Substance for detecting or assisting in detecting grain number per ear of wheat and application of substance

Abstract

The invention discloses a substance for detecting or assisting in detecting the grain number per ear of wheat and application of the substance. The invention aims to solve the problem of how to quickly and accurately identify or assist in identifying the grain number of wheat ears. According to the application, a substance for detecting the polymorphism or genotype of an SNP site in a wheat genome is applied to identification or auxiliary identification of the wheat grain number per ear, the SNP site is located in a wheat 7B chromosome TaFT-B1 gene, a single nucleotide polymorphism (SNP) site exists at the + 19 bp position of a coding region of the TaFT-B1 gene, amino acid in the coding region is changed, and therefore the function of TaFT-B1 protein is affected. The nucleotide is C or G, and is the 364th nucleotide of the sequence 1 in the sequence table. The marker is good in repeatability, convenient to detect and low in cost, and has a good application prospect in auxiliary selection and molecular design breeding for improving the grain number per ear of wheat.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a substance for detecting or assisting in detecting the number of wheat ears and an application thereof. Background Art

[0002] wheat( Triticum aestivum As one of the most important food crops in the world, its yield directly affects human food security. The number of grains per ear is one of the key factors determining wheat yield. Therefore, exploring the gene for the number of grains per ear and developing effective molecular markers are of great significance to improving wheat yield.

[0003] In recent years, the rapid development of molecular biology and genomics has enabled us to have a deeper understanding of the molecular regulatory mechanism of wheat grain number. Through the study of wheat genetic resources, scientists have found that multiple genes and their regulatory networks play an important role in the formation of wheat grain number. For example, overexpression TaPAP2 , TaVRS1 It can shorten the time of spikelet differentiation, reduce the number of spikelets, florets and grains per spike (Wang et al., 2017). LEAFY and WAPO 1 interact and jointly regulate the number of spikelets and floret development in wheat (Paraiso et al., 2024). In addition TaMYB30-A1 The mutation in the promoter region affected the transcriptional inhibition of WFZP. TaMYB30-A 1 The superior haplotype has a higher expression level, increases spike length, the number of fertile spikelets and the number of grains per spike, and is selected during wheat domestication and breeding (lin et al., 2024). TaSus1 After gene mutation, compared with the wild-type Fielder, the mutant's ear length, ear weight, number of fertile spikelets, number of grains per ear, and thousand-grain weight were significantly reduced (Shen et al., 2024).

[0004] In addition to the above genes, the molecular mechanism of regulating wheat grain number also involves the regulation of plant hormone signaling pathways. Studies have shown that hormones such as abscisic acid (ABA) and cytokinin (CK) play a key role in the development of grains. Abscisic acid is closely related to the abortion of wheat florets; while cytokinin indirectly affects the development of grain number by regulating the cell cycle and prolonging cell growth time (Dai et al., 1998).

[0005] In addition to genetic factors and hormone regulation, the influence of environmental factors on wheat grain number cannot be ignored. For example, under drought conditions, the number of wheat grains tends to decrease significantly, which is closely related to changes in hormone levels and regulation of gene expression in plants (Zhang et al., 2020). Therefore, future research should focus more on the interaction between environmental factors and molecular mechanisms to fully understand the regulatory mechanism of wheat grain number.

[0006] In summary, the regulatory mechanism of wheat grain number per ear is a complex network involving the interaction of genes, hormones and transcription factors. Future research can further improve wheat yield through methods such as gene editing and molecular breeding. In addition, the combination of advanced technologies such as marker-assisted selection (MAS) and genomic selection (GS) is expected to achieve more efficient genome improvement in wheat breeding. Therefore, the discovery of wheat grain number genes and the study of their molecular mechanisms will provide an important theoretical basis and practical guidance for achieving high-yield, high-quality and stress-resistant wheat breeding. Summary of the invention

[0007] The technical problem to be solved by the present invention is how to quickly and accurately identify and screen the number of wheat grains.

[0008] In order to solve the above problems, the present invention first provides an application of a substance for detecting the polymorphism or genotype of a SNP site in a wheat genome, wherein the SNP site is a site in the wheat genome, the nucleotide type of which is C or G, and is the 364th nucleotide of sequence 1 in the sequence table; the application is any of the following: A1) Use of the substance in identifying or assisting in identifying the number of grains per ear of wheat, A2) Application of the substance in wheat breeding, A3) Use of the substance in the preparation of a product for identifying or assisting in identifying the number of grains in a wheat ear, A4) Use of the substance in preparing wheat breeding products.

[0009] The substance may be the following B1), B2) or B3): B1) the substance is a primer composition for amplifying a wheat genomic DNA fragment including the SNP site, B2) The substance is a PCR reagent containing the primer combination described in B1), B3) The substance is a kit containing the primer composition described in B1) or the PCR reagent described in B2).

[0010] The SNP single nucleotide polymorphism site is a SNP in the wheat genome, which is the 364th nucleotide of SEQ ID No.1 in the sequence list, which is C or G; located on chromosome 7B of wheat TaFT-B1 In genes, TaFT-B1 Genes linked to grain number per ear in wheat.

[0011] The wheat grain number per ear refers to the number of wheat grains per ear of wheat.

[0012] In the above applications, the primer composition may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Markers include, but are not limited to, dyes; radioactive labels, such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight measurement, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The marker can include a nucleic acid sequence or a protein sequence or a combination thereof, as long as the sequence containing the marker is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly reading the sequence) without a marker.

[0013] The present invention also provides a method for identifying or assisting in identifying the number of wheat grains per ear, the method comprising detecting the genotype of the above-mentioned SNP site in the genome of the wheat to be tested, and identifying or assisting in identifying the number of wheat grains per ear according to the genotype.

[0014] In the above-mentioned method for identifying or assisting in identifying the number of wheat grains per ear, the genotype is CC or GG, the CC is the homozygous type when the SNP site is C, and the GG is the homozygous type when the SNP site is G. According to the genotype, the identification or assisting in identifying the number of wheat grains per ear can be that the average number of grains per ear of the wheat to be tested with the genotype of CC is higher than that of the wheat to be tested with the genotype of GG.

[0015] The present invention also provides a method for wheat breeding, which comprises detecting the genotype of the above-mentioned SNP site in the wheat genome, and selecting wheat whose genotype of the SNP site is CC or GG as a parent for breeding, wherein the CC is a homozygous type when the SNP site is C, and the GG is a homozygous type when the SNP site is G.

[0016] In the above-mentioned wheat breeding method, the wheat breeding index includes the number of wheat grains per ear.

[0017] Furthermore, the purpose of wheat breeding includes cultivating wheat with a high number of wheat grains per ear. The high number of wheat grains per ear means that the average number of wheat grains per ear is higher than that of the parent.

[0018] In the above method for identifying or assisting in identifying the number of wheat grains per ear and the above method for wheat breeding, the wheat may be an inbred line or a pure line.

[0019] The invention also provides application of the method in wheat breeding.

[0020] The present invention also provides a product for detecting the polymorphism or genotype of a SNP site in a wheat genome, wherein the SNP site is the above-mentioned SNP site, and the product contains the substance described above, and the product may be any of the following: C1) Products for detecting or assisting in detecting single nucleotide polymorphisms or genotypes related to wheat grain number. C2) Products that identify or assist in identifying the number of grains per ear of wheat. C3) Products used in wheat breeding.

[0021] Specifically, the product is as follows: D1), D2) or D3): D1) The product is a primer combination for amplifying a wheat genomic DNA fragment including the SNP site, D2) The product is a PCR reagent containing the primer combination described in D1), D3) The product is a kit containing the primer combination described in D1) or the PCR reagent described in D2).

[0022] In the above, the primer composition may be as follows (E1), E2) or E3): E1) The primer combination is a first-round PCR primer, E2) The primer combination is a second-round PCR primer, E3) the primer combination is a first-round PCR primer and a second-round PCR primer; The one-round PCR primers consist of a forward primer TaFT-B1-F1 and a reverse primer TaFT-B1-R1, wherein the forward primer TaFT-B1-F1 is a single-stranded DNA molecule of sequence 2 in the sequence list, and the reverse primer TaFT-B1-R1 is a single-stranded DNA molecule of sequence 3 in the sequence list; the specific sequence is: FT-B1-F1: 5'-GGTCTCGCTTCTGCCTGTG-3'; FT-B1-R1: 5'-TCATTGCCGCCCACCTC-3'.

[0023] The second round of PCR primers consist of a forward primer TaFT-B1-F2 and a reverse primer TaFT-B1-R2, wherein the forward primer TaFT-B1-F2 is a single-stranded DNA molecule of sequence 4 in the sequence list, and the reverse primer TaFT-B1-R2 is a single-stranded DNA molecule of sequence 5 in the sequence list; the specific sequence is: FT-B1-F2: 5'-CCACTTCCTCAATTCACAGC-3'; FT-B1-R2: 5'-ACCCTGCCAACCACCAGCGGAT-3'.

[0024] In the above applications and methods, the substance for detecting SNP polymorphism or genotype can be a nucleotide type of the SNP site in the wheat genome determined by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography and SNP chip. Among them, SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction endonuclease reaction, a chip based on protein DNA binding reaction, and a chip based on fluorescent molecule DNA binding reaction.

[0025] The present invention also provides a DNA molecule, the nucleotide sequence of the DNA molecule is sequence 1 in the sequence list.

[0026] The application of the above DNA molecules is also within the scope of protection of the present invention, and the application may be any of the following: F1) Application of the DNA molecule in identifying or assisting in identifying the number of grains per ear of wheat, F2) application of the DNA molecule in wheat breeding, F3) Use of the DNA molecule in the preparation of a product for identifying or assisting in identifying the number of grains per ear of wheat, F4) Use of the DNA molecule in preparing wheat breeding products.

[0027] The product may be a kit.

[0028] The kit comprises a primer composition for amplifying a wheat genomic DNA fragment including the SNP site and / or Fok Ⅰ.

[0029] The present invention is based on population genetics and association analysis, and is obtained by cloning Chinese Spring cDNA. TaFT-B1 By using different wheat materials TaFT-B1 Sequence polymorphism analysis of the coding region revealed that TaFT-B1 There is a SNP site (G / C) at the +19 bp position downstream of the "ATG" in the coding region, which causes amino acid changes in the coding region, thereby affecting the function of the TaFT-B1 protein. Based on this SNP site, a dCAPS marker was developed to form two alleles, and 348 wheat materials were scanned simultaneously. The association analysis between genotype and multi-year multi-point spike number found TaFT-B1 The _SNP+19-dCAPS marker was significantly correlated with wheat grain number per ear, indicating that this marker can assist in molecular design breeding of wheat grain number per ear. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 for TaFT-B1Variant sites and two alleles in the coding region.

[0031] Figure 2 for TaFT-B1_SNP+19 Mark dCAPS assay.

[0032] Figure 3 for TaFT-B1_SNP+19 Marker association analysis. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0035] The wheat varieties in the following examples are commercial varieties, which can be found on the China Crop Germplasm Information Network (http: / / www.cgris.net / ).

[0036] Embodiment 1, TaFT-B1 The role of cDNA SNP sites in identifying wheat grain number per ear 1. TaFT-B1_SNP+19 Discovery and genotyping 1.1 TaFT-B1 SNP site discovery and primer design in cDNA In the previous experiments, candidate genes related to wheat grain number were found. TaFT-B1 The coding sequence of this gene is located on chromosome 7B of wheat. TaFT-B1 Sequencing of the coding region revealed TaFT-B1 There is a single nucleotide polymorphism (SNP) site at the +19 bp position in the coding region, which leads to changes in the amino acids in the coding region, thus affecting the function of TaFT-B1 protein.

[0037] Using the wheat Chinese spring reference genome to obtain wheat TaFT-B1 Sequence information, by sequencing wheat materials, the SNP site of the gene was obtained and the SNP site was named TaFT-B1 _SNP+19, this site has C / G polymorphism. TaFT- B1 _SNP+19 is at position 364 of sequence 1, and its nucleotide type is C or G. Based on this site, a TaFT-B1_SNP+19-dCAPS marker was used to scan the natural wheat population. Agarose gel electrophoresis showed that the population can be basically divided into two types. The 154 bp band is TaFT-B1(G) Type, the band of 118bp is TaFT-B1(C) Combining genotype data with spikelet number phenotypic data for significant difference analysis, it was found that TaFT-B1(C) The number of grains per ear in the type material was significantly higher than that in the TaFT-B1(G) Type material, indicating TaFT-B1 _SNP+19 allele variation is significantly associated with wheat grain number. TaFT-B1 _The homozygous type of SNP+19 is named as genotype GG or TaFT-B1(G) ,Will TaFT-B1 _SNP+19 is homozygous for C and is named genotype CC or TaFT-B1(C) .

[0038] 1.2 TaFT-B1_SNP+19 Genotyping Specific dCAPS primers were designed using the dCAPS Finder 2.0 website according to the variation of the site.

[0039] 1) One round of PCR amplification: Extract wheat leaf genomic DNA and dissolve it in 200ulTE. Use 1% agarose gel electrophoresis to test DNA quality. The extracted DNA must be free of obvious impurities, clear bands, and no degradation. After measuring the DNA concentration, dilute it to 50ng / μl and use the diluted wheat genomic DNA as a template for PCR amplification.

[0040] The first round of PCR primers included forward primer TaFT-B1-F1 and reverse primer TaFT-B1-R1.

[0041] FT-B1-F1: 5'-GGTCTCGCTTCTGCCTGTG-3' (SEQ ID NO: 2); FT-B1-R1: 5'-TCATTGCCGCCCACCTC-3' (SEQ ID NO: 3).

[0042] The experiment used 2×Mix enzyme, and the PCR amplification system was as follows:

[0043] The PCR amplification program was as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 sec, 58°C annealing for 30 sec, 72°C extension (1 kb min -1), 30 cycles; 72℃ extension for 10 min, 12℃ end reaction, and obtain a round of PCR amplification products. A round of PCR amplification products contains SNP sites TaFT-B1 _SNP+19, which is sequence 1 in the sequence list (SEQ ID NO: 1, 524 bp), is as follows: 5'--3'.

[0044] 2) Second round of PCR amplification In the second round of PCR, a single base A mutation was introduced 2 bp upstream of the SNP site. The second round of PCR primers included specific forward and reverse primers TaFT-B1-F2 and TaFT-B1-R2, and formed an endonuclease Fok The recognition site of Ⅰ is "3'-CCTAC-5'".

[0045] FT-B1-F2: 5'-CCACTTCCTCAATTCACAGC-3' (SEQ ID NO: 4); FT-B1-R2: 5'-ACCCTGCCAACCACCAGCGGAT-3' (SEQ ID NO: 5).

[0046] The adenine base A at position 21 of FT-B1-R2 is the introduced mismatch site.

[0047] The first round of PCR products was diluted 200 times to obtain the first round of PCR diluted products as the amplification template, the primers were TaFT-B1-F2 and TaFT-B1-R2, and then 2×Mix was used for the second round of PCR amplification. The PCR amplification program was as follows: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 72℃ extension for 10 sec, 35 cycles; 72℃ extension for 10 min, 12℃ end reaction, and the second round of PCR amplification products were obtained. The second round of PCR amplification products were recovered by 4% agarose gel electrophoresis and sequenced to obtain sequence 6 (SEQ ID NO: 6, 154bp) and sequence 7 (SEQ ID NO: 7, 154bp) in the sequence list. The system is as follows:

[0048] The specific products of the second round of PCR amplification are as follows: TaFT-B1(C) (SEQ ID NO: 6):

[0049] TaFT-B1(G) (SEQ ID NO: 7):

[0050] Note: Double underlines are variant sites, boxed bases are introduced mismatched bases, and underlines are endonucleases. Fok ⅠRecognition site.

[0051] When the second round of PCR amplification products TaFT-B1 _SNP+19 forms endonuclease Fok Ⅰ recognition site "3'-CCTAC-5'", the second round of PCR amplification products can be Fok I is cut into two fragments; when the second round of PCR amplification products TaFT-B1 _SNP+19 forms the site "3´-CCTAG-5´", which cannot be removed by endonuclease Fok Identify and cut. By detecting the size of the second-round PCR enzyme digestion product by electrophoresis, it can be determined that the band of 154 bp is TaFT-B1(G) Type, the band of 118bp is TaFT-B1(C) type. 3) Enzyme digestion reaction Using endonucleases Fok Ⅰ The second-round PCR products were digested with enzymes. Fok Ⅰ The single enzyme digestion reaction system is as follows:

[0052] Reaction procedure: Enzyme digestion in a constant temperature water bath at 37°C for 4 h.

[0053] 4) Agarose gel electrophoresis TaFT-B1_SNP+19 Genotyping The digested products were electrophoresed in 4% agarose gel. The electrophoresis results showed that the second-round PCR amplification products were digested by endonuclease Fok The genotype of the material cut by Ⅰ is CC (also known as TaFT-B1(C) ), the second round of PCR amplification products cannot be cleaved by endonuclease Fok The genotype of the material cut by Ⅰ is GG (also known as TaFT-B1(G) ). Figure 2 The genotypes of 4 of the 348 wheat varieties in step 2 are TaFT-B1(G) and The genotypes of the four TaFT-B1(C) Electrophoresis results of the materials.

[0054] 2. TaFT-B1 _Functional validation of SNP+19 A natural population of 348 wheat accessions was used for the study. TaFT-B1_SNP+19 These materials were planted in Luoyang, Henan in 2002 and 2005, and in Shunyi, Beijing in 2010.

[0055] A completely randomized block design was used with three replicates, 4 rows, 2 m long and 0.25 m wide. 40 seeds were evenly sown in each row. The number of grains per ear was counted after maturity. The 348 wheat samples were tested according to the method in 1.2. TaFT-B1_ SNP+19 Then the genotyping data were associated with the phenotypic values ​​of grain number per ear (GNS) in multiple years and multiple points (Luoyang, Henan in 2002, Luoyang, Henan in 2005 and Shunyi, Beijing in 2010) and the data were processed using SPSS25 statistical software. The experimental results were expressed as mean ± standard deviation and tested using one-way ANOVA. P <0.01(**)indicates a highly significant difference. The results show that in multiple environments, TaFT-B1( C ) The average number of grains per ear in the material was significantly higher than that in the carrying TaFT-B1(G) The average number of grains per ear of the material ( Figure 3 ), 2002LY represents the average number of grains per ear of wheat materials planted in Luoyang, Henan in 2002; 2005LY represents the average number of grains per ear of wheat materials planted in Luoyang, Henan in 2005; 2010SY represents the average number of grains per ear of wheat materials planted in Shunyi, Beijing in 2010. Among them, the average number of grains per ear of wheat materials planted in Shunyi, Beijing in 2010 is shown in Table 1.

[0056] The above results show the effectiveness of this marker and it can be used as a functional marker for screening wheat grain number.

[0057] Table 1 Information on some genotypes and spikelet numbers of 348 wheat accessions

[0058]

[0059]

[0060] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application of a substance for detecting polymorphism or genotype of a SNP site in a wheat genome, characterized in that: The SNP site is a site in the wheat genome, the nucleotide type of which is C or G, and is the 364th nucleotide of sequence 1 in the sequence list; the application is any of the following: A1) Use of the substance in identifying or assisting in identifying the number of grains per ear of wheat, A2) Application of the substance in wheat breeding, A3) Use of the substance in the preparation of a product for identifying or assisting in identifying the number of grains in a wheat ear, A4) Use of the substance in preparing wheat breeding products.

2. The use according to claim 1, characterized in that: The substance is as follows (B1), B2) or B3): B1) the substance is a primer composition for amplifying a wheat genomic DNA fragment including the SNP site, B2) The substance is a PCR reagent containing the primer combination described in B1), B3) The substance is a kit containing the primer composition described in B1) or the PCR reagent described in B2).

3. A method for identifying or assisting in identifying the number of wheat grains per ear, characterized in that: The method comprises detecting the genotype of the SNP site in the genome of the wheat to be tested, and identifying or assisting in identifying the number of wheat grains per ear according to the genotype, wherein the SNP site is the SNP site described in claim 1.

4. Application of the method according to claim 3 in wheat breeding.

5. A method for wheat breeding, characterized in that: The method comprises detecting the genotype of the SNP site in claim 1 in the wheat genome, and selecting wheat whose genotype of the SNP site is CC or GG as a parent for breeding, wherein the CC is a homozygous type when the SNP site is C, and the GG is a homozygous type when the SNP site is G.

6. A product for detecting polymorphism or genotype of SNP sites in wheat genome, characterized in that: The SNP site is the SNP site described in claim 1, the product contains the substance described in claim 1, and the product is any one of the following: C1) Products for detecting or assisting in detecting single nucleotide polymorphisms or genotypes related to wheat grain number. C2) Products that identify or assist in identifying the number of grains per ear of wheat. C3) Products used in wheat breeding.

7. The product according to claim 6, characterized in that: The product is as follows (D1), D2) or D3): D1) The product is a primer combination for amplifying a wheat genomic DNA fragment including the SNP site, D2) The product is a PCR reagent containing the primer combination described in D1), D3) The product is a kit containing the primer combination described in D1) or the PCR reagent described in D2).

8. The use according to claim 2 or the product according to claim 7, characterized in that: The primer composition is as follows: E1), E2) or E3): E1) The primer combination is a first-round PCR primer, E2) The primer combination is a second-round PCR primer, E3) the primer combination is a first-round PCR primer and a second-round PCR primer; The one-round PCR primers are composed of a forward primer TaFT-B1-F1 and a reverse primer TaFT-B1-R1, wherein the forward primer TaFT-B1-F1 is a single-stranded DNA molecule of sequence 2 in the sequence list, and the reverse primer TaFT-B1-R1 is a single-stranded DNA molecule of sequence 3 in the sequence list; The second round PCR primers consist of a forward primer TaFT-B1-F2 and a reverse primer TaFT-B1-R2, wherein the forward primer TaFT-B1-F2 is a single-stranded DNA molecule of sequence 4 in the sequence list, and the reverse primer TaFT-B1-R2 is a single-stranded DNA molecule of sequence 5 in the sequence list.

9. A DNA molecule, characterized in that: The nucleotide sequence of the DNA molecule is sequence 1 in the sequence list.

10. The use of the DNA molecule according to claim 9, wherein the use is any of the following: F1) Application of the DNA molecule in identifying or assisting in identifying the number of grains per ear of wheat, F2) application of the DNA molecule in wheat breeding, F3) Use of the DNA molecule in the preparation of a product for identifying or assisting in identifying the number of grains per ear of wheat, F4) Use of the DNA molecule in preparing wheat breeding products.

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