A fragment of a SNP molecular marker of a thousand-grain weight trait of wheat, a CAPS molecular marker primer pair and application thereof

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a fragment containing a wheat thousand-grain weight trait SNP molecular marker, a CAPS molecular marker primer pair, and their applications. This invention provides a fragment containing a wheat thousand-grain weight trait SNP molecular marker, wherein the fragment contains the TaAOS-4B gene promoter region; the nucleotide sequence of the TaAOS-4B gene promoter region is shown in SEQ ID NO.1. This invention detects allelic variations in the wheat TaAOS-4B gene promoter region, enabling the differentiation between high and low thousand-grain weight wheat, improving the screening efficiency of thousand-grain weight wheat, and benefiting wheat germplasm breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a fragment containing a wheat thousand-grain weight trait SNP molecular marker, a CAPS molecular marker primer pair, and their applications. Background Technology

[0002] Wheat is an important food crop in my country, and increasing wheat yield plays a vital role in ensuring national food security. The number of spikes per unit area, the number of grains per spike, and the thousand-grain weight are key factors affecting wheat grain yield. Jasmonic acid (JA) and its metabolite methyl jasmonic acid (MeJA) are endogenous plant growth regulators that participate in regulating growth and development processes such as wheat grain development [Chen et al. Cloning of wheat keto-acyl thiolase 2B reveals a role of jasmonic acid in grain weight determination. Nature Communications, 2020, 11(1): 6266-6266]. Allene oxide synthase (AOS) is a key enzyme in the biosynthetic pathway of jasmonic acid.Studies have shown that the AOS gene is involved in pollen male sterility, cold resistance, disease and pest resistance, and other growth, development, and stress tolerance [Bae et al. Transgenic rice plants carrying RNA interference constructs of AOS (allene oxide synthase) genes show severe malesterility. Plant Breeding, 2010, 129(6): 647-651; Wang et al. Transcription factor SlWRKY50 enhances cold tolerance in tomato by activating the jasmonic acid signaling. Plant Physiology, 2023, 194(2): 1075-1090; Takao et al. CRISPR / Cas9-mediated disruption of ALLENE OXIDE SYNTHASE results in defective 12-oxo-phytodienoic acid accumulation and reduced defense against spider mite (Tetranychus urticae) in liverwort (Marchantia polymorpha). Plant Biotechnology (Tokyo, Japan), 2022, 39(2): 191-194; An et al. Bioinformatics, expression analysis, and functional verification of allene oxide synthase gene HvnAOS1 and HvnAOS2 in qingke. Open Life Sciences, 2024, 19(1): 20220855-20220855], However, the effect of AOS genes on grain weight has not been reported, especially in wheat. Therefore, developing molecular markers of AOS genes associated with wheat thousand-grain weight is expected to provide new theoretical basis and technical means for high-yield wheat breeding, and has important scientific value and application prospects. Summary of the Invention

[0003] The purpose of this invention is to provide a fragment containing a wheat thousand-grain weight trait SNP molecular marker, a CAPS molecular marker primer pair and their application, to detect allelic variations in the promoter region of the wheat TaAOS-4B gene, thereby improving the screening efficiency of thousand-grain weight wheat.

[0004] This invention provides a fragment containing a molecular marker for the wheat thousand-grain weight trait SNP, the fragment containing the promoter region of the TaAOS-4B gene; the nucleotide sequence of the TaAOS-4B gene promoter region is shown in SEQ ID NO.1.

[0005] As a preferred embodiment, the polymorphic site of the SNP molecular marker is the first 486th base of the sequence shown in SEQ ID NO.1, and the polymorphism is A / G.

[0006] The present invention also provides a CAPS molecular marker primer pair for identifying the fragment described in the above scheme, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.3.

[0007] The present invention also provides a kit for detecting the fragment described in the above scheme, comprising the CAPS molecular marker primer pair described in the above scheme.

[0008] This invention also provides a method for detecting superior allelic variations in the TaAOS-4B gene promoter in wheat, comprising the following steps:

[0009] The genomic DNA of the test material was amplified by PCR using the CAPS molecular marker primer pair or the kit described above to obtain the PCR amplification product.

[0010] The PCR amplification product was digested with restriction endonuclease Xho I, and the polymorphism of the digested product was detected.

[0011] When the enzyme digestion product includes two fragments of 612 bp and 26 bp, the allelic variant type is TaAOS-4B-G; when the enzyme digestion product is a 630 bp fragment, the allelic variant type is TaAOS-4B-A, with the homozygous AA genotype being the superior allelic variant genotype.

[0012] This invention also provides the application of the fragment described in the above scheme, the CAPS molecular marker primer pair, the kit described, or the method described therein in wheat breeding.

[0013] This invention also provides the application of the fragment described in the above scheme, the CAPS molecular marker primer pair, the kit described, or the method described in wheat-assisted breeding.

[0014] This invention also provides the application of the fragment described in the above scheme, the CAPS molecular marker primer pair, the kit described, or the method described in identifying wheat thousand-grain weight type.

[0015] This invention also provides the application of the fragment described in the above scheme, the CAPS molecular marker primer pair, the kit described, or the method described in screening high-grain-weight wheat.

[0016] This invention also provides a method for molecular marker-assisted breeding of high-thousand-grain-weight wheat. The method involves using the CAPS molecular marker primers or the kit described above to perform molecular markers on the target wheat, and screening for germplasm with polymorphism A at the first 486th base of the promoter region sequence of the wheat TaAOS-4B gene for subsequent screening.

[0017] Beneficial effects:

[0018] This invention provides a fragment containing a wheat 1000-grain weight SNP molecular marker, the fragment comprising the promoter region of the TaAOS-4B gene; the nucleotide sequence of the TaAOS-4B gene promoter region is shown in SEQ ID NO.1. This invention detects allelic variations in the wheat TaAOS-4B gene promoter region, enabling the differentiation between high-1000-grain-weight wheat and low-1000-grain-weight wheat, improving the screening efficiency of 1000-grain-weight wheat, and benefiting wheat germplasm breeding.

[0019] This invention uses CASP molecular marker primer pairs to detect allelic variations in the promoter region of the TaAOS-4B gene, which is related to thousand-grain weight in wheat. The identification of samples can be completed simply by DNA extraction, PCR-specific amplification, and enzymatic digestion, which facilitates the detection and screening of wheat varieties or lines with high thousand-grain weight and can greatly accelerate the breeding process of high-yielding wheat varieties. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 Figure 1 shows the amplification results of the TaAOS-4B gene promoter and its coding region; Figure A is a schematic diagram of primer design for the TaAOS-4B gene promoter and coding region; Figure B shows the partial amplification results of the TaAOS-4B gene promoter of different varieties; Figure C shows the partial amplification results of the TaAOS-4B gene coding region of different varieties; In Figures B and C, M is the marker; Lanes 1 to 9 from left to right represent Zhoumai 11, Kenong 9204, Yanzhan 4110, Yunong 202, Jinhe 9123, Mianyang 20, Zhengmai 366, Xinmai 9, and Yunong 416 respectively.

[0022] Figure 2The results of sequence alignment of the TaAOS-4B gene promoter in different varieties;

[0023] Figure 3 Sequence alignment results of the coding region of the TaAOS-4B gene in different varieties;

[0024] Figure 4 Sequence alignment results of the coding region of the TaAOS-4B gene in different varieties;

[0025] Figure 5 For TaAOS-4B gene haplotype analysis;

[0026] Figure 6 Distinguishing superior haplotypes of the high thousand-grain weight TaAOS-4B variety; where A is a schematic diagram of thousand-grain weight haplotype differentiation in 2015; and B is a schematic diagram of thousand-grain weight haplotype differentiation in 2016.

[0027] Figure 7 A schematic diagram illustrating the differentiation of different haplotype restriction sites in TaAOS-4B;

[0028] Figure 8 Electrophoresis images show the amplification of different haplotypes of TaAOS-4B and their corresponding sequences after Xho I and Sac II restriction enzyme digestion. A shows the electrophoresis image of the CAPS-486 sequence amplification of different haplotypes of the TaAOS-4B gene; B shows the electrophoresis image of the CAPS-486 sequence gel recovery product after Xho I single-enzyme digestion; C shows the electrophoresis image of the corresponding plasmid after Xho I and Sac II restriction enzyme digestion following the ligation of the CAPS-486 sequence into the 19T vector; M is the marker; lanes 1-3 from left to right represent the corresponding varieties in Hap1: Zhoumai 11, Kenong 9204, and Yanzhan 4l10; lanes 4-6 from left to right represent the corresponding varieties in Hap2: Yunong 202, Jinhe 9123, and Mianyang 20; lanes 7-9 from left to right represent the corresponding varieties in Hap3: Zhengmai 366, Xinmai 9, and Yunong 416. Detailed Implementation

[0029] This invention provides a fragment containing a molecular marker for the wheat thousand-grain weight trait SNP, the fragment containing the promoter region of the TaAOS-4B gene; the nucleotide sequence of the TaAOS-4B gene promoter region is shown in SEQ ID NO.1:

[0030] In one embodiment, the polymorphic site of the SNP molecular marker is the first 486th base of the sequence shown in SEQ ID NO.1, and the polymorphism is A / G. This invention can determine the thousand-grain weight property of wheat by identifying and genotyping the SNP molecular marker: when the polymorphic site is an A base, the thousand-grain weight trait of the wheat is high.

[0031] This invention also provides a CAPS molecular marker primer pair for identifying the fragment described in the above-described scheme, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2. and downstream primers with nucleotide sequences as shown in SEQ ID NO.3. The CAPS molecular marker primer pair described in this invention can be used to detect allelic variations in the promoter region of the TaAOS-4B gene, which is related to thousand-grain weight in wheat, and to detect and screen wheat varieties or lines with high thousand-grain weight, which can greatly accelerate the breeding process of high-yielding wheat varieties.

[0032] The present invention also provides a kit for detecting the fragment described in the above scheme, comprising the CAPS molecular marker primer pair described in the above scheme.

[0033] This invention also provides a method for detecting superior allelic variations in the TaAOS-4B gene promoter in wheat, comprising the following steps:

[0034] The genomic DNA of the test material was amplified by PCR using the CAPS molecular marker primer pair or the kit described above to obtain the PCR amplification product.

[0035] The PCR amplification product was digested with restriction endonuclease Xho I, and the polymorphism of the digested product was detected.

[0036] When the enzyme digestion product includes two fragments of 612 bp and 26 bp, the allelic variant type is TaAOS-4B-G; when the enzyme digestion product is a 630 bp fragment, the allelic variant type is TaAOS-4B-A, with the homozygous AA genotype being the superior allelic variant genotype.

[0037] This invention first extracts genomic DNA from the material to be tested. As one embodiment, the material to be tested is wheat; this invention does not have specific requirements regarding the type of wheat, and any type of wheat can be used. This invention does not have specific limitations on the method for extracting the genomic DNA from the wheat to be tested; conventional plant cell genome extraction methods in the art can be used. In the specific implementation of this invention, wheat leaves or other tissues to be tested are used as materials, and extraction is performed using the CTAB method. The CTAB method described in this invention is performed according to conventional methods.

[0038] After obtaining the genomic DNA, PCR amplification of the genomic DNA of the test material is performed using the CAPS molecular marker primer pair or the kit described above to obtain PCR amplification products. As one embodiment, the PCR amplification reaction system, in 20 μL volumes, includes: 10 μL of 2×3G Taq Master Mix for PAGE (Red Dye), 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of DNA template, and the remainder ddH2O. Using the wheat genomic DNA to be tested as a template, PCR amplification is performed using the primer pair to obtain PCR amplification products. As one embodiment, the PCR amplification reaction program is: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 61℃ annealing for 15 s, 72℃ extension for 30 s, 32 cycles; 72℃ final extension for 5 min.

[0039] As one implementation method, after the PCR amplification is completed, the PCR amplification product is stored at 4°C. This invention utilizes the primer pair to amplify the promoter fragment of the TaAOS-4B gene in different haplotypes of wheat, and the PCR amplification product obtained contains the DNA fragment containing the target SNP site.

[0040] After obtaining the PCR amplification product, the present invention digests the PCR amplification product with the restriction endonuclease Xho I. The present invention does not specifically limit the source of the restriction endonuclease Xho I, and the digestion system can be set using conventional methods in the art.

[0041] After obtaining the enzyme digestion products, this invention detects the polymorphism of the digestion products and determines the allelic variation type of the TaAOS-4B gene promoter in wheat based on the detection results. As one embodiment, the detection includes analysis using agarose gel electrophoresis. This invention detects allelic variations in the TaAOS-4B gene promoter in wheat based on the fragment size of the enzyme digestion products, and the type of allelic variation can determine the thousand-grain weight trait of wheat. When the enzyme digestion products include two fragments of 612 bp and 26 bp, the allelic variation type is TaAOS-4B-G; when the enzyme digestion products are 630 bp fragments, the allelic variation type is TaAOS-4B-A, with the homozygous AA genotype being the superior allelic variation genotype.

[0042] This invention also provides the application of the fragment described in the above scheme, the CAPS molecular marker primer pair, the kit described, or the method described therein in wheat breeding.

[0043] This invention also provides the application of the fragment described in the above scheme, the CAPS molecular marker primer pair, the kit described, or the method described in wheat-assisted breeding.

[0044] This invention also provides the application of the fragment described in the above scheme, the CAPS molecular marker primer pair, the kit described, or the method described in identifying wheat thousand-grain weight type.

[0045] This invention also provides the application of the fragment described in the above scheme, the CAPS molecular marker primer pair, the kit described, or the method described in screening high-grain-weight wheat.

[0046] This invention also provides a method for molecular marker-assisted breeding of high-thousand-grain-weight wheat. The method involves using the CAPS molecular marker primers or the kit described above to perform molecular markers on the target wheat, and screening for germplasm with polymorphism A at the first 486th base of the promoter region sequence of the wheat TaAOS-4B gene for subsequent screening.

[0047] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0048] The 2×3G Taq Master Mix for PAGE (Red Dye) used for PCR amplification in this invention was purchased from Novizan Biotechnology Co., Ltd., the T vector pMD19-T Vector for cloning was purchased from Baori Biotechnology (Beijing) Co., Ltd., Xho I and Sac II restriction endonucleases were purchased from NEB, and other reagents were domestically produced analytical grade.

[0049] Example 1: Screening for superior haplotypes of the TaAOS gene in high thousand-grain weight

[0050] (1) Primer design

[0051] Based on the TaAOS gene sequence in the wheat genome database (https: / / plants.ensembl.org / ), copies A (TraesCS4A02G061900), B (TraesCS4B02G237600), and D (TraesCS4D02G238800) were obtained. Referring to the expVIP website (https: / / www.wheat-expression.com / ), the gene expression levels during grain filling were found to be 23.08, 91.22, and 24.37, respectively. Copy 4B showed the highest expression level, and the wheat B genome exhibited higher genomic diversity compared to the A and D genomes; therefore, copy B was selected for subsequent experiments. Primers TaAOS-4B-Promoter and TaAOS-4B-CDS (Table 1) were designed for the promoter and coding regions of copy 4B. The primer distribution is as follows: Figure 1As shown in Table A, primer synthesis and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd., with 'Chinese Spring' as the comparison sequence. The number of amplified bases is shown in Table 1.

[0052] Table 1 Primer sequences used for TaAOS-4B amplification

[0053]

[0054] (2) Extraction of total DNA from wheat

[0055] Seventy-eight samples of common wheat varieties preserved in the laboratory (Laboratory 317, South Building, First Experimental Building, College of Agriculture, Henan Agricultural University) were selected, and total DNA was extracted from the wheat using the CTAB method. First, wheat leaves were flash-frozen and ground in liquid nitrogen, and 1 mL of preheated CTAB extraction buffer was added. The mixture was incubated at 65°C for 1 hour. Then, the mixture was centrifuged at 12000g for 10 minutes, and the supernatant was collected and mixed with an equal volume of chloroform:isoamyl alcohol (24:1 volume ratio). The mixture was then centrifuged again at 12000g for 10 minutes, and the supernatant was transferred to a new 1.5 mL centrifuge tube. An equal volume of pre-chilled isopropanol was added, and the mixture was incubated at -20°C for 30 minutes. The mixture was centrifuged again, the supernatant was discarded, and the sample was washed twice with 200 μL of 75% ethanol. The sample was then air-dried in a fume hood, and sterile water was added to dissolve the DNA to a concentration of 200 ng / μL.

[0056] The wheat varieties used include: Wen 9519, Yumai 58, Yunong 949, Yujiao 5, Yanzhan 4110, Zhengnong 17, Zhengzhou 3, Zhengmai 9023, Yubao 1, Lunxuan 126, Zhoumai 11, Yumai 18, Yunong 982, Wen 9629, Xinmai 18, Lunxuan 988, Kenong 9204, Zhongmai 578, Jimai 20, Wanyuanxuan 9, Shixin 828, Ningmai 16, Lankao 86, Luohan 13, Wenmai 8, Baiquan 3039, Huaimai 29, Jimai 30, Lu 98421, Jing 9428, Gaoyou 2018, Hanmai 11, Zhongfan 4, Jimai 20, Yanfu 188, Weimai 8, Luohan 6, Jinmai 47, and Mianyang. No. 20, Chuanmai 22, Yunong 202, 04zhong 36, Jinhe 9123, Bainong 207, Yunong 416, Lunxuan 66, Zhoumai 32, Luohan 2, Zhoumai 17, Yumai 18-99, Luomai 26, Yumai 70-36, Lumai 15, Xinong 979, Zhongmai 8, Xuzhou 25, Ji 5265, Cangmai 6005, Chuanong 16, Henong 822, Wanmai 50, Chuanmai 24, Kenong 199, Jimai 19, Jing 411, Zhongyu 10, Baomai 2, Xinmai 21, Yannong 21, Yangmai 16, Zhongyu 12, Yumai 18, Kexing 3302, Xinmai 26, Xiaoyan 54, Zhengmai 366, Xinmai 9, Jimai 22.

[0057] (3) Amplification and sequencing of the TaAOS-4B gene promoter and CDS region

[0058] Using TaAOS-4B-Promoter and TaAOS-4B-CDS primers, and with 78 hexaploid wheat DNA samples from step (2) as templates, the promoter and CDS sequences of TaAOS-4B were amplified using two primer pairs: TaAOS-4B-Promoter-F / R and TaAOS-4B-CDS-F / R. The PCR system is shown in Table 2, and the amplification program is shown in Table 3. The amplification results are as follows: Figure 1 As shown in B and C, B represents the agarose gel electrophoresis result of PCR amplification of the promoter region, and C represents the agarose gel electrophoresis result of PCR amplification of the CDS region.

[0059] Table 2 PCR amplification system

[0060] Components Total system (20 μL) 2×3G Taq Master Mix for PAGE(Red Dye) 10μL Forward primer F 1μL Reverse primer R 1μL DNA template 1μL <![CDATA[ddH2O]]> 7μL

[0061] Table 3 PCR amplification program

[0062] temperature time Cycle number 95℃ 5min / 95℃ 15s 30c 61℃ 15s 30c 72℃ 30s 30c 72℃ 5min /

[0063] Figure 1 Figures B and C indicate that the primer design was reasonable, the reaction conditions were optimized, and the PCR system was suitable. The target fragments amplified by the above PCR were recovered and sequenced using TaAOS-4B-Promoter-F (SEQ ID NO.4) or TaAOS-4B-CDS-F (SEQ ID NO.6).

[0064] (4) Sequence alignment and haplotype analysis of the TaAOS-4B gene promoter and CDS region

[0065] The sequencing results were aligned using DNAMAN software. The alignment results for the promoter region are shown below. Figure 2 As shown, the CDS region sequence alignment results are as follows: Figures 3-4 As shown in the figure. The results indicate that 13 SNP polymorphic variant sites exist in the promoter region (-650bp to -1bp). Haplotype identification of the promoter was performed using DnaSP 5.10 software (http: / / www.ub.edu / DnaSP), and the results are shown in the figure. Figure 5 As shown, the results revealed that these 13 SNPs are closely linked to form three haplotypes, named TaAOS-4B-Hap1, TaAOS-4B-Hap2, and TaAOSS-4B-Hap3, which will be abbreviated as Hap1, Hap2, and Hap3, respectively.

[0066] (5) Comparison of thousand-grain weight among wheat varieties with different haplotypes of the TaAOS-4B gene

[0067] In step (2), the haplotypes of 78 wheat varieties and the thousand-grain weight of hexaploid wheat over two consecutive years were statistically analyzed. After threshing, the grains of each variety were naturally dried, and 1000 seeds were randomly selected to measure their weight. The thousand-grain weight was used to represent the grain weight, and the average value was used for data analysis. The results are shown in Table 4. The average thousand-grain weight of the haplotypes was analyzed, and the results are shown in Tables 5 and 6. Figure 6 As shown.

[0068] Table 4. Thousand-grain weight and haplotype classification of 78 wheat varieties

[0069]

[0070]

[0071] Table 5. Average 1000-grain weight of TaAOS-4B haplotype

[0072] 2015 Thousand-grain weight (TKW(g)) 2016 Thousand-grain weight (TKW(g)) Hap1+Hap2 52.77±0.85a 51.15±0.85a Hap3 45.21±0.70b 45.09±0.48b

[0073] According to Tables 4-5 and Figure 6 It can be seen that the average thousand-grain weight of TaAOS-4B-Hapl+Hap2 was significantly higher than that of TaAOS-4B-Hap3 by 16.7% and 13.4% over two years, respectively, further indicating that the Hap1+Hap2 haplotype contained in the TaAOS gene is an excellent haplotype for high thousand-grain weight wheat.

[0074] Example 2: Development of CAPS molecular markers for the TaAOS-4B gene

[0075] Sequence differences between different haplotypes were analyzed using dCAPS Finder 2.0 (http: / / helix.wustl.edu / dcaps / dcaps.html). Referring to the Chinese spring gene sequence, it was found that the TaAOS-4B-promoter-486 sequence exists in both A and G forms. Figure 7 As shown in Figure A, Hap1 and Hap2 contain the following variety sequences: The sequence of Hap3 type wheat varieties is: To distinguish the above differences, a C mutation was made to A at position -487 and a G mutation was made to T at position -490 of the above sequences. A dCAPS forward primer (SEQ ID NO.2) and a dCAPS reverse primer (SEQ ID NO.3) containing the restriction endonuclease Xho I (CTCGAG) cleavage site were designed to differentiate Hap1, Hap2, and Hap3. Hap3 contains the CTCGAG sequence, which can be recognized and cleaved by the restriction endonuclease Xho I, while Hap1 and Hap2 contain the CTCGAA sequence and therefore cannot be cleaved by the restriction endonuclease Xho I. Figure 7(B) Based on the sequence difference at this location, it is believed that the dCAPS molecular marker is used to distinguish Hap3 from other haplotypes, and this marker is named dCAPS-486. This will help differentiate between different haplotypes and provide a basis for subsequent screening of high-grain-weight varieties.

[0076] To verify the practical applicability of the identified dCAPS-486 molecular marker, genomic DNA was extracted from wheat varieties containing different haplotypes. Specifically, Hap1 included varieties Zhoumai 11, Kenong 9204, and Yanzhan 4110; Hap2 included varieties Yunong 202, Jinhe 9123, and Mianyang 20; and Hap3 included varieties Zhengmai 366, Xinmai 9, and Yunong 416.

[0077] First, the promoter fragment was amplified using TaAOS-4B-Promoter primers; then, using the PCR product as a template, the dCAPS-486 molecular marker was amplified using forward and reverse primers labeled with dCAPS-486. The PCR system and reaction procedure were the same as those in Table 2. The results are as follows: Figure 8 As shown in Figure A, Hap1 includes varieties Zhoumai 11, Kenong 9204, and Yanzhan 4110 (lanes 1-3); Hap2 includes varieties Yunong 202, Jinhe 9123, and Mianyang 20 (lanes 4-6); and Hap3 includes varieties Zhengmai 366, Xinmai 9, and Yunong 416 (lanes 7-9). The TaAOS-4B fragment (-630bp to -1bp) in different haplotype wheat all yielded a single amplified band (around 630bp).

[0078] The amplified PCR products were recovered and digested with the restriction endonuclease Xho I. Electrophoresis results were obtained as follows: Figure 8 As shown in Figure B, the results showed that the transformation products of Hap1 and Hap2 haplotype wheat varieties remained as single bands of approximately 630 bp after enzyme digestion.

[0079]

[0080] The amplification product of the Hap3 haplotype material, after single enzyme digestion, split into two fragments, 612bp and 26bp, respectively. This indicates that the molecular marker dCAPS-486 can distinguish between the Hap3 and Hap1+Hap2 haplotypes and can be applied to molecular design breeding for screening high-thousand-grain-weight wheat varieties.

[0081] Because the 26bp band was relatively small, to facilitate result visualization, the amplified PCR product was recovered, ligated into the 19T vector, and transformed into the DH5α strain. After overnight culture, positive clones were picked, and plasmids were extracted. The plasmids were then digested with Xho I and Sac II enzymes, and the electrophoresis results are shown below. Figure 8 As shown in C.

[0082] The results showed that the transformation products of Hap1 and Hap2 haplotype wheat varieties remained a single band of approximately 3366 bp after enzyme digestion (containing 2736 bp of the vector and 630 bp of the fragment), while the amplification product of the Hap3 haplotype material split into two fragments after double enzyme digestion: 2988 bp (vector + fragment) and 386 bp. This indicates that the molecular marker dCAPS-486 can distinguish between Hap3 and Hap1+Hap2 haplotypes and can be applied to molecular design breeding for screening high-thousand-grain-weight wheat varieties.

[0083] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fragment containing a molecular marker for the SNP of the thousand-grain weight trait in wheat, a CAPS molecular marker primer pair, a kit, or a method for detecting SNPs in wheat. TaAOS-4B The application of the method of superior allelic variation of gene promoters is characterized by, The applications include at least one of the following: (1) wheat thousand-grain weight breeding; (2) wheat thousand-grain weight-assisted breeding; (3) identification of wheat thousand-grain weight types; (4) screening for high thousand-grain weight wheat; The fragment contains TaAOS-4B Gene promoter region; TaAOS-4B The nucleotide sequence of the gene promoter region is shown in SEQ ID NO.1; The polymorphic site of the SNP molecular marker is the -486th base of the sequence shown in SEQ ID NO.1, and the polymorphism is A / G; The CAPS molecular marker primer pair is used to identify the fragment containing the wheat thousand-grain weight trait SNP molecular marker, and the CAPS molecular marker primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.3; The kit is used to detect the fragment containing the wheat thousand-grain weight trait SNP molecular marker, including the CAPS molecular marker primer pair; The detection of wheat TaAOS-4B Methods for identifying superior allelic variations in gene promoters include the following steps: PCR amplification was performed on the genomic DNA of the test material using the CAPS molecular marker primer pair or the kit to obtain PCR amplification products. Using restriction endonucleases Xho I. The PCR amplification products were digested with enzymes, and the polymorphism of the digested products was detected. When the enzyme digestion product includes two fragments, 612 bp and 26 bp, the allelic variation type is: TaAOS-4B -G; When the enzyme digestion product is a 630 bp fragment, the allelic variation type is TaAOS-4B -A, with the homozygous AA genotype being the superior allelic variant genotype.

2. A method for molecular marker-assisted breeding of high-thousand-grain-weight wheat, characterized in that, Using the CAPS molecular marker primers or the kit described in claim 1, target wheat is molecularly labeled and wheat is screened. TaAOS-4B Germplasm with polymorphism A at the -486th base of the gene promoter region sequence was selected for further screening.

Images