A wheat tandem translocation chromosome, molecular markers thereof and application thereof

Abstract

The present application relates to the field of crop breeding technology, and specifically discloses a wheat tandem translocation chromosome, a molecular marker thereof and application, wherein the translocation chromosome comprises a segment of 637-812 Mb at the end of the long arm of a wheat 2B chromosome replaced by the long arm of a rye 2R chromosome and the 2S chromosome of a variable goat grass v to form a 2BS.2BL-2RL-2S v L tandem translocation chromosome. The present application creates a tandem translocation chromosome containing a powdery mildew resistance gene Pm2R QL and a stripe rust resistance gene YrAev , and develops a supporting molecular marker, solves the problem that after endogenous disease resistance genes are aggregated in wheat breeding, the identification of exogenous chromosome fragments relies on time-consuming and laborious cytological methods and is difficult to be applied in high-throughput, and quickly and accurately distinguishes between 2BS.2BL-2RL-2S v L tandem translocation chromosome carriers and non-carriers, replaces traditional cytological identification, supports molecular marker-assisted breeding, and accelerates the breeding process of new varieties of multi-resistant wheat.

Description

Technical Field

[0001] This invention belongs to the field of crop breeding technology, specifically relating to a wheat tandem translocation chromosome, its molecular markers, and their applications. Background Technology

[0002] Stripe rust and powdery mildew are important fungal diseases that threaten the safe production of wheat. Breeding and applying resistant varieties is an economical and efficient technical means to control these diseases. To date, a large number of wheat genes resistant to stripe rust and powdery mildew have been discovered and identified both domestically and internationally. Some genes possess resistance to multiple diseases, including stripe rust and powdery mildew, which can effectively improve the efficiency of disease resistance breeding; however, such broad-spectrum multi-resistance gene resources are still relatively scarce. Furthermore, the resistance of disease resistance genes is easily lost with variations in the physiological races of pathogens. Therefore, continuously exploring new disease-resistant germplasm resources is an important research area that urgently needs to be advanced in the field of wheat disease resistance breeding.

[0003] Wheat-exogenous translocation is an effective approach for creating multi-resistant breeding materials, and closely related species are important donors for wheat disease resistance gene discovery. However, due to genetic differentiation among species, natural genetic recombination is difficult to occur between wheat and closely related chromosomes. Therefore, chromosome engineering techniques are needed to create wheat-exogenous translocation materials to precisely introduce superior exogenous disease resistance genes into the wheat genome. Exogenous chromosome segments introduced into the wheat background typically do not recombine with wheat chromosomes, and the target disease resistance genes and linked genes they carry exhibit a fully linked inheritance pattern, displaying single-gene locus inheritance characteristics. Based on this genetic characteristic, recombination of chromosomal segments containing exogenous disease resistance genes from different closely related species and resistant to different diseases onto the same wheat chromosome can yield multi-resistant breeding elements. Because the tandem exogenous chromosome segments carrying different disease resistance genes are tightly linked and do not recombine with wheat chromosomes, simultaneous aggregation selection of multiple genes can be achieved during breeding, significantly improving the efficiency of multi-resistant wheat breeding.

[0004] Mutable Goatgrass ( Aegilops variabilis 2n=4x=28, UUS v S v AS116 exhibits high resistance to wheat stripe rust in mature plants. Its 2S... v 2S chromosomes were introduced into common wheat susceptible to stripe rust to form 2S v / 2B、2S v / 2D replacement system and 2S v The addition series all showed high resistance to stripe rust, indicating that 2S v Chromosomes may carry genes that resist wheat stripe rust. These genes can be induced using chromosome engineering techniques to develop 2S... v -2B recombination, yielding 30 2S units. v / 2B translocation system. It contains 2S. vThe smallest translocation chromosome segment of the L chromosome (chromosome conformation 2BS.2BL-2S) v L) co-segregates with stripe rust resistance, therefore the 2S chromosome of this translocation... v The chromosome segment contains a gene for resistance to stripe rust and is named YrAev. Transcriptome sequencing of the translocation line, combined with analysis of the Aegilops TL05 reference genome, revealed the 2S chromosome of the translocation chromosome. v The L-recombination breakpoint is defined as 844.2-844.6 Mb. Therefore, [the breakpoint is defined as follows]. YrAev It is located in the range from 844.6 Mb to approximately 7.5 Mb at the end of the long arm.

[0005] Qinling Rye ( Secale cereale L., 2n=2x=14, RR) is highly resistant to powdery mildew. The wheat-rye 2R addendum line obtained by crossing Qinling rye with powdery mildew-susceptible wheat is resistant to powdery mildew throughout its entire growth period, indicating that 2R carries the powdery mildew resistance gene. It was named Wei Pm2R QL Thirty-eight primary translocation lines were obtained by inducing 2B-2R recombination using chromosome engineering techniques. Molecular marker analysis was used to identify the size of the 2R fragment in different primary translocations. Combined with the powdery mildew resistance expression of different translocation lines, the powdery mildew resistance gene of Qinling rye was identified. Pm2R QL It is located between 833 Mb and 835 Mb in the 2RL (referencing the Lo7 genome).

[0006] Yr2S v and Pm2R QL This provides new genetic resources for wheat disease resistance breeding, but all of them were introduced into the long arm of wheat chromosome 2B. This means that in breeding applications, it is impossible to obtain genes that simultaneously carry these genes. YrAev and Pm2R QL New varieties (lines) of genes. Therefore, there is an urgent need to develop a method that can aggregate exogenous disease resistance genes. YrAev and Pm2R QL To identify chromosome structural variants that no longer separate, and to develop efficient identification techniques that can be used for molecular marker-assisted selection, in order to overcome the above-mentioned defects in existing breeding techniques and accelerate the breeding process of new wheat varieties that are resistant to both stripe rust and powdery mildew. Summary of the Invention

[0007] To address the aforementioned problems, the purpose of this invention is to provide a wheat tandem translocation chromosome, its molecular markers, and their applications, by creating a chromosome containing... YrAev and Pm2R QLThe study aims to identify tandem translocation chromosomes and develop corresponding molecular markers to address the current problems in wheat breeding, such as the lack of genes that resist multiple diseases and the reliance on time-consuming and laborious cytological methods for identifying exogenous multi-gene polymers, which are difficult to apply at high throughput. This will enable the rapid and accurate differentiation of translocation homozygotes, heterozygotes, and non-carriers, replacing traditional cytological identification, supporting molecular marker-assisted breeding, and accelerating the breeding process of new wheat varieties that resist multiple diseases.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: a wheat tandem translocation chromosome, comprising 2BS.2BL-2RL-2S v L-tandem translocation chromosome, namely 2BS.2BL-2RL-2S v The L-tandem translocation chromosome is formed by the long arm of the 2R chromosome of rye and the 2S chromosome of Aegilops variegata. v It is formed by replacing a segment of the long arm of the wheat 2B chromosome from 637 to 812 Mb at the end of the long arm.

[0009] Furthermore, the 2R chromosome segment contains a gene for resistance to powdery mildew. Pm2R QL The 2S v Chromosomal segments containing stripe rust resistance genes YrAev .

[0010] Furthermore, the 2BS.2BL-2RL-2S v The location of L-tandem translocation chromosome segments is based on the IWGSC wheat reference genome version 2.1 and the rye Lo7 reference genome.

[0011] Furthermore, the 2BS.2BL-2RL-2S v The recombination breakpoint of chromosome 2B in the L-tandem translocation is located in the 637-812 Mb region of chromosome 2B, namely 2BS.2BL-2RL-2S. v The recombination breakpoint of chromosome 2R in L tandem translocation is located in the 637-812 Mb region of chromosome 2R.

[0012] Furthermore, the 2BS.2BL-2RL-2S v L-tandem translocation chromosomes are obtained by utilizing... ph1b Gene-induced carrying of powdery mildew resistance gene Pm2R QL The 2RL chromosome segment of the 2BS.2BL-2RL translocation chromosome is associated with a gene carrying resistance to stripe rust. YrAev 2BS.2BL-2S v L translocation chromosome 2S v Obtained by partial homologous recombination of L chromosome segments.

[0013] This invention also provides a molecular marker for wheat tandem translocation chromosomes. This includes a specific molecular marker 2R-9 and a 2S in the 2R chromosome segment of a tandem translocation. v The specific molecular marker 2S-1 for chromosome segments, with the following primer sequences: 2R-9 forward primer 2R-1-F: 5'-CCTCGTTCCGTTAGCTCTGG-3'; 2R-9 reverse primer 2R-1-R: 5'-CGCATGAAGAGTGTGTGTGG-3'; 2S-1 forward primer 2S-1-F: 5'-CCCCTCAACCTCAACCTGTC-3'; 2S-1 reverse primer 2S-1-R: 5'-ACCTCCATTCATGTCGGCTG-3'.

[0014] This invention also provides an application of wheat tandem translocation chromosomes in wheat breeding for the selection of new wheat varieties that are resistant to both powdery mildew and stripe rust.

[0015] The present invention also provides the application of molecular markers of wheat tandem translocation chromosomes in wheat breeding, which can be used to assist in the selection of breeding materials carrying the translocation chromosomes.

[0016] Furthermore, the genomic DNA of the material to be tested is amplified by PCR using the molecular markers: if the 2R-9 marker can amplify a 717bp band or the 2S-1 marker can amplify a 249bp band, the material is determined to carry the tandem translocation chromosome; if neither molecular marker can amplify a band of the target size, the material is determined not to carry the tandem translocation chromosome.

[0017] Furthermore, the PCR amplification reaction program is as follows: pre-denaturation at 94℃ for 5 minutes; amplification for 35 cycles, wherein each cycle consists of: denaturation at 94℃ for 30 seconds, annealing at 57℃ for 30 seconds, extension at 72℃ for 30 seconds; and final extension at 72℃ for 10 minutes.

[0018] Compared with the prior art, the present invention can achieve the following technical effects: 1. This molecular marker is based on PCR technology to target 2BS.2BL-2RL-2S v The detection of L-tandem translocation chromosomes is simple to operate and has a rapid reaction. It does not rely on complex cytological techniques such as fluorescence in situ hybridization, thus eliminating the dependence on high-end experimental equipment and professional operating skills. It can quickly complete the screening of large-scale breeding populations, significantly reducing identification time and labor costs, and solving the bottleneck of low efficiency in traditional translocation line identification.

[0019] 2. 2R-9 targets the 2R segment of tandem translocation chromosomes, and 2S-1 targets the 2S segment of tandem translocation chromosomes. v At the ends of chromosome fragments, the two pairs of labeled primers can only amplify bands when the material carries the specific translocation chromosome. The band sizes are 717bp and 249bp, respectively. Non-carrying materials do not produce this product, which can effectively avoid false positive and false negative results.

[0020] 3. This marker can be used to track 2BS.2BL-2RL-2S throughout all generations of wheat breeding. v The transmission and segregation of L-tandem translocation chromosomes enable the targeting of chromosomes carrying powdery mildew resistance genes. Pm2R QL and stripe rust resistance gene YrAev Targeted screening avoids the loss of desirable traits, significantly improves breeding selection efficiency, and accelerates the breeding process of new wheat varieties that are both resistant to disease and disease.

[0021] 4. Matching 2BS.2BL-2RL-2S v The L-tandem translocation chromosome itself carries a gene for resistance to powdery mildew. Pm2R QL and stripe rust resistance gene YrAev This molecular marker provides a key tool for its rapid introduction and stable inheritance in breeding, promoting the transformation of this novel germplasm resource resistant to two diseases into practical breeding results, and providing core technical support for the cultivation of wheat varieties resistant to both diseases. Attached Figure Description

[0022] Figure 1 This invention is 2BS.2BL-2RL-2S v Fluorescence in situ hybridization diagram of L-tandem translocation chromosomes.

[0023] Figure 2 This invention contains 2BS.2BL-2RL-2S v Seedling powdery mildew resistance phenotype of L-tandem translocation chromosome lines.

[0024] Figure 3 This invention is 2BS.2BL-2RL-2S v Identification of breakpoints in L-tandem translocation chromosomes.

[0025] Figure 4 The present invention is based on the needle 2BS.2BL-2RL-2S. v Amplification bands of specific PCR markers 2R-9 and 2S-1 developed from L-tandem translocation chromosomes in parental DNA.

[0026] Figure 5 The present invention is based on the needle 2BS.2BL-2RL-2S. vSpecific PCR markers 2R-9 and 2S-1, developed from L-tandem translocation chromosomes, were used in chromosomes containing a single 2BS.2BL-2RL-2S chromosome. v Amplified banding results in the self-crossed progeny population of L translocation chromosomes.

[0027] Figure 6 For the present invention, a 2BS.2BL-2RL-2S is included. v Cytological identification results of single plants that amplify the target band in the self-crossed progeny population of L translocation chromosome.

[0028] Figure 7 This invention is aimed at 2BS.2BL-2RL-2S v Specific PCR markers 2R-9 and 2S-1, developed from L-tandem translocation chromosomes, were used in chromosomes containing a single 2BS.2BL-2RL-2S chromosome. v The amplified banding results of a single plant with L translocation chromosome in the F1 population of a cross between a common wheat variety, Shumai 2548 and a single plant.

[0029] Figure 8 This invention is aimed at 2BS.2BL-2RL-2S v Specific PCR markers 2R-9 and 2S-1, developed from L-tandem translocation chromosomes, were used in chromosomes containing a single 2BS.2BL-2RL-2S chromosome. v Cytological identification results of the target band amplified in the F1 population of a single plant with L translocation chromosome and a common wheat variety, Shumai 2548. Detailed Implementation

[0030] The following will describe the implementation of the present invention in detail with reference to the embodiments, so as to fully understand how the present invention uses technical means to solve technical problems and achieve technical effects and to implement it accordingly.

[0031] The specific implementation method is described below with reference to the accompanying drawings.

[0032] Example 1 This embodiment provides a wheat tandem translocation chromosome, including 2BS.2BL-2RL-2S v L-tandem translocation chromosome, namely 2BS.2BL-2RL-2S v The L-tandem translocation chromosome is formed by the long arm of the 2R chromosome of rye and the 2S chromosome of Aegilops variegata. v It is formed by replacing a segment of the long arm of the wheat 2B chromosome from 637 to 812 Mb at the end of the long arm.

[0033] The principle of the basic scheme is: based on the 2BS.2BL-2RL-2S created in this invention. vThe specific structural features of the L-tandem translocation chromosome (the chromosome in which a segment of the long arm of wheat chromosome 2B, 637-812 Mb, is replaced by the long arm of rye chromosome 2R and the 2S chromosome of Aegilops variegata). v (Formed at the long arm of the chromosome). This tandem translocation chromosome 2RL and 2S... v The L segment is completely linked, therefore only one chromosomal segment needs to be tested. Specific molecular markers for 2RL are designed to target the 2RL chromosomal segment and the 2S segment. v L-specific molecular markers target 2S v L chromosome ends. When wheat material carries this specific tandem translocation chromosome, the 2RL-specific molecular marker can amplify a DNA sequence fragment with a band size of 717 bp, 2S v The specific molecular marker L can amplify a DNA sequence fragment with a band size of 249 bp; however, materials without this tandem translocation chromosome cannot amplify a specific band with either marker. The use of either marker can achieve accurate identification of the tandem translocation chromosome.

[0034] Furthermore, the 2R chromosome segment contains a gene for resistance to powdery mildew. Pm2R QL The 2S v Chromosomal segments containing stripe rust resistance genes YrAev .

[0035] Furthermore, the 2BS.2BL-2RL-2S v The location of L-tandem translocation chromosome segments is based on the IWGSC wheat reference genome version 2.1 and the rye Lo7 reference genome.

[0036] Furthermore, the 2BS.2BL-2RL-2S v The recombination breakpoint of chromosome 2B in the L-tandem translocation is located in the 637-812 Mb region of chromosome 2B, namely 2BS.2BL-2RL-2S. v The recombination breakpoint of chromosome 2R in L tandem translocation is located in the 637-812 Mb region of chromosome 2R.

[0037] Furthermore, the 2BS.2BL-2RL-2S v L-tandem translocation chromosomes are obtained by utilizing... ph1b Gene-induced carrying of powdery mildew resistance gene Pm2R QL The 2RL chromosome segment of the 2BS.2BL-2RL translocation chromosome is associated with a gene carrying resistance to stripe rust. YrAev 2BS.2BL-2S v L translocation chromosome 2S v Obtained by partial homologous recombination of L chromosome segments.

[0038] This embodiment also provides a molecular marker for wheat tandem translocation chromosomes. This includes a specific molecular marker 2R-9 and a 2S in the 2R chromosome segment of a tandem translocation. v The specific molecular marker 2S-1 for chromosome segments, with the following primer sequences: 2R-9 forward primer 2R-1-F: 5'-CCTCGTTCCGTTAGCTCTGG-3'; 2R-9 reverse primer 2R-1-R: 5'-CGCATGAAGAGTGTGTGTGG-3'; 2S-1 forward primer 2S-1-F: 5'-CCCCTCAACCTCAACCTGTC-3'; 2S-1 reverse primer 2S-1-R: 5'-ACCTCCATTCATGTCGGCTG-3'.

[0039] This embodiment also provides an application of wheat tandem translocation chromosomes in wheat breeding, for the selection of new wheat varieties that are resistant to both powdery mildew and stripe rust.

[0040] This embodiment also provides the application of molecular markers of wheat tandem translocation chromosomes in wheat breeding, which can be used to assist in the selection of breeding materials carrying the translocation chromosomes.

[0041] Furthermore, the genomic DNA of the material to be tested is amplified by PCR using the molecular markers: if the 2R-9 marker can amplify a 717bp band or the 2S-1 marker can amplify a 249bp band, the material is determined to carry the tandem translocation chromosome; if neither molecular marker can amplify a band of the target size, the material is determined not to carry the tandem translocation chromosome.

[0042] Furthermore, the PCR amplification reaction program is as follows: pre-denaturation at 94℃ for 5 minutes; amplification for 35 cycles, wherein each cycle consists of: denaturation at 94℃ for 30 seconds, annealing at 57℃ for 30 seconds, extension at 72℃ for 30 seconds; and final extension at 72℃ for 10 minutes.

[0043] The experimental procedures for preparing the above translocation chromosomes and specific molecular markers are as follows: 1. Will carry the powdery mildew resistance gene Pm2R QL 2BS.2BL-2R translocation system and carrier YrAev 2BS.2BL-2S v Translocation lines were crossed and self-crossed to obtain the F2 population. Using... Ph1 / ph1b Screening using specific molecular markers Xwgc2111 and Xwgc2049 ph1bA single, genetically homozygous plant. Selected through cytological screening. ph1b The genetically homozygous material contained one 2BS.2BL-2R translocation chromosome and one 2BS.2BL-2S translocation chromosome. v Individual plants with translocated chromosomes. These individuals contain 2RL chromosome segments and 2S chromosome segments. v The L chromosome segment undergoes partial homologous chromosome pairing and recombination during meiosis to obtain 2RL-2S. v L-position screening group; 2. 2RL-2S v Translocation screening populations were selected by cytological screening for individuals containing 2BS.2BL-2RL-2S. v Single plants with L-recombinant chromosomes were further self-crossed, and their offspring were screened cytologically for 2BS.2BL-2RL-2S. v Materials homozygous for L-tandem translocation chromosomes (see) Figure 1 ); 3. Based on the rye Lo7 reference genome, a specific molecular marker 2R-9 was designed at 843.93 Mb on the long arm of chromosome 2R. The amplified DNA sequence fragment size of this marker was 717 bp. Based on the reference genome of Aegilops TL05, a specific molecular marker 2R-9 was designed at 843.93 Mb on the long arm of chromosome 2R. v A specific marker, 2S-1, was designed at the 852.1 Mb region of the long arm of the chromosome. The amplified DNA sequence fragment from this marker was 249 bp. 2R-9 and 2S-1 were used to target 2BS.2BL-2RL-2S... v The offspring of crosses between L-tandem translocation chromosomes and common wheat were identified, and chromosomes containing 2BS.2BL-2RL-2S were screened. v Materials from L-tandem translocation chromosomes. Further cytological identification clarified the identification results of 2R-9 and 2S-1.

[0044] The PCR reaction system and procedure are shown in Table 1 and Table 2.

[0045] Table 1. PCR reaction system Table 2. PCR reaction procedure Preparation experiment results: A segment of the long arm of wheat chromosome 2B, 637-812 Mb, was created and replaced with the long arm of rye chromosome 2R and the 2S chromosome of Aegilops variegata. v A novel 2BS.2BL-2RL-2S chromosome formed at the end of its long arm. v L-tandem translocation chromosome.

[0046] Figure 1 This invention is 2BS.2BL-2RL-2S vFluorescence in situ hybridization diagram of L-tandem translocation chromosomes. Figure 1 A in the middle is made using rye ( S. cereale L, 2n=2x=14, RR) and Aegilops variegata ( Ae. variabilis 2n=4x=28, UUS v S v ) probe, common wheat ( Triticum aestivum L, 2n=6x=42, AABBDD) Chinese spring was used as a barrier, and genomic in situ hybridization was performed to identify the homozygous primary translocation line 2BS.2BL-2RL and 2BS.2BL-2S. v Karyotype of F1 from the L homozygous primary translocation line. Red and green represent the genomes of rye and Aegilops variegata, respectively, and blue represents the genome of common wheat. Figure 1 B represents the fluorescent in situ hybridization karyotype of rye genomic DNA, Aegilops variegata genomic DNA, and Oligo-pSc119.2 probe. The white arrow points to 2BS.2BL-2RL-2S. v L-type tandem translocation chromosome. Fluorescence in situ hybridization (FISH) identification showed that this translocation chromosome was formed by the replacement of the long arm of a wheat genome chromosome with segments of rye and Aegilops genome chromosomes. Based on the FISH karyotype and Oligo-pSc119.2 signal characteristics, the Oligo-pSc119.2 signal characteristics of the wheat genome chromosome of this tandem translocation chromosome were consistent with those of chromosome 2B. Since the donor parents of the tandem translocation chromosome are the already identified 2BS.2BL-2RL and 2BS.2BL-2S... v L translocation lineage, therefore the newly formed tandem translocation chromosomes are composed of 2RL and 2S v It is formed by recombination and exchange at the end of the long arm of the L chromosome.

[0047] Figure 2 This invention contains 2BS.2BL-2RL-2S v Seedling powdery mildew resistance phenotype in L-tandem translocation chromosome lines. (For 2BS.2BL-2RL-2S) v L-tandem translocation chromosome and 2BS.2BL-2S v Seedling resistance to powdery mildew was assessed by segregating F2 populations of L chromosome. S represents the presence of two chromosomes 2BS.2BL-2S. v L chromosome, T indicates the presence of two 2BS.2BL-2RL-2S chromosomes. v L-linked translocation chromosome, H indicates the presence of one 2BS.2BL-2S chromosome. v L chromosome and one 2BS.2BL-2RL-2S chromosome v L-linked translocation chromosome. H and T materials showed resistance to powdery mildew, while S material was susceptible, indicating the newly formed 2BS.2BL-2RL-2S chromosome.v L-tandem translocation chromosomes include Pm2R QL It has resistance to powdery mildew.

[0048] Figure 3 This invention is 2BS.2BL-2RL-2S v Identification of breakpoints in L-tandem translocation chromosomes. Using the breakpoint located at 2BS.2BL-2S... v L chromosome 2S v Markers 2S-HM1740-left-1 and 2S-HM1740-left-2, located to the left of the chromosome breakpoint, were used to amplify E20-29 and its progeny. Both markers amplified corresponding bands, indicating that E20-29 carries a tandem translocation chromosome 2S... v Chromosomal fragments larger than YrAev The localization region extends from 844.6 Mb to the long arm (refer to the TL05 genome), therefore it contains... YrAev Gene.

[0049] Figure 4 The present invention is based on the needle 2BS.2BL-2RL-2S. v Amplification bands of specific PCR markers 2R-9 and 2S-1 developed from L-tandem translocation chromosomes in parental DNA.

[0050] Experimental Example 1 The basics are as follows: Figure 5 and Figure 6 As shown: For 2BS.2BL-2RL-2S v Experiments to validate L-tandem translocation chromosome-specific molecular markers in self-crossing segregating populations of translocation lines.

[0051] The experimental procedure is as follows: 1. Molecular cytological identification yielded a chromosome containing one 2BS.2BL-2RL chromosome and one 2BS.2BL-2RL-2S chromosome from the donor parent. v F2 was obtained by self-crossing heterozygous single plants with L-tandem translocation chromosomes; 2. Utilize 2BS.2BL-2RL-2S to obtain the 4 F2s. v PCR amplification and agarose gel electrophoresis were performed using the chromosome-specific molecular markers 2R-9 and 2S-1 for L-tandem translocation. Figure 5 As shown, three of the single plants (E20-29-3, E20-29-4 and E20-29-8) amplified specific bands of 717bp and 249bp with 2R-9 and 2S-1 markers, respectively, while the remaining single plant (E20-29-5) only amplified a specific band of 717bp with 2R-9 marker. 3. Three single plants exhibiting both 2R-9 and 2S-1 target bands during electrophoretic amplification were analyzed using rye (S. cereale L, 2n=2x=14, RR) and Aegilops variegata ( Ae. variabilis 2n=4x=28, UUS v S v ) probe, common wheat ( Triticum aestivum L, 2n=6x=42, AABBDD) Chinese spring was used as the blocking agent, and Oligo-pSc119.2 was used as the probe for fluorescence in situ hybridization. The results showed that E20-29-4 and E20-29-8 contained a 2BS.2BL-2RL-2S line. v L-tandem translocation chromosome, E20-29-3 contains two 2BS.2BL-2RL-2S chromosomes. v L-tandem translocation chromosome (see also) Figure 6 (This further illustrates the design of the 2BS.2BL-2RL-2S invention.) v L-tandem translocation chromosome-specific molecular marker primers provide accurate identification, with specificity and stability.

[0052] Experiment Example 2 The difference from the above experimental examples is that, for example, Figure 7 and Figure 8 As shown: Validation of 2BS.2BL-2RL-2S in different backgrounds v Experiments on the specificity and stability of L-tandem translocation chromosome tracking markers.

[0053] The experimental procedure is as follows: Step 1, Molecular cytological identification, obtained a 2BS.2BL-2RL chromosome and a 2BS.2BL-2RL-2S chromosome from the donor parent. v A heterozygous plant with L-tandem translocation chromosomes was crossed with common wheat Shumai 2548 to obtain two hybrid seeds (E20-29-1 and E20-29-7). Step 2: Use 2BS.2BL-2RL-2S for 2 seeds. v PCR amplification and agarose gel electrophoresis were performed using the chromosome-specific molecular markers 2R-9 and 2S-1 for L-tandem translocation. Figure 7 As shown, both E20-29-1 individual plants 2R-9 and 2S-1 amplified specific bands, with band sizes of 717 bp and 249 bp, respectively, indicating that they carry 2BS.2BL-2RL-2S. v L-tandem translocation chromosome; while E20-29-7 only showed a band amplified in 2R-9, indicating that it carries the 2BS.2BL-2RL chromosome from the donor parent; Step 3: For the single plant (E20-29-1) carrying the target bands of 717 bp and 249 bp for both 2R-9 and 2S-1, use rye ( S. cerealeL, 2n=2x=14, RR) genomic DNA and Aegilops variegata ( Ae. variabilis 2n=4x=28, UUS v S v Using genomic DNA as a probe, common wheat ( Triticum aestivum L, 2n=6x=42, AABBDD) Chinese spring was used as a barrier for fluorescent in situ hybridization. E20-29-1 contains 2BS.2BL-2RL-2S v L-tandem translocation chromosome (see also) Figure 8 This describes the 2BS.2BL-2RL-2S design of the present invention. v The 2R-9 and 2S-1 primers, molecular markers for L-tandem translocation chromosome breakpoints, provide accurate and stable identification.

[0054] The foregoing description illustrates and describes several preferred embodiments of the invention. However, as previously stated, it should be understood that the invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the inventive concept described herein through the foregoing teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the invention should be within the protection scope of the appended claims.

Claims

1. A wheat tandem translocation chromosome, characterized in that, Including 2BS.2BL-2RL-2S v L-tandem translocation chromosome, namely 2BS.2BL-2RL-2S v The L-tandem translocation chromosome is formed by the long arm of the 2R chromosome of rye and the 2S chromosome of Aegilops variegata. v It is formed by replacing a segment of the long arm of the wheat 2B chromosome from 637 to 812 Mb at the end of the long arm.

2. The wheat tandem translocation chromosome according to claim 1, characterized in that, The 2R chromosome segment contains a gene for resistance to powdery mildew. Pm2R QL The 2S v Chromosomal segments containing stripe rust resistance genes YrAev .

3. The wheat tandem translocation chromosome according to claim 2, characterized in that, The 2BS.2BL-2RL-2S v The location of L-tandem translocation chromosome segments is based on the IWGSC wheat reference genome version 2.1 and the rye Lo7 reference genome.

4. The wheat tandem translocation chromosome according to claim 3, characterized in that, The 2BS.2BL-2RL-2S v The recombination breakpoint of chromosome 2B in the L-tandem translocation is located in the 637-812 Mb region of chromosome 2B, namely 2BS.2BL-2RL-2S. v The recombination breakpoint of chromosome 2R in L tandem translocation is located in the 637-812 Mb region of chromosome 2R.

5. A wheat tandem translocation chromosome according to claim 4, characterized in that, The 2BS.2BL-2RL-2S v L-tandem translocation chromosomes are obtained by utilizing... ph1b Gene-induced carrying of powdery mildew resistance gene Pm2R QL The 2RL chromosome segment of the 2BS.2BL-2RL translocation chromosome is associated with a gene carrying resistance to stripe rust. YrAev 2BS.2BL-2S v L translocation chromosome 2S v Obtained by partial homologous recombination of L chromosome segments.

6. A molecular marker for wheat tandem translocation chromosomes, characterized in that, This includes a specific molecular marker 2R-9 and a 2S in the 2R chromosome segment of a tandem translocation. v The specific molecular marker 2S-1 for chromosome segments, with the following primer sequences: 2R-9 forward primer 2R-1-F: 5'-CCTCGTTCCGTTAGCTCTGG-3'; 2R-9 reverse primer 2R-1-R: 5'-CGCATGAAGAGTGTGTGTGG-3'; 2S-1 forward primer 2S-1-F: 5'-CCCCTCAACCTCAACCTGTC-3'; 2S-1 reverse primer 2S-1-R: 5'-ACCTCCATTCATGTCGGCTG-3'.

7. The application of tandem translocation chromosomes according to any one of claims 1-5 in wheat breeding, characterized in that, Breeding new wheat varieties resistant to both powdery mildew and stripe rust.

8. The application of the molecular marker in wheat breeding according to claim 6, characterized in that, Used to assist in the selection of breeding materials carrying the tandem translocation chromosomes.

9. The application of the molecular marker according to claim 8 in wheat breeding, characterized in that, The genomic DNA of the material to be tested is amplified by PCR using the molecular markers: if the 2R-9 marker can amplify a 717bp band or the 2S-1 marker can amplify a 249bp band, the material is determined to carry the tandem translocation chromosome; if neither molecular marker can amplify a band of the target size, the material is determined not to carry the tandem translocation chromosome.

10. The application of the molecular marker according to claim 9 in wheat breeding, characterized in that, The PCR amplification reaction procedure is as follows: pre-denaturation at 94℃ for 5 minutes; amplification for 35 cycles, each cycle consisting of: denaturation at 94℃ for 30 seconds, annealing at 57℃ for 30 seconds, extension at 72℃ for 30 seconds; and final extension at 72℃ for 10 minutes.

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