Development of Polymorphic Primers for SNP and InDel Molecular Markers of Camellia vietnamensis Resources and Their Application Methods

Abstract

The present invention provides a method for developing polymorphic primers for SNP and InDel molecular markers of Camellia vietnamensis resources and its application method. The SNP and InDel molecular marker primers for Camellia vietnamensis resources include 5 pairs of primers with high polymorphism. The present invention has important application significance for scientifically protecting the diversity of Camellia vietnamensis germplasm resources, studying the genetic protection and genetic evolution of Camellia vietnamensis resources, and the genetic breeding of Camellia vietnamensis. The SNP and InDel molecular marker primers developed by the present invention have the advantages of simple method, high efficiency and specificity, and high sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of genetic breeding of woody oil crops in plant germplasm resources, and particularly relates to the development of polymorphic primers for SNP and InDel molecular markers of Camellia vietnamensis resources and their application methods. Background Art

[0002] Camellia vietnamensis is one of the species in the genus Camellia of the family Theaceae, and is also a characteristic woody oil crop widely planted in China. It has a wide ecological adaptability, tall tree body, lush branches and leaves, and can be used for afforestation of barren mountains, soil and water conservation, and water source conservation; in addition, its kernel has a high oil content and high single plant yield, and high yields can be obtained in a suitable ecological environment. The resources of Camellia vietnamensis are diverse. Especially the local Camellia vietnamensis in Hainan, its resources are a special ecological type of Camellia vietnamensis with rich genetic resources. In order to better preserve and compare the biological, ecological characteristics and economic traits of Camellia vietnamensis germplasm resources, it is necessary to collect and preserve the germplasm resources. Therefore, scientifically protecting the diversity of Camellia vietnamensis germplasm resources, studying the genetic protection and genetic evolution of Camellia vietnamensis resources, and the genetic breeding of Camellia vietnamensis have important application significance.

[0003] At present, there is a need to provide simple and efficient technologies or systems for the identification and protection of Camellia vietnamensis germplasm resources. The present invention provides polymorphic primers for SNP and InDel molecular markers of the chloroplast genome of Camellia vietnamensis, and provides application methods for the polymorphic primers of SNP and InDel molecular markers of the chloroplast genome of Camellia vietnamensis, which are helpful for the identification of Camellia vietnamensis varieties, the analysis of the population genetic structure and resource genetic diversity of Camellia vietnamensis. Summary of the Invention

[0004] The present invention obtains SNP and InDel polymorphic sites in Camellia vietnamensis germplasm resources from different regions through chloroplast whole-genome sequencing and genome comparative analysis of Camellia vietnamensis germplasm distributed in 5 different regions, and designs primers for SNP and InDel molecular markers of Camellia vietnamensis germplasm resources according to the SNP and InDel polymorphic sites. The present invention provides the following technical solutions:

[0005] Polymorphic primers for SNP molecular markers of Camellia vietnamensis resources, which are composed of polymorphic primer pairs YNSC01, YNSC03, YNSC04, and YNSC05, and their primer nucleotide sequences are as follows:

[0006] YNSC01 upstream primer: 5'-AGGTTCACAAGCAGCCGAAT-3';

[0007] YNSC01 downstream primer: 5'-AGAAGCCATTGCAATTGCCG-3';

[0008] YNSC03 upstream primer: 5'-CTAATTCGTCCCCGCCTACG-3';

[0009] YNSC03 downstream primer: 5'-GTCGACCGGGTCTACGAATC-3';

[0010] YNSC04 upstream primer: 5'-TGGTCCATTCTCAGCCTTGG-3';

[0011] YNSC04 downstream primer: 5'-ATCATGGCTTGAAGCGGGTT-3';

[0012] YNSC05 upstream primer: 5'-CGATCTTGATCCAGGCCTCA-3';

[0013] YNSC05 downstream primer: 5'-AGATTCGAATCACCAAATTGGCA-3'.

[0014] The polymorphic primers for InDel molecular markers of Camellia vietnamensis resources consist of YNSC02 polymorphic primer pairs, and their primer nucleotide sequences are as follows:

[0015] YNSC02 upstream primer: 5'-GGAGTCTTGGAATCTAGGTTTGGA-3';

[0016] YNSC02 downstream primer: 5'-TGGTAGCGCGTTTGTTTTGG-3'.

[0017] The application method of polymorphic primers for SNP and InDel molecular markers of Camellia vietnamensis resources includes the following steps:

[0018] (1) Extract the DNA of the sample to be analyzed: Extract the total genomic DNA of the Camellia vietnamensis germplasm material to be tested;

[0019] (2) PCR amplification reaction: Use the YNSC01 primer pair, the YNSC02 primer pair, the YNSC03 primer pair, the YNSC04 primer pair, and the YNSC05 primer pair to perform PCR amplification reactions on the genomic DNA in step (1);

[0020] (3) Detection of PCR amplification products: Electrophorese the PCR amplification products on a 1.5% agarose gel containing 10% Goldview nucleic acid dye, electrophorese at a voltage of 120 V for 40 min in 0.5×TBE buffer, and take pictures and record the running gel results in a gel imaging system;

[0021] (4) Detection of PCR amplification products: Sequence the PCR amplification products, and determine the gene polymorphism sites according to the nucleotide sequence information at the corresponding positions.

[0022] Further, the PCR amplification reaction program described in step (2) is as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min, for 40 cycles; extension at 72°C for 10 min.

[0023] Further, the PCR amplification reaction system described in step (2) is as follows: 20 ng of Camellia vietnamensis DNA, 0.20 mmol / L of dNTPs, 0.20 μmol / L of polymorphic primers, 2.00 U of Taq DNA polymerase, and ddH2O is added to make the total volume 30 μL.

[0024] Further, the genomic sequencing described in step (3) is Sanger first-generation conventional sequencing.

[0025] Advantages of the present invention:

[0026] The present invention provides SNP and InDel molecular marker primers for Camellia vietnamensis resources and their application methods. The SNP and InDel molecular marker primers for Camellia vietnamensis resources include 5 pairs of primers with high polymorphism. The present invention has important application significance for scientifically protecting the diversity of Camellia vietnamensis germplasm resources, studying the genetic protection and genetic evolution of Camellia vietnamensis resources, and the genetic breeding of Camellia vietnamensis. The SNP and InDel molecular marker primers developed by the present invention have the advantages of simple method, high efficiency and specificity, and high sensitivity. Description of the drawings

[0027] Figure 1 It is the electrophoresis result of the amplification products of primer YNSC01 in 4 Camellia vietnamensis materials;

[0028] Figure 2 It is the electrophoresis result of the amplification products of primer YNSC02 in 4 Camellia vietnamensis materials;

[0029] Figure 3 It is the electrophoresis result of the amplification products of primer YNSC03 in 4 Camellia vietnamensis materials;

[0030] Figure 4 It is the electrophoresis result of the amplification products of primer YNSC04 in 4 Camellia vietnamensis materials;

[0031] Figure 5 It is the electrophoresis result of the amplification products of primer YNSC05 in 4 Camellia vietnamensis materials;

[0032] Figure 6 It is the polymorphism sequence alignment result of primer YNSC01 in 4 Camellia vietnamensis materials;

[0033] Figure 7 Polymorphism sequence alignment results of primer YNSC02 in 4 Camellia vietnamensis materials;

[0034] Figure 8 Polymorphism sequence alignment results of primer YNSC03 in 4 Camellia vietnamensis materials;

[0035] Figure 9 Polymorphism sequence alignment results of primer YNSC04 in 4 Camellia vietnamensis materials;

[0036] Figure 10 Polymorphism sequence alignment results of primer YNSC05 in 4 Camellia vietnamensis materials. Detailed implementation manners

[0037] The following describes the detailed implementation manners of the present invention to facilitate those skilled in the art of the present technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the detailed implementation manners. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions made using the concept of the present invention are within the scope of protection.

[0038] Example 1: Development of SNP and InDel molecular marker primers

[0039] (1) Extract the genomic DNA of Camellia vietnamensis germplasms distributed in 5 different regions, respectively from 2 populations in Hainan, sample BP from Lingao, Hainan and sample QKY from Baisha, Hainan, sample HTDL from Luchuan County, Yulin, Guangxi, sample YN from Mingning County, Chongzuo, Guangxi, and sample WSG from Gaozhou City, Maoming, Guangdong. Use the Illumina second-generation sequencing technology to complete the total DNA sequencing of the samples, establish a sequencing library, and obtain SNP and InDel molecular markers of Camellia vietnamensis resources in different regions according to the chloroplast genome sequence of Camellia vietnamensis germplasms;

[0040] (2) According to the SNP and InDel molecular markers of Camellia vietnamensis resources in different regions obtained above, design 5 pairs of primers ( Figure 1 ), as Figure 1 shown, the primer sequences are:

[0041] YNSC01 forward primer: 5'-AGGTTCACAAGCAGCCGAAT-3';

[0042] YNSC01 reverse primer: 5'-AGAAGCCATTGCAATTGCCG-3';

[0043] YNSC02 upstream primer: 5'-GGAGTCTTGGAATCTAGGTTTGGA-3';

[0044] YNSC02 downstream primer: 5'-TGGTAGCGCGTTTGTTTTGG-3';

[0045] YNSC03 upstream primer: 5'-CTAATTCGTCCCCGCCTACG-3';

[0046] YNSC03 downstream primer: 5'-GTCGACCGGGTCTACGAATC-3';

[0047] YNSC04 upstream primer: 5'-TGGTCCATTCTCAGCCTTGG-3';

[0048] YNSC04 downstream primer: 5'-ATCATGGCTTGAAGCGGGTT-3';

[0049] YNSC05 upstream primer: 5'-CGATCTTGATCCAGGCCTCA-3';

[0050] YNSC05 downstream primer: 5'-AGATTCGAATCACCAAATTGGCA-3'.

[0051] Example 2: Application of SNP and Indel Molecular Marker Primers for Camellia vietnamensis Resources

[0052] (1) Select experimental materials: Select samples FCGS from Shangsi, Guangxi, sample MMGZ from Gaozhou City, Maoming, Guangdong, sample HKFY from Haikou, Hainan, and sample HNYC from Chengmai, Hainan, and the Camellia vietnamensis species as experimental materials for the verification of SNP and Indel molecular markers;

[0053] (2) Extract DNA of samples to be analyzed: Use the modified CTAB method to extract the total DNA of the above-mentioned experimental material Camellia vietnamensis samples;

[0054] (3) PCR amplification reaction: Using the extracted DNA of Camellia vietnamensis FCGS, MMGZ, HKFY, and HNYC as the amplification template, and the primers of the developed 5 pairs of SNP and Indel molecular markers as the amplification primers, perform PCR amplification. The PCR reaction system: 20 ng of Camellia vietnamensis DNA, 0.20 mmol / L dNTPs, 0.20 μmol / L polymorphic primers, 2.00 U of Taq DNA polymerase, supplemented with ddH2O to a total volume of 30 μL. The PCR reaction amplification program: Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min, for 40 cycles; finally, extension at 72°C for 10 min, and store at 4°C;

[0055] (4) Detection of PCR amplification products: The PCR amplification products are electrophoresed using 10% Goldview nucleic acid dye and 1.5% agarose gel in 0.5×TBE buffer at a voltage of 120 V for 40 min, and the gel running results are photographed and recorded in the gel imaging system;

[0056] (5) Detection of PCR amplification products: The PCR amplification products are subjected to Sanger first-generation conventional sequencing, and the gene polymorphism sites are determined according to the nucleotide sequence information at the corresponding positions.

[0057] The detection results are as follows:

[0058] As shown in Table 1, the starting and ending sites of the gene fragment sequences of the 5 pairs of polymorphic primers in the chloroplast genome are respectively: 60611 - 61556, 67895 - 69287, 82229 - 82928, 122714 - 129090.

[0059] Table 1 Nucleotide sequences of 5 pairs of primers

[0060]

[0061] As Figures 1 to 5 shown:

[0062] The amplified band sizes of primer YNSC01 in 4 Camellia vietnamensis materials are about 945 bp; the amplified band sizes of primer YNSC02 in 4 Camellia vietnamensis materials are about 1392 bp; the amplified band sizes of primer YNSC03 in 4 Camellia vietnamensis materials are about 699 bp; the amplified band sizes of primer YNSC04 in 4 Camellia vietnamensis materials are about 1300 bp; the amplified band sizes of primer YNSC05 in 4 Camellia vietnamensis materials are about 1035 bp.

[0063] Table 2 shows the analysis of the sequencing base results of polymorphic primers. The sequencing base results of 5 pairs of primers showed polymorphism. Among them, YNSC01-SNP, YNSC03-SNP, YNSC04-SNP, and YNSC05-SNP are SNP polymorphic base sites, and YNSC02-InDel is an InDel polymorphic base site.

[0064] Table 2 Analysis of the sequencing base results of polymorphic primers

[0065]

[0066] As Figures 6 to 10 shown: When using YNSC01 as a primer for PCR amplification, the base mutations in FCGS and MMGZ are "G", and the base mutations in HNYC and HKFY are "T"; when using YNSC02 as a primer for PCR amplification, the base sequence "ATTAAC" exists in FCGS and MMGZ, and the base sequence "ATTAAC" is missing in HNYC and HKFY; when using YNSC03 as a primer for PCR amplification, the base mutations in FCGS and MMGZ are "C", and the base mutations in HNYC and HKFY are "A"; when using YNSC04 as a primer for PCR amplification, the base mutations in FCGS and MMGZ are "T", and the base mutations in HNYC and HKFY are "G"; when using YNSC05 as a primer for PCR amplification, the base mutations in FCGS and MMGZ are "C", and the base mutations in HNYC and HKFY are "A". The 5 pairs of primers showed polymorphism in the base of Camellia vietnamensis.

[0067] In the present invention, specific embodiments are used to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation manner and application scope. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A primer set for identifying different Vietnamese camellia oleifera varieties, characterized in that: It includes a SNP molecular marker primer set and an InDel molecular marker primer set, and the primer set consists of YNSC01, YNSC02, YNSC03, YNSC04, and YNSC05, and its nucleotide sequence is as follows: YNSC01 upstream primer: 5′-AGGTTCACAAGCAGCCGAAT-3′; YNSC01 downstream primer: 5′-AGAAGCCATTGCAATTGCCG-3′; YNSC02 upstream primer: 5′-GGAGTCTTGGAATCTAGGTTTGGA-3′; YNSC02 downstream primer: 5′-TGGTAGCGCGTTTGTTTTGG-3′; YNSC03 upstream primer: 5′-CTAATTCGTCCCCGCCTACG-3′; YNSC03 downstream primer: 5′-GTCGACCGGGTCTACGAATC-3′; YNSC04 upstream primer: 5′-TGGTCCATTCTCAGCCTTGG-3′; YNSC04 downstream primer: 5′-ATCATGGCTTGAAGCGGGTT-3′; YNSC05 upstream primer: 5′-CGATCTTGATCCAGGCCTCA-3′; YNSC05 downstream primer: 5′-AGATTCGAATCACCAAATTGGCA-3′; The Vietnamese tea oil varieties include Vietnamese tea oil FCGS, Vietnamese tea oil MMGZ, Vietnamese tea oil HKFY and Vietnamese tea oil HNYC.

2. The method for identifying Vietnamese camellia oleifera materials using the primer set of claim 1, characterized in that: The steps include: (1) Extracting DNA from samples to be analyzed: Extracting total genomic DNA from the Vietnamese Camellia oleifera germplasm materials to be tested; (2) PCR amplification reaction: using the five pairs of primers in claim 1 to perform PCR amplification reaction on the genomic DNA in step (1); Among them, the site recognition sequence of the YNSC01 upstream primer and YNSC01 downstream primer identification site is: GAATAAAAAT[G / T]AAATCAAGTA; Site recognition sequence of the site identified by YNSC02 upstream primer and YNSC02 downstream primer: ATGAGTTAAAATTAAC-----ATTAA------CGTAA or ATGAGTTAAAA---------TTAAC----------------------------------------------------ATTAAC----GTAA; Site recognition sequence of the site identified by the YNSC03 upstream primer and YNSC03 downstream primer: CGCCCTTTTT[C / A]TTTTTTTTTT; Site recognition sequence for the YNSC04 upstream primer and YNSC04 downstream primer identification site: AAATATTCCA[T / G]CCGCCCAAGT; Site recognition sequence for the site identified by the YNSC05 upstream primer and the YNSC05 downstream primer: TACATAATAT[C / A]ACTTTTTCCT; (3) Detection of PCR amplification products: The PCR amplification products were electrophoresed on a 1.5% agarose gel containing 10% Goldview nucleic acid dye in 0.5× TBE buffer at 120 V for 40 min. The gel running results were photographed and recorded in a gel imaging system. (4) PCR amplification product detection: PCR amplification products are subjected to genome sequencing; The Vietnamese camellia oil materials include Vietnamese camellia oil FCGS, Vietnamese camellia oil MMGZ, Vietnamese camellia oil HKFY and Vietnamese camellia oil HNYC.

3. The method according to claim 2, characterized in that The PCR amplification reaction procedure described in step (2) is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min, for 40 cycles; extension at 72°C for 10 min.

4. The method according to claim 2, characterized in that: The PCR amplification reaction system described in step (2) is: 20 ng of Camellia oleifera DNA, 0.20 mmol / L of dNTPs, 0.20 μmol / L of polymorphic primers, 2.00 U of Taq DNA polymerase, and ddH2O added to a total volume of 30 μL.

5. The method according to claim 2, characterized in that: The genome sequencing described in step (3) is conventional Sanger first generation sequencing.

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