Molecular marker related to wheat curly mite resistance and application of molecular marker

Abstract

The invention discloses a molecular marker related to wheat curly mite resistance and application of the molecular marker, and belongs to the technical field of wheat breeding for disease resistance. According to the invention, a nucleotide fragment related to wheat curly mite resistance is obtained through screening, and based on a nucleotide sequence of the fragment, the invention also designs a molecular marker for specific detection of the nucleotide fragment. The nucleotide fragment and the detection molecular marker thereof are not limited by environmental conditions, PCR amplification is carried out through the primer pair, plant germplasm resources with wheat curly mite resistance can be accurately screened according to the existence of target bands, the breeding efficiency is improved, and the breeding process is accelerated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of wheat disease resistance breeding, and particularly relates to a molecular marker related to wheat curl mite resistance and application thereof. BACKGROUND

[0002] Wheat curl mite is a tiny phytophagous mite, which is widely distributed in the main wheat planting areas of the world. Wheat curl mite not only seriously affects the yield and quality of wheat, but also spreads a variety of plant viruses (such as wheat stripe mosaic virus and wheat mosaic virus), causing serious losses to wheat production.

[0003] At present, the main prevention and control measures for wheat curl mite include chemical control and biological control. The main miticides used in chemical control are chlorpyrifos, thiamethoxam and chlorfenapyr, etc. However, wheat curl mite can occupy hidden positions such as curled leaves, leaf sheaths, new leaves and leaf axils of plants, thereby effectively weakening the effect of chemical control. The biological characteristics of wheat curl mite make the chemical control effect poor. Biological control mainly uses predatory mites or fungi, but the effect is limited.

[0004] Planting wheat varieties resistant to wheat curl mite is considered to be the most economical and effective way to control the damage of wheat curl mite. However, the resistance resources available for wheat breeding are very scarce, which seriously restricts the application of resistance breeding in the prevention and control of wheat curl mite. SUMMARY

[0005] In view of the above prior art, the present application aims to provide a molecular marker related to wheat curl mite resistance and application thereof.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: In the first aspect of the present application, a nucleotide fragment related to wheat curl mite resistance is provided, the sequence of which is shown in SEQ ID NO. 1; and the specific sequence is as follows:

[0007] In a second aspect of the present application, the nucleotide fragment is used in (1) or (2) as follows: (1) identifying the resistance of plant germplasm resources to wheat curl mite; and (2) breeding a plant variety resistant to wheat curl mite.

[0008] In the above use, the plant variety is a plant in the family Poaceae; including but not limited to a wheat-Aegilops sharonensis short fragment translocation line and Aegilops sharonensis.

[0009] In a third aspect of the present application, a molecular marker for detecting the nucleotide fragment is provided, and the nucleotide sequences thereof are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively; and the molecular marker is specifically as follows: SEQ ID NO. 2: 5'-TTAATTGTCATTGATGACATATGGG-3'; SEQ ID NO. 3: 5'-CAGGAATCGCGTTCCAAAG-3'.

[0010] The sample to be tested is amplified by the above primers, and if a 1588bp target band shown in SEQ ID NO. 1 can be amplified, the sample to be tested is identified as resistant to wheat curl mite; and if the target band cannot be amplified, the sample to be tested is identified as not resistant to wheat curl mite.

[0011] In the third aspect of the present application, a kit containing the above molecular marker is provided.

[0012] Further, the kit further comprises a DNA template, Taq Master Mix and ddH2O.

[0013] In a fourth aspect of the present application, the molecular marker or the kit is used in (1) or (2) as follows: (1) identifying the resistance of plant germplasm resources to wheat curl mite; and (2) breeding a plant variety resistant to wheat curl mite.

[0014] In a fifth aspect of the present application, a method for identifying the resistance of plant germplasm resources to wheat curl mite is provided, comprising the following steps: The genomic DNA of the plant germplasm resource to be tested is used as a template, and the primers shown in SEQ ID NO. 2 and SEQ ID NO. 3 are used for PCR amplification, and if a 1588bp target band shown in SEQ ID NO. 1 is amplified, it is determined that the plant germplasm resource to be tested has resistance to wheat curl mite.

[0015] Further, the PCR amplification system comprises: 2 μL of DNA template, 7.5 μL of 2 × Taq Master Mix, 1 μL of each of the primers shown in SEQ ID NO. 2 and SEQ ID NO. 3 with a concentration of 10 μmol / L, and water is added to make up the total volume of 15 μL.

[0016] The PCR reaction condition is: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 2 min, 35 cycles; and 72 ℃ extension for 10 min.

[0017] The present application has the following beneficial effects: (1) The present application screens a nucleotide fragment related to the resistance of wheat C. cirsus, and based on the sequence information of the nucleotide fragment, the present application also designs a molecular marker for specifically detecting the fragment. The nucleotide fragment and the molecular marker thereof of the present application are not limited by environmental conditions, and by performing PCR amplification through the molecular marker, the presence or absence of the target band can be used to accurately screen plant germplasm resources with wheat C. cirsus resistance, thereby improving the breeding efficiency and accelerating the breeding process.

[0018] (2) The molecular marker of the present application is simple to use, has low requirements for experimental equipment and experimental conditions, can be used in most laboratories, can be directly used in production practice, and is conducive to popularization and application in wheat molecular breeding. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Phenotype identification results of wheat C. cirsus on wheat near-isogenic lines. Phenotype identification results of wheat C. cirsus sensitive near-isogenic lines (NIL-S) and phenotype identification results of wheat C. cirsus resistant near-isogenic lines (NIL-R).

[0020] Figure 2 : Part of the genotyping identification results of the molecular marker on the natural populations of Aegilops tauschii collected worldwide. The materials No. 1, 2, 3, 4, 8, 9, 15, 17, 18 and 21 have target bands, indicating that the sample to be tested is resistant to wheat C. cirsus; the materials No. 5, 6, 7, 10, 11, 12, 13, 14, 16 and 20 have no bands, indicating that the sample to be tested is not resistant to wheat C. cirsus. The material No. 19 is a water control without PCR amplification. (M, CASSUL-Gel ladder, the bands are 2000 bp, 1600 bp, 1005 bp, 765 bp, 525 bp, 285 bp and 125 bp in turn) DETAILED DESCRIPTION It should be noted that the following detailed description is illustrative only and is intended to provide further description in order to provide a thorough understanding of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0021] In order to enable persons skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific embodiments.

[0022] The test materials used in the embodiments of the present application are all conventional test materials in the art and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out according to the conventional test methods or according to the operation instructions recommended by the suppliers.

[0023] Example 1: Screening of nucleotide fragments related to resistance to wheat curl mite and determination of molecular markers The resistance sources of wheat curl mite are very scarce, and the currently located resistance genes are only five, which are Cmc1 , Cmc2 , Cmc3 , Cmc4 and CmcTAM112 . Haplotype analysis results show that Cmc4 and CmcTAM112 may be the same genotype or homologous genes. Analysis of the source of wheat curl mite resistance genes found that all resistance genes come from the close relatives of wheat, and no resistance genes have been found in wheat. The resistant material OK05312 carries Cmc4 resistance gene, which shows stable resistance to wheat curl mite, and is applied to wheat resistance breeding, which has high research value. Cmc4 The gene is derived from the close relative of wheat, Aegilops TA2379 material, and currently there is no reference genome for gene cloning and marker development. The reference genome sequencing material AL8 / 78 of Aegilops does not carry the resistance gene to wheat curl mite and shows susceptibility, so the reference genome cannot meet the current research needs.

[0024] The previous gene mapping results show that the gene is located between molecular markers SDOKSNP6314 and SDOKSNP2805between the two markers, the physical distance is ~523kb. According to the reference genome of A. thunbergii, there are 13 candidate genes in this region. Combined with the transcriptome data, one gene is finally determined as the resistance candidate gene. However, by aligning the homologous gene sequences of A. thunbergii and wheat, it is found that the gene is relatively conservative at the front end of the gene, but there is a big difference in the sequence at the rear end of the gene, so it is difficult to accurately predict the stop codon of the gene, which brings great challenges to the design of the rear primer. By aligning the existing wheat and A. thunbergii gene sequences, the sequences at the end of the gene which are relatively conservative are selected for primer design. And using the DNA of the wheat resistant parent OK05312 as a template, PCR amplification and electrophoresis detection are carried out by using different primer combinations. The bands that meet the size are cut and the nucleic acid is recovered. And through a large number of E. coli transformation, single clone detection and sequencing technology, the complete sequence of the target gene is finally obtained in a single clone. By sequence alignment, the difference between the sequence and the known reference gene sequence is compared. The sequence comparison result shows that the first exon of the gene has high homology with the reference gene sequence, and the sequence difference is not big, which is not easy to design specific detection markers. And the third and third exon sequences are short and have great difference with the reference gene. Therefore, the conservative sequence located at the start position of the second intron is finally selected as the front primer, and the nucleotide sequence is shown as SEQ ID NO. 2, which is specifically as follows: SEQ ID NO. 2: 5'-TTAATTGTCATTGATGACATATGGG-3'; The sequence specific to the gene located in the second intron is selected as the rear primer, and the nucleotide sequence is shown as SEQ ID NO. 3, which is specifically as follows: SEQ ID NO. 3: 5'-CAGGAATCGCGTTCCAAAG-3'.

[0025] SEQ ID NO. 2 and SEQ ID NO. 3 can be used as molecular markers for specific detection of the resistance gene related to wheat curl mite resistance. The length of the sequence amplified by SEQ ID NO. 2 and SEQ ID NO. 3 is 1588bp, which can ensure that general commercial nucleic acid amplification enzymes can be amplified, and the target band is relatively large, which can eliminate the interference of false positive signals such as primer dimers, and at the same time, the two primers are located on the exons of the gene, which can meet the detection of the gene at the genomic DNA level and the transcriptome RNA level at the same time.

[0026] Example 2: Identification of wheat near-isogenic line materials resistant to wheat curl mite by using molecular markers 1. Test materials: A pair of near-isogenic lines, NIL-S and NIL-R, were selected from the progeny of a cross between wheat varieties SD06165 and OK05312. SD06165 and NIL-S were susceptible to wheat curl mite, while OK05312 and NIL-R were resistant to wheat curl mite.

[0027] 2. Test method and results: According to the molecular marker associated with wheat curl mite resistance screened in Example 1, a pair of detection primers was designed, the sequences of which are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively. DNA was extracted from the near-isogenic lines NIL-S and NIL-R, respectively, and PCR amplification was performed using the primer pair shown in SEQ ID NO. 2 and SEQ ID NO. 3. Wheat curl mite resistance was identified according to whether the target band shown in SEQ ID NO. 1 could be amplified: if the 1588 bp target band shown in SEQ ID NO. 1 could be amplified, the sample to be tested was identified as resistant to wheat curl mite; if the target band could not be amplified, the sample to be tested was identified as not resistant to wheat curl mite. The details are as follows: (1) Molecular marker identification: 1) CTAB method was used to extract sample genomic DNA; The CTAB method was used to extract DNA from the wheat sample, and the specific steps were as follows: For the above-mentioned materials, 2-3 cm wheat leaves were cut at the 2-leaf stage and placed in a 2 mL centrifuge tube. One 3.2 mm stainless steel bead was added, and the material was ground into powder after liquid nitrogen freezing treatment. 650 μL of preheated 65°C CTAB extraction buffer was added, and the mixture was incubated at 65°C for 45 min. Then, 300 μL of chloroform:isopropyl alcohol=24:1 (V:V) was added, and the mixture was slowly mixed and incubated for 15 min. During this period, the mixture was slowly mixed 2-3 times, and then centrifuged at 12000 rpm for 10 min. 450 μL of supernatant was carefully removed, mixed with an equal volume of ice isopropyl alcohol, and incubated at 4°C for 30 min or overnight. Then, the mixture was centrifuged at 12000 rpm for 45 min, and the supernatant was discarded. The precipitate was washed twice with 500 μL of 70% ethanol, dried, and then dissolved in TE solution or sterile water. The concentration was diluted to 20 ng / μL, and the solution was stored at low temperature for later use.

[0028] 2) PCR amplification; The PCR reaction system used was as follows: 2 μL of DNA template, 7.5 μL of 2 × Taq Master Mix, 1 μL of each of the primers SEQ ID NO. 2 and SEQ ID NO. 3 with a concentration of 10 μmol / L, and water to make up to 15 μL.

[0029] PCR reaction conditions used were: 95℃ pre-denaturation 5 min; 94℃ annealing 30 sec, 55℃ annealing 30 sec, 72℃ extension 2 min, 35 cycles; 72℃ extension 10 min.

[0030] 3) 1% agarose gel detection of PCR products: The results show that: NIL-R can amplify 1588bp target band, identified as wheat curl mite resistance; NIL-S can not amplify the target band, identified as wheat curl mite susceptible.

[0031] (2) Phenotypic identification: Wheat curl mites were kept on the seedlings of susceptible wheat variety Jagger, which were grown in a plant growth chamber. Before inoculation, a dissecting microscope was used to examine the highly curled infested leaves to determine the mite density on each curled leaf. According to the density of wheat curl mites, the infested leaves were cut into approximately 0.5cm leaf segments, ensuring that there were at least 30 wheat curl mites on each leaf segment. The prepared leaf segments were inoculated onto the two-leaf stage wheat varieties to be tested, with the inoculation site being the angle between the emerging new leaf and the adjacent unfolded true leaf. After inoculation, the plants were not moved as much as possible to avoid the shedding of the inoculated leaf segments. After 12h of inoculation, the inoculated plants to be tested were moved to a plant growth chamber at 22 ± 2 °C for cultivation. Phenotypic identification was performed 12-14 days after inoculation. The susceptible wheat variety Jagger was used as the susceptible control.

[0032] The results of phenotypic identification are shown in Table 1. Figure 1 The results show that: NIL-R shows resistance to wheat curl mites; NIL-S shows susceptibility to wheat curl mites.

[0033] Therefore, it can be seen that the identification results of wheat curl mite resistance using the molecular marker of the present application are consistent with the actual phenotypic identification results.

[0034] Example 3: Identification of Aegilops tauschii natural population resistance to wheat curl mites using molecular markers 1. Test method: The molecular marker developed in Example 1 was used to identify the resistance of 106 Aegilops tauschii materials collected from all over the world to wheat curl mites. The methods of molecular marker identification and phenotypic identification were the same as in Example 2.

[0035] 2. Test results: Figure 2The results of gel electrophoresis detection of part of the rough goat grass materials are shown in the following table. The results of detection of the natural population of rough goat grass show that samples 1, 2, 3, 4, 8, 9, 15, 17, 18 and 21 have target bands, indicating wheat curl mite resistance; samples 5, 6, 7, 10, 11, 12, 13, 14, 16 and 20 have no target bands, indicating wheat curl mite susceptibility. Sample 19 is a water control without PCR amplification.

[0036] The molecular marker identification results of the above rough goat grass materials are consistent with the actual wheat curl mite resistance phenotype identification results. Therefore, the molecular marker of the present application can also accurately diagnose the resistance to wheat curl mite in the natural population.

[0037] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A nucleotide fragment associated with resistance to wheat curl mite, characterized in that, The nucleotide sequence of the nucleotide fragment is shown as SEQ ID NO.

1.

2. The nucleotide fragment of claim 1 is applied in (1) or (2) as follows: (1) identifying the resistance of plant germplasm resources to wheat curtiworm; (2) breeding plant varieties resistant to wheat curtiworm. The plant variety is a Gramineae plant.

3. Use according to claim 2, characterized in that, The plant variety is a wheat-Aegilops sharonensis short fragment translocation line and Aegilops sharonensis.

4. Use according to claim 3, characterized in that, The nucleotide sequences of the molecular markers are shown as SEQ ID NO. 2 and SEQ ID NO. 3 respectively.

5. A molecular marker detecting the nucleotide fragment of claim 1, characterized in that, 6. A kit containing the molecular marker of claim 5. The kit further comprises a DNA template, Taq Master Mix and ddH2O.

7. The kit of claim 6, wherein 8. The molecular marker of claim 5 or the kit of claim 6 or 7 is applied in (1) or (2) as follows: (1) identifying the resistance of plant germplasm resources to wheat curtiworm; (2) breeding plant varieties resistant to wheat curtiworm. The method comprises the following steps: If the 1588bp target band shown as SEQ ID NO. 1 is amplified, it is determined that the plant germplasm resource to be tested has resistance to wheat curtiworm.

9. A method of identifying plant germplasm resources for resistance to wheat curl mites, characterized in that, The PCR amplification system comprises 2μL of DNA template, 7.5μL of 2 × Taq Master Mix, 1μL of each of the primers shown as SEQ ID NO. 2 and SEQ ID NO. 3 with a concentration of 10μmol / L, and water to make up the total volume to 15μL; The PCR reaction conditions are as follows: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 2min, 35 cycles; 72℃ extension for 10min.

10. The method of claim 9, wherein, ​ ​

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