Specific molecular marker for wheat powdery mildew resistance gene pm68 and use thereof
By developing the tightly linked molecular marker Xdw08.9 (Pm68-M1), the problem of accurately tracing the Pm68 gene in durum wheat varieties was solved, enabling efficient and accurate breeding detection and improving the precision and efficiency of breeding.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGSU UNIV
- Filing Date
- 2022-12-16
- Publication Date
- 2026-06-05
AI Technical Summary
In existing technologies, it is difficult to accurately track the powdery mildew resistance gene Pm68 in durum wheat varieties, and the known molecular marker Xdw15 is too far genetically distant from it, leading to breeding difficulties.
A tightly linked molecular marker, Xdw08.9 (Pm68-M1), was developed. Through BSR-seq high-throughput sequencing data analysis, combined with PCR amplification and electrophoresis detection, the fine localization and detection of the Pm68 gene were achieved.
It enables precise tracking and detection of the Pm68 gene, improving the accuracy and efficiency of breeding, ensuring stable amplification and high resolution of the Pm68 gene under different wheat backgrounds, and reducing false positive results.
Smart Images
Figure CN115747374B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of molecular biology and genetic breeding science and technology, specifically involving the specific molecular marker of wheat powdery mildew resistance gene Pm68 and its applications. Background Technology
[0002] Wheat is the most widely cultivated food crop globally and a crucial source of plant protein for humans. Powdery mildew fungus causes wheat powdery mildew disease, leading to significant yield reductions and a decline in wheat quality. Therefore, it is urgent to discover and utilize superior powdery mildew-resistant genes in wheat production and breeding.
[0003] Durum wheat is a tetraploid wheat variety primarily used for making pasta. Durum wheat exhibits good resistance to leaf rust, stem rust, and stripe rust and has been used for wheat variety improvement. However, most durum wheat varieties are susceptible to powdery mildew. Over the past few decades, three powdery mildew resistance genes have been identified in this crop: Mld, Pm3h, and PmDR147. Mld is a recessive gene located on chromosome 4B, which, when combined with other powdery mildew resistance genes (such as Pm2), has been used in wheat breeding but quickly lost its resistance. Pm3h is a dominant resistance gene located on chromosome 1AS, identical to Pm3d at the nucleotide sequence level. PmDR147 is another dominant gene identified from chromosome 2AL of durum wheat DR 147. However, the current powdery mildew resistance in Chinese wheat varieties is not optimistic. Powdery mildew resistance genes are easily lost with the evolution of plants and fungal communities. Therefore, there is an urgent need in this field to discover more powdery mildew resistance genes and molecular markers for disease resistance breeding.
[0004] Pm68 is a gene discovered in durum wheat TRI 1796 that exhibits broad-spectrum resistance to different strains of wheat powdery mildew at the seedling stage. Through genetic analysis and molecular mapping, Pm68 has been identified as a dominant gene controlled by a single gene. Using BSR-seq technology, Pm68 was located at the end of chromosome 2B (He et al., Characterization of Pm68, a new powdery mildew resistance gene on chromosome 2BS of Greek durum wheat TRI1796. Theoretical and Applied Genetics. 2021, 134:53–62). Furthermore, as one of the direct ancestors of common wheat, durum wheat readily hybridizes with it and produces offspring; therefore, Pm68 in durum wheat TRI 1796 may also be used for resistance improvement in common wheat. Therefore, developing molecular markers that are tightly linked to this gene can be used to efficiently and accurately detect and track powdery mildew resistance genes. However, the currently known molecular marker Xdw15 is still far from Pm68, with a genetic distance of 0.44 cM, making it difficult to accurately track Pm68. Therefore, developing molecular markers that are more tightly linked is of great value and significance for its breeding. Summary of the Invention
[0005] The purpose of this invention is to provide a molecular marker that can accurately track the wheat powdery mildew resistance gene Pm68 and its applications.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] This invention combines BSR-seq high-throughput sequencing data and compares the genomes of two durum wheat varieties (Svevo and Kronos). Based on the gene TRITD2Bv1G010390, a specific molecular marker Xdw08.9 was developed and formally named Pm68-M1. This invention also experimentally demonstrates that this molecular marker can be used for fine mapping and detection of the wheat powdery mildew resistance gene Pm68, enabling precise selection during the breeding of powdery mildew-resistant wheat varieties or lines. This provides a novel, practical, and efficient molecular marker-assisted selection technology for breeding new powdery mildew-resistant wheat varieties.
[0008] The present invention first provides a molecular marker for the wheat powdery mildew resistance gene Pm68, the molecular marker being Xdw08.9 and named Pm68-M1, which is closely linked to the wheat powdery mildew resistance gene Pm68.
[0009] This invention also provides primers for amplifying the molecular marker, the nucleotide sequences of which are as follows:
[0010] Pm68-M1F: 5'-CTAGATGTCAGTCCAGATGTTG-3' (SEQ ID NO.1),
[0011] Pm68-M1R: 5'-GTTATGCTGCAGTAACTAACTCTC-3' (SEQ ID NO. 2).
[0012] The present invention also provides a product for identifying wheat powdery mildew resistance, the product comprising the primers.
[0013] The present invention also provides the application of the molecular marker, the primer, or the product in detecting whether wheat carries the powdery mildew resistance gene Pm68, identifying or assisting in the identification of whether wheat is resistant to powdery mildew, and / or in wheat breeding or assisting in wheat breeding.
[0014] Furthermore, the application includes the following steps:
[0015] The primers described above were used to perform PCR amplification of the genomic DNA of the test material; the amplification product was detected. If the PCR amplified a specific product of 156 bp, it meant that the test material carried the powdery mildew resistance gene Pm68, and that the wheat material was resistant to powdery mildew.
[0016] Furthermore, the PCR reaction system used in the PCR amplification procedure is as follows: a 25 μL reaction system containing approximately 50 ng template DNA, 1× PCR buffer, and 1.5 mmol / L... -1 MgCl2, 200 mmol L -1 dNTPs, with a final concentration of 2 mol / L for each primer. -1 Add 1 U Taq DNA polymerase and replenish the reaction system to 25 μL with sterile distilled water.
[0017] The PCR amplification reaction procedure is as follows:
[0018] Pre-denaturation at 94℃ for 3 minutes; denaturation at 94℃ for 20 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 60 seconds, 32 cycles; extension at 72℃ for 5 minutes; store at 4℃.
[0019] The beneficial effects of this invention are:
[0020] The molecular marker Xdw08.9 (Pm68-M1) provided by this invention is a molecular marker that can accurately track the wheat powdery mildew resistance gene Pm68. Using this molecular marker to detect genetic segregation populations, no exchange was detected in a population of 1382 individual plants, indicating that the molecular marker Xdw08.9 (Pm68-M1) co-segregates with Pm68 and is a tightly linked molecular marker.
[0021] Meanwhile, the molecular marker Pm68-M1 of the present invention has high specificity in tracking Pm68. Among the 40 wheat varieties tested, only the material carrying Pm68, TRI 1796, showed a specific band of Pm68-M1, while it did not appear in the other wheat varieties tested.
[0022] The molecular marker Pm68-M1 provided by this invention can effectively track Pm68 in the background of hexaploid wheat. Moreover, the molecular marker Pm68-M1 has the advantages of stable amplification of polymorphic bands, high resolution, and good reproducibility, which can greatly improve the accuracy and efficiency of breeding.
[0023] The molecular markers of this invention have important application value in the transgenic development of the powdery mildew resistance gene Pm68. Attached Figure Description
[0024] Figure 1 This is a fine genetic map of the powdery mildew resistance gene Pm68. The molecular marker Xdw08.9 in the map is the molecular marker Pm68-M1, which is co-segregated with Pm68 and provided by this invention.
[0025] Figure 2 The results of powdery mildew identification of the Pm68 gene-transferred wheat offspring are shown in the figure. In the figure, No. 1-6 are the offspring of recurrent hybridization between durum wheat TRI1796 (containing Pm68) and Yangmai 158; No. 7 is the powdery mildew-susceptible recurrent parent Yangmai 158.
[0026] Figure 3 The results of the detection of molecular marker Pm68-M1 in high-generation germplasm of durum wheat TRI 1796 / Yangmai 158 are shown in the figure. M is the DNA molecular marker DL2000, and numbers 1-20 are those containing... Pm68 The wheat transgenic progeny single plants are: No. 21 is Yangmai 158, No. 22 is powdery mildew-susceptible durum wheat PI 584832, and No. 23 is powdery mildew-resistant durum wheat TRI 1796 (containing Pm68); the arrows indicate the specific bands of materials containing the Pm68 gene.
[0027] Figure 4The results of detecting the molecular marker Pm68-M1 in domestic and foreign wheat varieties are shown in the figure. In the figure, M represents the DNA molecular weight marker DL2000. Varieties 1-30 are domestic wheat varieties, namely: Jimai 19, Chuanmai 42, Zhengmai 9023, Jimai 22, Yumai 34, Lumai 14, Yangmai 158, Yangmai 18, Yumai 18, Zhou 8425B, Liangxing 66, Liangxing 99, Tainong 18, Tainong 19, Shannong 17, Shannong 21, Yannong 19, Shi 4185, Shimai 15, Ji 5265, Henong 827, Kenong 199, Kenong 9204, Zhenmai 168, Ningmai 13, Aikang 58, Luomai 21, Zhengmai 366, Zhengyumai 9989, and Bima 1. Varieties 31-40 are foreign wheat varieties, namely: Chancellor, Ovest, Kolibri, Armada, and Coker. 747, Norin 10, Maris Huntsman, Ralle, Arthur, Suwon 92; No. 41 is Yangmai 158; No. 42 is powdery mildew-susceptible durum wheat PI 584832; No. 43 is powdery mildew-resistant durum wheat TRI 1796 (containing Pm68). Detailed Implementation
[0028] To enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention will be described in detail below. However, the following embodiments do not limit the scope of protection of the present invention.
[0029] In the embodiments of the present invention, unless otherwise described, conventional experimental methods were used. The processes involved in the embodiments, unless otherwise described, can be understood and easily implemented by those skilled in the art based on the product manual or basic knowledge in the field, and therefore will not be described in detail.
[0030] Example 1: Development of a specific molecular marker for the wheat powdery mildew resistance gene Pm68
[0031] In the inventors' previous research (He et al., Characterization of Pm68, a new powdery mildew resistance gene on chromosome 2BS of Greek durum wheat TRI1796. Theoretical and Applied Genetics. 2021, 134:53-62), Pm68 Preliminary localization to molecular markers Xdw04 and Xdw12 A 1.78 Mb chromosomal region containing 84 genes.
[0032] To achieve the powdery mildew resistance gene Pm68 For fine genetic mapping, this invention crosses powdery mildew-resistant durum wheat TRI 1796 (from the German Germplasm Bank: https: / / gbis.ipk-gatersleben.de / gbis2i / faces / index.jsf) with powdery mildew-susceptible durum wheat PI 584832 (from the US Germplasm Bank: https: / / npgsweb.ars-grin.gov / gringlobal / search). The resulting F2 genetic mapping population contains over 1382 individual plants. Using... Pm68 Known molecular markers on both sides of the site Xdw03 and Xdw15 (See: He et al., Characterization of Pm68, a new powdery mildew resistance gene on chromosome 2BS of Greek durum wheat TRI 1796. Theoretical and Applied Genetics. 2021, 134: 53–62) PCR amplification was performed, and exchanged single plants were directly screened from the mapping population. In this invention, 57 exchanged single plants were identified from 1382 single plants.
[0033] Comparative analysis of the genome sequences of two durum wheat varieties (Svevo and Kronos) in public databases (https: / / www.interomics.eu / durum-wheat-genome);
[0034] (https: / / opendata.earlham.ac.uk / opendata / data / Triticum_turgidum / EI / v1.1), developed polymorphic molecular markers, in which molecular markers Xdw03, Xdw04, Xdw05, Xdw06, Xdw07, Xdw08 Xdw09, Xdw10, Xdw11, Xdw12, Xdw15 The development of this gene has been reported (see: He et al., Characterization of Pm68, a new powdery mildew resistance gene on chromosome 2BS of Greek durumwheat TRI 1796. Theoretical and Applied Genetics. 2021, 134:53–62), and further screening was performed. Xdw08.9 ( Pm68-M1 Molecular markers, molecular markers Xdw08.9 ( Pm68-M1The primer was developed based on the durum wheat gene TRITD2Bv1G010390. The forward primer sequence is SEQ ID NO.1, and the reverse primer sequence is SEQ ID NO.2. Specific information is as follows:
[0035] Pm68-M1F: 5'-CTAGATGTCAGTCCAGATGTTG-3' (SEQ ID NO.1),
[0036] Pm68-M1R: 5'-GTTATGCTGCAGTAACTAACTCTC-3' (SEQ ID NO. 2).
[0037] Then, the 57 exchanged monocultures were genotyped using the aforementioned molecular markers, and combined with the powdery mildew resistance survey results of each monoculture (according to conventional methods in the field), the powdery mildew resistance genes were identified. Pm68 It was located to a 0.11 cM genetic region, based on Figure 1 The fine genetic map of the powdery mildew resistance gene Pm68 shown is marked on both sides of this interval as follows: Xdw05 / Xdw06 and Xdw09 / Xdw10 / Xdw11 They and Pm68 The genetic distances were 0.07 cM and 0.04 cM, respectively. Furthermore, this invention demonstrates through genetic analysis that molecular markers... Xdw07 , Xdw08 and Xdw08.9 ( Pm68-M1 () was co-separated from powdery mildew resistance. Among them, molecular markers Xdw09 The annotation gene corresponding to the Svevo reference genome of durum wheat, TRITD2Bv1G010400 (chr2B:22723631). - 22727432).
[0038] Further investigation during fine genetic mapping revealed existing molecular markers Xdw15 and Pm68 There is still a considerable distance to go, with a genetic distance of 0.44 cM, making precise tracking difficult. Pm68 Among the aforementioned population of 1382 individual plants, molecular markers developed based on the gene TRITD2Bv1G010390 were... Xdw08.9 ( Pm68-M1 )and Pm68 No exchange occurs between them, i.e., molecular markers. Xdw08.9 ( Pm68-M1 )and Pm68 It is co-separated, in Pm68 The transition to breeding is more precise.
[0039] This invention further detected three co-separated molecular markers. Xdw07 , Xdw08 and Xdw08.9 ( Pm68-M1 Tracking in the context of hexaploid wheat (publicly known and used) Pm68 The effectiveness was found to be limited to molecular markers. Xdw08.9 ( Pm68-M1 It has stable amplified polymorphic bands, high resolution, and good reproducibility, making it an ideal molecular breeding marker.
[0040] Specifically, PCR amplification was performed on hexaploid wheat DNA, including molecular markers. Xdw07 , Xdw08 For specific information and amplification primers, please refer to the aforementioned literature. 。 Amplification conditions were as follows: PCR reaction system: 25 μL reaction system containing approximately 50 ng template DNA, 1×PCR buffer, 1.5 mmol L⁻¹ MgCl₂, and 200 mmol L⁻¹ MgCl₂. -1 dNTPs, with final concentrations of 2 μmol / L for both upstream and downstream primers. -1 1 U Taq DNA polymerase was added, and the reaction mixture was brought to 25 μL with sterile distilled water. The PCR reaction program was: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 20 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 32 cycles; 72℃ extension for 5 minutes; storage at 4℃. PCR products were detected by 8% polyacrylamide gel electrophoresis (PAGE): PCR products were mixed with 5 μL of loading buffer and separated by electrophoresis on an 8% non-denaturing polyacrylamide gel at a constant voltage of 120 V for 2 hours, followed by silver staining. Only molecular markers were detected. Xdw08.9 ( Pm68-M1 It exhibits stable amplified polymorphic bands with high resolution, good repeatability, and an amplification product size of 156 bp.
[0041] Example 2: Carrying Pm68 Creation of new wheat germplasm and marker-assisted breeding
[0042] This invention uses Yangmai 158, a wheat variety susceptible to powdery mildew, as the wheat receptor, to generate a wheat variety carrying a powdery mildew resistance gene. Pm68 The durum wheat TRI 1796 was crossed with it, and then Yangmai 158 was used as the recurrent parent for multiple rounds of crosses, followed by self-pollination. The seedlings of the offspring from each backcross and the self-pollinated offspring were marked with molecular markers. Xdw08.9 ( Pm68-M1 PCR amplification was performed.
[0043] Amplification conditions were as follows: PCR reaction system: 25 μL reaction system containing approximately 50 ng template DNA, 1×PCR buffer, 1.5 mmol L⁻¹ MgCl₂, and 200 mmol L⁻¹ MgCl₂. -1dNTPs, with a final concentration of 2 μmol / L for each primer. -1 1 U Taq DNA polymerase was added to the reaction system with sterile distilled water to bring the volume to 25 μL. The PCR reaction program was as follows: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 20 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 32 cycles; 72℃ extension for 5 minutes; and storage at 4℃.
[0044] PCR products were detected using 8% polyacrylamide gel electrophoresis (PAGE): PCR products were mixed with 5 μL of loading buffer and separated by electrophoresis on an 8% non-denaturing polyacrylamide gel at a constant voltage of 120 V for 2 hours, followed by silver staining. Positive single plants were then transplanted and propagated to rapidly create the powdery mildew-resistant germplasm BC3F4. Powdery mildew-resistant BC3F4 was inoculated into mature plants using the shaking method, and powdery mildew identification results were observed after 10 days. The experiment showed that the obtained BC3F4 family exhibited good powdery mildew resistance. Further molecular marker analysis was performed... Pm68-M1 Tests showed that these plants all exhibited... Pm68 The specific bands indicate that the material has been transferred. Pm68 Gene.
[0045] like Figure 2 As shown, Figure 2 The results of powdery mildew identification of the Pm68 gene-transferred wheat offspring are shown in the figure. Nos. 1-6 are the offspring of recurrent crosses between durum wheat TRI 1796 (containing Pm68) and Yangmai 158; No. 7 is the powdery mildew-susceptible recurrent parent Yangmai 158. Figure 2 As can be seen, no powdery mildew occurred in the offspring of the recurrent cross between durum wheat TRI 1796 (including Pm68) and Yangmai 158, while the recurrent parent Yangmai 158 was susceptible to powdery mildew.
[0046] Figure 3 molecular markers Pm68-M1 The results of detection of high-generation durum wheat TRI 1796 / Yangmai 158 germplasm are shown in the figure. M represents the DNA molecular weight marker DL2000, and numbers 1-20 represent those containing... Pm68 The wheat transgenic progeny single plants are: No. 21 is Yangmai 158, No. 22 is powdery mildew-susceptible durum wheat PI 584832, and No. 23 is powdery mildew-resistant durum wheat TRI 1796 (including...). Pm68 (The arrow indicates that it contains) Pm68 Specific bands in the material of the gene. Figure 3 As can be seen, all the wheat plants tested, from No. 1 to No. 20, have already transitioned to the new growth stage. Pm68 Genes: Experiments have shown that the molecular markers of this invention can effectively detect whether wheat carries the powdery mildew resistance gene Pm68, identify or assist in the identification of whether wheat is resistant to powdery mildew, and / or be used for wheat breeding or to assist in wheat breeding.
[0047] Example 3: Molecular Markers Pm68-M1 Specificity analysis in different wheat varieties
[0048] To understand molecular markers Pm68-M1 In tracking Pm68 Whether it possesses high specificity, this invention uses 31 domestic wheat varieties (Jimai 19, Chuanmai 42, Zhengmai 9023, Jimai 22, Yumai 34, Lumai 14, Yangmai 158, Yangmai 18, Yumai 18, Zhou 8425B, Liangxing 66, Liangxing 99, Tainong 18, Tainong 19, Shannong 17, Shannong 21, Yannong 19, Shi 4185, Shimai 15, Ji 5265, Henong 827, Kenong 199, Kenong 9204, Zhenmai 168, Ningmai 13, Aikang 58, Luomai 21, Zhengmai 366, Zhengyumai 9989, Bima 1, Yangmai 158; all are publicly known and used, provided by Shandong Academy of Agricultural Sciences and other units) and 10 foreign wheat varieties (Chancellor, Ovest, Kolibri, Armada, Coker 747, Norin 10, Maris). Huntsman, Ralle, Arthur, and Suwon 92 (all publicly known and available from the U.S. Germplasm Bank: https: / / npgsweb.ars-grin.gov / gringlobal / search) were used as materials for PCR amplification and PAGE detection. The PCT system and reaction procedure were as described in Example 2. The detection results showed (e.g.) Figure 4 (as shown) Pm68-M1 Polymorphic bands are highly specific, appearing only in carriers. Pm68 In the material TRI 1796, no corresponding polymorphic bands were found in the 41 wheat varieties tested, indicating that the genetic variation in these materials is not significant. Pm68 At that time, molecular markers Pm68-M1 Effectively detect Pm68 Genes without false positives mean that co-segregation molecular markers Pm68-M1 It can be effectively used in different wheat backgrounds Pm68 Transformation and breeding.
Claims
1. The application of primers for amplifying the molecular marker of the wheat powdery mildew resistance gene Pm68, or products containing primers for amplifying the molecular marker of the wheat powdery mildew resistance gene Pm68, in detecting whether wheat carries the powdery mildew resistance gene Pm68, and in identifying or assisting in the identification of whether wheat is resistant to powdery mildew; The nucleotide sequence of the primer is as follows: Pm68-M1F: 5'-CTAGATGTCAGTCCAGATGTTG-3' (SEQ ID NO.1), Pm68-M1R: 5'-GTTATGCTGCAGTAACTAACTCTC-3' (SEQ ID NO. 2).
2. The application according to claim 1, characterized in that, The applications include: The genomic DNA of the test material was amplified by PCR using primers that amplify the molecular marker of the wheat powdery mildew resistance gene Pm68. The amplification products were detected. If a specific target product was obtained, the test material carried the powdery mildew resistance gene Pm68 and was therefore resistant to powdery mildew.
3. The application according to claim 2, characterized in that, The PCR reaction system used in the PCR amplification procedure includes: a 25 μL reaction system containing: 50 ng template DNA, 1× PCR buffer, and 1.5 mmol / L... -1 MgCl2, 200 mmol / L -1 dNTPs, final concentration 2 mol L -1 Amplification primers, 1 U Taq DNA polymerase, and sterile distilled water to make up the difference.
4. The application according to claim 2, characterized in that, The PCR amplification reaction procedure includes: Pre-denaturation at 94℃ for 3 minutes; denaturation at 94℃ for 20 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 60 seconds, 32 cycles; extension at 72℃ for 5 minutes; store at 4℃.
5. The application according to claim 2, characterized in that, The specific target product is a molecular marker for the wheat powdery mildew resistance gene Pm68, with a product size of 156 bp.
6. A method for detecting or assisting in the detection of wheat powdery mildew resistance, characterized in that, include: The genomic DNA of the test material was amplified by PCR using a primers that amplify the molecular marker of the wheat powdery mildew resistance gene Pm68. The amplification products were detected. If a specific target product was obtained, the test material carried the powdery mildew resistance gene Pm68 and was therefore resistant to powdery mildew. The nucleotide sequence of the primer is as follows: Pm68-M1F: 5'-CTAGATGTCAGTCCAGATGTTG-3' (SEQ ID NO.1), Pm68-M1R: 5'-GTTATGCTGCAGTAACTAACTCTC-3' (SEQ ID NO. 2).