Fragment containing SNP molecular marker of thousand grain weight character of wheat, CAPS molecular marker primer pair and application of CAPS molecular marker primer pair

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a fragment containing a wheat thousand grain weight character SNP molecular marker, a CAPS molecular marker primer pair and application of the CAPS molecular marker primer pair. The invention provides a fragment containing a wheat thousand grain weight character SNP (Single Nucleotide Polymorphism) molecular marker. The fragment comprises a TaAOS-4B gene promoter region; the nucleotide sequence of a promoter region of the TaAOS-4B gene is as shown in SEQ ID NO. 1. By detecting the allelic variation of the wheat TaAOS-4B gene promoter region, high-thousand-grain-weight wheat and low-thousand-grain-weight wheat can be distinguished, the screening efficiency of the thousand-grain-weight wheat is improved, and wheat germplasm breeding is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a fragment containing SNP molecular markers for the thousand-grain weight trait of wheat, a CAPS molecular marker primer pair and their applications. Background Art

[0002] Wheat is an important food crop in China, and increasing wheat yield plays a crucial role in ensuring national food security. The number of spikes per unit area, the number of grains per spike, and the 1000-grain weight are the key factors affecting wheat grain yield. Jasmonic acid (JA) and its metabolite methyl jasmonate (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 jasmonates.Studies have shown that the AOS gene is involved in growth and development processes such as pollen male sterility, cold tolerance, and resistance to pests and diseases [Bae et al. Transgenic rice plants carrying RNA interference constructs of AOS (allene oxide synthase) genes show severe male sterility. Plant Breeding, 2010, 129(6): 647-651; Wang et a1. 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 the AOS gene on grain weight has not been reported, especially in wheat. Therefore, developing molecular markers of the AOS gene associated with wheat thousand-grain weight is expected to provide new theoretical bases and technical means for wheat high-yield breeding, with important scientific value and application prospects. Summary of the Invention

[0003] The object of the present invention is to provide a fragment containing SNP molecular markers for the 1000-grain weight trait of wheat, CAPS molecular marker primer pairs, and their applications, which can detect allelic variations in the promoter region of the wheat TaAOS-4B gene and improve the screening efficiency of wheat with high 1000-grain weight.

[0004] The present invention provides a fragment containing SNP molecular markers for the 1000-grain weight trait of wheat, and the fragment contains the promoter region of the TaAOS-4B gene; the nucleotide sequence of the promoter region of the TaAOS-4B gene is as shown in SEQ ID NO.1.

[0005] As a preferred embodiment, 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.

[0006] The present invention also provides a pair of CAPS molecular marker primers for identifying the fragment described in the above scheme, including 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, including the pair of CAPS molecular marker primers described in the above scheme.

[0008] The present invention also provides a method for detecting excellent allelic variations in the promoter of the TaAOS-4B gene in wheat, including the following steps:

[0009] Performing PCR amplification on the genomic DNA of the material to be tested using the pair of CAPS molecular marker primers or the kit described in the above scheme to obtain a PCR amplification product;

[0010] Digesting the PCR amplification product with the restriction enzyme Xho I and detecting the polymorphism of the digestion product;

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

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

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

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

[0015] The present invention also provides the use of the fragment, the CAPS molecular marker primer pair, the kit or the method described in the above solution in screening for wheat with high thousand-grain weight.

[0016] The present invention also provides a method for molecular marker-assisted breeding of wheat with high thousand-grain weight. Using the CAPS molecular marker primer or the kit described in the above solution to perform molecular marking on the target wheat, and screening for germplasms with the 486th base in the promoter region of the wheat TaAOS-4B gene being A with polymorphism for subsequent screening.

[0017] Beneficial effects:

[0018] The present invention provides a fragment containing SNP molecular markers for the thousand-grain weight trait of wheat, and the fragment contains the promoter region of the TaAOS-4B gene; the nucleotide sequence of the promoter region of the TaAOS-4B gene is as shown in SEQ ID NO.1. The present invention detects allelic variations in the promoter region of the wheat TaAOS-4B gene, can distinguish high thousand-grain weight wheat from low thousand-grain weight wheat, improve the screening efficiency of high thousand-grain weight wheat, and is beneficial to wheat germplasm breeding.

[0019] The present invention uses the CASP molecular marker primer pair to detect allelic variations in the promoter region of the TaAOS-4B gene related to the thousand-grain weight in wheat. The identification of the sample can be completed only through simple DNA extraction, PCR specific amplification, and enzymatic digestion detection, which is convenient for detecting and screening wheat varieties or lines with high thousand-grain weight, and can greatly accelerate the breeding process of high-yield wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0021] Figure 1 It is the amplification result of the promoter and coding region of the TaAOS-4B gene; among them, A is the schematic diagram of the primer design for the promoter and coding region of the TaAOS-4B gene; B is the partial amplification result of the TaAOS-4B gene promoter of different varieties; C is the partial amplification result 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, Yanzhen 4110, Yunong 202, Jinhe 9123, Mianyang 20, Zhengmai 366, Xinmai 9, and Yunong 416 in sequence;

[0022] Figure 2Alignment results of TaAOS-4B gene promoter sequences in different varieties;

[0023] Figure 3 Alignment results of TaAOS-4B gene coding region sequences in different varieties;

[0024] Figure 4 Alignment results of TaAOS-4B gene coding region sequences in different varieties;

[0025] Figure 5 Haplotype analysis of TaAOS-4B gene;

[0026] Figure 6 Differentiation of excellent haplotypes of TaAOS-4B high thousand-grain weight varieties; where A is the schematic diagram of thousand-grain weight haplotype differentiation in 2015; B is the schematic diagram of thousand-grain weight haplotype differentiation in 2016;

[0027] Figure 7 Schematic diagram of differentiation of restriction enzyme sites of different types of TaAOS-4B haplotypes;

[0028] Figure 8 Amplification of different types of TaAOS-4B haplotypes and their corresponding Xho I and Sac II restriction enzyme electrophoresis maps; where A is the electrophoresis map of CAPS-486 sequence amplification of different types of TaAOS-4B gene haplotypes; B is the electrophoresis map of single restriction enzyme digestion of CAPS-486 sequence gel recovery product with Xho I; C is the electrophoresis map of Xho I and SacII restriction enzyme digestion of the corresponding plasmid after CAPS-486 sequence is ligated to the 19T vector; M is Marker; lanes 1-3 from left to right represent the corresponding varieties Zhoumai 11, Kenong 9204, and Yuzhan 4l10 in Hap1; lanes 4-6 from left to right represent the corresponding varieties Yunong 202, Jinhe 9123, and Mianyang 20 in Hap2; lanes 7-9 from left to right represent the corresponding varieties Zhengmai 366, Xinmai 9, and Yunong 416 in Hap3. Specific implementation manners

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

[0030] As an implementation manner, 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 present invention can determine the thousand-grain weight performance of the wheat to be tested by identifying and genotyping the SNP molecular marker: when the polymorphic site is the A base, the thousand-grain weight trait of the wheat is high thousand-grain weight.

[0031] The present invention also provides a pair of CAPS molecular marker primers for identifying the fragment described in the above scheme, including 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 Using the pair of CAPS molecular marker primers of the present invention can detect the allelic variation in the promoter region of the TaAOS-4B gene related to the thousand-grain weight in wheat, detect and screen wheat varieties or lines with high thousand-grain weight, and can greatly accelerate the breeding process of high-yield wheat varieties.

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

[0033] The present invention also provides a method for detecting excellent allelic variation in the promoter of the TaAOS-4B gene in wheat, including the following steps:

[0034] Using the pair of CAPS molecular marker primers or the kit described in the above scheme to perform PCR amplification on the genomic DNA of the material to be tested to obtain a PCR amplification product;

[0035] Using the restriction endonuclease Xho I to digest the PCR amplification product and detect the polymorphism of the digestion product;

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

[0037] The present invention first extracts the genomic DNA of the material to be tested. As an implementation manner, the material to be tested is wheat, and the present invention has no special requirements for the type of the wheat, and any type of wheat can be used. The present invention has no special limitation on the method for extracting the genomic DNA of the wheat to be tested, and a conventional method for extracting the genomic DNA of plant cells in the art can be used. In the specific implementation process of the present invention, the leaves or other tissues of the wheat to be tested are used as materials and extracted by the CTAB method. The CTAB method of the present invention is carried out according to the conventional method.

[0038] After obtaining the genomic DNA, the genomic DNA of the material to be tested is subjected to PCR amplification using the CAPS molecular marker primer pair described in the above scheme or the kit described above to obtain a PCR amplification product. As an implementation method, the reaction system for the PCR amplification is 20 μL in total, including: 10 μL of 2×3G Taq Master Mix for PAGE (Red Dye), 1 μL of the upstream primer, 1 μL of the downstream primer, 1 μL of the DNA template, and the balance of ddH2O. Using the genomic DNA of the wheat to be tested as a template, a PCR amplification reaction is carried out using the primer pair to obtain a PCR amplification product. As an implementation method, the reaction procedure for the PCR amplification is: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 15 s, annealing at 61 °C for 15 s, extension at 72 °C for 30 s, for 32 cycles; final extension at 72 °C for 5 min.

[0039] As an implementation method, after the PCR amplification is completed, the PCR amplification product is stored at 4 °C. The present invention can amplify the promoter fragment of the TaAOS-4B gene in different haplotype wheats using the primer pair, and the DNA fragment containing the target SNP site is contained in the PCR amplification product obtained by the PCR amplification reaction.

[0040] After obtaining the PCR amplification product, the present invention uses the restriction enzyme Xho I to digest the PCR amplification product. The present invention does not make special limitations on the source of the restriction enzyme Xho I, and the digestion system can adopt the conventional settings in the art.

[0041] After obtaining the digestion product, the present invention detects the polymorphism of the digestion product and judges the allelic variation type of the TaAOS-4B gene promoter in wheat according to the detection result. As an implementation method, the detection includes analysis by agarose gel electrophoresis. The present invention detects the allelic variation of the TaAOS-4B gene promoter in wheat according to the fragment size of the digestion product, and can determine the thousand-grain weight trait of wheat through the type of allelic variation. When the digestion product includes two fragments of 612 bp and 26 bp, the allelic variation type is TaAOS-4B-G; when the digestion product is a fragment of 630 bp, the allelic variation type is TaAOS-4B-A, and the homozygous AA genotype is the excellent allelic variation genotype.

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

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

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

[0045] The present invention also provides the use of the fragment, the CAPS molecular marker primer pair, the kit or the method described in the above solution in screening wheat with high thousand-grain weight.

[0046] The present invention also provides a method for molecular marker-assisted breeding of wheat with high thousand-grain weight. The target wheat is molecularly marked using the CAPS molecular marker primer or the kit described in the above solution, and germplasms with the 486th base in the promoter region sequence of the wheat TaAOS-4B gene being A with polymorphism are screened for subsequent screening.

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

[0048] The 2×3G Taq Master Mix for PAGE (Red Dye) used for PCR amplification in the present invention is purchased from Novoprotein Scientific Inc., the cloning T-vector pMD 19-T Vector is purchased from Takara Biotechnology (Beijing) Co., Ltd., the Xho I and Sac II restriction endonucleases are purchased from NEB Company, and other chemicals are of domestic analytical purity.

[0049] Example 1 Screening of TaAOS Gene in Excellent Haplotypes with High Thousand-Grain Weight

[0050] (1) Primer Design

[0051] According to the sequences of the TaAOS gene in the wheat genome database (https: / / plants.ensembl.org / ), the A copy (TraesCS4A02G061900), B copy (TraesCS4B02G237600), and D copy (TraesCS4D02G238800) were obtained by alignment. Referring to the expVIP website (https: / / www.wheat-expression.com / ), it was found that their gene expression levels during grain filling were 23.08, 91.22, and 24.37 respectively. Among them, the expression level of the 4B copy was the highest, and the wheat B genome had higher genomic diversity compared to the A and D genomes. Therefore, the B copy was selected for subsequent experiments. Primers TaAOS-4B-Promoter and TaAOS-4B-CDS (Table 1) were designed for the promoter region and coding region of the 4B copy in segments, and the primer distribution is as Figure 1As shown in A, primer synthesis and sequencing were completed by Sangon Biotechnology (Shanghai) Co., Ltd., with 'Chinese Spring' as the comparison sequence, and 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 wheat DNA

[0055] 78 common wheat varieties preserved in the laboratory (Laboratory 317, South of the First Laboratory Building, College of Agriculture, Henan Agricultural University) were selected and the total DNA of wheat was extracted by CTAB method. First, the wheat leaves were quickly frozen in liquid nitrogen and ground, and 1 mL of preheated CTAB extraction solution was added and the mixture was bathed at 65℃ for 1 h. Then centrifuged at 12000g for 10 min, the supernatant was added with an equal volume of chloroform:isoamyl alcohol (volume ratio of 24:1) and oscillated; centrifuged at 12000g for 10 min again, the supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of precooled isopropanol was added to mix, and the mixture was allowed to stand at -20℃ for 30 min; centrifuged as above, the supernatant was discarded, and the leaves were washed twice with 200 μL of 75% ethanol, placed in a fume hood to air dry, and sterile water was added to dissolve the DNA to 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, Handanmai 11, Zhongfan 4, Jimai 20, Yanfu 188, Weimai 8, Luohan 6, Jinmai 47, Mianyang No. 20, Sichuan wheat 22, Henan wheat 202, 04 Zhong 36, Jinhe 9123, Bainong 207, Henan wheat 416, Lunxuan 66, Zhou wheat 32, Luohan 2, Zhou wheat 17, Henan wheat 18-99, Luomai 26, Henan wheat 70-36, Lumai 15, Xinong 979, Zhongmai 8, Xuzhou 25, Ji 5265, Cangmai 6005, Sichuan wheat 16, Henan wheat 822, Anhui wheat 50, Sichuan wheat 24, Kenong 199, Jima wheat 19, Jing 411, Zhongyu 10, Baomai 2, Xinmai 21, Yannong 21, Yangmai 16, Zhongyu 12, Henan wheat 18, Kexing 3302, Xinmai 26, Xiaoyan 54, Zhengmai 366, Xinmai 9, Jima wheat 22.

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

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

[0059] Table 2 PCR amplification system

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

[0061] Table 3 PCR amplification program

[0062] Temperature Time Number of cycles 95℃ 5 min / 95℃ 15s 30c 61℃ 15s 30c 72℃ 30s 30c 72℃ 5 min /

[0063] Figure 1 B and C in the figure indicate that the primer design is reasonable, the reaction conditions are optimized, and the PCR system is appropriate. The target fragments of the above PCR amplifications were recovered respectively, and they were 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 promoter and CDS regions of the TaAOS-4B gene

[0065] The sequencing results were aligned using the DNAMAN software. Among them, the alignment results of the promoter region are as Figure 2 shown, and the sequence alignment results of the CDS region are as Figures 3 - 4 shown. The results show that there are 13 SNP polymorphic variation sites in the promoter region (-650bp to -1bp). The promoter was identified for haplotypes using the DnaSP 5.10 software (http: / / www.ub.edu / DnaSP). The results are as Figure 5 shown. It was found that these 13 SNPs are tightly linked to form three haplotypes, which were named TaAOS-4B-Hap1, TaAOS-4B-Hap2, and TaAOSS-4B-Hap3 respectively, and were subsequently abbreviated as Hap1, Hap2, and Hap3 for short.

[0066] (5) Comparison of 1000-grain weights of wheat varieties with different haplotypes of the TaAOS-4B gene

[0067] Statistically analyze the haplotypes of 78 wheat varieties in step (2) and the thousand-grain weight for two consecutive years in hexaploid wheat. After threshing the grains of each variety and natural drying, randomly select 1000 seeds from them to measure their weights. Use the thousand-grain weight to represent the grain weight, and take its average value for data analysis. The results are shown in Table 4. Analyze the average thousand-grain weight of the haplotypes, and the results are shown in Table 5 and Figure 6 as follows.

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

[0069]

[0070]

[0071] Table 5 Average thousand-grain weight of TaAOS-4B haplotypes

[0072] 1000-grain weight TKW (g) in 2015 1000-grain weight TKW (g) in 2016 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 for two years is significantly higher than that of TaAOS-4B-Hap3 by 16.7% and 13.4% respectively, further indicating that the TaAOS gene containing the Hap1 + Hap2 haplotype is an excellent haplotype for high thousand-grain weight in wheat.

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

[0075] Use dCAPS Finder2.0 (http: / / helix.wustl.edu / dcaps / dcaps.html) to analyze the sequence differences of different haplotypes. Referring to the Chinese Spring gene sequence, it is found that there are two forms of A / G at the -486 position of the TaAOS-4B-promoter sequence, as shown in Figure 7 A in it. The sequences of the varieties containing Hap1 and Hap2 are: The sequence of the Hap3 type wheat variety is: To distinguish the above differences, mutate C to A at the -487 position and G to T at the -490 position of the above sequence, and design 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 to distinguish Hap1, Hap2, and Hap3. Among them, Hap3 contains the CTCGAG sequence and can be recognized and cut by the restriction endonuclease Xho I, while Hap1 and Hap2 contain the sequence CTCGAA and thus cannot be cut by the restriction endonuclease XhoI ( Figure 7In B). According to the sequence differences at that place, it is considered that the dCAPS molecular marker is used to distinguish Hap3 from other haplotypes, and this marker is named dCAPS-486. It is used to distinguish different haplotypes and provide a basis for the subsequent screening of high thousand-grain weight varieties.

[0076] To verify the practical applicability of the determined dCAPS-486 molecular marker above. Select the above-mentioned wheat varieties containing different types of haplotypes and extract genomic DNA respectively. Among them, Hap1 includes varieties Zhoumai 11, Kenong 9204, and Yuzhan 4110; Hap2 includes varieties Yunong 202, Jinhe 9123, and Mianyang 20; Hap3 includes varieties Zhengmai 366, Xinmai 9, and Yunong 416.

[0077] First, use the TaAOS-4B-Promoter primer to amplify the promoter fragment; then, using the PCR product as a template, use the upstream and downstream primers of the dCAPS-486 marker to amplify the dCAPS-486 molecular marker respectively. The PCR system and reaction program are the same as those in Table 2. The results are as Figure 8 shown in A. Hap1 includes varieties Zhoumai 11, Kenong 9204, and Yuzhan 4110 (lanes 1-3); Hap2 includes varieties Yunong 202, Jinhe 9123, and Mianyang 20 (lanes 4-6); Hap3 includes varieties Zhengmai 366, Xinmai 9, and Yunong 416 (lanes 7-9). For the TaAOS-4B fragment (-630bp to -1bp) in different haplotype wheats, a single amplification band (about 630bp) can be obtained.

[0078] Recover the above-mentioned amplified PCR products and perform single enzyme digestion using the restriction enzyme Xho I. The electrophoresis detection results are as Figure 8 shown in B. The results show that the transformed products of Hap1 and Hap2 haplotype wheat varieties are still a single band of about 630bp after enzyme digestion

[0079]

[0080] However, the amplified products of the Hap3 haplotype material become two fragments after single enzyme digestion, which are 612bp and 26bp respectively. It shows that the molecular marker dCAPS-486 can distinguish Hap3 from Hap1 + Hap2 haplotypes and can be applied to the molecular design breeding for screening wheat varieties with high thousand-grain weight.

[0081] Since the 26bp band is relatively small, to facilitate the presentation of the results, furthermore, the above amplified PCR products were recovered, ligated into the 19T vector, and then transferred into DH5α strains. After overnight culture, positive clones were picked and plasmids were extracted; double digestion was performed using Xho I enzyme and Sac II enzyme, and the electrophoresis detection results are as Figure 8 shown in C.

[0082] The results showed that the transformation products of Hap1 and Hap2 haplotype wheat varieties were still a single band of about 3366bp (including the vector 2736bp + fragment 630bp) after digestion, while the amplified products of the Hap3 haplotype material became two fragments after double digestion, which were 2988bp (vector + fragment) and 386bp respectively. It shows that the molecular marker dCAPS-486 can distinguish Hap3 from Hap1 + Hap2 haplotypes and can be applied to the molecular design breeding for screening wheat varieties with high thousand-grain weight.

[0083] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fragment containing a SNP molecular marker for wheat thousand-grain weight trait, characterized in that: The fragment contains the promoter region of the TaAOS-4B gene; the nucleotide sequence of the promoter region of the TaAOS-4B gene is shown in SEQ ID NO.

1.

2. The fragment according to claim 1, characterized in that The polymorphic site of the SNP molecular marker is the first 486 bases of the sequence shown in SEQ ID NO.1, and the polymorphism is A / G.

3. A CAPS molecular marker primer pair for identifying the fragment according to claim 1 or 2, characterized in that: It 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.

4. A kit for detecting the fragment according to claim 1 or 2, characterized in that: It comprises the CAPS molecular marker primer pair described in claim 3.

5. A method for detecting superior allelic variation of the TaAOS-4B gene promoter in wheat, characterized in that: The steps include: Using the CAPS molecular marker primer pair described in claim 3 or the kit described in claim 4 to perform PCR amplification on the genomic DNA of the material to be tested, to obtain a PCR amplification product; The PCR amplification product is digested with restriction endonuclease Xho I to detect the polymorphism of the digestion product; When the enzyme cleavage product includes two fragments of 612bp and 26bp, the allelic variation type is TaAOS-4B-G; when the enzyme cleavage product is a fragment of 630bp, the allelic variation type is TaAOS-4B-A, and the homozygous AA genotype is the superior allelic variation genotype.

6. Use of the fragment according to claim 1 or 2, the CAPS molecular marker primer pair according to claim 3, the kit according to claim 4 or the method according to claim 5 in wheat breeding.

7. Use of the fragment according to claim 1 or 2, the CAPS molecular marker primer pair according to claim 3, the kit according to claim 4 or the method according to claim 5 in wheat assisted breeding.

8. Use of the fragment according to claim 1 or 2, the CAPS molecular marker primer pair according to claim 3, the kit according to claim 4 or the method according to claim 5 in identifying the type of wheat thousand-grain weight.

9. Use of the fragment according to claim 1 or 2, the CAPS molecular marker primer pair according to claim 3, the kit according to claim 4 or the method according to claim 5 in screening high thousand-grain weight wheat.

10. A method for molecular marker-assisted breeding of high thousand-grain weight wheat, characterized in that: The target wheat is molecularly marked using the CAPS molecular marker primers described in claim 3 or the kit described in claim 4, and the -486th base of the promoter region sequence of the wheat TaAOS-4B gene is screened, and germplasm with polymorphism A is subsequently screened.

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