Multiplex PCR (Polymerase Chain Reaction) molecular marker amplification system for identifying Waxy mutant gene of wheat, product and application thereof

Abstract

The invention relates to the technical field of molecular biology, in particular to a multiplex PCR (polymerase chain reaction) molecular marker amplification system for identifying Waxy mutant genes of wheat, a product and application of the multiplex PCR molecular marker amplification system. The multiple PCR molecular marker amplification system developed by the invention has the advantages of stable amplification and good specificity, realizes simultaneous genotype identification of three waxy genes Wx-A1, Wx-B1 and Wx-D1 through one-time PCR, is simple, convenient and rapid in detection process and high in flux, can accurately and efficiently screen out waxy wheat materials containing Waxy mutant genes, and has good application prospects. The waxy wheat breeding molecular marker-assisted selection efficiency and accuracy can be improved, the breeding process is shortened, and technical support can be provided for waxy wheat breeding work.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, in particular to a multiplex PCR molecular marker amplification system for identifying wheat Waxy mutant genes, a product and an application thereof. BACKGROUND

[0002] Starch is the main component of wheat kernels, which is composed of 20-30% amylose and 70-80% amylopectin. The whole waxy wheat has no or very low content of amylose, and has the characteristics of high water absorption, high peak viscosity, low gelatinization temperature, good freeze-thaw stability, etc. In food processing, waxy wheat flour is suitable for making cold storage, quick-frozen food and rice cake characteristic food. The use of waxy wheat flour can improve the comprehensive quality of noodles, improve the leavening characteristics of steamed buns, improve the yield and quality of brewing, effectively prolong the shelf life, etc. In industry, waxy wheat can play an important role in new resin, fuel ethanol, food packaging paper, industrial adhesive, etc.

[0003] Granule bound starch synthase I (GBSS I) is a key enzyme for controlling the synthesis of wheat kernel amylose, also known as Waxy protein. The wheat Waxy gene has three alleles of Wx-A1, Wx-B1 and Wx-D1, which are located on the 7AS, 4AL and 7DS chromosomes of the wheat genome, respectively. The absence of any one Waxy waxy gene directly affects the synthesis of GBSS, resulting in changes in the content of kernel amylose.

[0004] Multiplex PCR can identify multiple gene sites in the same reaction system, greatly saving time and reagents, and has the characteristics of high efficiency and economy. However, the primers of multiplex PCR are not a simple mixture of single PCR primers. When designing primers, the non-complementarity between primers, the complementarity between primers and DNA templates, the consistency of primer annealing temperature, and the difference of amplified fragments need to be considered, which further affects the application of multiplex PCR. According to the conservation and specificity of the wild type and mutant sequences of the three Waxy genes, a multiplex PCR system is designed, which can accurately, quickly and efficiently detect the waxy gene mutation of wheat and speed up the breeding process of waxy wheat in China.

[0005] Current waxy wheat molecular markers have the problems of multiple detection times, Wx-B1 site as a dominant marker, etc., which leads to low selection efficiency, high detection cost and poor marker effectiveness, and cannot detect the mutation of three waxy genes at one time, accurately and effectively. The present application aims to develop a multiplex PCR molecular marker system for identifying waxy wheat, which can genotype the three Waxy genes at the same time, so as to accurately, quickly and efficiently screen waxy wheat materials containing Waxy mutant genes and shorten the breeding process, in order to provide technical support for waxy wheat breeding. SUMMARY

[0006] The application aims to provide a multiplex PCR molecular marker amplification system for identifying a wheat Waxy mutant gene, a product and an application thereof, so as to solve the problems in the prior art.

[0007] To achieve the above-mentioned object, the application provides the following solutions.

[0008] The application provides a multiplex PCR molecular marker amplification system for identifying a wheat Waxy mutant gene, which comprises a Wx-ABD primer pair, a Wx-B primer pair and a Wx-D primer pair.

[0009] The Wx-ABD primer pair comprises Wx-ABD-F with a nucleotide sequence as shown in SEQ ID NO. 1 and Wx-ABD-R with a nucleotide sequence as shown in SEQ ID NO. 2.

[0010] The Wx-B primer pair comprises Wx-B-F with a nucleotide sequence as shown in SEQ ID NO. 3 and Wx-B-R with a nucleotide sequence as shown in SEQ ID NO. 4.

[0011] The Wx-D primer pair comprises Wx-D-1F with a nucleotide sequence as shown in SEQ ID NO. 5, Wx-D-2F with a nucleotide sequence as shown in SEQ ID NO. 6 and Wx-D-R with a nucleotide sequence as shown in SEQ ID NO. 7.

[0012] Further preferably, the Wx-ABD molecular marker is co-located with the wheat Wx-A1, Wx-B1 and Wx-D1 genes on the wheat 7A, 4A and 7D chromosomes respectively.

[0013] The Wx-B molecular marker is co-located with the wheat Wx-B1 gene on the wheat 4A chromosome.

[0014] The Wx-D molecular marker is co-located with the wheat Wx-D1 gene on the wheat 7D chromosome.

[0015] The application provides an application of the multiplex PCR molecular marker amplification system described above in the preparation of a product for identifying a wheat Waxy mutant gene.

[0016] Preferably, the product comprises reagents, kits and chips.

[0017] The application provides a product for identifying a wheat Waxy mutant gene, which comprises the multiplex PCR molecular marker amplification system described above.

[0018] Preferably, the product comprises reagents, kits and chips.

[0019] The application provides application of the multiplex PCR molecular marker amplification system or the product in any of the following aspects:

[0020] (1) identifying the content of amylose in wheat;

[0021] (2) identifying the type of waxy wheat;

[0022] (3) detecting the mutation of the Waxy gene in wheat;

[0023] (4) screening the type of waxy wheat;

[0024] (5) molecular marker assisted breeding of waxy wheat;

[0025] (6) improvement of germplasm resources of waxy wheat;

[0026] (7) breeding of wheat high amylopectin traits and other excellent traits of wheat;

[0027] (8) preparation of a product for identifying the content of amylose in wheat;

[0028] (9) preparation of a product for identifying the type of waxy wheat;

[0029] (10) preparation of a product for detecting the mutation of the Waxy gene in wheat;

[0030] (11) preparation of a product for screening the type of waxy wheat.

[0031] The application provides a method for identifying the content of amylose in wheat, comprising the following steps:

[0032] The genomic DNA of the wheat to be detected is used as a template, the template is subjected to PCR amplification by using the multiplex PCR molecular marker amplification system, and the content of amylose in the wheat to be detected is determined according to the PCR amplification result.

[0033] Preferably, the determination result is shown in the following table:

[0034]

[0035] The application provides a method for identifying the type of wheat, comprising the following steps:

[0036] The genomic DNA of the wheat to be detected is used as a template, the template is subjected to PCR amplification by using the multiplex PCR molecular marker amplification system, and the type of the wheat to be detected is determined according to the PCR amplification result.

[0037] Preferably, the determination result is shown in the following table:

[0038]

[0039] The present invention discloses the following technical effects:

[0040] This invention provides a multiplex PCR molecular marker amplification system for identifying the wheat Waxy mutant gene. The multiplex PCR molecular marker amplification system includes a Wx-ABD primer pair, a Wx-B primer pair, and a Wx-D primer pair. The Wx-ABD primer pair includes Wx-ABD-F with the nucleotide sequence shown in SEQ ID NO.1 and Wx-ABD-R with the nucleotide sequence shown in SEQ ID NO.2. The Wx-B primer pair includes Wx-BF with the nucleotide sequence shown in SEQ ID NO.3 and Wx-BR with the nucleotide sequence shown in SEQ ID NO.4. The Wx-D primer pair includes Wx-D-1F with the nucleotide sequence shown in SEQ ID NO.5, Wx-D-2F with the nucleotide sequence shown in SEQ ID NO.6, and Wx-DR with the nucleotide sequence shown in SEQ ID NO.7. The multiplex PCR molecular marker amplification system developed in this invention has the advantages of stable amplification and good specificity. It enables genotyping of three glutinous wheat genes, Wx-A1, Wx-B1 and Wx-D1, in a single PCR. The detection process is simple, rapid and has high throughput. It can accurately and efficiently screen glutinous wheat materials containing Waxy mutant genes, improve the efficiency and accuracy of molecular marker-assisted selection in glutinous wheat breeding, shorten the breeding process, and provide technical support for glutinous wheat breeding. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 The image shows the genotyping results of Example 1; where M is the DNA marker, samples 1-14 are 14 near-isogenic lines of the Waxy gene, and sample 15 is Shijiazhuang No. 8.

[0043] Figure 2 The image shows the genotyping results of the self-crossed offspring in Example 2; where M is the marker and the remaining lanes represent the partial self-crossed offspring. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0048] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0049] Example 1: Obtaining and validating the genotyping effect of multiplex PCR molecular markers

[0050] 1. Material Background

[0051] This invention utilizes a near-isogenic Waxy line with the background of Shijiazhuang No. 8 as material. This material was provided by the Germplasm Resource Bank of Hebei Agricultural University, which has committed to distributing it to the public for 20 years from the date of application. It comprises a total of 8 genotypes (AAA, AAB, ABA, BAA, ABB, BBA, BAB, and BBB). The wild-type (AAA) of Shijiazhuang No. 8 has an amylose content of 21.5%.

[0052] 2. Tag Development

[0053] Based on the conservation and specificity of the wild-type and mutant sequences of Wx-A1, Wx-B1, and Wx-D1 genes, and using the three Waxy gene sequences on the Chinese Spring (IWGSC RefSeq v2.1) Chr7A, Chr4A, and Chr7D chromosomes as reference sequences, multiple sets of PCR primers were designed using Primer Premier5 software to establish a multiplex PCR molecular marker amplification system containing 3 sets (Wx-ABD, Wx-B, and Wx-D) and 7 primers. The nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.7, respectively.

[0054] Wx-ABD-F: 5'-ACCTCGTGTTCGTCGGC-3', SEQ ID NO.1;

[0055] Wx-ABD-R: 5'-GCGTCCTTGTACTGGTCGTAG-3', SEQ ID NO.2;

[0056] Wx-BF: 5'-TGGTTGTTTTCCCATGGCAT-3', SEQ ID NO.3;

[0057] Wx-BR: 5'-GTGGTTATCAGAGTTGCACAGT-3', SEQ ID NO.4;

[0058] Wx-D-1F: 5'-CTCTGGTCTGGTTTAGGATGCA-3', SEQ ID NO.5;

[0059] Wx-D-2F: 5'-TGCTATTGGTTCTCCGTGTTT-3', SEQ ID NO.6;

[0060] Wx-DR: 5'-TTGGCGGTCATCTGTCATTT-3', SEQ ID NO.7.

[0061] 3. Primer synthesis

[0062] The primers were sent to Beijing Liuhe BGI Genomics Co., Ltd. for primer synthesis.

[0063] 4. Verification of the typing effect of the multiplex PCR molecular marker amplification system

[0064] The near-isogenic line of Shijiazhuang No. 8 was detected using a developed multiplex PCR molecular marker amplification system. The specific method is as follows:

[0065] (1) DNA extraction

[0066] Using Shijiazhuang No. 8 and 14 near-isogenic lines of the Waxy gene from the background of Shijiazhuang No. 8 (samples 1-14) as materials, genomic DNA was extracted from wheat seedling leaves of each material. The DNA concentration and purity were detected using a micro-visible / ultraviolet spectrophotometer (NanoDrop 2000), and the OD was measured. 260 / 280 A ratio between 1.7 and 2.2 indicates high DNA quality; dilute to 100 ng / μL template for later use.

[0067] (2) Preparation of PCR amplification reaction system

[0068] PCR amplification system preparation (15 μL): 1 μL 100 ng / μL template DNA, 7.5 μL 2×Taq PCR Master Mix, 1.47 μL primer system, and ddH2O water added to a total volume of 15 μL; the primer system is as follows: Wx-ABD-F 0.25 μL, Wx-ABD-R 0.25 μL, Wx-BF 0.3 μL, Wx-BR 0.3 μL, Wx-D-1F 0.12 μL, Wx-D-2F 0.1 μL, and Wx-DR 0.15 μL, with each primer concentration of 100 μM.

[0069] (3) Perform PCR amplification on the genomic DNA to be tested.

[0070] The prepared system was subjected to PCR amplification. The PCR amplification reaction program was as follows: 94℃ heat activation for 5 min; 94℃ denaturation for 30 s, 63-60℃ annealing for 50 s, 72℃ extension for 30 s (10 touch-down cycles, decreasing the temperature by 0.3℃ each cycle); 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 20 cycles, and a final extension at 72℃ for 7 min, then stored at 24℃.

[0071] (4) Judgment of typing results

[0072] The PCR products were analyzed by 6% polyacrylamide gel electrophoresis for 65 min. After gel development, the gels were observed and photographed under a light box. The genotyping results are as follows: Figure 1As shown, the electrophoretic products (lane 15) of parent P of Shijiazhuang No. 8 show four bands of 227bp, 244bp, 271bp, and 235bp, indicating that the three waxy genes Wx-A1, Wx-B1, and Wx-D1 of this wheat are all wild-type, and this genotype is denoted as AAA. The electrophoretic products (lanes 1, 2, and 6) of near-isogenic lines 1, 2, and 6 show four bands of 208bp, 311bp, 185bp, and 235bp, indicating that the three waxy genes Wx-A1, Wx-B1, and Wx-D1 of this wheat are all homozygous mutants, and this near-isogenic line genotype is denoted as BBB. The electrophoretic products (lanes 3 and 9) of near-isogenic lines 3 and 9 show bands of 227bp, 244bp, 271bp, 235bp, 244bp, 271 ... If four bands of 71bp and 235bp are observed in the electrophoretic products of near-isogenic lines 4, 7, and 11 (lanes 4, 7, and 11) are observed, indicating that the three glutinous genes Wx-A1, Wx-B1, and Wx-D1 are all wild-type, the genotype of this near-isogenic line is AAA. If four bands of 227bp, 311bp, 185bp, and 235bp are observed in the electrophoretic products of near-isogenic lines 5, 7, and 13 (lanes 5 and 13), indicating that the three glutinous genes Wx-A1, Wx-B1, and Wx-D1 are wild-type, the genotype of this near-isogenic line is ABB. If four bands of 208bp, 244bp, 185bp, and 235bp are observed in the electrophoretic products of near-isogenic lines 5 and 13 (lanes 5 and 13), the three glutinous genes Wx-A1, Wx-B1, and Wx-D1 are wild-type, the genotype of this near-isogenic line is ABB. The wheat Wx-B1 glutinous gene is identified as wild-type, and both Wx-A1 and Wx-D1 glutinous genes are mutants. The genotype of this near-isogenic line is denoted as BAB. The electrophoretic products (lane 8) of near-isogenic line plant 8 show four bands of 208bp, 311bp, 271bp, and 235bp. Therefore, the Wx-D1 glutinous gene is identified as wild-type, and both Wx-A1 and Wx-B1 glutinous genes are mutants. The genotype of this near-isogenic line is denoted as BBA. The electrophoretic products (lane 10) of near-isogenic line plant 10 show four bands of 227bp, 311bp, 271bp, and 235bp. Therefore, both Wx-A1 and Wx-D1 glutinous genes are identified as wild-type. The x-B1 glutinous gene is a mutant, and the genotype of this near-isogenic line is denoted as ABA; the electrophoretic products (lane 12) of the near-isogenic line plant 12 show four bands of different sizes: 208bp, 244bp, 271bp, and 235bp. Therefore, the Wx-A1 glutinous gene of this wheat is determined to be a mutant, while the Wx-B1 and Wx-D1 glutinous genes are both wild-type. The genotype of this near-isogenic line is denoted as BAA; the electrophoretic products (lane 14) of the near-isogenic line plant 14 show four bands of different sizes: 227bp, 244bp, 185bp, and 235bp. Therefore, the Wx-D1 glutinous gene of this wheat is determined to be a mutant, while the Wx-A1 and Wx-B1 glutinous genes are wild-type. The genotype of this near-isogenic line is denoted as AAB.

[0073] (5) Determination of amylose content

[0074] The grains of the near-isogenic line Shijiazhuang No. 8 were ground into flour, and the amylose content was determined using an amylose analyzer as follows: Shijiazhuang No. 8 parental line 21.5%, whole glutinous wheat BBB 1.4%, near-isogenic line AAA 21.3%, BAA 19.9%, AAB 19.8%, ABA 19.5%, BAB 16.6%, BBA 15.6%, and ABB 14.0%.

[0075] The multiplex PCR molecular markers developed in this invention produce excellent genotyping results with clear delineation of different genotypes and high resolution. Measurements of amylose content in different near-isogenic lines show that the markers produce completely consistent genotyping results for both waxy wheat and the parent variety Shijiazhuang 8, effectively distinguishing the Wx genotype. This demonstrates the successful development of the multiplex PCR molecular marker amplification system, which can be used to identify the amylose content of wheat and to determine wheat type. Furthermore, this multiplex PCR molecular marker amplification system can be further applied to high-throughput detection of breeding materials and marker-assisted breeding in large-scale production.

[0076] Example 2: Application of Multiplex PCR Molecular Marker System

[0077] Using Tiannuo 158, a glutinous wheat material, as the donor of the glutinous gene, and the improved variety Shimai 22 as the recurrent parent, hybridization was carried out. The multiplex PCR molecular marker amplification system established in Example 1 was used to test the hybrid offspring. Single plants containing the three glutinous mutant genes Wx-A1, Wx-B1, and Wx-D1 were retained. These were then backcrossed with Shimai 22. Each backcross offspring was tested using the multiplex PCR molecular marker amplification system of Example 1. Single plants containing the three glutinous mutant genes were retained. After three generations of backcrossing, the single plants containing the three glutinous mutant genes were self-pollinated. The self-pollinated seeds were tested using the multiplex PCR molecular marker amplification system of Example 1. Figure 2 By selecting single plants with three homozygous genotypes of glutinous mutants, and after field planting, propagation and agronomic trait evaluation, the Shimai 22 variety was successfully improved into glutinous wheat, and is named Shimai 22 glutinous wheat.

[0078] After grinding the grains of Shimai 22 parent and Shimai 22 glutinous wheat, the amylose content was measured using an amylose analyzer. The amylose content of Shimai 22 parent was 21.4%, and the amylose content of Shimai 22 glutinous wheat was 1.2%, with almost no amylose. This indicates that the multiplex PCR molecular marker amplification system developed in this invention has successfully directionally improved Shimai 22 into a fully glutinous wheat.

[0079] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A multiplex PCR molecular marker amplification system for identifying the wheat Waxy mutant gene, characterized in that, The multiplex PCR molecular marker amplification system includes Wx-ABD primer pairs, Wx-B primer pairs, and Wx-D primer pairs; The Wx-ABD primer pair includes Wx-ABD-F with the nucleotide sequence shown in SEQ ID NO.1 and Wx-ABD-R with the nucleotide sequence shown in SEQ ID NO.2; The Wx-B primer pair includes Wx-BF with the nucleotide sequence shown in SEQ ID NO.3 and Wx-BR with the nucleotide sequence shown in SEQ ID NO.4; The Wx-D primer pair includes Wx-D-1F with nucleotide sequence as shown in SEQ ID NO.5, Wx-D-2F with nucleotide sequence as shown in SEQ ID NO.6, and Wx-DR with nucleotide sequence as shown in SEQ ID NO.

7.

2. The application of the multiplex PCR molecular marker amplification system according to claim 1 in the preparation of products for identifying wheat Waxy mutant genes.

3. The application according to claim 2, characterized in that, The products include reagents, reagent kits, and chips.

4. A product for identifying wheat Waxy mutant genes, characterized in that, The product includes the multiplex PCR molecular marker amplification system as described in claim 1.

5. The product according to claim 4, characterized in that, The products include reagents, reagent kits, and chips.

6. The use of the multiplex PCR molecular marker amplification system of claim 1 or the product of claim 4 or 5 in any of the following: (1) To determine the level of amylose content in wheat; (2) Identify the type of glutinous wheat; (3) Detect the mutation status of the wheat Waxy gene; (4) Screening for different types of glutinous wheat; (5) Molecular marker-assisted breeding of glutinous wheat; (6) Germplasm resource improvement of glutinous wheat; (7) Polymerization breeding between wheat high amylopectin trait and other desirable traits in wheat; (8) Prepare products for identifying the amylose content of wheat; (9) Prepare products for identifying types of glutinous wheat; (10) Prepare products for detecting mutations in the wheat Waxy gene; (11) Prepare products for screening glutinous wheat types.

7. A method for determining the level of amylose content in wheat, characterized in that, Includes the following steps: Using the genomic DNA of the wheat to be tested as a template, the template is amplified by PCR using the multiplex PCR molecular marker amplification system described in claim 1, and the amylose content of the wheat to be tested is determined by the PCR amplification results.

8. The method according to claim 7, characterized in that, The judgment results are shown in the table below: 。 9. A method for identifying wheat types, characterized in that, Includes the following steps: Using the genomic DNA of the wheat to be tested as a template, the template is amplified by PCR using the multiplex PCR molecular marker amplification system described in claim 1, and the type of wheat to be tested is determined by the PCR amplification results.

10. The method according to claim 9, characterized in that, The judgment results are shown in the table below: 。

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