Camellia tonkinensis CdS-RNase gene insertion mutation and molecular identification method and application thereof
By developing specific primer pairs and optimizing PCR conditions, the 64bp insertion mutation of the CdS-RNase gene of Vietnamese camellia was quickly screened, which solved the problem of screening CdSm-RNase mutants in the existing technology, improved the self-pollination fruit setting rate, and promoted the breeding and promotion of Vietnamese camellia.
Patent Information
- Application Number
- CN202510924542.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-07-19
- Filing Date
- 2025-07-04
- Publication Date
- 2025-10-28
AI Technical Summary
The lack of existing technologies for rapidly screening CdSm-RNase mutants in Vietnamese Camellia oleifera germplasm resources leads to low self-pollination fruit set rate, hindering its breeding and promotion.
Specific primer pairs were developed, and based on PCR amplification technology, the 64bp insertion mutation of the CdS-RNase gene was detected, the PCR reaction system and amplification conditions were optimized, and rapid screening of CdS-RNase gene mutants was achieved.
The screening efficiency of CdS-RNase mutants, the pistil-determining factor of self-incompatibility in Vietnamese tea oil, was improved, the self-pollination fruit setting rate was improved, the field parent matching was guided, and the fruit setting rate of Vietnamese tea oil was increased.
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Figure CN120843556A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of molecular biology and plant molecular breeding technology, specifically relating to the CdS-RNase gene insertion mutation in Camellia oleifera from Vietnam and its molecular identification methods and applications. Background Technology
[0002] Camellia drupifera, commonly known as Vietnamese camellia, is the third most widely cultivated camellia species in my country, primarily distributed in Guangdong, Guangxi, and Hainan provinces. It is renowned for its large, heavy fruit, high seed yield, and high oil content. The cultivation and planting of Vietnamese camellia is an important component of tropical high-efficiency agriculture, playing a vital role in increasing farmers' income and ensuring national food and oil security. However, Vietnamese camellia is a strictly cross-pollinated plant, with an extremely low self-pollination fruit set rate (<5%). This self-incompatibility limitation has resulted in a generally low yield, a problem that has long plagued Vietnamese camellia breeders and growers, hindering its rapid promotion and negatively impacting the improvement of quality and efficiency in tropical high-efficiency agriculture.
[0003] Previous studies have shown that self-incompatibility in Camellia oleifera var. praecox belongs to the late-acting self-incompatibility (LSI) type, mainly controlled by the self-incompatibility locus, and may also be regulated by other self-incompatibility-related factors. Our team previously identified the CdS-RNase gene, a pistil-determining factor at the S-locus of self-incompatibility in Camellia oleifera var. praecox. This not only confirmed that crossbreeding between Camellia oleifera var. praecox germplasm of different CdS-RNase protein types significantly improved the fruit setting rate, but also discovered the presence of a mutant CdS-RNase gene (CdS...). m The self-pollination and fruit setting rate of Camellia oleifera var. chinensis in Vietnam was significantly improved by CdS-RNase. Subsequently, our team analyzed the diversity of CdS-RNase genes and their encoded proteins in 78 Camellia oleifera var. chinensis germplasm resources from Hainan Island, finding that the CdS-RNase gene and its encoded protein diversity in Camellia oleifera var. chinensis var. chinensis were rich in CdS-RNase genes and their encoded proteins. Based on the CdS-RNase protein type, the Camellia oleifera var. chinensis germplasm was divided into three categories: Type I, where only normal CdS-RNase protein was detected; Type II, where both normal CdS-RNase protein and mutant CdS-RNase protein were detected. m -RNase protein; Type III, only mutant CdS detected m -RNase protein. In Vietnamese Camellia oleifera germplasm resources, the self-pollination fruit set rate of type I germplasm was significantly lower than that of type II, and the self-pollination fruit set rate of type II germplasm was significantly lower than that of type III, indicating that the CdS mutation... m -RNase protein can improve the self-pollination fruit setting rate of Camellia oleifera in Vietnam to a certain extent.
[0004] Therefore, rapid screening for CdS mCdS-RNase gene mutations in Vietnamese Camellia oleifera germplasm are of positive significance for guiding the field pairing of different CdS-RNase genotypes in Vietnamese Camellia oleifera and improving the low self-pollination fruit set rate. However, there is still no information on how to quickly screen Vietnamese Camellia oleifera germplasm containing CdS-RNase. m Methods for RNase mutants reported. Summary of the Invention
[0005] This invention, through CdS-RNase gene diversity analysis and haplotype classification in two Camellia oleifera germplasms from Vietnam, identified two CdS-RNase genes containing a 64bp insertion fragment in two Camellia oleifera germplasms. m -RNase type, encoding a truncated mutant CdS m -RNase protein type. This invention develops specific primer pairs based on a 64bp insertion mutation in the CdS-RNase gene, identifying the CdS-RNase gene mutation according to the size of the PCR amplified fragment. Based on optimized PCR reaction system and amplification conditions, a method for rapid detection of the 64bp insertion mutation in the CdS-RNase gene based on PCR amplification has been developed, improving the efficiency of screening for CdS-RNase gene mutants, a key factor in self-incompatibility and pistil determination in Camellia oleifera from Vietnam.
[0006] In order to achieve the above object, the present invention provides the following technical solutions:
[0007] The first aspect of this invention provides a 64bp insertion mutation in the CdS-RNase gene, a self-incompatible pistil-determining factor in Camellia oleifera from Vietnam. The nucleotide sequence of the insertion mutation is as follows: GTGAG AATGT GCCTT AATCT CTCTC TCTCTCTCTC TCTCT CTCAG CTTGG AACTT TGGCT GCAG; GTGAG AATGT GCCTT AATCT CTCTC TCTCTCTCTC TCTCT CTAAA CTTGG AACTT TGGCT GCAG.
[0008] A second aspect of this invention provides a primer pair for detecting a 64bp insertion mutation in the CdS-RNase gene, a pistil-determining factor in self-incompatibility in Camellia oleifera from Vietnam, comprising the following primer sequences:
[0009] Forward primer F: 5'-TGCAAYGCCTAGCAGTTC-3';
[0010] Reverse primer R: 5'-TGGRTAAGTGCCGTCGTT-3';
[0011] Where Y represents T / C degeneracy and R represents G / A degeneracy, used to amplify the 64bp insertion region gene sequence of the CdS-RNase gene.
[0012] The third aspect of the present invention provides a method for analyzing and identifying the 64bp insertion mutation of the CdS-RNase gene, a self-incompatible pistil-determining factor in the above-mentioned Camellia oleifera from Vietnam. The method uses the cDNA of Camellia oleifera from Vietnam to be identified as an amplification template, performs PCR amplification on the template using the above-mentioned primer pair, and determines the 64bp insertion mutation of the CdS-RNase gene, a self-incompatible pistil-determining factor in the above-mentioned Camellia oleifera from Vietnam to be identified, based on the size of the amplification product.
[0013] Furthermore, the PCR amplification reaction system is 20 μL: 10 μL of 2×PhantaMax MasterMix, 0.5 μL each of 10 μM forward and reverse primers, 1 μL of cDNA template, and 8 μL of ddH2O;
[0014] The PCR amplification reaction program was 95℃ for 3 min; 95℃ for 15 sec, 53℃ for 15 sec, 72℃ for 20 sec, for 35 cycles; 72℃ for 5 min.
[0015] Furthermore, the method for determining the 64bp insertion mutation in the CdS-RNase gene, the pistil-determining factor for self-incompatibility in Camellia oleifera from Vietnam, was electrophoresis detection.
[0016] Furthermore, the cDNA of the Vietnamese Camellia oleifera to be identified was obtained by extracting RNA from the style of the Camellia oleifera to be identified and reverse transcribing it.
[0017] Furthermore, the basis for determining the size of the amplification product is as follows:
[0018] When the PCR amplification product fragment length is 243bp, it is determined to contain a 64bp CdS mutation. m -RNase Vietnamese Camellia oleifera germplasm;
[0019] When the PCR amplification product fragment length is 179bp, it is determined to be a normal CdS-RNase Vietnamese Camellia oleifera germplasm without the 64bp mutation.
[0020] The fourth aspect of this invention provides an application in screening for CdS-RNase gene mutants, which are the pistil determinants of self-incompatibility in Camellia oleifera from Vietnam.
[0021] The beneficial effects of this invention are:
[0022] This invention, based on a newly discovered CdS-RNase mutation containing a 64bp insert fragment, rationally designed primer pairs and optimized PCR systems and amplification conditions to develop a rapid detection method for this mutation type. An example demonstrates its effectiveness in detecting CdS-RNase mutants containing the 64bp insert fragment in Vietnamese Camellia oleifera. This invention can rapidly screen Vietnamese Camellia oleifera germplasm resources containing CdS-RNase. m The mutant of CdS-RNase can improve the screening efficiency of CdS-RNase mutants, which are the key determinants of self-incompatibility in Camellia oleifera in Vietnam. This is of positive significance for guiding the matching of different CdS-RNase genotypes among parents in Camellia oleifera in Vietnam and improving the low self-pollination fruit setting rate of Camellia oleifera in Vietnam. Attached Figure Description
[0023] Figure 1 Sequence alignment of 10 CdS-RNase haplotypes (Hap1 to Hap10) in Vietnamese Camellia oleifera: 64bp insertion fragments detected in Hap9 and Hap10 are marked in red, differences between the two 64bp insertion sequences are marked in yellow, and degenerate bases in the primer pairs of this invention are marked in yellow.
[0024] Figure 2 A schematic diagram of the primer pairs for amplifying the 64bp insertion fragment of the CdS-RNase gene and the length of its PCR product.
[0025] Figure 3 Electrophoretic detection of PCR products of the 64bp insertion fragment of the CdS-RNase gene.
[0026] Figure 4 Electrophoresis diagram showing the detection and validation effect of the 64bp insertion fragment of the CdS-RNase gene. Detailed Implementation
[0027] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.
[0028] Example 1: Discovery of a 64bp insertion mutation in the CdS-RNase gene
[0029] 1. The styles of 13 Vietnamese Camellia oleifera germplasms were selected (Table 1). Total RNA was extracted and reversed into cDNA. The full-length CdS-RNase gene was amplified by PCR, ligated into the blunt-end sequencing vector PeasyBlunt, and sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing to obtain the full-length CdS-RNase gene sequence.
[0030] 2. The reliability of sequencing mutation sites was assembled and verified using the ContigExpress module of VectorNTI Advance 11.5.2 software. All full-length CdS-RNase gene sequences were aligned using the AlignX module. Based on the alignment results, the coding sequences of CdS-RNase genes were organized. The CdS-RNase gene coding sequences from 13 Vietnamese Camellia oleifera germplasm accessions were then classified into haplotypes (Hap1–Hap10) using DnaSP5 (version 5.10) software. Figure 1 Among them, the Hap9 and Hap10 haplotypes contain a 64bp insertion fragment, while the Hap1 to Hap8 haplotypes do not contain a 64bp insertion fragment.
[0031] The nucleotide sequence of the 64bp insert is as follows: GTGAG AATGT GCCTT AATCT CTCTC TCTCTCTCTC TCTCT CTCAG CTTGG AACTT TGGCT GCAG;
[0032] GTGAG AATGT GCCTT AATCT CTCTC TCTCT CTCTC TCTCT CTAAA CTTGG AACTTTGGCT GCAG (SEQ ID NO. 1).
[0033] Table 1. Materials for CdS-RNase gene sequence diversity analysis of Vietnamese Camellia oleifera germplasm in this invention.
[0034]
[0035] Example 2: Development of primer pairs for amplification of the 64bp insert fragment of the CdS-RNase gene
[0036] Using Primer Premier 6.0 software, primers were designed upstream and downstream of the 64bp insert fragment using Hap9 and Hap10 sequences as templates. Figure 2 The final designed primer pair sequences are: F(5'-3'): TGCAAYGCCTAGCAGTTC (SEQ ID NO.2); R(5'-3'):
[0037] TGGRTAAGTGCCGTCGTT (SEQ ID NO.3); where Y represents T / C degeneracy and R represents G / A degeneracy, used to amplify the 64bp insertion region gene sequence of the CdS-RNase gene.
[0038] Example 3: PCR amplification conditions for screening the 64bp insertion fragment of the CdS-RNase gene
[0039] PCR reaction system (20 μL):
[0040]
[0041] PCR amplification procedure:
[0042]
[0043] Example 4: Electrophoretic detection of PCR products from the 64bp CdS-RNase gene insert.
[0044] PCR products were detected by electrophoresis on a 2% agarose gel (containing 100 mL / 5 μL of 5% Goldview novel nucleic acid dye) at 120 V for 40 min in 0.5×TBE buffer. A DYY-7C electrophoresis system (Beijing Liuyi Biotechnology Co., Ltd.) was used, and gel imaging was performed using a Gel Doc™ XR+ (Bio-Rad).
[0045] Example 5: Verification of the effect of PCR electrophoresis detection of the 64bp insertion fragment of the CdS-RNase gene.
[0046] Twenty-four new Vietnamese Camellia oleifera germplasm materials were selected. Using the style cDNA of these 24 Vietnamese Camellia oleifera germplasm materials as templates, the primer pairs, PCR amplification system, and PCR amplification conditions of this invention were used. The size of the corresponding PCR amplified fragments was detected by 2% agarose gel electrophoresis. Figure 4 The detection results showed fragments of length 243bp and 179bp, indicating that the detection method of the present invention is feasible and can rapidly detect the 64bp insertion fragment of the CdS-RNase gene in Camellia oleifera from Vietnam.
[0047] Example 6: Association Analysis of 64bp CdS-RNase Gene Insertion with Self-Incompatibility
[0048] To evaluate the effect of the 64bp CdS-RNase gene insertion on the self-pollination fruit set rate of Camellia oleifera var. chinensis in Vietnam, 24 Camellia oleifera var. chinensis germplasm materials provided in Example 5 were selected for a statistical experiment on self-pollination fruit set rate. The results of the self-pollination fruit set rate experiment are shown in Table 2. The self-pollination fruit set rate of Camellia oleifera var. chinensis germplasm containing the 64bp insertion fragment in the CdS-RNase gene was significantly higher. Figure 4The materials corresponding to bands 1 and 2 in the middle band have a significantly higher self-pollination fruit set rate than the other 22 CdS-RNase genes that do not contain the 64bp insertion fragment, indicating that CdS-RNase gene mutation can improve the self-pollination fruit set rate of Vietnamese Camellia oleifera to a certain extent.
[0049] Table 2. Validation materials for the 64bp CdS-RNase gene insertion and their self-pollination and fruit set rates.
[0050]
[0051] Note: When comparing Duncan's self-pollination fruit set rates, the same letter indicates no significant difference (P>0.05), and different letters indicate significant differences (P<0.05). The self-pollination fruit set rates of various Vietnamese Camellia oleifera germplasms were statistically analyzed using a self-pollination experiment with 50 flowers and three independent replicates.
[0052] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A 64bp insertion mutation in the CdS-RNase gene, a pistil-determining factor in self-incompatibility in Camellia oleifera from Vietnam, characterized by: The nucleotide sequences of the insertion mutation are as follows: GTGAG AATGT GCCTT AATCT CTCTC TCTCT CTCTC TCTCTCTCAG CTTGG AACTT TGGCT GCAG; GTGAG AATGT GCCTT AATCT CTCTC TCTCT CTCTC TCTCTCTAAA CTTGG AACTT TGGCT GCAG.
2. A primer pair for detecting a 64bp insertion mutation in the CdS-RNase gene, a self-incompatible pistil-determining factor, of the Vietnamese Camellia oleifera as described in claim 1, characterized in that, Including the following primer sequences, Forward primer F: 5'-TGCAAYGCCTAGCAGTTC-3'; Reverse primer R: 5'-TGGRTAAGTGCCGTCGTT-3'; Where Y represents T / C degeneracy and R represents G / A degeneracy, used to amplify the 64bp insertion region gene sequence of the CdS-RNase gene.
3. A method for analyzing and identifying a 64bp insertion mutation in the CdS-RNase gene, a self-incompatible pistil-determining factor, of the Vietnamese Camellia oleifera as described in claim 1, characterized in that, This method uses the cDNA of the Vietnamese Camellia oleifera to be identified as an amplification template, and performs PCR amplification on the template using the primer pair of claim 2. The 64bp insertion mutation of the CdS-RNase gene, the self-incompatible pistil determinant of the Vietnamese Camellia oleifera to be identified, is determined based on the size of the amplification product.
4. The analytical identification method as described in claim 3, characterized in that, The PCR amplification reaction system consisted of 20 μL: 10 μL of 2×Phanta Max MasterMix, 0.5 μL each of 10 μM forward and reverse primers, 1 μL of cDNA template, and 8 μL of ddH2O. The PCR amplification reaction program was 95℃ for 3 min; 95℃ for 15 sec, 53℃ for 15 sec, 72℃ for 20 sec, for 35 cycles; 72℃ for 5 min.
5. The analytical identification method as described in claim 3, characterized in that, The 64bp insertion mutation in the CdS-RNase gene, a pistil-determining factor in self-incompatibility of Camellia oleifera in Vietnam, was determined by electrophoresis.
6. The analytical identification method as described in claim 3, characterized in that, The cDNA of the Vietnamese Camellia oleifera to be identified was obtained by extracting RNA from the style of the Camellia oleifera to be identified and reverse transcribing it.
7. The analytical identification method as described in claim 3, characterized in that, The basis for determining the size of the amplification product is as follows: When the PCR amplification product fragment length is 243bp, it is determined to contain a 64bp CdS mutation. m -RNase Vietnamese Camellia oleifera germplasm; When the PCR amplification product fragment length is 179bp, it is determined to be a normal CdS-RNase Vietnamese Camellia oleifera germplasm without the 64bp mutation.
8. The application of the analytical identification method according to any one of claims 3 to 7 in screening for CdS-RNase gene mutants, which are the pistil determinants of self-incompatibility in Camellia oleifera from Vietnam.