Primer pair and method for identifying hardness type of wheat grains and application

Abstract

The invention relates to the technical field of wheat genetic breeding, and particularly discloses a primer pair and method for identifying the hardness type of wheat grains and application. The marker is developed on the basis of an SNP (Single Nucleotide Polymorphism) site AX-109988793 which is closely linked with a new grain hardness site qTaHa-5DL on a 5DL chromosome, and a primer sequence of the marker is as shown in SEQ ID NO. 1 and SEQ ID NO. 2. When the marker is used, the functional molecular markers of Pina and Pinb are used for screening out a wheat material of which both are wild types, and then the marker is used for PCR amplification and agarose gel electrophoresis detection: the wheat material which can amplify about 200bp bands is of a soft type, and the wheat material which does not amplify about 200bp bands is of a mixed type. The marker detection equipment is simple, convenient to operate, stable in result and easy to popularize and apply in breeding units, and can be used for auxiliary breeding of pure soft wheat varieties.

Description

Technical Field

[0001] This invention belongs to the field of wheat genetics and breeding technology, specifically relating to a primer pair, method, and application for identifying wheat grain hardness types. Background Technology

[0002] Grain hardness is an important quality trait of wheat and a key indicator for wheat quality classification and grading. Currently, the Single Kernel Characterization System (SKCS) has become the main method for determining wheat grain hardness due to its ease of operation, rapid detection, reliable data, and good repeatability. Based on the Hardness Index (HI) value measured by SKCS, wheat is generally classified into three categories: soft (HI < 40), mixed (HI 40–60), and hard (HI > 60).

[0003] From a genetic perspective, wheat grain hardness is mainly influenced by two major genes at the Ha locus on the short arm of chromosome 5D— puroindoline a ( Pina )and puroindoline b ( Pinb ) control. When Pina and Pinb When all genes are wild-type, wheat grains are soft; if any gene is deleted or has a base mutation, the grain hardness increases, resulting in hard or mixed grains. Currently, research is focused on... Pina and Pinb Research on gene allelic variation detection, functional molecular marker development, and germplasm resource evaluation has been widely carried out both domestically and internationally. Various molecular markers, including the KASP (competitive allelic PCR) marker suitable for high-throughput typing, have been developed, which has greatly promoted the progress of wheat molecular breeding.

[0004] However, grain hardness is a typical quantitative trait, except for the major gene. Pina and Pinb In addition, it is regulated by multiple minor genes or quantitative trait loci (QTLs). Although multiple QTLs related to grain hardness have been identified across the entire genome, except for... Pina / Pinb Furthermore, most QTLs have not yet been thoroughly studied or applied in practical breeding.

[0005] In actual breeding work, it has been found that even Pina and PinbAll were wild-type, but some wheat varieties or higher-generation lines still exhibited moderate grain hardness (approximately 45–50), indicating a mixed type, suggesting the presence of regulatory effects from other hardness-related genes in the genetic background. Recent studies, through genome-wide association analysis (GWAS) and linkage mapping, identified a novel, environmentally stable, and significantly effective grain hardness locus *qTaHa-5DL* on chromosome 5DL, and developed a corresponding KASP marker (K-Ha5DL) to validate its effect. The results indicate that in Pina and Pinb In a wild-type genetic background, this locus can significantly increase grain hardness, raising it from soft (SKCS hardness value around 25) to mixed (SKCS hardness value around 45), providing a new molecular tool for eliminating mixed materials in the breeding of soft wheat.

[0006] Although KASP markers offer advantages such as high throughput and automation, their reliance on specialized fluorescence detection equipment, coupled with expensive instruments and demanding operational skills, limits their widespread application in most grassroots breeding units. Therefore, there is an urgent need to develop a site-specific molecular marker that can achieve rapid and accurate genotyping based on conventional PCR and agarose gel electrophoresis, to promote the widespread application of the *qTaHa-5DL* site in practical breeding. Summary of the Invention

[0007] This invention provides a primer pair, method, and application for identifying wheat grain hardness types, which can be used in... Pina and Pinb Given a genetic background where all genes are wild-type, conventional PCR and electrophoresis techniques can be used to quickly and accurately distinguish between soft and mixed wheat types, offering advantages such as simple equipment and ease of promotion.

[0008] On the one hand, the present invention provides a primer pair for identifying wheat grain hardness types, using the following technical solution: A primer pair for identifying wheat grain hardness type, the primer pair consisting of a forward primer with sequence SEQ ID NO.1 and a reverse primer with sequence SEQ ID NO.2; Primer pairs are used in Pina Genes and Pinb In wheat materials where all genes are wild-type, detection was performed via PCR amplification*. qTaHa-5DL * Allele type at the locus.

[0009] On the other hand, the present invention also provides a method for identifying wheat grain hardness type based on primer pairs, using the following technical solution: A method for identifying wheat grain hardness type based on primer pairs includes the following steps: (1) Extract genomic DNA from the wheat material to be tested; (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using primer pairs; (3) Perform electrophoretic detection on the amplification products of step (2); If the electrophoresis results show a DNA fragment of approximately 200 bp, then the wheat material being tested is considered * qTaHa-5DL *The locus indicates a soft allele; if this DNA fragment is not present, it is determined to be a mixed allele.

[0010] Preferably, in step (2), the PCR amplification reaction program includes: pre-denaturation at 94°C for 3 minutes; followed by 30 cycles, each cycle including 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds; and finally extension at 72°C for 5 minutes.

[0011] This invention also provides a method for assisting in the breeding of soft wheat, comprising the following steps: (a) Obtain the wheat population to be screened; (b) Utilizing targets Pina and Pinb Functional molecular markers of genes were screened from wheat populations. Pina Genes and Pinb Candidate materials with all genes being wild-type; (c) The candidate materials selected in step (b) are tested to identify the type of wheat grain hardness; (d) Select the option that was determined to be * in step (c). qTaHa-5DL *The locus is the material with the soft allele, used for the breeding of soft wheat.

[0012] The present invention also provides a detection kit for identifying wheat grain hardness type, comprising the primer pair described above for identifying wheat grain hardness type.

[0013] Preferably, the use of a primer pair in the preparation of a product for identifying wheat grain hardness type.

[0014] Preferably, a kit is used in the preparation of a product for identifying the type of wheat grain hardness.

[0015] In summary, the beneficial effects of the present invention are as follows: The most significant technical effect of this invention lies in simplifying and popularizing detection technology. Existing methods based on the same QTL locus ( qTaHa-5DLWhile KASP markers are accurate, their reliance on expensive dedicated instruments severely limits their application. The JAAS-Ha5DL marker developed in this invention, however, transforms detection into a routine PCR amplification combined with agarose gel electrophoresis, requiring only basic equipment in a typical molecular biology laboratory. This significantly lowers the technical barrier and detection cost, enabling the marker to be widely applied in breeding units at all levels and solving a bottleneck problem in the promotion of key technologies.

[0016] In breeding applications, this invention provides a precise and efficient auxiliary selection tool. When utilizing... Pina and Pinb After screening for "double wild-type" materials using major gene markers, the JAAS-Ha5DL marker can further effectively distinguish between materials that ultimately exhibit mixed characteristics (average hardness index ~44) and truly soft materials (average hardness index ~22). This allows breeders to quickly and accurately eliminate materials carrying genes unfavorable to hardness in the genetic background at early generations, thereby concentrating resources on breeding pure soft wheat varieties and significantly improving the breeding efficiency and selection accuracy of high-quality soft wheat. Attached Figure Description

[0017] Figure 1 Electrophoresis diagram of the amplification of molecular marker JAAS-Ha5DL in different subtypes of SNP site AX-109988793; Figure 2 In the background Pina and Pinb Box plot of wheat grain hardness difference between the two bands of molecular marker JAAS-Ha5DL when both are wild type. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the embodiments.

[0019] Example Example 1 The specific steps for converting QTL-associated SNPs into practical PCR molecular marker experiments are as follows: S1. For SNPs (A / G), prioritize the design of allele-specific primers.

[0020] Forward primer design: Designed approximately 20-25 bp upstream of the SNP. The 3' terminal base of the primer is designed to be a "C" that perfectly matches allele G. To improve specificity, a mismatched base (optimized) is often artificially introduced at the second or third to last position of the 3' end to disrupt binding with the non-target allele (A). Reverse primer design: Designed approximately 100-300 bp downstream of the SNP, ensuring an appropriate amplified fragment size. Primer properties (Tm value, dimer, hairpin structure) are evaluated using software such as Primer Premier 5, and their specificity in the wheat genome is verified using BLAST. Forward primer SEQ ID NO.1 and reverse primer SEQ ID NO.2 were obtained.

[0021] Table 1. PCR molecular marker sequences

[0022] S2. Using eight wheat materials (Ningmai 3, Ningmai 9, Ningmai 10, Yangmai 158, Ningmai 11, Ningmai 15, Ningmai 21, and Ningmai 23) with known *qTaHa-5DL* locus genotypes (known through sequencing), amplification and electrophoresis were performed strictly following the optimized JAAS-Ha5DL standardized PCR procedure. All materials known to have the G / G genotype (Ningmai 3, Ningmai 9, Ningmai 10, and Yangmai 158) should show a single, clear band at approximately 200 bp; all materials known to have the A / A genotype (Ningmai 11, Ningmai 15, Ningmai 21, and Ningmai 23) should not show this band. The concordance rate should reach 100%. Any discrepancies require investigation of primer specificity or PCR conditions.

[0023] Three aliquots of A / A and G / G genotype materials were selected, and PCR was performed using template DNA at concentrations of 10 ng / µL, 50 ng / µL, 100 ng / µL, and 200 ng / µL, respectively. The same DNA sample was used to perform at least three independent experiments using different batches of PCR Mix. The reproducibility of the labeling results was assessed to ensure the labeling was stable and reliable.

[0024] S3. Using optimal reaction components, concentrations, and PCR program parameters: 0.5 µL each of 10 µM primers, 5 µL of 2×Taq PCR premix, 2 µL of DNA, and ddH2O to a final volume of 10 µL. Program: 94℃ for 3 min; 30 cycles: 94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s; 72℃ for 5 min. Take 5 µL of PCR product and electrophoresis on a 1.0-1.5% agarose gel (150 V, 10-15 min), stain with EB, and observe using a gel imaging system.

[0025] like Figure 1As shown, lane M represents the DNA molecular weight standard (DL2000), and lanes 1 to 8 represent Ningmai 3, Ningmai 9, Ningmai 10, Yangmai 158, Ningmai 11, Ningmai 15, Ningmai 21, and Ningmai 23, respectively. Electrophoresis results showed that four varieties (Ningmai 3, Ningmai 9, Ningmai 10, and Yangmai 158) exhibited a clear band at approximately 200 bp, while the other four varieties (Ningmai 11, Ningmai 15, Ningmai 21, and Ningmai 23) did not show this specific band. This clear difference in the presence / absence of banding patterns directly demonstrates that the JAAS-Ha5DL marker can reliably distinguish different allelic genotypes of wheat at the *qTaHa-5DL* locus, providing direct technical evidence for its subsequent application in identifying grain hardness types.

[0026] Example 2 The effectiveness and practicality of the molecular marker JAAS-Ha5DL were verified in a set of breeding materials with known grain hardness phenotypes and Pina / Pinb being double wild-type. The concordance rate between marker banding (present / absent) and hardness phenotype (soft / mixed) was statistically analyzed, and a t-test was performed to confirm whether the difference in hardness index between the two banding patterns reached a highly significant level. The specific steps are as follows: S1. Using SKCS, 368 wheat accessions harvested in 2025 were analyzed. These 386 accessions included 214 varieties from national, provincial, and consortium regional trials, and 172 high-generation breeding lines. At least 300 grains were analyzed for each material, and the average value was calculated. For all materials, the analysis was first performed using... Pina / Pinb Functional marker identification was used to screen out "double wild-type" materials (Group B). Materials in Group B were tested using JAAS-Ha5DL and divided into JAAS-Ha5DL positive group (with a 200bp band) and JAAS-Ha5DL negative group (no band) based on electrophoresis results.

[0027] Compare the mean hardness index of the JAAS-Ha5DL positive group and the JAAS-Ha5DL negative group within Group B. Calculate the overall concordance rate between the JAAS-Ha5DL genotyping results and the SKCS phenotypic classification results according to the SKCS classification criteria.

[0028] Table 2. 368 wheat samples

[0029] S2. Perform the entire process from DNA analysis to result interpretation for 100 samples, recording the total time, manual operation time, and required technical training difficulty. Compared with the KASP labeling process, highlight the advantages of not requiring special fluorescence equipment and having results that can be interpreted visually. Calculate the detection cost per sample (including primers, reagents, and consumables) and compare it with the KASP detection cost.

[0030] In the early generations of breeding (such as F2 or F3 populations), 100 individual plants are randomly selected and subjected to a "screening" process. Pina / Pinb The process involved a two-step screening procedure using JAAS-Ha5DL. The smoothness of the process, the success rate of labeling under different single-strain DNA quality, and the proportion of "double-superior" single strains ultimately selected were recorded.

[0031] like Figure 2 As shown, this study demonstrates the association between the results of detection using the molecular marker JAAS-Ha5DL and the corresponding wheat grain hardness phenotype in a genetic background where both Pina and Pinb genes are wild-type. The results showed 128 wheat accessions with both Pina and Pinb being wild-type. Further detection using the JAAS-Ha5DL marker in this wild-type background revealed that 73 accessions amplified a 200bp band, with an average grain hardness of 23.51 (soft type), while 55 accessions failed to amplify a 200bp band, with an average grain hardness of 44.89 (mixed type). The difference between the two types was highly significant, directly verifying that the JAAS-Ha5DL marker can effectively distinguish between soft and mixed wheat types when Pina and Pinb are wild-type, providing clear phenotypic evidence for the use of this marker in breeding-assisted selection.

[0032] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A primer pair for identifying wheat grain hardness types, characterized in that, The primer pair consists of a forward primer with the sequence shown in SEQ ID NO.1 and a reverse primer with the sequence shown in SEQ ID NO.2; The primer pair is used for... Pina Genes and Pinb In wheat materials where all genes are wild-type, detection was performed via PCR amplification*. qTaHa-5DL * Allele type at the locus.

2. A method for identifying wheat grain hardness type based on the primer pair described in claim 1, characterized in that, Includes the following steps: (1) Extract genomic DNA from the wheat material to be tested; (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using primer pairs; (3) Perform electrophoretic detection on the amplification products of step (2); If the electrophoresis results show a DNA fragment of approximately 200 bp, then the wheat material being tested is considered * qTaHa- 5DL *The locus indicates a soft allele; if the DNA fragment is not present, it is determined to be a mixed allele.

3. The method for identifying wheat grain hardness type according to claim 2, characterized in that, In step (2), the PCR amplification reaction program includes: pre-denaturation at 94°C for 3 minutes; followed by 30 cycles, each cycle including 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds; and finally extension at 72°C for 5 minutes.

4. A method for identifying wheat grain hardness types based on the methods described in claims 2-3, used to assist in the breeding of soft wheat, characterized in that, Includes the following steps: (a) Obtain the wheat population to be screened; (b) Utilizing targets Pina and Pinb Functional molecular markers of genes were screened from the wheat population. Pina Genes and Pinb Candidate materials with all genes being wild-type; (c) The candidate materials selected in step (b) are tested to identify the type of wheat grain hardness; (d) Select the option that was determined to be * in step (c). qTaHa-5DL *The locus is the material with the soft allele, used for the breeding of soft wheat.

5. A test kit for identifying the type of wheat kernel hardness, characterized in that, The kit contains the primer pair for identifying wheat grain hardness type as described in claim 1.

6. The application of the primer pair according to claim 1 in the preparation of a product for identifying wheat grain hardness type.

7. The use of a kit based on claim 5 in the preparation of a product for identifying wheat grain hardness type.

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