Detection method of single nucleotide polymorphism of rice TAC4 gene by ARMS-PCR and application

Abstract

This invention provides a rice TAC4 A primer set for detecting single nucleotide polymorphisms (SNPs) of a gene contains four ARMS-PCR primers: two outer primers with nucleotide sequences as shown in SEQ ID NO. 1-2, and two inner primers with nucleotide sequences as shown in SEQ ID NO. 3-4. This invention also provides a rice... TAC4 A method for detecting single nucleotide polymorphisms (SNPs). The primer set detection method of this invention exhibits high specificity and accuracy, and is rapid and low-cost, enabling rapid selection of rice plant types and accelerating the process of breeding superior rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of agricultural molecular biology technology, specifically relating to an ARMS-PCR detection method and its application for single nucleotide polymorphisms in the rice TAC4 gene. Background Technology

[0002] Rice is a grass crop with multiple tillers. The tillering angle affects planting density, photosynthetic efficiency, and disease resistance, and is an important component of rice plant architecture, playing a decisive role in ensuring food security and increasing yield. Tiller Angle Control 4 (TAC4), a tillering angle regulating gene discovered in 2020, encodes a plant-specific, evolutionarily conserved protein that plays a crucial role in controlling rice plant architecture.

[0003] In cultivated rice, TAC4 is mainly divided into two haplotypes: Hap1 (with a T base at position 1793 in the coding region) and Hap2 (with a C base at position 1793 in the coding region). Rice varieties with the Hap1 haplotype have smaller tillering angles, while those with the Hap2 haplotype have larger tillering angles. Therefore, developing functional molecular markers for these two haplotypes of the TAC4 gene in cultivated rice and using them for marker-assisted selection of tillering angle traits can greatly improve the efficiency of rice plant architecture breeding, especially for japonica rice.

[0004] Currently, the molecular marker technology for distinguishing the two haplotypes at the 1793rd base of the rice TAC4 gene coding region is available in Chinese Patent 202311598888.9. This patent utilizes the KASP marker, developed to differentiate between the Hap1 and Hap2 haplotypes of TAC4 in different rice varieties, for the detection of these two haplotypes. However, the KASP marker detection method requires fluorescently labeled primers and corresponding detection equipment, which is relatively expensive. This makes it unsuitable for widespread use in many small and medium-sized laboratories and research institutions lacking the necessary equipment (e.g., municipal agricultural research institutes). Furthermore, the cost is high when testing small numbers of samples (e.g., only a few to a few dozen samples).

[0005] Four-primer amplification refractory mutation system (ARMS-PCR) is a technique developed from conventional PCR for detecting single nucleotide mutations. It offers advantages such as speed, simplicity, and low cost. Therefore, to accelerate the breeding process for rice plant architecture improvement, there is an urgent need to develop an accurate ARMS-PCR method for detecting TAC4 that can be applied to numerous small and medium-sized laboratories and research institutions. Summary of the Invention

[0006] The purpose of this invention is to provide an ARMS-PCR detection method for single nucleotide polymorphisms in the rice TAC4 gene, which has high accuracy, low detection cost, and low requirements for experimental equipment.

[0007] To achieve the above objectives, the present invention provides a primer set for detecting single nucleotide polymorphisms in the rice TAC4 gene, which contains four ARMS-PCR primers: two outer primers with nucleotide sequences as shown in SEQ ID NO. 1-2, and two inner primers with nucleotide sequences as shown in SEQ ID NO. 3-4.

[0008] Forward outer primer TAC4-ef: 5'-GATGGGTCATTCTGGAGTGAG-3' (SEQ ID NO.1);

[0009] Reverse outer primer TAC4-er: 5'-CTCTGCTTGTTTGCGACCT-3' (SEQ ID NO.2);

[0010] The forward inner primer TAC4-if5: 5'-TTGGTGCGATAAATTCGACAT-3' (SEQ ID NO.3);

[0011] Reverse inner primer TAC4-ir5: 5'-GGCAGCGCCCTCGTCAG-3' (SEQ ID NO.4).

[0012] This invention also provides a method for detecting single nucleotide polymorphisms in the rice TAC4 gene, which is an ARMS-PCR method developed based on the T>C mutation at base 1793 in the coding region of the TAC4 gene, comprising the following steps:

[0013] (1) Extract genomic DNA from the rice sample to be tested;

[0014] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the above primer set to obtain PCR products;

[0015] (3) Perform agarose gel electrophoresis on the PCR products and determine the genotype of the single nucleotide polymorphism site of the TAC4 gene based on the electrophoresis results.

[0016] The method for judging based on electrophoresis results is as follows:

[0017] If two bands of 494bp and 351bp are amplified, then the haplotype of the rice sample TAC4 to be tested is Hap1;

[0018] If two bands of 494bp and 180bp are amplified, then the haplotype of the rice sample TAC4 to be tested is Hap2;

[0019] If three bands of 494bp, 351bp, and 180bp are amplified, then the haplotype of the rice sample TAC4 is a heterozygote of Hap1 and Hap2.

[0020] Hap1: The 1793rd base is a homozygous T / T;

[0021] Hap2: The 1793rd base is a homozygous C / C;

[0022] The hybrid of Hap1 and Hap2: the base at position 1793 is a hybrid T / C.

[0023] In step (2),

[0024] The PCR amplification reaction system is as follows: 20 μL reaction system includes: 1 μL of genomic DNA (5-50 ng / μL); 0.2 μL of DNA polymerase (5 U / μL); 2 μL of 10× DNA polymerase buffer; 1 μL of 2.5 mM dNTPs; 1.5 μL of 25 mM MgCl2; 0.1 μL each of the 10 μM outer primers shown in SEQ ID NO. 1-2; 0.3 μL each of the 10 μM inner primers shown in SEQ ID NO. 3-4; ddH2O is used to bring the reaction system to 20 μL.

[0025] The PCR amplification reaction procedure is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 5 min.

[0026] The rice samples included: rice varieties C418 (one of the representative varieties with the Hap1 haplotype of TAC4), Zhonghua 17 (one of the representative varieties with the Hap2 haplotype of TAC4), and F1 generation plants of the two hybrids (plants with the Hap1 / Hap2 heterozygous haplotype).

[0027] The present invention also provides a detection kit for single nucleotide polymorphism of rice TAC4 gene, which includes the above-mentioned primer set.

[0028] This invention also provides the application of the above-mentioned primer set or kit in molecular marker-assisted selection of rice plant type.

[0029] The inventors discovered in their research that when using ARMS-PCR technology for TAC4 genotyping, the four primers directly designed based on polymorphic sites have poor specificity and cannot distinguish SNP sites. Through extensive experimental screening, they found that only by artificially introducing a mismatched G base at the 5th base from the end of the 3' terminal of both the forward and reverse inner primers can a better genotyping effect be achieved. Furthermore, this artificially mismatched design is placed within the TAC4 gene, co-segregating with it, and the detection results directly reflect the TAC4 genotype, avoiding misidentification of the TAC4 genotype due to genetic exchange, resulting in more accurate detection results.

[0030] This invention successfully achieved ARMS-PCR specific amplification of two haplotypes of TAC4, Hap1 (the coding region has a T base at position 1793) and Hap2 (the coding region has a C base at position 1793). The designed primers have good specificity and high accuracy. The TAC4 gene can be genotyped by ordinary agarose gel electrophoresis. This invention can be used as a molecular marker to assist in the selection of rice plant type and provide a basis for the breeding of superior rice varieties.

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the embodiments do not limit the present invention in any way. For those skilled in the art, any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention. Attached Figure Description

[0032] Figure 1 The results show the amplification of Zhonghua 17 (haap2), C418 (haap1), and the F1 generation of the cross between C418 and Zhonghua 17 (heterozygous haap1 / Hap2) using molecular markers TAC4-J3 to TAC4-J7. M: DL2000 DNA marker; 1 and 2: two replicates of rice variety Zhonghua 17; 3 and 4: two replicates of rice variety C418; 5 and 6: two replicates of the F1 generation of the cross between C418 and Zhonghua 17.

[0033] Figure 2The results of TAC4 genotyping of 12 individual plants from the F2 generation of C418 and Zhonghua 17 in Example 2 are shown. M: DL2000 DNA marker; 1: Zhonghua 17 (haap2); 2: C418 (haap1); 3-14 are F2 generation individual plants, of which 5, 9, and 13 are haap1 (homozygous T / T genotype), 3 and 10 are haap2 (homozygous C / C genotype), and 4, 6, 7, 8, 11, 12, and 14 are heterozygous Hap1 and Hap2 plants (heterozygous T / C genotype). Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited thereto.

[0035] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0036] Example 1: ARMS-PCR primer set design for TAC4 rice plant architecture regulatory genes

[0037] I. Primer design

[0038] ARMS-PCR technology requires primers to have extremely high allele specificity in order to accurately distinguish SNP sites and eliminate typing errors caused by non-specific amplification. On the other hand, the four-primer system must be balanced in the same reaction to avoid imbalances in the intensity of electrophoretic bands, which can lead to uneven brightness or even missing bands and ultimately typing failure.

[0039] This invention designs ARMS-PCR primers based on the single nucleotide polymorphism (SNP) at position 1793 (T / C) of the coding region of the rice plant architecture regulatory gene TAC4. To obtain ARMS molecular markers with better amplification efficiency, the inventors artificially introduced mismatched bases at multiple different positions in both the forward and reverse inner primers, some of which are listed below:

[0040] The primer set contains four primers: a forward outer primer, a reverse outer primer, a forward inner primer, and a reverse inner primer. The forward outer primer and the reverse outer primer are located upstream and downstream of the 1793rd SNP in the TAC4 gene coding region, respectively; the 3' ends of the forward inner primer and the reverse inner primer terminate at the 1793rd SNP in the TAC4 gene coding region.

[0041] 1. TAC4-J3 primer set: A mismatched base G is artificially introduced at the third-to-last base position (G') of the 3' end of both the forward and reverse inner primers. The forward inner primer introduces a G's base interchange at the third-to-last base position (G''), and the reverse inner primer also introduces a G's base interchange at the third-to-last base position (G'''). The primer sequence for TAC4-J3 is as follows (underlined bases are the artificially introduced mismatched bases):

[0042] Forward lateral primer, TAC4 -ef: SEQ ID NO.1;

[0043] Reverse outer primer, TAC4 -er: SEQ ID NO.2;

[0044] Forward inner primer, TAC4-if3: 5'-TTGGTGCGATAAATTCAA G AT-3' (SEQ ID NO.5);

[0045] Reverse inner primer, TAC4-ir3: 5'-GGCAGCGCCCTCAT G AG-3' (SEQ ID NO.6).

[0046] 2. TAC4-J4 primer set: A mismatched base G is artificially introduced at the fourth-to-last base from the 3' end of both the forward and reverse inner primers. The forward inner primer introduces an A-G base swap at the fourth-to-last base from the 3' end, while the reverse inner primer introduces a TG base swap at the fourth-to-last base from the 3' end. The primer sequence for TAC4-J4 is as follows (underlined bases are the artificially introduced mismatched bases):

[0047] Forward lateral primer, TAC4 -ef: SEQ ID NO.1;

[0048] Reverse outer primer, TAC4 -er: SEQ ID NO.2;

[0049] Forward inner primer, TAC4-if4:5'-TTGGTGCGATAAATTCA G CAT-3' (SEQ ID NO.7);

[0050] Reverse inner primer, TAC4-ir4: 5'-GGCAGCGCCCTCA G CAG-3' (SEQ ID NO.8).

[0051] 3. TAC4-J5 primer set: A mismatched base G is artificially introduced at the fifth base from the end of the 3' terminal of both the forward and reverse inner primers. The forward inner primer introduces an A / G base swap at the fifth base from the end of the 3' terminal, and the reverse inner primer also introduces an A / G base swap at the fifth base from the end of the 3' terminal. The primer sequence for TAC4-J5 is as follows (underlined bases are the artificially introduced mismatched bases):

[0052] Forward lateral primer, TAC4 -ef: SEQ ID NO.1;

[0053] Reverse outer primer, TAC4 -er: SEQ ID NO.2;

[0054] Forward inner primer, TAC4-if5: SEQ ID NO.3;

[0055] Reverse inner primer, TAC4-ir5: SEQ ID NO.4.

[0056] 4. TAC4-J6 primer set: A mismatched base G is artificially introduced at the sixth base from the end of the 3' terminal of both the forward and reverse inner primers. The forward inner primer introduces a base swap of G / C at the sixth base from the end of the 3' terminal, and the reverse inner primer also introduces a base swap of G / C at the sixth base from the end of the 3' terminal. The primer sequence for TAC4-J6 is as follows (underlined bases are the artificially introduced mismatched bases):

[0057] Forward lateral primer, TAC4 -ef: SEQ ID NO.1;

[0058] Reverse outer primer, TAC4 -er: SEQ ID NO.2;

[0059] Forward inner primer, TAC4-if6:5'-TTGGTGCGATAAATT G AACAT-3' (SEQ ID NO.9);

[0060] Reverse inner primer, TAC4-ir6: 5'-GGCAGCGCCCT G ATCAG-3' (SEQ ID NO. 10).

[0061] 5. TAC4-J7 primer set: A mismatched base G is artificially introduced at the 7th base from the end of the 3' terminal of both the forward and reverse inner primers. The forward inner primer introduces a TG base exchange at the 7th base from the end of the 3' terminal, and the reverse inner primer also introduces a TG base exchange at the 7th base from the end of the 3' terminal. The primer sequence for TAC4-J7 is as follows (underlined bases are the artificially introduced mismatched bases):

[0062] Forward lateral primer, TAC4 -ef: SEQ ID NO.1;

[0063] Reverse outer primer, TAC4 -er: SEQ ID NO.2;

[0064] Forward inner primer, TAC4-if7:5'-TTGGTGCGATAAAT G CAACAT-3' (SEQ ID NO. 11);

[0065] Reverse inner primer, TAC4-ir7: 5'-GGCAGCGCCC G CATCAG-3' (SEQ ID NO. 12).

[0066] II. PCR Amplification and Electrophoresis

[0067] The primers for the five pairs of ARMS molecular markers, namely TAC4-J3 to TAC4-J7, were added to the same PCR reaction system in a ratio of forward outer primer: reverse outer primer: forward inner primer: reverse inner primer = 1:1:3:3. Using rice varieties C418 (homozygous T / T genotype, i.e., haplotype Hap1), Zhonghua 17 (homozygous C / C genotype, i.e., haplotype Hap2), and F1 generation plants of C418 and Zhonghua 17 (heterozygous T / C genotype, i.e., heterozygous haplotypes Hap1 / Hap2) as DNA templates, ARMS-PCR amplification was performed.

[0068] The ARMS-PCR reaction program used was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 5 min. The genotyping effect of the five ARMS molecular markers was detected by agarose gel electrophoresis. The ideal genotyping results were:

[0069] 1. When the haplotype of the rice sample TAC4 to be tested is Hap1 (the base at position 1793 in the coding region is homozygous T / T), only a common band of 494bp and a haplotype Hap1-specific band of 351bp can be amplified, for a total of two bands. The haplotype Hap2-specific band of 180bp cannot be amplified.

[0070] 2. When the haplotype of the rice sample TAC4 to be tested is Hap2 (the base at position 1793 in the coding region is homozygous C / C), only a common band of 494bp and a haplotype Hap2-specific band of 180bp can be amplified, for a total of two bands. The haplotype Hap1-specific band of 351bp cannot be amplified.

[0071] 3. When the haplotype of the rice sample TAC4 to be tested is heterozygous Hap1 and Hap2 (the base at position 1793 in the coding region is heterozygous T / C), a total of three bands can be amplified: a common band of 494 bp, a haplotype-specific band of 351 bp and a haplotype-specific band of 180 bp.

[0072] The 494bp common band is amplified by the forward and reverse outer primers, and this common band should be amplified in every sample. The 351bp band is a Hap1-specific band amplified by the forward and reverse outer primers, and the 180bp band is a Hap2-specific band amplified by the reverse inner primer and the forward outer primers.

[0073] The results of agarose gel electrophoresis are shown in [the table]. Figure 1 .

[0074] according to Figure 1 It can be seen that the amplification effects of using five different mismatched ARMS primer sets varied considerably. Among them, TAC4-J5 showed the best genotyping effect. When amplifying rice variety C418 (haap1), only two bands of 494bp and 351bp were amplified; when amplifying rice variety Zhonghua 17 (haap2), only two bands of 494bp and 180bp were amplified; when amplifying F1 plants of C418 and Zhonghua 17 (heterozygous haap1 / Hap2), three bands of 494bp, 351bp, and 180bp were amplified. Figure 1 and Figure 2 Furthermore, the brightness of the amplified common band and each specific band is most uniform.

[0075] Using primer sets other than TAC4-J5 results in either the 351bp specific band of haplotype Hap1 failing to be effectively amplified (e.g., TAC4–J3 and J6; even when using primer set J3, the 494bp common band that should have been amplified cannot appear stably due to primer interference and decreased PCR amplification stability), or the 180bp specific band of haplotype Hap2 being amplified in both parents (TAC4–J4), or the 351bp and 180bp bands of heterozygous haplotypes Hap1 / Hap2 failing to be effectively amplified or appearing faint (TAC4–J4 and J7). These primer sets cannot accurately distinguish between the three genotypes and cannot meet the needs of actual detection.

[0076] It can be seen that only by using the ARMS-PCR primer set TAC4-J5 designed in this invention can a good genotyping effect be achieved. If the mismatch site or type of the primer is not properly selected, the TAC4 haplotype cannot be effectively distinguished.

[0077] Therefore, this invention selects TAC4-J5 as the ARMS molecular marker for detecting TAC4 haplotypes.

[0078] The following experimental examples illustrate the beneficial effects of the present invention:

[0079] Experimental Example 1: Application of ARMS molecular marker TAC4-J5 in the detection of TAC4 haplotypes in rice

[0080] Rice varieties C418 (homozygous T / T genotype, i.e., haplotype Hap1) and Zhonghua 17 (homozygous C / C genotype, i.e. haplotype Hap2) were crossed. The F1 generation was self-pollinated to produce the F2 generation population. Genomic DNA was extracted from C418, Zhonghua 17 and 12 F2 generation individual plants.

[0081] The primers for the ARMS molecular marker TAC4-J5 were added to the same PCR reaction system in the following ratio (forward outer primer: reverse outer primer: forward inner primer: reverse inner primer = 1:1:3:3) for ARMS-PCR amplification.

[0082] The ARMS-PCR reaction system is 20 μL, and the specific components include: 1 μL genomic DNA (5-50 ng / μL); 0.2 μL DNA polymerase (5 U / μL); 2 μL DNA polymerase buffer (10×); 1 μL dNTP (2.5 mM); 1.5 μL MgCl2 (25 mM); 0.1 μL forward outer primer (10 μM); 0.1 μL reverse outer primer (10 μM); 0.3 μL forward inner primer (10 μM); 0.3 μL reverse inner primer (10 μM); and ddH2O to bring the reaction system to 20 μL.

[0083] The ARMS-PCR reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 5 min.

[0084] Prepare a 1.5% agarose gel and perform electrophoresis at 120V for 20 min. Observe and record the images using a gel imaging system. Determine the genotype of TAC4 based on the amplified bands. If the sample amplifies two bands (494bp and 351bp), the haplotype of the rice sample TAC4 is Hap1 (homozygous T / T genotype); if two bands (494bp and 180bp) are amplified, the haplotype of the rice sample TAC4 is Hap2 (homozygous C / C genotype); if three bands (494bp, 351bp, and 180bp) are amplified, the haplotype of the rice sample TAC4 is a heterozygote of Hap1 and Hap2 (heterozygous T / C genotype). The genotyping results are shown below. Figure 2 .

[0085] have Figure 2 It is known that the primer set of this invention can accurately detect haplotypes of rice TAC4.

[0086] In summary, the primer set with artificial mismatch and ARMS-PCR method introduced in this invention has high specificity and accuracy. The difference between heterozygous and homozygous genotypes of TAC4 can be distinguished by simple agarose gel electrophoresis. The detection results are reliable, easy to interpret and low in cost, and are suitable for accurate typing of the rice TAC4 gene.

Claims

1. A type of rice TAC4 A primer set for detecting single nucleotide polymorphisms of genes, characterized in that, It contains four ARMS-PCR primers: two outer primers with nucleotide sequences as shown in SEQ ID NO. 1-2, and two inner primers with nucleotide sequences as shown in SEQ ID NO. 3-4.

2. A type of rice TAC4 A method for detecting single nucleotide polymorphisms in genes, characterized in that, It is based on TAC4 The ARMS-PCR method developed using the T>C mutation at base 1793 in the coding region of a gene includes the following steps: (1) Extract genomic DNA from the rice sample to be tested; (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the primer set described in claim 1 to obtain PCR products; (3) Perform agarose gel electrophoresis on the PCR products and judge the results based on the electrophoresis results. TAC4 Genotype of a single nucleotide polymorphism site.

3. The detection method according to claim 2, characterized in that, The method for judging based on electrophoresis results is as follows: If two bands, 494bp and 351bp, are amplified, then the rice sample to be tested... TAC4 The haplotype is Hap1; If two bands, 494bp and 180bp, are amplified, then the rice sample to be tested... TAC4 The haplotype is Hap2; If three bands of 494bp, 351bp, and 180bp are amplified, then the rice sample to be tested... TAC4 The haplotype is a hybrid of Hap1 and Hap2.

4. The detection method according to claim 2, characterized in that, In step (2), The PCR amplification reaction system is as follows: A 20 μL reaction system includes: 1 μL of genomic DNA (5-50 ng / μL); 0.2 μL of DNA polymerase (5 U / μL); 2 μL of 10× DNA polymerase buffer; 1 μL of 2.5 mM dNTPs; 1.5 μL of 25 mM MgCl2; 0.1 μL each of the 10 μM outer primers shown in SEQ ID NO. 1-2; 0.3 μL each of the 10 μM inner primers shown in SEQ ID NO. 3-4; and ddH2O to bring the reaction system to 20 μL. The PCR amplification reaction procedure is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 5 min.

5. The detection method according to claim 2, characterized in that, The rice samples were: rice varieties C418, Zhonghua 17, and F1 generation plants of their hybridization.

6. A type of rice TAC4 A single nucleotide polymorphism (SNP) detection kit is characterized by, It includes the primer set as described in claim 1.

7. The application of the primer set of claim 1 or the kit of claim 6 in molecular marker-assisted selection of rice plant types.

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