阅读说明：本技术 一种与小麦抗白粉病基因Pm37连锁的分子标记及其应用 (Molecular marker linked with wheat powdery mildew resistance gene Pm37 and application thereof ) 是由 马朋涛 刘成 何华纲 杜文晓 贾梦淑 张旭 王文瑞 于 2019-12-11 设计创作，主要内容包括：本发明公开了一种与小麦抗白粉病基因<I>Pm37</I>连锁的分子标记及其应用；所述分子标记YTURGA的上游引物为YTURGA-F,其核苷酸序列如SEQ ID NO:1所示；所述分子标记YTURGA的下游引物为YTURGA-R,其核苷酸序列如SEQ ID NO:2所示；以所述分子标记YTURGA的标记引物对小麦基因组DNA进行PCR扩增,将扩增产物电泳分离,获得对应的扩增产物分子量为296bp,即为与小麦抗白粉病基因<I>Pm37</I>连锁的分子标记。本发明开发的与<I>Pm37</I>连锁的分子标记YTURGA,可高效检测作图群体,应用于<I>Pm37</I>的分子标记定位和图位克隆；可高效检测育种群体,提高转移抗病基因<I>Pm37</I>基因的效率,加快<I>Pm37</I>基因不同遗传背景抗白粉病小麦新品种的培育,提高小麦抗白粉病育种的效率。(The invention discloses a powdery mildew resistance gene of wheat Pm37 Linked molecular markers and uses thereof; the upstream primer of the molecular marker YTURGA is YTURGA-F, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1; the downstream primer of the molecular marker YTURGA is YTURGA-R, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2; carrying out PCR amplification on wheat genome DNA by using the marker primer of the molecular marker YTURGA, and carrying out electrophoretic separation on the amplification product to obtain a corresponding amplification product with the molecular weight of 296bp, namely the gene which is resistant to powdery mildew of wheat Pm37 Linked molecular markers. The invention was developed with Pm37 The linked molecular marker YTURGA can be used for efficiently detecting mapping population and is applied to Pm37 The molecular marker positioning and map location cloning; can efficiently detect breeding groups and improve transferred disease-resistant genes Pm37 Efficiency of gene, acceleration of Pm37 Cultivation of new wheat variety with gene different genetic backgrounds for resisting powdery mildew and improving wheat qualityThe efficiency of breeding against powdery mildew.)

1. Wheat powdery mildew resistance gene Pm37A linked molecular marker, wherein the molecular marker is YTURGA; the upstream primer of the molecular marker YTURGA is YTURGA-F, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1; the downstream primer of the molecular marker YTURGA is YTURGA-R, and the nucleotide sequence of the downstream primer is shown as SEQ ID N: 2; performing PCR amplification on wheat genome DNA by using an upstream primer and a downstream primer of the molecular marker YTURGA, and performing electrophoretic separation on an amplification product to obtain a corresponding amplification product with the molecular weight of 296bp, namely the molecular weight of the amplification product is the powdery mildew resistance gene of wheat Pm37Linked molecular markers.

2. The powdery mildew resistance gene of wheat according to claim 1 Pm37The linked molecular marker is characterized in that the PCR amplification system is 10 mu L, and comprises 1.0 mu L of wheat genome DNA with the concentration of 30 ng/mu L, 1.0 mu L of 10 XPCR buffer, 0.2 mu L of 10 MdNTP and 10mM MgCl ₂1.0. mu.L, 5U Taq polymerase 0.2. mu.L, 5. mu.M upstream primer 0.4. mu.L, 5. mu.M downstream primer 0.4. mu.L and sterile deionized water 5.8. mu.L.

3. The powdery mildew resistance gene of wheat according to claim 1 or 2 Pm37Linked molecular markers ofCharacterized in that the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 8 min; denaturation at 94 ℃ for 30 seconds, annealing at 57 ℃ for 40 seconds, extension for 40 seconds, and 36 cycles; extending for 10min at 72 ℃; storing at 4 ℃.

4. The powdery mildew resistance gene of wheat according to claim 3 Pm37The linked molecular marker is characterized in that the amplification product electrophoresis refers to electrophoresis for 3h on denaturing polyacrylamide gel with the mass percentage concentration of 8% under the constant voltage of 280V.

5. The powdery mildew resistance gene of any one of claims 1 to 4 for wheat Pm37Linkage molecular marker in wheat powdery mildew resistance gene Pm37In localization, detection or map-based cloning.

6. Use according to claim 5, characterized in that the wheat powdery mildew resistance gene is detected Pm37The method comprises the following steps:

(1) extracting wheat genome DNA from young and tender leaves of a sample to be detected;

(2) carrying out PCR amplification on the genome DNA by using an upstream primer and a downstream primer of a molecular marker YTURGA to obtain an amplification product; the primers of the molecular marker YTURGA comprise an upstream primer YTURGA-F and a downstream primer YTURGA-R; the nucleotide sequence of the upstream primer YTURGA-F is shown as SEQ ID NO. 1; the nucleotide sequence of the downstream primer YTURGA-R is shown as SEQ ID NO. 2;

(3) and (3) detecting a PCR amplification product: performing electrophoresis on 8 mass percent modified polyacrylamide gel, mixing the amplification product with 6 mu L of sample loading buffer solution, spotting 2 mu L of mixture, performing electrophoresis at constant voltage of 280V for 3h, taking a picture after silver nitrate dyeing, and if a 296bp specific strip can be amplified, indicating that powdery mildew resistant gene exists in the wheat germplasm to be detected Pm37Otherwise, the wheat powdery mildew resistance gene does not exist in the wheat germplasm to be detected Pm37。

7. Use according to claim 6, wherein the PCR is amplifiedThe system is 10 μ L, and comprises 30 ng/. mu.L wheat genome DNA 1.0 μ L, 10 XPCR buffer1.0 μ L, 10mM dNTP 0.2 μ L, 10mM MgCl ₂1.0 mul, 5U Taq polymerase 0.2 mul, 5 mul upstream primer 0.4 mul, 5 mul downstream primer 0.4 mul and sterile deionized water 5.8 mul; the procedure of PCR amplification is as follows: pre-denaturation at 94 ℃ for 8 min; denaturation at 94 ℃ for 30 seconds, annealing at 57 ℃ for 40 seconds, extension for 40 seconds, and 36 cycles; extending for 10min at 72 ℃; storing at 4 ℃.

8. The powdery mildew resistance gene of any one of claims 1 to 4 for wheat Pm37The application of the linked molecular marker in wheat molecular marker-assisted selective breeding.

Technical Field

The invention relates to the field of wheat molecular biotechnology and breeding application, in particular to a powdery mildew resistance gene of wheat Pm37Linked molecular markers and uses thereof.

Background

Wheat powdery mildew is one of three most serious diseases in wheat diseases (Ma et al. Characteriationoff a powder wheat resistance gene in wheat drying line 10V-2 and availability in marker-associated selection. Plant disease 2018, 102:925 and 931.) seriously threatens wheat safety production. Of the numerous wheat powdery mildew control strategies, the use of variety resistance is the most economical, effective and environmentally friendly strategy (Petersen et al, Mapping of powder mill resistance gene) Pm53introgressed from Aegilopsspeltoidesinto soft red winter wheat.Theoretical and Applied Genetics. 2015, 128:303-312.）。

The disease-resistant gene is the basis for breeding disease-resistant varieties. Up to now, more than 70 formally named wheat powdery mildew resistance genes have been found, distributed over 53 sites (McIntosh et al Catalogue of gene systems for wheat:2017 supplement. http:// www.shigen.nig.ac.jp/wheat/komugi/genes/symbolClassList. jsp. 2017). However, many disease-resistant genes lose resistance to powdery mildew gradually due to variety-flora interaction, and some effective disease-resistant genes greatly affect the application efficiency in breeding because of lack of available breeding available markers (Lihongjie et AL, resistance reaction of Chinese wheat varieties to powdery mildew and detection of disease-resistant genes. Proc. Rev. crops 2011, 37: 943. 954; Li et AL. Characterisation of Pm65, a new powder modern resistance gene on chromosome 2AL of a robust wheat gene var. Theoretica and Applied genetics 2019, 132: 2625. 2632). Therefore, the wheat powdery mildew can be effectively prevented and controlled by enhancing the high-efficiency utilization of broad-spectrum powdery mildew resistant genes in production.

Traditional breeding of wheat has been a notable achievement in the past few decades. However, traditional breeding is often blind and cannot keep a steady increase. Using molecular breeding, unfavorable genes can be removed in fewer backcross generations, and the breeding process can be accelerated (jelly et al. Development of robust molecular markers for marker-assisted selection of leaf seed resistance gene) Lr23in common and durmwhen curing programs, Molecular curing 2017, 37: 21). In the breeding process of wheat disease-resistant molecules, the development of genetic markers and the construction of genetic linkage maps play a vital role. Genetic maps of tetraploid and hexaploid wheat varieties have been published to date, with constant perfection of The international wheat Genome Sequencing Consortium. Shifting The limits in wheat research and in wheat breeding using a wheat mutated gene [ J]. Science, 2018, 361(6403): eaar7191；Ling et al. Genome sequence of the progenitor of wheat Asubgenome Triticumurartu[J]. Nature, 2018, 557:424-428；Zhao et al. The Aegilopstauschiigenome reveals multiple impacts of transposons[J]Naturepels, 2017, 3: 946-955), the development efficiency of the wheat excellent gene Molecular marker is greatly improved, which effectively promotes the breeding process of the wheat disease-resistant molecule (Semagn et al, Single nucleotide polymorphism using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement. Molecular Breeding, 2016,33: 1-14.).

Wheat powdery mildew resistance gene Pm37Is a kind of wheat (A) derived from the plant Timofetil TriticumtimopheeviiAAGG), currently transformed into the background of common wheat line NC99BGTAG11, to be located on the wheat 7AL chromosome (peregrini et AL). Pm37, a new broadly effective powdery mildewresistance gene from Triticumtimopheevii. Theoreticaland Applied genetics.2008, 116: 417-. The anti-spectrum analysis of 27 different toxic powdery mildew strains in the laboratory shows that, Pm37the gene is immune to all strains and is an excellent powdery mildew resistance gene with broad-spectrum resistance, but Pm37At present, no marker report available for breeding exists, which greatly influences Pm37The method is applied to molecular breeding. Therefore, if can be developed and Pm37linked molecular markers, and can be used for efficiently detecting and tracking the powdery mildew resistance gene, and realizing Pm37Has very important value and significance for efficient breeding and utilization.

Disclosure of Invention

The invention aims to provide a powdery mildew resistance gene for wheat Pm37A linked molecular marker and application thereof, which are used for efficiently detecting powdery mildew resistance genes of wheat Pm37Provides a convenient tool, and is applied to gene cloning and powdery mildew resistant molecular marker assisted selective breeding.

The invention is realized by the following method: wheat powdery mildew resistance gene Pm37A linked molecular marker which is a dominant marker YTURGA;

the upstream primer of the molecular marker YTURGA is YTURGA-F, and the nucleotide sequence of the upstream primer is shown as follows:

YTURGA-F：5' - 3'：TGAGCTCCACTACTTCATCATCCAG；

the downstream primer of the molecular marker YTURGA is YTURGA-R, and the nucleotide sequence of the downstream primer is shown as follows;

YTURGA-R：5' - 3'：AGTGTATTTCCTCCATCACTGACT；

performing PCR amplification on wheat genome DNA by using an upstream primer and a downstream primer of the molecular marker YTURGA, and performing electrophoretic separation on an amplification product to obtain an amplification product with the molecular weight of 296bp, wherein the amplification product is the segment of DNA which is amplified by using the upstream primer and the downstream primer of the molecular marker YTURGA Pm37Linked molecular markers.

The PCR amplification system is a 10 mu L system: 30 ng/. mu.L of wheat genome DNA 1.0. mu.L, 10 XPCR buffer 1.0. mu.L, 10mM dNTP 0.2. mu.L, 10mM MgCl ₂1.0 μ L, 5U Taq polymerase 0.2 μ L, 5 μ M upstream primer 0.4 μ L, 5 μ M downstream primer 0.4 μ L and sterile deionized water 5.8 μ L.

The PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 8 min; denaturation at 94 ℃ for 30 seconds, annealing at 57 ℃ for 40 seconds, extension for 40 seconds, and 36 cycles; extending for 10min at 72 ℃; after amplification, the amplification product was stored at 4 ℃.

And carrying out electrophoretic separation on the amplification product on a denaturing polyacrylamide gel with the mass percentage concentration of 8%, wherein the amplification voltage is 280V constant voltage, and the electrophoresis time is 3 h.

The gene provided by the invention and resisting wheat powdery mildew Pm37Linkage molecular marker in wheat powdery mildew resistance gene Pm37Detection and map-based cloning.

The gene provided by the invention and resisting wheat powdery mildew Pm37Linked molecular markers for detection Pm37The method comprises the following steps:

(1) extracting genome DNA from leaves of a wheat sample to be detected;

(2) carrying out PCR amplification on the genome DNA by using a primer of a molecular marker YTURGA; the primers of the molecular marker YTURGA comprise an upstream primer YTURGA-F and a downstream primer YTURGA-R; the nucleotide sequence of the upstream primer YTURGA-F is shown as SEQ ID NO. 1; the nucleotide sequence of the downstream primer YTURGA-R is shown as SEQ ID NO. 2;

(3) and (3) detecting a PCR amplification product: carrying out electrophoresis detection on the obtained amplification product on a denaturing polyacrylamide gel with the mass percentage concentration of 8%, mixing the amplification product with 6 mu L of a sample loading buffer solution, carrying out sample application on 2.0 mu L of the mixture, carrying out electrophoresis at a constant voltage of 280V for 3h, carrying out silver nitrate staining, and photographing, wherein if a band of 296bp can be amplified, the existence of the powdery mildew resistance gene in the germplasm to be detected is indicated Pm37Otherwise, if the specific band of 296bp can not be amplified, it indicates that the wheat germplasm for testing does not have wheat powdery mildew resistant gene Pm37。

The amplification system of the PCR amplification in the detection method is as follows: 30 ng/. mu.L of wheat genome DNA 1.0. mu.L, 10 XPCR buffer 1.0. mu.L, 10mM dNTP 0.2. mu.L, 10mM MgCl ₂1.0 muL, 0.2 muL of 5U Taq polymerase, 0.4 muL of 5 muM upstream primer, 0.4 muL of 5 muM downstream primer and 5.8 muL of sterile deionized water, and the total amount is 10 muL; the amplification procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 8 min;denaturation at 94 ℃ for 30 seconds, annealing at 57 ℃ for 40 seconds, extension for 40 seconds, and 36 cycles; extension at 72 ℃ for 10 min; storing at 4 ℃.

The wheat powdery mildew resistance gene of the invention Pm37The application of the linked molecular marker YTURGA in wheat molecular marker assisted selective breeding.

The invention carries a single dominant gene through a common wheat strain NC99BGTAG11 Pm37Is positioned on wheat 7AL chromosome by means of Chinese spring reference genome sequence Pm37The molecular marker YTURGA is firstly developed in the initial positioning interval, and the research finds that the molecular marker YTURGA is detected by genetic segregation population and genes Pm37Has a genetic distance of 9.0cM, and Pm37linkage, can detect efficiently Pm37Large population of genetic maps of Pm37The fine positioning and map-based cloning of (2) can also be carried out using the markers Pm37The molecular marker assisted selective breeding has very important value and significance for breeding powdery mildew resistant wheat varieties.

Drawings

FIG. 1 shows the molecular marker YTURGA detection NC99BGTAG11, Ningmai 13 and F thereof ₂Banding pattern of the isolated population.

In the figure: m: DNA marker pUC19 MspI; 1: NC99BGTAG11 (powdery mildew resistant variety, carrier) Pm37) (ii) a 2: ningmai 13 (variety of erysiphe cichoracearum); 3-7: disease-resistant F of NC99BGTAG11 and Ningmai 13 ₂Carrying out single plant cultivation; 8-12: susceptibility F of NC99BGTAG11 and Ningmai 13 ₂Carrying out single plant cultivation; the arrows indicate that tracing is possible Pm37296bp specific band.

FIG. 2 shows the result of detecting the breeding availability of partial disease-sensitive main cultivars by using the molecular marker YTURGA.

In the figure: m: DNA marker pUC19 MspI; 1: NC99BGTAG11 (powdery mildew resistant variety, carrier) Pm37) (ii) a 2: ningmai 13 (variety of erysiphe cichoracearum); 3: handan wheat 13; 4: tylonone 1014; 5: a good star 619; 6: 20 of economic wheat; 7: stone dust 633; 8: pumai 28; 9: smoke 187; 10: midwifery 1311; 11: FC 009; 12: new Loxono. 4; black arrows to track Pm37The specific band of (1).

Detailed Description

The following examples serve to illustrate the invention in further detail, but without restricting it in any way.