CN117385094A - Molecular marker primer for amplifying wheat powdery mildew resistance gene PmJM809 and application thereof - Google Patents
Molecular marker primer for amplifying wheat powdery mildew resistance gene PmJM809 and application thereof Download PDFInfo
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Abstract
The invention discloses an amplified wheat powdery mildew resistance genePmJM809The molecular marker primer comprises an upstream primer YTU103-79-F and a downstream primer YTU103-79-R, wherein the nucleotide sequence of the upstream primer YTU103-79-F is shown as SEQ ID NO. 1; the nucleotide sequence of the downstream primer YTU103-79-R is shown in SEQ ID NO. 2. The invention also discloses a method for detecting genes in wheat varieties by using the primerPmJM809Is a method of (2). The method provided by the invention is rapid and accurate in screening, free from environmental influence, clear in selection target, and capable of saving production cost, greatly improving the selection efficiency of new wheat varieties or strains resistant to powdery mildew, and can be widely applied to identification of the powdery mildew resistant characters of wheat and breeding.
Description
Technical Field
The invention relates to the field of biotechnology and agricultural breeding, in particular to an amplified wheat powdery mildew resistance genePmJM809Is provided.
Background
Wheat is one of the important grain crops, and its high and stable yield is especially important for grain safety. Wheat yield and quality are affected by a variety of diseases, among which, blackground powdery mildew @ is usedBlumeria graminisf. sp.tritici,Bgt) Wheat powdery mildew caused by infection is one of the most serious diseases, and seriously threatens the safe production of wheat. Wheat can generally result in a 10-15% yield loss after powdery mildew infection, and can even result in up to 50% yield loss during severe epidemics (Jia et AL, 2018. Jia AL, ren R, gao FM, et AL Mapping and validation of a new QTL for adult-plant resistance to powdery mildew in Chinese elite bread wheat line Zhou8425B [ J)]Theoretical and Applied Genetics, 2018, 131:1063-1071). Therefore, the wheat can be effectively prevented and controlledThe occurrence and prevalence of powdery mildew are essential. At present, three measures of agricultural control, chemical control and disease-resistant variety breeding are mainly adopted for preventing and controlling wheat powdery mildew, and compared with the former two measures, the method has the advantages of most efficient, safe and environment-friendly effects of excavating and reasonably utilizing disease-resistant genes and cultivating disease-resistant varieties.
Up to now 69 formally named wheat powdery mildew resistance genes have been identified at 64 sitesPm1-Pm69，Pm8=Pm17，Pm18=Pm1c，Pm22=Pm1e，Pm23=Pm4c，Pm31=Pm21) And 30 more transiently named genes (Li et al, 2022; mcIntosh et al, 2019. Li YH, wei ZZ, sela HN, et al Long-read genome sequencing accelerated the cloning of)Pm69by resolving the complexity of a rapidly evolving resistance gene cluster in wheat [J]. 2022. BioRxiv. McIntosh RA, Dubcovsky J, Rogers WJ, et al. “Catalogue2 of gene symbols for wheat: 2019 supplement” in Annual Wheat Newsletter [J]Ed. W.J. Raupp (Manhattan, N.Y. The Wheat Genetic and Genomic Resources at Kansas State University), 2019, 98-113). Wherein the method comprises the steps ofPm1、Pm2、Pm3、Pm4、Pm5、Pm24AndPm60is a multiple allele site. However, the disease-resistant genes mostly have one cause of linkage encumbrance and disadvantage, onlyPm2、Pm4a、Pm4b、Pm6、Pm8、Pm21AndPm52and a few genes are widely used for breeding. Powdery mildew resistance gene due to genetic linkage encumbering, production singleization, overuse, competition lag and adverse one-cause multiple effectPm3a、Pm3b、Pm3f、Pm4a、Pm6、Pm8AndPm17etc. have lost resistance in some areas (Cowger et al, 2018. Cowger C, mehra L, arellano C, et al Virulence differences in)Blumeria graminisf. sp.triticifrom the central and eastern United States[J]. Phytopathology, 2018, 108: 402-411.）。Pm1、Pm3、Pm4、Pm5、Pm8A kind of electronic device with high-pressure air-conditioning systemPm17Has been partially or completely deprived of resistance (Zeller et al, 2002. Zeller FJ, kong L, hartl L, et al. Chromosomal)ocation of genes for resistance to powdery mildew in common wheat (Triticum aestivum L. em Thell.) 7. GenePm29in line Pova[J]Euphytica, 2002, 123 (2): 187-194.). Therefore, there is a need to find new broad-spectrum powdery mildew resistance genes in breeding to balance the layout of the disease resistance genes, improve the resistance of wheat to powdery mildew, and provide durable support for preventing and controlling the powdery mildew of wheat.
The developed wheat powdery mildew resistance gene mainly comes from common wheat and related species. Compared with the disease resistance genes from exogenous species or farmyard species, the powdery mildew resistance genes from the bred variety/strain are easier to directly utilize in production, and have higher breeding utilization value. Therefore, the development and utilization of new powdery mildew resistance genes from common wheat varieties/strains are of more practical significance. After successful development of genes with excellent resistance, how to apply their high effect to wheat disease-resistant breeding is important. In recent years, with the continuous development of wheat breeding technology, molecular marker assisted selective breeding has become an important means for wheat powdery mildew resistance breeding. The high-efficiency, accurate and easy-to-use molecular marker is a precondition for carrying out molecular marker assisted selection on the target gene, and the target gene is detected by utilizing molecular marker tracking, so that the breeding process can be greatly shortened, and the breeding efficiency is improved. Therefore, new powdery mildew resistance genes are continuously searched and positioned in the existing variety with excellent performance, molecular markers and primers of the new powdery mildew resistance genes are developed, and the new powdery mildew resistance genes are applied to wheat powdery mildew resistance breeding to enrich wheat powdery mildew antigens, so that the new powdery mildew resistance genes have important significance.
Disclosure of Invention
The invention aims to provide an amplified wheat powdery mildew resistance genePmJM809Molecular marker primer of (2) and application thereof, so as to amplify powdery mildew resistance gene of wheat by using primer amplification molecular markerPmJM809Positioning 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 technical scheme: a molecular marker primer for amplifying a wheat powdery mildew resistance gene PmJM809, wherein the molecular marker primer comprises an upstream primer YTU103-79-F and a downstream primer YTU103-79-R;
the nucleotide sequence of the upstream primer YTU103-79-F is 5'-TGGGATTTTGAGGACCCACG-3', as shown in SEQ ID NO. 1,
the nucleotide sequence of the downstream primer YTU103-79-R is 5'-GGTCAAGGTGGACAAGGAGG-3', and is shown as SEQ ID NO. 2.
The amplified wheat powdery mildew resistance genePmJM809Molecular marker primer in genePmJM809Is applied to detection, identification and auxiliary identification of powdery mildew resistance of wheat.
The amplified wheat powdery mildew resistance genePmJM809Molecular marker primer of (2) has powdery mildew resistance gene in breedingPmJM809Is used in wheat.
The invention also provides a method for detecting whether the wheat variety carries wheat powdery mildew resistance genesPmJM809Comprising 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 of an amplified wheat powdery mildew resistance gene PmJM809 to obtain an amplification product; the molecular marker primer comprises an upstream primer YTU-79-F and a downstream primer YTU-79-R; the nucleotide sequence of the upstream primer YTU103-79-F is 5'-TGGGATTTTGAGGACCCACG-3', shown in SEQ ID NO. 1, and the nucleotide sequence of the downstream primer YTU103-79-R is 5'-GGTCAAGGTGGACAAGGAGG-3', shown in SEQ ID NO. 2;
(3) Electrophoresis and detection are carried out on the amplified product, if the specific band of 466 bp can be amplified, the detection shows that the wheat sample to be detected carries powdery mildew resistance genesPmJM809The method comprises the steps of carrying out a first treatment on the surface of the Otherwise, the wheat to be tested does not carry powdery mildew resistance genesPmJM809。
The PCR amplification system in the method of the invention is 10 mu L, 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.
The PCR amplification procedure in the method of the invention is: pre-denaturation at 94℃for 3 min; denaturation at 94℃for 15 s, annealing at 55℃for 15 s, extension for 40 s,30 cycles; extending at 72 ℃ for 10 min; preserving at 4 ℃.
The invention shows that the resistance of Jimai 809 seedling stage to powdery mildew epidemic strain E09 is controlled by a single dominant gene, named gene, through genetic analysis and molecular marker detection of the seedling stage powdery mildew resistancePmJM809. Ejimai 809, susceptible wheat variety Junda159 and F using Jimai 809 XJunda159 are marked with 321 molecules uniformly distributed over the genome 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, 16 pairs of markers exhibit consistent polymorphisms in the anti-susceptibility parent and anti-susceptibility pool, and then 121 Jimai 809 XJuda 159F pairs are used with these markers 2:3 Genotyping the family and typing the genesPmJM809Preliminary mapping is within the interval of wheat 2BL chromosome 704.2-708.2 Mb. Further according to the sequence of the Chinese spring wheat reference genome in the interval, primer5.0 software is utilized to design and largely screen primer pairs to obtain the gene and the genePmJM809Closely linked Simple Sequence Repeat (SSR) markers, designated YTU103-79. The invention adopts wheat powdery mildew resistance genePmJM809Molecular marker primer pair genes of (2)PmJM809Amplifying, detecting genetic segregation population, and marking and genePmJM809Is only 1.3 cM, and is related to the genePmJM809Closely linked, can efficiently and accurately detect genesPmJM809Is a large population. Experiments prove that the amplified wheat powdery mildew resistance gene provided by the inventionPmJM809The closely linked molecular marker primer can be widely applied to genesPmJM809Is used for detecting, identifying and assisting in identifying powdery mildew resistance character and powdery mildew resistance gene of wheatPmJM809And (5) breeding wheat. The application method is rapid and accurate in screening, free of environmental influence, clear in selection target, low in production cost and capable of greatly improving the selection efficiency of new wheat varieties or strains resistant to powdery mildew.
Drawings
FIG. 1 shows F derived from primers labeled YTU103-79 in part Jimai 809 XJuda 159 2:3 Amplification results in pedigree.
The samples corresponding to each lane in the figure are as follows: m: marker pUC19/Msp I;1: jimai 809 (disease resistant parent); 2: junda 159 (susceptible parent); 3-17: jimai 809 XJunda 159F 2:3 Family, wherein 3-7: homozygous disease-resistant family, 8-12: anti-sensory separation pedigree, 13-17: homozygous disease family; arrows are specific bands labeled YTU-79.
FIG. 2 shows the partial amplification of markers YTU103-79 in 40 parts of susceptible wheat variety.
The samples corresponding to each lane in the figure are as follows: m: marker pUC19/Msp I;1: jimai 809 (disease resistant parent); 2: junda 159 (susceptible parent); 3-17: handan wheat 13, huai wheat 0226, zhou Mai, tobacco 1212, xenong979, zhongyu 1311, ji wheat 268, tainong 1014, ji wheat 229, ji wheat 21, ji wheat 20, dai wheat 2173, zhongmai 1751, liangxing 619 and Dan Mai. The arrow indicates the specific band amplified by the primer of the label YTU, 103-79.
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 obtaining powdery mildew resistant Gene of wheatPmJM809Molecular markers YTU103-79 and primers thereof
1. Material
The disease resistant parent Jimai 809 is provided by the Shandong agricultural sciences wheat breeding team. The susceptible parent Junda 159 is a commercial variety of wheat and is obtained from commercial sources.
Hybridization of Jimai 809 and Junda 159 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 10 min, 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 10 min, 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 wheat seedling powdery mildew resistance was done in the greenhouse. The disease-resistant parent Jimai 809 and the disease-sensitive parent Junda 159 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 in families, and the infected control Tainong 18 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 susceptibility control tenone 18 was fully developed. 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: jimai 809 shows high resistance (it=0) to powdery mildew strain E09, junda 159 shows high sensation (it=4), F 1 Plants all show disease resistance (IT is 0-1), which indicates that Jimai 809 carries dominant 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 86:35, and the chi-square test accords with the separation ratio of single dominant gene of 3:1 (χ 2 =0.80,P=0.37), 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 infectious disease families is 30:56:35, and the chi-square test accords with the separation ratio of single dominant genes of 1:2:1 (χ 2 =1.08,P=0.58). In conclusion, the resistance of Jimai 809 to powdery mildew strain E09 is controlled by a single dominant gene, and the powdery mildew resistance gene is named asPmJM809。
4、PmJM809Molecular 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 is carried out on Jimai 809, jimai variety Junda 159, disease resistance pool and disease resistance pool by 321 molecular markers uniformly distributed in whole genome, 16 pairs of markers show consistent polymorphism in anti-disease parent and anti-disease pool, and then F of 121 Jimai 809 XJunda 159 is utilized by the markers 2:3 Genotyping the familiesPmJM809Preliminary mapping is within the interval of wheat 2BL chromosome 704.2-708.2 Mb.
5. And (3) withPmJM809Development of closely linked molecular markers
According to the sequence information of the Chinese spring wheat reference genome in the candidate interval 704.2-708.2 Mb, primers 5.0 software is used for designing Simple Sequence Repeat (SSR) marked primers, and finally a pair of primers comprising an upstream primer and a downstream primer are screened out:
nucleotide sequence of the upstream primer YTU 103-79-F: 5'-TGGGATTTTGAGGACCCACG-3' (SEQ ID NO: 1);
nucleotide sequence of the downstream primer YTU 103-79-R: 5'-GGTCAAGGTGGACAAGGAGG-3' (SEQ ID NO: 2).
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 3 min; denaturation at 94℃for 15 s, annealing at 55℃for 15 s, extension for 40 s,30 cycles; extending at 72 ℃ for 10 min; 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.
By F of Jimai 809 XJuda 159 2:3 Genotyping the family, and finally obtaining the genePmJM809Closely linked SSR markers YTU103-79, with genetic distances of only 1.3 cM, are shown in FIG. 1. FIG. 1 shows the results of partial amplification of markers YTU103-79 in a partial Jimei 809 XJunda 159 derived F2:3 family. In the figure, M: pUC19/Msp I;1: jimai 809 (disease resistant parent); 2: junda 159 (susceptible parent); 3-17: jimai 809 XJunda 159F 2:3 Family, wherein 3-7: homozygous disease-resistant family, 8-12: anti-sensory separation pedigree, 13-17: homozygous disease family; the arrow is the genePmJM809Is a specific band of (a). The amplification result shows that the markers YTU-79 amplify specific bands of 466 bp in the disease-resistant parent Jimai 809 and the disease-resistant family, and do not amplify the target bands in the disease-resistant parent Junda 159 and the disease-resistant family.
Example 2 wheat powdery mildew resistance GenePmJM809Application of molecular marker YTU-79 primer
The sample to be tested comprises disease-resistant parent Jimai 809, disease-resistant parent Junda 159 and 40 parts of non-disease-resistant genePmJM809The wheat DNA extraction method to be tested was the same as in example 1 (Handan wheat 13, huai wheat 0226, zhou Mai, tobacco 1212, west pesticide 979, middle breeding 1311, ji wheat 268, tainong 1014, ji wheat 229, ji wheat 21, ji wheat 20, dai wheat 2173, zhongmai 1751, zhongmai 9398, liangxing 619, dan Mai, xinlong 4, qing wheat 6, zheng wheat 0856, shannong 202, wu Nong 6, taimai 1918, gin red, wheat 28, zhongxin wheat 77, dan Mai, tainong 18, tobacco 15, tobacco 17, tobacco 23, tobacco 24, tobacco 301, tobacco 390, tobacco 745, tobacco 836, tobacco 2415, tobacco 5158, tobacco 999, tobacco 215 and tobacco 199).
The genomic DNA of the above materials is used as a PCR amplification template, and the primers of the molecular markers YTU103-79 developed by the invention are used for amplification:
nucleotide sequence of the upstream primer YTU 103-79-F: 5'-TGGGATTTTGAGGACCCACG-3';
nucleotide sequence of the downstream primer YTU 103-79-R: 5'-GGTCAAGGTGGACAAGGAGG-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 3 min; denaturation at 94℃for 15 s, annealing at 55℃for 15 s, extension for 40 s,30 cycles; extending at 72 ℃ for 10 min; 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 detection result of the molecular marker is shown in figure 2. FIG. 2 shows the partial amplification of markers YTU103-79 in 40 parts of material. In the figure, M: pUC19/Msp I;1: jimai 809 (disease resistant parent); 2: junda 159 (susceptible parent); 3-17: handan wheat 13, huai wheat 0226, zhou Mai, tobacco 1212, xenong979, zhongyu 1311, ji wheat 268, tainong 1014, ji wheat 229, ji wheat 21, ji wheat 20, dai wheat 2173, zhongmai 1751, liangxing 619 and Dan Mai. The arrow isPmJM809Is a specific band of (a). The amplification result shows that the markers YTU103-79 only amplify the specific band of 466 bp in the disease-resistant parent Jimai 809, and do not amplify the target band in the disease-resistant parent Junda 159 and the disease-resistant wheat variety. No specific band of 466 bp was amplified in 40 susceptible wheat varieties, indicating that none of the varieties of Junda 159 and 40 contained genesPmJM809All belong to the wheat variety with the disease.
Powdery mildew resistant gene of wheatPmJM809The wheat strain Jimai 809 is derived from the regional test of China, and is a novel gene with excellent resistance. At present, the gene has no report related to localization, map cloning and molecular breeding. The molecular markers YTU103-79 provided by the invention can be used for detecting genetic mapping large populations and helping to realize genesPmJM809Fine localization and map cloning of (2); will bePmJM809Introduced into the main variety of the wheat with infectious diseases, the molecular markers YTU103-79 developed by the invention can efficiently and accurately detect and select powdery mildew resistancePopulation, greatly improving transfer disease-resistant genePmJM809To the efficiency and precision ofPmJM809Has very important significance in high-efficiency transformation and deep analysis of disease-resistant mechanisms.
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 (6)
1. Amplified wheat powdery mildew resistance genePmJM809Is characterized in that the molecular marker primer comprises an upstream primer YTU-79-F and a downstream primer YTU-79-R; the nucleotide sequence of the upstream primer YTU103-79-F is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer YTU-79-R is shown as SEQ ID NO. 2.
2. An amplified wheat powdery mildew resistance gene according to claim 1PmJM809Molecular marker primer in genePmJM809Is applied to detection, identification and auxiliary identification of powdery mildew resistance of wheat.
3. An amplified wheat powdery mildew resistance gene according to claim 1PmJM809Molecular marker primer of (2) in breeding powdery mildew resistance genePmJM809Application in wheat.
4. Detection of whether wheat powdery mildew resistance gene is carried in wheat varietyPmJM809Is characterized by comprising the following steps:
(1) Extracting genome DNA of a wheat sample to be detected;
(2) Amplified wheat powdery mildew resistance genePmJM809Carrying out PCR amplification on genome DNA of a wheat sample to be detected to obtain an amplification product; the molecular marker primer comprises an upstream primer YTU-79-F and a downstream primer YTU-79-R; the nucleotide sequence of the upstream primer YTU103-79-F is shown as SEQ ID NO. 1, and the downstream primer YTU103-79-R has a nucleotide sequence shown in SEQ ID NO. 2;
(3) Electrophoresis and detection are carried out on the amplified product, if the specific band of 466 bp can be amplified, the detection shows that the wheat sample to be detected carries powdery mildew resistance genesPmJM809The 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 genesPmJM809。
5. The method of claim 4, 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.
6. The method of claim 5, wherein the PCR amplification procedure is: pre-denaturation at 94℃for 3 min; denaturation at 94℃for 15 s, annealing at 55℃for 15 s, extension for 40 s,30 cycles; extending at 72 ℃ for 10 min; preserving at 4 ℃.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104313021A (en) * | 2014-10-21 | 2015-01-28 | 山西省农业科学院作物科学研究所 | Molecular marker of wheat powdery mildew disease-resistant genes Pm51 and application of molecular marker |
| CN117025812A (en) * | 2023-02-21 | 2023-11-10 | 烟台大学 | Primer for amplifying molecular marker closely linked with wheat powdery mildew resistance gene PmCG15-009 and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104313021A (en) * | 2014-10-21 | 2015-01-28 | 山西省农业科学院作物科学研究所 | Molecular marker of wheat powdery mildew disease-resistant genes Pm51 and application of molecular marker |
| CN117025812A (en) * | 2023-02-21 | 2023-11-10 | 烟台大学 | Primer for amplifying molecular marker closely linked with wheat powdery mildew resistance gene PmCG15-009 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| YA ZHAO等: "Characterization and identification of the powdery mildew resistance gene in wheat breeding line Jimai 809", RESEARCH SQUARE, pages 24 - 25 * |
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