CN117025812A - Primer for amplifying molecular marker closely linked with wheat powdery mildew resistance gene PmCG15-009 and application thereof - Google Patents

Primer for amplifying molecular marker closely linked with wheat powdery mildew resistance gene PmCG15-009 and application thereof Download PDF

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CN117025812A
CN117025812A CN202310143762.6A CN202310143762A CN117025812A CN 117025812 A CN117025812 A CN 117025812A CN 202310143762 A CN202310143762 A CN 202310143762A CN 117025812 A CN117025812 A CN 117025812A
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靳玉丽
马朋涛
张文静
刘开昌
刘成
李林志
王江春
刘伟
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Yantai University
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Abstract

The invention discloses a primer for amplifying a molecular marker closely linked with a wheat powdery mildew resistance gene PmCG15-009, wherein the molecular marker is YTU103-101; the labeled primers of the molecular markers YTU-101 comprise an upstream primer YTU-101-F and a downstream primer YTU-103-101-R. The molecular marker YTU-101 marker primer is adopted to carry out PCR amplification on the genomic DNA of the wheat to be detected, and the corresponding amplified product has a molecular weight of 435bp, so that the molecular marker primer is applied to detection, map cloning and molecular marker assisted breeding of the powdery mildew resistance gene PmCG15-009 of the wheat, the breeding efficiency can be greatly improved, the breeding period can be shortened, and the wheat disease resistance breeding can be served.

Description

Primer for amplifying molecular marker closely linked with wheat powdery mildew resistance gene PmCG15-009 and application thereof
Technical Field
The invention relates to the technical field of biological gene engineering, in particular to a primer for amplifying a molecular marker closely linked with a wheat powdery mildew resistance gene PmCG15-009 and application thereof.
Background
Wheat (Triticum aestivum l., 2n=6x=42, aabbdd) is an important food crop in our country, and its high and stable yield is critical to national food safety. The wheat powdery mildew caused by the infection of the powdery mildew of the Brucella seriously threatens the safe production of wheat. The wheat can generally cause 10-15% yield loss after powdery mildew infection, and the yield reduction in serious years can reach more than 40%. (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 control of powdery mildew is urgent. Compared with agricultural control and chemical control, the reasonable utilization of disease-resistant genes and cultivation of disease-resistant varieties are one of the most powerful measures for controlling wheat powdery mildew.
To date, 68 formally named wheat powdery mildew resistance genes (Pm 1-Pm68, pm8=pm17, pm18=pm1c, pm22=pm1e, pm23=pm4c, pm31=pm21) and 30 more temporally named wheat powdery mildew resistance genes (He et al, 2021;McIntosh et al, 2019.He,HG,Liu,RK,Ma,PT,et al.Characterization of Pm68,a new powdery mildew resistance gene on chromosome 2BS of Greek durum wheat TRI 1796[J ]. Theoretical and Applied Genetics,2021,134:53-62.McIntosh RA,Dubcovsky J,Rogers WJ,et al), "catalyst 2 of gene symbols for wheat:2019 supplement"in Annual Wheat Newsletter[J ]. Ed.w. j. Raupp (Manhattan, NY: the Wheat Genetic and Genomic Resources at Kansas State University), 2019,98-113.) have been identified at 63 sites. Wherein Pm1, pm2, pm3, pm4, pm5, pm24 and Pm60 are multiple allele sites. Most of these disease-resistant genes are race-specific resistance and are prone to loss of resistance with large-scale use in production and evolution of pathogenic bacteria. Studies have shown that powdery mildew resistance genes Pm3a, pm3b, pm3f, pm4a, pm6, pm8, and Pm17, etc. have lost resistance even throughout the United states in some areas of the United states due to production singleness, overuse of the resistance genes (Cowger et al, 2018.Cowger C,Mehra L,Arellano C,et al.Virulence differences in Blumeria graminis f.sp.tritici from the central and eastern United States[J ]. Phytopathogy, 2018, 108:402-411.). Pm1, pm3, pm4, pm5, pm8 and Pm17 have lost resistance partially or completely in our country (Zeller et al 2002.Zeller FJ,Kong L,Hartl L,et al.Chromosomal location of genes for resistance to powdery mildew in common wheat (Triticum aestivum l. Em. Shell.) 7.Gene Pm29 in line Pova[J ]. Euphytica,2002,123 (2): 187-194.). Therefore, there is a need to find new sources of resistance to increase the resistance of wheat to powdery mildew in order to continuously address the challenges presented by powdery mildew of wheat.
The developed wheat powdery mildew resistance gene sources mainly comprise common wheat and related species. For example, pm21 (He et al 2018.He HG,Zhu SY,Zhao RH,et al.Pm21,Encoding a typical CC-NBS-LRR protein, related broad-spectrum resistance to wheat powdery mildew disease [ J ]. Molecular Plant 2018,11 (6): 879-882) from Hamamelis villosa (Dasypyrum villosum), pm36 (Blanco et al 2008.Blanco A,Gadaleta A,Cenci A,et al.Molecular mapping of the novel powdery mildew resistance gene Pm36 introgressed from Triticum turgidum var.dicoccoides in durum wheat[J ]. Theoretical and Applied Genetics,2008, 117:135-142) from wild-type two-grain wheat (T.dicoccoides), pm60 (Zou et al 2018.Zou SH,Wang H,Li YW,et al.The NB-LRR gene Pm60 confers powdery mildew resistance in wheat [ J ]. New Phylloglossant, 2018,218 (1): 298-309) from wheat A genome ancestor Ulare wheat (T.harartu), etc., which are often linked to adverse traits, and materials carrying these disease resistance genes cannot be used directly as disease resistance breeding parents for wheat, requiring multiple backcross with the wheat host cultivar, and selfing breaking linkage to the disease resistance parent. Whereas nearly half of the developed powdery mildew resistance genes are derived from common wheat, e.g., powdery mildew resistance gene Pm65 is derived from new wheat 208, a wheat cultivar in China (Li et al 2019.Li GQ,Cowger C,Wang XW,et al.Characterization of Pm65,a new powdery mildew resistance gene on chromosome 2AL of a facultative wheat cultivar[J, theoretical and Applied Genetics,2019, 132:2625-2632.), pm5e is derived from rejuvenation 30, a farm cultivar in China (Huang et al, 2003.Huang XQ,Wang LX,Xu MX,et al.Microsatellite mapping of the powdery mildew resistance gene Pm5e in common wheat (Triticum aestivum L.) [ J ]. Theoretical and Applied Genetics,2003,106:858-865 ]. Pm59 is derived from afgham local variety PI181356 (Tan et al 2018.Tan CC,Li GQ,Cowger C,et al.Characterization of Pm59,a novel powdery mildew resistance gene in Afghanistan wheat landrace PI181356[J ]. Theoretical and Applied Genetics,2018a, 131:1145-1152.). The disease-resistant genes from common wheat are easier to use and can be directly applied to disease-resistant breeding by the traditional hybridization and backcross technology (Li et al 2019;Li GQ,Cowger C,Wang XW,et al.Characterization of Pm65,a new powdery mildew resistance gene on chromosome 2AL of a facultative wheat cultivar[J ]. Theoretical and Applied Genetics,2019, 132:2625-2632). Therefore, the development and utilization of new powdery mildew resistance gene resources from common wheat are of great significance.
After a gene with excellent resistance is discovered, how to efficiently apply the gene 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 a main means of wheat powdery mildew resistance breeding. The efficient, accurate and well-used molecular marker is a precondition for carrying out molecular marker assisted selection on the target gene. The molecular marker is utilized to trace and detect the target gene, so that the breeding process can be greatly shortened, and the breeding efficiency can be improved.
Dan Mai CG15-009 is a provincial region test wheat strain, has excellent agronomic characteristics, and good resistance to powdery mildew in multi-year and multi-point field identification, and is an excellent powdery mildew resistant germplasm resource of wheat. The genetic analysis of the powdery mildew resistance in the seedling stage and the detection of molecular markers show that the resistance of Dan Mai CG15-009 to the powdery mildew epidemic strain E09 in the seedling stage is controlled by a pair of dominant genes PmCG15-009, and the gene is positioned on a wheat 2BL chromosome, thus being a novel wheat powdery mildew resistance gene/allele. Therefore, the development of the molecular marker closely linked with the gene PmCG15-009 is applied to the molecular marker assisted selection breeding of the wheat powdery mildew resistance gene PmCG15-009, and has very important significance for realizing the breeding of powdery mildew resistance wheat varieties and further effectively preventing and controlling the wheat powdery mildew.
Disclosure of Invention
The invention aims to provide a primer for amplifying a molecular marker closely linked with a wheat powdery mildew resistance gene PmCG15-009 and application thereof, so that the molecular marker is amplified by the primer to locate and detect the wheat powdery mildew resistance gene PmCG15-009, parents of the wheat powdery mildew resistance gene PmCG15-009 are purposefully selected in wheat breeding, and a guiding basis is provided for breeding new varieties of wheat of powdery mildew resistance types.
The invention is realized by the following steps: a primer for amplifying molecular markers closely linked with wheat powdery mildew resistance gene PmCG15-009 and application thereof, wherein the molecular markers are co-dominant SSR markers YTU103-101;
the upstream primer of the molecular marker YTU103-101 is YTU103-101-F, and the nucleotide sequence is:
5'-GGGAGAGCCGTCAAAGAACA-3', as shown in SEQ ID NO. 1;
the downstream primer of the molecular marker YTU103-101 is YTU103-101-R, and the nucleotide sequence is:
5'-CTTCTCATTTTCTCCGCGCG-3', as shown in SEQ ID NO. 2;
and carrying out PCR (polymerase chain reaction) amplification on the genomic DNA of the wheat to be detected by adopting the molecular marker YTU-101 marker primer to obtain a corresponding molecular weight of 435bp of an amplified product, namely the molecular marker closely linked with the wheat powdery mildew resistance gene PmCG15-009.
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 molecular marker closely linked with the wheat powdery mildew resistance gene PmCG15-009 provided by the invention is applied to the positioning, map-based cloning and molecular marker assisted selection breeding of the wheat powdery mildew resistance gene PmCG15-009.
The application of the invention for detecting whether the wheat powdery mildew resistance gene PmCG15-009 is carried in the variety to be detected mainly comprises the following steps:
(1) Extracting genome DNA from fresh leaves of a wheat sample to be tested;
(2) Performing PCR (polymerase chain reaction) amplification on the wheat genome DNA by using a primer of a molecular marker YTU103-101 to obtain an amplification product;
(3) If a 435bp specific band can be amplified, the existence of powdery mildew resistance genes PmCG15-009 in the wheat to be detected is indicated; otherwise, the wheat powdery mildew resistance gene PmCG15-009 does not exist in the wheat to be detected.
In the application, the primers of the molecular marker YTU, 103 and 101 in the step (2) comprise an upstream primer YTU, 103 and 101-F and a downstream primer YTU, 103 and 101-R, wherein the nucleotide sequence of the upstream primer YTU, 103 and 101-F is shown as SEQ ID NO. 1, namely: YTU103-101-F:5'-GGGAGAGCCGTCAAAGAACA-3'; the downstream primer of the molecular marker YTU103-101 is YTU103-101-R, and the nucleotide sequence of the downstream primer is shown as follows; the nucleotide sequence of the downstream primer YTU103-101-R is shown in SEQ ID NO. 2, YTU103-101-R:5'-CTTCTCATTTTCTCCGCGCG-3'.
The PCR amplification system is 10 mu L, and 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 55℃for 15s, extension for 40s,30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
PCR amplification product detection: electrophoresis is carried out on non-denaturing polyacrylamide gel with the mass-volume ratio concentration of 8%, after the amplification product and 2 mu L of 6 times of loading buffer solution are mixed, 2 mu L of mixture is taken for sample application, electrophoresis is carried out for 2.5-3h under the constant pressure of 180V, silver nitrate is dyed, photographing is carried out, if 435bp specific bands can be amplified, the existence of powdery mildew resistance genes PmCG15-009 in the germplasm of the wheat to be detected is indicated, otherwise, the existence of the powdery mildew resistance genes PmCG15-009 in the germplasm of the wheat to be detected is not indicated.
According to the invention, through genetic analysis of powdery mildew resistance in the seedling stage and detection of molecular markers, the resistance of Dan Mai CG15-009 to powdery mildew epidemic strain E09 in the seedling stage is controlled by a single dominant gene, and is named as PmCG15-009. 321 pairs of molecular markers Dan Mai CG15-009, susceptible wheat variety Nicotine 21 and Dan Mai CG 15-009X F of Nicotine 21 distributed uniformly throughout 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, 10 pairs of markers show consistent polymorphism in anti-susceptibility parent and anti-susceptibility pool, and then 115 pairs of Dan Mai CG 15-009X Nicotine 21F are used by the markers 2:3 Genotyping was performed in the family and the gene PmCG15-009 was initially located in the region of the wheat 2BL chromosome 688.67-723.48 Mb. Further according to the sequence of the Chinese spring wheat reference genome in the interval, primer5.0 software is utilized to design and screen and obtain Simple Sequence Repeat (SSR) markers YTU-101 closely linked with the gene PmCG15-009. The molecular marker YTU-101 of the wheat powdery mildew resistant gene PmCG15-009 provided by the invention is subjected to genetic segregation population detection, the genetic distance between the molecular marker YTU-101 and the gene PmCG15-009 is only 1.2cM, and the molecular marker is closely linked with the gene PmCG15-009, so that the genetic mapping large population of the gene PmCG15-009 can be efficiently and accurately detected, and the molecular marker is applied to fine positioning and map cloning of the gene PmCG15-009.
The molecular marker closely linked with the wheat powdery mildew resistance gene PmCG15-009 is applied to powdery mildew resistance wheat breeding, and has the advantages of rapid and accurate screening, no environmental influence, clear selection target, production cost saving and great improvement of the selection efficiency and quality of high-quality powdery mildew resistance wheat varieties or strains.
Drawings
FIG. 1 shows F derived from the markers YTU103-101 at 115 Dan Mai CG 15-009X Nicotinone 21 2:3 Partial amplification results in the family.
In the figure, M: pUC18Msp I;1: dan Mai CG15-009 (disease-resistant parent); 2: tobacco grower 21 (susceptible parent); 3-17: dan Mai CG 15-009X F of Ningnong 21 2:3 Family, wherein 3-7: homozygous disease-resistant family, 8-12: anti-sensory separationFamily, 13-17: homozygous disease family; white arrow is specific band of PmCG15-009.
FIG. 2 shows the partial amplification of markers YTU103-101 in 40 parts of susceptible wheat variety.
In the figure, M: pUC18Msp I;1: dan Mai CG15-009 (disease-resistant parent); 2: tobacco grower 21 (susceptible parent); 3-17: shannon 1538, handan wheat 13, huaimai 0226, zhou Mai, smoke 1212, xinong 979, lu Mai 185, zhongyu 1311, jimai 268, tainong 1014, jimai 229, jimai 21, jimai 20, daimai 2173 and Zhongmai 1751. White arrow is specific band of PmCG15-009.
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 development of molecular marker YTU103-101 of wheat powdery mildew resistance gene PmCG15-009
1. Material
The disease-resistant parent Dan Mai CG15-009 is a provincial test wheat strain, and the disease-resistant parent Ningnong 21 is a national audit wheat variety. Dan Mai CG15-009 hybridizes with Ningnong 21 to give 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 2mL EP tube;
2) Adding 600-800 μl of CTAB extract, and mixing in water bath at 65deg.C for 1 hr;
3) Adding chloroform with equal volume, and mixing on a shaking table for 15min;
4) Centrifuging at 12000rpm at room temperature for 10min, collecting 400 μl supernatant, adding 3 times of pre-cooled absolute ethanol into 1.5mL EP tube, mixing, and settling at-20deg.C for 2 hr;
5) Centrifuging at 12000rpm 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 wheat seedling powdery mildew resistance was done in the greenhouse. The disease-resistant parent Dan Mai CG15-009 and the disease-resistant parent Ningnong 21 and F 1 Hybrid seeds, F 2 Population and F 2:3 Family is planted in a 128-hole tray (3.2X3.2X4.2 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 are controlled at 18-20 ℃ and the relative humidity is 80%, and the photoperiod is 14h illumination/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 (Sitao Min et al, 1987).
The investigation result shows that: dan Mai CG15-009 shows high resistance to powdery mildew strain E09 (IT=0), nicotiana 21 shows high perception (IT=4), F 1 Plants all show disease resistance (IT is 0-1), which indicates that Dan Mai CG15-009 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 79:36, and the chi-square test accords with the separation ratio of single dominant gene of 3:1 (chi 2 =2.11, p=0.15), further F 2:3 The family identification result shows that the disease-resistant family is homozygous: anti-sensory separation family: the isolation ratio of homozygous susceptible families was 22:57:36, and chi-square test was consistent with the isolation ratio of the single dominant gene of 1:2:1 (χ 2 =3.42, p=0.18). In conclusion, the resistance of Dan Mai CG15-009 to powdery mildew strain E09 is controlled by a single dominant gene, and the powdery mildew resistance gene is named as PmCG15-009.
4. Molecular marker initial positioning of PmCG15-009
Identifying knots based on phenotypeAnd respectively selecting 20 homozygous disease-resistant families and 20 homozygous disease-sensitive families to construct a disease-resistant pool and a disease-sensitive pool. Polymorphism detection is carried out on the pair of molecular markers Dan Mai CG15-009, the susceptible wheat variety Nicotine 21, the disease resistance pool and the susceptible pool by utilizing 321 evenly distributed in the whole genome, 10 pairs of markers show consistent polymorphism in the susceptible parent and the susceptible pool, and then 115 pairs of markers Dan Mai CG 15-009X Nicotine 21F are utilized 2:3 Genotyping was performed in the family and PmCG15-009 was initially located in the region of wheat 2BL chromosome 688.67-723.48 Mb.
5. Development of molecular marker closely linked with PmCG15-009
According to the sequence information of the Chinese spring wheat reference genome in the candidate interval 688.67-723.48Mb, primer5.0 software is used for designing a Simple Sequence Repeat (SSR) marked primer, and the primer pair Dan Mai CG 15-009X F of Nicotiana grower 21 2:3 Genotyping is carried out on the family, and finally SSR markers YTU103-101 closely linked with the genes PmCG15-009 are obtained, wherein the genetic distance is only 1.2cM.
The primers of molecular markers YTU-101 include an upstream primer and a downstream primer:
nucleotide sequence of the upstream primer YTU103-101-F:5'-GGGAGAGCCGTCAAAGAACA-3';
nucleotide sequence of the downstream primer YTU103-101-R:5'-CTTCTCATTTTCTCCGCGCG-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 55℃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 concentration 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. If 435bp specific band can be amplified, it indicates that powdery mildew resistance gene PmCG15-009 exists in the germplasm of the wheat to be detected, otherwise, no powdery mildew resistance gene PmCG15-009 exists in the germplasm of the wheat to be detected.
Example 2 application of wheat powdery mildew resistance gene PmCG15-009 molecular marker YTU103-101 primer
The sample to be tested comprises disease-resistant parent Dan Mai CG15-009, disease-resistant parent Nicotine 21, 115 Dan Mai CG 15-009X Nicotine 21 derived F 2:3 The method for extracting DNA of wheat to be tested was the same as in example 1, except that the family and 40 parts of susceptible wheat varieties (shannon 1538, handan wheat 13, huai wheat 0226, zhou Mai, tobacco 1212, xi nong 979, lu Mai 185, zhongyu 1311, jimai 268, tainong 1014, jimai 229, jimai 21, jimai 20, daima 2173, zhongmai 1751, jinan 17, zhongmai 9398, vortex wheat 8, liangxing 619, dan Mai 15, xindan 4, qing wheat 6, zhengmai 0856, wu Nong 6, hui nong red, wheat 28, zhongxin wheat 77, tobacco 15, tobacco 17, tobacco 23, tobacco 24, tobacco 161, tobacco 301, tobacco 390, tobacco 745, tobacco 836, tobacco 5158, tobacco 999, tobacco 215 and tobacco 199) were used.
The genomic DNA of the materials is used as a PCR amplification template, and the primers of the molecular markers YTU103-101 developed by the invention are used for amplification:
nucleotide sequence of the upstream primer YTU103-101-F:5'-GGGAGAGCCGTCAAAGAACA-3';
nucleotide sequence of the downstream primer YTU103-101-R:5'-CTTCTCATTTTCTCCGCGCG-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 55℃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 concentration 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 fig. 1 and 2. Wherein FIG. 1 is a chart of F derived from the markers YTU103-101 at 115 Dan Mai CG 15-009X Nicotiana grower 21 2:3 Partial amplification results in the family. In the figure, M: pUC18Msp I;1: dan Mai CG15-009 (disease-resistant parent); 2: tobacco grower 21 (susceptible parent); 3-17: dan Mai CG 15-009X F of Ningnong 21 2:3 Family, wherein 3-7: homozygous disease-resistant family, 8-12: anti-sensory separation pedigree, 13-17: homozygous disease family; the white arrow is the specific band of the gene PmCG15-009. The amplification result shows that the markers YTU103-101 amplify specific bands of 435bp in the disease-resistant parents Dan Mai CG15-009 and the disease-resistant families, and do not amplify the target bands in the disease-resistant parents Nicotiana tabacum 21 and the disease-resistant families.
FIG. 2 shows the partial amplification of markers YTU103-101 in 40 parts of susceptible wheat variety. In the figure, M: pUC18Msp I;1: dan Mai CG15-009 (disease-resistant parent); 2: tobacco grower 21 (susceptible parent); 3-17: shannon 1538, handan wheat 13, huaimai 0226, zhou Mai, smoke 1212, xinong 979, lu Mai 185, zhongyu 1311, jimai 268, tainong 1014, jimai 229, jimai 21, jimai 20, daimai 2173 and Zhongmai 1751. White arrow is specific band of PmCG15-009. The amplification result shows that the markers YTU-101 only amplify 435bp specific bands in the disease-resistant parent Dan Mai CG15-009, and the target bands are not amplified in the disease-resistant parent tobacco grower 21 and the disease-resistant wheat varieties, which means that the tobacco grower 21 and 15 wheat varieties do not contain the gene PmCG15-009, and the wheat varieties belong to the disease-resistant wheat varieties. No 435bp specific band was amplified in 40 wheat varieties, 15 materials were selected as the amplification schematic, and the results of the remaining 25 materials were identical to 15 in the graph.
The wheat powdery mildew resistance gene PmCG15-009 is derived from wheat-series stone wheat CG15-009 of a regional test in China, is a novel gene with excellent resistance, and has no relevant reports of localization, map cloning and molecular breeding at present. The molecular marker YTU103-101 provided by the invention is used for detecting a large population of genetic mapping, and is beneficial to realizing fine positioning and map cloning of the gene PmCG15-009; the molecular marker YTU-101 developed by the invention can efficiently and accurately detect a large population, greatly improve the efficiency and precision of transferring the disease-resistant gene PmCG15-009, and has very important significance for efficient transfer of the gene PmCG15-009 and deep analysis of a disease-resistant mechanism.
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 of the invention will occur to those skilled in the art without departing from the spirit and principles of the invention as described herein.

Claims (6)

1. A primer for amplifying a molecular marker closely linked with a wheat powdery mildew resistance gene PmCG15-009, which is characterized in that the molecular marker is YTU103-101; the labeling primer of the molecular label YTU103-101 comprises an upstream primer YTU-101-F and a downstream primer YTU103-101-R, and the nucleotide sequence of the upstream primer YTU103-101-F is shown as SEQ ID NO. 1; the nucleotide sequence of the downstream primer YTU103-101-R is shown in SEQ ID NO. 2; and (3) carrying out PCR (polymerase chain reaction) amplification on the genomic DNA of the wheat to be detected by using the labeled primer of the molecular marker YTU-101 to obtain a corresponding molecular weight of 435bp of an amplified product, namely the molecular marker closely linked with the powdery mildew resistance gene PmCG15-009 of the wheat.
2. An application of a primer of a molecular marker closely linked with the wheat powdery mildew resistance gene PmCG15-009 in detection, map-based cloning and molecular marker assisted breeding of the wheat powdery mildew resistance gene PmCG15-009 in claim 1.
3. The method for detecting whether the wheat powdery mildew resistance gene PmCG15-009 is carried in the variety to be detected is characterized by comprising the following steps:
(1) Extracting genome DNA of a wheat sample to be detected;
(2) Carrying out PCR amplification on the genomic DNA of the material of the wheat sample to be detected by using a marked primer of the molecular marker YTU-101 to obtain an amplified product;
(3) Electrophoresis and detection are carried out on the amplification product, if 435bp specific bands can be amplified, the existence of powdery mildew resistance genes PmCG15-009 in the wheat sample to be detected is indicated; otherwise, the wheat powdery mildew resistance gene PmCG15-009 does not exist in the wheat to be detected.
4. The method of claim 3, wherein the labeled primers of the molecular markers YTU, 103-101 of step (2) comprise an upstream primer YTU, 103-101-F and a downstream primer YTU, 103-101-R, wherein the nucleotide sequence of the upstream primer YTU, 103-101-F is shown as SEQ ID NO. 1; the nucleotide sequence of the downstream primer YTU103-101-R is shown as SEQ ID NO. 2.
5. The method of claim 3 or 4, wherein the PCR amplification system is 10 μl, comprising: 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.
6. The method of claim 5, 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 ℃.
CN202310143762.6A 2023-02-21 2023-02-21 Primer for amplifying molecular marker closely linked with wheat powdery mildew resistance gene PmCG15-009 and application thereof Pending CN117025812A (en)

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CN117385094A (en) * 2023-12-06 2024-01-12 山东省烟台市农业科学研究院(山东省农业科学院烟台市分院) Molecular marker primer for amplifying wheat powdery mildew resistance gene PmJM809 and application thereof
CN117757983A (en) * 2024-02-22 2024-03-26 山东省烟台市农业科学研究院(山东省农业科学院烟台市分院) molecular marker primer coseparated with powdery mildew resistance gene PmYD001 of durum wheat and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117385094A (en) * 2023-12-06 2024-01-12 山东省烟台市农业科学研究院(山东省农业科学院烟台市分院) Molecular marker primer for amplifying wheat powdery mildew resistance gene PmJM809 and application thereof
CN117757983A (en) * 2024-02-22 2024-03-26 山东省烟台市农业科学研究院(山东省农业科学院烟台市分院) molecular marker primer coseparated with powdery mildew resistance gene PmYD001 of durum wheat and application thereof
CN117757983B (en) * 2024-02-22 2024-04-19 山东省烟台市农业科学研究院(山东省农业科学院烟台市分院) Molecular marker primer co-separated from powdery mildew resistance gene PmYD-001 of durum wheat and application thereof

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