KASP primer for short arm of 2d chromosome of aegilops tauschii and application thereof

Abstract

The application discloses KASP primers of the short arm of 2D chromosome of Aegilops tauschii and application thereof, and belongs to the technical field of wheat molecular breeding. The KASP primers of the short arm of 2D chromosome of Aegilops tauschii comprise five groups of primers and can be used for identifying fragments derived from the short arm of 2D chromosome of Aegilops tauschii. The identification method comprises the following steps: (1) extracting wheat genomic DNA; (2) mixing the five groups of primers with the wheat genomic DNA and KASP reaction liquid respectively, and performing KASP amplification and reading fluorescence signals; and (3) judging whether the detected wheat genomic DNA contains fragments derived from the short arm of 2D chromosome of Aegilops tauschii according to the fluorescence signals. The KASP primers can be used for rapidly screening breeding materials with high photosynthetic efficiency and high yield potential in weak light areas.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of wheat molecular breeding, and particularly relates to KASP primers of the short arm of chromosome 2D of Aegilops tauschii and application thereof. BACKGROUND

[0002] Triticum turgidum ssp. Durum (chromosome number 2N=4x=28, genome AABB) is widely distributed in Europe and the United States, and is mainly used for producing pasta. Aegilops tauschii (chromosome number 2N=2x=14, genome DD) is mainly divided into Tauschii and Strangulata types, and contains many excellent genes such as high quality, disease resistance and stress tolerance that hard wheat does not have, and only a small part of Aegilops tauschii is involved in the formation of hexaploid common wheat through allopolyploidization. Therefore, it is an important genetic improvement method to introduce Aegilops tauschii excellent genes into hard wheat or common wheat. The prior art hybridizes waxy hard wheat LM with Strangulata type non-waxy Aegilops tauschii AT23 to obtain artificial wheat (chromosome number 2N=6x=42, genome AABBDD), and then hybridizes the artificial wheat with LM to obtain F1 pentaploid hybrid (chromosome number 2N=5x=35). Through continuous selfing to F7 generation, about 13.8 Mb fragment of the short arm of chromosome 2D of Aegilops tauschii AT23 is successfully used to replace about 16.1 Mb fragment of the short arm of chromosome 2A of hard wheat LM, and an LM 2DS-2AS chromosome small fragment translocation line (chromosome number 2N=4x=28) is created, which shows no wax on the surface of stems, leaves and spikes.

[0003] Wax is a general term for the lipid components on the surface of plant cuticle, which forms a crystal similar to white frost covering the surface of plant cuticle, and plays an important role in various physiological functions and development processes of plants, such as limiting transpiration of plants, reducing loss of non-stomatal water, and being an important mechanism to ensure the survival of plants in water-deficient environment, protecting plants from ultraviolet damage, resisting pests and diseases, etc. Therefore, cuticular wax is considered as one of the key adaptive evolutionary traits of terrestrial plants. Previous studies on wheat have shown that wax can reduce transpiration and improve water use efficiency. Non-waxy bread wheat can reduce solar reflection and enhance photosynthesis in weak light production areas, which is beneficial to photosynthetic product synthesis during the grain filling period and significantly improves yield.

[0004] Wheat epicuticular wax traits are mainly controlled by two sets of dominant genes, including wax formation gene Wax 1 (W1) and wax inhibitor gene (inhibitor of wax, Iw1) located on 2BS, and W2 and Iw2 on 2DS. A single Iw allele present in the genome can inhibit the function of W1 or W2 gene, resulting in a waxless phenotype. Iw3 and Ws sites that regulate the wax phenotype of the spike are located on 1BS and 1AS, respectively.

[0005] The exogenous 2DS fragment of the LM 2DS-2AS chromosome small fragment translocation line carries the wax inhibitor gene Iw2, and therefore exhibits a waxless phenotype. This mutant is crucial for creating breeding materials with high photosynthetic efficiency and high yield potential in weak light areas. SUMMARY

[0006] In view of the above prior art, the KASP primer of the short arm of the 2D chromosome of Aegilops tauschii and the application thereof provided by the present application can be used for screening and breeding breeding materials with high photosynthetic efficiency and high yield potential in weak light areas.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is to provide a KASP primer of the short arm of the 2D chromosome of Aegilops tauschii, which comprises 5 groups of primers, and the sequence information is as follows:

[0008] The forward primer KASP-2D-115108-A is shown as SEQ ID No. 1 in the sequence table;

[0009] The forward primer KASP-2D-115108-B is shown as SEQ ID No. 2 in the sequence table;

[0010] The reverse primer KASP-2D-115108-R is shown as SEQ ID No. 3 in the sequence table;

[0011] The forward primer KASP-2D-1051718-A is shown as SEQ ID No. 4 in the sequence table;

[0012] The forward primer KASP-2D-1051718-B is shown as SEQ ID No. 5 in the sequence table;

[0013] The reverse primer KASP-2D-1051718-R is shown as SEQ ID No. 6 in the sequence table;

[0014] The forward primer KASP-2D-1253779-A is shown as SEQ ID No. 7 in the sequence table;

[0015] The forward primer KASP-2D-1253779-B is shown as SEQ ID No. 8 in the sequence table;

[0016] Reverse primer KASP-2D-1253779-R is shown as SEQ ID No. 9 in the sequence listing;

[0017] Forward primer KASP-2D-1253802-A is shown as SEQ ID No. 10 in the sequence listing;

[0018] Forward primer KASP-2D-1253802-B is shown as SEQ ID No. 11 in the sequence listing;

[0019] Reverse primer KASP-2D-1253802-R is shown as SEQ ID No. 12 in the sequence listing;

[0020] Forward primer KASP-2D-7469401-A is shown as SEQ ID No. 13 in the sequence listing;

[0021] Forward primer KASP-2D-7469401-B is shown as SEQ ID No. 14 in the sequence listing;

[0022] Reverse primer KASP-2D-7469401-R is shown as SEQ ID No. 15 in the sequence listing.

[0023] On the basis of the above technical solutions, the application can be further improved as follows.

[0024] Further, the 5' end of the forward primer is connected with a fluorescent label, and the two forward primers in the same group are respectively connected with different colors of fluorescent labels.

[0025] Further, the application of the KASP primer of the short arm of the 2D chromosome of the Aegilops tauschii can identify the fragment derived from the short arm of the 2D chromosome of the Aegilops tauschii.

[0026] Further, the breeding material containing the fragment derived from the short arm of the 2D chromosome of the Aegilops tauschii and the epidermal trait of the wheat showing no waxy is identified.

[0027] Further, the method for identifying the fragment derived from the short arm of the 2D chromosome of the Aegilops tauschii comprises the following steps:

[0028] (1) Extracting wheat genomic DNA;

[0029] (2) Mixing the 5 groups of primers with the wheat genomic DNA and KASP reaction liquid respectively, and performing KASP amplification, and reading the fluorescent signal;

[0030] (3) According to the fluorescent signal, it is judged whether the detected wheat genome contains the fragment derived from the short arm of the 2D chromosome of the Aegilops tauschii.

[0031] Further, the reaction system for KASP amplification is: 0.3 μL of forward primer with a concentration of 100 μM, 0.8 μL of reverse primer with a concentration of 100 μM, 3.6 μL of genomic DNA with a concentration of 30 ng / μL and 5 μL of KASP reaction solution are added respectively to form a KASP total reaction system.

[0032] Further, the reaction conditions for KASP amplification are: pre-denaturation, 95 °C for 10 min; first round: 95 °C for 20 s, 61 °C for 1 min, 10 cycles, and each cycle is reduced by 0.6 °C; second round: 95 °C for 20 s, 55 °C for 40 s, 30 cycles; 4 °C termination.

[0033] The beneficial effects of the present application are: (1) Compared with the AB genome of wheat, the specific molecular markers developed based on the D genome are less. The specific markers developed in the present application not only enrich the D genome marker library, but also identify the genetic material derived from excellent germplasm resources, such as Stragulata type Aegilops sharonensis, which helps to quickly screen breeding materials with high yield potential in the hybrid offspring prepared by using Aegilops sharonensis or waxless LM 2DS-2AS chromosome small fragment translocation line and Durum wheat, and helps to quickly identify breeding materials containing Aegilops sharonensis AT23 2D chromosome short arm fragment (carrying excellent genes not yet introduced into common wheat) in the hybrid offspring prepared by using Aegilops sharonensis and common wheat. (2) Through molecular marker-assisted screening of waxless breeding intermediate materials, not only the production cost is saved, the selection efficiency is improved, but also the breeding cycle of high light efficiency wheat varieties in the oligo-sun area is shortened. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The KASP marker development technical solution;

[0035] Figure 2Five marker genotyping results; wherein, A, B, C, D and E respectively correspond to the results of KASP-2D-115108, KASP-2D-1051718, KASP-2D-1253779, KASP-2D-1253802 and KASP-2D-7469401; the blue dot in A, B, C, E represents the AT23 2DS-2AS chromosome small fragment translocation line, the AT23 genotyping result of Aegilops tauschii, the red dot represents the genotyping result of 25 hexaploid common wheat varieties, the red cross represents no amplification of 3 tetraploid durum wheat, and the black square dot represents no amplification of 2 negative controls; the blue dot in D represents the genotyping result of 25 hexaploid common wheat varieties, the red dot represents the AT23 genotyping result of the wax-free LM-AT23 2DS-2AS chromosome small fragment translocation line, the red cross represents no amplification of 3 tetraploid durum wheat, and the black square dot represents no amplification of 2 negative controls. DETAILED DESCRIPTION

[0036] The specific embodiments of the present application will be described in detail below with reference to the examples.

[0037] Example 1

[0038] The development of five pairs of KASP markers, the technical scheme is as shown in Figure 1

[0039] (1) The genomic resequencing results of the wax-free LM-AT23 2DS-2AS chromosome small fragment translocation line are compared with the reference genome of durum wheat LM and Aegilops tauschii, and the 2D chromosome short arm translocation fragment SNPs are screened and KASP markers are developed.

[0040] ​(2) According to the above scheme, based on the LM-AT23 2DS-2AS chromosome translocation fragment of the short arm of the AT23 2D chromosome of Aegilops tauschii, a total of 5 pairs of molecular markers are developed, which are named as KASP-2D-115108, KASP-2D-1051718, KASP-2D-1253779, KASP-2D-1253802 and KASP-2D-7469401, respectively, corresponding to the 2DS physical locations 115108 kb, 1051718 kb, 1253779 kb, 1253802 kb and 7469401 kb. The sequence of the 5' end of the forward primer-FAM sequence of the KASP marker primer group is 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO. 16), and the sequence of the 5' end of the forward primer-HEX sequence is 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO. 17). The primer group information is shown in Table 1, which is synthesized by Shanghai Yingjie Biotechnology Co., Ltd.

[0041] Table 1 KASP marker information

[0042]

[0043] Example 2

[0044] The developed KASP markers are used for genotyping of 3 tetraploid wheat species (LM, CC20, YP) of LM-AT23 2DS-2AS chromosome small fragment translocation line, Aegilops tauschii AT23, LM, and 25 hexaploid wheat species (Zhongguochun, Chuanmai 30, Chuanmai 42, Chuanmai 43, Chuanmai 56, Chuanmai 58, Chuanmai 60, Chuanmai 61, Chuanmai 64, Chuanmai 80, Chuanmai 81, Chuanmai 96, Chuanmai 98, Chuanmai 602, Chuanmai 19, Chuanmai 20, Chuanmai 8, Chuanmai 24, Mianmai 51, Mianmai 112, Mianmai 228, Mianmai 285, Mianmai 367, Zhongkemai 138, Zhongkemai 1816) of LM, to verify the specificity of the KASP markers.

[0045] (1) Collect the leaf samples of the wheat to be identified to extract genomic DNA;

[0046] (2) Using the DNA extracted in step (1) as a template, using the primers of the KASP markers KASP-2D-115108, KASP-2D-1051718, KASP-2D-1253779, KASP-2D-1253802 and KASP-2D-7469401 designed above, using the reaction system in Table 2 and the reaction conditions in Table 3, PCR amplification is carried out in a LightCycle 96 PCR instrument;

[0047] Table 2 PCR reaction system

[0048]

[0049] Table 3 PCR amplification conditions

[0050]

[0051] (3) Amplification product clustering typing: using a fluorescence scanner and data analysis software (Chengdu Hanchen Guangyi Science and Technology Co., Ltd.), the PCR amplification products of 5 KASP markers of 30 wheat materials were scanned and analyzed, and the genotyping results are shown in Table 3. Figure 2 As can be seen from the results, the 5 pairs of KASP marker primers can identify the genotypes of the short arm fragment of 2D chromosome derived from Aegilops AT23 and other common wheat 2D chromosomes, and the hard wheat material without D genome does not amplify.

[0052] Although the specific embodiments of the present application are described in detail in combination with the embodiments, it should not be understood as limiting the protection scope of the patent. Various modifications and variations made by those skilled in the art within the scope described in the claims are still within the protection scope of the patent.

Claims

1. A KASP primer for the short arm of the Aegilops tauschi i AT23 2D chromosome, characterized in that, The KASP primers include five groups, and the sequence information is as follows: The forward primer KASP-2D-115108-A is shown as SEQ ID No. 1 in the sequence listing; The forward primer KASP-2D-115108-B is shown as SEQ ID No. 2 in the sequence listing; The reverse primer KASP-2D-115108-R is shown as SEQ ID No. 3 in the sequence listing; The forward primer KASP-2D-1051718-A is shown as SEQ ID No. 4 in the sequence listing; The forward primer KASP-2D-1051718-B is shown as SEQ ID No. 5 in the sequence listing; The reverse primer KASP-2D-1051718-R is shown as SEQ ID No. 6 in the sequence listing; The forward primer KASP-2D-1253779-A is shown as SEQ ID No. 7 in the sequence listing; The forward primer KASP-2D-1253779-B is shown as SEQ ID No. 8 in the sequence listing; The reverse primer KASP-2D-1253779-R is shown as SEQ ID No. 9 in the sequence listing; The forward primer KASP-2D-1253802-A is shown as SEQ ID No. 10 in the sequence listing; The forward primer KASP-2D-1253802-B is shown as SEQ ID No. 11 in the sequence listing; The reverse primer KASP-2D-1253802-R is shown as SEQ ID No. 12 in the sequence listing; The forward primer KASP-2D-7469401-A is shown as SEQ ID No. 13 in the sequence listing; The forward primer KASP-2D-7469401-B is shown as SEQ ID No. 14 in the sequence listing; The reverse primer KASP-2D-7469401-R is shown as SEQ ID No. 15 in the sequence listing; The 5' end of the forward primer is connected with a fluorescent label, and the two forward primers in the same group are respectively connected with different colors of fluorescent labels.

2. Use of a KASP primer for the short arm of the Aegilops tauschii AT23 2D chromosome according to claim 1, characterized in that: The KASP primer of the short arm of the 2D chromosome of Aegilops tauschii AT23 is used to identify the fragment derived from the short arm of the 2D chromosome of Aegilops tauschii AT23.

3. Use according to claim 2, characterized in that: The breeding material containing the fragment derived from the short arm of the 2D chromosome of Aegilops tauschii AT23 and the epidermal trait of wheat showing no waxy is identified.

4. Use according to claim 3, characterized in that, The method for identifying the fragment derived from the short arm of the 2D chromosome of Aegilops tauschii AT23 comprises the following steps: (1) Extracting wheat genomic DNA; (2) Mixing the five groups of primers with the wheat genomic DNA and KASP reaction liquid respectively, and performing KASP amplification, and reading the fluorescence signal; (3) According to the fluorescence signal, it is judged whether the detected wheat genome contains the fragment derived from the short arm of the 2D chromosome of Aegilops tauschii AT23.

5. Use according to claim 4, characterized in that, The reaction system for KASP amplification is: 0.3 μL of forward primer with a concentration of 100 μM, 0.8 μL of reverse primer with a concentration of 100 μM, 3.6 μL of genomic DNA with a concentration of 30 ng / μL and 5 μL of KASP reaction liquid are added to form the KASP total reaction system.

6. Use according to claim 4, characterized in that, Reaction conditions for KASP amplification: pre-denaturation, 95°C for 10 min; first round: 95°C for 20 s, 61°C for 1 min, 10 cycles, each cycle decreasing by 0.6°C; second round: 95°C for 20 s, 55°C for 40 s, 30 cycles; 4°C termination.

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