CN113215299B - SNP closely linked with control of capsaicine content of capsicum fruits, specific primer and application thereof - Google Patents
SNP closely linked with control of capsaicine content of capsicum fruits, specific primer and application thereof Download PDFInfo
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- CN113215299B CN113215299B CN202110607899.3A CN202110607899A CN113215299B CN 113215299 B CN113215299 B CN 113215299B CN 202110607899 A CN202110607899 A CN 202110607899A CN 113215299 B CN113215299 B CN 113215299B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention relates to the field of molecular markers, in particular to SNP closely linked with control of capsaicin content of pepper fruits, a specific primer and application thereof. According to the invention, 311 annual pepper materials are subjected to whole genome association analysis, SNP sites which are positioned on chromosome 6 and are obviously related to capsaicin content control are found, a candidate gene Capana06g001204 is determined, 1 SNP site which is obviously related to capsaicin content is positioned on the gene, and related CAPS primers are designed and applied. The research utilizes a large sample size to carry out correlation analysis, so that the SNP related molecular marker has a wider application range.
Description
Technical Field
The invention relates to the field of molecular markers, in particular to SNP closely linked with control of capsaicin content of pepper fruits, a specific primer and application thereof.
Background
Due to synthesis of capsaicin substances, the capsaicin substance content shows quantitative trait inheritance in pepper, former people report that the capsaicin substance content is positioned to more Quantitative Trait Loci (QTL), and Yarnes and the like (2012) position the capsaicin substance content to 12 QTL loci on 6 chromosomes; ben Chaim et al (2006) mapped it to chromosome 4; blum et al mapped a major QTL to chromosome 7 through BSA (bulked segregant analysis), Zhu et al (2019) also mapped a major QTL controlling capsaicin content on chromosome 7, and determined a candidate gene Pu 3, which is transcription factor MYB 31; nimmayala et al (2016) found 14 SNPs with significant correlations to capsaicin and dihydrocapsaicin content by Genome Wide Associated Study (GWAS) using 94 parts of pepper material; lee et al (2016) mapped the QTL sites for capsaicin content to chromosomes 3 and 6 and mapped the QTL sites for dihydrocapsaicin content to chromosome 2. According to previous reports, the inheritance of the content of capsaicin substances is complex, the content of the capsaicin substances has great difference under different genetic backgrounds, genes for controlling related traits are arranged in a plurality of segments of a plurality of chromosomes, and the utilization of the genes related to the genetic traits and molecular markers needs to be combined with different genetic backgrounds.
Disclosure of Invention
The invention aims to provide application of SNP closely linked with control of capsaicin content in pepper fruits.
It is still another object of the present invention to provide specific primers for SNPs closely linked to control capsaicin content in capsicum fruits.
Still another object of the present invention is to provide the use of the specific primer mentioned above for closely linking to the SNP for controlling the capsaicin content in capsicum fruits.
Still another object of the present invention is to provide a method for identifying capsaicin content in pepper fruits.
The invention provides application of SNP (single nucleotide polymorphism) closely linked with control of capsaicin content of pepper fruits, wherein when the base at a 22820065bp locus of a chromosome is T, the capsaicin content is less than 2000 mg/kg; when the base group is C, the content of capsaicin is more than 2000 mg/kg.
The nucleotide sequence of the specific primer closely linked with the control of the capsaicin content of the capsicum fruit according to the invention is as follows:
the front primer is F: 5 'CCTTCTGCTTAGGAGAGACACGACA 3', the rear primer is R: 5 'CCCACATTCTGCCTTTGCCATGAAT 3'.
The invention provides application of the specific primer, and the specific primer is used for identifying the capsaicin content of pepper fruits.
The invention provides a method for identifying capsaicin content of pepper fruits, which comprises the step of performing PCR identification by using a specific primer of SNP closely linked with the control of capsaicin content of pepper fruits, wherein the nucleotide sequence of the specific primer is as follows:
the front primer is F: 5 'CCTTCTGCTTAGGAGAGACACGACA 3', the rear primer is R: 5 'CCCACATTCTGCCTTTGCCATGAAT 3'.
According to the method for identifying the capsaicin content of the pepper fruits, disclosed by the invention, a PCR amplification product is 140bp, the PCR product is subjected to SspI enzyme digestion, when the SNP site is a base T, the product can be subjected to enzyme incision, and when the enzyme digestion product presents 2 strips, the capsaicin content is less than 2000 mg/kg; when the SNP locus is a variation type base, namely C, the SNP locus cannot be cut by enzyme, the enzyme digestion product presents 1 strip by agarose gel electrophoresis, and the content of capsaicin is more than 2000 mg/kg.
According to the invention, 311 annual pepper materials are subjected to whole genome association analysis, SNP sites which are positioned on chromosome 6 and are obviously related to capsaicin content control are found, a candidate gene Capana06g001204 is determined, 1 SNP site which is obviously related to capsaicin content is positioned on the gene, and related CAPS primers are designed and applied. The research utilizes a large sample size to carry out correlation analysis, so that the SNP related molecular marker has a wider application range.
Drawings
FIG. 1 shows the result of PCR amplification, wherein, a. agarose electrophoresis PCR amplification product, M, marker D2000, PCR amplification product length is 140 bp; b. performing PCR amplification on polyacrylamide gel electrophoresis, and performing SspI enzyme digestion on the product, wherein M, marker D2000, 1-6 are reference genome genotypes, and 7-11 are mutant types.
Detailed Description
Example 1
Re-sequencing 311 annual peppers (Capsicum annuum L.), wherein the sequencing platform is Illumina Solexa, processing original data by using Trimmomatic v0.33, removing a linker, poly-N and a low-quality fragment to obtain clean data, comparing the clean data to Zunla-1 genome data (Qin et al, 2014) by using BWA0.75, and setting parameters to be aln-o 1-e 10-t 4-L32-i 15-q 10; by utilizing Samtools 0.1.19(Li et al, 2009), setting a parameter-q 1-C50-S-D-m 2-F0.002-u by adopting a Bayesian algorithm, operating a command mpieup, searching for SNP, filtering SNP, and setting the minimum gene allelic frequency to be more than 0.01 and the deletion rate to be less than 0.1.
Removing seeds and fruit stalks of 311 annual hot peppers (Capsicum annuum L.), drying at 55-60 ℃, grinding into powder by using a grinder, determining the capsaicin content by adopting ultra-high performance liquid chromatography (UPLC), performing whole genome association analysis on the SNP obtained by filtering and capsaicin content data by adopting a linear mixed model in Gemma (Zhou and Stephens 2012) by adopting a capsaicin extraction and determination method according to agricultural industry standards, searching genes near the site, and determining the genes with higher expression in the placenta of the Capana06g001204 in the capsaicin synthesis period, thereby determining the Capana06g001204 as a candidate gene for controlling the capsaicin content.
On this gene Chr 06: 22820065bp has nonsynonymous mutation SNP obviously related to capsaicin content, the base of the reference genome is T, and the variant base is C.
The content of capsaicin in 142 parts of pepper material is counted and calculated according to the weight ratio of the pepper material in Chr 06: 22820065bp base type, the locus genotype has better corresponding relation with the hot taste. When the capsaicin content is 2000mg/kg as a threshold value, 88.64% of materials with the capsaicin content being less than 2000mg/kg have T at the site; more than 2000mg/kg of material 100% of which are C at this site; overall statistics show that Chr 06: the identification accuracy of the 22820065bp base pair material with high capsaicin content is 89.44%.
Designing dCAPS primer for the SNP locus, wherein the primer is F: CCTTCTGCTTAGGAGAGACACGACA, the rear primer is R: CCCACATTCTGCCTTTGCCATGAAT, PCR the amplification product was 140bp and the agarose gel electrophoresis was performed as shown in a in FIG. 1. B in figure 1 shows that polyacrylamide gel electrophoresis PCR is amplified and the products are subjected to SspI enzyme digestion, 1-6 are reference genome genotypes, 7-11 are mutant types, SNP loci are reference genome types, namely T, the products can be subjected to enzyme cutting, the enzyme digestion products show 2 bands through agarose gel electrophoresis, and capsaicin is less than 2000 mg/kg; when the SNP locus is a variation type base, namely C, the enzyme can not be cut, the enzyme digestion product presents 1 strip by agarose gel electrophoresis, and the content of capsaicin is more than 2000 mg/kg.
The molecular marker designed aiming at the mutation site is a candidate gene internal molecular marker, the identification accuracy of the gene is 100 percent, and the method has important significance for molecular marker-assisted selective breeding.
Sequence listing
<110> vegetable and flower institute of Chinese academy of agricultural sciences
<120> SNP closely linked with control of capsaicin content in pepper fruits, and specific primer and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ccttctgctt aggagagaca cgaca 25
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cccacattct gcctttgcca tgaat 25
Claims (1)
1. A method for identifying capsaicin content in pepper fruits, comprising the step of performing PCR identification using specific primers for SNPs closely linked to control capsaicin content in pepper fruits, wherein,
the SNP closely linked with the control of the capsaicin content of the pepper fruits is positioned at 22820065bp locus of No. 6 chromosome of the pepper, when the basic group is T, the capsaicin content is less than 2000mg/kg, when the basic group is C, the capsaicin content is more than 2000mg/kg,
the nucleotide sequence of the specific primer is as follows:
the front primer is F: 5 'CCTTCTGCTTAGGAGAGACACGACA 3', the rear primer is R: 5 'CCCACATTCTGCCTTTGCCATGAAT 3' of the reaction mixture,
the PCR amplification product is 140bp, 2 strips are obtained after the PCR amplification product is subjected to SspI enzyme digestion, and capsaicin is less than 2000 mg/kg; the PCR product can not be cut by SspI enzyme, the enzyme digestion product presents 1 strip, and the content of capsaicin is more than 2000 mg/kg.
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