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

Abstract

The invention discloses a KASP primer of a 2D chromosome short arm of aegilops tauschii and application of the KASP primer, and belongs to the technical field of wheat molecular breeding. The KASP primer for the short arm of the 2D chromosome of the aegilops tauschii comprises five groups of primers and can be used for identifying fragments derived from the short arm of the 2D chromosome of the aegilops tauschii. The identification method comprises the following steps: (1) extracting wheat genome DNA; (2) respectively mixing the five groups of primers with wheat genome DNA and a KASP reaction solution, carrying out KASP amplification, and reading a fluorescence signal; and (3) judging whether the detected wheat genome contains a fragment derived from the short arm of the 2D chromosome of the aegilops tauschii or not according to the fluorescence signal. The breeding material with high photosynthetic efficiency and high yield potential in a weak illumination region can be rapidly screened through the KASP primer.

Description

Technical Field

[0001] The invention belongs to the technical field of wheat molecular breeding, and particularly relates to a KASP primer of the short arm of Aegilops tauschii 2D chromosome and an application thereof. Background Art

[0002] Tetraploid durum wheat (Triticum turgidum ssp. Durum, chromosome number 2N=4x=28, genome AABB) is widely distributed in Europe and the United States, primarily used for pasta production. Diploid wheat (Aegilopstauschii, chromosome number 2N=2x=14, genome DD), primarily divided into two types, Tauschii and Strangulata, contains many superior genes lacking in durum wheat, such as high-quality, disease resistance, and stress tolerance. Furthermore, only a small fraction of Aegilopstauschii participates in the allopolyploidization that forms hexaploid common wheat. Therefore, introducing superior Aegilopstauschii genes into durum or common wheat is an important genetic improvement method. The existing technology uses artificial wheat (chromosome number 2N=6x=42, genome AABBDD) obtained by hybridizing waxy durum wheat LM with Strangulata type non-waxy Aegilops AT23, and then hybridizing it with LM to obtain an F1 pentaploid hybrid (chromosome number 2N=5x=35). After continuous self-pollination to the F7 generation, the approximately 13.8Mb fragment of the short arm of the 2D chromosome of Aegilops AT23 was successfully used to replace the approximately 16.1Mb fragment of the short arm of the 2A chromosome of durum wheat LM, creating the LM 2DS-2AS chromosome small fragment translocation line (chromosome number 2N=4x=28), which is characterized by the absence of wax on the epidermis of stems, leaves, ears, etc.

[0003] Wax is a general term for the lipid components of the plant cuticle. Forming frost-like crystals that cover the plant cuticle, wax plays a vital role in various physiological functions and developmental processes. For example, it limits transpiration and reduces non-stomatal water loss, a crucial mechanism for plant survival in water-scarce environments. It also protects plants from UV damage and pests and diseases. Therefore, cuticular wax is considered a key adaptive evolutionary trait in terrestrial plants. Previous studies on wheat have shown that wax can reduce transpiration and improve water use efficiency. Wax-free bread wheat can reduce solar reflection and enhance photosynthesis in low-light production areas, facilitating photosynthate synthesis during the grain-filling period and significantly increasing yield.

[0004] The waxy epidermal trait of wheat is primarily controlled by two sets of dominant genes: the wax formation gene Wax 1 (W1) and the wax inhibitor gene Iw1, located on the 2BS, and the W2 and Iw2 genes on the 2DS. A single Iw allele in the genome can suppress the function of either W1 or W2, resulting in a non-waxy phenotype. The Iw3 and Ws loci, which regulate the waxy ear phenotype, are located on the 1BS and 1AS, respectively.

[0005] The LM 2DS-2AS small chromosome translocation line, in which the exogenous 2DS fragment carries the wax suppressor gene Iw2, exhibits a wax-free phenotype. This mutant is crucial for developing breeding materials with high photosynthetic efficiency and high yield potential for low-light environments. Summary of the Invention

[0006] In response to the above-mentioned prior art, the present invention provides KASP primers for the short arm of Aegilops tauschii 2D chromosome and applications thereof, which can be used to screen and cultivate breeding materials with high photosynthetic efficiency and high yield potential in low-light areas.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a KASP primer for the short arm of the 2D chromosome of Aegilops tauschii, including 5 sets of primers, the sequence information of which 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.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, the 5' end of the forward primer is connected to a fluorescent marker, and the two forward primers in the same group are respectively connected to fluorescent markers of different colors.

[0010] Furthermore, the KASP primers for the short arm of Aegilops tauschii 2D chromosome are used to identify fragments derived from the short arm of Aegilops tauschii 2D chromosome.

[0011] Furthermore, breeding materials containing a fragment derived from the short arm of Aegilops tauschii 2D chromosome and exhibiting a wax-free wheat epidermis trait were identified.

[0012] Furthermore, the method for identifying the fragment derived from the short arm of Aegilops tauschii chromosome 2D comprises the following steps: (1) Extraction of wheat genomic DNA; (2) mixing the five sets of primers with the wheat genomic DNA and the KASP reaction solution, performing KASP amplification, and reading the fluorescence signal; (3) Determine whether the detected wheat genome contains a fragment derived from the short arm of Aegilops tauschii 2D chromosome based on the fluorescence signal.

[0013] Furthermore, the reaction system for KASP amplification was as follows: 0.3 μL of 100 μM forward primer, 0.8 μL of 100 μM reverse primer, 3.6 μL of 30 ng / μL genomic DNA and 5 μL of KASP reaction solution were added to form a total KASP reaction system.

[0014] Furthermore, the reaction conditions for KASP amplification were as follows: pre-denaturation, 95°C for 10 min; first round: 95°C for 20 s, 61°C for 1 min, 10 cycles, decreasing 0.6°C each cycle; second round: 95°C for 20 s, 55°C for 40 s, 30 cycles; termination at 4°C.

[0015] The beneficial effects of the present invention are as follows: (1) Compared with the wheat AB genome, there are fewer specific molecular markers developed based on the D genome. The specific markers developed by the present invention can not only enrich the D genome marker library, but also identify genetic materials from excellent germplasm resources, such as the Stragulata type of Aegilops tauschii. This helps to quickly screen breeding materials with high yield potential through molecular markers in the hybrid offspring prepared by using Aegilops tauschii or the wax-free LM 2DS-2AS chromosome small segment translocation line and durum wheat; and helps to quickly identify breeding materials containing the Aegilops tauschii AT23 2D chromosome short arm segment (carrying excellent genes that have not yet been introduced into common wheat) in the hybrid offspring prepared by using Aegilops tauschii and common wheat through molecular markers. (2) The molecular marker-assisted screening of wax-free breeding intermediate materials not only saves production costs and improves selection efficiency, but also shortens the breeding cycle of high-light-efficiency wheat varieties in low-sunlight areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Develop technical solutions for KASP labeling; Figure 2 The five marker typing results are shown in Figure 2. A, B, C, D, and E correspond to the results of KASP-2D-115108, KASP-2D-1051718, KASP-2D-1253779, KASP-2D-1253802, and KASP-2D-7469401, respectively. The blue dots in A, B, C, and E represent the typing results of the wax-free LM-AT23 2DS-2AS chromosome small segment translocation line and Aegilops tauschii AT23, the red dots represent the typing results of 25 hexaploid common wheat varieties, the red cross represents the absence of amplification in 3 tetraploid durum wheat varieties, and the black square represents the absence of amplification in 2 negative controls. The blue dots in D represent the typing results of 25 hexaploid common wheat varieties, the red dots represent the absence of amplification in LM-AT23, and the red dots represent the absence of amplification in LM-AT23. Typing results of the 2DS-2AS chromosome small segment translocation line and Aegilops tauschii AT23. The red cross represents the absence of amplification in three tetraploid durum wheat samples, and the black square represents the absence of amplification in two negative controls. DETAILED DESCRIPTION

[0017] The specific implementation methods of the present invention are described in detail below with reference to the embodiments.

[0018] Example 1 The development of five pairs of KASP markers, technical solutions such as Figure 1 As shown: (1) The genome resequencing results of the wax-free LM-AT23 2DS-2AS chromosome small segment translocation line were compared with the durum wheat LM and Aegilops tauschii AT23 reference genomes to screen for SNPs in the translocation fragment of the short arm of chromosome 2D and develop KASP markers.

[0019] (2) According to the above scheme, five pairs of molecular markers were developed based on the LM-AT23 2DS-2AS chromosome translocation fragment of the short arm of the 2D chromosome of Aegilops tauschii AT23. They were named: KASP-2D-115108, KASP-2D-1051718, KASP-2D-1253779, KASP-2D-1253802 and KASP-2D-7469401, corresponding to the 2DS physical positions of 115108 kb, 1051718 kb, 1253779 kb, 1253802 kb and 7469401 kb, respectively. The KASP-labeled primer set has a fluorescent probe labeled group at the 5' end of the forward primer-FAM sequence: 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO. 16). The forward primer-HEX sequence has a fluorescent probe labeled group at the 5' end: 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO. 17). Primer set information is shown in Table 1. These primers were synthesized by Shanghai Yingjie Biotechnology Co., Ltd.

[0020] Table 1 KASP tag information

[0021] Example 2 The developed KASP markers were used to genotype the wax-free LM-AT23 2DS-2AS small chromosome segment translocation line, Aegilops tauschii AT23, three tetraploid wheat varieties including LM (LM, CC20, and YP), and 25 hexaploid wheat varieties including Chuanmai 42 (China Spring, 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, Chuannong 19, Chuanyu 20, Chuanfu 8, Chuanfu 24, Mianmai 51, Mianmai 112, Mianmai 228, Mianmai 285, Mianmai 367, Zhongkemai 138, and Zhongkemai 1816) to verify the specificity of the KASP markers.

[0022] (1) Collect wheat leaf samples to be identified and extract genomic DNA; (2) Using the DNA extracted in step (1) as a template, PCR amplification was performed in a LightCycle 96 PCR instrument using the above-designed KASP-labeled primers KASP-2D-115108, KASP-2D-1051718, KASP-2D-1253779, KASP-2D-1253802, and KASP-2D-7469401, using the reaction system in Table 2 and the reaction conditions in Table 3; Table 2 PCR reaction system

[0023] Table 3 PCR amplification conditions

[0024] (3) Cluster typing of amplification products: Using a fluorescence scanner and data analysis software (Chengdu Hanchen Guangyi Technology Co., Ltd.), the 5 KASP marker PCR amplification products of 30 wheat materials were scanned and analyzed. The genotyping results are as follows: Figure 2 The results showed that all five pairs of KASP marker primers could identify the genotypes of the 2D chromosome short arm fragment from Aegilops tauschii AT23 and the 2D chromosome of other common wheat, and durum wheat materials without a D genome were not amplified.

[0025] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A KASP primer for the short arm of Aegilops tauschii chromosome 2D, characterized in that: Includes 5 sets of primers, 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.

2. The KASP primer for the short arm of Aegilops tauschii chromosome 2D according to claim 1, characterized in that: The 5' end of the forward primer is connected to a fluorescent marker, and the two forward primers in the same group are respectively connected to fluorescent markers of different colors.

3. Use of the KASP primer for the short arm of Aegilops tauschii chromosome 2D according to claim 1 or 2, characterized in that: The KASP primers for the short arm of Aegilops tauschii chromosome 2D were used to identify the fragments derived from the short arm of Aegilops tauschii chromosome 2D.

4. The use according to claim 3, characterized in that: Identify breeding materials containing a fragment derived from the short arm of chromosome 2D of Aegilops tauschii and exhibiting a wax-free wheat epidermis trait.

5. The use according to claim 4, characterized in that The method for identifying a fragment derived from the short arm of Aegilops tauschii chromosome 2D comprises the following steps: (1) Extraction of wheat genomic DNA; (2) mixing the five sets of primers with the wheat genomic DNA and the KASP reaction solution, performing KASP amplification, and reading the fluorescence signal; (3) Determine whether the detected wheat genome contains a fragment derived from the short arm of Aegilops tauschii chromosome 2D based on the fluorescence signal.

6. The use according to claim 5, characterized in that The reaction system for KASP amplification was as follows: 0.3 μL of 100 μM forward primer, 0.8 μL of 100 μM reverse primer, 3.6 μL of 30 ng / μL genomic DNA and 5 μL of KASP reaction solution were added to form the total KASP reaction system.

7. The use according to claim 5, characterized in that The reaction conditions for KASP amplification were as follows: pre-denaturation, 95°C for 10 min; first round: 95°C for 20 s, 61°C for 1 min, 10 cycles, decreasing 0.6°C each cycle; second round: 95°C for 20 s, 55°C for 40 s, 30 cycles; termination at 4°C.

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