation.
Of which 4 BC2F2The group is Chinese cabbage susceptible material Y510-9 (P)1As female parent) and the disease-resistant material European turnip ECD01 (P)2As the male parent), the two materials are hybridized to obtain F1Generation of seed, F1Then with P1Backcrossing to obtain BC1F1，BC1F1Then with P1Backcrossing to obtain BC2F1，BC2F1Selfing the material to obtain BC2F2Generation seed, which will be numbered 1e177BC respectively2F2-5、1e177BC2F2-24、1e177BC2F2BC of-25 and 1e177BC2F2-262F2The seeds were cultured to obtain 156 plants shown in Table 3.
The results are shown in the following table. The results show that the coincidence rate between the genotype and the disease-resistant and susceptible phenotypes of each individual plant is 91.3-100%, the average coincidence rate is 97.8%, the accuracy is very high, and the method can be used for molecular marker-assisted selection of a new clubroot disease-resistant gene Crr 5.
TABLE 3 BC2F2Genotyping and disease-level of 156 individuals of a population
TABLE 4 marker Crr5-KASP1 at 4 BC2F2Verification in a population
The invention provides an SNP locus Crr5-1 (T/C SNP variation occurs on 101 site of a sequence shown as a sequence 1 on a Chinese cabbage genome) closely linked with a clubroot resistance gene Crr5, and develops KASP Crr5-KASP1, wherein the marker can detect Crr5 genotype at high flux, can be applied to molecular marker assisted breeding of a new variety with clubroot resistance of Chinese cabbage at high efficiency and low cost, and has very important significance.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
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Claims (10)
1, Crr5-1, in the identification or auxiliary identification of clubroot resistance of Chinese cabbage; crr5-1 is a SNP locus of the genome of the Chinese cabbage, is the 101 th nucleotide of SEQ ID No.1, and is C or T.
2. The application of the substance for detecting Crr5-1 polymorphism or genotype in Chinese cabbage genome in identifying or assisting in identifying the clubroot resistance of Chinese cabbage; crr5-1 is an SNP locus on the genome of the Chinese cabbage, Crr5-1 is an SNP locus on the genome of the Chinese cabbage, is the 101 th nucleotide of SEQ ID No.1, and is C or T.
3. Use according to claim 2, characterized in that: the substance for detecting the polymorphism or the genotype of Crr5-1 in the genome of the Chinese cabbage contains a primer for amplifying the DNA fragment of the genome of the Chinese cabbage including Crr 5-1.
4. Use according to claim 3, characterized in that: the primer is a primer composition consisting of single-stranded DNA shown in SEQ ID No.2, single-stranded DNA shown in SEQ ID No.3 and single-stranded DNA shown in SEQ ID No. 4.
5. A method for identifying or assisting in identifying the clubroot resistance of Chinese cabbage, which comprises detecting the Crr5-1 genotype of claim 1 of the Chinese cabbage to be detected, and identifying or assisting in identifying the clubroot resistance of the Chinese cabbage according to the genotype of the Chinese cabbage to be detected.
6. Use of Crr5-1 of claim 1 in Chinese cabbage breeding.
7. The method for identifying or assisting in identifying the clubroot resistance of Chinese cabbage as claimed in claim 4, which is applied to breeding of Chinese cabbage.
8. Use according to claim 7, characterized in that: the Chinese cabbage breeding is to select and breed the clubroot-resistant Chinese cabbage.
9. The clubroot disease resisting Chinese cabbage breeding process includes the following steps: detecting the Crr5-1 genotype of claim 1 in the genome of the Chinese cabbage, and selecting the Chinese cabbage with the genotype of Crr5-1 and CC in the genome of the Chinese cabbage as a parent breeding.
10. The primer set forth in claim 3 or 4.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112831594A (en) * | 2021-03-17 | 2021-05-25 | 西北农林科技大学 | Functional molecular marker of clubroot-resistant gene CRa of Chinese cabbage and application of functional molecular marker |
| CN114381463A (en) * | 2022-02-23 | 2022-04-22 | 华中农业大学 | Application of BraA08g039212E gene from European turnip ECD04 in improvement of plasmodiophora tumefaciens resistance |
| CN114774569A (en) * | 2022-03-18 | 2022-07-22 | 华中农业大学 | Molecular marker linked with Chinese cabbage DW anti-clubroot site CRA8.1 and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110119038A (en) * | 2010-04-26 | 2011-11-02 | 충남대학교산학협력단 | Species-specific primers to chinese cabbage clubroot pathogen spore and method for detecting or separating using thereof |
| KR20140126207A (en) * | 2013-04-22 | 2014-10-30 | 대한민국(농촌진흥청장) | SNP Maker for identifying of clubroot resistance in Cabbage and uses thereof |
| CN105543391A (en) * | 2016-02-05 | 2016-05-04 | 北京市农林科学院 | InDel molecular marker for identifying clubroot-resistant QTL (quantitative trait locus) located on Chinese cabbage A03 chromosome and application thereof |
| KR101704382B1 (en) * | 2015-11-06 | 2017-02-08 | 충남대학교산학협력단 | Specific primer set for discrimination of cabbage clubroot disease-resistant cultivar and uses thereof |
| CN106591457A (en) * | 2016-12-23 | 2017-04-26 | 北京市农林科学院 | Development and application of SNP molecular marker for identifying clubroot resistance QTL located on A03 chromosome of Chinese cabbage |
| CN107937599A (en) * | 2018-01-17 | 2018-04-20 | 天津科润农业科技股份有限公司 | A kind of molecular labeling KB2, primer and application for identifying the anti-clubroot of Chinese cabbage |
| US20180305774A1 (en) * | 2015-04-30 | 2018-10-25 | Monsanto Technology Llc | Methods for Producing Canola Plants with Clubroot Resistance and Compositions Thereof |
| CN109295247A (en) * | 2018-10-22 | 2019-02-01 | 沈阳农业大学 | Compact linkage molecule label yau403, primer and the application of the anti-clubroot CRd gene of Chinese cabbage |
| CN110734999A (en) * | 2019-11-12 | 2020-01-31 | 北京市农林科学院 | SNP molecular marker tightly linked with new clubroot-resistant gene CRw of Chinese cabbage and application thereof |
-
2020
- 2020-05-15 CN CN202010411279.8A patent/CN112143823B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110119038A (en) * | 2010-04-26 | 2011-11-02 | 충남대학교산학협력단 | Species-specific primers to chinese cabbage clubroot pathogen spore and method for detecting or separating using thereof |
| KR20140126207A (en) * | 2013-04-22 | 2014-10-30 | 대한민국(농촌진흥청장) | SNP Maker for identifying of clubroot resistance in Cabbage and uses thereof |
| US20180305774A1 (en) * | 2015-04-30 | 2018-10-25 | Monsanto Technology Llc | Methods for Producing Canola Plants with Clubroot Resistance and Compositions Thereof |
| KR101704382B1 (en) * | 2015-11-06 | 2017-02-08 | 충남대학교산학협력단 | Specific primer set for discrimination of cabbage clubroot disease-resistant cultivar and uses thereof |
| CN105543391A (en) * | 2016-02-05 | 2016-05-04 | 北京市农林科学院 | InDel molecular marker for identifying clubroot-resistant QTL (quantitative trait locus) located on Chinese cabbage A03 chromosome and application thereof |
| CN106591457A (en) * | 2016-12-23 | 2017-04-26 | 北京市农林科学院 | Development and application of SNP molecular marker for identifying clubroot resistance QTL located on A03 chromosome of Chinese cabbage |
| CN107937599A (en) * | 2018-01-17 | 2018-04-20 | 天津科润农业科技股份有限公司 | A kind of molecular labeling KB2, primer and application for identifying the anti-clubroot of Chinese cabbage |
| CN109295247A (en) * | 2018-10-22 | 2019-02-01 | 沈阳农业大学 | Compact linkage molecule label yau403, primer and the application of the anti-clubroot CRd gene of Chinese cabbage |
| CN110734999A (en) * | 2019-11-12 | 2020-01-31 | 北京市农林科学院 | SNP molecular marker tightly linked with new clubroot-resistant gene CRw of Chinese cabbage and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| ADRIAN CHANG ET AL.: "Clubroot resistance gene Rcr6 in Brassica nigra resides in a genomic region homologous to chromosome A08 in B. rapa", 《BMC PLANT BIOLOGY》 * |
| 宋莹等: "大白菜抗根肿病基因定位及分子标记研究进展", 《分子植物育种》 * |
| 杨双娟等: "大白菜KASP反应体系的优化与建立", 《园艺学报》 * |
| 杨双娟等: "大白菜抗根肿病基因 BraA.Pb.8.4 的定位及KASP 标记开发", 《园艺学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112831594A (en) * | 2021-03-17 | 2021-05-25 | 西北农林科技大学 | Functional molecular marker of clubroot-resistant gene CRa of Chinese cabbage and application of functional molecular marker |
| CN114381463A (en) * | 2022-02-23 | 2022-04-22 | 华中农业大学 | Application of BraA08g039212E gene from European turnip ECD04 in improvement of plasmodiophora tumefaciens resistance |
| CN114381463B (en) * | 2022-02-23 | 2023-11-07 | 华中农业大学 | Application of BraA08g039212E gene derived from European turnip ECD04 in improving resistance of rhizopus |
| CN114774569A (en) * | 2022-03-18 | 2022-07-22 | 华中农业大学 | Molecular marker linked with Chinese cabbage DW anti-clubroot site CRA8.1 and application thereof |
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