InDel molecular marker on wheat TaTT8 gene promoter and application of InDel molecular marker
By designing PCR primer pairs A and B, the promoter type of the wheat TaTT8 gene was identified using PCR amplification technology, which solved the problem of distinguishing between purple and white wheat in the seedling stage and enabled rapid and accurate identification of germplasm resources and breeding guidance.
Patent Information
- Application Number
- CN202511855337.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-10
- Publication Date
- 2026-02-06
AI Technical Summary
Existing technologies make it difficult to quickly distinguish between purple and white wheat during the seedling stage, and there is a lack of effective molecular markers to identify the type of TaTT8 gene promoter, which affects the innovative breeding of new germplasm resources of purple wheat.
This invention provides a method to detect the InDel molecular marker on the promoter of the wheat TaTT8 gene. By designing specific PCR primer pairs A and B, the type of the TaTT8 gene promoter can be identified using PCR amplification technology. Combined with the size and sequence analysis of the PCR product, purple-grained and white-grained wheat can be distinguished.
This technology enables rapid and accurate identification of purple and white wheat during the seedling stage, improving the breeding efficiency of new germplasm resources and ensuring the accuracy of anthocyanin content and expression levels.
Smart Images
Figure CN121472455A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to wheat. TaTT8 InDel molecular markers on gene promoters and their applications. Background Technology
[0002] wheat( Triticum aestivum Purple wheat is one of the world's three major staple foods, and improving its nutritional quality has become a key goal of modern breeding. The unique characteristic of purple wheat lies in the accumulation of anthocyanins in its seed coat, which gives the wheat grains their purple color. Anthocyanins are water-soluble flavonoid compounds found in plants and are secondary metabolites. Besides providing vibrant color, anthocyanins also possess powerful antioxidant, anti-inflammatory, and cell-damage-repair-promoting functions.
[0003] Currently, research on purple wheat mainly focuses on the determination of anthocyanin content and types, the isolation of genes related to anthocyanin synthesis, and chromosome localization. The unique anthocyanin components abundant in purple wheat have great potential and far-reaching research value in promoting human health.
[0004] Developing molecular markers that can be used to quickly distinguish between purple wheat and white wheat varieties (white wheat) in the seedling stage can provide strong technical support and resource reserves for the innovative breeding of new germplasm resources. Summary of the Invention
[0005] To quickly identify purple wheat during the seedling stage, this invention first provides a method for detecting wheat... TaTT8 The substance marked with InDel molecules on a gene promoter, wherein the InDel molecular marker is a DNA molecule with a nucleotide sequence as shown in positions 37-1048 of SEQ ID No. 3.
[0006] The substance is either A or B as follows: A. The substance contains PCR primer pair A for amplifying a wheat genomic DNA fragment including the DNA molecule shown in positions 37-1048 of SEQ ID No. 3; B. The substance contains PCR primer pair B for amplifying a wheat genomic DNA fragment including the DNA molecule shown at positions 37-1332 of SEQ ID No. 3.
[0007] The PCR primer pair A consists of a forward primer and a reverse primer. The forward primer is a single-stranded DNA that specifically binds to the upstream of the double-stranded DNA shown in positions 37-1048 of SEQ ID No. 3 in wheat genomic DNA, and the reverse primer is a single-stranded DNA that specifically binds to the downstream of the double-stranded DNA shown in positions 37-1048 of SEQ ID No. 3 in wheat genomic DNA.
[0008] The PCR primer pair B consists of a forward primer and a reverse primer. The forward primer is a single-stranded DNA that specifically binds to the upstream of the double-stranded DNA shown in SEQ ID No. 3, positions 37-1332 of wheat genomic DNA. The reverse primer is a single-stranded DNA that specifically binds to the downstream of the double-stranded DNA shown in SEQ ID No. 3, positions 37-1332 of wheat genomic DNA.
[0009] In the above-mentioned substances, the forward primer in primer pair A or primer pair B is a single-stranded DNA with the nucleotide sequence of SEQ ID No. 3, and the reverse primer is a single-stranded DNA with the nucleotide sequence of SEQ ID No. 4.
[0010] The above substances are for detecting wheat TaTT8 The InDel molecular marker system on the gene promoter consists of X1 and X2; wherein X1 is primer pair A or primer pair B, and X2 is reagents and / or instruments required for PCR amplification.
[0011] In the above system, the reagents required for PCR amplification may contain dNTPs of dATP, dTTP, dCTP and dGTP, DNA polymerase and / or PCR reaction buffer, or may only be the dNTP mixture, the DNA polymerase and / or the PCR reaction buffer; the instrument required for PCR amplification may be a PCR instrument.
[0012] In the above system, primer pair A or primer pair B and the reagents required for PCR amplification can all be packaged independently. The two single-stranded DNA molecules in primer pair A or primer pair B can be packaged independently. All reagents required for PCR amplification can be packaged independently.
[0013] To address the aforementioned technical problems, the present invention also provides a method for identifying wheat. TaTT8 Methods for gene promoter types.
[0014] The wheat identification method provided by this invention TaTT8 The method for gene promoter type, wherein the wheat to be tested is a homozygous inbred line, and the TaTT8 The gene promoter type is TT8-P or TT8-W, and the method is as follows: I or II: I. Including K1) and K2): K1) Using the wheat genomic DNA to be tested as a template, the substance was used to perform PCR amplification to obtain PCR products; K2) Detect the PCR product obtained in step K1), and determine the wheat to be tested based on the PCR product. TaTT8 Gene promoter types: The PCR product contains the InDel molecular marker in the wheat being tested. TaTT8 The gene promoter type is TT8-P; the PCR product does not contain the InDel molecular marker in the tested wheat. TaTT8 Gene promoter type TT8-W; II. Including L1) and L2): L1) Using the wheat genomic DNA to be tested as a template, PCR amplification was performed using a primer pair consisting of a forward primer and a reverse primer to obtain the PCR product; the forward primer is the single-stranded DNA shown in SEQ ID No:3 of the sequence listing, and the reverse primer is the single-stranded DNA shown in SEQ ID No:4 of the sequence listing; L2) (L21) or L22 below): L21) Detect the size of the PCR product obtained in step L1), and determine the wheat to be tested based on the size of the PCR product. TaTT8 Gene promoter types: The PCR product contained only a 1332bp DNA fragment of the wheat being tested. TaTT8 The gene promoter type is TT8-P; the PCR product contains only a 320bp DNA fragment of the wheat being tested. TaTT8 The gene promoter type is TT8-W; L22) Detect the sequence of the PCR product obtained in step L1), and determine the promoter type of the wheat TaTT8 gene to be tested based on the PCR product: The PCR product contains only the DNA fragment shown in positions 1 to 1332 of SEQ ID No:1, and does not contain the DNA fragment shown in positions 1 to 320 of SEQ ID No:2, from the wheat being tested. TaTT8 The gene promoter type is TT8-P; the PCR product contains only the DNA fragment shown in positions 1 to 320 of SEQ ID No:2, and does not contain the DNA fragment shown in positions 1 to 1332 of SEQ ID No:1, of the wheat being tested. TaTT8 The gene promoter type is TT8-W.
[0015] The above identification TaTT8 In the gene promoter type method, the PCR amplification system for performing the PCR amplification may contain dNTPs of dATP, dTTP, dCTP and dGTP, DNA polymerase and / or PCR reaction buffer.
[0016] The above identification TaTT8In the gene promoter type method, the reaction conditions for PCR amplification can be: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 15 s, 59℃ annealing for 10 s, 68℃ extension at a rate of 10 s / kb, 36 cycles; 72℃ extension for 6 min, and 12℃ incubation.
[0017] The above identification TaTT8 In the method of gene promoter type, the TaTT8 Wheat with TT8-P gene promoter type and TaTT8 Compared to wheat with the TT8-W gene promoter type, TaTT8 The gene expression level is high, and the anthocyanin content is high. TaTT8 Wheat with the TT8-P gene promoter type is or is a candidate for purple-grained wheat. TaTT8 Wheat with the TT8-W gene promoter type is or is a candidate for white-grained wheat.
[0018] To address the aforementioned technical problems, the present invention also provides a method for identifying or assisting in the identification of wheat grain color, the method comprising the following steps S1 and S2: S1. Identify the wheat to be tested using the method described above. TaTT8 Gene promoter type; S2, according to TaTT8 Gene promoter type determines the grain color of the wheat being tested: TaTT8 Wheat with the TT8-P gene promoter type is or is a candidate for purple-grained wheat. TaTT8 Wheat with the TT8-W gene promoter type is or is a candidate for white-grained wheat.
[0019] The PCR amplification system used in the above-described method for identifying or assisting in the identification of homozygous wheat grain color can be used for the above-described identification. TaTT8 The system for PCR amplification using the gene promoter type method, and the reaction conditions for PCR amplification can be as described above for identification. TaTT8 The reaction conditions for performing the PCR amplification in the gene promoter type method.
[0020] To address the aforementioned technical problems, this invention also provides methods for identifying or assisting in the identification of wheat. TaTT8 Methods for measuring the expression level and / or anthocyanin content in seeds.
[0021] The present invention provides identification or auxiliary identification of wheat. TaTT8 Methods for determining the expression level and / or anthocyanin content in seeds, including steps T1 and T2: T1. Identify the wheat to be tested using the method described above. TaTT8 Gene promoter type; T2, according to TaTT8Gene promoter type determines the wheat being tested TaTT8 Expression levels and / or anthocyanin content in seeds: TaTT8 Wheat with the TT8-P gene promoter type, and TaTT8 Compared to wheat with the TT8-W gene promoter type, TaTT8 It has a high expression level and a high anthocyanin content in the grains.
[0022] The above-mentioned identification or auxiliary identification of homozygous wheat TaTT8 The PCR amplification system used in the method for determining the expression level and / or anthocyanin content of seeds can be used for the above identification. TaTT8 The system for PCR amplification using the gene promoter type method, and the reaction conditions for PCR amplification can be as described above for identification. TaTT8 The reaction conditions for performing the PCR amplification in the gene promoter type method.
[0023] To address the aforementioned technical problems, this invention also provides primer pairs for identifying or assisting in the identification of wheat grain color.
[0024] The primer pair provided by this invention for identifying or assisting in the identification of wheat grain color is primer pair A or primer pair B.
[0025] To address the aforementioned technical problems, this invention also provides methods for identifying or assisting in the identification of wheat. TaTT8 Primer pairs for expression levels and / or anthocyanin content in seeds.
[0026] The identification or auxiliary identification of wheat provided by this invention TaTT8 The primer pair for determining the expression level and / or anthocyanin content of the seeds is either primer pair A or primer pair B.
[0027] To address the aforementioned technical problems, the present invention also provides any one of the following applications H1-H17: H1, The molecular markers are used in the identification or auxiliary identification of wheat. TaTT8 Applications in gene promoter types; H2. The molecular markers are used in the identification or auxiliary identification of wheat. TaTT8 Application in grain color; H3. The molecular markers are used in the identification or auxiliary identification of wheat. TaTT8 Application in expression levels and / or anthocyanin content in grains; H4. Application of the molecular markers in wheat breeding; H5. The substance described herein is used in the identification or auxiliary identification of wheat. TaTT8 Applications in gene promoter types; H6. The substance described herein is used in the identification or auxiliary identification of wheat. TaTT8 Application in grain color; H7. The substance described herein is used in the identification or auxiliary identification of wheat. TaTT8 Application in expression levels and / or anthocyanin content in grains; H8. Application of the aforementioned substances and molecular markers in wheat breeding; H9, the identification of wheat TaTT8 Application of gene promoter type methods in identifying or assisting in the identification of wheat grain color; H10, the identification of wheat TaTT8 Methods for identifying or assisting in the identification of wheat promoter types TaTT8 Application in expression levels and / or anthocyanin content in grains; H11, the identification of wheat TaTT8 Application of gene promoter type methods in wheat breeding; H12. The application of the method for identifying or assisting in identifying wheat grain color in wheat breeding; H13, the identification or auxiliary identification of wheat TaTT8 Application of methods for measuring anthocyanin expression levels and / or grain anthocyanin content in wheat breeding; H14, The primer pair is used in the preparation, identification, or auxiliary identification of wheat. TaTT8 Application in reagents or kits for determining expression levels and / or anthocyanin content and / or seed color; H15, the primer pair is used in the identification or auxiliary identification of wheat. TaTT8 Applications in gene promoter types; H16, the primer pair is used in the identification or auxiliary identification of wheat. TaTT8 Application in the expression level and / or anthocyanin content and / or grain color; H17. Application of the primer pair in wheat breeding.
[0028] In the above applications, the TaTT8 Gene promoter type is TT8-P wheat, and TaTT8 Gene promoter type is TT8-W Compared to wheat, TaTT8 The expression level is high, the anthocyanin content in the grains is high, and the grains are purple.
[0029] To address the aforementioned technical problems, this invention also provides a wheat breeding method.
[0030] The wheat breeding method provided by this invention, according to the identification... TaTT8 Methods for identifying wheat gene promoter types TaTT8 Gene promoter type, selection TaTT8 Gene promoter type is TT8-W Wheat was used as a parent for breeding.
[0031] The wheat breeding described in this article aims to select purple-grained wheat and / or wheat with high anthocyanin content.
[0032] In this invention, the molar ratio of the two single-stranded DNA molecules in primer pair A or primer pair B can be 1:1.
[0033] In this invention, the size of the PCR product can be detected by electrophoresis. A PCR product containing a 1332 bp DNA fragment can show a band between 1200 bp and 2000 bp. A PCR product containing a 320 bp DNA fragment can show a band between 200 bp and 500 bp.
[0034] In this invention, the wheat to be tested is a homozygous self-crossing line.
[0035] Experiments have shown that the molecular markers of this invention can be used for identification or auxiliary identification during the seedling stage. TaTT8 Gene promoter type / TaTT8 The expression level and / or anthocyanin content and / or grain color, to have different TaTT8 Gene promoter type / TaTT8 Using the wheat genome as a template, and employing the primer pairs of this invention for PCR amplification, two PCR products are obtained corresponding to different expression levels and / or anthocyanin content and / or grain color. TaTT8 Gene promoter type / TaTT8 The expression level and / or anthocyanin content and / or grain color of wheat, when the PCR product of wheat contains only the DNA fragment shown in positions 37-1048 of SEQ ID No:1, wheat exhibits [characteristics]. TaTT8 Gene promoter type TT8-P and / or TaTT8 When the expression level of the protein is high and / or the anthocyanin content in the grains is high and / or the grain color is purple, and the PCR product of wheat does not contain the DNA fragment shown in positions 37-1048 of SEQ ID No:1, the wheat exhibits the following characteristics: TaTT8 Gene promoter type TT8-W and / or TaTT8 The expression level is low and / or the anthocyanin content in the grains is low and / or the grain color is white. Attached Figure Description
[0036] Picture 1 This is a graph showing the GWAS correlation analysis results in Example 1 of this application. Picture 1 A is a GWAS Manhattan plot, with the threshold set to p<10. -6 ; Picture 1 B is the threshold where p < 10 -8 Genes within the SNP locus region.
[0037] Picture 2 The results show the expression levels of seven genes within the GWAS screening region in Example 1 of this application in six white-grained wheat materials and six purple-grained wheat materials.
[0038] Picture 3 In Example 1 of this application TaTT8 Fragment gene analysis and clone electrophoresis diagram. Picture 3 A represents the value of A in the TAIR Arabidopsis database. TraesCS2A02G409400 The results of the analysis and comparison; Picture 3 B is TaTT8 Electrophoretic strips.
[0039] Picture 4 For example, the purple wheat gene and white wheat gene in Example 1 of this application. TaTT8 Sequence alignment of the gene and its upstream genes. Picture 4 A is a comparison of purple wheat and white wheat (white wheat) using DNAMAN software. TaTT8 Gene coding region; Picture 4 B is a DNAMAN software comparison of purple wheat. TaTT8 The upstream sequence of the gene promoter corresponds to the sequence of white wheat (white wheat). Picture 4 C represents the molecular marker size display for purple wheat and white wheat; Picture 4 D represents the molecular marker model for purple wheat and white wheat, and F and R are the up and down indices of the molecular marker sites, respectively. In the figure, purple wheat is labeled as... TT8 proP, corresponding to TaTT8 Gene promoter type is TT8-P White wheat is labeled as TT8 proW, corresponding TaTT8 Gene promoter type is TT8-W .
[0040] Picture 5 For the molecular marker identification or auxiliary identification of wheat in Embodiment 2 of this application. TaTT8 Results of gene expression levels. Picture 5 A is TaTT8 The high-altitude 115 gene with the TT8-P promoter type (i.e., GY115) and TaTT8 Fielder genes with TT8-W promoter type in different tissues TaTT8 Gene expression level detection; Picture 5 B is TaTT8 Yuzhou Rye No. 1 (i.e., YH1) with TT8-P promoter type and TaTT8 Jimai 22 (i.e., JM22) with TT8-W gene promoter type in different tissues TaTT8 Gene expression level detection; Picture 5C is in 6 TaTT8 Wheat with a TT8-P gene promoter type and 6 TaTT8 In the coleoptile of wheat with the TT8-W gene promoter type TaTT8 Expression level detection; Picture 5 D is TaTT8 In 6 TaTT8 Wheat with a TT8-P gene promoter type and 6 TaTT8 In wheat leaves with the TT8-W gene promoter type TaTT8 Expression level detection. Data are expressed as mean ± SD of three biological replicates. Different letters above the bars represent significant differences. p< 0.05. The significance analysis was performed using a t-test, with *** indicating a significant difference. p <0.001, ** indicates that the result of the significance analysis is not significant. p <0.01, * indicates that the result of the significance analysis is not significant. p <0.05.
[0041] Picture 6 The differences in Embodiment 2 of this application are as follows TaTT8Anthocyanin content in wheat grains of gene promoter types. In the figure, Anthocyanidin_15: cyanidin-3-O-sophoroside; Anthocyanidin_11: cyanidin-3-O-glucoside; Anthocyanidin_05: cyanidin-3-O-(6-O-malonyl-β-D-glucoside); Anthocyanidin_149: cyanidin-3-O-sinosinolate-(diglucoside); Anthocyanidin_30: delphinidin-3-O-morula disaccharide; Anthocya nidin_32: delphinidin-3-O-rutin-5-O-glucoside; Anthocyanidin_31: delphinidin-3-O-sophoroside; Anthocyanidin_29: delphinidin-3-O-rutinoside; Anthocyanidin_27: delphinidin-3-O-glucoside; Anthocyanidin_47: malvidin-3-(6-Op-coumaroyl)-glucoside; Anthocyanidin_51: malvidin-3 -O-glucoside; Anthocyanidin_396: pelargonidin-3,5-O-digalactoside; Anthocyanidin_68: pelargonidin-3-O-glucoside; Anthocyanidin_88: peonidin-3-O-rutinoside; Anthocyanidin_78: peonidin-3,5-O-diglucoside; Anthocyanidin_86: peonidin-3-O-glucoside; Anthocyanidin_320: Paeoniflorin-3-O-(6''-O-acetyl)galactoside; Anthocyanidin_80: Paeoniflorin-3-(6-Op-p-coumaryl)-glucoside; Anthocyanidin_98: Petunidin-3-O-glucoside; Anthocyanidin_99: Petunidin-3-O-rutinoside; Anthocyanidin_107: Proanthocyanidin B3; Anthocyanidin_41: Quercetin-3-O-glucoside (isoquercetin). Detailed Implementation
[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0043] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.
[0044] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.
[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0046] The white wheat Fielder described in the following examples is described in the non-patent literature “Zhang S, Zhang R, Gao J, Song G, Li J, Li W, Qi Y, Li Y, Li G. CRISPR / Cas9-mediated genome editing for wheat grain quality improvement. Plant Biotechnol J. 2021 Sep;19(9):1684-1686. doi: 10.1111 / pbi.13647. Epub 2021 Jul 5. PMID: 34143557; PMCID:PMC8428824.”, which can be obtained by the public from the applicant to repeat the experiments of the present invention.
[0047] The white-grained wheat Zhengmai 7698 described in the following examples is described in the non-patent literature "Zhao Weiqin, Wang Haifeng, Wu Changcheng, et al. Production technology of original seed of strong gluten wheat Zhengmai 7698 [J]. China Seed Industry, 2020, (08): 81-83. DOI: 10.19462 / j.cnki.1671-895x.2020.08.025.", which can be obtained from the applicant to repeat the experiment of the present invention.
[0048] The white-grained wheat variety Bainong 607 described in the following examples is described in the non-patent literature “Li Xinhua, Qiao Hong, Ou Xingqi, et al. Breeding and application of short-stalked, stress-resistant, and high-yielding wheat variety Bainong 607 [J]. Agricultural Science and Technology Communications, 2024, (07): 183-185.” The public can obtain it from the applicant to repeat the experiments of this invention.
[0049] The white-grained wheat Jimai 22 described in the following examples is described in the non-patent literature “Shang Juanhua, Yin Qilin, Chen Lei, et al. Preparation of protoplasts and establishment of transient expression system for ‘Jimai 22’ [J]. Molecular Plant Breeding, 2025, 23(13):4363-4367.DOI:10.13271 / j.mpb.023.004363.”, which can be obtained from the applicant to repeat the experiments of this invention.
[0050] The white wheat variety Anke 1802 described in the following examples has the approval number Wan Shenmai 20210049 and is available for public purchase.
[0051] The white wheat variety Luomai 55 described in the following examples has the approval number Guoshenmai 20253010 and is available for public purchase.
[0052] The white-grained wheat varieties Luomai 50, Zhengmai 1905, Zhaofeng 35, Pumai 157, Tianmai 189, and Xinmai 67 described in the following examples are described in the non-patent literature “Song Zhijun, Xue Zhiwei, Shen Weimin. Comprehensive evaluation of new wheat varieties in the Huanghuai winter wheat region based on the grey multidimensional comprehensive membership method [J]. Heilongjiang Agricultural Sciences, 2025(5):31-36. DOI:10.11942 / j.issn1002-2767.2025.05.0031.”, which can be obtained from the applicant to repeat the experiments of this invention.
[0053] The purple-grained wheat variety Yuzhou Black Wheat No. 1 described in the following examples is recorded in the non-patent literature “Zhang Kunpu, Bi Huihui, Shi Cuilan, et al. Characteristics and cultivation techniques of the new colored wheat variety Yuzhou Black Wheat No. 1 [J]. Modern Agricultural Science and Technology, 2025, (15): 183-185+189.” The public can obtain it from the applicant to repeat the experiments of the present invention.
[0054] The purple wheat plateau 115 described in the following examples is described in the non-patent literature "Ye Guangji, Zhang Bo, Chen Wenjie, et al. Cloning and functional analysis of R2R3-MYB transcription factor TaMYB3-4A in purple wheat plateau 115 [J]. Molecular Plant Breeding, 2016, 14(08):1940-1947.DOI:10.13271 / j.mpb.014.001940.", which can be obtained by the public from the applicant to repeat the experiments of the present invention.
[0055] In the following examples, the approval number of the purple wheat variety Xinping No. 1 is Yu Shen Mai 20243003, and it is available for purchase by the public.
[0056] The purple-grained wheat variety Jiheimai No. 1 described in the following examples is recorded in the non-patent literature “Peng Tao, Zhao Weifeng, Gao Yan, et al. Breeding and cultivation techniques of the new specialty wheat variety Jiheimai No. 1 [J]. Seed Industry Guide, 2025, (01): 36-39.” The public can obtain it from the applicant to repeat the experiments of this invention.
[0057] The purple wheat variety Taike Rye No. 1 described in the following examples is described in the non-patent literature “Zhang Zhonghua. New specialty wheat variety Taike Rye No. 1 [J]. Agricultural Knowledge, 2020, (18): 28-29.” The public can obtain it from the applicant to repeat the experiment of the present invention.
[0058] The purple-grained wheat variety Ziyou 1 described in the following examples is documented in the non-patent literature “Zhang Wei, Zhang Pengfei, Zhang Shuai. Comprehensive evaluation of aphid resistance and photosynthetic characteristics of 32 wheat germplasm resources [J]. Agricultural Science and Technology Communications, 2016(9):165-170. DOI:10.3969 / j.issn.1000-6400.2016.09.062.”, which can be obtained by the public from the applicant to repeat the experiments of this invention.
[0059] The purple-grained wheat variety Ziyou 1 described in the following examples is documented in the non-patent literature “Zhang Wei, Zhang Pengfei, Zhang Shuai. Comprehensive evaluation of aphid resistance and photosynthetic characteristics of 32 wheat germplasm resources [J]. Agricultural Science and Technology Communications, 2016(9):165-170. DOI:10.3969 / j.issn.1000-6400.2016.09.062.”, which can be obtained by the public from the applicant to repeat the experiments of this invention.
[0060] The purple wheat variety Luoheimai No. 1 in the following examples has the approval number Yu Shen Mai 20230043 and is available for public purchase.
[0061] Example 1: Key genes for anthocyanin synthesis in purple wheat TATT8 and its molecular markers 1. Genes related to anthocyanin synthesis in purple wheat The laboratory previously collected 60 purple wheat accessions and 20 white wheat accessions and constructed natural populations. Using a 55K microarray combined with genome-wide association analysis (GWAS), gene regions regulating the phenotype of purple wheat were located. A threshold was set at... p <10 -6 The results showed that the significantly associated region was located on chromosome 2A, which contained eight SNP loci ( Picture 1 A). Due to the extremely high significance of the first two SNP sites ( p <10 -8 Since the regions are geographically close, this area was chosen for preliminary analysis. Considering potential signal attenuation, the analysis region was extended by 0.5 Mb at both ends. Combined with the average genetic distance between 55 K chip markers being 0.1 cM, the genomic difference region between purple and white wheat was ultimately narrowed down to 1.11 Mb, containing a total of 7 genes (…). Picture 1 B).
[0062] For the above 7 genes, RNA was extracted from 6 white-grained wheat varieties (Luomai 55, Pumai 127, Tianmai 157, Luomai 50, Pumai 175, Annong 188) and 6 purple-grained wheat varieties (Xinping 1, Jiheimai 1, Luoheimai 1, Dongheimai 1, Yunonghei 925, Yuzhouheimai 1) using RNAex Pro RNA reagent SYBR® Green Pro TaqHS Premix (catalog number AG21101) from Akeri Biotechnology Co., Ltd., and reverse transcribed into cDNA for qRT-PCR detection (using a LightCycler 480 real-time PCR instrument, Roche). The internal control gene used was TaActin. Among them, for the genes specifically mentioned above... TraesCS2A02G409400 The primers used for the gene (synthesized by Beijing Qingke Biotechnology Co., Ltd.) are q-TraesCS2A02G409400.1-F and q-TraesCS2A02G409400.1-R: q-TraesCS2A02G409400.1-F: 5'-GAGAGCTATTCGCAGGCCAT-3'; q-TraesCS2A02G409400.1-R: 5'-GGCTTCTGCATCTAGGCAATG-3'.
[0063] The primers used for the internal reference gene TaActin are q-TaActin-F and q-TaActin-R: q-TaActin-F: 5'-TACTCCCTCACAACAACC-3'; q-TaActin-R: 5'-GCTCCTGCTCATAATCAAG-3'. The results show that only TraesCS2A02G409400 Gene expression is more stable in all purple wheat than in white wheat. Picture 2 ).
[0064] Through the TAIR Arabidopsis database ( https: / / www.arabidopsis.org / The comparison revealed that it is similar to At4G09820.1 ( AtTT8 The highest homology is shown in the figure (marked as TT8). Picture 3 A). A previous study reported... AtTT8 This gene is associated with the synthesis and accumulation of anthocyanins in Arabidopsis seed coats; mutations in this gene result in white seeds in Arabidopsis. It is speculated that this gene is also associated with the synthesis and accumulation of anthocyanins in purple wheat. TraesCS2A02G409400 Named TaTT8 And based on the Ensembl Plants database ( https: / / plants.ensembl.org / index.htmL The encoded region sequence (1707 bp) downloaded from ).
[0065] 2. Purple wheat and white wheat TaTT8 Gene sequence alignment 2.1 Purple wheat and white wheat TaTT8 CDS sequence comparison TaTT8 It is closely related to the color of wheat seed coat. When TaTT8 When the gene is highly expressed, the seed coat of white wheat exhibits a purple seed phenotype. Therefore, it is hypothesized that there may be mutations or deletions in the base sequence of this gene between purple and white wheat, leading to changes in the protein sequence and ultimately causing phenotypic differences. To verify this, 33 purple-grained and 24 white-grained varieties were collected, and... TaTT8 Base sequencing and alignment were performed on the coding region of the gene.
[0066] PCR products were analyzed by agarose gel electrophoresis (1% concentration gel) to determine band size. DNA fragments were then extracted from the gel and recovered, following the instructions of the Tiangen Agarose Gel DNA Recovery Kit. Wheat samples were sequenced by Beijing Qingke Biotechnology Co., Ltd. TaTT8 The CDS sequence of the gene showed that in all wheat varieties TaTT8 The coding region of the gene has the same base sequence, with no differences. Picture 1 A).
[0067] 2.2 Purple wheat and white wheat TaTT8 Comparison of promoter sequences Sequencing alignment TaTT8 Upstream sequences revealed that white-grained wheat... TaTT8 There are no differences in the sequence promoter regions, and the differences between purple wheat varieties are not significant. TaTT8 There were no differences in the sequence promoter regions. However, purple wheat TaTT8 The upstream sequence is shown in SEQ ID No:1, white wheat. TaTT8 The upstream sequence is shown in SEQ ID No:2, in which the purple wheat variety has a 261 bp long base sequence, which is repeated 4 times consecutively in purple wheat and once incompletely repeated at a length of 207 bp. Picture 1 The 261 bp base sequence appears only once in white-grained wheat varieties (Section B). The difference between the two is that, relative to SEQ ID No:1, SEQ ID No:2 lacks 1012 bp between positions 37 and 1048 of SEQ ID No:1. This is considered to be a distinguishing feature from purple-grained wheat. TaTT8 Promoters and white wheat TaTT8The promoter is marked with the InDel molecular marker. Sequence alignment of the promoter outside the differential regions showed no differences between different wheat varieties. Therefore, the sequence from positions 37 to 1048 of SEQ ID No:1 can serve as one of the characteristics distinguishing purple wheat from white wheat.
[0068] Based on the above results, primers targeting this molecular marker were designed: the forward primer binding sites are located in purple wheat and white wheat, respectively. TaTT8 The first 1737 bp and 725 bp of the gene start codon (ATG) (marked as F in the figure); the reverse primer binding site is located at TaTT8 The first 405 bp of the gene start codon (ATG) (marked as R in the figure) were used to design the forward primer TaTT8-pP / WF and the reverse primer TaTT8-pP / WR.
[0069] TaTT8-pP / WF: 5'-CAAGACTTTCGCAGTTACACT-3' (as shown in SEQ ID No:3, it is the same as the first to 21st positions of SEQ ID No:1, and the same as the first to 21st positions of SEQ ID No:2); TaTT8-pP / WR: 5'-ACGCAACATCAAAGTACTGTG -3' (as shown in SEQ ID No:4, it is the reverse complementary bit to bits 1312 to 1332 of SEQ ID No:1, and the reverse complementary bit to bits 300 to 320 of SEQ ID No:2).
[0070] The PCR products were analyzed by agarose gel electrophoresis (1% concentration gel) to determine band size. Purple wheat was expected to produce a band of approximately 1332 bp, with its PCR product sequence corresponding to positions 1-1332 of SEQ ID No:1, including 1012 bp from positions 37-1048 of SEQ ID No:1, indicating an insertion type. This promoter type was named TT8-P. White wheat was expected to produce a band of 320 bp, with its PCR product sequence corresponding to positions 1-320 of SEQ ID No:2, excluding 1012 bp from positions 37-1048 of SEQ ID No:1, indicating a deletion type. This promoter type was named TT8-W; see [link to relevant documentation]. Picture 4 C and Picture 4 The D. This molecular marker system allows for direct molecular identification during the seedling stage, distinguishing between purple and white wheat, without relying on long reproductive periods to observe grain phenotypes.
[0071] Example 2: Application of Molecular Markers 1. Identification or auxiliary identification of purple wheat using molecular markers. Using 33 purple-grained wheat and 24 white-grained wheat seedlings as materials, genomic DNA was extracted from the seedlings. This DNA was then used as a template for PCR amplification using primers TaTT8-pP / WF and TaTT8-pP / WR. The PCR products were analyzed by agarose gel electrophoresis (1% concentration gel) to determine band size; a band of approximately 1332 bp was expected for purple-grained wheat. TaTT8 The gene promoter type is TT8-P; a band of approximately 320 bp is expected to be obtained from white-grained wheat. TaTT8 The gene promoter type is TT8-W; The PCR system is shown in Table 1, and the PCR reaction procedure is shown in Table 2. Table 1 PCR system
[0072] Among them, 2×KOD One PCR Master Mix (Catalog No.: KMM-201) is a 2× premixed reagent developed based on modified KOD DNA polymerase (UKOD) and is a product of Toyobo (Shanghai) Biotechnology Co., Ltd.
[0073] Table 2 PCR reaction procedure
[0074] The results are shown in Table 3: Table 3 Results of molecular marker identification of wheat seedlings
[0075] 2. TaTT8 Wheat with different gene promoter types TaTT8 Differences in gene expression levels To explore TaTT8 Does the difference in gene promoter type lead to TaTT8 Differences in gene expression levels were selected from those in Table 3. TaTT8 Yuzhou rye 1 and Gaoyuan 115, whose gene promoter types are TT8-P, and TaTT8 RNA was extracted from roots, stems, leaves, embryos, endosperm, and seed coats of Fielder and Jimai 22 varieties with the TT8-W gene promoter type 15-17 days after flowering, and detected by qRT-PCR. TaTT8 Gene expression levels.
[0076] The results showed that, TaTT8In various parts of the TT8-P gene promoter type (Yuzhou Rye No. 1 and Gaoyuan 115), the seed coat TaTT8 Expression was high, with the most significant differences observed in the seed coat region. Meanwhile... TaTT8 In gene promoters of type TT8-W (Fielder and Jimai 22), various parts TaTT8 Low expression level ( Picture 5 A and Picture 5 (B), and this phenomenon is consistent with the phenotype of wheat grain color.
[0077] To further verify the molecular marker differentiation TaTT8 Gene promoter type pair TaTT8 The effect of expression levels was investigated by qRT-PCR analysis on six purple wheat varieties (Kezi 1, Xinping 1, Jiheimai 1, Taikeheimai 1, Ziyou 1, and Luoheimai 1) and six white wheat varieties (Shangnong 5, Kenong 9204, Luomai 54, Bainong 607, Annong 1802, and Pumai 157). TaTT8 Differences in gene expression. The results showed that... TaTT8 In wheat with the TT8-P gene promoter type TaTT8 The expression level was significantly higher than TaTT8 Wheat with a gene promoter type of TT8-W ( Picture 5 (C). And in leaf tissue, both... TaTT8 There was no difference in gene expression levels. Picture 5 D).
[0078] 3. TaTT8 Anthocyanin content of wheat with TT8-P gene promoter type Select 20 from Table 3 TaTT8 Wheat grains with the TT8-P gene promoter type (Shu Zimai 1801, Ke Zi 1, Xinping 1, Mian Zimai 830, Ji Heimai 1, Tai Ke Heimai 1, Luo Heimai 1, Ziyou 5, Yuzhou Heimai 1, Yong Heimai 1, Nongda 375, Shang Zi 1, Cun Heimai 1, Dong Hei 1206, Yunong Hei 924, Yunong Hei 925, Yuzhou Heimai 2, Zhong Zimai 3, Keda Q124, Zheng Heimai 6131) and 3 others TaTT8 Wheat grains (Zhengmai 7698, Jimai 22, and Bainong 207) with the TT8-W gene promoter type were analyzed for metabolites.
[0079] The results are as follows Picture 6As shown, nine anthocyanins were detected in 20 varieties. Among them, the anthocyanins shared among varieties and present in relatively high amounts included: cyanidin-3-O-glucoside, cyanidin-3-O-(6-O-malonyl-β-D-glucoside), malvidin-3-(6-Op-p-coumaryl)-glucoside, and peonidin-3-(6-Op-p-coumaryl)-glucoside. (3) TaTT8 Wheat grains with the TT8-W gene promoter type contain lower levels of anthocyanins, mainly malvidin-3-(6-Op-p-coumaryl)-glucoside and peonidin-3-(6-Op-p-coumaryl)-glucoside.
[0080] Analysis results show that the molecular markers of the present invention can identify or assist in the identification of wheat grain color and anthocyanin content, and can also identify or assist in the identification of wheat. TaTT8 Gene expression levels.
[0081] In summary, TaTT8 The gene is a key gene in the production and accumulation of anthocyanins in the seed coat of purple wheat, promoting the biosynthesis and accumulation of anthocyanins, thus giving the seed coat its purple color. Utilizing... TaTT8 Molecular markers upstream of the gene can be used to screen for purple-grained wheat during the seedling stage, significantly improving breeding efficiency.
[0082] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. Testing wheat TaTT8 The substance marked with InDel molecules on the gene promoter is characterized by: The InDel molecular marker is a DNA molecule with a nucleotide sequence as shown in positions 37-1048 of SEQ ID No.
3.
2. The substance according to claim 1, characterized in that: The substance is either A or B as follows: A. The substance contains PCR primer pair A for amplifying a wheat genomic DNA fragment including the DNA molecule shown in positions 37-1048 of SEQ ID No. 3; B. The substance contains PCR primer pair B for amplifying a wheat genomic DNA fragment including the DNA molecule shown at positions 37-1332 of SEQ ID No.
3.
3. The substance according to claim 2, characterized in that: The PCR primer pair A consists of a forward primer and a reverse primer. The forward primer is a single-stranded DNA that specifically binds to the upstream of the double-stranded DNA shown in positions 37-1048 of SEQ ID No. 3 in wheat genomic DNA, and the reverse primer is a single-stranded DNA that specifically binds to the downstream of the double-stranded DNA shown in positions 37-1048 of SEQ ID No. 3 in wheat genomic DNA. The PCR primer pair B consists of a forward primer and a reverse primer. The forward primer is a single-stranded DNA that specifically binds to the upstream of the double-stranded DNA shown in positions 37-1332 of SEQ ID No. 3 in wheat genomic DNA, and the reverse primer is a single-stranded DNA that specifically binds to the downstream of the double-stranded DNA shown in positions 37-1332 of SEQ ID No. 3 in wheat genomic DNA.
4. The substance according to claim 3, characterized in that: The forward primer in primer pair A or primer pair B is a single-stranded DNA with the nucleotide sequence of SEQ ID No. 3, and the reverse primer is a single-stranded DNA with the nucleotide sequence of SEQ ID No.
4.
5. Identifying wheat TaTT8 The method for gene promoter types is characterized by: The wheat to be tested is a homozygous inbred line. TaTT8 The gene promoter type is TT8-P or TT8-W, and the method is as follows: I or II: I. Including K1) and K2): K1) Using the wheat genomic DNA to be tested as a template, PCR amplification was performed using any of the substances described in claims 1-4 to obtain PCR products; K2) Detect the PCR product obtained in step K1), and determine the wheat to be tested based on the PCR product. TaTT8 Gene promoter types: The PCR product contains the InDel molecular marker of the wheat sample as described in claim 1. TaTT8 The gene promoter type is TT8-P; The PCR product does not contain the InDel molecular marker described in claim 1 in the tested wheat. TaTT8 Gene promoter type TT8-W; II. Including L1) and L2): L1) Using the wheat genomic DNA to be tested as a template, PCR amplification was performed using a primer pair consisting of a forward primer and a reverse primer to obtain the PCR product; the forward primer is the single-stranded DNA shown in SEQ ID No:3 of the sequence listing, and the reverse primer is the single-stranded DNA shown in SEQ ID No:4 of the sequence listing; L2) (L21) or L22 below): L21) Detect the size of the PCR product obtained in step L1), and determine the wheat to be tested based on the size of the PCR product. TaTT8 Gene promoter types: The PCR product is a DNA fragment of the wheat being tested, ranging from greater than 1200 bp to less than 2000 bp (e.g., 1332 bp). TaTT8 The gene promoter type is TT8-P; The PCR product is a DNA fragment of the wheat being tested, ranging from a value greater than 200 bp to a value less than 500 bp (e.g., 320 bp). TaTT8 The gene promoter type is TT8-W; L22) Detect the sequence of the PCR product obtained in step L1), and determine the promoter type of the wheat TaTT8 gene to be tested based on the PCR product: The PCR product contains only the DNA fragment shown in positions 1 to 1332 of SEQ ID No:1, and does not contain the DNA fragment shown in positions 1 to 320 of SEQ ID No:2, from the wheat being tested. TaTT8 The gene promoter type is TT8-P; the PCR product contains only the DNA fragment shown in positions 1 to 320 of SEQ ID No:2, and does not contain the DNA fragment shown in positions 1 to 1332 of SEQ ID No:1, of the wheat being tested. TaTT8 The gene promoter type is TT8-W.
6. A method for identifying or assisting in the identification of wheat grain color, characterized in that: The method includes the following steps S1 and S2: S1. Identifying the wheat to be tested according to the method of claim 5. TaTT8 Gene promoter type; S2, according to TaTT8 Gene promoter type determines the grain color of the wheat being tested: TaTT8 Wheat with the TT8-P gene promoter type is or is a candidate for purple-grained wheat. TaTT8 Wheat with the TT8-W gene promoter type is or is a candidate for white-grained wheat.
7. Identification or auxiliary identification of wheat TaTT8 The method for determining the expression level and / or anthocyanin content in seeds, characterized in that: The method includes steps T1 and T2: T1. Identifying the wheat to be tested according to the method of claim 5. TaTT8 Gene promoter type; T2, according to TaTT8 Gene promoter type determines the wheat being tested TaTT8 Expression levels and / or anthocyanin content in seeds: TaTT8 Wheat with the TT8-P gene promoter type, and TaTT8 Compared to wheat with the TT8-W gene promoter type, TaTT8 It has a high expression level and a high anthocyanin content in the grains.
8. Primer pairs for identifying or assisting in the identification of wheat grain color, or for identifying or assisting in the identification of wheat... TaTT8 Primer pairs for expression levels and / or anthocyanin content in seeds, characterized in that: The primer pair is either primer pair A or primer pair B as described in any of claims 2-4.
9. Any application from H1 to H18: H1. The molecular marker described in claim 1 in the identification or auxiliary identification of wheat TaTT8 Applications in gene promoter types; H2. The molecular marker described in claim 1 in the identification or auxiliary identification of wheat TaTT8 Application in grain color; H3. The molecular marker described in claim 1 in the identification or auxiliary identification of wheat TaTT8 Application in expression levels and / or anthocyanin content in grains; H4. The application of the molecular markers described in claim 1 in wheat breeding; H5. The substance described in any one of claims 1-4 in the identification or auxiliary identification of wheat TaTT8 Applications in gene promoter types; H6. The substance described in any one of claims 1-4 in the identification or auxiliary identification of wheat TaTT8 Application in grain color; H7. The substance described in any one of claims 1-4 in the identification or auxiliary identification of wheat TaTT8 Application in expression levels and / or anthocyanin content in grains; H8. The application of the molecular markers described in any one of claims 1-4 in wheat breeding; H9, the wheat identification method according to claim 5 TaTT8 Application of gene promoter type methods in identifying or assisting in the identification of wheat grain color; H10, the wheat identification method according to claim 5 TaTT8 Methods for identifying or assisting in the identification of wheat promoter types TaTT8 Application in expression levels and / or anthocyanin content in grains; H11, the wheat identification method according to claim 5 TaTT8 Application of gene promoter type methods in wheat breeding; H12. The application of the method for identifying or assisting in the identification of wheat grain color as described in claim 6 in wheat breeding; H13, the identification or auxiliary identification of wheat as described in claim 7 TaTT8 Application of methods for measuring anthocyanin expression levels and / or grain anthocyanin content in wheat breeding; H14, The primer pair of claim 8 is used in the preparation, identification, or auxiliary identification of wheat. TaTT8 Application in reagents or kits for determining expression levels and / or anthocyanin content and / or seed color; H15, The primer pair according to claim 8 is used in the identification or auxiliary identification of wheat. TaTT8 Applications in gene promoter types; H16, The primer pair of claim 8 is used in the identification or auxiliary identification of wheat. TaTT8 Application in the expression level and / or anthocyanin content and / or grain color; H17. Application of the primer pair according to claim 8 in wheat breeding.
10. A wheat breeding method, characterized by: The identification according to claim 5 TaTT8 Methods for identifying wheat gene promoter types TaTT8 Gene promoter type, selection TaTT8 Gene promoter type is TT8-W Wheat was used as a parent for breeding.