Development method and application of KASP marker closely associated with pungency degree of pepper

Abstract

The invention relates to a development method and application of a KASP marker closely related to the pungency degree of hot peppers, and the development method comprises the following steps: constructing two hot pepper pungency degree difference extreme mixing pools by utilizing an F2 segregation population obtained by hybridizing HJ01 of high-pungency hot peppers and HJ02 of low-pungency hot peppers; according to a BSA-seq whole genome sequencing result, a candidate region for regulating and controlling the pungency degree of hot peppers is preliminarily screened out, correlation analysis is carried out on genes in the candidate region in combination with RNA-seq, it is found that only two significant intervals are located, QTL intervals are located on a chromosome 2, the starting position range and the ending position range of the QTL intervals are 135670001-136670000 and 136800001-152640000, and the QTL intervals are located on a chromosome 2. The method is used for the interval range of a key gene causing the difference between the capsaicin content in the 'HJ01' high-spicy wild type material and the capsaicin content in the 'HJ02' low-spicy mutant material. KASP molecular markers are developed on the basis of variation sites, target site genotype detection is carried out on high-spicy materials and low-spicy materials, the high-spicy materials are C: C type materials, the low-spicy materials are G: G type materials, and the markers are co-dominant; kASP is used for carrying out genotype identification on the F2 population, and the accuracy rate reaches 89.3%.

Description

Technical Field

[0001] The invention relates to the technical field of pepper genetic breeding and molecular biology, and more specifically to a method for developing a KASP marker closely related to the spiciness of pepper and its application. Background Art

[0002] The spiciness of peppers is a quantitative value that indicates the spiciness of peppers. It is determined by the content of capsaicinoids in pepper fruits. Capsaicinoids are unique to pepper fruits and have medical and health effects such as anti-cancer, analgesia and weight loss. PUN1 , Pun2 , Pun3, Putative aminotransferase gene ( putative aminotransferase , pAMT ) and putative ketoacyl ACP reductase gene ( putative ketoacyl-ACP reductase , CaKR1 ) etc. Pun1 , pAMT and CaKR1 Capsaicin biosynthesis pathway-related genes, Pun3 It is the pepper MYB transcription factor gene, Pun2 Found in wild species of pepper, but not yet seen Pun2 Reports of relevant candidate genes. Pun1 Acyltransferase, which encodes the catalytic enzyme in the last step of capsaicin biosynthesis, affects other structural genes and plays a leading role in the synthesis and accumulation of capsaicin.

[0003] At present, there are two main methods for identifying the spiciness of peppers: sensory testing and instrument testing. Sensory testing relies on people to identify the spiciness of peppers through taste. This method has disadvantages such as low accuracy, inability to quantify spiciness data, and non-repeatable testing. Instrument testing is mainly carried out through equipment such as spiciness meters. This method has disadvantages such as long testing time, high cost, and high technical difficulty. Moreover, both methods require the fruit to be ripe before testing.

[0004] A Chinese patent with publication number CN 113151568A discloses a SNP site tightly linked to the capsaicin content of peppers, its CAPS molecular marker and its application. A genome-wide association analysis was performed on the capsaicin content of 311 annual pepper materials, and a SNP site (SNP 6:22748122) on chromosome 6 that was significantly correlated with controlling the capsaicin content was found. The relevant CAPS molecular marker and its amplification primer were designed based on the SNP site.

[0005] The above patents are based on the resequencing of 311 annual peppers, the extraction and determination of capsaicin, which is labor-intensive and has low screening efficiency. In addition, CAPS markers usually require electrophoresis and enzyme digestion, which are complex operations and not suitable for high-throughput screening.

[0006] In addition, the existing development of Capana02g002340 is only a development of low-spicy chili pepper spiciness. Figure 1 As shown, the PUN1 gene of low-spiciness material 1, low-spiciness material 2, high-spiciness material 61, and high-spiciness material 62 was verified and found to be 2.5 kb in all four materials, and no deletion occurred. Therefore, the development of a KASP marker closely related to the spiciness of peppers and its application are of great significance. Summary of the invention

[0007] The present invention provides a method for developing a KASP marker closely related to the spiciness of peppers and its application, using the F hybrid of high-spiciness HJ01 and low-spiciness HJ02 to 2 Separate the population and construct two mixed pools with extreme differences in pepper spiciness; based on the BSA-seq whole genome sequencing results and RNA-Seq association analysis, screen out candidate genes that regulate pepper spiciness; develop KASP molecular markers based on variant sites, and use KASP to 2 The genotype of the population was identified with an accuracy rate of 89.3%.

[0008] The present invention adopts the following technical solution: In a first aspect, the present invention provides a method for developing a KASP marker closely related to the spiciness of peppers, comprising the following steps: (1) The F hybrid of the high-spicy 'Yong'an yellow pepper' (HJ01) and the low-spicy 'Yong'an yellow pepper' mutant (HJ02) was used to generate 2 Separate the groups and construct two mixed pools with extreme differences in pepper spiciness; (2) Collect pepper fruits of the parents 'HJ01' and 'HJ02' in step (1) respectively, and extract genomic DNA. According to the results of BSA-seq whole genome sequencing, combined with RNA-seq, the genes in the candidate region are subjected to association analysis, and the candidate gene regions that regulate the spiciness of peppers are preliminarily screened; (3) Based on the candidate gene regions of the BSA-Seq whole genome resequencing data of HJ01 and HJ02, KASP markers were designed using the online platform Polymarker website; (4) According to the primer design results on the website, add the universal fluorescent linker sequences FAM (5'-GAAGGTGACCAAGTTCATGCT-3') and HEX (5'-GAAGGTCGGAGTCAACGGATT-3') to the 5' end of the two forward primers, then perform PCR reaction, and then read the final fluorescence data in an ELISA reader. Finally, import the data into Klustercaller software for genotyping.

[0009] In the above step (5), the primers synthesized into the universal fluorescent linker sequence are dissolved and configured into a primer mix, wherein the forward primer is 12 mM and the reverse primer is 30 mM.

[0010] The PCR reaction procedure in the above step (5) is as follows: denaturation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing at 65°C for 60 s, decreasing 1.0°C each time for 8 cycles; denaturation at 95°C for 20 s, annealing at 57°C for 60 s, for 30 cycles.

[0011] In a second aspect, the present invention also provides an application of a KASP marker closely related to the spiciness of peppers in the genetic detection of the spiciness of peppers.

[0012] When the KASP molecular marker was used to detect the target site genotype of the high-spicy material 'HJ01' and the low-spicy material 'HJ02', the high-spicy material showed C:C type, and the low-spicy materials all showed G:G type. The phenotypic spiciness was co-dominantly separated with the molecular marker.

[0013] Furthermore, KASP molecular markers were used to identify the F1 hybrid strain of the high-spicy 'Yong'an Huangjiao' (HJ01) and the low-spicy 'Yong'an Huangjiao' mutant (HJ02). 2 Genotyping of the population.

[0014] It can be seen from the above description of the present invention that, compared with the prior art, the present invention has the following advantages: 1. The present invention uses the F hybrid of the high-spicy 'Yong'an yellow pepper' (HJ01) and the low-spicy 'Yong'an yellow pepper' mutant (HJ02) 2Separate populations, and construct two extreme mixed pools of pepper spiciness differences; Based on the results of BSA-seq whole genome sequencing, preliminary screening of candidate regions regulating pepper spiciness, and combined with RNA-seq to conduct association analysis on genes in the candidate regions, it was determined that Capana02g002340 is the key gene that causes the difference in capsaicin content in the 'HJ01' high-spiciness wild-type material and the 'HJ02' low-spiciness mutant material. Developing KASP markers for mutation sites in gene sequences related to pepper spiciness can quickly distinguish the difference between high and low spiciness peppers at the seedling stage, and the cost is low. For pepper breeders, it can greatly reduce the workload of identifying high-spiciness and low-spiciness pepper germplasm resources and improve the breeding process of high-spiciness peppers.

[0015] 2. The KASP marker used in the present invention has many advantages over the CAPS marker: KASP marker technology can achieve high-throughput detection and is suitable for rapid detection of a large number of samples; KASP marker technology does not require electrophoresis, and the detection results are intuitive. Different genotypes can be distinguished by color and the corresponding materials can be directly located, which greatly simplifies the operation process and reduces the complexity and time cost of the experiment; KASP marker technology can achieve accurate double allele detection of SNPs and insertion and deletion (InDels) sequences at specific sites, and its genotyping error rate is low, which is significantly better than some traditional methods; the cost of using KASP technology is about 80% lower than that of using TaqMan probe method, which makes KASP marker more economical in large-scale applications; KASP marker technology avoids the use of toxic chemicals such as nucleic acid dyes, reducing potential harm to the human body and the environment. KASP marker technology has significant advantages over CAPS markers in terms of high throughput, low cost, simple operation, high accuracy, flexibility and environmental friendliness.

[0016] 3. The present invention is aimed at high-spicy yellow pepper varieties, and the marker developed for the spiciness of peppers is different from the previous marker development of Capana02g002340 gene (a marker developed for the presence or absence of spiciness of low-spicy peppers), and has different application scenarios and application opportunities, which is more realistic. In addition, the pepper spiciness survey results (high, low) of the present invention are basically consistent with the genotyping results, with a matching rate of 89.3%. Therefore, the developed KASP marker has a high accuracy and can efficiently identify the genotype of the offspring single plant at the target point. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Electropherogram for marker validation of the existing development Capana02g002340.

[0018] Figure 2 This is a genome-wide distribution diagram of the G value of an embodiment of the present invention.

[0019] Figure 3 It is the PCA graph of HJ01 and HJ02 in 6 samples of the embodiment of the present invention.

[0020] Figure 4 It is a heat map of the correlation between samples in the embodiment of the present invention.

[0021] Figure 5 Schematic diagram of the comparison of differentially expressed genes in the transcriptome of the embodiment of the present invention.

[0022] Figure 6 This is a scatter plot of differentially up-regulated and down-regulated genes in the examples of the present invention.

[0023] Figure 7 This is a diagram of enrichment analysis of differentially expressed genes in an embodiment of the present invention.

[0024] Figure 8 This is a correlation analysis diagram of BSA-Seq and RNA-Seq of the present invention.

[0025] Fig. 9 This is a distribution diagram of signal points of some samples in which the KASP marker is used to detect the genotype of the target site of a single plant in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The specific implementation of the present invention is described below with reference to the accompanying drawings. In order to fully understand the present invention, many details are described below, but for those skilled in the art, the present invention can be implemented without these details. For well-known components, methods and processes, they are not described in detail below.

[0027] Development of KASP markers closely related to the spiciness of chili peppers 1.1 Materials In 2016, the Vegetable Research Institute of Sanming Academy of Agricultural Sciences collected the local variety 'Yong'an Yellow Pepper' (HJ01) in Yong'an Town, Sanming City. After five consecutive generations of self-pollination and purification, in the autumn of 2018, when the capsaicin content of the Sanming local variety 'Yong'an Yellow Pepper' population was detected, a low-spicy 'Yong'an Yellow Pepper' mutant was found in its offspring. Its fruits showed low spiciness from the green fruit stage to the old fruit stage, and the capsaicin content in the fruits of the collected Sanming local variety 'Yong'an Yellow Pepper' was extremely significantly different. After self-pollination and harvesting in the spring of 2019, the agronomic traits of the mutant were identified in the field in the autumn of 2019 and the F 2 The first-generation population showed high purity and the capsaicin content test found that the mutant had a very significant difference from the local variety 'Yong'an Yellow Pepper' in the capsaicin content, and it could be stably inherited. In the spring of 2020, the mutant strain of the local variety 'Yong'an Yellow Pepper' in Sanming City was named 'HJ02'. In the autumn of 2021, 'HJ01' was used as the female parent and 'HJ02' as the male parent, and hybrid seeds were harvested. In the spring of 2022, hybrid F 1In the autumn of 2022, F 2 group.

[0028] 1.2 Extraction and content detection of capsaicin Accurately weigh appropriate amounts of capsaicin standard material (Alta Technology Co., Ltd.) with a purity of ≥95% and dihydrocapsaicin standard material (Alta Technology Co., Ltd.) with a purity of ≥90%, add methanol to dissolve, and prepare a mixed standard solution of capsaicin and dihydrocapsaicin with appropriate concentrations; place the sample in a 60°C oven to dry until the moisture content is ≤15%, then crush it with an electric grinder, pass it through a 50-mesh sieve, weigh 0.5g of the sample into a 50ml centrifuge tube. Add about 25ml of methanol-tetrahydrofuran (1:1) mixed solvent, extract it with ultrasound (power 100 W) in a 60°C water bath for 20min, filter, repeat the extraction, combine the two filtrates, and make up to 50ml. After filtering through a 0.45µm filter membrane, chromatographic analysis was performed (chromatographic column: Zorbax SB-C18 4.6mm×250mm 5µm; mobile phase: methanol + water (65+35); injection volume: 10µL; flow rate: 1mL / min; detection wavelength: 280nm; column temperature: 30℃); the dilution factor can be appropriately adjusted according to the total amount of capsaicinoids in the sample and the sensitivity of the detector.

[0029] The spiciness of the 'HJ01' and 'HJ02' materials was measured, and it was found that the spiciness of 'HJ01' reached 83060 SHU, and the spiciness of 'HJ02' reached 5228 SHU. Among them, the low-spiciness material 'HJ02' pepper material showed low spiciness characteristics from the green fruit stage to the old fruit stage, and there was a significant difference in spiciness with the 'HJ01' pepper material. The capsaicin content detection and analysis results showed that the hybrid F of the high-spicy 'HJ01' and the low-spicy 'HJ02' 1 The spiciness of the pepper fruit is 53066SHU, indicating that 1 The capsaicin content in the pepper fruit of the first generation reached the average value of the male and female parents. 2 It was found that 88 individual plants in the generation population had a spiciness of more than 50,000 SHU, 326 individual plants had a spiciness of 5,000-50,000 SHU, and 88 individual plants had a spiciness of less than 5,000 SHU, as shown in Table 1.

[0030] Table 1 Spiciness analysis results 1.3 DNA Extraction and BSA-Seq analyze Each offspring population was planted in the experimental field of the pepper breeding base of the Vegetable Research Institute of Sanming Academy of Agricultural Sciences. Five pepper fruits were randomly selected from each parent and offspring population to determine the capsaicin content in mature pepper fruits. Thirty plants with the highest and lowest spiciness were selected to construct the high-spiciness pool (H-pool) and low-spiciness pool (L-pool), and pepper fruits of the parents 'HJ01' and 'HJ02' were collected to extract genomic DNA for Illumina sequencing.

[0031] Total genomic DNA was extracted, and paired-end DNA sample preparation kit (Illumina Inc., San Diego, CA, USA) was used to construct paired ends with an insert size of 500 bp using at least 3 μg of genomic DNA. Four stringent filtering criteria were used: 1) reads with unidentified nucleotides (N) ≥ 10% were removed; 2) phred quality scores ≤ 20 were removed; 3) reads aligned to barcode adapters were removed. Calculation of G statistic: The following formula was used to calculate the G statistic for each SNP.

[0032] in, O To observe AD (AD r ,AD d ), E is the expected AD under the null hypothesis, calculated in the same way as the original G statistic, and ln represents the natural logarithm.

[0033] The average G value was calculated using an XXX-kb sliding window with a step size of XX-kb. Windows with SNP / Indel < XX were removed, and then windows with an average G value greater than the XXth percentile of the average G value of the whole genome were taken as significant windows. The overlapping parts of adjacent significant windows were merged into a large significant genomic region, and the genes within the interval were taken as candidate genes.

[0034] After the genomic DNA of the parents and the extreme trait progeny pool was sequenced on the Illumina platform (as shown in Table 2), the high-spicy wild-type material 'HJ01' generated 100857477600bp of raw data (Q20 ≥ 97.31%, Q30 ≥ 92.23%, GC: 36.03%); the low-spicy mutant material 'HJ02' generated 109693800600bp of raw data (Q20 ≥ 97.45%, Q30 ≥ 92.63%, GC: 36.06%). The two progeny pools H-pool and L-pool generated 94906207800bp (Q20≥97.82%, Q30≥93.59%, GC: 34.61%) and 96892266300bp (Q20≥97.77%, Q30≥93.48%, GC: 35.72%) of raw data, respectively. After sorting and filtering, at least 95.53% of the sequencing data of each group of samples were successfully aligned to the pepper reference genome 'ZUNLA-1'. The sequencing data of each sample was sufficient and of good quality, and the alignment results of all samples were normal and could be used for subsequent analysis.

[0035] Table 2 Statistics of base information before and after filtering After filtering, the G-statistic method was used to select candidate regions related to spiciness. In order to reduce background noise, the calculated G value was fitted according to the sliding window method, and the average value in the window was taken as the fitted value. The window size used for sliding window was 1000kb, the step size used was 10kb, the number of SNPs in the window was greater than or equal to 10, and then the number of effective windows for each chromosome and genome was counted (as shown in Table 3).

[0036] Table 3. G value sliding window statistics effective window number table The sequencing data from H-pool and L-pool were aligned, molecular marker detected and annotated by BWA\GATK and ANNOVAR, respectively, and a total of 8,804,977 SNPs and 691,834 InDels were obtained. The Gst algorithm was used and the confidence threshold of the genomic region was set to be greater than 99% ( p <0.01), the candidate genomic regions 135670001-136670000 and 136800001-152640000 were located in Ch2 (e.g. Figure 2 It is worth noting that the candidate segment of Chr.02 contains the gene Capana02g002340 ( Pun1 ).

[0037] According to the significant intervals obtained by 99% quantile screening and the candidate genes within the intervals (Table 4), it was found that only two significant intervals were located, and both QTL intervals were located on chromosome 2. The starting and ending positions of the QTL intervals ranged from 135670001 to 136670000 and 136800001 to 152640000. The reverse direction was determined to be greater than or equal to the threshold, that is, the positive direction of Δ (SNP-index).

[0038] Table 4 G-value significant intervals and genes within the intervals 1.4 RNA Extraction and RNA-Seq analyze RNA extraction, transcriptome sequencing, and data analysis were performed by Kidio (Guangzhou, China). Total RNA was extracted using the Trizol method (Invitrogen). The concentration and purity of RNA were determined using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 assay kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Two cDNA libraries of pepper high SHU groups (HJ01-1, HJ01-2, and HJ01-3) and low SHU groups (HJ02-1, HJ02-2, and HJ02-3) were constructed and quality tested and sequenced using the Illumina HiSeq platform. The annotation of high-quality clean reads was completed using the pepper genome database (http: / / peppersequence.genomics.cn / ) as the reference sequence. The Benjamini-Hochberg method was used for multiple hypothesis testing, and the probability of hypothesis testing (p value) was calibrated to obtain the false discovery rate (FDR). EdgeR was used for differential expression analysis of the two samples, and FDR < 0.01 and fold change ≥ 2 were set as the thresholds for significant differential expression. The differential expression, functional annotation, and functional enrichment of genes in non-spicy and spicy samples were analyzed.

[0039] A total of 6 samples were tested by RNA-seq, of which 3 samples were selected for transcriptome sequencing analysis from the high-spiciness wild-type material 'HJ01' and the low-spiciness mutant material 'HJ02' (as shown in Table 5). An average of Gb of high-quality Clean read data was obtained for each sample, with Q20 at 97.86% and above, Q30 at 93.64% and above, and GC content at 42.59% and above, indicating that the sequencing quality was ideal and could be used for subsequent analysis.

[0040] Table 5 Transcriptome data comparison statistics Among the six samples tested by RNA-seq, principal component analysis was used to study the distance relationship between samples using the idea of ​​dimensionality reduction. The analysis results showed that the high-spicy wild-type material 'HJ01' and the low-spicy mutant material 'HJ02' were distributed on both sides of the distance in the PCA graph (such as Figure 3 As shown in the figure). The correlation heat map analysis between samples shows that the colors in the correlation heat maps of the ‘HJ01’ group and the ‘HJ02’ group are close to blue, indicating that the greater the correlation between the respective groups; the colors of the ‘HJ01’ group and the ‘HJ02’ group in the correlation heat map are close to white, indicating that the correlation is smaller (such as Figure 4 as shown).

[0041] In the comparative transcriptome of pepper fruit between ‘HJ01’ and ‘HJ02’, a total of 3478 differentially expressed genes were detected, including 1765 up-regulated genes and 1713 down-regulated genes (e.g. Figure 5 GO analysis showed that in biological processes, the main annotations were metabolic processes, cellular processes, and single-organism processes; in cellular components, the main annotations were cells, cell parts, and organelles; in molecular functions, the main annotations were catalytic activity, binding, nucleic acid binding, transcription factor activity, etc. (e.g. Figure 6 as shown).

[0042] The results of KEGG enrichment analysis showed that ( Figure 7 The differentially expressed gene enrichment pathways mainly include phenylpropanoid metabolism, fatty acid chain elongation and galactose metabolism biosynthesis, indicating that there are great differences in the biosynthesis of capsaicinoids between the high-spiciness wild-type material 'HJ01' and the low-spiciness mutant material 'HJ02'.

[0043] In order to further explore the key genes that affect the spiciness of peppers, the results obtained by BSA-Seq and RNA-Seq were correlated (e.g. Figure 8 as shown).

[0044] The candidate genes obtained by BSA-Seq and the 3478 DEGs obtained by RNA-Seq were compared with the Wayne plot at a 99% threshold to obtain their common genes. Results A total of 21 genes were differentially expressed in parents with different spiciness. Based on the 'HJ02' low-spiciness mutant material, this study focused on analyzing differentially expressed genes that may be related to capsaicin content. A total of 21 candidate genes (Capana02g002261, Capana02g002374, Capana02g002149, Capana02g002264, Capana02g002166, Capana02g002340, Capana02g002229, Capana02g002396, Capana02g002158, Capana02g002318, C apana02g002373, Capana02g002286, Capana02g002147, Capana02g002254, Capana02g002191, Capana02g 002383, Capana02g002188, Capana02g002164, Capana02g002372, Capana02g002168, Capana02g002314). Among them, Capana02g002340 had significant differences in HJ01_mean (0.001) and HJ02_mean (55.18967), and the log2FC value (15.75211) was the highest among the 21 candidate genes. Therefore, Capana02g002340 was determined to be the key gene causing the difference in capsaicin content between the 'HJ01' high-spicy wild-type material and the 'HJ02' low-spicy mutant material. Further combined with homologous gene functional annotation, gene expression and bioinformatics analysis, it was found that a key gene for capsaicin synthesis, Capana02g002340 ( PUN1 ).

[0045] 1.5 Development and validation of KASP molecular markers Based on the BSA-Seq whole genome resequencing data of HJ01 and HJ02, the KASP marker was designed using the online platform Polymarker website. According to the primer design results of the website, the universal fluorescent linker sequences FAM (5'-GAAGGTGACCAAGTTCATGCT-3') and HEX (5'-GAAGGTCGGAGTCAACGGATT-3') were added to the 5' end of the two forward primers. The primers were synthesized by Guangzhou Kidio Biotechnology Co., Ltd. After the primers were synthesized and dissolved, they needed to be configured into primer mix (two forward primers were 12 mM and the reverse primer was 30 mM). The KASP test was performed according to the steps provided by LGC Genomics; the DNA template concentration was about 30 ng / µL; the 384 plate reaction system was 2.0 µL of dried DNA, followed by 2.0 µL of 2×KASP V4.0 Mastermix, 0.045 µL of primer mix, and 1.924 µL of water. The PCR reaction program was as follows: denaturation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing at 65°C for 60 s, decreasing 1.0°C per cycle, for 8 cycles; denaturation at 95°C for 20 s, annealing at 57°C for 60 s, for 30 cycles. After the PCR reaction was completed, the final fluorescence data was read in an autofocus fluorescence multifunctional microplate reader (PHERAstarplus SNP, BMG LABTECH), and then the data was imported into Klustercaller v3.4 software (LGC, Hoddesdon, UK) for genotyping.

[0046] Based on the resequencing data, 10 pairs of KASP markers were opened (as shown in Table 6), and then these markers were used to detect the target site genotype of the high-spiciness material 'HJ01' and the low-spiciness material 'HJ02'. The results showed that the signal point of the developed sixth KASP marker was red, aggregated near the y-axis, and the primer with the FAM fluorescent label sequence at the 5' end was competitively amplified, and the genotype was T; T; the signal point of the low-spiciness material 'HJ02' was blue, aggregated near the x-axis, and the primer with the HEX fluorescent label sequence at the 5' end was competitively amplified, and the genotype was C; C.

[0047] Table 6 Resequencing results of 10 pairs of KASP markers To verify the accuracy of KASP markers, a hybrid combination of high-spiciness material 'HJ01' and low-spiciness material 'HJ02' was configured and self-fertilized to obtain F 2 Generation, for F 2The 56 plants in the group were numbered and leaf samples were collected at the mature stage to extract DNA. The KASP marker was used to detect the target point genotype of each plant. The distribution of signal points of some samples was as follows: Fig. 9 As shown. The sample genotypes were clearly divided into three categories. The red signal point samples aggregated near the y-axis detected competitive amplification of the primer linked to the FAM fluorescent label sequence, which was a T:T genotype; the green signal point samples aggregated in the middle detected both FAM and HEX fluorescent signals, which was a heterozygous C:T genotype, and the KASP marker was co-segregated with the pepper spiciness phenotype; the blue signal point samples aggregated near the x-axis detected competitive amplification of the primer linked to the FAM fluorescent label sequence, which was a C:C genotype. After the fruit matured, the capsaicin content was detected by HPLC, and the results showed that 56 F 2 There were 10 high-spiciness types, 23 medium-spiciness types, and 23 low-spiciness types in the population. Comparison revealed that 18 samples at blue signal points were all low-spiciness types; 23 samples at green signal points were medium-spiciness types; and 9 samples at red signal points were all high-spiciness types. The results of the pepper spiciness survey were basically consistent with the genotyping results, with a matching rate of 89.3%. Therefore, the developed KASP marker has a high accuracy and can efficiently identify the genotype of the offspring individual plants at the target point.

[0048] Table 7 Comparison of genotype and phenotype The above is only a specific implementation of the present invention, but the design concept of the present invention is not limited thereto. Any non-substantial changes to the present invention using this concept shall be deemed as an infringement of the protection scope of the present invention.

Claims

1. A method for developing a KASP marker closely related to the spiciness of peppers, characterized in that: The steps include: (1) Using the F2 segregating population of the high-spiciness 'Yong'an yellow pepper' (HJ01) and the low-spiciness 'Yong'an yellow pepper' mutant (HJ02), two mixed pools with extreme differences in pepper spiciness were constructed; (2) Collect pepper fruits of the parents 'HJ01' and 'HJ02' in step (1) respectively, and extract genomic DNA. According to the results of BSA-seq whole genome sequencing, combined with RNA-seq, the genes in the candidate region are subjected to association analysis, and the candidate gene regions that regulate the spiciness of peppers are preliminarily screened; (3) Based on the candidate gene regions of the BSA-Seq whole genome resequencing data of HJ01 and HJ02, KASP markers were designed using the online platform Polymarker website; (4) According to the primer design results on the website, add the universal fluorescent linker sequences FAM (5'-GAAGGTGACCAAGTTCATGCT-3') and HEX (5'-GAAGGTCGGAGTCAACGGATT-3') to the 5' end of the two forward primers, then perform PCR reaction, and then read the final fluorescence data in an ELISA reader. Finally, import the data into Klustercaller software for genotyping.

2. The method for developing a KASP marker closely associated with the spiciness of pepper as claimed in claim 1, characterized in that: In the step (4), the primers synthesized into the universal fluorescent linker sequence are dissolved and configured into a primer mix, wherein the forward primer is 12 mM and the reverse primer is 30 mM.

3. The method for developing a KASP marker closely related to the spiciness of pepper as claimed in claim 1, characterized in that: The procedure of the PCR reaction in step (4) is as follows: denaturation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing at 65°C for 60 s, decreasing 1.0°C each time for 8 cycles; denaturation at 95°C for 20 s, annealing at 57°C for 60 s, for 30 cycles.

4. An application of a KASP marker closely related to the spiciness of peppers, characterized in that: The method for developing a KASP marker closely related to the spiciness of peppers as described in claim 1 is used, and the KASP marker is used in genetic detection of the spiciness of peppers.

5. The use of a KASP marker closely related to the spiciness of peppers as claimed in claim 4, characterized in that: The KASP molecular marker was used to detect the target site genotype of the high-spiciness material 'HJ01' and the low-spiciness material 'HJ02'. The detected high-spiciness materials showed C:C type, and the low-spiciness materials all showed G:G type. The phenotypic spiciness was co-dominantly separated with the molecular marker.

6. The use of a KASP marker closely related to the spiciness of peppers as claimed in claim 4, characterized in that: The KASP molecular marker was used to identify the genotype of the F2 population formed by the cross between the high-spicy 'Yong'an Yellow Pepper' (HJ01) and the low-spicy 'Yong'an Yellow Pepper' mutant (HJ02).

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