Specific molecular marker of wheat stripe rust physiological race V5 and application

Abstract

The invention discloses a specific molecular marker of wheat stripe rust physiological race V5 and application, and belongs to the technical field of agricultural biology. The nucleotide sequence of the RAPD primer is as shown in SEQ ID NO. 1, and the nucleotide sequences of the SCAR molecular marker are as shown in SEQ ID NO. 2 and SEQ ID NO. 3. The technical scheme provided by the invention has relatively high specificity and sensitivity. The invention further provides application of the reagent for detecting the specific molecular marker of the wheat stripe rust physiological race V5 in field detection, and based on detection of the molecular marker, 3.6% of the V5 race is detected from leaf samples collected from a wheat field. The method can be used as a molecular tool for rapidly and accurately detecting the V5 race, and is used for knowing the population dynamic of the V5 race and predicting the prevalence of the V5 race.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and relates to a specific molecular marker, primer set and application of wheat stripe rust race V5. Background Technology

[0002] Wheat (Triticum aestivum) has long been one of the world's most important food crops, making a significant contribution to global food supply. However, wheat cultivation and production are plagued by various wheat diseases, severely impacting yield and quality. Among these, wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most serious wheat diseases. Reports indicate that wheat stripe rust occurs in more than 60 countries worldwide, causing yield losses of 10% to 30%, and under favorable conditions, even total crop failure. It remains a limiting factor for wheat production in many countries.

[0003] Planting disease-resistant varieties is the most effective, economical, and environmentally friendly measure to control stripe rust. However, due to the frequent mutations in the virulence of wheat stripe rust fungus, which constantly generate new strains, disease-resistant varieties lose their rust resistance shortly after being introduced due to the emergence of new strains. Therefore, monitoring new strains is a very important basic research task.

[0004] The Yr5 resistance gene was first reported in 1966 and has been widely used in wheat breeding due to its 61 prominent resistances to stripe rust worldwide. The virulence of this gene was first reported in India, followed by Australia. In recent years, a strain (V5) virulent to Yr5 has been identified in China. V5 exhibits a broader virulence spectrum and higher parasitic ability than the currently dominant races, including CYR34, CYR33, and CYR32, revealing a high potential risk to wheat. Therefore, monitoring emerging V5 strains is crucial for understanding the population dynamics of the stripe rust pathogen, predicting stripe rust epidemics, and guiding wheat breeding for stripe rust resistance.

[0005] Random Amplified Polymorphic DNA (RAPD) technology is a PCR-based molecular biology technique that helps distinguish different physiological races of the same pathogen, especially when traditional morphological methods cannot differentiate them. Specific Characteristic Amplification Region (SCAR) markers are developed based on RAPD technology. The target RAPD fragment is cloned and its ends sequenced. Specific primers are designed based on the sequences at both ends of the RAPD fragment, and the gene DNA fragment is then specifically amplified by PCR to identify a single site corresponding to the original RAPD fragment. Therefore, converting specific RAPD bands into SCAR markers provides an efficient method and multiple safeguards for the identification and monitoring of V5 bacterial strains. Summary of the Invention

[0006] This invention provides a specific molecular marker, primer set, and application for wheat stripe rust race V5. This method offers a molecular biology approach, utilizing specific molecular marker technology to provide a new pathway for strain identification. By combining PCR with agarose gel electrophoresis, rapid detection in the early stages of growth and development of infected wheat plants can be achieved. Furthermore, combining phenotypic identification can improve the accuracy and reliability of the identification.

[0007] The technical solution is as follows:

[0008] First, the present invention provides a RAPD primer S115 that can screen for specific fragments of wheat stripe rust race V5, the nucleotide sequence of which is shown in SEQ ID NO.1 (5'-GTCGGCTTCA-3').

[0009] Second, the present invention provides a SCAR primer set V5F / R for specific detection of V5. The primer set is the specific primer pair for detecting the specific molecular marker of wheat stripe rust race V5 as described in claim 1. The primer pair includes a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.2 (5'-CTGCTCATCGTAAGCCATCT-3'), and the sequence of the reverse primer is shown in SEQ ID NO.3 (5'-CGAACGCCATAGGAACATAG-3').

[0010] Third, this invention verifies specificity and sensitivity. Using common fungi and wheat stripe rust races as references, it can specifically detect race V5. The lowest detectable DNA concentration is 100 pg / μL. Using 8 μL of this solution yields a concentration of 2.4 × 10⁸ pg / mL. 4It can be detected on day 5 after inoculation with a suspension of urediniospores, and on day 6 after inoculation with a single spore.

[0011] Fourth, this invention provides an application of the primer set described herein in screening for V5 races in the field and detecting their occurrence frequency. The occurrence frequency of wheat stripe rust from Shaanxi, Gansu, Qinghai, Xinjiang, and Tibet was 3.6%.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] (1) This invention employs molecular marker technology to identify the newly generated V5 strain. Traditional morphological identification is easily affected by the quality of the germplasm, has high environmental requirements, and a long identification cycle. In contrast, this invention is fast, easy, and highly stable, offering significant advantages for large-scale identification. For small-scale identification, both methods can be used to corroborate each other, improving accuracy.

[0014] (2) Traditional RAPD molecular marker technology is highly sensitive to changes in experimental procedures and conditions, and has low stability. In contrast, SCAR markers have high stability and good reproducibility. Furthermore, because they are codominantly inherited, differences between the DNA samples being tested can be directly indicated by the presence or absence of amplification products. Therefore, this invention converts RAPD results into SCAR markers, enabling more accurate identification of the V5 bacterial strain.

[0015] (3) The molecular marker technology of the present invention has high sensitivity, with a minimum detectable DNA concentration of 100 pg / μL, and can be detected as early as 5 or 6 days after infection, which can effectively predict the occurrence frequency of V5 in natural populations. Attached Figure Description

[0016] Figure 1 Agarose gel electrophoresis pattern of PCR amplification products of various physiological races of wheat stripe rust and other fungal DNA using RAPD primer S115; CK1 and CK2: sterile water and healthy wheat leaves as negative controls; M: DL2000 Marker; Lanes 1-15: wheat powdery mildew (Blumeria graminis f. sp. tritici), wheat leaf rust (P. triticina), wheat stem rust (P. graminis f. sp. tritici), barley stripe rust (P. striiformis f. sp. hordei), barley leaf rust (P. hordei), barley stem rust (P. graminis f. sp. hordei), CYR23, CYR26, CYR29, CYR30, CYR31, CYR32, CYR33, CYR34, V5-1; the 610 bp fragment in lane 15 is a specific band;

[0017] Figure 2 Agarose gel electrophoresis patterns of PCR amplification products of various physiological races of wheat stripe rust and other fungal DNA using SCAR primers; CK1 and CK2: sterile water and healthy wheat leaves as negative controls; M: DL2000 Marker; A: Lanes 1-16: Wheat powdery mildew (Blumeria graminis f. sp. tritici), wheat leaf rust (P. triticina), wheat stem rust (P. graminis f. sp. tritici), barley stripe rust (P. striiformis f. sp. hordei), barley leaf rust (P. hordei), barley stem rust (P. graminis f. sp. hordei), CYR23, CYR26, CYR29, CYR30, CYR31, CYR32, CYR33, CYR34, V5-1, V5-2, M: DL2000 Marker; B: Lanes 1-8: Eight V5 physiological subtypes, V5-1 to V5-8;

[0018] Figure 3 : PCR amplification products using SCAR primers at different DNA concentrations of race V5; M: DL 2000 DNA Marker; CK: sterile water as a negative control; lanes 1-7 were 1 pg / μL, 10 pg / μL, 100 pg / μL, 500 pg / μL, 1 ng / μL, 10 ng / μL, and 100 ng / μL, respectively.

[0019] Figure 4 : Use 8 μL of 2.4×10 4 A urediniospore suspension of 192 urediniospores per mL (equivalent to 192 urediniospores on a 1 cm long leaf) and single urediniospores were inoculated onto leaves of wheat variety Mingxian 169; AB, chlorosis appeared on wheat leaves inoculated with urediniospore suspension on day 5 post-inoculation, and bands were first detected; CD, chlorosis appeared on leaves inoculated with single urediniospores on day 6 post-inoculation, and bands were first detected. Figure 4 Lanes 1-12 in B and Figure 4 Lanes 1-11 in D represent leaf samples collected daily after inoculation; CK1 and CK2 represent sterile water and healthy leaves as negative controls, respectively. Detailed Implementation

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Unless otherwise specified, the experimental and detection methods in the following embodiments are conventional methods; the reagents and materials mentioned are commercially available unless otherwise specified; and the index data are measured using conventional methods unless otherwise specified.

[0022] Example 1

[0023] In this embodiment, 300 random RAPD primers were used to perform PCR amplification and agarose gel electrophoresis separation on different tested stripe rust species.

[0024] 1. Material Preparation

[0025] The tested fungal strains were *Blumeria graminis* f. sp. tritici, *P. triticina*, *P. graminis* f. sp. tritici, *P. striiformis* f. sp. hordei, *P. hordei*, *P. graminis* f. sp. hordei, and *P. graminis* races 23 (CYR23), 26 (CYR26), 29 (CYR29), 30 (CYR30), 31 (CYR31), 32 (CYR32), 33 (CYR33), and 34 (CYR34) of wheat stripe rust, all provided by the Southeast Kiln Experimental Station of Northwest A&F University. Each strain was extensively propagated on the susceptible wheat variety Mingxian 169, and fresh urediniospores were collected for the experiments. Field samples were collected from Shaanxi, Gansu, Qinghai, Xinjiang, and Tibet.

[0026] 2. Extraction of genomic DNA from wheat stripe rust fungus

[0027] Referring to the strain and the identified V5 strain, cut 3-4 diseased leaf samples about 1 cm long and put them into a 2 mL grinding tube. Add a steel ball, freeze in liquid nitrogen, and grind into powder using a grinder. Then extract DNA using the BevoFungal Genomic DNA Extraction Kit (GD2416) according to the instructions.

[0028] 3. RAPD molecular markers

[0029] Three hundred 10bp random RAPD primers were purchased from Beijing Solarbio Science & Technology Co., Ltd., and were used to amplify the physiological races of the tested stripe rust fungi by PCR.

[0030] The total reaction volume was 25 µL: 12.5 μL of 2×Es Taq MasterMix, 1.0 μL of primer (10 μmol / L), 1.0 μL of template DNA (30 ng / μL), and ddH2O to 25.0 μL.

[0031] The PCR program was performed on a PCR instrument. The amplification program was as follows: 94℃ for 5 min; 94℃ for 30 s, 36℃ for 30 s, 72℃ for 90 s, for 40 cycles; 72℃ for 7 min, and stored at 16℃.

[0032] The amplified products were separated by electrophoresis using a 2% agarose gel, and the RAPD band patterns were photographed and recorded using a gel imaging system.

[0033] The results are as follows Figure 1 As shown, a total of 300 random primers with 10 base pairs were screened. Among them, primer S115 (5'-GTCGGCTTCA-3') amplified a specific band of 610 bp from V5 stripe rust fungus. Multiple RAPD analyses showed that the amplification of this band was large and stable, and no such amplification product was found in other stripe rust fungus strains tested.

[0034] Example 2

[0035] This embodiment provides the recovery, cloning, and sequencing of a RAPD-specific fragment from V5 wheat stripe rust, which is then converted into a SCAR marker.

[0036] 1. Specific fragment recovery and purification

[0037] Specific bands were excised from agarose gels under long-wave ultraviolet light and recovered and purified using a DNA purification and recovery kit (MegBio).

[0038] 2. Specific fragment cloning and transformation into competent E. coli cells.

[0039] The recovered product was ligated into the pMD18-T vector (TaKaRa, Japan), heat-shocked into DH-5α Escherichia coli competent cells, and gently spread onto LB medium containing ampicillin (50 μg / mL) using a sterile spreader. The cells were then incubated in the dark at 37°C for 12 h. Single colonies were picked for colony PCR identification. Positive strains were inoculated into liquid LB medium containing ampicillin and cultured overnight with shaking.

[0040] 3. Sequencing and conversion into SCAR markers.

[0041] The samples were sent to Sangon Biotech Co., Ltd., where bidirectional sequencing and assembly were performed using the universal primers M13-48 (5'-GAGCGGATAACAATTTCACAC-3') and M13-47 (5'-AGGGTTTTCCCAGTCACG-3') for the pMD18-T vector.

[0042] Based on the sequencing results of the fragment, specific primers V5F (5'-CTGCTCATCGTAAGCCATCT-3') and V5R (5'-CGAACGCCATAGGAACATAG-3') were designed.

[0043] Optimized PCR reaction system: 12.5 μL of 2×Es Taq MasterMix, 1.0 μL of primer (10 μmol / L), 1.0 μL of template DNA (30 ng / μL), and ddH2O added to 25.0 μL.

[0044] PCR reaction conditions: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 40 s, 30 cycles; 72℃ for 7 min, store at 16℃.

[0045] Example 3

[0046] This embodiment verifies the specificity and sensitivity of the SCAR primers.

[0047] 1. Verify primer specificity

[0048] Using the designed SCAR primers, PCR amplification was performed on the tested physiological races of stripe rust and the DNA of common fungi, respectively. Figure 2 (A), and simultaneously, PCR amplification was performed on the DNA of eight V5 races with different virulences on a group of 25 Yr single-gene lines for identification ( Figure 2 (B) The amplified products were separated by electrophoresis using a 2% agarose gel, and the band patterns were photographed and recorded using a gel imaging system.

[0049] Figure 2 In study A, this primer was able to amplify a specific target band of 610 bp from the V5 genome, while no band was amplified from the genomic DNA of other tested physiological races and wheat leaves. Figure 2 In B, all eight V5 races amplified a specific target band of 610 bp, indicating that the primers are specific to the V5 genome and can be used as specific molecular markers for detecting V5 races.

[0050] 2. Verify primer sensitivity

[0051] The minimum DNA concentration for detection was verified using a DNA gradient solution of the V5 race. Based on the PCR amplification results, the 610 bp target band appeared at DNA concentrations of 100 ng / μL, 10 ng / μL, 1 ng / μL, 500 pg / μL, and 100 pg / μL, but did not appear at DNA concentrations of 10 pg / μL, 1 pg / μL, or as a negative control in sterile water. Figure 3 This indicates that the effective minimum detectable DNA concentration is 100 pg / μL.

[0052] Example 4

[0053] This embodiment verifies the earliest detectable number of days after infection.

[0054] To determine the earliest detectable time of the V5 physiological race in wheat leaves after infection, the highly susceptible wheat variety Mingxian 169 was planted in a rust-free growth chamber located in a controlled greenhouse with standard temperature and light settings.

[0055] Use 8 μL of urediniospore suspension (concentration 2.4 × 10⁸) 4 A single urediniospore (approximately 24 urediniospores / μL) and a single urediniospore of race V5 were inoculated onto the leaf surface of 10-day-old wheat seedlings and labeled. Infected leaf segments (5 cm and 1 cm in length, respectively) were collected until the initial appearance of uredinia. DNA solutions were amplified using SCAR markers to ensure effective detection of race V5 in the early incubation period after infection. Healthy leaves and sterile water were used as negative controls.

[0056] Based on V5F / R marker detection, leaf samples inoculated with urediniospore suspension failed to amplify any bands before 4 days post-inoculation, but the target bands appeared from day 5 onwards. However, sporulation was not observed until day 8. Figure 4 A and Figure 4 (B). Compared to leaves inoculated with urediniospore suspension, leaf samples infected with single urediniospores failed to amplify the target band before day 5, but were detectable from day 6 to day 11. However, obvious uredinia formation on the leaf surface was not observed until day 10. Figure 4 C and Figure 4 (D). In all tests, sterilized water and healthy leaves, used as negative controls, did not amplify any bands ( Figure 4 B and Figure 4 (of D).

[0057] Example 5

[0058] This embodiment measured the occurrence frequency of race V5 in the field.

[0059] Wheat leaves with uredinia were collected, and DNA was extracted from each sample. Amplification was performed using the SCAR marker V5F / V5R. Sterile water and healthy wheat leaves were used as negative controls for all assays. Positive amplification bands were recorded, and the frequency of V5 physiological races was calculated.

[0060] Molecular detection based on V5F / R markers was performed on 140 wheat stripe rust leaf samples collected from Shaanxi, Gansu, Qinghai, Xinjiang, and Tibet in China. The results showed that five V5 physiological races were detected in all isolates, with a total frequency of 3.6% (Table 1). Among these V5 strains, two were detected in 40 samples from Gansu, with a frequency of 5.0%, the highest. One was detected in 20 samples from Shaanxi, with a frequency of 5.0%, the same as Xinjiang. Only one V5 physiological race was detected in each of the 30 samples from Qinghai and Tibet, with frequencies of 3.3%, respectively. However, no V5 physiological races were detected in the 20 samples from Xinjiang.

[0061] In summary, this invention discloses a method for detecting wheat stripe rust race V5. The nucleotide sequences of RAPD primers for V5 race are shown in SEQ ID NO.1, and these are converted into stable SCAR primers as shown in SEQ ID NO.2 and SEQ ID NO.3. These primers specifically amplify a 610 bp band of V5 race. The sensitivity was verified, with a detection limit of 100 pg / μL of DNA, detectable as early as day 5 or 6 (chlorosis stage) of wheat stripe rust infection. This invention provides technical support for the rapid detection of wheat stripe rust race V5. Using this invention, field strains from Gansu, Shaanxi, Xinjiang, Tibet, and Qinghai provinces were tested, revealing a V5 race occurrence frequency of 3.6%. Compared to traditional methods, molecular marker-based detection greatly facilitates the identification of specific races in a short time. More importantly, molecular detection not only supports real-time monitoring of the dynamics of a specific race but also helps in the early prediction of the asymptomatic period of wheat stripe rust in the field.

Claims

1. A RAPD primer for screening wheat stripe rust race V5, characterized in that, The nucleotide sequence of the primer is shown in SEQ ID NO.

1.

2. A pair of SCAR primers for the specific amplification of wheat stripe rust race V5, characterized in that, It includes a forward primer and a reverse primer, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

3. A kit for detecting wheat stripe rust physiological race V5, characterized in that, It includes the SCAR primer set as described in claim 2.

4. A method for detecting wheat stripe rust physiological race V5 using the kit described in claim 3, characterized in that, Includes the following steps: Using the genomic DNA of the sample to be tested as a template, PCR amplification was performed using the SCAR primer set in the kit.

5. The method according to claim 4, characterized in that, The method can detect genomic DNA at a minimum concentration of 100 pg / μL.

6. The use of the SCAR primer set of claim 2, the kit of claim 3, or the method of any one of claims 4-5 in screening or identifying wheat stripe rust race V5.

7. The application according to claim 6, characterized in that, include: Wheat field leaf samples were collected, genomic DNA was extracted, and PCR detection was performed using the kit described in claim 3 and / or the method described in any of claims 4-5 to determine the occurrence frequency of wheat stripe rust physiological race V5.

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