CAPS-153 marker associated with wheat starch content and kernel weight and detection method thereof
By developing the CAPS-153 marker and utilizing specific SNP sites and the restriction endonuclease Taa I, the problems of large errors and complex operations in wheat starch content determination were solved, enabling efficient screening of new wheat varieties with high thousand-grain weight and improving breeding results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN FENGDEKANG SEED IND CO LTD
- Filing Date
- 2025-10-31
- Publication Date
- 2026-06-26
AI Technical Summary
Existing methods for determining wheat starch content suffer from large errors, complex operations, and poor repeatability, which affect wheat breeding results and lack effective molecular marker-assisted selection methods.
The CAPS-153 marker was developed. By detecting specific SNP sites in the tae-miR1121-2A gene, the CAPS molecular marker primer pair and restriction endonuclease Taa I were used to distinguish the haplotypes Hap1 and Hap2 of the thousand-grain weight trait in wheat. Specific primers were designed for PCR amplification and enzyme digestion to achieve efficient prediction of the thousand-grain weight of wheat.
It enables efficient and accurate prediction of wheat grain thousand-grain weight, improving the accuracy and efficiency of wheat breeding and enabling the screening of new wheat varieties with high starch content or high thousand-grain weight.
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Figure CN121137246B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genotyping technology, specifically relating to a CAPS-153 marker and detection method related to wheat starch content and grain weight. Background Technology
[0002] As an important food crop, ensuring stable wheat yield is of great significance, and exploring wheat yield potential remains the primary task of breeding work. Wheat yield is composed of three factors: number of ears per unit area, number of grains per ear, and thousand-grain weight. Among these, starch accounts for 65% to 75% of the dry weight of wheat grains and significantly affects the thousand-grain weight and final grain yield.
[0003] Wheat yield is a complex quantitative trait, influenced by the interaction of genetic and environmental factors. Grain weight (measured as 1,000 grains) is the most genetically stable of the three yield factors, primarily affected by genetic factors and controlled by additive gene effects. Therefore, under the premise of stable number of spikes per unit area and number of grains per spike, increasing grain weight plays an important role in improving wheat yield.
[0004] Starch is the most important component of wheat grains, and its content largely reflects changes in the thousand-grain weight. Current methods for determining amylose content primarily use colorimetry, based on the spectrophotometric reaction of iodide ions with amylose and amylopectin to form a colorimetric product. However, amylopectin can bind with iodine to form a purple complex, interfering with the determination of amylose and causing errors, thus affecting the total starch content. Furthermore, this method is time-consuming, some experimental steps are difficult to perform, and the repeatability and reproducibility are poor. Therefore, the thousand-grain weight can be used indirectly to measure wheat starch content. This simple method for determining wheat starch content or thousand-grain weight is of great significance for wheat breeding and selection. Summary of the Invention
[0005] This invention provides a CAPS-153 marker and detection method related to wheat starch content and grain weight. The CAPS molecular marker helps wheat marker-assisted selection breeding and cost reduction and efficiency improvement.
[0006] The present invention provides a sequence containing a tightly linked SNP site for the thousand-grain weight trait of wheat grains, the sequence comprising the promoter region of tae-miR1121-2A and the precursor sequence of tae-miR1121-2A;
[0007] The nucleotide sequence of the promoter region is shown in SEQ ID No. 1. In the sequence shown in SEQ ID No. 1, there are A and G polymorphisms at 1040 bp, A and G polymorphisms at 1863 bp, A and G polymorphisms at 1869 bp, A and G polymorphisms at 1881 bp, and C and T polymorphisms at 1883 bp.
[0008] The nucleotide sequence of the precursor sequence is shown in SEQ ID No. 2. In the sequence shown in SEQ ID No. 2, there are G and A polymorphisms at position 29 bp, C and deletion polymorphisms at position 32 bp, and A and T polymorphisms at position 49 bp.
[0009] The present invention also provides the application of primer pairs for detecting the above sequences in the preparation of a kit for predicting the thousand-grain weight trait of wheat grains.
[0010] The present invention also provides a method for predicting the thousand-grain weight trait of wheat using CAPS molecular markers, including detecting the above-mentioned sequence and predicting the thousand-grain weight of wheat based on the haplotypes shown in the detection results;
[0011] The thousand-grain weight of wheat grains with haplotype Hap1 was lower than that of wheat grains with haplotype Hap2.
[0012] The haplotype Hap1 has the following characteristic sequence in the promoter region shown in SEQ ID No. 1: A at position 1040bp, A at position 1863bp, A at position 1869bp, A at position 1881bp, and C at position 1883bp; and in the precursor sequence shown in SEQ ID No. 2, it has the following characteristic sequence: G at position 29bp, C at position 32bp, and A at position 49bp.
[0013] The haplotype Hap2 has the following characteristic sequence in the promoter region shown in SEQ ID No. 1: G at 1040bp, G at 1863bp, G at 1869bp, G at 1881bp, and T at 1883bp; and in the precursor sequence shown in SEQ ID No. 2, it has the following characteristic sequence: A at 29bp, a deletion at 32bp, and T at 49bp.
[0014] The present invention also provides a CAPS molecular marker primer pair for detecting the thousand-grain weight trait of wheat grains, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID No. 3 and a downstream primer shown in SEQ ID No. 4.
[0015] The present invention also provides a kit for detecting the thousand-grain weight trait of wheat grains, comprising the above-mentioned CAPS molecular marker primer pair.
[0016] In a preferred embodiment of the present invention, the kit further includes the restriction endonuclease Taa I.
[0017] The present invention also provides a method for detecting wheat grain haplotypes, comprising using the wheat genomic DNA to be tested as a template and amplifying it using the above-mentioned CAPS molecular marker primer pair or the CAPS molecular marker primer pair in the above-mentioned kit;
[0018] The amplification products were digested with the restriction endonuclease Taa I. When only one product was obtained, it was the Hap1 haplotype; when two products were obtained, it was the Hap2 haplotype.
[0019] The haplotype Hap1 has the following characteristic sequence in the promoter region shown in SEQ ID No. 1: A at position 1040bp, A at position 1863bp, A at position 1869bp, A at position 1881bp, and C at position 1883bp; and in the precursor sequence shown in SEQ ID No. 2, it has the following characteristic sequence: G at position 29bp, C at position 32bp, and A at position 49bp.
[0020] The haplotype Hap2 has the following characteristic sequence in the promoter region shown in SEQ ID No. 1: G at 1040bp, G at 1863bp, G at 1869bp, G at 1881bp, and T at 1883bp; and in the precursor sequence shown in SEQ ID No. 2, it has the following characteristic sequence: A at 29bp, a deletion at 32bp, and T at 49bp.
[0021] In a preferred embodiment of the present invention, the amplification includes amplification using PCR, and the PCR program includes: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 54°C for 10 s, extension at 72°C for 20 s, 35 cycles; and further extension at 72°C for 5 min.
[0022] This invention also provides the application of the above-mentioned sequences, the above-mentioned CAPS molecular marker primer pairs, or the above-mentioned kits in molecular marker-assisted breeding of wheat.
[0023] This invention also provides the application of the above sequence, the above CAPS molecular marker primer pair, or the above kit in screening wheat germplasm with high thousand-grain weight.
[0024] Beneficial effects: By comparing the sequence differences of tae-miR1121 among different wheat varieties, this invention found three SNP sites (29G / A, 32C / -, 49A / T) in the precursor sequence (0 bp-98 bp) of tae-miR1121-2A; and five SNP sites (-976A / G, -153A / G, -147A / G, -135A / G, -133C / T) in the promoter region (-2015 bp-0 bp). Haplotype identification of these eight SNP sites was performed using DnaSP 5.10 software, and the results showed that these eight SNPs could be closely linked to form two haplotypes, named Hap1 and Hap2, respectively. Statistical analysis of the thousand-grain weight of wheat revealed that the average thousand-grain weight of the Hap1 wheat variety was 40.94±0.43 g, while that of the Hap2 wheat variety was 45.4±0.52 g. The difference between the two varieties was highly significant, with Hap2 being a superior haplotype with a high thousand-grain weight.
[0025] This invention developed a CAPS functional marker to distinguish between wheat haplotypes Hap1 and Hap2, and named the marker CAPS-153. Specific upstream and downstream amplification primers were designed based on the sequence recognized by the CAPS-153 functional marker on the tae-miR1121-2A gene. Amplification of the tae-miR1121-2A gene fragments from different wheat haplotypes using these primers yielded a single amplified band. The purified PCR products were then digested with Taa I. Electrophoresis results showed that the amplified product from the Hap1 haplotype wheat variety remained a single band after digestion, while the amplified product from the Hap2 haplotype wheat variety was digested into two fragments. Therefore, the CAPS-153 functional marker described in this invention can distinguish between the two tae-miR1121-2A haplotype wheat varieties and can be subsequently applied to marker-assisted selection breeding of new wheat varieties with high starch content. Attached Figure Description
[0026] Figure 1 This is a sequence alignment diagram of promoters from some wheat varieties with the tae-miR1121-2A name.
[0027] Figure 2 This is a sequence alignment diagram of precursors of tae-miR1121-2A in some wheat varieties;
[0028] Figure 3 Figure showing the identification results of SNPs on the tae-miR1121-2A sequence among wheat varieties;
[0029] Figure 4 A graph showing the thousand-grain weight of two haplotype wheat varieties;
[0030] Figure 5The diagram shows the development of the tae-miR1121-2A functional marker. In the diagram, A: specific amplification diagram, B: restriction site, and C: restriction digestion result diagram.
[0031] Figure 6 The diagram shows the mechanism of high starch content in wheat varieties with superior haplotype tae-miR1121-2A. In the diagram, A represents the relative expression level of tae-miR1121, and B represents the activity of AGPase enzyme. Detailed Implementation
[0032] The present invention provides a sequence containing a tightly linked SNP site for the thousand-grain weight trait of wheat grains, the sequence comprising the promoter region of tae-miR1121-2A and the precursor sequence of tae-miR1121-2A;
[0033] The nucleotide sequence of the promoter region is shown in SEQ ID No. 1. In the sequence shown in SEQ ID No. 1, there are A and G polymorphisms at 1040 bp, A and G polymorphisms at 1863 bp, A and G polymorphisms at 1869 bp, A and G polymorphisms at 1881 bp, and C and T polymorphisms at 1883 bp.
[0034] The nucleotide sequence of the precursor sequence is shown in SEQ ID No. 2. In the sequence shown in SEQ ID No. 2, there are G and A polymorphisms at position 29 bp, C and deletion polymorphisms at position 32 bp, and A and T polymorphisms at position 49 bp.
[0035] The promoter sequence of tae-miR1121-2A described in this invention is shown in SEQ ID No. 1.
[0036] Upon comparison, five SNP sites (-976A / G, -153A / G, -147A / G, -135A / G, -133C / T) were found in the promoter region (-2015 bp-0 bp) shown in SEQ ID No.1 among different wheat varieties.
[0037] The precursor sequence of tae-miR1121-2A described in this invention is shown in SEQ ID No. 2. Upon comparison, three SNP sites (29G / A, 32C / -, 49A / T) were found in the precursor sequence (0 bp-98 bp) shown in SEQ ID No. 2.
[0038] In the embodiments described in this invention, the amplification of the promoter and coding region of the tae-miR1121-2A gene is carried out using primer pairs tae-miR1121-2A-F1 / R1 (SEQ ID No. 5 / SEQ ID No. 6), tae-miR1121-2A-F2 / R2 (SEQ ID No. 7 / SEQ ID No. 8), and tae-miR1121-2A-F3 / R3 (SEQ ID No. 9 / SEQ ID No. 10) and 150 wheat variety DNA samples as templates to amplify the promoter and precursor sequences of tae-miR1121-2A in segments.
[0039] Table 1 Primer information involved in this invention
[0040]
[0041] The present invention also provides the application of primer pairs for detecting the above sequences in the preparation of a kit for predicting the thousand-grain weight trait of wheat grains.
[0042] In this embodiment of the invention, it was found that the eight SNPs can be closely linked to form two haplotypes, Hap1 and Hap2, and that Hap1 and Hap2 are significantly associated with the thousand-grain weight trait of wheat grains, and that the thousand-grain weight of wheat grains of haplotype Hap2 is significantly higher than that of haplotype Hap1.
[0043] In this invention, AGPase, which catalyzes the first step of starch synthesis, is considered the rate-limiting enzyme in starch biosynthesis. Increasing the enzyme activity of AGPase can effectively accelerate the transport of reducing sugars in starch synthesis, thereby increasing starch synthesis and yield in wheat. In the embodiments of this invention, it was also found that in wheat with the Hap2 haplotype, the AGPase activity was significantly higher than that of the Hap1 haplotype, corresponding to the thousand-grain weight of wheat grains. Therefore, the detection of the two haplotypes composed of the eight SNPs described in this invention can be closely correlated with the thousand-grain weight trait of wheat grains.
[0044] The present invention also provides a method for predicting the thousand-grain weight trait of wheat using CAPS molecular markers, including detecting the above-mentioned sequence and predicting the thousand-grain weight of wheat based on the haplotypes shown in the detection results;
[0045] The thousand-grain weight of wheat grains with haplotype Hap1 was lower than that of wheat grains with haplotype Hap2.
[0046] The haplotype Hap1 has the following characteristic sequence in the promoter region shown in SEQ ID No. 1: A at position 1040bp, A at position 1863bp, A at position 1869bp, A at position 1881bp, and C at position 1883bp; and in the precursor sequence shown in SEQ ID No. 2, it has the following characteristic sequence: G at position 29bp, C at position 32bp, and A at position 49bp.
[0047] The haplotype Hap2 has the following characteristic sequence in the promoter region shown in SEQ ID No. 1: G at 1040bp, G at 1863bp, G at 1869bp, G at 1881bp, and T at 1883bp; and in the precursor sequence shown in SEQ ID No. 2, it has the following characteristic sequence: A at 29bp, a deletion at 32bp, and T at 49bp.
[0048] In one embodiment of the present invention, a pair of specific primers, CAPS-F and CAPS-R, were constructed based on the CAPS molecular marker, wherein the nucleotide sequence of CAPS is shown in SEQ ID No. 3 and the nucleotide sequence of CAPS is shown in SEQ ID No. 4.
[0049] The present invention also provides a CAPS molecular marker primer pair for detecting the thousand-grain weight trait of wheat grains, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID No. 3 and a downstream primer shown in SEQ ID No. 4.
[0050] The present invention also provides a kit for detecting the thousand-grain weight trait of wheat grains, comprising the above-mentioned CAPS molecular marker primer pair.
[0051] The kit described in this invention also includes the restriction endonuclease Taa I, as well as other reagents for PCR amplification.
[0052] The present invention also provides a method for detecting wheat grain haplotypes, comprising using the wheat genomic DNA to be tested as a template and amplifying it using the above-mentioned CAPS molecular marker primer pair or the CAPS molecular marker primer pair in the above-mentioned kit;
[0053] The amplification products were digested with the restriction endonuclease Taa I. When only one product was obtained, it was the Hap1 haplotype; when two products were obtained, it was the Hap2 haplotype.
[0054] The haplotype Hap1 has the following characteristic sequence in the promoter region shown in SEQ ID No. 1: A at position 1040bp, A at position 1863bp, A at position 1869bp, A at position 1881bp, and C at position 1883bp; and in the precursor sequence shown in SEQ ID No. 2, it has the following characteristic sequence: G at position 29bp, C at position 32bp, and A at position 49bp.
[0055] The haplotype Hap2 has the following characteristic sequence in the promoter region shown in SEQ ID No. 1: G at 1040bp, G at 1863bp, G at 1869bp, G at 1881bp, and T at 1883bp; and in the precursor sequence shown in SEQ ID No. 2, it has the following characteristic sequence: A at 29bp, a deletion at 32bp, and T at 49bp.
[0056] The amplification described in this invention includes PCR amplification, the PCR program of which includes: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 54℃ annealing for 10 s, 72℃ extension for 20 s, 35 cycles; and a final extension at 72℃ for 5 min. Using the method described in this invention, a single 321 bp amplification band can be obtained from different haplotype wheat varieties. The purified PCR product was then digested with Taa I. Electrophoresis results showed that the amplification product of haplotype Hap1 wheat remained a single band after digestion, while the amplification product of haplotype Hap2 wheat was digested into two fragments, 253 bp and 68 bp, respectively. This indicates that the CAPS-153 functional marker described in this invention can distinguish between the two haplotype wheat varieties of tae-miR1121-2A, and can subsequently be applied to molecular marker-assisted selection breeding of new wheat varieties with high starch content or high thousand-grain weight.
[0057] This invention also provides the application of the above-mentioned sequences, the above-mentioned CAPS molecular marker primer pairs, or the above-mentioned kits in molecular marker-assisted breeding of wheat.
[0058] This invention also provides the application of the above sequence, the above CAPS molecular marker primer pair, or the above kit in screening wheat germplasm with high thousand-grain weight.
[0059] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a CAPS-153 labeling and detection method related to wheat starch content and grain weight provided by the present invention, should not be construed as limiting the scope of protection of the present invention.
[0060] Example 1
[0061] 1. Materials
[0062] The study utilized 150 wheat varieties approved in recent years, as shown in Table 2. The 2×Taq Plus Master Mix used for PCR amplification was purchased from Vazyme, and the Taa I restriction endonuclease was purchased from TaKaRa.
[0063] Table 2. Haplotypes and 1000-grain weight of 150 wheat varieties
[0064]
[0065]
[0066]
[0067]
[0068] 2. Primer design
[0069] The precursor sequence pre-miR1121 (SEQ ID No. 2) of tae-miR1121 was found in the miRbase database. Sequence differences among the three copies of the tae-miR1121 gene were then analyzed in a wheat database. The A copy was divided into three segments, and specific primers were designed for amplification (tae-miR1121-2A-F1 / R1, tae-miR1121-2A-F2 / R2, tae-miR1121-2A-F3 / R3). Primer synthesis and sequencing were performed by Henan Shangya Biotechnology Co., Ltd.
[0070] 3. Extraction of total DNA from wheat
[0071] DNA was extracted from wheat tissue or fresh grain samples using the CTAB method.
[0072] 4. Amplification of the tae-miR1121-2A gene promoter and precursor
[0073] The target fragment was amplified using 2×Taq Plus Master Mix. The promoter and precursor sequences of tae-miR1121-2A were amplified in segments using tae-miR1121-2A-F1 / R1, tae-miR1121-2A-F2 / R2, and tae-miR1121-2A-F3 / R3 as templates from 150 wheat cultivar DNA samples.
[0074] 5. Sequence alignment and haplotype analysis of the tae-miR1121-2A gene promoter
[0075] DNAMAN software was used to assemble and align the sequencing results. Figure 1 , Figure 2The results showed that three SNPs (29G / A, 32C / -, 49A / T) were present in the precursor sequence of tae-miR1121-2A (0 bp-98 bp); and five SNPs (-976A / G, -153A / G, -147A / G, -135A / G, -133C / T) were present in the promoter region (-2015 bp-0 bp). Haplotype identification of these eight SNPs using DnaSP 5.10 software revealed that these eight SNPs were tightly linked to form two haplotypes (…). Figure 3 They were named Hap1 and Hap2 respectively.
[0076] 6. Correlation analysis between tae-miR1121-2A haplotype and thousand-grain weight
[0077] Correlation analysis between the two haplotypes of tae-miR1121-2A and the thousand-grain weight revealed that the average thousand-grain weight of the Hap1 wheat variety was 40.94 ± 0.43 g, and that of the Hap2 wheat variety was 45.4 ± 0.52 g. Figure 4 The difference between the two was highly significant, with Hap2 being a superior haplotype with high thousand-grain weight.
[0078] 7. Development of CAPS molecular markers
[0079] Sequence differences between two haplotypes of tae-miR1121-2A were analyzed using dCAPSFinder 2.0 to predict SNP sites and restriction endonucleases for the development of enzyme digestion amplification polymorphism (CAPS) markers. Sequence analysis at -153 bp in the promoter region of the two haplotypes revealed that the SNP (G / A) between the two haplotypes is located within the sequences ACTAT (Hap1) and ACTGT (Hap2). ACTGT can be recognized and cleaved by the restriction endonuclease Taa I, while ACTAT cannot be cleaved. Figure 5 (B). Based on the sequence differences here, a CAPS functional marker can be developed to distinguish between the two haplotypes, and this marker is named CAPS-153.
[0080] Specific upstream and downstream amplification primers (CAPS-F / R) were designed based on the sequence recognized by the CAPS-153 functional marker on the tae-miR1121-2A gene. Amplification of the tae-miR1121-2A gene fragment from different haplotypes of wheat using these primers yielded a single amplified band of 321 bp in length. Figure 5(A). The purified PCR product was then digested with Taa I. Electrophoresis results showed that the amplification product of the Hap1 haplotype wheat variety remained a single band after digestion, while the amplification product of the Hap2 haplotype wheat variety was digested into two fragments, 253 bp and 68 bp respectively. Figure 5 (C). The experimental results show that the CAPS-153 functional marker can distinguish between two haplotype wheat varieties of tae-miR1121-2A, and can be applied to molecular marker-assisted selection breeding of new wheat varieties with high starch content.
[0081] 8. Mechanism of high starch content in superior haplotype wheat varieties tae-miR1121-2A
[0082] In this invention, four wheat varieties of each of the two haplotypes of tae-miR1121-2A were randomly selected (Hap1: Yunong 908, Fusui 3, Xinmai 45 and Bainong 307; Hap2: Xinhuamai 818, Zhengyumai 9987, Xinmai 60 and Zhoumai 36). Total RNA was extracted and reverse transcribed. Then, the expression level of tae-miR1121-2A between the two haplotype wheat varieties was detected. The primers used are shown in Table 1: tae-miR1121F (SEQ ID No. 11) and tae-miR1121R (SEQ ID No. 12).
[0083] The results showed that on day 14 after grain filling, the expression level of the tae-miR1121-2A gene in the Hap1 wheat variety was significantly higher than that in the Hap2 wheat variety. On day 21 after grain filling, the expression level of the tae-miR1121-2A gene in the Hap1 wheat variety was also significantly higher than that in the Hap2 wheat variety, indicating that the superior haplotype of tae-miR1121-2A wheat variety has a lower tae-miR1121-2A gene expression level. Figure 6 (A)
[0084] This invention simultaneously detected the difference in AGPase enzyme activity caused by allelic variations in the tae-miR1121-2A gene. The enzyme activity detection results for the two haplotypes are as follows: Figure 6As shown in Figure B, on day 14 after grain filling, the AGPase activities of Hap1 and Hap2 were 18.32±0.036 U / g and 24.87±3.00 U / g, respectively, with a highly significant difference between them. On day 21 after grain filling, the activities of Hap1 and Hap2 were 11.07±0.38 U / g and 36.40±4.14 U / g, respectively, also with a highly significant difference between them. This indicates that the superior haplotype wheat variety tae-miR1121-2A has higher AGPase activity, which is consistent with the results of the thousand-grain weight correlation analysis. AGPase catalyzes the first step of starch synthesis and is considered the rate-limiting enzyme in starch biosynthesis. Increasing the activity of AGPase can effectively accelerate the transport of reducing sugars in starch synthesis, thereby increasing starch synthesis and yield in wheat.
[0085] 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 nucleic acid fragment tae-miR1121-2A associated with the thousand-grain weight trait of wheat grains, characterized in that, The nucleic acid fragment tae-miR1121-2A consists of a promoter region and a precursor sequence; The nucleotide sequence of the promoter region is shown in SEQ ID No. 1, and the nucleotide sequence of the precursor sequence is shown in SEQ ID No.
2.
2. A method for predicting the thousand-grain weight trait of wheat using primer pairs marked with CAPS molecular markers, characterized in that, Using wheat genomic DNA as a template, PCR amplification was performed using primer pairs with CAPS molecular markers as shown in SEQ ID No. 3-4; the amplification products were digested with restriction endonuclease Taa I. When only one product was obtained, it was haplotype Hap1; when two products were obtained, it was haplotype Hap2. The thousand-grain weight of wheat grains with haplotype Hap1 was lower than that of wheat grains with haplotype Hap2. The haplotype Hap1 has the following positions in the promoter region shown in SEQ ID No. 1: A at 1040 bp, A at 1863 bp, A at 1869 bp, A at 1881 bp, and C at 1883 bp; and in the precursor sequence shown in SEQ ID No. 2: G at 29 bp, C at 32 bp, and A at 49 bp. The haplotype Hap2 has G at position 1040bp, G at position 1863bp, G at position 1869bp, G at position 1881bp, and T at position 1883bp in the promoter region shown in SEQ ID No. 1; and in the precursor sequence shown in SEQ ID No. 2, A at position 29bp, a deletion at position 32bp, and T at position 49bp.
3. The method according to claim 2, characterized in that, The PCR amplification program includes: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 54℃ annealing for 10 s, 72℃ extension for 20 s, 35 cycles; 72℃ extension for 5 min.
4. A kit for detecting the thousand-grain weight trait of wheat grains, characterized in that, The primer pair includes a CAPS molecular marker and a restriction endonuclease Taa I, wherein the CAPS molecular marker primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID No. 3 and a downstream primer as shown in SEQ ID No.
4.
5. The application of the nucleic acid fragment tae-miR1121-2A according to claim 1 and the kit according to claim 4 in molecular marker-assisted breeding of wheat grain thousand-grain weight, characterized in that, The tae-miR1121-2A was detected using the kit, and the thousand-grain weight of wheat was predicted based on the haplotypes shown in the detection results. The thousand-grain weight of wheat grains with haplotype Hap1 was lower than that of wheat grains with haplotype Hap2. The haplotype Hap1 is defined as follows: in the promoter region shown in SEQ ID No. 1, A is at position 1040, A is at position 1863, A is at position 1869, A is at position 1881, and C is at position 1883; and in the precursor sequence shown in SEQ ID No. 2, G is at position 29, C is at position 32, and A is at position 49. The haplotype Hap2 has G at position 1040bp, G at position 1863bp, G at position 1869bp, G at position 1881bp, and T at position 1883bp in the promoter region shown in SEQ ID No. 1; and in the precursor sequence shown in SEQ ID No. 2, A at position 29bp, a deletion at position 32bp, and T at position 49bp.
6. The application of the nucleic acid fragment tae-miR1121-2A according to claim 1 and the kit according to claim 4 in screening wheat germplasm with high thousand-grain weight, characterized in that, The kit was used to detect tae-miR1121-2A, and wheat germplasm with high thousand-grain weight was screened based on the haplotypes shown in the detection results. The thousand-grain weight of wheat grains with haplotype Hap1 was lower than that of wheat grains with haplotype Hap2. The promoter region of the detected tae-miR1121-2A was identified and cleaved using the restriction endonuclease Taa I. The region that was identified and cleaved by the restriction endonuclease Taa I was identified as haplotype Hap2, while the region that was not identified and cleaved by the restriction endonuclease Taa I was identified as haplotype Hap1. The haplotype Hap1 is defined as follows: in the promoter region shown in SEQ ID No. 1, A is at position 1040, A is at position 1863, A is at position 1869, A is at position 1881, and C is at position 1883; and in the precursor sequence shown in SEQ ID No. 2, G is at position 29, C is at position 32, and A is at position 49. The haplotype Hap2 has G at position 1040bp, G at position 1863bp, G at position 1869bp, G at position 1881bp, and T at position 1883bp in the promoter region shown in SEQ ID No. 1; and in the precursor sequence shown in SEQ ID No. 2, it has A at position 29bp, a deletion at position 32bp, and T at position 49bp.