CN113234852B - Molecular marker and primer group for identifying wheat powdery mildew resistance and application - Google Patents
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Abstract
The invention discloses a molecular marker for identifying wheat powdery mildew resistance, a primer group and application. The molecular marker is two pairs of KASP specific linkage markers developed by using QPm. Saas-4AS flanking SNP probes AX-110406692 and AX-109926787, wherein the linkage markers are KASP markers of KASP-AX-110406692 and KASP markers of KASP-AX-109926787. The molecular marker, the primer set and the application for identifying the wheat powdery mildew resistance provided by the invention develop the molecular marker which can be used for the auxiliary selection of the wheat powdery mildew resistance, can quickly screen the wheat variety or the line with high powdery mildew resistance containing the gene for the disease-resistant breeding utilization of wheat, and obtains the wheat variety containing the wheat powdery mildew resistance gene, thereby not only saving the production cost, but also greatly improving the selection efficiency of wheat materials with powdery mildew resistance and greatly shortening the breeding cycle of the wheat disease-resistant variety; and the detection is convenient and quick and is not influenced by the environment.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a molecular marker and a primer group for identifying wheat powdery mildew resistance and application thereof.
Background
Wheat powdery mildew is a disease caused by powdery mildew, buchner burley of the Gramineae family, which occurs on wheat. Mainly harms leaves, and in severe cases, leaf sheaths, stalks and spikes are all infected. Yellow spots are seen in the disease part at the early stage of disease onset, the spots gradually develop into spots with the disease condition being serious, the spots are oval or round, a powdery mildew layer is arranged on the surface, the spots develop into white gray at the middle stage and light brown at the later stage, and an encystal shell is generated. When the disease condition is mild, the mildew spots are distributed dispersedly, and gradually expand into pieces as the disease condition worsens, and finally cover the whole leaves; the lower and surrounding tissues of the lesion are faded, the diseased leaves are yellow and withered early, and if the disease affects the stem and leaf sheath, the whole plant can be fallen down; the plants are short, small and weak, the ears are small, the thousand kernel weight is obviously reduced, and the wheat yield is finally influenced. Therefore, the discovery and the utilization of the new wheat powdery mildew resistance gene have important significance for wheat powdery mildew resistance breeding.
The research on wheat powdery mildew resistance genes in the prior art is not deep; molecular markers for identifying wheat powdery mildew resistance are not reported, and breeding selection for wheat powdery mildew resistance by using the molecular markers is not reported.
Disclosure of Invention
The invention aims to provide a molecular marker for identifying wheat powdery mildew resistance, a primer group and application thereof, and aims to solve the technical problem that the prior art is lack of breeding selection for wheat powdery mildew resistance by utilizing the molecular marker.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a molecular marker for identifying wheat powdery mildew resistance based on QPm.saas-4AS gene, wherein QPm.saas-4AS is positioned in the interval of 1.06Mb on the end part of chromosome 4AS, the molecular marker is two pairs of KASP specific linkage markers developed by utilizing QPm.saas-4AS gene flanking SNP probes AX-110406692 and AX-109926787, and the linkage markers are KASP markers of KASP-AX-110406692 and KASP markers of KASP-AX-109926787; the nucleotide sequences of the primer sets of KASP-AX-110406692 and KASP-AX-109926787 are respectively,
the first group, primer sequence of KASP-AX-110406692 is as follows:
FAM：GAAGGTGACCAAGTTCATGCTACTGAATAGTCTTTTTGGTGACAAA，
HEX：GAAGGTCGGAGTCAACGGATTCTGAATAGTCTTTTTGGTGACAAG，
COM：TTACATGTACCCATCCCATGAACC；
the second set of KASP-AX-109926787 primers had the following sequences:
FAM：GAAGGTGACCAAGTTCATGCTTCCCCTTCTCCTTTTGTTTTCCTC，
HEX：GAAGGTCGGAGTCAACGGATTTCCCCTTCTCCTTTTGTTTTCCTT，
COM：AAAATCAGAGGAAATGAAAACATC。
furthermore, KASP-AX-110406692 and KASP-AX-109926787 are respectively designed according to the probe sequences of AX-110406692 and AX-109926787, and the probe sequences of AX-110406692 and AX-109926787 are respectively as follows:
AX-110406692：
CTGTAGTTGTAACTGAATAGTCTTTTTGGTGACAA[A/G]GGTTCATGGGATGGGTACATGTAATTGATCACACG；
AX-109926787：
TATCAGTTTTTATCCCCTTCTCCTTTTGTTTTCCT[C/T]GATGTTTTCATTTCCTCTGATTTTTTGGTTTTGTT。
the preparation method of the molecular marker for identifying the wheat powdery mildew resistance provided by the invention comprises the following steps:
(1) Construction of high-generation recombinant inbred line genetic population
Constructing a high-generation recombinant inbred line genetic population F8 by adopting a common cultivated wheat variety Sichuan wheat 104 with high powdery mildew resistance and a wheat variety Baimao with high susceptibility to powdery mildew;
(2) Construction of genetic linkage maps
Performing whole genome scanning on the population by using a 50K high-density SNP chip, constructing a genetic linkage map, performing multi-point powdery mildew resistance identification on the genetic population and parents for many years, and positioning a powdery mildew resistance gene QPm.saas-4AS;
(3) Genetic mapping
Genetic localization is carried out on powdery mildew resistant gene QPm.saas-4AS, and the probe intervals are AX-110406692 and AX-109926787;
(4) Obtaining molecular marker for identifying wheat powdery mildew resistance gene
Two pairs of KASP specific linkage markers are developed by using QPm, saas-4AS site flanking probes AX-110406692 and AX-109926787, and the linkage markers are KASP-AX-110406692 and KASP-AX-109926787;
the probe sequences of the AX-110406692 and the AX-109926787 are respectively as follows:
AX-110406692：
CTGTAGTTGTAACTGAATAGTCTTTTTGGTGACAA[A/G]GGTTCATGGGATGGGTACATGTAATTGATCACACG；
AX-109926787：
TATCAGTTTTTATCCCCTTCTCCTTTTGTTTTCCT[C/T]GATGTTTTCATTTCCTCTGATTTTTTGGTTTTGTT。
further, in the step (3), the method for genetic localization of powdery mildew resistance gene qpm.
The primer group provided by the invention for identifying the molecular marker has the nucleotide sequences as follows:
the first group, primer sequence of KASP-AX-110406692 is as follows:
FAM：GAAGGTGACCAAGTTCATGCTACTGAATAGTCTTTTTGGTGACAAA，
HEX：GAAGGTCGGAGTCAACGGATTCTGAATAGTCTTTTTGGTGACAAG，
COM：TTACATGTACCCATCCCATGAACC；
the second set of KASP-AX-109926787 primers had the following sequences:
FAM：GAAGGTGACCAAGTTCATGCTTCCCCTTCTCCTTTTGTTTTCCTC，
HEX：GAAGGTCGGAGTCAACGGATTTCCCCTTCTCCTTTTGTTTTCCTT，
COM：AAAATCAGAGGAAATGAAAACATC。
the invention provides a method for identifying wheat powdery mildew resistance genes, which comprises the following steps:
(1) Extracting wheat genome DNA to be identified;
(2) Carrying out quantitative PCR amplification on the DNA extracted in the step (1) by adopting a primer group of KASP-AX-86175290 and KASP-AX-86172908;
(3) And identifying the amplification product.
Further, in the step (2), when the quantitative PCR amplification is performed:
and (3) PCR reaction system: 2 μ l of DNA; KASP Master mix 5. Mu.l; 3 μ l of KASP primer set, and 3 primers of primer set are mixed at equal concentration according to the volume ratio of FAM: HEX: COM = 1; total reaction volume 10. Mu.l;
PCR reaction procedure: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20sec, gradient renaturation/elongation at 61-55 ℃ for 60sec, reduction per cycle of 0.6 ℃, for 10 cycles; denaturation at 94 ℃ for 20sec, renaturation/elongation at 55 ℃ for 60sec, for 26 cycles; the KaSP typing fluorescence data were read at 40 ℃ or lower.
The molecular marker provided by the invention is applied to identification of wheat powdery mildew resistance.
Based on the technical scheme, the embodiment of the invention can at least produce the following technical effects:
the molecular marker, the primer set and the application for identifying the wheat powdery mildew resistance provided by the invention develop the molecular marker which can be used for auxiliary selection of the wheat powdery mildew resistance, greatly improve the utilization efficiency of an important gene resource, and further can quickly screen the wheat variety or the strain with high powdery mildew resistance containing the gene for disease-resistant breeding utilization of wheat, so as to obtain the wheat variety containing the wheat powdery mildew resistance gene, thereby not only saving the production cost, but also greatly improving the selection efficiency of wheat materials with powdery mildew resistance and greatly shortening the breeding cycle of the wheat disease-resistant variety; and the detection is convenient and quick and is not influenced by the environment.
Drawings
FIG. 1 is a technical roadmap for an embodiment of the present invention;
FIG. 2 shows the resistance reaction of the highly susceptible powdery mildew material barley mallow selected in example 1 and the highly powdery mildew resistant variety Chuan wheat 104 against wheat powdery mildew;
FIG. 3 is a typing chart of the detection of the specific molecular marker KASP-AX-110406692 by the fluorescent quantitative PCR instrument in example 2 of the present invention;
FIG. 4 is a typing chart showing the detection of the specific molecular marker KASP-AX-109926787 by the fluorescent quantitative PCR instrument in example 2 of the present invention.
Detailed Description
As shown in fig. 1-4:
the powdery mildew resistance gene QPm.saas-4AS of the present invention is disclosed in Liu Z, wang Q, wan H, et al, QTL mapping for adult-plant resistance to powder family in Chinese elite common wheat Chuanmai104[ J ]. Central Research Communications, 2020, 49 (12): 1-10.
Example 1:
the preparation method of the molecular marker for identifying the wheat powdery mildew resistance provided by the invention comprises the following steps:
(1) Construction of high-generation recombinant inbred line genetic population
Constructing a high-generation recombinant inbred line genetic population F8 by adopting a common cultivated wheat variety Sichuan wheat 104 with high powdery mildew resistance and a wheat variety Baimao with high susceptibility to powdery mildew;
(2) Construction of genetic linkage maps
Performing whole genome scanning on the population by using a 50K high-density SNP chip, constructing a genetic linkage map, performing multi-point powdery mildew resistance identification on the genetic population and parents for many years, and positioning a powdery mildew resistance gene QPm.saas-4AS;
(3) Genetic localization
Genetic localization is carried out on powdery mildew resistance gene QPm.saas-4AS through a QTL localization method, and the probe intervals are AX-110406692 and AX-109926787;
(4) Obtaining molecular marker for identifying wheat powdery mildew resistance gene
Two pairs of KASP specific linkage markers were developed using QPm. Saas-4AS site flanking probes AX-110406692 and AX-109926787, said linkage markers being KASP-AX-110406692 and KASP-AX-109926787.
Example 2:
the KASP-AX-110406692 and KASP-AX-109926787 in example 1 were verified, and the KASP-labeled primer detection reagent was a mix reagent from Egypsy technologies, inc., and the quantitative PCR instrument used a CFX384 Real-Time System from BIO-RAD; the phenotype identification of the powdery mildew resistance is to carry out grading identification on the infection degree of each material leaf by pathogenic bacteria; the detection of the specific marker is the typing detection through a fluorescent quantitative PCR instrument; the method specifically comprises the following steps:
(1) Extracting the wheat genome DNA to be identified and subpackaging the wheat genome DNA to a quantitative PCR reaction plate;
(2) Carrying out quantitative PCR amplification on the DNA extracted in the step (1) by adopting a primer group of KASP-AX-86175290 and KASP-AX-86172908;
the nucleotide sequences of the primer groups are respectively as follows:
the first group, primer sequence of KASP-AX-110406692 is as follows:
FAM：GAAGGTGACCAAGTTCATGCTACTGAATAGTCTTTTTGGTGACAAA，
HEX：GAAGGTCGGAGTCAACGGATTCTGAATAGTCTTTTTGGTGACAAG，
COM：TTACATGTACCCATCCCATGAACC；
the second group of KASP-AX-109926787 primer sequences are as follows:
FAM：GAAGGTGACCAAGTTCATGCTTCCCCTTCTCCTTTTGTTTTCCTC，
HEX：GAAGGTCGGAGTCAACGGATTTCCCCTTCTCCTTTTGTTTTCCTT，
COM：AAAATCAGAGGAAATGAAAACATC；
the quantitative PCR amplification method comprises the following steps: PCR amplification reaction and data observation and analysis were carried out using a fluorescent quantitative PCR instrument manufactured by BIO-RAD, and PCR amplification was carried out using Master mix reaction solution for KASP marker detection provided by LGC Biotechnology.
And (3) PCR reaction system: 2 μ l of DNA; KASP Master mix 5. Mu.l; 3 μ l of KASP primer set, and 3 primers of primer set are mixed at equal concentration according to the volume ratio of FAM: HEX: COM = 1; total reaction volume 10. Mu.l;
PCR reaction procedure: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20sec, gradient renaturation/elongation at 61-55 ℃ for 60sec, reduction per cycle of 0.6 ℃, for 10 cycles; denaturation at 94 ℃ for 20sec, renaturation/elongation at 55 ℃ for 60sec, for 26 cycles; the KaSP-typed fluorescence data were read at 40 ℃ or lower.
(3) And identifying the amplification product.
According to the wheat powdery mildew resistance grading identification standard, the concrete grading standard is 0 grade and 0 grade; stage, stage 1, stage 2, stage 3, stage 4; wherein, the 0 grade is that the leaves have no infection trace and are represented as immunity, 0; grade 1 is that the leaves have no fresh spores but a small amount of necrotic spots, grade 1 is that the leaves have a small amount of fresh spores but show high powdery mildew resistance, grade 2 is that the leaves have a certain proportion of fresh spores but show medium powdery mildew resistance, grade 3 is that the leaves have more fresh spores, show medium powdery mildew resistance, and grade 4 is that the leaves are completely infected and show high powdery mildew resistance. The results of phenotype grading identification and detection of the infected degree of the parental leaves are shown in fig. 2, and show that the results of KASP marker detection are stable and reliable according to phenotype analysis and positioning results, and can be used for molecular detection of gene locus QPm.
Claims (4)
1. A molecular marker for identifying wheat powdery mildew resistance based on the gene locus qpm saas-4AS, which is located within the interval of 1.06Mb on the end of chromosome 4AS, characterized in that: the molecular marker is two pairs of KASP specific linkage markers developed by QPm. Saas-4AS gene flanking SNP probes AX-110406692 and AX-109926787, and the linkage markers are KASP markers of KASP-AX-110406692 and KASP markers of KASP-AX-109926787; the nucleotide sequences of the primer sets of KASP-AX-110406692 and KASP-AX-109926787 are respectively,
the first group of KASP-AX-110406692 primers had the following sequences:
FAM：GAAGGTGACCAAGTTCATGCTACTGAATAGTCTTTTTGGTGACAAA，
HEX：GAAGGTCGGAGTCAACGGATTCTGAATAGTCTTTTTGGTGACAAG，
COM：TTACATGTACCCATCCCATGAACC；
the second set of KASP-AX-109926787 primers had the following sequences:
FAM：GAAGGTGACCAAGTTCATGCTTCCCCTTCTCCTTTTGTTTTCCTC，
HEX：GAAGGTCGGAGTCAACGGATTTCCCCTTCTCCTTTTGTTTTCCTT，
COM：AAAATCAGAGGAAATGAAAACATC；
the probe sequences of the AX-110406692 and the AX-109926787 are respectively as follows:
AX-110406692：
CTGTAGTTGTAACTGAATAGTCTTTTTGGTGACAA[A/G]GGTTCATGGGATGGGTACATGTAATTGATCACACG；
AX-109926787：
TATCAGTTTTTATCCCCTTCTCCTTTTGTTTTCCT[C/T]GATGTTTTCATTTCCTCTGATTTTTTGGTTTTGTT。
2. a primer set for identifying the molecular marker of claim 1, wherein: the nucleotide sequences of the primer groups are respectively as follows:
the first group, primer sequence of KASP-AX-110406692 is as follows:
FAM：GAAGGTGACCAAGTTCATGCTACTGAATAGTCTTTTTGGTGACAAA，
HEX：GAAGGTCGGAGTCAACGGATTCTGAATAGTCTTTTTGGTGACAAG，
COM：TTACATGTACCCATCCCATGAACC；
the second set of KASP-AX-109926787 primers had the following sequences:
FAM：GAAGGTGACCAAGTTCATGCTTCCCCTTCTCCTTTTGTTTTCCTC，
HEX：GAAGGTCGGAGTCAACGGATTTCCCCTTCTCCTTTTGTTTTCCTT，
COM：AAAATCAGAGGAAATGAAAACATC。
3. a method for identifying a wheat powdery mildew resistance gene, which is characterized in that: the method comprises the following steps:
(1) Extracting wheat genome DNA to be identified;
(2) Carrying out quantitative PCR amplification on the DNA extracted in the step (1) by adopting a primer group of KASP-AX-110406692 and KASP-AX-109926787;
the primer sequence of KASP-AX-110406692 is as follows:
FAM：GAAGGTGACCAAGTTCATGCTACTGAATAGTCTTTTTGGTGACAAA，
HEX：GAAGGTCGGAGTCAACGGATTCTGAATAGTCTTTTTGGTGACAAG，
COM：TTACATGTACCCATCCCATGAACC；
the KASP-AX-109926787 primer sequence is as follows:
FAM：GAAGGTGACCAAGTTCATGCTTCCCCTTCTCCTTTTGTTTTCCTC，
HEX：GAAGGTCGGAGTCAACGGATTTCCCCTTCTCCTTTTGTTTTCCTT，
COM：AAAATCAGAGGAAATGAAAACATC；
when quantitative PCR amplification is performed:
and (3) PCR reaction system: 2 μ l of DNA; KASP Master mix 5. Mu.l; 3 μ l of KASP primer set, and 3 primers of primer set are mixed at equal concentration according to the volume ratio of FAM: HEX: COM = 1; total reaction volume 10. Mu.l;
PCR reaction procedure: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20sec, gradient renaturation/elongation at 61-55 ℃ for 60sec, reduction of 0.6 ℃ per cycle, and 10 cycles in total; denaturation at 94 ℃ for 20sec and renaturation/elongation at 55 ℃ for 60sec, and the total number of cycles is 26; reading fluorescence data of KASP typing is carried out below 40 ℃;
(3) And identifying the amplification product.
4. Use of the molecular marker of claim 1 for identifying wheat powdery mildew resistance.
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