CN107151709A - The SNP marker related to capsicum pungent and its application - Google Patents

Abstract

The invention discloses a SNP marker related to hot pepper taste and application thereof. The application provided by the present invention is specifically the single nucleotide polymorphism of the following SNP site in the pepper genome or the material used to detect the single nucleotide polymorphism of the following SNP site in the pepper genome in identifying or assisting in the identification of pepper Application in spicy traits; the SNP site corresponds to the 24th position of the nucleotide sequence shown in Sequence 4 in the sequence listing in the capsicum genome; the nucleotide at the SNP site is A or C. The present invention does not need to identify the maturity stage of pepper fruit, but only detects the DNA of pepper plants at the seedling stage to judge whether the hot pepper is hot or not, and can be widely applied to molecular marker assisted breeding. Moreover, the marker can be detected in large quantities by KASPar genotyping technology, which can improve efficiency and shorten identification time.

Description

SNP markers related to hot pepper taste and its application

technical field

The invention belongs to the field of molecular biology, and relates to a SNP marker related to hot pepper taste and application thereof.

Background technique

The "spicy taste" of pepper (Capsicum spp.) is the burning sensation caused by the unique capsaicin in the pepper fruit. The presence or absence of hot pepper taste is a quality trait controlled by the dominant Pun (indicated by C at the earliest) gene locus, and the Pun gene The mutation in the chili pepper causes a loss of its spiciness, resulting in what is known as a bell pepper. In capsicum genetics and breeding, capsicum or sweet pepper breeding materials are often selected according to different breeding objectives, but it is often time-consuming to wait until the capsicum fruit is mature.

Molecular marker-assisted breeding screens germplasm resources for genotypes, avoids blindness in phenotypic selection, greatly shortens the breeding process, and improves breeding efficiency. Obtaining molecular markers linked to hot pepper traits, especially functional molecular markers developed for key mutation sites of target genes, is an important condition for the use of molecular markers to assist hot pepper transformation and achieve germplasm innovation.

Although molecular marker-assisted selection based on PCR and electrophoresis technology can greatly improve the efficiency, there are still problems such as high cost and low throughput. The high sensitivity and high throughput of SNP molecular markers of KASPar technology, as well as the advantages of low cost and easy operation have unique advantages in molecular marker-assisted selection.

Contents of the invention

The purpose of the present invention is to provide a SNP marker related to hot pepper and its application.

The application provided by the present invention is specifically the single nucleotide polymorphism of the following SNP site in the pepper genome or the material used to detect the single nucleotide polymorphism of the following SNP site in the pepper genome in identifying or assisting in the identification of pepper Application in spicy traits;

The SNP site corresponds to the 24th position of the nucleotide sequence shown in Sequence 4 in the sequence listing in the pepper genome; the nucleotide at the SNP site is A or C.

The invention also provides a reagent for identifying or assisting in identifying the hot pepper character.

The reagent provided by the present invention for identifying or assisting in the identification of hot pepper traits is specifically a substance for detecting the single nucleotide polymorphism of the following SNP site in the pepper genome; the SNP site is in the pepper genome It corresponds to the 24th position of the nucleotide sequence shown in sequence 4 in the sequence listing; the nucleotide at the SNP site is A or C.

Specifically, the "material for detecting the single nucleotide polymorphism of the following SNP site in the pepper genome" can be as follows a) or b):

a) A complete set of single-stranded DNA A, which is as follows (a1) or (a2):

(a1) by the single-stranded DNA shown in the 22nd-45th nucleotide of sequence 1 in the sequence listing, the single-stranded DNA shown in the 22nd-45th nucleotide of sequence 2 in the sequence listing, and in the sequence listing A set of single-stranded DNA composed of the single-stranded DNA shown in sequence 3.

(a2) the 22-45 nucleotides of sequence 1 in the sequence listing, the 22-45 nucleotides of sequence 2 in the sequence listing and sequence 3 are replaced by one or several nucleotides and/or A set of single-stranded DNA composed of three single-stranded DNA molecules shown in the sequence obtained after deletion and/or addition, and having the same function as the set of single-stranded DNA described in (a1).

b) Complete set of single-stranded DNA B, which is as follows (b1) or (b2):

(b1) A set of single-stranded DNAs consisting of the single-stranded DNA shown in sequence 1 in the sequence listing, the single-stranded DNA shown in sequence 2 in the sequence listing, and the single-stranded DNA shown in sequence 3 in the sequence listing.

(b2) consists of three single-stranded DNA molecules shown in the sequence obtained by substituting and/or deleting and/or adding one or several nucleotides to Sequence 1, Sequence 2 and Sequence 3 in the sequence listing, and is compatible with The set of single-stranded DNAs described in (b1) has the same function as the set of single-stranded DNAs.

A kit containing the reagent for identifying or assisting in identifying the hot pepper trait also belongs to the protection scope of the present invention.

Specifically, the kit also contains fluorescent probe A, fluorescent probe B, quenching probe A and quenching probe B;

The nucleotide sequence of the fluorescent probe A is the 1-21st position of sequence 1 in the sequence listing, and the 5' end is connected with the fluorescent group A; the nucleotide sequence of the quenching probe A is the sequence in the sequence listing The reverse complementary sequence of positions 1-21 of 1, and a quenching group is connected to the 3' end;

The nucleotide sequence of the fluorescent probe B is the 1-21st position of sequence 2 in the sequence listing, and the 5' end is connected with the fluorescent group B; the nucleotide sequence of the quenching probe B is the sequence in the sequence listing The reverse complementary sequence of positions 1-21 of 2, and a quenching group is connected to the 3' end.

In the present invention, the fluorescent group A is specifically FAM; the fluorescent group B is specifically HEX; and the quenching group is specifically Q.

In the present invention, the fluorescent probe A, the fluorescent probe B, the quenching probe A and the quenching probe B are present in KASP V4.0 2×Master mix 96/384, Low In Rox, wherein said KASP V4.0 2×Mastermix 96/384, Low Rox is a product of UK LGC company, and its catalog number is KBS-1016-017.

The application of the reagent or the kit in identifying or assisting the identification of the hot pepper trait also belongs to the protection scope of the present invention.

The invention also provides a method for identifying or assisting in identifying whether the chili to be tested is hot pepper or non-spicy pepper.

The method provided by the present invention for identifying or assisting in identifying whether the pepper to be tested is hot pepper or non-spicy pepper may specifically include the following steps: detecting whether the nucleotide at the following SNP site in the genome of the pepper to be tested is A or C or A and C, to determine whether the genotype of the capsicum to be tested is A:A or C:C or A:C, according to the genotype of the capsicum to be tested, it is determined as follows that the capsicum to be tested has a spicy taste Capsicum or non-pungent capsicum: if described capsicum to be tested is A:A genotype or A:C genotype, then described capsicum to be tested is or candidate is to have spicy capsicum; If described capsicum to be tested is C: C genotype, then the pepper to be tested is or a candidate for non-spicy pepper.

The SNP site corresponds to the 24th position of the nucleotide sequence shown in Sequence 4 in the sequence listing in the pepper genome; the nucleotide at the SNP site is A or C.

The A:A genotype is a homozygous type in which the 24th position of the nucleotide sequence corresponding to sequence 4 in the sequence listing is A in the pepper genome.

The C:C genotype is a homozygous type in which the 24th position of the nucleotide sequence corresponding to sequence 4 in the sequence listing is C in the pepper genome.

The A:C genotype is a heterozygous type in which the 24th position of the nucleotide sequence corresponding to sequence 4 in the sequence listing is A and C in the pepper genome.

In the method, the method of "detecting whether the nucleotide at the following SNP site in the genome of the pepper to be tested is A or C or A and C" can specifically be the KASPar detection method.

The KASPar detection method may specifically include the following two steps: KASPar amplification and fluorescent scanning of the KASPar amplified product to determine whether the nucleotide at the SNP site is A or C or A and C; The template for the KASPar amplification is the genomic DNA of the pepper to be tested, and the KASPar primers used meet the following conditions: the sequence of the amplified product obtained by performing KASPar amplification with the genomic DNA of the pepper to be tested as a template contains the sequence in the sequence table 4.

Further, in the KASPar detection method, the KASPar primer is the set of single-stranded DNA B (a set of single-stranded DNA composed of the single-stranded DNAs shown in Sequence 1, Sequence 2 and Sequence 3 in the Sequence Listing).

More specifically, the KASPar detection method includes the following steps: using the genomic DNA of the pepper to be tested as a template, using the kit (KASPar amplification primers used are the complete set of single-stranded DNA B, and the amplification reagent contains Fluorescent probe A, fluorescent probe B, quenching probe A and quenching probe B; the nucleotide sequence of the fluorescent probe A is the 1-21st position of sequence 1 in the sequence listing, and the 5' end is connected Fluorescent group FAM; the nucleotide sequence of the quenching probe A is the reverse complementary sequence of No. 1-21 of sequence 1 in the sequence listing, and the 3' end is connected with a quenching group Q; the fluorescent probe The nucleotide sequence of B is the 1-21st position of sequence 1 in the sequence listing, and the fluorescent group HEX is connected to the 5' end; the nucleotide sequence of the quenching probe B is the 1st-21st of sequence 1 in the sequence listing. The reverse complementary sequence at position 21, the 3' end is connected to the quenching group (Q) for KASPar amplification, and then RocheLC480 is used for data reading, and the nucleoside at the SNP site in the pepper genome to be tested is determined as follows Whether the acid is A or C or A and C: if the fluorescent signal data of the amplified product of the pepper to be tested is analyzed by Roche LC480 and presents blue (Allele X) in the obtained scoring type cluster diagram, then the pepper to be tested is The nucleotide at the SNP site in the genome is A; if the fluorescent signal data of the amplified product of the capsicum to be tested is analyzed by Roche LC480 and presents green (Allele Y) in the resulting scoring cluster diagram, then the The nucleotide at the SNP site in the pepper genome to be tested is C; if the fluorescent signal data of the amplified product of the pepper to be tested is analyzed by Roche LC480, it will appear red (BothAlleles) , then the nucleotides at the SNP site in the pepper genome to be tested are A and C.

The application of the reagent or the kit or the method in capsicum breeding also belongs to the protection scope of the present invention.

The following application of the reagent or the test kit or the method in (a) or (b) also belongs to the protection scope of the present invention:

(a) Screening with hot pepper;

(b) Purity-aided identification of hybrids with hot and non-hot pepper parents.

The method of following (A) or (B) also belongs to protection scope of the present invention:

(A) A method for cultivating hot pepper varieties, including the step of selecting A:A genotype peppers as parents for breeding; the A:A genotype is corresponding to the nucleosides shown in sequence 4 in the sequence table in the pepper genome The 24th position of the acid sequence is homozygous for A.

(B) A method for cultivating non-spicy pepper varieties, including the step of selecting a C:C genotype pepper as a parent for breeding; the C:C genotype is corresponding to the nucleoside shown in sequence 4 in the sequence table in the pepper genome The 24th position of the acid sequence is homozygous for C.

In an embodiment of the present invention, the pepper is specifically selected from the following materials or hybrid offspring (such as F1 generation): CM334, perennial, small Marseille, H3, 77013A, 0601M, 83-60, 77013A×CM334, 77013A×perennial, 77013A×Little Marseille, 77013A×H3, 77013A×0601M, 83-60×H3, 0819, KLS, 83-163, 0516, Carolinawonder, DH330, 0818, 7714, Yiduhong, Jizejiao, Pig Intestine, Eggplant , Wangdujiao, 04Q-29, 04Q-15, 04Q-29×04Q-15.

The advantage of the invention is that it is not necessary to identify the maturity stage of pepper fruit, but only the DNA detection of pepper plants at the seedling stage can determine whether the hot pepper is hot or not, and can be widely used in molecular marker assisted breeding. Moreover, the marker can be detected in large quantities by KASPar genotyping technology, which can improve efficiency and shorten identification time.

The molecular marker provided by the invention realizes the screening of hot peppers and the identification of the purity of hybrids at the seedling stage, greatly shortens the breeding time, and saves manpower and material resources. By utilizing the specific primers provided by the present invention and adopting the KASPar genotyping technology, with the published whole genome sequence with hot pepper CM334 and Zunla_1 as a control, with the same genotype and heterozygous genotype of hot pepper CM334 and Zunla_1 It has a spicy taste, and the genotypes different from the hot pepper CM334 and Zunla_1 do not have a spicy taste. The molecular marker can be used to screen hot pepper materials and identify the purity of hybrids, thereby improving breeding efficiency. Compared with other molecular markers found before, the SNP marker 2_25 of the present invention is located in the capsicum CA02g19240 gene interval and belongs to the candidate gene molecular marker.

Description of drawings

Fig. 1 is the amplification result of KASPar marker 2_25 in the hybrid combination. The blue dots are the same genotype as the spicy parent (A:A genotype), the green dots are the non-spicy materials 0601M and 77013A (C:C genotype), and the red dots are the heterozygous individual plants ( A: C genotype).

Fig. 2 is the amplification result of KASPar marker 2_25 in 18 pepper materials. The blue dots are homozygous plants with spicy taste (A:A genotype), and the green dots are non-spicy single plants (C:C genotype).

Fig. 3 is the amplification result of KASPar marker 2_25 in the hybrid progeny of hot pepper and non-hot pepper. The blue dots are non-spicy parents and pseudo-hybrid F1 plants (A:A genotype), and the red dots are F1 hybrid plants (A:C genotype).

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The peppers involved in the following examples are as follows: CM334, perennial, small Marseille, H3, 77013A, 0601M, 83-60, 0819, KLS, 83-163, 0516, Carolina wonder, DH330, 0818, 7714, benefit Duhong, Jizejiao, Pig Intestine, Tomato, Wangdujiao, 04Q-29 and 04Q-15. The literature describing each material is as follows.

Example 1. Acquisition of SNP sites related to pepper pungency traits and determination of corresponding KASPar primers

1. Test materials

In this embodiment, the hot peppers CM334, perennial, Marseillaise, H3 and non-hot peppers 77013A, 0601M, _83-60 , and F1 thereof are used as test materials.

2. Phenotype identification

The identification of each pepper material and its F1 phenotype was identified by the method of mouth-tasting the mature fruit of pepper. Take 3 plants for each material, pick 3 fruits from each plant, and hand them over to different personnel for trial. A single plant with more than 2 spicy fruits is a spicy single plant, and the material of 3 spicy single plants is a spicy material.

3. 77013A genome resequencing

The 77013A genome was resequenced using PE150 and Hiseq4000, with an average sequencing depth of 42X. Taking CM334 (pepper.v.1.55, http://peppergenome.snu.ac.kr/.) as the reference genome, Samtools software was used to predict the SNP loci. KASPar primers were designed online using Primer3 (http://bioinfo.ut.ee/primer3).

4. Screening of polymorphic SNP markers

1. KASPar mark

Each pair of KASPar molecular markers used in the present invention contains three primer sequences: A1, A2, and C.

Among them, A1 and A2 are only differences in the SNP sites at the ends, and different fluorescent label sequences are added to the 5' ends of A1 and A2:

5'-GAAGGTGACCAAGTTCATGCT-3'(FAM fluorescent tag sequence);

5'-GAAGGTCGGAGTCAACGGATT-3' (HEX fluorescent tag sequence).

These two sequences are complementary to the fluorescent sequence in KASP V4.0 2X Master mix 96/384, Low Rox, and will excite fluorescence after pairing.

C is the reverse complementary sequence.

(1) KASPar-labeled PCR amplification system (4.055 μL):

DNA template (5ng·μL ^-1 ) 2μL KASP V4.0 2X Master mix 96/384,Low Rox 2μL primer mix 0.055μL

Among them, KASP V4.0 2×Master mix 96/384, Low Rox are products of UK LGC Company, and their catalog number is KBS-1016-017. KASP V4.0 2×Master mix 96/384, Low Rox consists of fluorescent probe A, fluorescent probe B, quenching probe A and quenching probe B, as well as high-fidelity Taq enzyme, dNTP, etc. The sequence of fluorescent probe A is 5'-GAAGGTGACCAAGTTCATGCT-3', a fluorescent group FAM is connected to the 5' end; the sequence of fluorescent probe B is 5'-GAAGGTCGGAGTCAACGGATT-3', and a fluorescent group is connected to the 5' end HEX; the sequence of quenching probe A is 5'-AGCATGAACTTGGTCACCTTC-3', the 3' end is connected to the quenching group Q; the sequence of quenching probe B is 5'-AATCCGTTGACTCCGACCTTC-3', the 3' end is connected to the quenching group Group Q

(2) Primer mixture system (50 μL): the primer concentration is 100 μmol L ^-1

C 15μL A1 6μL A2 6μL Sterilized H ₂ O 23μL

(3) 384-well PCR plate amplification, the procedure is as follows:

(4) Detection of PCR products

After the KASPar PCR amplification reaction, you need to use Roche 480 fluorescence quantitative instrument to perform KASParGenotying to read the fluorescence data. If the genotyping effect is not satisfactory, you need to add the program: 94°C for 20s, 57°C for 60s, 3 cycles; 37°C 1min, and then read the data.

Instructions for analysis on the Roche 480 Fluorometer are provided by LGC Corporation, available at www.lgcgenomics.com. The X-axis is the FAM (465-510) channel, and the Y-axis is the HEX (533-580) channel. According to the analysis results, the specific genotype of the gene to be tested is determined as follows: the genotype of the blue sample aggregated near the X axis is the allele type connected to the FAM fluorescent tag sequence, and the green sample aggregated near the Y axis The genotype of the sample is the allelic type connected with the HEX fluorescent label sequence, the genotype of the sample shown in red in the middle is the heterozygous type of two alleles, and the sample shown in black in the lower left corner is the blank with H ₂ O replacing the template control.

2. Acquisition of SNP markers of pepper Pun candidate gene and determination of corresponding KASPar primers

KASPar primers located in the pepper Pun candidate gene interval were designed, and five different hybridization combinations were used for polymorphism screening. As a result, a KASPar primer combination 2_25 with the same phenotype as the Pun genotype was found in the pepper CA02g19240 gene interval (see Table 1 for primer sequence information , phenotype and genotype identification results are shown in Table 2 and Figure 1), which can be used for the SNP molecular marker-assisted selection of pepper spicy materials.

The corresponding SNP site is specifically the 24th position in the capsicum genome corresponding to the nucleotide sequence shown in Sequence 4 in the Sequence Listing; the nucleotide at the SNP site is A or C.

When the phenotype of the pepper is "spicy", and the corresponding Pun genotype is Pun/Pun, the SNP sites are all A:A genotypes (that is, in the pepper genome corresponding to sequence 4 in the sequence table The 24th position of the nucleotide sequence is homozygous for A);

When the phenotype of the pepper is "spicy", and the corresponding Pun genotype is Pun/pun, the SNP sites are all A:C genotypes (that is, in the pepper genome corresponding to sequence 4 in the sequence table The 24th position of the nucleotide sequence is heterozygous for A and C);

When the phenotype of the pepper is "no spicy taste", and the corresponding Pun genotype is pun/pun, the SNP sites are all C:C genotypes (that is, in the pepper genome corresponding to sequence 4 in the sequence table The homozygous type whose 24th position of the nucleotide sequence is C is shown).

Table 1 KASPar primer 2_25 nucleotide sequence information

Table ₂ Molecular marker 2_25 in parent and F1 generation typing of pepper

Embodiment 2, utilize mark 2_25 of the present invention to screen spicy taste material from natural population

1. Test materials

18 parts chili material (table 3).

2. KASPar mark

See Step 4 of Example 1.

3. Test results

The results are referring to Table 3 and Fig. 2, and it can be seen that: when the phenotype of the tested capsicum is "spicy" (corresponding Pun genotype is Pun/Pun), the SNP sites corresponding to molecular marker 2_25 are all A:A genes type; when the phenotype of the tested pepper is "no spicy taste" (the corresponding Pun genotype is pun/pun), the SNP loci corresponding to molecular marker 2_25 are all C:C genotypes. Therefore, the marker 2_25 of the present invention can be used to screen spicy materials from natural populations.

Table 3 Typing of molecular marker 2_25 in natural populations of pepper

Embodiment 3, utilize mark 2_25 of the present invention to carry out the identification of the purity of the offspring of hybridization of hot pepper and non-hot pepper (sweet pepper)

1. Test materials

1) 5 female control plants (04Q-29) with spicy taste; 2) 88 plants in the sampling population of pepper (04Q-29)×sweet pepper (04Q-15) hybrids, and the genomic DNA was extracted respectively.

2. Phenotype identification

Plant pepper×sweet pepper hybrid progenies and their female parents in the field, and manage them with conventional management measures. At the adult plant stage, the phenotypes are identified one by one according to the characteristics of the varieties, and the pseudo F ₁ offspring similar to the female parent are marked, and the purity is calculated.

3. KASPar mark

See Step 4 of Example 1.

4. Test results

Seedling phenotype identification found that 8 plants were pseudo F ₁ generation in 88 individual plant samples, and the purity was 91%. Marker 2_25 analysis showed that 8 of the 88 individual plant samples were of the maternal genotype (Fig. 3). The results show that the marker of the present invention can quickly carry out molecular marker-assisted identification of the selfed offspring of the female parent in the offspring of the pepper x sweet pepper hybrid, and can be used for the purity identification of the pepper x sweet pepper hybrid in production or scientific research practice.

<110> Vegetable and Flower Research Institute, Chinese Academy of Agricultural Sciences

<120> SNP markers related to hot pepper taste and its application

<130> GNCLN171368

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<170> PatentIn version 3.5

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Claims (10)

1. the SNP of following SNP site or following SNP in capsicum genome for detecting in capsicum genome Application of the material of the SNP of point in identifying or aiding in identification capsicum pungent character；

The SNP site corresponds to the 24th of nucleotide sequence shown in sequence 4 in sequence table in capsicum genome；It is described Nucleotides at SNP site is A or C.

2. the reagent for identifying or aiding in identification capsicum pungent character, is to be used to detect following SNP site in capsicum genome SNP material；The SNP site corresponds to nucleotides shown in sequence 4 in sequence table in capsicum genome The 24th of sequence；Nucleotides at the SNP site is A or C；

Specifically, described " being used for the material for detecting the SNP of following SNP site in capsicum genome " is as follows A) or b)：

A) complete single stranded DNA first, is following (a1) or (a2)：

(a1) as the 22-45 of sequence 2 in the single stranded DNA shown in 22-45 nucleotides of sequence in sequence table 1, sequence table The complete single stranded DNA of single stranded DNA composition in single stranded DNA and sequence table shown in the nucleotides of position shown in sequence 3；

(a2) in 22-45 nucleotides, sequence tables by sequence in sequence table 1 sequence 2 22-45 nucleotides and sequence Three single stranded DNAs point of the row 3 after the substitution of one or several nucleotides and/or missing and/or addition shown in gained sequence It is molecular, and with the complete complete single stranded DNA of single stranded DNA function identical described in (a1)；

B) complete single stranded DNA second, is following (b1) or (b2)：

(b1) as sequence in the single stranded DNA shown in sequence 2 in the single stranded DNA shown in sequence in sequence table 1, sequence table and sequence table The complete single stranded DNA of single stranded DNA composition shown in row 3；

(b2) by by sequence in sequence table 1, sequence 2 and sequence 3 by one or several nucleotides substitution and/or missing and/ Or three single strand dnas composition after addition shown in gained sequence, and it is same with complete single stranded DNA function phase described in (b1) Complete single stranded DNA.

3. the kit for identifying or aiding in identification capsicum pungent character, contains the reagent described in claim 2；

Specifically, also containing fluorescence probe A, fluorescence probe B, quenching probes A and quenching probes B in the kit；

The nucleotides sequence of the fluorescence probe A is classified as 1-21 of sequence 1 in sequence table, 5 ' ends connection fluorophor A；Institute The nucleotides sequence for stating quenching probes A is classified as the reverse complementary sequence of 1-21 of sequence 1 in sequence table, and the connection of 3 ' ends is quenched Group；

The nucleotides sequence of the fluorescence probe B is classified as 1-21 of sequence 2 in sequence table, 5 ' ends connection fluorophor B；Institute The nucleotides sequence for stating quenching probes B is classified as the reverse complementary sequence of 1-21 of sequence 2 in sequence table, and the connection of 3 ' ends is quenched Group；

More specific, the fluorophor A is FAM；The fluorophor B is HEX；The quenching group is Q.

4. kit described in reagent described in claim 2 or claim 3 is in identifying or aiding in identification capsicum pungent character Using.

5. a kind of identify or aid in identify that capsicum to be measured is the method for having pungent capsicum still without pungent capsicum, including following step Suddenly：It is A or C or A and C to detect the nucleotides in the genome of capsicum to be measured at following SNP site, described to be measured to determine The genotype of capsicum is A:A or C:C or A:C, it is described to be measured according to being identified below according to the genotype of the capsicum to be measured Capsicum is to have pungent capsicum still without pungent capsicum：If the capsicum to be measured is A:A genotype or A:C genotype, then it is described Capsicum to be measured is or candidate is to have pungent capsicum；If the capsicum to be measured is C:C genotype, then the capsicum to be measured be or candidate For without pungent capsicum；

The SNP site corresponds to the 24th of nucleotide sequence shown in sequence 4 in sequence table in capsicum genome；It is described Nucleotides at SNP site is A or C；

The A:A genotype is that to correspond to the 24th of nucleotide sequence shown in sequence 4 in sequence table in capsicum genome be A's It is homozygous；

The C:C genotype is that to correspond to the 24th of nucleotide sequence shown in sequence 4 in sequence table in capsicum genome be C's It is homozygous；

The A:C genotype be correspond in capsicum genome the 24th of nucleotide sequence shown in sequence 4 in sequence table be A and C heterozygous.

6. method according to claim 5, it is characterised in that：It is described " following SNP in the genome of detection capsicum to be measured Nucleotides at point is that A or C or A and C " method are KASPar detection methods；

The KASPar detection methods include following two steps：KASPar is expanded and KASPar amplification products therefroms is carried out Fluorescent scanning is so that it is determined that the nucleotides at the SNP site is A or C or A and C；The template of KASPar amplification is The genomic DNA of the capsicum to be measured, KASPar primers used meet following condition：With the genome of the capsicum to be measured DNA is that template carries out the sequence of KASPar amplification gained amplified productions containing sequence 4 in ordered list.

7. method according to claim 6, it is characterised in that：In the KASPar detection methods, the KASPar primers For the complete single stranded DNA second described in claim 2.

8. any methods described is peppery in reagent described in claim 2 or the kit described in claim 3 or claim 5-7 Application in green pepper breeding.

9. any methods described is such as in reagent described in claim 2 or the kit described in claim 3 or claim 5-7 Under application in (a) or (b)：

(a) there is the screening of pungent capsicum；

(b) there is the purity auxiliary identification of pungent capsicum parent and the cenospecies without pungent capsicum parent.

The method of following 10. (A) or (B)：

(A) method for having pungent capsicum variety, including selection A are cultivated:The capsicum of A genotype carries out the step of breeding as parent Suddenly；The A:A genotype is that to correspond to the 24th of nucleotide sequence shown in sequence 4 in sequence table in capsicum genome be A's It is homozygous；

(B) method without pungent capsicum variety, including selection C are cultivated:The capsicum of C genotype carries out the step of breeding as parent Suddenly；The C:C genotype is that to correspond to the 24th of nucleotide sequence shown in sequence 4 in sequence table in capsicum genome be C's It is homozygous.