Primer group, kit and method for detecting waxy gene of corn
By designing the primer sets WaxyD7-F and WaxyD7-R, combined with melting curve analysis and agarose gel electrophoresis, the accuracy and cost issues of corn waxy genotype detection on ordinary PCR instruments were solved, and low-cost and efficient genotype differentiation was achieved.
Patent Information
- Application Number
- CN202510789452.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-13
- Publication Date
- 2025-09-12
AI Technical Summary
In the existing technology, molecular marker analysis based on high-resolution melting curves requires dedicated equipment, which limits its widespread application. How to achieve accurate and low-cost detection of the corn waxy gene on an ordinary quantitative PCR instrument?
Specific primer sets WaxyD7-F and WaxyD7-R were designed, and the amplified fragments were 84 bp and 54 bp, respectively. The waxy genotypes of maize were distinguished using a conventional PCR instrument in combination with melting curve analysis and agarose gel electrophoresis.
The accurate differentiation of maize waxy genotypes on a common PCR instrument was achieved with low cost, good specificity, and large differences in the melting temperatures of the amplified products, and the results were consistent with existing technologies.
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Figure CN120624699A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of molecular biological detection technology, and more specifically to a primer set, a kit, and a method for detecting the waxy gene of corn. Background Art
[0002] The present application belongs to the field of agricultural biotechnology, and specifically relates to a method for detecting the functional marker of the waxy gene of waxy corn using a melting curve.
[0003] Waxy corn (Zeamays), also known as waxy corn, is nutritious and highly economically valuable. The waxy genotype of waxy corn is a waxy mutation, characterized by a loss of amylose in the seed endosperm starch. This waxy mutation results in an amylopectin content approaching 100%, compared to 70%-85% in standard corn. The maize waxy gene is located on the short arm of chromosome 9. The wild-type Wx gene sequence is 3718 base pairs long and consists of 14 exons and 13 introns. Long-term research has uncovered over 50 wx alleles, including unstable waxy mutations caused by the same transposon inserting at different locations or by different transposons inserting at the same location, as well as stable waxy mutations. Some of these stable waxy mutations are deletion mutations, such as the wx-D7 (D7) and wx-D10 (D10) mutations in waxy corn. wx-D7 is a deletion of the last 30 bases of exon 7 of the wx gene. Transcriptional splicing fails at this site, and the remaining 7th intron is retained in the transcript, resulting in the premature appearance of the terminator TAA, premature interruption of translation, and loss of GBSSI protein function.
[0004] Molecular marker-assisted identification is an important means of detecting genotype mutations and a key means of improving breeding efficiency. Currently, commonly used molecular markers mainly include conventional molecular markers based on electrophoresis, molecular markers based on high-resolution melting curves, and fluorescent markers based on competitive amplification of alleles. Electrophoresis-based molecular markers are accurate and reliable, but there is a risk of contamination and low analysis efficiency. They are only suitable for analyzing sites with large differences in amplified fragments and are difficult to apply on a large scale. Fluorescent markers based on competitive amplification of alleles, such as KASP, have high analysis efficiency and can analyze polymorphic sites including SNPs, but the analysis cost is high and they rely on specialized reagents provided by commercial companies. Molecular labeling based on high-resolution melting curves is accurate, efficient, and low-cost. However, high-resolution melting curve analysis (HRM) typically requires specialized equipment (such as the LightScanner and Rotor-Gene 6000). These devices feature high-precision temperature control (e.g., collecting 40-120 data points per degree Celsius) and low inter-well temperature differences (<0.1°C). Conventional quantitative PCR instruments (such as the QuantStudio™ 3) can have inter-well temperature differences of up to 1.3°C, making them generally considered unsuitable for HRM analysis. Therefore, the specialized nature of the equipment limits the widespread application of this method.
[0005] Therefore, how to break through the technical bottleneck that only professional equipment can be used to analyze melting curves and provide a primer set, kit and method for detecting the maize waxy gene by analyzing melting curves using a common quantitative PCR instrument is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention
[0006] In view of this, the present application provides a primer set, a kit and a method for detecting the waxy gene in corn.
[0007] In order to achieve the above objectives, this application adopts the following technical solutions:
[0008] The first object of the present application is to provide: a primer set for detecting the waxy gene of corn, the nucleotide sequence of the primer set is as follows:
[0009] WaxyD7-F: 5'-ACTACCAGTCCCACGGCATCT-3', SEQ ID NO.1;
[0010] WaxyD7-R: 5'-TACGAGCATGGAGAACGAA-3', SEQ ID NO. 2.
[0011] Another object of the present application is to provide: a kit for detecting the waxy gene in corn, comprising the primer set described above.
[0012] Another object of the present application is to provide: an application of the above primer set or the above kit, wherein the application is in any of the following directions:
[0013] 1) Application in detection of waxy genotype in maize;
[0014] 2) Application in screening D7 mutants of the waxy gene in maize;
[0015] 3) Application in waxy corn genetic breeding;
[0016] 4) Application in screening of sweet and sticky double hidden materials.
[0017] Another object of the present application is to provide: a method for detecting the waxy genotype of corn, using the above-mentioned primer set or the above-mentioned kit for detection, comprising the following steps: extracting DNA of the sample to be tested as a template, performing PCR amplification using the above-mentioned primer set or the above-mentioned kit, and analyzing the amplified product by melting curve analysis and / or agarose gel electrophoresis to identify the waxy genotype of the corn sample to be tested.
[0018] As a preferred technical solution, the PCR amplification system is as follows:
[0019] 10×PCR Buffer 1μL, 2.5mM each dNTP 0.8μL, 10μM WaxyD7-F and WaxyD7-R 0.1μL each, 5U / μL TaKaRataq 0.1μL, 20×Evagreen 0.2μL, 20-100ng / μL corn sample DNA 1μL, add dH2O to make up to 10μL.
[0020] As a preferred technical solution, the PCR amplification program is as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 34 cycles; and extension at 72°C for 5 minutes.
[0021] As a preferred technical solution, the process of analyzing the amplified product using melting curve analysis is as follows: when the melting temperature range of the melting curve is 81.5-82°C and the fluorescence intensity is the highest, the corn sample to be tested is judged to be the wild type of the Waxy gene, and is a sweet corn kernel;
[0022] When the melting temperature range of the melting curve is 80.5-81.5℃ and the fluorescence intensity is the lowest, the corn sample to be tested is judged to be a Waxy gene mutant type. If it appears as sweet corn kernels, it is a sweet and waxy double hidden material.
[0023] When the fluorescence intensity value is between the two, it is a heterozygous type.
[0024] As a preferred technical solution, the process of analyzing the amplified product by agarose gel electrophoresis is as follows:
[0025] When an 84 bp band is amplified, the sample to be tested is the wild type of the Waxy gene; when a 54 bp band is amplified, the sample to be tested is the D7 mutant type of the Waxy gene; when both 84 bp and 54 bp bands are amplified, the sample to be tested is the heterozygous type of the Waxy gene.
[0026] Another object of the present application is to provide: application of the above method in detecting the waxy genotype of corn.
[0027] It can be seen from the above technical solutions that compared with the prior art, this application has the following beneficial effects:
[0028] The present invention targets the waxy maize mutation wx-D7, targeting a 30-base sequence deletion within the region bordering the seventh exon and eighth intron of the waxy gene. Amplification primers, WaxyD7-F and WaxyD7-R, were designed. These primers closely follow the mutation site, amplifying small fragments of 84 and 54 bp, respectively. The Tm values differ significantly, resulting in distinct melting curves for different genotypes. This allows accurate differentiation between mutant, wild-type, and heterozygous forms of the waxy gene D7 using the melting temperature analysis function of a conventional quantitative PCR instrument. Compared to existing techniques, the disclosed detection method offers low cost, high specificity, and a large difference in the melting temperatures of the amplified products, enabling accurate differentiation between different genotypes.
[0029] Furthermore, agarose gel electrophoresis of the primer-amplified products provided by the present invention can also distinguish different genotypes of Waxy, and the typing results are consistent with the results of melting curve analysis. Laboratories or institutions without quantitative PCR can also use agarose gel electrophoresis to perform genotyping of Waxy. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without any creative work.
[0031] Figure 1 For: Development and design of primer marker site map.
[0032] Figure 2 Melting curve analysis diagram.
[0033] Figure 3 Agarose gel electrophoresis. DETAILED DESCRIPTION
[0034] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.
[0035] Example 1
[0036] Development and design of primer markers
[0037] The genome sequences of Wannuo 2000 (D7 mutant of Waxy gene) and JR86 (provided by the Institute of Crop Science, Fujian Academy of Agricultural Sciences) (wild type of Waxy gene) were compared. The functional site of the 30 bp base sequence deletion in the seventh exon and eighth intron of the Waxy gene (located on the broken arm of chromosome 9, accession number 541854) was selected. Figure 1 ) as the target, a functional marker for identifying the Waxy genotype of maize was designed at 11 bases and 3 bases on both sides of the mutation site, respectively. The functional marker consists of the following two primers:
[0038] WaxyD7-F:5'-ACTACCAGTCCCACGGCATCT-3', SEQ ID NO.1;
[0039] WaxyD7-R: 5'-TACGAGCATGGAGAACGAA-3', SEQ ID NO.2;
[0040] The lengths of the amplified products of this functional marker for wild-type corn with Waxy gene and D7 mutant corn with Waxy gene were 84 bp and 54 bp, respectively. Products of 84 bp and 54 bp were amplified for heterozygous corn with Waxy gene at the same time.
[0041] Example 2
[0042] A method for detecting waxy genotype of corn
[0043] (1) Using JR86 (wild type of Waxy gene) as the female parent and Wannuo 2000 (commercially available) (D7 mutant of Waxy gene) as the male parent, hybridization was performed, and self-pollination was performed to harvest the self-pollinated F2 generation corn;
[0044] (2) DNA was extracted from more than 5,000 maize plants of Wannuo 2000, JR86, and their self-pollinated F2 generations. The specific DNA extraction method is as follows:
[0045] Extraction of DNA from corn leaves using the CTAB method:
[0046] 1) A 1 cm section of a three-leaf, one-heart stage corn leaf was placed in a 2 mL centrifuge tube. A 5 mm diameter steel ball was added, followed by 500 μL of 2× CTAB solution. The corn leaf was then ground in a grinder (High Throughput Tissue Grinder, Shanghai Jingxin) at 50 Hz for 60 s.
[0047] 2) Place in a water bath at 65°C for 1 hour, stirring gently every 20 minutes.
[0048] 3) After cooling to room temperature in a water bath, add an equal volume of chloroform / isoamyl alcohol to CTAB (volume ratio 24:1), shake thoroughly, and centrifuge at 12,000 rpm for 10 minutes.
[0049] 4) Take 200 μL of the supernatant and place it in a 1.5 mL centrifuge tube. Add 400 μL of pre-chilled anhydrous ethanol, shake well, place in a 20°C refrigerator for 1 hour, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, and invert on a paper towel for 10 minutes.
[0050] 5) Add 500 μL of 75% ethanol and shake gently to make the DNA precipitate float. Centrifuge and pour off the supernatant. Place the tube back in the clean bench. After the ethanol has completely evaporated, add 100 μL of TE solution and warm it to 65°C to dissolve and inactivate the DNA enzyme. After returning to room temperature, place it in a 4°C refrigerator (for short-term storage) or a 20°C refrigerator (for long-term storage).
[0051] (3) PCR amplification system and procedure: 10× PCR buffer 1 μL, 2.5 mM each dNTP 0.8 μL, 10 μM forward and reverse primers 0.1 μL each, 5 U / μL TaKaRaTaq enzyme 0.1 μL, 20× Evagreen 0.2 μL, DNA template 20-100 ng, add dH2O to make up to 10 μL;
[0052] The PCR reaction parameters were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 34 cycles; and extension at 72°C for 5 min.
[0053] (4) Melting curve analysis
[0054] After PCR, the amplified product was centrifuged at 1000 rpm for 30 seconds to eliminate bubbles and the reaction solution attached to the wall. The reaction solution was then placed in a quantitative PCR instrument, QuantStudioTM3, and heated at a rate of 0.2℃ / s in the range of 60-95℃. The melting curve detection function was used to obtain the melting curve and corresponding TM value of each reaction tube. The results are as follows: Figure 2 shown.
[0055] Result analysis: Figure 2 As you can see, there are three different types of melting curves, and different genotypes can be easily identified from the graphs:
[0056] 1) When the melting temperature range of the melting curve is 81.5-82°C and the fluorescence intensity is the highest, the corn sample to be tested is determined to be the wild type of the Waxy gene and the sweet corn kernel;
[0057] 2) When the melting temperature range of the melting curve is 80.5-81.5°C and the fluorescence intensity is the lowest, the corn sample to be tested is judged to be a Waxy gene mutant type. If it appears as sweet corn kernels, it is a sweet and waxy double-hidden material;
[0058] 3) When the fluorescence intensity is between the two, it is a heterozygous type.
[0059] (5) Agarose gel electrophoresis analysis
[0060] After the melting curve analysis, the PCR products were electrophoresed on 3% agarose gel. Figure 3 As shown, the amplified band corresponding to the wild-type corn of the Waxy gene is 84 bp, the amplified band corresponding to the D7 mutant corn of the Waxy gene is 54 bp, and the amplified bands of 84 bp and 54 bp are simultaneously amplified in the heterozygous corn of the Waxy gene.
[0061] Analysis of results: The amplified band size ranged from 50 to 100 bp, meeting design expectations. The amplified band size of individuals with different Waxy genotypes was clearly distinguishable, and genotypes could be determined based on band size.
[0062] Combined with the comparison of melting temperature detection results, it was found that electrophoresis showed that larger fragments corresponded to samples with high TM values, smaller fragments corresponded to samples with low TM values, and hybrid bands corresponded to intermediate TM values.
[0063] The primer amplification products provided in this application were used to distinguish different genotypes of Waxy using agarose gel electrophoresis, which was consistent with the results of melting curve analysis. Laboratories or institutions without quantitative PCR can also use agarose gel electrophoresis to perform genotyping of Waxy.
[0064] Example 3
[0065] Screening of sweet and sticky double hidden materials
[0066] The F2 generation, produced by hybridization between JR86 and Wannuo 2000, showed differences at the D7 locus in the Waxy gene, resulting in varying waxy properties. To confirm the genetic effects of the developed functional markers, over 5,000 F2 plants from the selfed hybrids were harvested from southern Hainan. Using the epistatic effect of the sweet gene in the sweet-waxy recessive gene, the sweet-grain phenotype was selected based on phenotype. This method was then used to screen for homozygous Waxy D7 mutations. Phenotypic analysis confirmed these recessive sweet-waxy mutations (consistent with molecular marker validation results), resulting in approximately 300 plants, representing approximately 1 / 16 of the 5,000 total, consistent with Mendel's law of segregation.
[0067] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0068] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present application. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A primer set for detecting the waxy gene in corn, characterized in that: The nucleotide sequence of the primer set is as follows: WaxyD7-F: 5'-ACTACCAGTCCCACGGCATCT-3', SEQ ID NO.1; WaxyD7-R: 5'-TACGAGCATGGAGAACGAA-3', SEQ ID NO.
2.
2. A kit for detecting the waxy gene in corn, characterized in that: Comprising the primer set according to claim 1.
3. Use of the primer set according to claim 1 or the kit according to claim 2, characterized in that: The application is any of the following: 1) Application in detection of waxy genotype in maize; 2) Application in screening D7 mutants of the waxy gene in maize; 3) Application in waxy corn genetic breeding; 4) Application in screening of sweet and sticky double hidden materials.
4. A method for detecting the waxy genotype of corn, characterized in that: The detection is performed using the primer set according to claim 1 or the kit according to claim 2, comprising the following steps: extracting DNA of a sample to be tested as a template, performing PCR amplification using the primer set according to claim 1 or the kit according to claim 2, and analyzing the amplified product by melting curve analysis and / or agarose gel electrophoresis, thereby identifying the waxy genotype of the corn sample to be tested.
5. The method for detecting the waxy genotype of corn according to claim 4, wherein: The amplification system of the PCR is as follows: 10×PCR Buffer 1μL, 2.5mM each dNTP 0.8μL, 10μM WaxyD7-F and WaxyD7-R 0.1μL each, 5U / μL TaKaRataq 0.1μL, 20×Evagreen 0.2μL, 20-100ng / μL corn sample DNA 1μL, add dH2O to make up to 10μL.
6. The method for detecting the waxy genotype of corn according to claim 4, wherein: The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 34 cycles; and extension at 72°C for 5 min.
7. The method for detecting the waxy genotype of corn according to claim 4, wherein: The process of analyzing the amplified product using melting curve analysis is as follows: when the melting temperature range of the melting curve is 81.5-82° C. and the fluorescence intensity is the highest, the corn sample to be tested is determined to be a wild type of the Waxy gene, a sweet corn kernel; When the melting temperature range of the melting curve is 80.5-81.5℃ and the fluorescence intensity is the lowest, the corn sample to be tested is judged to be a Waxy gene mutant type. If it appears as sweet corn kernels, it is a sweet and waxy double hidden material. When the fluorescence intensity value is between the two, it is heterozygous.
8. The method for detecting the waxy genotype of corn according to claim 4, wherein: The process of analyzing the amplified product by agarose gel electrophoresis is as follows: When an 84 bp band is amplified, the sample to be tested is the wild type of the Waxy gene; when a 54 bp band is amplified, the sample to be tested is the D7 mutant type of the Waxy gene; when both 84 bp and 54 bp bands are amplified, the sample to be tested is the heterozygous type of the Waxy gene.
9. Use of the method according to any one of claims 4 to 8 in detecting the waxy genotype of maize.