CN117344054B - Molecular marker primer for amplifying wheat powdery mildew resistance gene pmXQ-0508 and application thereof - Google Patents
Molecular marker primer for amplifying wheat powdery mildew resistance gene pmXQ-0508 and application thereof Download PDFInfo
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Abstract
The invention discloses an amplified wheat powdery mildew resistance genepmXQ‑0508The molecular marker primer includes an upstream primer 69-2B-025-F and a downstream primer 69-2B-025-R; the nucleotide sequence of the upstream primer 69-2B-025-F is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer 69-2B-025-R is shown as SEQ ID NO. 2. The invention also discloses the molecular marker primer in genepmXQ‑0508Is used for detecting, identifying and assisting in identifying powdery mildew resistance character of wheat and molecular marker assisted breeding. The primer and the application thereof provided by the invention are not affected by the environment, the selection target is clear, the detection is rapid and accurate, the cost is saved, and the breeding efficiency of powdery mildew resistant wheat varieties is greatly improved.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to an amplified wheat powdery mildew resistance genePmXQ-0508Is provided.
Background
Wheat is one of the important grain crops, and its high and stable yield is especially important for grain safety. Wheat powdery mildew caused by the infection of the powdery mildew of the wheat causes the yield reduction of the wheat, and seriously threatens the safe production of the wheat. The measures for preventing and controlling wheat powdery mildew mainly comprise chemical agent prevention and control and breeding and popularization of disease-resistant varieties. Compared with the traditional chemical agent control, the method has the advantages that the disease-resistant genes are excavated for efficient breeding selection, the disease-resistant varieties are widely popularized and used in production, and the method is economical, effective and environment-friendly.
In recent years, more than 100 wheat powdery mildew resistance genes or their alleles have been identified and molecularly located, including 64 formally named genes. However, most of these disease-resistant genes are race-specific resistance and are liable to lose resistance with large-scale use in production and evolution of pathogenic bacteria. Therefore, it is necessary to constantly find and locate new powdery mildew resistance genes and apply the genes to wheat powdery mildew resistance breeding to enrich wheat powdery mildew antigens.
Disclosure of Invention
The invention aims to provide an amplified wheat powdery mildew resistance genepmXQ-0508Molecular marker primer of (2) and application thereof, so as to amplify powdery mildew resistance gene of wheat by using primer amplification molecular markerpmXQ-0508Positioning and detection are carried out, and the parents of the wheat are purposefully selected in the wheat breeding process, so that a guiding basis is provided for breeding new wheat varieties resistant to powdery mildew.
The invention is realized by the following steps: amplified wheat powdery mildew resistance genepmXQ-0508The molecular marker primer includes an upstream primer 69-2B-025-F and a downstream primer 69-2B-025-R; the nucleotide sequence of the upstream primer 69-2B-025-F is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer 69-2B-025-R is shown as SEQ ID NO:2.
The invention provides an amplified wheat powdery mildew resistance genepmXQ-0508Molecular marker primer in genepmXQ- 0508Is used for detecting, identifying and assisting in identifying powdery mildew resistance character of wheat and molecular marker assisted breeding.
The invention also provides a method for detecting whether the wheat variety carries the wheat powdery mildew resistance genepmXQ-0508Comprising the steps of:
(1) Extracting genome DNA of a wheat sample to be detected;
(2) Carrying out PCR amplification on genome DNA of a wheat sample to be detected by using a molecular marker primer to obtain an amplification product; the molecular marker primer comprises an upstream primer 69-2B-025-F and a downstream primer 69-2B-025-R; the nucleotide sequence of the upstream primer 69-2B-025-F was 5'-AACATAAATGCGTTCGACAC-3' (SEQ ID NO: 1), and the nucleotide sequence of the downstream primer 69-2B-025-R was 5'-TTAAAGCATTGCACCAAAGG-3' (SEQ ID NO: 2);
(3) Electrophoresis and detection are carried out on the amplified product, if the specific band of 156bp can be amplified, the detection shows that the wheat sample to be detected carries powdery mildew resistance genespmXQ-0508The method comprises the steps of carrying out a first treatment on the surface of the Otherwise, the wheat sample to be tested does not carry powdery mildew resistance genespmXQ- 0508。
The PCR amplification system in the method provided by the invention is 10 mu L, and comprises the following steps: 50 ng/. Mu.L wheat genomic DNA 1.0. Mu.L, PCR Master mix 4. Mu.L, 5. Mu.M upstream primer 0.5. Mu.L, 5. Mu.M downstream primer 0.5. Mu.L, sterile deionized water 4. Mu.L.
The PCR amplification procedure in the method provided by the invention is as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 15s, annealing at 55℃for 15s, extension for 40s,30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
Through multi-point field identification for many years, the wheat XQ-0508 has good performance on powdery mildew resistance and is an excellent powdery mildew resistance germplasm resource of wheat. Genetic analysis and molecular marker detection of powdery mildew resistance in seedling stage show that the resistance of wheat XQ-0508 seedling stage to powdery mildew epidemic strain E09 is controlled by a single recessive gene and is named aspmXQ-0508. 118 with uniform distribution of whole genomeMolecular marker pair XQ-0508, susceptible wheat variety SH3566, and F using XQ-0508×SH3566 2:3 Polymorphism detection of disease resistance pool and disease susceptibility pool composed of 20 homozygous disease resistance and 20 homozygous disease susceptibility families in population, 9 pairs of markers exhibit consistent polymorphism in the anti-susceptibility parent and anti-susceptibility pool, and then 138 XQ-0508 XSH 3566F pairs are used with these markers 2:3 Genotyping the family and typing the genespmXQ-0508Preliminary mapping was within the interval of wheat 2BS chromosome 26.35-29.59 Mb. Further according to the sequence of the Chinese spring wheat reference genome in the interval, the primer5.0 software is utilized to design and screen to obtain the genepmXQ-0508Closely linked Indel markers 69-2B-025. The invention provides a wheat powdery mildew resistance genepmXQ-0508The molecular marker 69-2B-025 of (B) is subjected to genetic segregation population detection and genepmXQ-0508Co-separation can efficiently and accurately detect genespmXQ-0508Is applied to the genetic mapping large population of genespmXQ-0508And (3) detecting, identifying and assisting in identifying powdery mildew resistance characters of wheat and assisting in selecting and breeding by using molecular markers. Amplification of powdery mildew resistance Gene of wheatpmXQ-0508The molecular marker primer is applied to powdery mildew resistant wheat breeding, is rapid and accurate in detection, is not influenced by environment, has definite selection targets, saves cost, and greatly improves the breeding efficiency of powdery mildew resistant wheat varieties or strains.
Drawings
Fig. 1: labeled 69-2B-025 primer at XQ-0508 XSH 3566, part F 2:3 Amplification results in pedigree.
In the figure, M: pUC 18-MspI, a step of I;1: XQ-0508 (disease resistant parent); 2: SH3566 (susceptible parent); 3-17: f of XQ-0508 XSH 3566 2:3 Family, wherein 3-7: homozygous disease-resistant family, 8-12: anti-sensory separation pedigree, 13-17: homozygous disease family; the arrow indicates the amplified genepmXQ-0508Is a specific band of (a).
Fig. 2: amplification results of marker 69-2B-025 in partially diseased wheat varieties.
In the figure, M: pUC 18-MspI, a step of I;1: XQ-0508 (disease resistant parent); 2: SH3566 (susceptible parent); 3-17: shannong 1538, handan wheat 13, zhou Mai, smoke 1212, middle-breeding1311. Tennong 1014, jimai 229, jimai 21, jimai 20, dai wheat 2173, zhong mai 1751, zhong mai 9398, liangxing 619, green wheat 6 and zheng 0856; the arrow ispmXQ-0508Is a specific band of (a).
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials, reagents and the like used in the examples are all commercially available unless otherwise specified.
Example 1 wheat powdery mildew resistance GenepmXQ-0508Development of molecular marker 69-2B-025
Material
Wheat XQ-0508 is provided by the university of smoke desk wheat breeding team and is highly resistant to wheat powdery mildew. SH3566 was obtained from the national crop germplasm pool. Hybridization of XQ-0508 with SH3566 to obtain F 1 Selfing to obtain F 2 Group and corresponding F 2:3 Family.
2. Extraction of wheat genomic DNA
The CTAB method for extracting the wheat genome DNA comprises the following steps:
1) Taking young leaves of wheat materials, quick-freezing in liquid nitrogen, grinding into powder, and filling into a 2 mL EP tube;
2) Adding 600-800 μl of CTAB extract, and mixing in water bath 1 h at 65deg.C for several times;
3) Adding chloroform with equal volume, and mixing on a shaking table for 15 min;
4) Centrifuging at 12000 rpm at room temperature for 10min, collecting 400 μl supernatant, adding 3 times of pre-cooled absolute ethanol into EP tube of 1.5 and mL, mixing, and settling at-20deg.C for 2 h;
5) Centrifuging at 12000 rpm at room temperature for 10min, discarding supernatant, and adding 800 μl of 70% ethanol for washing 3 times;
6) Air-drying the precipitate, adding 50. Mu.L of 1 XTE or ddH 2 O is dissolved.
7) The DNA stock solution was diluted to 50 ng/. Mu.L with sterile deionized water as working solution for use.
3. Identification of wheat seedling powdery mildew resistance and genetic analysis of resistance
Identification of powdery mildew resistance in wheat seedling stage is completed in an intelligent greenhouse. The disease-resistant parent XQ-0508 and the disease-resistant parent SH3566 and F 1 Hybrid seeds, F 2 Population and F 2:3 Family is planted in 128 holes of plug tray (3.2×3.2×4.2 cm), parent and F 1 At least 20 seeds were identified, each F 2:3 At least 25 seeds are identified from families, and the susceptible control Mingxian 169 is randomly sown and is distinguished by inserting a label. The greenhouse conditions were controlled at 18-20 ℃ with a relative humidity of 80% and a photoperiod of 14 h light/10 h darkness. Powdery mildew strain E09 was inoculated by a brushing method during the one-leaf period. After 10-14 days, phenotypes were investigated when the sensory control Mingxian 169 was fully ill. The type of reaction (IT) is described in the 0-4 level standard. Disease resistance grade division: the 0-2 grade is disease-resistant type, and the 3-4 grade is disease-sensitive type.
The results show that: XQ-0508 shows high resistance (it=0) to powdery mildew strain E09, SH3566 shows high feel (it=4), F 1 The plants all show disease (IT is 3-4), which indicates that XQ-0508 carries recessive disease resistance genes; f to the combination 2 The population is subjected to resistance identification, and the result shows that the anti-susceptibility separation ratio is 32:106, and the chi-square test accords with the separation ratio of single recessive genes of 1:3 (chi 2 =0.24,P=0.62), further F 2:3 The family identification result shows that the disease-resistant family is homozygous: anti-sensory separation family: the separation ratio of homozygous susceptible families is 32:73:33, and chi-square test accords with the separation ratio of single recessive genes of 1:2:1 (χ 2 =0.49,P=0.79). In conclusion, the resistance of XQ-0508 to powdery mildew strain E09 is controlled by a single recessive gene, which is named powdery mildew resistance genepmXQ-0508。
4、pmXQ-0508Molecular marker initial positioning of (2)
And respectively selecting 20 homozygous disease-resistant families and 20 homozygous disease-sensitive families to construct a disease-resistant pool and a disease-sensitive pool according to the phenotype identification result. Polymorphism detection of XQ-0508, SH3566 and anti-disease pools using 118 uniformly distributed molecular markers throughout the genome, 9 pairs of markers exhibited consistent polymorphisms in anti-disease parent and anti-disease pools, followed by F of 138 XQ-0508 XSH 3566 pairs using these markers 2:3 Genotyping the familiespmXQ-0508Preliminary mapping was within the interval of wheat 2BS chromosome 26.35-29.59 Mb.
5. And (3) withpmXQ-0508Development of closely linked molecular markers
According to the sequence information of the Chinese spring wheat reference genome in the candidate interval 26.35-29.59 and Mb, primer5.0 software is used for designing a primer marked by Indel, and F of XQ-0508×SH3566 is used for 2:3 Genotyping the family, and finally obtaining the genepmXQ-0508Co-isolated Indel markers 69-2B-025.
The primers of molecular marker 69-2B-025 included an upstream primer and a downstream primer:
nucleotide sequence of the upstream primer 69-2B-025-F: 5'-AACATAAATGCGTTCGACAC-3';
nucleotide sequence of the downstream primer 69-2B-025-R: 5'-TTAAAGCATTGCACCAAAGG-3'.
The PCR amplification system was 10. Mu.L, and it comprises: 50 ng/. Mu.L wheat genomic DNA 1.0. Mu.L, 4. Mu.L PCR Master mix, 5. Mu.M upstream primer 0.5. Mu.L, 5. Mu.M downstream primer 0.5. Mu.L, sterile deionized water 4. Mu.L.
The PCR amplification procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 15s, annealing at 54℃for 15s, extension for 40s,30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
The electrophoresis separation procedure of the amplification product is as follows: electrophoresis is carried out on non-denaturing polyacrylamide gel with the mass volume percentage concentration of 8%, after the amplification product is mixed with 2 mu L of 6 Xloading buffer solution, 2 mu L of mixture is taken for sample application, electrophoresis is carried out for 2.5-3h under the constant pressure of 180V, and after silver nitrate staining, photographing is carried out. If the specific band of 156bp can be amplified, the detection of powdery mildew resistance gene carried in the wheat sample to be detected is indicatedpmXQ-0508Otherwise, the wheat sample to be tested does not carry the wheat powdery mildew resistance genepmXQ- 0508。
The molecular marker 69-2B-025 is part F of XQ-0508 XSH 3566 2:3 The typing results in the family are shown in FIG. 1. Wherein: m: pUC 18-MspI, a step of I;1: XQ-0508 (disease resistant parent); 2: SH3566 (susceptible parent); 3-17: f of XQ-0508 XSH 3566 2:3 Family of 3-7: homozygous disease-resistant family, 8-12: anti-sensory separation pedigree, 13-17: homozygous disease family; the arrow is the genepmXQ-0508Is a specific band of (a). The amplification result shows that the marker 69-2B-025 amplifies the specific band of 156bp in the disease-resistant parent XQ-0508 and the disease-resistant family, and does not amplify the target band in the disease-resistant parent SH3566 and the disease-resistant family.
Example 2 wheat powdery mildew resistance GenepmXQ-0508Application of molecular marker primer
The sample to be tested comprises disease-resistant parents XQ-0508, disease-resistant parents SH3566 and 138 XQ-0508 XSH 3566 derived F 2:3 Family and 35 parts of non-carrying disease-resistant genepmXQ-0508The wheat DNA extraction method to be tested was the same as in example 1 (shannon 1538, handan wheat 13, zhou Mai, tobacco 1212, middle breeding 1311, tainong 1014, jimai 229, jimai 21, jimai 20, dai wheat 2173, middle wheat 1751, middle wheat 9398, star 619, green wheat 6, zhengmai 0856, lander 677, wu Nong 16, taimai 1918, psing red, wheat 28, zhongxin wheat 77, tobacco 15, tobacco 17, tobacco 23, tobacco 24, tobacco 161, tobacco 191, tobacco 301, tobacco 390, tobacco 572, tobacco 745, tobacco 836, tobacco 5158, tobacco 215 and tobacco 199).
The genomic DNA of the materials is used as a PCR amplification template, and the primers of the molecular marker 69-2B-025 developed by the invention are used for amplification:
nucleotide sequence of the upstream primer 69-2B-025-F: 5'-AACATAAATGCGTTCGACAC-3';
nucleotide sequence of the downstream primer 69-2B-025-R: 5'-TTAAAGCATTGCACCAAAGG-3'.
The PCR amplification system was 10. Mu.L, including: 50 ng/. Mu.L wheat genomic DNA 1.0. Mu.L, 4. Mu.L PCR Master mix, 5. Mu.M upstream primer 0.5. Mu.L, 5. Mu.M downstream primer 0.5. Mu.L, sterile deionized water 4. Mu.L.
The PCR amplification procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 15s, annealing at 54℃for 15s, extension for 40s,30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
The electrophoresis separation procedure of the amplification product is as follows: electrophoresis is carried out on non-denaturing polyacrylamide gel with the mass and volume percentage of 8%, after the amplification product is mixed with 2 mu L of 6 times of loading buffer solution, 2 mu L of mixture is taken for sample application, electrophoresis is carried out for 2.5-3h under the constant pressure of 180V, and after silver nitrate staining, photographing is carried out.
The results of partial amplification of the molecular marker 69-2B-025 primer in a partially diseased wheat variety are shown in FIG. 2. Wherein: m: pUC 18-MspI, a step of I;1: XQ-0508 (disease resistant parent); 2: SH3566 (susceptible parent); 3-17: shannon 1538, handan wheat 13, zhou Mai, tobacco 1212, zhongyu 1311, tainong 1014, ji mai 229, ji mai 21, ji mai 20, dai wheat 2173, zhongmai 1751, zhongmai 9398, liangxing 619, qing mai 6 and Zhengmai 0856. The arrow ispmXQ-0508Is a specific band of (a). The amplification result shows that the marker 69-2B-025 only amplifies the specific band of 156bp in the disease-resistant parent XQ-0508, and the target band is not amplified in the disease-resistant parent SH3566 and the disease-resistant wheat variety. No specific band of 156bp was amplified in 35 parts of the susceptible wheat varieties, only 15 materials were cut out as an amplification schematic diagram, and the results of the other 20 parts are completely consistent with the 15 parts, which indicates that SH3566 and 15 of the wheat varieties do not contain genespmXQ-0508All belong to the wheat variety with the disease.
Powdery mildew resistant gene of wheatpmXQ-0508Is a novel powdery mildew resistant gene, and has not been reported in the related fields of molecular localization, map cloning and molecular breeding at present. The molecular marker 69-2B-025 primer amplification provided by the invention is used for detecting a large population with genetic mapping, and is beneficial to realizing genespmXQ-0508The detection, identification and auxiliary identification of the powdery mildew resistance character of the wheat and the molecular marker auxiliary selective breeding can greatly acceleratepmXQ-0508The breeding and utilization in production enriches the genetic basis of wheat powdery mildew resistance, and can permanently and effectively prevent and control the wheat powdery mildew.
The above examples are of preferred embodiments of the present invention and are intended to be illustrative of the invention and not limiting thereof. Modifications and equivalents may be made by those skilled in the art without departing from the spirit and principles of the embodiments of the invention, which are intended to be within the scope of the invention as claimed.
Claims (3)
1. A method for detecting whether wheat powdery mildew resistance gene pmXQ-0508 is carried in a wheat variety, which is characterized by comprising the following steps:
(1) Extracting genome DNA of a wheat sample to be detected;
(2) Carrying out PCR amplification on genome DNA of a wheat sample to be detected by using a molecular marker primer to obtain an amplification product; the molecular marker primer comprises an upstream primer 69-2B-025-F and a downstream primer 69-2B-025-R; the nucleotide sequence of the upstream primer 69-2B-025-F is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer 69-2B-025-R is shown as SEQ ID NO. 2;
(3) Carrying out electrophoresis detection on the amplification product, if a specific band of 156bp can be amplified, the detection shows that the wheat sample to be detected carries powdery mildew resistance gene pmXQ-0508; otherwise, the wheat sample to be detected does not carry the wheat powdery mildew resistance gene pmXQ-0508.
2. The method of claim 1, wherein the PCR amplification system is 10 μl, comprising: 50 ng/. Mu.L wheat genomic DNA 1.0. Mu.L, PCR Master mix 4. Mu.L, 5. Mu.M upstream primer 0.5. Mu.L, 5. Mu.M downstream primer 0.5. Mu.L, sterile deionized water 4. Mu.L.
3. The method of claim 2, wherein the PCR amplification procedure is: pre-denaturation at 94℃for 3min; denaturation at 94℃for 15s, annealing at 55℃for 15s, extension for 40s,30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
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| CN110777218A (en) * | 2019-12-11 | 2020-02-11 | 烟台大学 | Molecular marker linked with wheat powdery mildew resistance gene Pm37 and application thereof |
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