CN113881802B - SNP molecular marker KQ8-3918 linked with gene for controlling Vc content of pepper fruits, application and special primer - Google Patents
SNP molecular marker KQ8-3918 linked with gene for controlling Vc content of pepper fruits, application and special primer Download PDFInfo
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Abstract
The invention belongs to the technical field of pepper variety breeding, and relates to an SNP molecular marker KQ8-3918 closely linked with a gene for controlling the Vc content of pepper fruits, a special primer and a kit for amplifying the molecular marker, and application thereof. According to the invention, a high-density complete linkage genetic map of hot pepper is constructed for the RILs population containing 252 strains by using the SLAF-seq technology, and the main effect QTL for controlling the Vc content of fruits is positioned to chromosome 1 by combining the phenotypic data of three seasons of the Vc content of the fruits of each RILs strain, so that a closely linked SNP molecular marker KQ8-3918 is obtained, wherein the marker is positioned on chromosome 1 32485915 bp. The specific amplification primer developed by the molecular marker can be used for efficiently and accurately identifying whether the pepper material or variety belongs to the material or variety with high fruit Vc content, and the breeding efficiency can be obviously improved.
Description
Technical Field
The invention relates to the technical field of pepper variety breeding, in particular to an SNP molecular marker KQ8-3918 linked with a gene for controlling the Vc content of pepper fruits, a special primer and a kit for amplifying the molecular marker and application thereof.
Background
The pepper is an important vegetable and economic crop widely cultivated in the world and is also a main economic prop crop in many areas of China. With the continuous improvement of the living standard of people, the demand of consumers on high-quality agricultural products is more urgent, and the quality of vegetables is receiving more and more attention of people. The quality improvement of pepper has become an important research topic of pepper breeding work.
The pepper is recognized as a vegetable with the highest content of fruit vitamin C (Vc), and the content of fruit Vc is an important nutritional quality of the pepper. However, the difference of Vc content of fruits among different pepper varieties is very obvious. The breeding of the variety with high Vc content in fruits is an important direction for pepper quality breeding. The selection of the target genotype plant by the conventional vegetable breeding technology depends on the plant phenotype, and the quantitative characters such as fruit Vc content, capsorubin content, capsaicin content and the like can not be selected by the plant phenotype. By utilizing a molecular Marker Assisted Selection (MAS) breeding technology and combining a haploid breeding technology with a traditional breeding method, the selection efficiency can be greatly improved, the process of material innovation and variety breeding is greatly accelerated, and the MAS breeding method is a necessary way for narrowing the pepper breeding gap between China and developed countries. However, metabolic pathways of the capsicum frutescens involved in fruit Vc biosynthesis, circulation and degradation are not clear, the research on the capsicum frutescens Vc is limited only in the aspects of genetic rules and the like, and the research on the molecular level is slow. No molecular marker which can be used for breeding pepper varieties with high fruit Vc content exists so far, so that the development and control of the molecular marker closely linked with the Vc content gene of pepper fruits and the establishment of a molecular marker assisted breeding technical system are particularly important for accelerating the breeding of the pepper varieties with high fruit vitamin C content.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide an SNP molecular marker KQ8-3918 linked with a gene for controlling the Vc content of pepper fruits. The molecular marker provides a new approach for breeding the pepper variety with high Vc content.
The invention also aims to provide a KASP specific primer for amplifying the SNP molecular marker KQ8-3918 linked with the gene for controlling the Vc content of pepper fruits or determining the genotype corresponding to the SNP molecular marker KQ 8-3918.
The third object of the present invention is to provide a kit comprising the above-mentioned KASP-specific primer.
The fourth purpose of the invention is to provide the SNP molecular marker KQ8-3918, a KASP specific primer and the application of the kit.
The fifth purpose of the invention is to provide a method for identifying the Vc content of pepper fruits.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides an SNP molecular marker KQ8-3918 linked with a gene controlling Vc content in pepper fruits, wherein the molecular marker is located at 32485915bp of No.1 chromosome, and the basic group is A or G; the genotype corresponding to the molecular marker is as follows: g is genotype of high Vc content fruit of hot pepper; a is the genotype of low Vc content in pepper.
In a second aspect, the invention also provides a KASP specific primer for amplifying the SNP molecular marker KQ8-3918 linked with the gene for controlling the Vc content of pepper fruits or for determining the genotype corresponding to the SNP molecular marker KQ8-3918, wherein the specific primer comprises a first forward primer, a second forward primer and a universal reverse primer.
Preferably, the base sequence of the KASP specific primer is shown in a sequence table SEQ ID NO. 1; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO. 2; the base sequence of the universal reverse primer is shown in a sequence table SEQ ID NO. 3.
Preferably, the KASP specific primers of the present invention have different fluorescent linker sequences attached to the 5' ends of the first forward primer and the second forward primer, respectively.
Preferably, the KASP-specific primer of the invention, the fluorescent linker sequence is selected from one of FAM, HEX, FITC, RED, TET, JOE, R110.
In a third aspect, the invention also provides a kit comprising the above KASP specific primers and PCR reaction reagents.
Preferably, in the kit of the present invention, the PCR reaction reagent is Touch-down PCR reaction reagent.
In a fourth aspect, the invention also provides the use of the SNP molecular marker KQ8-3918 linked to the gene controlling the Vc content in pepper fruits, or the KASP specific primer, or the kit in any one of the following aspects:
(a) auxiliary breeding of a pepper variety with high fruit Vc content;
(b) identifying the Vc content of the pepper fruits.
In a fifth aspect, the invention also provides a method for identifying the Vc content of pepper fruits, which comprises the following steps: extracting the genome DNA of a sample to be detected;
taking the genome DNA of a sample to be detected as a template, and carrying out PCR amplification by using the KASP specific primer;
performing fluorescence detection and analysis on the amplification product to obtain the genotype of the sample to be detected, thereby determining the Vc content level of the sample to be detected;
when the genotypes are G: G and A: G, the sample to be detected is a variety with high fruit Vc content; when the genotype is A: A, the sample to be detected is a variety with low fruit Vc content.
Preferably, in the method of the present invention, the conditions for PCR amplification are: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 s; annealing at 61-55 deg.C for 60s for 10 cycles, 61 deg.C being the annealing temperature of the first cycle, and then reducing the annealing temperature by 0.6 deg.C in each cycle; denaturation at 94 ℃ for 20 s; renaturation/elongation at 55 ℃ for 60s for 26 cycles.
The invention has the beneficial effects that: the invention utilizes two pepper inbred lines with extremely obvious difference of Vc content of fruits and an RILs population which is constructed by taking the two materials as parents and comprises 252 strains as test materials, constructs a pepper high-density genetic map by the SLAF-seq technology, and positions a main effect QTL for controlling the Vc content of the fruits to a No.1 chromosome by combining phenotypic data of three seasons of the Vc content of the fruits of the RILs strains, and obtains a closely linked SNP molecular marker KQ8-3918 which is positioned on the No.1 chromosome 32485915 bp. The research result of the invention lays a good foundation for fine positioning and cloning of the gene, and provides a powerful technical support for molecular breeding of pepper fruits with high Vc content. The specific amplification primer developed by the molecular marker can be used for efficiently and accurately identifying whether the pepper material or variety belongs to the material or variety with high fruit Vc content, and the breeding efficiency can be obviously improved.
Drawings
FIG. 1 is a high density genetic map of Capsicum annuum;
FIG. 2 shows the QTL positioning result of Vc content of pepper control fruits;
FIG. 3 is a graph showing the results of genotyping or KASP amplifying 100 parts of pepper material using the molecular marker KQ8-3918 of the present invention, wherein A is a pepper single strain having the same genotype as the parent with low Vc content (A: A genotype, red); the B is a heterozygous single plant (A: G genotype, green); at C is a pepper individual with the same genotype as the high Vc content parent (G: G genotype, blue).
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The following examples are provided as the basis for further modifications and applications by those skilled in the art and are not to be construed as limiting the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise indicated, and may be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. Materials, reagents and the like used in the following examples are conventionally commercially available unless otherwise specified.
The SNP molecular marker KQ8-3918 linked with the gene for controlling the Vc content of pepper fruits provided by the invention is positioned on chromosome 1, the physical position is 32485915, and the basic group in the high Vc parent Z5 is G; the base at this position of the low Vc parent Z6 is A.
Genotype corresponding to molecular marker: g, G and A, G genotype is the genotype with high Vc content in pepper fruit, and A, A genotype is the genotype with low Vc content in pepper fruit.
Two inbred parents used in the invention: the Vc content of the semi-wild germplasm Z5 fruit is 390mg/100g fresh weight, and the Vc content of the sweet pepper inbred line Z6 fruit is 25mg/100g fresh weight. In general, in the industry, the Vc content of pepper fruits is less than 100mg/100g of fresh weight and is a variety with low Vc content, and the Vc content of pepper fruits is not less than 100mg/100g of fresh weight and is a variety with high Vc content.
The invention also provides a KASP specific primer which can be used for amplifying the SNP molecular marker KQ8-3918 linked with the gene for controlling the Vc content of pepper fruits or determining the genotype corresponding to the SNP molecular marker KQ8-3918, and the specific primer comprises a first forward primer, a second forward primer and a universal reverse primer.
Preferably, the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.1, specifically 5'-ATAAACTATAGCTAATAGCAAGCGCG-3';
the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.2, specifically 5'-CATAAACTATAGCTAATAGCAAGCGCA-3';
the base sequence of the universal reverse primer is shown as a sequence table SEQ ID NO.3, and is specifically 5'-GCTTGGTGGGCTTAGTTTAGAAATAAAG-3'.
In order to facilitate the detection of PCR products, the 5' ends of the first forward primer and the second forward primer are respectively connected with different fluorescent adaptor sequences. The fluorescent linker sequence is selected from one of FAM, HEX, FITC, RED, TET, JOE and R110.
In the embodiment of the invention, FAM is selected as a fluorescent linker sequence for the first forward primer, HEX is selected as a fluorescent linker sequence for the second forward primer, the base sequence of the first forward primer with a linker connected to the 5 'end is shown in SEQ ID NO.4 of the sequence table, and the base sequence of the second forward primer with a linker connected to the 5' end is shown in SEQ ID NO.5 of the sequence table.
When the amplified product is subjected to fluorescence detection, if the PCR product of the sample only detects a fluorescent signal corresponding to the first forward primer connected with the fluorescent linker sequence, the corresponding genotype is G.G, and the pepper fruit is judged to have high fruit Vc content; if the PCR product of the sample only detects a fluorescent signal corresponding to the second forward primer connected with the fluorescent linker sequence, the corresponding genotype is A: A, and the pepper fruit is judged to have low fruit Vc content; if the fluorescent signal corresponding to the first forward primer and the fluorescent signal corresponding to the second forward primer are detected simultaneously, the corresponding genotype is A: G (or G: A), and the pepper fruit is judged to have high Vc content.
The kit provided by the invention comprises the KASP specific primer and a PCR reaction reagent, wherein the PCR reaction reagent is a Touch-down PCR reaction reagent, such as 2x KASP Master mix.
The molecular marker KQ8-3918 linked to the gene for controlling the Vc content in the pepper fruits, or the KASP specific primer, or the kit can be used for:
(a) auxiliary breeding of a pepper variety with high fruit Vc content;
(b) identifying the Vc content of the pepper fruits.
The invention also provides a method for identifying the Vc content of the pepper fruits, which comprises the following steps:
extracting genome DNA of a sample to be detected;
taking the genome DNA of a sample to be detected as a template, and carrying out PCR amplification by using the KASP specific primer;
performing fluorescence detection and analysis on the amplification product to obtain the genotype of the sample to be detected so as to determine the Vc content level of the sample to be detected;
when the genotypes are G: G and A: G, the content of Vc in the sample to be detected is high; when the genotype is A: A, the Vc content of the sample to be detected is low.
The PCR amplification conditions are as follows in sequence: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 s; annealing at 61-55 deg.C for 60s for 10 cycles, with 61 deg.C being the annealing temperature of the first cycle, and then reducing the annealing temperature by 0.6 deg.C for each cycle; denaturation at 94 ℃ for 20 s; renaturation/elongation at 55 ℃ for 60s for 26 cycles.
The present invention will be described in further detail with reference to examples.
Example 1 obtaining of molecular marker KQ8-3918 linked to Gene controlling Vc content in Capsicum fruit
1. Construction of the population
The F10 generation recombinant inbred line which is constructed by taking the inbred line Z5 and the inbred line Z6 as parents and comprises 252 lines is used as a test material. The Vc content of the semi-wild germplasm Z5 fruit is 390mg/100g fresh weight, and the Vc content of the sweet pepper inbred line Z6 fruit is 25mg/100g fresh weight.
2. And (3) measuring the content of Vc:
and (3) when the pepper fruits enter the red-ripe stage, mixing and sampling the fruits of each strain in the red-ripe stage, wherein at least 15g of pepper fruits in each strain is detected by using a chromatograph.
3. DNA extraction and SLAF data analysis:
extracting the genome DNA of each strain of the parental and recombinant inbred line population by adopting a radix asparagi plant genome DNA extraction kit.
The original sequencing read length of the SLAF-seq library was PE125 bp. In order to ensure the information analysis quality, original sequencing data is filtered before analysis, and the original data is filtered according to the standard that reads containing a linker sequence needs to be filtered; when the content of N contained in the read exceeds 10% of the length proportion of the read, the pair of reads needs to be removed.
And (3) performing sequence alignment on reads generated by sequencing through BWA software to obtain a bam file, and then performing SNPcalling by using GATK software. According to the parental condition of each SNP label, encoding for parent typing is firstly carried out, and then the genotype of the filial generation is determined according to the consistency of the sequenced sequence of the filial generation and the parents. Filtering the successfully coded SNP markers, and carrying out subsequent map construction and analysis for obtaining high-quality SNP markers, wherein the criteria of marker filtering are as follows:
1) filtering parental sequencing depth under 10X.
2) And (5) performing subtype correction on offspring. If the SNP depth of the offspring is less than 2x, the depth is too low, and the offspring is corrected to be deletion.
3) And (4) filtering the integrity. The markers are screened for genotypes covering at least 90% of all progeny.
4) Partial separation marker (chi-square test P < 0.05) filtration.
4. And (3) genetic map construction:
the genetic map construction is completed by adopting HighMap mapping software independently developed by Baimaike, firstly, the recombination rate and the MLOD value between markers are calculated, and then, the molecular markers are divided into different linkage groups according to the MLOD value, wherein each linkage group is a chromosome. The method comprises the steps of constructing a genetic map by using a maximum likelihood method by taking a chromosome as a unit to obtain a marked initial version sequence, correcting typing according to the map sequence due to certain typing errors of molecular markers, performing map arrangement, correcting, re-arranging the map, and finally obtaining a high-quality genetic map in the correction cycle. Wherein the plotting function is performed using a Kosambi function. QTL positioning selects a met algorithm of ICImapping software to scan and position multi-environment data, ICImapping uses a complete interval mapping method, and a manual threshold value of 2.5 is used for positioning.
And (2) sequencing simplified genomes with the parent average sequencing depth of 109.43X and the offspring average sequencing depth of 16.26X by using a high-generation RILs (Rils populations containing 252 strains and constructed by taking Z5 and Z6 as parents as materials through an SLAF-seq technology to obtain 1,377,197,015 data and 2,098,184 SNP labels, and finally obtaining 8,061 SNP labels for mapping through screening. Calculating MLOD values between every two labels, setting the number of labels in a minimum group and a maximum group, presetting an MLOD value interval, arranging the labels from small to large according to the MLOD values of the labels, dividing the labels with the highest MLOD values among the labels into the same linkage group, filtering out the labels with the MLOD values lower than 5 of other SNP labels, and determining the labels as a mark (Marker). Finally, 6,345 are marked on the picture, and 12 linkage groups are constructed.
And (2) analyzing by using a HighMap software to obtain linear arrangement of the markers in the linkage group by taking the linkage group as a unit, and estimating genetic distance between adjacent markers to finally obtain a high-density complete linkage pepper genetic map with the total map distance of 1,552.20cM and the average map distance of 0.25cM (see figure 1).
5. Fruit Vc content QTL location
Combining genetic map Marker information obtained by sequencing and fruit Vc content phenotype data of 3 seasons of RILs population, applying ICIMapping software, setting a manual threshold of 2.5 for positioning, obtaining 12 QTL intervals in total, distributing the QTL intervals on 6 chromosomes (such as figure 2 and table 1) of 1, 2, 5, 8, 9 and 11, wherein the main QTL interval with the highest contribution rate is positioned between Marker4486 and Marker3918 on chromosome 1, the contribution rate is 15.1 percent, and the interval size is 2.30 Mb.
TABLE 1 QTL location results for Vc content of pepper control fruits
6. Development of molecular marker linked with Vc content gene of control fruit in hot pepper
Sequencing sequences of 70bp before and after marking Marker3918 at the right end of the main effect positioning interval shows that the allelic gene bases at the position of the high Vc parent Z5 are G, and the allelic gene bases at the position of the low Vc parent Z6 are A. This SNP was developed as KASP-specific primer KQ8-3918 based on the sequencing results, see Table 2 for primer sequences.
TABLE 2 primer sequences designed for the molecular marker KQ8-3918
In Table 2, the underlined parts are fluorescent linker sequences attached to the 5 'ends of the respective forward primers, the linker of the first forward primer 5' is FAM, and the linker of the second forward primer is HEX.
Example 2 verification of the reliability of molecular markers linked to genes controlling Vc content in fruits in Capsicum annuum L
Further population validation was performed on the molecular markers developed in example 1. 100 parts of pepper inbred lines bred by the subject group of the inventor are taken as verification materials, PCR amplification is carried out by using the specific primer of the embodiment 1, and the accuracy of the molecular marker KQ8-3918 which is closely linked with the gene for controlling the Vc content of the fruit and is applied to molecular marker assisted breeding is verified.
The PCR reaction was performed on a Hydrocycler water bath PCR instrument, and the fluorescence detection was performed on the BMG LABTECH GMbH platform.
The PCR amplification system is as follows: 2.5ul of genomic DNA (20 ng/. mu.L), 2.5. mu.L of 2xKASP Master mix (from GenBank Co.); 0.07. mu.L of KBD Assay mix (i.e., a mixture of primers, each primer concentration being 10. mu. mol/. mu.L, first forward primer, second forward primer and universal reverse primer mixed in a molar ratio of 2:2: 5);
the amplification conditions were in order: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 s; annealing at 61-55 deg.C for 60s for 10 cycles, with 61 deg.C being the annealing temperature of the first cycle, and then reducing the annealing temperature by 0.6 deg.C for each cycle; denaturation at 94 ℃ for 20 s; renaturation/elongation at 55 ℃ for 60s for 26 cycles.
When the amplified product is subjected to fluorescence detection, if the PCR product of the sample only detects a fluorescence signal (blue fluorescence) corresponding to the first forward primer connected with the fluorescent linker sequence, the corresponding genotype is G: G, and the pepper fruit is judged to have high Vc content; if the PCR product of the sample only detects a fluorescent signal (red fluorescence) corresponding to the second forward primer connected with the fluorescent linker sequence, the corresponding genotype is A: A, and the pepper fruit is judged to have low Vc content; and if the fluorescent signal corresponding to the first forward primer and the fluorescent signal corresponding to the second forward primer (namely green) are detected at the same time, the corresponding genotype is A: G, and the pepper fruit is judged to have high Vc content.
The Vc content of the fruits of 100 parts of the pepper material selected in the example was determined according to the Vc content measurement method of the 2 nd part of the example 1, and the specific content is shown in Table 3.
According to the research result of the existing genetic rule of the Vc content of the pepper fruits, the Vc content of the pepper ripe fruits is controlled by a pair of additive-dominant major gene + additive-dominant-epistatic polygene (D-0) (Zhangfen and the like, the change rule and genetic analysis of the Vc content of the pepper fruits, nuclear agriculture bulletin, 2015, 12), and 100 parts of pepper materials are divided into two groups aiming at a molecular marker KQ8-3918, namely, a group with the same genotype as that of a low Vc parent KASP typing (A: A) and a group with the same genotype as that of a high Vc parent KASP typing (G: G or A: G). T-test tests the KASP type and Vc content of fruits of the two groups of lines gave P values of 1.74E-16, indicating that there was a very significant difference between the two groups (Table 3).
According to the definition of high and low Vc content of pepper fruits in the general industry, the accuracy of the result obtained by molecular detection by using a molecular marker KQ8-3918 is judged according to the fact that the Vc content of the pepper fruits is lower than 100mg/100G, namely, the fresh weight of the pepper fruits is a low-Vc-content variety, and not lower than 100mg/100G, namely, the Vc content of the pepper fruits is high, the genotyping result of each pepper material is compared with the actually measured Vc content of the pepper fruits, if the genotyping of the material is G: G or A: G, the actually measured Vc content of the pepper fruits of the material is not lower than 100mg/100G, the identification result is determined to be accurate, otherwise, the identification result is determined to be inaccurate; if the gene typing of the strain is A: A, and the actually measured Vc content of the pepper fruit of the strain is lower than 100mg/100g fresh weight, the identification result is determined to be accurate, otherwise, the identification result is determined to be inaccurate. The data in the table 3 show that the accuracy of molecular detection of the molecular marker KQ8-3918 is 91%, and the method can be applied to auxiliary selective breeding of pepper molecular markers.
TABLE 3 accuracy verification of the markers KQ8-3918 with 100 parts of material
Sequence listing
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Claims (8)
1. A KASP specific primer is used for identifying the Vc content of pepper fruits, and comprises a first forward primer, a second forward primer and a universal reverse primer,
the base sequence of the first forward primer is shown in a sequence table SEQ ID NO. 1;
the base sequence of the second forward primer is shown as a sequence table SEQ ID NO. 2;
the base sequence of the universal reverse primer is shown in a sequence table SEQ ID NO. 3.
2. The KASP-specific primer of claim 1, wherein the first forward primer and the second forward primer have different fluorescent linker sequences attached to their 5' ends.
3. The KASP-specific primer of claim 2, wherein the fluorescent linker sequence is selected from one of FAM, HEX, FITC, RED, TET, JOE, R110.
4. A kit comprising KASP specific primers of any one of claims 1 to 3 and PCR reaction reagents.
5. The kit according to claim 4, wherein the PCR reaction reagent is Touch-down PCR reaction reagent.
6. Use of a KASP specific primer according to any one of claims 1 to 3, or a kit according to any one of claims 4 to 5, for any one of:
(a) auxiliary breeding of a pepper variety with high fruit Vc content;
(b) identifying the Vc content of the pepper fruits.
7. A method for identifying the Vc content of pepper fruits comprises the following steps:
extracting the genome DNA of a sample to be detected;
using the genomic DNA of the sample to be tested as a template, and carrying out PCR amplification by using the KASP specific primer of any one of claims 1-3;
performing fluorescence detection and analysis on the amplification product to obtain the genotype of the sample to be detected, and determining the level of Vc content in the fruit of the sample to be detected;
when the genotypes are G: G and A: G, the sample to be detected is a variety with high fruit Vc content; when the genotype is A: A, the sample to be detected is a variety with low fruit Vc content.
8. The method of claim 7, wherein the PCR amplification conditions are, in order: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 s; annealing at 61-55 deg.C for 60s for 10 cycles, with 61 deg.C being the annealing temperature of the first cycle, and then reducing the annealing temperature by 0.6 deg.C for each cycle; denaturation at 94 ℃ for 20 s; renaturation/elongation at 55 ℃ for 60s for 26 cycles.
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| CN113151568A (en) * | 2021-05-20 | 2021-07-23 | 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 |
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| CN113151568A (en) * | 2021-05-20 | 2021-07-23 | 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 |
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