Molecular marker primer for specific identification of wheat-taishanmillet t2ds·2vl translocation line and application thereof

Abstract

This invention discloses molecular marker primers for the specific identification of the wheat-Triticum aestivum T2DS·2V#4L translocation line and their applications, belonging to the field of agricultural biotechnology. Using the wheat variety *Chinese Spring* as a background, the long arm of the *Triticum aestivum* 2V#4L chromosome was introduced to obtain the wheat-Triticum aestivum T2DS·2V#4L translocation line. Identification results showed that this translocation line exhibited significantly enhanced resistance to sheath blight, and the resistance gene was located on the long arm of chromosome 2V#4L. The genomic sequence of the long arm of the *Triticum aestivum* 2V#4L chromosome was obtained using flow cytometry chromosome sorting and high-throughput sequencing. The *Triticum aestivum*-specific SNP site XM000010_C1161A was identified in the gene XM000010 sequence, and a set of high-throughput KASP markers was developed. The primers described in this invention can be used for high-throughput screening of wheat-Triticum aestivum T2DS·2V#4L translocation lines resistant to sheath blight.

Description

Technical Field

[0001] This invention relates to molecular marker primers for the specific identification of the wheat-Triticum aestivum T2DS·2V#4L translocation line and their applications, which can be applied to molecular marker-assisted breeding and belong to the field of agricultural biotechnology. Background Technology

[0002] Wheat sharp eyespot (WSH) is a global soil-borne fungal disease of wheat caused by *Rhizoctonia cerealis*. As the world's largest endemic area, my country suffers severe damage to wheat production from WSH. From 2011 to 2019, WSH occurred at a moderate to severe level in my country, with an average annual affected area of ​​approximately 130 million mu (15 million hectares), making it one of the major diseases affecting wheat production, especially in the Huang-Huai and Yangtze River mid-lower reaches regions. WSH can cause basal stem rot in wheat, reducing lodging resistance, and in severe cases, leading to seedling death and whiteheads. Studies show that the field disease incidence of WSH is generally 10%-20%, reaching 60%-80% in severely affected fields, generally resulting in a yield reduction of 10%-30%, and even more than 50% in severely affected areas. Because WSH is a soil-borne disease, agronomical cultivation methods are difficult to control, and chemical control can easily cause environmental pollution. Therefore, studying the mechanism of wheat resistance to sheath blight, exploring and utilizing disease-resistant gene resources, and breeding and promoting disease-resistant varieties are the most economical and environmentally friendly measures to control the occurrence of the disease and reduce yield losses.

[0003] To date, numerous domestic research institutions have conducted sheath blight resistance assessments on thousands of wheat germplasm accessions. Results show that most accessions are susceptible, with very few resistant varieties, and no immune germplasm resources have been identified. For example, Liu et al. (2015) assessed sheath blight resistance in 3500 domestic and international wheat varieties, finding only 32 materials to be moderately resistant. Currently, it is generally believed that there are significant differences in sheath blight resistance among different wheat varieties. Most varieties are highly susceptible or moderately susceptible, a few have good resistance, but no varieties are completely immune. Widely used wheat varieties and their core parents generally exhibit poor sheath blight resistance. The scarcity of superior resistance sources is a major challenge currently facing wheat sheath blight resistance breeding.

[0004] Among closely related genera and species of wheat, some exhibit high resistance or immunity to sheath blight. Yuan Hongxia et al. (1998) identified the sheath blight resistance of 26 closely related wheat genera and found that 5 materials showed immunity, including triticale and Roegneria kamoji. Li Hongjie et al. (2013) identified sheath blight in hybrid progeny of wheat-Leymus chinensis and wheat-Leymus chinensis hybrids and found that the wheat-Leymus chinensis 4Ai#2 or 4Ai#2S addition lines, substitution lines, and translocation lines, as well as the wheat-Leymus chinensis 4J chromosome substitution line, all significantly reduced the sheath blight index, indicating that the Leymus chinensis 4Ai#2 chromosome and the Leymus chinensis 4J chromosome may be related to the reduction in the sheath blight disease index. Li Qiang et al. (2000) identified wheat-Triticum aestivum germplasm against wheat sheath blight and found that some materials were highly resistant to the disease, but they did not further report the chromosomal location of the resistance gene. Therefore, discovering wheat sheath blight resistance gene resources from wild closely related genera of wheat is one of the important ways to solve the current lack of resistance sources.

[0005] *Dasypyrum villosum* (L.) P. Candargy, 2n=2x=14, VV, is an annual diploid wild species mainly distributed in the Mediterranean coastal region and the Caucasus Mountains. *Dasypyrum villosum* exhibits strong resistance to wheat sheath blight and several other diseases, making it an important tertiary gene source for wheat genetic improvement. The common wheat-*Dasypyrum villosum* T2DS·2V#4L translocation line is a Robertson translocation line produced from the offspring of a cross between a Sub2V(2D) substitution line (Chinese Spring background) and a Chinese Spring wheat variety (Zhang et al., 2015). Five consecutive years of sheath blight resistance testing showed that the T2DS·2V#4L translocation line exhibited superior resistance to the resistant control variety Niavt 14, demonstrating good breeding potential. The resistance gene was located on the long arm of chromosome 2V#4L.

[0006] Traditional methods for tracing exogenous chromosomes rely on cytological techniques, specifically fluorescence in situ hybridization (FISH). The main procedure involves taking root tip tissue from the sample after rooting, pretreating it with ethanol-acetic acid fixative, performing hybridization with a specialized probe, and then observing the results under a fluorescence microscope. This method is relatively inefficient, identifying only one sample at a time. With the development of molecular marker technology, high-throughput detection can be performed using exogenous chromosome-specific molecular markers, significantly improving detection efficiency while maintaining accuracy. This lays the foundation for large-scale introduction of superior exogenous genes into common wheat varieties.

[0007] Technical solution

[0008] To address the aforementioned technical problems, this invention provides a set of molecular marker primers for the specific identification and screening of wheat-Triticum aestivum T2DS·2V#4L translocation lines. These primers are used for high-throughput screening of wheat-Triticum aestivum translocation lines resistant to sheath blight, laying the foundation for large-scale introduction of superior sheath blight resistance genes from Triticum aestivum 2V#4L into common wheat varieties and promoting wheat breeding for sheath blight resistance.

[0009] The technical solution provided by this invention is as follows:

[0010] A set of molecular marker primers specifically for identifying the wheat-Triticum aestivum T2DS·2V#4L translocation line, wherein the nucleotide sequence of the molecular marker is shown in SEQ ID NO:1, and the primer sequence is as follows:

[0011] KASP_XM000010_C1161A front primer 1:

[0012] FAM-5'-GTCGTCCGTGTCGTGTTCTA-3' (SEQ ID NO: 2);

[0013] KASP_XM000010_C1161A front primer 2:

[0014] HEX-5'-GTCGTCGGTTTGTCGTGTTCTC-3' (SEQ ID NO: 3);

[0015] KASP_XM000010_C1161A back primer:

[0016] 5'-CAGAAGCCCACATGCAGCAA-3' (SEQ ID NO: 4).

[0017] Furthermore, the molecular marker is the KASP marker KASP_XM000010_C1161A located at the 1161st nucleotide of the XM000010 sequence of the Triticum aestivum gene.

[0018] The primer is the KASP marker KASP_XM000010_C1161A located at nucleotide 1161 of the XM000010 sequence of the Triticum aestivum gene.

[0019] The present invention also provides the application of the above-mentioned molecular marker primers in the identification or screening of wheat-truncatella T2DS·2V#4L translocation lines resistant to sheath blight.

[0020] This invention also provides the application of the above-mentioned molecular markers in any of the following:

[0021] (1) Application in identifying or assisting in the identification of the 1161st nucleotide-specific SNP site in the XM000010 sequence of the long arm gene on chromosome 2V#4L of Triticum aestivum;

[0022] (2) Application in identifying or assisting in the identification of Robertsonian translocation lines containing the long arm of the tufted wheat 2V#4L chromosome in a wheat background.

[0023] A method for identifying the wheat-Triticum aestivum T2DS·2V#4L translocation line involves PCR amplification of wheat leaf DNA using the aforementioned primers, and selection of varieties whose KASP_XM000010_C1161A marker detection results are HEX genotypes.

[0024] Furthermore, the obtained amplification products were subjected to fluorescence signal scanning, and the scan data were analyzed using Kluster Caller software to obtain a scatter plot. When the genotype appeared in the X-axis region, it indicated that the wheat was a FAM genotype variety; when the genotype appeared in the Y-axis region of the scatter plot, it indicated that the wheat was a HEX genotype variety.

[0025] Furthermore, the reaction system for PCR amplification is as follows:

[0026] Template (~20 ng / μl) 2.5 μl, KASP assay mix 0.07 μl, 2×KASP master mix 2.5 μl; the KASP assay mix (100 μl) includes: 12 μl (100 μM) of each allele-specific front primer, 30 μl (100 μM) of the back primer, and 46 μl of Tris-HCl (10 mM, pH 8.3).

[0027] The PCR reaction cycle program is as follows: pre-denaturation at 94℃ for 15 min, then 10 cycles of "94℃, 20s; 65-57℃, 60s (decreasing by 0.8℃ per cycle)"; finally, 32 cycles of amplification of "94℃, 20s; 57℃, 60s".

[0028] Fluorescence analysis of PCR products was performed using a PHERAstar (LGC Genomics) instrument, and finally, KlusterCaller (LGC Genomics) software was used for genotyping.

[0029] The present invention also provides a detection kit for the specific identification of the wheat-flesh wheat T2DS·2V#4L translocation line, wherein the detection kit contains the above-mentioned primers.

[0030] The application of the aforementioned molecular marker primers involves using a set of molecular marker primers specifically designed for identifying the wheat-Triticum aestivum T2DS·2V#4L translocation line to amplify wheat plant DNA via PCR. If the HEX genotype with the KASP_XM000010_C1161A marker appears, it indicates that the plant is a wheat-Triticum aestivum T2DS·2V#4L translocation line. This application targets wheat-Triticum aestivum T2DS·2V#4L translocation lines containing the long arm of the Triticum aestivum 2V#4L chromosome and their recombinant inbred lines for amplifying wheat plant DNA.

[0031] Beneficial effects

[0032] Sheath blight is a serious disease affecting wheat production. This invention utilizes molecular marker methods to develop a set of molecular markers specifically for identifying the wheat-Triticum aestivum T2DS·2V#4L translocation line, which has significant value in wheat breeding practice and disease resistance theory research. Its advantages can be summarized in the following three points:

[0033] (1) The set of molecular markers for the specific identification of the wheat-truncatella T2DS·2V#4L translocation line in this invention is a new marker obtained by sequencing analysis of the long arm genome of the truncatella 2V#4L chromosome. It is stable and can be used to specifically identify wheat-truncatella T2DS·2V#4L translocation line material.

[0034] (2) Using the wheat variety *Chinese Spring* as a background, the long arm of chromosome 2V#4L of *Triticum aestivum* was introduced to obtain the wheat-*Triticum aestivum* T2DS·2V#4L translocation line. Identification results showed that the wheat-*Triticum aestivum* T2DS·2V#4L translocation line exhibited significantly enhanced resistance to sheath blight, and the resistance gene was located on the long arm of chromosome 2V#4L, contributed by *Triticum aestivum*. The *Triticum aestivum* 2V#4L material used in this study is a novel source of resistance to sheath blight, and based on this molecular marker, it is hoped that new sheath blight resistance genes can be discovered.

[0035] (3) This marker is a stable KASP marker, which can be directly used to specifically identify wheat-Triticum aestivum T2DS·2V#4L translocation lines, accelerating the research and breeding application of introducing exogenous chromosome fragments from Triticum aestivum 2V#4L into wheat. Traditional fluorescence in situ hybridization identification methods require a cumbersome slide preparation process, needing overnight storage, and fluorescence microscopy examination can only be performed on individual slides sequentially, resulting in low efficiency for large-scale identification. This invention utilizes molecular marker methods for large-scale detection, significantly improving identification efficiency while maintaining accuracy compared to existing techniques. Attached Figure Description

[0036] Figure 1The results show the BLAST alignment of the *Triticum aestivum* gene XM000010 with the *Wheat* Chinese Spring reference genome. The results indicate that the XM000010 gene homologous sequence exists only on chromosomes 2A, 2B, and 2D in wheat.

[0037] Figure 2 The results of BLAST alignment of the *Triticum aestivum* gene XM000010 with three homologous sequences on chromosomes 2A, 2B, and 2D of the wheat *Triticum aestivum* Chinese spring reference genome are shown. The 1161st amino acid in gene XM000010 is C, while the corresponding amino acid in the three homologous sequences in wheat is either A or G.

[0038] Figure 3 The KASP_XM000010_C1161A marker was used to detect the wheat variety *Chinese Spring* and the *Wheat-Triticum aestivum* T2DS·2V#4L translocation line with a *Chinese Spring* background. The HEX genotype indicates that the sample is a *Wheat-Triticum aestivum* T2DS·2V#4L translocation line.

[0039] Figure 4 The results of testing the KASP_XM000010_C1161A marker on the dwarf-resistant 58 / NAU2V-8BC2F4 population are shown. The HEX genotype indicates that this sample is a wheat-tufted wheat T2DS·2V#4L translocation line.

[0040] Figure 5 The results are from fluorescence in situ hybridization identification of NAU2V-8. The green line represents the long arm of chromosome 2V#4L, while the rest are common wheat chromosomes.

[0041] Figure 6 The results of the identification of sheath blight resistance in materials containing the long arm of chromosome 2V#4L of Triticum aestivum var. 2V#4L and materials without the long arm of chromosome 2V#4L from the dwarf resistant 58 / NAU2V-8BC2F4 population. Detailed Implementation

[0042] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0043] Example 1

[0044] Acquisition of a set of molecular markers specifically for identifying the wheat-Triticum aestivum T2DS·2V#4L translocation system

[0045] First, the chromosome of the wheat-T2DS·2V#4L translocation line containing the long arm of the *Triticum aestivum* 2V#4L chromosome was sorted by flow cytometry. The high-purity chromosomal DNA obtained was then subjected to high-throughput sequencing and assembled to obtain the genome sequence of the long arm of chromosome 2V#4L. The genome sequence was annotated, and the gene information on the long arm of the *Triticum aestivum* 2V#4L chromosome was compared with the wheat reference genome using BLAST to analyze sequence differences.

[0046] After aligning the *Triticum aestivum* gene XM000010 with the wheat genome sequence, three homologous sequences were obtained on wheat chromosomes 2A, 2B, and 2D. Figure 1 Further alignment of XM000010 with the three homologous sequences revealed a gene-specific SNP site at amino acid 1161, which was distinct from all three homologous sequences. Figure 2 Using the software Primer3web, this specific SNP marker was successfully designed as a KASP marker. Testing showed that the designed KASP marker could detect polymorphism between the two parents. Figure 3 ).

[0047] The newly developed marker information is shown in Table 1, and the primer sequences used to amplify the molecular markers are shown in Table 2.

[0048] Table 1 Newly Developed Tagging Information

[0049]

[0050] Table 2. Primer sequences used for amplifying the molecular markers.

[0051]

[0052] Example 2

[0053] Application of molecular marker KASP_XM000010_C1161A in the specific identification of wheat-Triticum aestivum T2DS·2V#4L translocation system

[0054] To verify the feasibility of using the KASP_XM000010_C1161A marker for specific identification of the wheat-Triticum aestivum T2DS·2V#4L translocation line, and to compare the efficiency and accuracy of KASP molecular marker identification and traditional fluorescence in situ hybridization identification, this example tested the dwarf-resistant 58 / NAU2V-8BC2F4 population.

[0055] 1. Comparison of the efficiency of KASP molecular marker detection and fluorescence in situ hybridization detection.

[0056] KASP molecular marker detection includes steps such as DNA extraction, PCR detection, and result interpretation. The specific detection method is as follows:

[0057] Methods for extracting genomic DNA:

[0058] Total DNA was extracted from young leaves using the CTAB extraction method described by Maquire et al., and stored at -20°C for later use. This step took approximately 1 hour and 15 minutes.

[0059] Wheat leaf DNA was amplified by PCR using the primers described in Table 2. The PCR amplification reaction system was as follows:

[0060] Template (~20 ng / μl) 2.5 μl, KASP assay mix 0.07 μl, 2×KASP master mix 2.5 μl; the KASP assay mix (100 μl) includes: 12 μl (100 μM) of each allele-specific front primer, 30 μl (100 μM) of the back primer, and 46 μl of Tris-HCl (10 mM, pH 8.3).

[0061] The PCR reaction cycling program is as follows: pre-denaturation at 94℃ for 15 min, followed by 10 cycles of "94℃, 20 s; 65-57℃, 60 s (decreasing by 0.8℃ per cycle)"; finally, 32 cycles of amplification are performed using the "94℃, 20 s; 57℃, 60 s" method. This step takes approximately 1 hour and 40 minutes.

[0062] The amplified products were scanned for fluorescence signals using a PHERAstar (LGC Genomics) instrument. The scanned data were analyzed using Kluster Caller (LGC Genomics) software to obtain scatter plots. When the genotype appeared in the X-axis region of the scatter plot, it indicated that the wheat was a FAM genotype variety; when the genotype appeared in the Y-axis region, it indicated that the wheat was a HEX genotype variety; and varieties falling between the two regions were heterozygous. Figure 4 This step takes approximately 5 minutes.

[0063] In summary, the KASP molecular marker method takes approximately 3 hours to identify a single sample. The total time does not increase exponentially when testing a large number of samples. For example, testing 384 samples takes approximately 7 hours.

[0064] Traditional fluorescence in situ hybridization (FISH) identification methods utilize different fluorescent probes to reflect chromosome characteristics from different sources. The main steps include preparing the hybridization solution, slide preparation and denaturation, hybridization, washing, staining, and microscopic examination. The specific experimental steps are as follows:

[0065] (1) Preparation of hybridization solution (1 slide):

[0066] dFA 7.5μl 20×SSC 1.5μl 50% Dextran Sulfate 1μl Salinon sperm DNA 0.5μl gDNA Probe 1.5μl Oligo DNA (1 μg / μl) 2μl <![CDATA[TTC (10) ]]> 1μl Total volume 15μl

[0067] Note: Add TTC (10) To avoid GAA (10) Signal pollution.

[0068] After mixing and centrifuging, the hybridization solution was placed in a 105℃ heating block for denaturation for 13 minutes, and then placed at -20℃ for freezing for more than 10 minutes.

[0069] (2) Slide denaturation: After dehydration, the slides are denatured in a 70% alcohol solution of 0.15 mol / L NaOH at 37°C for 4-5 min, and then dehydrated in 70%, 70%, and 100% alcohol for 3 min, 3 min, and 5 min respectively, and then air-dried.

[0070] (3) Hybridization: Add 15 μl of hybridization solution treated with ice bath to each slide, cover with a coverslip, and place in a humidified box for hybridization at 37°C in the dark for more than 6 hours.

[0071] (4) Washing the slides after hybridization (42℃ water bath):

[0072] 2×SSC 5min 2 times

[0073] ddH2O 2min 1 time

[0074] Drain the film.

[0075] (5) Staining and microscopic examination

[0076] Add 7 μl of H1200 (VECTA) anti-fluorescence quencher containing DAPI, and cover with a 24 mm × 24 mm coverslip. Observe under an Olympus BX60 fluorescence microscope and capture images using a SPOT CCD (SPOT Cooled Color Digital, DP72, Olympus, Japan).

[0077] In summary, the fluorescence in situ hybridization method takes approximately 7.5 hours to identify a single sample, which is more than twice that of the KASP molecular detection method. This method is still usable when the amount of sample to be tested is small; however, when the amount of sample to be tested is large, the time required will increase significantly because microscopic examination can only be performed on a single sample at a time.

[0078] The above comparison shows that the KASP molecular marker method requires half the time of traditional fluorescence in situ hybridization to detect a single sample, and is more efficient and has a more obvious advantage when detecting large-scale samples.

[0079] 2. Comparison of the accuracy of KASP molecular marker detection and fluorescence in situ hybridization detection.

[0080] Preliminary cytological identification results showed that among the 57 lines of the dwarf-resistant 58 / NAU2V-8 BC2F4 population, 11 lines had normal wheat chromosomes, lacking the long arm of the tufted wheat 2V#4L chromosome; 40 lines had both long arms of the 2DL chromosome replaced by 2V#4L; the remaining 6 lines were heterozygous, meaning one 2DL chromosome arm was replaced by the tufted wheat 2V#4L chromosome arm, while the other had a normal wheat 2DL chromosome long arm. The identification results of the parental NAU2V-8 are shown below. Figure 5 KASP detection results showed that the genotypes obtained using the KASP_XM000010_C1161A marker were completely consistent with the cytological identification results, meaning that samples with the FAM genotype were samples containing normal wheat chromosomes. Figure 4 The sample is designated as 2DL / 2DL, and the HEX genotype sample is the sample carrying the long arm of chromosome 2V#4L of Triticum aestivum. Figure 4 The sample, denoted as 2V#4L / 2V#4L, that detected both FAM and HEX signals is a heterozygous sample containing both the 2DL chromosome arm and the 2V#4L chromosome arm. Figure 4 (represented as 2DL / 2V#4L in Chinese).

[0081] 3. Application of KASP molecular marker detection in screening wheat resistant to sheath blight carrying the long arm of chromosome 2V#4L.

[0082] To evaluate the effect of the long arm of chromosome 2V#4L on resistance to sheath blight, resistance to sheath blight was identified in 57 lines from the aforementioned dwarf resistant 58 / NAU2V-8BC2F4 population. The toothpick embedding method was used for sheath blight inoculation sampling, and the specific method is as follows:

[0083] Soak wooden toothpicks in distilled water for 24 hours, arrange them neatly in an aluminum box, and sterilize. Pour sterilized, non-solidified PDA culture medium up to one-third the height of the toothpicks, cool, and inoculate with *Rhizoctonia solani* R0301. Incubate at 25℃ until the mycelium completely covers the area around the toothpicks before field planting. When the basal internodes are fixed at the wheat jointing stage, select leaf sheaths close to the ground, gently insert toothpicks between the leaf sheath and the stem, and keep moist until the disease is fully developed. At the milk stage, investigate the incidence of *Rhizoctonia solani* on the inoculated stems according to a 0-5 grade system.

[0084] Grade 0: The entire plant is disease-free;

[0085] Grade 1: The leaf sheaths have typical sheath blight lesions, but the stem is not affected;

[0086] Level 2: The pathogen invades the stem, and the width of the lesion does not exceed 1 / 4 of the stem circumference;

[0087] Level 3: The pathogen invades the stem, and the width of the lesions on the stem is between 1 / 4 and 1 / 2 of the stem circumference;

[0088] Level 4: The pathogen invades the stem, and the lesions on the stem cover 1 / 2 to 3 / 4 of the stem;

[0089] Level 5: Pathogens invade the stem, and lesions cover more than 3 / 4 of the stem, or soft rot of the stem, withered ears, or withered white ears appear.

[0090] The disease index (DI) was used to evaluate the sheath blight resistance of each strain. The disease index was calculated using the following formula:

[0091]

[0092] In the formula, i represents a certain disease level, and x i The number of stems for this disease grade is denoted by N, and N is the total number of stems surveyed. At least 30 stems should be surveyed for each material.

[0093] The disease resistance levels corresponding to the disease severity index are shown in Table 3.

[0094] Table 3. Correspondence between the disease index and disease resistance level of sheath blight

[0095] 0.00% ≤ DI ≤ 20.00% High resistance (HR) 20.00% < DI ≤ 40.00% Anti(R) 40.00% < DI ≤ 60.00% Mid-Range Antibody (MR) 60.00% < DI ≤ 80.00% Feeling (S) 80.00% < DI ≤ 100.00% High Sensitivity (HS)

[0096] The results of the sheath blight resistance identification showed that the disease index of lines without the 2V#4L chromosome arm of *Triticum aestivum* was 75.60% on average, and the disease resistance level was susceptible (S). In contrast, the resistance of lines containing this chromosome arm was significantly enhanced, with a disease index of 47.96% on average, and the disease resistance level was moderately resistant (MR) (Table 4). Figure 6 ).

[0097] Table 4. Effects of the long arm of chromosome 2V#4L on the resistance of the dwarf resistant 58 / NAU2V-8BC2F4 population to sheath blight.

[0098] Contains 2V#4L 47.96 Mid-Range Antibody (MR) Excluding 2V#4L 75.60 Sickness (S)

[0099] This result indicates that the KASP_XM000010_C1161A marker can be used to detect the wheat-Triticum aestivum T2DS·2V#4L translocation line. Since materials containing the long arm of chromosome 2V#4L carry the sheath blight resistance gene, rapid screening of sheath blight resistant materials was achieved. This result also suggests that the KASP_XM000010_C1161A marker can be applied to the hybridization and transfer of superior Triticum aestivum genes into wheat varieties.

Claims

1. Molecular marker primers for specific identification of the wheat-Triticum aestivum T2DS·2V#4L translocation line, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO:1, and its nucleotide at position 1161 is C / A; the primer sequence is as follows: KASP_XM000010_C1161A Pre-primer 1: FAM-5'-gtcgtcCGtgtcgtgttctA-3'; KASP_XM000010_C1161A Pre-primer 2: HEX-5'-gtcgtcGGTtgtcgtgttctC-3'; KASP_XM000010_C1161A Post-primer: 5'-cAGaagcccacatgcagcaa-3'.

2. The molecular marker primers for specific identification of the wheat tufted wheat T2DS·2V#4L translocation line according to claim 1, characterized in that, The molecular marker is the tufted wheat gene located at SEQ ID NO:

1. XM000010 KASP marker for nucleotide 1161 of the sequence.

3. The application of the molecular marker primers as described in claim 1 in the identification or screening of wheat-flesh blight resistant wheat T2DS·2V#4L translocation line materials.

4. A method for identifying the wheat-Triticum aestivum T2DS·2V#4L translocation line, characterized in that, Wheat leaf DNA was amplified by PCR using the primers described in claim 1, and selected... KASP_XM000010_C1161A The marker detection result indicates that the variety carries the long arm of chromosome 2V#4L of *Triticum aestivum*.

5. The method for identifying the wheat-Triticum aestivum T2DS·2V#4L translocation line according to claim 4, characterized in that, The amplified products were scanned for fluorescence signals, and the scanned data were analyzed using Kluster Caller software to obtain a scatter plot. When the genotype appeared in the X-axis region of the scatter plot, it indicated that the wheat was a variety without the long arm of the tufted wheat 2V#4L chromosome; when the genotype appeared in the Y-axis region, it indicated that the wheat was a variety carrying the long arm of the tufted wheat 2V#4L chromosome.

6. The method for identifying the wheat-Triticum aestivum T2DS·2V#4L translocation line according to claim 5, characterized in that, The reaction system for PCR amplification is as follows: Template 2.5 µl, KASP assay mix 0.07 µl, 2×KASP master mix 2.5 µl; the KASPassay mix includes: 12 µl of each allele-specific front primer, 30 µl of the back primer, and 46 µl of Tris-HCl.

7. The method for identifying the wheat-Triticum aestivum T2DS·2V#4L translocation line according to claim 4, characterized in that, The PCR reaction cycle program is as follows: pre-denaturation at 94℃ for 15 min, then 10 cycles of 94℃ for 20 s and 65-57℃ for 60 s; finally, 32 cycles of amplification at 94℃ for 20 s and 57℃ for 60 s.

8. A detection kit for identifying the wheat-Triticum aestivum T2DS·2V#4L translocation line, characterized in that, The detection kit contains the primers as described in claim 1.

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