InDel molecular marker for distinguishing wheat haplotype and / or identifying high-thousand-grain-weight wheat and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to an InDel molecular marker for distinguishing wheat haplotype and / or identifying wheat with high thousand kernel weight and application thereof. The application takes 332 hexaploid wheat varieties as research objects, performs polymorphism analysis, finds that Hap2 compared with Hap1, there is one InDel: -475bp contains 67bp base deletion, which is named InDel-541 / -475, and the deleted nucleotide sequence is specifically shown as SEQ ID NO. 1. Based on the site, Hap1 and Hap2 can be distinguished, and wheat varieties with high thousand kernel weight can be identified. Based on the InDel molecular marker of the application, a primer is designed, and only simple PCR specific amplification is needed to distinguish the wheat haplotype, complete the identification of wheat with high thousand kernel weight, facilitate the detection and screening of wheat varieties or lines with high thousand kernel weight, and can greatly accelerate the breeding process of wheat varieties with high thousand kernel weight.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to an InDel molecular marker for distinguishing wheat haplotypes and / or identifying high 1000-grain weight wheat and an application thereof. Background Art

[0002] Wheat is an important staple food crop, playing a crucial role in ensuring the nation's absolute food security. Starch is the primary storage component of the wheat endosperm, accounting for 60%-70% of the total grain mass. Depending on the connection method, starch in crop grains can be divided into amylose and amylopectin.

[0003] Studies have shown that the activity of starch synthesis enzymes directly influences grain starch content and determines grain weight. AGPL or AGPS deletion mutants result in reduced seed starch content and grain weight. Upregulating AGPL or AGPS expression in rice, maize, and wheat has been shown to increase grain starch content, grain weight, and yield. BRITTLE1 (BT1) belongs to the mitochondrial transport protein family, primarily responsible for transporting small molecules between mitochondria and the cytoplasm. Studies have shown that TaBT1 is responsible for transmembrane transport of ADP-glucose, directly affecting starch synthesis and leading to reduced grain weight in wheat. TaBT1-6B possesses a superior haplotype that is positively correlated with starch synthesis and grain weight, providing valuable genetic and germplasm resources for high-yield wheat breeding. Therefore, regulating the expression of starch synthesis enzymes could potentially increase wheat grain starch content and grain weight. Screening for superior haplotypes of related genes and developing molecular markers could provide valuable genetic resources for breeding and significantly accelerate the advancement of high-yield wheat breeding.

[0004] The BTB domain was first discovered in Drosophila. Its protein sequence is highly homologous to the Drosophila ttk (tramtrack) and BR-C (Broad-Complex) proteins, hence the name BTB (Bric-a-Brac / Tramtrack / Broad Complex). Furthermore, because many poxvirus proteins also contain this motif, it is also known as POZ (pox virus and zinc finger). The BTB domain is evolutionarily conserved, with BTB proteins found in yeast, humans, and plants. BTB proteins typically contain one or more conserved BTB domains. Some BTB proteins also contain one or more kletch repeats or zinc finger structures, MATH (meprin and TRAF-C homology) domains, ankyrin repeats, TAZ (transcriptional adapter zinc finger), or NPH3 (Nonphototropic Hypocotyl 3) domains. BTB proteins directly or indirectly participate in plant organ growth and differentiation by influencing cell differentiation and division. Pumilio protein 24 (APUM24) in Arabidopsis thaliana reduces the mRNA stability of BTB / POZMATH family genes in vivo, affecting seed shape and grain weight. However, no studies have linked the BTB domain to wheat genotypes and 1000-grain weight, making it urgent to understand the functions of this family of genes in wheat. Summary of the Invention

[0005] The purpose of the present invention is to provide an InDel molecular marker for distinguishing wheat haplotypes and / or identifying high 1000-grain weight wheat varieties and its application, to distinguish wheat haplotypes and screen high 1000-grain weight wheat.

[0006] The present invention provides an InDel molecular marker for distinguishing wheat haplotypes and / or identifying high 1000-grain weight wheat varieties, wherein the InDel molecular marker includes InDel-541 / -475;

[0007] The InDel-541 / -475 is located at -475bp in the promoter region of the wheat TaBTB-1D gene and contains a 67bp base deletion.

[0008] The 67 bp nucleotide sequence is shown as SEQ ID NO.1.

[0009] The present invention also provides a primer for detecting the InDel molecular marker described in the above technical solution, wherein the primer comprises an upstream primer TaBTB-1D-SF and a downstream primer TaBTB-1D-SR;

[0010] The upstream primer TaBTB-1D-SF includes the nucleotide sequence shown in SEQ ID NO.2;

[0011] The downstream primer TaBTB-1D-SR includes the nucleotide sequence shown in SEQ ID NO.3.

[0012] The present invention also provides the use of the InDel molecular markers or primers described in the above technical solution in distinguishing wheat haplotypes and / or identifying high thousand-grain weight wheat varieties.

[0013] Preferably, the wheat haplotype includes TaBTB-1D-Hap1 haplotype and / or TaBTB-1D-Hap2 haplotype.

[0014] The present invention also provides the use of the InDel molecular marker or primer described in the above technical solution in wheat breeding.

[0015] Preferably, the wheat breeding includes breeding of high thousand-kernel weight wheat.

[0016] The present invention also provides a method for distinguishing wheat haplotypes and / or identifying high 1000-grain weight wheat varieties, comprising the following steps:

[0017] PCR amplification is performed on the genomic DNA of the wheat to be tested using the primers described in the above technical solution to obtain a PCR amplification product;

[0018] Detecting the size of the PCR amplification product; if the size of the PCR amplification product is 370 bp, the wheat to be tested is of the TaBTB-1D-Hap1 haplotype, a low 1000-grain weight wheat variety;

[0019] If the size of the PCR amplification product is 303 bp, the wheat to be tested is of the TaBTB-1D-Hap2 haplotype, a high 1000-grain weight wheat variety.

[0020] Preferably, the PCR amplification system, based on 50 μL, includes 25 μL of 2×KOD One™ PCR MasterMix, 0.2-0.5 μL of 10-20 μM upstream primer, 0.2-0.5 μL of 10-20 μM downstream primer, 200 ng of genomic DNA and the balance of water.

[0021] Preferably, the PCR amplification program is: 94°C for 3 min; 98°C for 10 s, 52°C for 20 s, 68°C for 3 min, 35 cycles; 68°C for 5 min.

[0022] Preferably, the size of the PCR amplification product is detected by agarose gel electrophoresis or sequencing.

[0023] Beneficial effects:

[0024] The present invention used 332 hexaploid wheat varieties as research subjects and conducted polymorphism analysis. It was discovered that Hap2, compared to Hap1, has an InDel: -475bp containing a 67bp base deletion, designated InDel-541 / -475. The specific nucleotide sequence of the deletion is shown in SEQ ID NO. 1. Based on this site, it can be used to distinguish Hap1 from Hap2 and identify wheat varieties with high 1000-grain weight. Primers designed based on the InDel molecular markers of the present invention only require simple PCR-specific amplification to distinguish wheat haplotypes and complete the identification of wheat with high 1000-grain weight. This facilitates the detection and screening of wheat varieties or lines with high 1000-grain weight and can greatly accelerate the breeding process of wheat varieties with high 1000-grain weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0026] Figure 1 The results of agarose gel electrophoresis detection are shown in Figure 2. (a) shows the detection results of the TaBTB-1D promoter region, from left to right they are M, Xuke 718, Xuke 718, Wenmai 18, and Wenmai 18; (b) shows the detection results of the TaBTB-1D coding region, from left to right they are M, Xuke 718, Xuke 718, Wenmai 18, and Wenmai 18;

[0027] Figure 2 The mutation sites and two haplotypes of the TaBTB-1D promoter region in common hexaploid wheat;

[0028] Figure 3 The comparison results of TaBTB-1D promoter sequences in two haplotypes of wheat are shown;

[0029] Figure 4 is the thousand-grain weight of TaBTB-1D in two haplotype wheat varieties; different lowercase letters indicate significant differences in the thousand-grain weight levels of different haplotype wheat varieties (p < 0.01);

[0030] Figure 5 The identification results of wheat using the molecular markers developed for the present invention; from left to right are 2000M, Xuke 718, Xuke 718, Wenmai 18 and Wenmai 18. DETAILED DESCRIPTION

[0031] The present invention provides an InDel molecular marker for distinguishing wheat haplotypes and / or identifying high 1000-grain weight wheat varieties, wherein the InDel molecular marker includes InDel-541 / -475;

[0032] The InDel-541 / -475 is located at -475bp in the promoter region of the wheat TaBTB-1D gene and contains a 67bp base deletion.

[0033] The 67 bp nucleotide sequence is shown as SEQ ID NO.1.

[0034] The present invention is based on the TaBTB-1D gene (TraesCS1D02G449100) in the wheat genome database, using 332 hexaploid wheat varieties as research objects. Based on the sequence differences of different wheat varieties in the wheat genome database, polymorphism analysis of the TaBTB-1D gene promoter region and coding region of different wheat varieties was performed. It was found that Hap2 contains a 67bp base deletion at -475bp in the TaBTB-1D gene promoter region compared to Hap1. The nucleotide sequence of the deletion is shown in SEQ ID NO. 1. Based on this site, it can be used to distinguish Hap1 from Hap2 and identify high 1000-grain weight wheat varieties. The nucleotide sequence of the promoter region of the TaBTB-1D gene in the wheat genome database is shown in SEQ ID NO. 4, and the nucleotide sequence of the coding region is shown in SEQ ID NO. 5.

[0035] The present invention also provides a primer for detecting the InDel molecular marker described in the above technical solution, wherein the primer comprises an upstream primer TaBTB-1D-SF and a downstream primer TaBTB-1D-SR;

[0036] The upstream primer TaBTB-1D-SF includes the nucleotide sequence shown in SEQ ID NO.2;

[0037] The downstream primer TaBTB-1D-SR includes the nucleotide sequence shown in SEQ ID NO.3.

[0038] The nucleotide sequences shown in SEQ ID NOs. 1 to 5 of the present invention are as follows:

[0039] SEQ ID NO.1: 5'-AACTAGTACCCAAGACACCGCTTGGACTAAGCGGTTAGCTATCTCAAAAACAATCAACAACTCAAAA-3';

[0040] SEQ ID NO.2: 5'-TGGTGGCGTTACATCGAT-3';

[0041] SEQ ID NO.3:5'-TTATGCAATGCATGCAACGA-3';

[0042]

[0043]

[0044] The present invention also provides the use of the InDel molecular markers or primers described in the above technical solution in distinguishing wheat haplotypes and / or identifying high 1000-grain weight wheat varieties. In the present invention, the wheat haplotype preferably includes the TaBTB-1D-Hap1 haplotype and / or the TaBTB-1D-Hap2 haplotype.

[0045] The present invention also provides the use of the InDel molecular marker or primer described in the above technical solution in wheat breeding. In the present invention, the wheat breeding preferably includes high thousand-grain weight wheat breeding.

[0046] The present invention also provides a method for distinguishing wheat haplotypes and / or identifying high 1000-grain weight wheat varieties, comprising the following steps:

[0047] PCR amplification is performed on the genomic DNA of the wheat to be tested using the primers described in the above technical solution to obtain a PCR amplification product;

[0048] Detecting the size of the PCR amplification product; if the size of the PCR amplification product is 370 bp, the wheat to be tested is of the TaBTB-1D-Hap1 haplotype, a low 1000-grain weight wheat variety;

[0049] If the size of the PCR amplification product is 303 bp, the wheat to be tested is of the TaBTB-1D-Hap2 haplotype, a high 1000-grain weight wheat variety.

[0050] In the present invention, the PCR amplification system preferably includes 25 μL of 2×KOD One™ PCR Master Mix, 0.2-0.5 μL of 10-20 μM upstream primer, 0.2-0.5 μL of 10-20 μM downstream primer, 200 ng of genomic DNA, and the balance of water, and further preferably includes 25 μL of 2×KOD One™ PCR Master Mix, 0.3 μL of 10 μM upstream primer, 0.3 μL of 10 μM downstream primer, 200 ng of genomic DNA, and the balance of water. The PCR amplification program of the present invention is preferably: 94°C for 3 min; 98°C for 10 s, 52°C for 20 s, 68°C for 3 min, 35 cycles; 68°C for 5 min. The method for detecting the size of the PCR amplification product of the present invention preferably includes agarose gel electrophoresis detection or sequencing.

[0051] Based on the above-mentioned InDel molecular markers or primers, the present invention only requires simple PCR-specific amplification to distinguish wheat haplotypes and complete the identification of high 1000-grain weight wheat, which facilitates the detection and screening of wheat varieties or lines with high 1000-grain weight and can greatly accelerate the breeding process of wheat varieties with high 1000-grain weight.

[0052] To further illustrate the present invention, the following detailed description of an InDel molecular marker for distinguishing wheat haplotypes and / or identifying high 1000-grain weight wheat and its application is provided by the present invention, in conjunction with the accompanying drawings and examples. However, these should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] 1. Materials

[0055] Study subjects: 332 hexaploid wheat varieties, with detailed information on varieties shown in Table 1;

[0056] Consumables: KOD One™ PCR Master Mix, a high-fidelity PCR enzyme used for PCR amplification, was purchased from Toyobo Bio.

[0057] 2. Primer Design

[0058] Based on the sequence differences of the TaBTB-1D gene in the wheat genome database, specific promoter amplification primers and coding region amplification primers were designed. The promoter amplification primers included the upstream primer TaBTB-1D-Promoter-SF and the downstream primer TaBTB-1D-Promoter-SR; the coding region amplification primers included the upstream primer TaBTB-1D-CDS-SF and the downstream primer TaBTB-1D-CDS-SF.

[0059] The above primers were synthesized by Henan Shangya Biotechnology Co., Ltd. The specific primer information is as follows:

[0060] TaBTB-1D-Promoter-SF: 5'-TGGTGGCGTTACATCGAT-3' (SEQ ID NO. 6);

[0061] TaBTB-1D-Promoter-SR: 5'-TCCTTGGCTCTTGAATAT-3' (SEQ ID NO. 7);

[0062] TaBTB-1D-CDS-SF: 5'-ATGGCAGAGCAATGCAAG-3' (SEQ ID NO. 8);

[0063] TaBTB-1D-CDS-SR: 5'-TCACATTTTCATGACAACCTCC-3' (SEQ ID NO. 9).

[0064] 3. Extraction of Wheat Genomic DNA

[0065] Total wheat DNA was extracted using the CTAB method: First, quick-freeze wheat leaves or other tissues in liquid nitrogen and grind them. Add 1 mL of preheated CTAB extraction buffer and incubate in a 65°C water bath for 1 hour. Centrifuge at 12,000 g for 10 minutes at room temperature. Remove the supernatant and add an equal volume of chloroform and isoamyl alcohol (chloroform:isoamyl alcohol ratio: 24:1 (v / v)) to the supernatant, then shake. Centrifuge at 12,000 g for 10 minutes, transfer the supernatant to a fresh tube, add 2 volumes of anhydrous ethanol, mix thoroughly, and let stand at -20°C for 30 minutes. Centrifuge again to remove the supernatant, wash twice with 75% ethanol, air-dry, and dissolve the DNA in 100 μL of sterile water to obtain total wheat DNA.

[0066] 4. Amplification and Sequencing of the TaBTB-1D Gene Promoter and Coding Region

[0067] The target fragment was amplified using the high-fidelity PCR enzyme KOD One™ PCR Master Mix. The TaBTB-1D promoter and coding region sequences were amplified using two primer pairs, TaBTB-1D-Promoter-SF / SR and TaBTB-1D-CDS-SF / SR, using total DNA from 332 hexaploid wheat lines as templates.

[0068] PCR system: 25 μL KOD One™ PCR Master Mix (2×), 0.3 μL each of upstream and downstream primers (10 μM), 200 ng DNA template, supplemented with ddH2O to a total volume of 50 μL;

[0069] PCR reaction program: first step, 94°C for 3 min; second step, 98°C for 10 s, 52°C for 20 s, 68°C for 3 min, 35 cycles; third step, 68°C for 5 min.

[0070] After the PCR was completed, agarose gel electrophoresis was performed, and some amplification results were as follows Figure 1 As shown. Figure 1 It can be seen that the promoter region of TaBTB-1D ( Figure 1 (a)) and coding region ( Figure 1 In (b), a single band was amplified.

[0071] 5. Sequence alignment and haplotype analysis of the TaBTB-1D gene promoter and coding region

[0072] Henan Shangya Biotechnology Co., Ltd. was commissioned to sequence the PCR amplification products. DNAMAN software (http: / / www.lynnon.com) was used to splice and align the sequencing results. The results showed that the coding region of TaBTB-1D in 332 wheat varieties was 1089 bp in length. In the promoter region, there were 30 SNP polymorphic variation sites (-605 bp to -120 bp), of which a large fragment was deleted from -541 bp to -475 bp. Some site information is as follows: Figure 2 Haplotype identification of the promoter was performed using DNAMAN software (http: / / www.ub.edu / DnaSP). The results showed that these 30 SNPs were closely linked to form two haplotypes, named TaBTB-1D-Hap1 and TaBTB-1D-Hap2. The wheat variety information corresponding to the haplotypes is shown in Table 1. The nucleotide sequence of TaBTB-1D-Hap1 is shown in SEQ ID NO.10; the nucleotide sequence of TaBTB-1D-Hap2 is shown in SEQ ID NO.11. The sequence differences between TaBTB-1D-Hap1 and TaBTB-1D-Hap2 are shown in Table 1. Figure 3 .

[0073]

[0074]

[0075] 6. Analysis of Thousand Kernel Weight (TKW) of Different Haplotypes of Wheat Varieties in Hexaploid Wheat

[0076] The thousand-grain weight of wheat varieties with different haplotypes was statistically analyzed. Detailed information is shown in Table 1. The data were analyzed and collated. The results showed that the average thousand-grain weight of wheat varieties with the Hap2 haplotype was significantly higher than that of the Hap1 haplotype, indicating that Hap2 is an excellent haplotype with high thousand-grain weight (Tables 2 and Figure 4 ).

[0077] Table 1 Wheat varieties and detailed information

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] Table 2 Correspondence between wheat haplotypes and 1000-grain weight

[0088]

[0089] 7. InDel molecular marker development

[0090] Sequence differences between the two TaBTB-1D haplotypes were analyzed based on insertion / deletion (InDel) differences. Compared to Hap1, Hap2 contains one InDel: a 67-bp deletion at -475bp, designated InDel-541 / -475. Therefore, InDel-541 / -475 can be used to distinguish Hap1 from Hap2, indicating a 67-bp difference between Hap1 and Hap2. The specific sequence is shown in SEQ ID NO. 1.

[0091] Based on the sequence differences here, specific molecular marker primers were designed (TaBTB-1D-SF: 5'-TGGTGGCGTTACATCGAT-3', SEQ ID NO.2; TaBTB-1D-SR: 5'-TTATGCAATGCATGCAACGA-3', SEQ ID NO.3;

[0092] Using DNA of different haplotype wheat varieties as templates, PCR amplification was performed using the method in step 4 to amplify TaBTB-1D fragments of different haplotype wheat varieties, and agarose gel electrophoresis was performed to detect the results. Figure 5 shown.

[0093] according to Figure 5 It can be seen that the amplification products of the Hap1 wheat variety and the Hap2 wheat variety can obtain single amplification bands, the band size of the Hap1 wheat variety is 370bp, and the band size of the Hap2 wheat variety is 303bp. The Hap1 wheat variety and the Hap2 wheat variety can be distinguished according to the band size.

[0094] Based on the above, it can be seen that the molecular markers developed in the present invention can distinguish wheat Hap1 and Hap2, and can be applied to the molecular design breeding of new wheat varieties with high 1000-grain weight.

[0095] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of InDel molecular markers or their detection primers in identifying high 1000-kernel weight wheat; The InDel molecular marker is InDel-541 / -475; the InDel-541 / -475 is located at -475 bp in the promoter region of the wheat TaBTB-1D gene, and has a 67 bp base deletion; the 67 bp nucleotide sequence is shown in SEQ ID NO.1; The detection primers include an upstream primer TaBTB-1D-SF and a downstream primer TaBTB-1D-SR; the nucleotide sequence of the upstream primer TaBTB-1D-SF is shown in SEQ ID NO.2; the nucleotide sequence of the downstream primer TaBTB-1D-SR is shown in SEQ ID NO.

3.

2. Application of InDel molecular markers or their detection primers in high 1000-grain weight wheat breeding; The InDel molecular marker is InDel-541 / -475; the InDel-541 / -475 is located at -475 bp in the promoter region of the wheat TaBTB-1D gene, and has a 67 bp base deletion; the 67 bp nucleotide sequence is shown in SEQ ID NO.1; The detection primers include an upstream primer TaBTB-1D-SF and a downstream primer TaBTB-1D-SR; the nucleotide sequence of the upstream primer TaBTB-1D-SF is shown in SEQ ID NO.2; the nucleotide sequence of the downstream primer TaBTB-1D-SR is shown in SEQ ID NO.

3.

3. A method for identifying high thousand-grain weight wheat varieties, characterized in that: The steps include: PCR amplification is performed on the genomic DNA of the wheat to be tested using the primers to obtain a PCR amplification product; Detecting the size of the PCR amplification product; if the size of the PCR amplification product is 370 bp, the wheat to be tested is a low thousand-grain weight wheat variety; If the size of the PCR amplification product is 303 bp, the wheat to be tested is a high thousand-grain weight wheat variety; The primers include an upstream primer TaBTB-1D-SF and a downstream primer TaBTB-1D-SR; The nucleotide sequence of the upstream primer TaBTB-1D-SF is shown in SEQ ID NO.2; the nucleotide sequence of the downstream primer TaBTB-1D-SR is shown in SEQ ID NO.

3.

4. The method according to claim 3, characterized in that The PCR amplification system, based on 50 μL, includes 25 μL of 2×KOD One™ PCR Master Mix, 0.2-0.5 μL of 10-20 μM upstream primer, 0.2-0.5 μL of 10-20 μM downstream primer, 200 ng of genomic DNA, and the remainder of water.

5. The method according to claim 3 or 4, characterized in that The PCR amplification program was as follows: 94°C for 3 min; 98°C for 10 s, 52°C for 20 s, 68°C for 3 min, 35 cycles; and 68°C for 5 min.

6. The method according to claim 3, characterized in that Methods for detecting the size of the PCR amplification product include agarose gel electrophoresis detection or sequencing.

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