CN114774570A - Molecular marker closely linked with wheat stem basal rot resistance QTL and application thereof - Google Patents
Molecular marker closely linked with wheat stem basal rot resistance QTL and application thereof Download PDFInfo
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Abstract
The invention relates to a molecular marker closely linked with a wheat basal stem rot resistance QTL and application thereof. The molecular marker provided by the invention is SNP1869, wherein the SNP1869 is tightly linked with wheat stem-root rot resistance QTL Qfcr. sicau.1B-5, and the two are co-located on wheat 1B chromosome. The detection and analysis show that the molecular marker SNP1869 can accurately track the wheat stem basal rot resistance QTL and predict the wheat stem basal rot resistance, thereby facilitating the molecular design breeding. The invention also provides application of the molecular marker SNP1869 in wheat breeding. The method provided by the invention can enhance the accuracy of the resistance prediction of the wheat stem basal rot, so that the wheat variety or strain with the QTL of high resistance to the wheat stem basal rot can be quickly screened out for breeding, and the breeding process of the high-yield wheat variety can be greatly accelerated.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to a molecular marker closely linked with wheat stem basal rot resistance QTL Qfcr.
Background
Wheat (Triticum aestivum L) is one of the most important food crops in the world, not only the major source of starch and energy, but also providers of various ingredients essential to health, especially proteins, vitamins and dietary fibers. Wheat basal rot (FCR) has been a soil-borne disease that has been found to seriously compromise wheat production. In recent years, the major wheat yield is spread and the harm is increased, and researchers are urgently required to strengthen the work of resistance source screening and disease-resistant gene mining to improve the disease resistance of major cultivars.
In recent years, QTL sites related to stem-based rot have been continuously discovered. Wallwork et al found a QTL conferring FCR disease resistance on chromosome 4B near dwarfing gene Rht1 (Wallwork, H., Butt, M., Cheng, J.P.E., et al.Resistence to crown in a shallow identified threaded and improved method for screening adult plants [ J ]. Australian Plant Pathology,2004,33: 1-7.); collard et AL, based on seedling experiments, used "2-49" to detect two QTLs conferring FCR disease resistance on the 1DL and 1AL chromosomes, respectively, accounting for 21% and 10% phenotypic variation (Collard, B.C.Y., Grams, R.A., Boville, W.D., et AL, development of molecular markers for crown rot resistance in a book: mapping of ls for a segmented resistance in a ' 2-49 ' × Janz ' position [ J ]. Plant Breeding,2005,124, 6) (QT 532) 537); bovil et al detected 2 QTLs on 5D and 2D based on seedling experiments using "W21 MMT 70", accounting for up to 28% of phenotypic variation but not detected in all three reported assays (Bovil, W.D., Ma, W., Ritter, K., Collad, B.C.Y., et al.identification of novel QTL for resistance to crown rotation in the double-hatched work position 'W21 MMT 70' X 'Mendos' J. Plant Breeding,2010,125(6): 538-543); ma et al, using "CSCR 6" and the susceptible material "Lang", mapped to 2 major QTL sites Qcrs. cpi-3B and Qcrs. cpi-4B by seedling greenhouse inoculation identification, where Qcrs. cpi-3B accounted for up to 48.8% of phenotypic variation, detectable in different genetic backgrounds, was the FCR disease-resistant site with the greatest effect so far (Ma, J., Li, H.B., Zhang, C.Y., et al., identification and identification of a major QTL containment crown resistance in Applied Genetics [ J ]. 120 (1119); 1128.).
Local wheat varieties are also called farmer varieties and are products of long-term natural selection and artificial selection. The local wheat variety can be well adapted to natural environment and cultivation conditions, so that the possessed genetic material resource is very rich; meanwhile, local varieties of wheat also become a valuable gene bank for wheat improvement because the local varieties have rich agronomic characters and genetic diversity and are potential gene resources for enriching the genetic variation of wheat (dune Gem, Zhengpalahi, China wheat genetic resources [ M ]. China agriculture Press, 2000.). Therefore, 361 parts of local wheat are subjected to Genome-wide association analysis (GWAS), the wheat stem-root rot resistance locus is further positioned, genes with high stem-root rot resistance are excavated, closely linked molecular markers are searched, the map-based cloning of the stem-root rot resistance genes is promoted, new gene resources are provided for the creation and high-yield breeding of wheat high-stem-root rot resistance materials, the molecular markers are further utilized for assisting selection, the accuracy of prediction of the stem-root rot disease resistance is enhanced, the breeding efficiency is improved, and the aim of increasing the single yield of the wheat is accelerated.
The molecular marker assisted selection is not dependent on phenotype selection, namely is not influenced by various factors such as environmental conditions, gene interaction, genotype and environment interaction and the like, but directly selects the genotype, so that the breeding efficiency can be greatly improved. Competitive Allele-Specific PCR (Kompetitive Allele Specific PCR) allows for accurate biallelic detection of SNPs and indels at Specific sites in a wide range of genomic DNA samples. The detection method has the advantages of simplicity and convenience in operation, good specificity, high flux, rapidness, low detection cost, accurate result and the like, and realizes real closed-tube operation, so that the detection method is generally concerned. Therefore, the molecular marker which is closely linked with the wheat stem rot resistance QTL and is suitable for the KASP technology of the fluorescent quantitative PCR platform is screened out, so that the wheat stem rot resistance gene can be selected, the disease resistance of wheat is effectively improved, the normal growth and fructification of wheat are ensured, the selection flux, the speed and the accuracy are improved, the technical bottleneck of large-scale popularization and application is solved, and the molecular marker has important significance for improving the quality and the yield of wheat breeding populations on a large scale.
Disclosure of Invention
The invention aims to provide a molecular marker SNP1869 closely linked with a wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 and application of the molecular marker in wheat breeding.
In a first aspect, the invention provides a molecular marker closely linked with wheat stem basal rot resistance QTL Qfcr. sicau.1B-5, which is a molecular marker SNP1869, and a nucleotide sequence SEQ ID NO.1 (5 '-CGTCGTTCGGTTGGTTATGTTTCAAGTAGGTTTCAATTNTAGCAATCGAAATGAAATTACCCG-3'; wherein N is A or G.), namely, the polymorphism of the 39 th base of the sequence is A/G, and the polymorphism is related with wheat stem basal rot resistance.
The molecular marker SNP1869 is closely connected with the wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 and is co-located in an 11.01Mb zone between the markers SNP1868 and SNP1918 on the wheat 1B chromosome, and the molecular marker SNP1869 is located in a confidence interval of the wheat stem basal rot resistance QTL Qfcr. sicau.1B-5.
Specifically, the wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 provided by the invention can obviously increase the wheat stem basal rot resistance, the LOD value is 7.29 at most, and 8.29% of phenotypic variation is explained.
More specifically, the single base difference site of the molecular marker SNP1869 provided by the invention is G, and the resistance of the corresponding wheat stem basal rot is strong; the single base difference site of the molecular marker SNP1869 is A, and the resistance of the single base difference site to the wheat stem rot is weak.
In a second aspect, the invention also provides a specific primer set for the fluorescent quantitative PCR amplification of the molecular marker SNP 1869.
One skilled in the art can design primers for amplifying the molecular marker SNP1869 based on KASP detection platform technology, preferably, the specific primer pair includes primers with sequences shown in SEQ ID nos. 2-4. Wherein, the 5' ends of the primers shown in SEQ ID NO.2 and SEQ ID NO.3 are respectively connected with different fluorescent probes.
SNP1869-1:5’-GAAGGTGACCAAGTTCATGCTGCCCATTAAAGTAAAGCTAACGATA-3’；(SEQ ID NO.2)
SNP1869-2:5’-GAAGGTCGGAGTCAACGGATTGCCCATTAAAGTAAAGCTAACGATG-3’；(SEQ ID NO.3)
SNP1869-3:5’-GCAGCAAGCCAACCAATACA-3’；(SEQ ID NO.4)
Moreover, the 5' ends of the primers SNP1869-1 and SNP1869-2 are respectively connected with different fluorescent probes;
the sequence of the fluorescent probe is as follows:
f, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO.5), which in the examples of the present invention bind FAM fluorophore.
H, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO.6) in an embodiment of the invention the probe binds to a HEX fluorophore.
The invention provides a molecular marker SNP1869 or any one of the following applications of the specific primers:
(1) the application in identification of wheat stem basal rot resistance QTL Qfcr.sicau.1B-5;
(2) the application in screening or identifying wheat varieties with resistance to the stem base rot of wheat;
(3) the application in wheat molecular marker assisted breeding;
(4) application in improving wheat germplasm resources.
In a third aspect, the invention also provides a method for identifying the wheat stem-base rot resistance QTL Qfcr.sicau.1B-5, which comprises the steps of carrying out fluorescent quantitative PCR amplification by using the genome DNA of wheat to be detected as a template and adopting the specific primer group, and carrying out genotyping on the wheat to be detected according to the PCR amplification result.
Preferably, the reaction system for the fluorescent quantitative PCR amplification comprises: 2 XKASP Mastermix 5. mu.L, KASP Assay Mix 0.14. mu.L, template DNA 50ng, DNase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the nucleotide sequence of the primer contained in the KASP Assay Mix is shown in SEQ ID NO.2-4, and the volume ratio of the three primers is 2:2: 5.
In the examples of the present invention, the fluorescent quantitative PCR procedure: activating at 95 deg.C for 10 min; denaturation at 95 ℃ for 20s, annealing and extension at 65 ℃ for 60s, and circulating for 10 times, wherein the annealing and extension temperature is reduced by 1 ℃ every time; denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 60s, and circulating for 36 times; fluorescence signals were collected at 37 ℃ for 60 s.
The judgment standard of the method for identifying the wheat stem-base rot resistance QTL Qfcr.sicau.1B-5 provided by the invention is as follows: wheat varieties containing the wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 all generate fluorescent signals identical to the fluorescent probes marked by the primers shown in SEQ ID NO.3, and wheat varieties not containing the wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 all generate fluorescent signals obviously different from the fluorescent probes marked by the primers shown in SEQ ID NO. 3.
In the invention, the wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 and the molecular marker SNP1869 are obtained by the following method:
(1) 361 local wheat varieties are taken as genetic location groups.
(2) Extracting DNA of each strain of the genetic population by using a CTAB method, and genotyping 361 parts of materials by using a DArT chip technology to obtain genotype data of the population.
(3) The wheat seedling stage greenhouse soaking inoculation method identifies the Disease grade of the stem-based rot of 361 wheat local varieties, and calculates the Disease Index (DI).
(4) Combining 361 parts of local wheat variety molecular marker data and phenotype data, carrying out Genome-wide association analysis (GWAS) by using TASSEL v5.0 association analysis software under a mixed linear model, and positioning the wheat stem basal rot resistance QTL Qfcr.sicau.1B-5 in the interval of SNP 1868-SNP 1918 on the 1B chromosome.
(5) And converting SNP sites in the target section into fluorescent quantitative PCR primers for subsequent screening. The PolyMarker website was used to design fluorescent quantitative PCR primer 8 pairs (table 1). Design standard of fluorescent quantitative PCR primer: the length of the amplification primer is 18-25 bp, the length of the amplification product is 45-60bp, the annealing temperature is 57-62 ℃, and the GC content is 40-60%.
The sequence of the synthetic primer is as follows:
forward primer 1: f probe + amplification primer sequence
Forward primer 2: h probe + amplification primer sequence
Reverse primer: amplification primer sequences
F, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (conjugated FAM fluorophores)
H, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (binding HEX fluorophore)
TABLE 18 pairs of KASP primer sequences and amplified fragment lengths
(6) Competitive allele specific PCR (KASP) assay
a) Screening of polymorphic molecular markers: the 8 pairs of primers are selected, and the DNA of local wheat stem basal rot extremely-resistant materials (Lemai, Shishou wheat and paw teeth) and extremely-susceptible materials (white rye wheat, Sanyuehuang and Maoyang wheat) are used as templates to carry out PCR amplification, so that 1 pair of molecular marker primers with good effect is obtained, wherein the primers shown in SEQ ID NO.2-4 are named as SNP1869-1/2/3 (the nucleotide sequences are respectively shown in SEQ ID NO. 2-4). The amplification product is a molecular marker SNP1869 with polymorphism, a primer shown as SEQ ID NO.2-4 is used, and the nucleotide sequence obtained by amplification is shown as SEQ ID NO. 1.
b) KASP analysis of the local wheat population: and amplifying DNA of a local wheat population by using the PCR primer of the molecular marker SNP1869 with polymorphism obtained in the steps, and carrying out genotype identification to obtain molecular marker data. The type of extreme disease-resistant materials (Lei Mai, Shi shou Mai, paw) of the stem base rot is marked as A, the size of the amplified fragment is 63bp, and the single base difference site is G. The type of extreme disease material (barley grass, yellow of three months, red barley grass) is marked as B, the length of the amplified fragment is 63bp, and the single base difference site is A. The genotype of local wheat group strain is consistent with that of extreme disease-resistant materials (Lepidium stringoides, Pandali and Calophyllum) and is marked as A, and the genotype of local wheat group strain is consistent with that of extreme disease-sensitive materials (Amylum pratense, Trigonella foenum-graecum and Malabar sieboldii) and is marked as B.
The beneficial effects of the invention at least comprise:
the invention discloses a wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 positioned in local wheat for the first time, which is positioned on a wheat 1B chromosome, and the disease resistance of the wheat stem basal rot is obviously improved. The QTL has higher utilization value in wheat yield (improving the disease resistance of wheat stem basal rot) breeding. The invention discloses a molecular marker SNP1869 for accurately detecting wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 based on a fluorescent quantitative PCR platform for the first time, and the marker is a codominant marker, and has the advantages of accurate and efficient detection and convenient and stable amplification.
The molecular marker SNP1869 disclosed by the invention is obviously related to wheat stem basal rot resistance QTL Qfcr. sicau.1B-5, presents coseparation marker characteristics, has high accuracy when used for molecular marker-assisted selection, improves the selection and identification efficiency of specific wheat stem basal rot disease-resistant varieties suitable for different environments, and has high success rate.
Drawings
FIG. 1 is a linkage genetic map of the wheat stem rot resistance QTL Qfcr. sicau.1B-5 on chromosome 1B and a molecular marker SNP 1869.
FIG. 2 shows the results of genotyping DNA of leaves of endemic wheat stem basal rot extreme disease-resistant materials (Lepidium striolatum, Panicum miliaceum, and Torula hispidium) and extreme disease-sensitive materials (Amylum Tritici testa, Triesbiota sanguinea, and Malabar hispidium) in trefoil stage using fluorescent quantitative PCR primers in example 2 of the present invention.
FIG. 3 shows the results of genotyping by fluorescent quantitative PCR primers for the local wheat population in example 2 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 acquisition of wheat stalk rot resistance QTL Qfcr. sicau.1B-5 and molecular marker SNP1869
In the invention, the wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 and the molecular marker SNP1869 are obtained by the following method:
(1) 361 local wheat varieties are taken as genetic location groups.
(2) Extracting DNA of each strain of the genetic population by using a CTAB method, and genotyping 361 parts of materials by using a DArT chip technology to obtain genotype data of the population.
(3) The wheat seedling stage greenhouse soaking inoculation method identifies the Disease grade of the stem basal rot of 361 wheat local varieties, and calculates Disease Index (DI).
(4) Combining 361 parts of local wheat variety molecular marker data and phenotype data, carrying out Genome-wide association analysis (GWAS) by using TASSEL v5.0 association analysis software under a mixed linear model, and positioning the wheat stem basal rot resistance QTL Qfcr.sicau.1B-5 in the interval of SNP 1868-SNP 1918 on the 1B chromosome.
(5) And converting the SNP locus in the target section into a fluorescent quantitative PCR primer for subsequent screening. The PolyMarker website was used to design fluorescent quantitative PCR primer 8 pairs (table 1). Design standard of fluorescent quantitative PCR primer: the length of the amplification primer is 18-25 bp, the length of the amplification product is 45-60bp, the annealing temperature is 57-62 ℃, and the GC content is 40-60%.
The sequence of the synthetic primer is as follows:
forward primer 1: f probe + amplification primer sequence
Forward primer 2: h probe + amplification primer sequence
Reverse primer: amplification primer sequences
F, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (conjugated FAM fluorophores)
H, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (binding HEX fluorophore)
(6) Competitive allele specific PCR (KASP) assay
a) Screening of polymorphic molecular markers: the 8 pairs of primers are selected, and the DNA of local wheat stem basal rot extremely-resistant materials (Lemai, Shishou wheat and paw teeth) and extremely-susceptible materials (white rye wheat, Sanyuehuang and Maoyang wheat) are used as templates to carry out PCR amplification, so that 1 pair of molecular marker primers with good effect is obtained, and the molecular marker primers are named as SNP1869-1/2/3 (the nucleotide sequences are respectively shown in SEQ ID NO. 2-4). The amplification product is a molecular marker SNP1869 with polymorphism, and the nucleotide sequence is shown as SEQ ID NO. 1.
b) KASP analysis of the local wheat population: and amplifying DNA of a local wheat population by using the PCR primer of the molecular marker SNP1869 with polymorphism obtained in the steps, and carrying out genotype identification to obtain molecular marker data. The type of extreme disease-resistant materials (Lemai, Shishou mai and paw) of the stem base rot is marked as A, the size of the amplified fragment is 63bp, and the single base difference site is G. The type of extreme disease material (white brown wheat, yellow croaker and rough red wheat) is marked as B, the length of the amplified fragment is 63bp, and the single base difference site is A. The genotype of local wheat group strain is consistent with that of extreme disease-resistant materials (Lepidium stringoides, Pandali and Calophyllum) and is marked as A, and the genotype of local wheat group strain is consistent with that of extreme disease-sensitive materials (Amylum pratense, Trigonella foenum-graecum and Malabar sieboldii) and is marked as B.
c) By using a mixed linear model calculation method of software TASSEL v5.0 and combining local wheat population stem basal rot resistance phenotype data, a molecular marker SNP1869 is found to be closely linked with a wheat stem basal rot resistance QTL Qfcr.sicau.1B-5 locus, and the result is shown in FIG. 1. From FIG. 1, it can be seen that the wheat stalk rot resistance QTL Qfcr. sicau.1B-5 is located between markers SNP1868 and SNP1918, while molecular marker SNP1869 is tightly linked with QTL.
The position of the wheat stem rot resistance QTL Qfcr. sicau.1B-5 on the 1B chromosome and the linkage genetic map between the molecular marker SNP1869 are shown in figure 1.
Example 2 application of molecular marker SNP1869 closely linked with wheat stem basal rot resistance QTL Qfcr. sicau.1B-5
1. DNA extraction
The test materials are selected from Lepidium barbarum, Spanish wheat, claw teeth, white brown wheat, Martin Elaeagnus and Haematococcus, wherein Lepidium barbarum, Spanish wheat and claw teeth are disease-resistant varieties of wheat stem base rot, and white brown wheat, Martin Elaeagnus and Haematococcus is susceptible varieties of wheat stem base rot. And (3) extracting the leaf DNA of the wheat sample in the trefoil stage by adopting a CTAB method.
2. Screening of primers for detecting wheat stem-base rot resistance QTL Qfcr. sicau.1B-5
2.1 primer design
SNP marker SNP1869 is converted to design a fluorescent quantitative PCR primer for subsequent screening. The Polymarker website was used to design the pair of fluorescent quantitative PCR primers 8 (Table 1). Design standard of fluorescent quantitative PCR primer: the length of the amplification primer is 18-25 bp, the length of the amplification product is 45-60bp, the annealing temperature is 57-62 ℃, and the GC content is 40-60%. The sequence of the synthetic primer is as follows:
forward primer 1: f probe + amplification primer sequence
Forward primer 2: h probe + amplification primer sequence
Reverse primer: amplification primer sequences
F, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (bindable FAM fluorophore)
H, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (bindable HEX fluorophore)
2.2 fluorescent quantitative PCR platform test primers and differences between their parents
(1) Extracting leaf DNA of local wheat stem basal rot extreme disease-resistant materials (Lemai, Shishou wheat and paw teeth) and extreme disease-sensitive materials (white rough wheat, Sanyuehuang and Maohuo wheat) in three leaf stage.
(2) Using genome DNA of wheat to be detected as a template, designing a primer based on a KASP detection platform technology, and carrying out fluorescent quantitative PCR amplification;
wherein, the primer sequence of the step 2 is as follows:
SNP1869-1:5’-GAAGGTGACCAAGTTCATGCTGCCCATTAAAGTAAAGCTAACGATA-3’；
SNP1869-2:5’-GAAGGTCGGAGTCAACGGATTGCCCATTAAAGTAAAGCTAACGATG-3’；
SNP1869-3:5’-GCAGCAAGCCAACCAATACA-3’；
moreover, the 5' ends of the primers SNP1869-1 and SNP1869-2 are respectively connected with different fluorescent probes;
the sequence of the fluorescent probe is as follows:
f, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (conjugated FAM fluorophores)
H, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (bindable HEX fluorophore)
(3) A fluorescent quantitative PCR amplification reaction system: 2 XKASP Mastermix 5. mu.L, KASP Assay Mix 0.14. mu.L, template DNA 50ng, DNase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the KASP Assay Mix contains primers SNP1869-1, SNP1869-2 and SNP1869-3 with the volume ratio of 2:2: 5. That is, in KASP Assay Mix, primers SNP1869-1, SNP1869-2, and SNP1869-3 at a concentration of 100. mu.M were mixed at a volume ratio of 2:2: 5.
(4) Fluorescent quantitative PCR procedure: activating at 95 deg.C for 10 min; denaturation at 95 ℃ for 20s, annealing and extension at 65 ℃ for 60s, and circulating for 10 times, wherein the annealing and extension temperature is reduced by 1 ℃ every time; denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 60s, and circulating for 36 times; fluorescence signals were collected at 37 ℃ for 60 s.
(5) The specific method for analyzing PCR products is as follows: wheat samples containing the wheat stem basal rot resistance QTL Qfcr.sicau.1B-5 all have the type A of extreme disease-resistant materials (beard wheat, hand-applying wheat and claw teeth) of the stem basal rot, and wheat samples not containing the wheat stem basal rot resistance QTL Qfcr.sicau.1B-5 all have fluorescence signals which are obviously different from the extreme disease-resistant materials (beard wheat, hand-applying wheat and claw teeth) of the wheat stem basal rot, such as extreme disease-resistant materials (white brown wheat, yellow in three months and ruddy wheat) and have the type B of the extreme disease-resistant materials. The results of genotyping Hordeum vulgare, Ardisia mamillata, Trigonella foenum-graecum, and Hordeum majus using KASP primers are shown in FIG. 2.
3. Applicability of primer sequence SNP1869-1/2/3 in population detection process
(1) And hybridizing by taking Shichimai as a female parent and Sanyuehuang as a male parent to obtain F1, and selfing by F1 to obtain F2 as a verification population. And extracting the leaf DNA of each strain in the three-leaf stage in the population.
(2) And (2) performing fluorescent quantitative PCR amplification by using the DNA obtained in the step (1) as a template and using the primer provided by the invention, wherein the fluorescent dye is SsoFast EvaGreen.
(3) A fluorescent quantitative PCR amplification reaction system: 2 XKASP Mastermix 5. mu.L, KASP Assay Mix 0.14. mu.L, template DNA 50ng, DNase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the KASP Assay Mix contains primers SNP1869-1, SNP1869-2 and SNP1869-3, and the volume ratio is 2:2: 5. That is, in KASP Assay Mix, 100. mu.M primers SNP1869-1, SNP1869-2, and SNP1869-3 were mixed at a volume ratio of 2:2:5, and then 0.14. mu.L of the mixture was aspirated.
(4) Fluorescent quantitative PCR procedure: activating at 95 deg.C for 10 min; denaturation at 95 ℃ for 20s, annealing and extension at 65 ℃ for 60s, and circulating for 10 times, wherein the annealing and extension temperature is reduced by 1 ℃ every time; denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 60s, and circulating for 36 times; fluorescence signals were collected at 37 ℃ for 60 s.
(5) The specific method for analyzing the PCR product is as follows: wheat samples containing the wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 all have a type A which is marked as the type of wheat applied with the extreme disease-resistant material for the stem basal rot, and wheat samples not containing the wheat stem basal rot resistance QTL Qfcr.sicau.1B-5 all have fluorescent signals which are obviously different from the type B which is marked as the type of wheat applied with the extreme disease-resistant material for the stem basal rot, such as the extreme disease material yellow in three months, and the result is shown in figure 3. Randomly spot-checking 73 strains, and 35 strains can amplify segments of the same type as the stem rot resistance QTL Qfcr. sicau.1B-5 of wheat, and predicting the strong stem rot resistance of the strains. 38 plants can be amplified to obtain B-type fragments which are the same as those of the common yellows in the three months and are plants which do not contain the wheat stem rot resistance QTL Qfcr.
(6) The disease-resistant grade of the stem-base rot of 73F 2 plants is identified in a greenhouse of wheat seedling stage, the result is shown in table 2, and the molecular marker SNP1869 of the wheat stem-base rot resistance QTL Qfcr. The disease resistance of the average stem rot of the plants with the same type as the hand-applied wheat is 1.23, which is obviously lower than the disease resistance of the average stem rot of the plants with the same type as the marchand yellow, and is 2.42. The actual result is consistent with the expected result, which shows that the wheat stem basal rot resistance QTL Qfcr. sicau.1B-5 of the invention has the effect of remarkably improving the wheat stem basal rot resistance, and the molecular marker SNP1869 can be used for tracking and identifying the wheat stem basal rot resistance QTL Qfcr. sicau.1B-5.
TABLE 2
Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (10)
1. The molecular marker SNP1869 closely linked with the wheat stalk base rot resistance QTL Qfcr. sicau.1B-5 is characterized in that the nucleotide sequence of the molecular marker SNP1869 is shown as SEQ ID No.1, and the 39 th base polymorphism of the nucleotide sequence is A or G.
2. The molecular marker SNP1869 according to claim 1, wherein the molecular marker SNP1869 is co-localized with the wheat stalk rot resistance QTL Qfcr. sicau.1B-5 in the 11.01Mb region between the markers SNP1868 and SNP1918 on the wheat 1B chromosome, and the molecular marker SNP1869 is located in the wheat stalk rot resistance QTL Qfcr.sicau.1B-5 confidence region.
3. The molecular marker SNP1869 according to claim 1 or 2, characterized in that the wheat stalk rot resistance QTL qfcr. sicau.1b-5 significantly increases wheat stalk rot resistance with LOD values up to 7.29 accounting for 8.29% of the phenotypic variation.
4. The molecular marker SNP1869 according to claim 3, wherein when the single base difference site of the molecular marker SNP1869 is G, wheat contains QTL Qfcr.sicau.1B-5, which is highly resistant to wheat stalk base rot; when the single base difference site of the molecular marker SNP1869 is A, the wheat does not contain QTL Qfcr. sicau.1B-5, and the resistance of the wheat stem basal rot is weak.
5. A specific primer set for amplifying the molecular marker of any one of claims 1 to 4.
6. The specific primer group of claim 5, wherein the specific primer group comprises primers with sequences shown as SEQ ID No. 2-4.
7. The specific primer set of claim 6, wherein the primers shown in SEQ ID No.2 and SEQ ID No.3 have different fluorescent probes attached to their 5' ends, respectively.
8. The molecular marker of any one of claims 1 to 4 or the specific primer set of any one of claims 5 to 7, wherein any one of the following applications:
(1) the application in identifying wheat stem basal rot resistance QTL Qfcr. sicau.1B-5;
(2) the application in screening or identifying wheat varieties with high stem basal rot resistance;
(3) the application in wheat molecular marker assisted breeding;
(4) application in improving wheat germplasm resources.
9. A method for identifying wheat stem basal rot resistance QTL Qfcr.sicau.1B-5 is characterized by using genome DNA of wheat to be detected as a template, adopting the specific primer group of any one of claims 5-7 to perform fluorescent quantitative PCR amplification, and genotyping the wheat to be detected according to the PCR amplification result.
10. The method according to claim 9, wherein the wheat variety containing the QTL qfcr. sicau.1b-5 for stalk rot resistance of wheat exhibits the same fluorescent signal as the fluorescent probe attached to the primer shown in SEQ ID No.3, and the wheat variety not containing the QTL qfcr. sicau.1b-5 for stalk rot resistance of wheat exhibits a fluorescent signal that is significantly different from the fluorescent probe attached to the primer shown in SEQ ID No. 3.
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| CN117512180A (en) * | 2023-12-01 | 2024-02-06 | 中国农业大学 | KASP molecular marker of wheat stem-based rot disease-resistant site Qfcr.cau.2A and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115961081A (en) * | 2022-12-26 | 2023-04-14 | 四川农业大学 | Molecular marker closely linked with corn basal rot resistance gene locus qFCR9 and application thereof |
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| CN117512177A (en) * | 2023-11-30 | 2024-02-06 | 中国农业大学 | KASP (KASP-labeled primer) related to wheat stem-based rot resistance and application thereof |
| CN117512177B (en) * | 2023-11-30 | 2024-04-02 | 中国农业大学 | KASP (KASP-labeled primer) related to wheat stem-based rot resistance and application thereof |
| CN117512180A (en) * | 2023-12-01 | 2024-02-06 | 中国农业大学 | KASP molecular marker of wheat stem-based rot disease-resistant site Qfcr.cau.2A and application thereof |
| CN117512180B (en) * | 2023-12-01 | 2024-06-07 | 中国农业大学 | KASP molecular marker of wheat stem-based rot disease-resistant site Qfcr.cau.2A and application thereof |
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