CN112725520B - Specific CAPS primer of SNP molecular marker related to control of fruit capsaicin content and application of specific CAPS primer - Google Patents

Specific CAPS primer of SNP molecular marker related to control of fruit capsaicin content and application of specific CAPS primer Download PDF

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CN112725520B
CN112725520B CN202110227094.6A CN202110227094A CN112725520B CN 112725520 B CN112725520 B CN 112725520B CN 202110227094 A CN202110227094 A CN 202110227094A CN 112725520 B CN112725520 B CN 112725520B
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primer
capsaicin
content
capsaicin content
molecular marker
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CN112725520A (en
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曹亚从
程锋
王立浩
张亢
于海龙
张宝玺
张正海
景雅欣
靳远
张伟丽
陈姝敏
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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Abstract

The invention relates to the field of molecular markers, in particular to a specific CAPS primer of an SNP molecular marker related to controlling the content of fruit capsaicin 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.

Description

Specific CAPS primer of SNP molecular marker related to control of fruit capsaicin content and application of specific CAPS primer
Technical Field
The invention relates to the field of molecular markers, in particular to a specific CAPS primer of an SNP molecular marker related to controlling the content of fruit capsaicin and application thereof.
Background
The formation of the peppery taste of pepper fruits is characterized in that due to the synthesis of capsaicin substances, the capsaicin substance content shows Quantitative trait inheritance in pepper, the capsaicin substance content is reported to be positioned to more Quantitative Trait Loci (QTL), and Yarnes and the like position the capsaicin substance content to 12 QTL loci on 6 chromosomes; ben Chaim et al mapped it to chromosome 4; blum et al mapped a major QTL to chromosome 7 through BSA (bulked segregant analysis), Zhu et al also mapped a major QTL controlling the content of capsaicinoids on chromosome 7, and determined candidate gene Pu 3, which is transcription factor MYB 31; lee et al 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 the existing reports, the inheritance of the content of capsaicin substances is complex, the capsaicin substances have 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 and molecular markers related to the genetic traits needs to be combined with different genetic backgrounds.
Disclosure of Invention
The invention aims to provide a specific CAPS primer of a SNP molecular marker related to controlling the content of fruit capsaicin.
Still another objective of the invention is to provide application of the CAPS primer for controlling the SNP molecular marker related to the capsaicin content of the fruit.
It is still another object of the present invention to provide a method for identifying capsaicin content in pepper fruits.
The specific CAPS primer of the SNP molecular marker related to controlling the fruit capsaicin content has the following nucleotide sequence:
the front primer is F: 5 'ACCTGTCAGCTCCTTTACACA 3' of the composition,
the rear primer is R: 5 'GGCAAGATCCAGACATGTCAC 3'.
The method for identifying capsaicin content in pepper fruits according to the invention comprises the step of amplifying by using the following specific primers,
the front primer is F: 5 'ACCTGTCAGCTCCTTTACACA 3' of the composition,
the rear primer is R: 5 'GGCAAGATCCAGACATGTCAC 3'.
According to the method for identifying the capsaicin content of the pepper fruits, when the capsaicin content is 2000mg/kg as a threshold value, 92.82% of bases at the site in materials smaller than 2000mg/kg are G, a PCR product is subjected to enzyme digestion by using ScrFI and can be subjected to enzyme digestion, the enzyme digestion products are 496bp and 78bp respectively, and the SNP site is a variation type base;
in the material with the SNP site of more than 2000mg/kg, the base of the site is A, the PCR product is subjected to enzyme digestion by ScrFI, the enzyme digestion cannot be carried out, and the band is still 574bp when the PCR product is of a mutation type.
The invention provides application of the specific CAPS primer of the SNP molecular marker related to controlling the capsaicin content of the fruits in pepper breeding.
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 content of capsaicin of 192 parts of pepper materials is counted and calculated according to the weight ratio of the capsaicin in Chr 06: 22821795bp base type, the locus genotype has better corresponding relation with the hot taste. When the capsaicin content is 2000mg/kg as a threshold value, the basic group of the material with the content less than 2000mg/kg at the position is G; more than 2000mg/kg of material has a base A at this site; overall statistics show that Chr 06: 22821795bp base can accurately identify the content of capsaicin in the material. The invention utilizes large sample size to carry out correlation analysis, so that the SNP related molecular marker has wider application range. 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.
Drawings
FIG. 1 is a Manhattan chart of genome-wide association analysis of capsaicin content.
FIG. 2 shows agarose electrophoresis PCR amplification product using the CAPS primers of the present invention, M, marker D2000, with a PCR amplification product length of 574 bp;
FIG. 3 shows agarose electrophoresis PCR amplification and cleavage of the product, M, marker D2000.
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 referring to agricultural industry standards (NY/T1381-2007) by adopting a capsaicin extraction and determination method, and determining that the Capana06g001204 has a gene with higher expression in a placenta in a capsaicin synthesis period as shown in figure 1 and has a significant related signal on a No. 6 chromosome, so that the Capana06g001204 is determined as a candidate gene for controlling the capsaicin content.
FIG. 1 is a genome-wide association analysis Manhattan map of capsaicin content, in which Chr 06: 22821795bp has nonsynonymous mutation SNP obviously related to capsaicin content, the base of the reference genome is G, and the variant base is A. The content of capsaicin of 192 parts of pepper materials is counted and calculated according to the weight ratio of the capsaicin in Chr 06: 22821795bp base type, the locus genotype has better corresponding relation with the hot taste. When the capsaicin content is 2000mg/kg as a threshold value, the basic group of the material with the content less than 2000mg/kg at the position is G; more than 2000mg/kg of material has a base A at this site; overall statistics show that Chr 06: 22821795bp base can accurately identify the content of capsaicin in the material.
Designing CAPS primers for the SNP locus, wherein the front primers are F: ACCTGTCAGCTCCTTTACACA, the rear primer is R: GGCAAGATCCAGACATGTCAC, PCR the amplified product is 574bp, agarose gel electrophoresis is shown in FIG. 2. Carrying out enzyme digestion on the PCR product by using ScrFI, wherein when the SNP site is of a reference genome type, namely G, the SNP site can be cut by enzyme, and the enzyme digestion products are 496bp and 78bp respectively; when the SNP site is a mutation type base, namely A, it cannot be enzymatically cleaved. The enzyme digestion product is subjected to agarose gel electrophoresis, and when the enzyme digestion product is a reference genome, a bright band is obvious at 496bp, and when the enzyme digestion product is a variant type, a band is still 574bp and is obviously longer than the enzyme digestion product fragment of the reference genome (figure 3). The band after enzyme digestion is 496bp, and the mutant genotype band is 574 bp.
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> specific CAPS primer of SNP molecular marker related to controlling fruit capsaicin content and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acctgtcagc tcctttacac a 21
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggcaagatcc agacatgtca c 21

Claims (4)

1. The specific CAPS primer of the SNP molecular marker related to controlling the fruit capsaicin content is characterized by comprising the following nucleotide sequences:
the front primer is F: 5 'ACCTGTCAGCTCCTTTACACA 3' of the composition,
the rear primer is R: 5 'GGCAAGATCCAGACATGTCAC 3'.
2. A method for identifying capsaicin content in pepper fruits, which comprises the steps of amplifying by using the following specific primers,
the front primer is F: 5 'ACCTGTCAGCTCCTTTACACA 3' of the composition,
the rear primer is R: 5 'GGCAAGATCCAGACATGTCAC 3'.
3. The method for identifying the capsaicin content of the pepper fruits as claimed in claim 2, wherein the PCR product is subjected to enzyme digestion by ScrFI, and when the PCR product is cut open and the enzyme digestion products are 496bp and 78bp respectively, the capsaicin content of the sample is less than 2000 mg/kg; when the PCR product is not cut and the electrophoresis band is still 574bp, the capsaicin content of the sample is more than 2000mg/kg of material.
4. The use of the CAPS primer specific for the SNP molecular marker related to the control of fruit capsaicin content according to claim 1 in pepper breeding.
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CN113151568B (en) * 2021-05-20 2022-05-17 中国农业科学院蔬菜花卉研究所 SNP (Single nucleotide polymorphism) site with closely linked capsaicin content, CAPS (cleaved amplified polymorphic sequence) molecular marker of SNP site and application of CAPS molecular marker
CN113215299B (en) * 2021-06-01 2022-09-27 中国农业科学院蔬菜花卉研究所 SNP closely linked with control of capsaicine content of capsicum fruits, specific primer and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107151709A (en) * 2017-07-13 2017-09-12 中国农业科学院蔬菜花卉研究所 The SNP marker related to capsicum pungent and its application

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107151709A (en) * 2017-07-13 2017-09-12 中国农业科学院蔬菜花卉研究所 The SNP marker related to capsicum pungent and its application

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
Genetic variability of Pun1 gene (Capsaicin synthase) in pungent cultivars of Capsicum annuum of Northern Mexico;Alberto González Zamora等;《Tropical and Subtropical Agroecosystems》;20200331;第23卷;全文 *
Identification of Gene-Specific Polymorphisms and Association with Capsaicin Pathway Metabolites in Capsicum annuum L. Collections;Umesh K. Reddy等;《PLOS ONE》;20140131;第9卷(第期);全文 *
IdentiWcation, validation and survey of a single nucleotide polymorphism (SNP) associated with pungency in Capsicum spp.;Ana Garcés-Claver等;《Theor Appl Genet》;20070919;第115卷;全文 *
Pepper variome reveals the history and key loci associated with fruit domestication and diversification;Yacong Cao等;《Research Square》;20211028;全文 *
QTL mapping for capsaicin and dihydrocapsaicin content in a population of Capsicum annuum ‘NB1’ 3 Capsicum chinense ‘Bhut Jolokia’;Jandae Lee等;《Plant Breeding》;20161231;第135卷;全文 *
一个与辣椒素含量高低紧密连锁的KASP标记及应用;欧立军等;《分子植物育种》;20191231(第24期);全文 *
不同栽培条件下辣椒果实辣椒素含量的分析与QTL定位;王宁等;《中国蔬菜》;20161231(第03期);全文 *
基于SNPs全基因组测序技术对泰国辣椒地方品种遗传多样性分析和辣椒素含量关联分析;Wassana Kethom等;《辣椒杂志》;20190620(第02期);全文 *
辣椒素类物质生物合成研究进展;张西露等;《辣椒杂志》;20190620(第02期);全文 *

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