KASP molecular marker for identifying pepper floral leaf generation and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker for identifying pepper floral leaf generation and application of the KASP molecular marker. According to the invention, the strongest candidate gene for controlling the character of the pepper flower leaf is determined through construction of a genetic map linked with the character of the pepper flower leaf and development of a molecular marker, and a KASP marker and a primer capable of identifying the generation of the pepper flower leaf are obtained through research and can be used for detecting or screening different pepper leaf types, identifying the pepper flower leaf and performing pepper leaf type typing; meanwhile, the invention provides a method for pepper mosaic typing or pepper normal leaf plant breeding, a large amount of genotype sequencing analysis is not needed, plants with different leaf types can be identified and distinguished through conventional detection amplification in a laboratory, the method is convenient and easy to operate, pepper mosaic screening and identification can be carried out in advance, the breeding screening time is shortened, and the breeding efficiency is improved. Meanwhile, more excellent pepper germplasm resources are provided, and technical support is provided for pepper germplasm identification and strain breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular marker breeding. More specifically, it relates to a KASP molecular marker for identifying pepper mosaic and its application. Background Art

[0002] Pepper (Capsicum annuum L.) is an annual herbaceous plant of the genus Capsicum in the Solanaceae family. It is native to the tropical regions of Central and South America, with extensive cultivation in Mexico. It was introduced into China in the late 16th century and is now cultivated throughout China, widely distributed in Yunnan. The cultivated area of pepper in China is 2 million hectares, ranking first among vegetables. Pepper is an important vegetable crop and can also be used as an ornamental plant. Identifying and screening excellent pepper germplasm materials is of great significance for breeding leafy pepper varieties, ornamental pepper varieties, and improving pepper yield, resistance, etc. There is a variegated leaf phenomenon in pepper, that is, the leaves of the pepper plants of this germplasm resource will show irregular white chlorosis, also known as pepper mosaic. The main reason for this phenomenon is the mutation of chloroplast-related genes, which ultimately affects the photosynthesis of the plant, resulting in the mosaic phenomenon of the plant, affecting the photosynthesis of the plant, reducing the yield, and also reducing the ornamental value of the leaves, seriously affecting the breeding of excellent pepper germplasm resources.

[0003] At present, it is difficult to detect and screen pepper mosaic in advance. The breeding cycle of identifying pepper mosaic through traditional field methods is relatively long, and the screening accuracy is not high. With the progress of modern molecular biotechnology, molecular marker-assisted selection breeding has also been applied to the selection of excellent traits of pepper germplasm resources. Single nucleotide polymorphisms (SNP) markers are distributed on chromosomes and have advantages such as rich polymorphism and good genetic stability. They are mostly used in research such as species genetic diversity analysis, core germplasm bank construction, genetic map construction, and variety identification, and are important markers for molecular breeding applications. In the prior art, only studies on molecular markers linked to pepper leaf yellowing genes and SNP molecular markers related to pepper leaf yellow-green traits have been reported, which are used to screen and identify pepper plants with yellow-green leaves. So far, there are few research reports on molecular markers related to the occurrence of pepper mosaic (showing irregular white chlorosis) and genes related to mosaic chlorosis. In order to reduce the occurrence of pepper mosaic and screen out pepper plants with normal leaves in advance, more molecular markers need to be developed for the breeding of excellent pepper germplasm resources, providing technical support and more means for pepper germplasm identification and the breeding of healthy strains. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing pepper breeding, which cannot screen out pepper variegated leaves in advance and avoid the occurrence of mosaic leaves, and to provide a KASP molecular marker for identifying the occurrence of pepper mosaic leaves and its application.

[0005] The object of the present invention is to provide a KASP molecular marker for identifying the occurrence of pepper mosaic leaves.

[0006] Another object of the present invention is a specific primer set for detecting the KASP molecular marker.

[0007] Another object of the present invention is to provide the application of the KASP molecular marker for identifying the occurrence of pepper mosaic leaves and its detection primer set.

[0008] Another object of the present invention is to provide a kit for detecting different leaf types of peppers.

[0009] Another object of the present invention is to provide a method for screening or identifying the occurrence of pepper mosaic leaves.

[0010] Another object of the present invention is to provide a method for classifying pepper mosaic leaves or breeding pepper plants with normal leaves.

[0011] The above objects of the present invention are achieved by the following technical solutions:

[0012] The present invention provides a KASP molecular marker for identifying the occurrence of pepper mosaic leaves. This KASP molecular marker is located at the 122883926 bp locus on chromosome 12 of pepper. The base at this locus for normal leaves is T, and the base at this locus for mosaic leaves is A.

[0013] The present invention constructs a linkage genetic map of pepper mosaic traits based on different pepper leaf type (normal leaf, mosaic leaf) segregation populations, locates the strongest candidate gene controlling pepper mosaic traits, develops a KASP molecular marker for identifying the occurrence of pepper mosaic leaves, and conducts molecular marker-assisted breeding based on the kompetitive allele specific PCR (KASP) marker platform; provides a method for classifying pepper mosaic leaves or breeding pepper plants with normal leaves. By detecting with KASP marker primers, different pepper leaf type plants can be classified, and mosaic leaf and normal leaf plants can be identified in both parental and offspring populations. Combining with field phenotype analysis, the coincidence rate reaches 84.21%, which has important application value for pepper germplasm identification and healthy strain breeding.

[0014] The present invention provides a specific primer set for detecting and identifying KASP molecular markers for pepper mosaic occurrence. This primer set includes Primer 1: 5-’GAAGGTGACCAAGTTCATGCTACATCGCCTCGTTTTACCACAT-3’, Primer 2: 5-’GAAGGTCGGAGTCAACGGATTACATCGCCTCGTTTTACCACAA-3’, and Primer 3: 5-’TGGGAATGATTGAGGAAACTAGCA-3’.

[0015] The present invention provides the application of KASP molecular markers for identifying pepper mosaic occurrence, or their detection primer sets, in detecting different leaf types of peppers, screening or identifying pepper mosaic occurrence, pepper mosaic typing, or in breeding pepper plants with normal leaves, or in preparing a kit for detecting different leaf types of peppers.

[0016] The present invention provides a kit for detecting different leaf types of peppers, and the kit contains a specific primer set for detecting and identifying KASP molecular markers for pepper mosaic occurrence.

[0017] Preferably, the primer set includes Primer 1: 5-’GAAGGTGACCAAGTTCATGCTACATCGCCTCGTTTTACCACAT-3’, Primer 2: 5-’GAAGGTCGGAGTCAACGGATTACATCGCCTCGTTTTACCACAA-3’, and Primer 3: 5-’TGGGAATGATTGAGGAAACTAGCA-3’.

[0018] Preferably, the kit further contains reagents for extracting DNA from the sample to be tested.

[0019] The present invention provides a method for screening or identifying pepper mosaic occurrence. Detect the 122883926bp site on chromosome 12 of the plant to be tested. Plants with base A at this site have pepper mosaic, and plants with base T at this site have normal leaves.

[0020] The present invention also provides a method for pepper mosaic typing or breeding pepper plants with normal leaves, which includes the following steps:

[0021] S1. Extract DNA from the sample to be tested;

[0022] S2. Using the sample extracted in step S1 as a template, perform qPCR detection with a specific primer set for detecting and identifying KASP molecular markers for pepper mosaic occurrence.

[0023] S3. Analyze the test results in step S2 with qPCR genotyping software; if the genotyping result is close to the abscissa, mark it as X; if the genotyping result is in the middle position between the abscissa and the ordinate, mark it as XY; if the genotyping result is close to the ordinate, mark it as Y.

[0024] Further, among the identification results in step S3, those with the result of Y are pepper mosaic plants, and those with the results of XY and X are pepper normal leaf plants.

[0025] Preferably, the qPCR detection system is: 5 μL of 2×PARMS Pro SNP Gentyping PCR Mix, 0.075 μL of F-Primer1 (primer 1), 0.075 μL of F-Primer 2 (primer 2), 0.2 μL of R-Primer 3 (primer 3), 2 μL of DNA, and ddH2O to 5 μL.

[0026] Preferably, the qPCR reaction program is: 94°C for 15 min; 94°C for 20 s, 78°C for 10 s, 62°C for 1 min; cycle 10 times; 94°C for 20 s, 57°C for 1 min; cycle 30 times; 25°C for 1 min.

[0027] More preferably, in step S3, use the built-in genotyping software BioRadCFXmanager of qPCR for genotyping and view the results.

[0028] The present invention has the following beneficial effects:

[0029] The present invention provides a KASP molecular marker for identifying pepper mosaic and its application. Based on different pepper leaf type (normal leaf, mosaic leaf) segregating populations, a linkage genetic map of pepper mosaic traits is constructed, the strongest candidate gene controlling pepper mosaic traits is located, and a KASP molecular marker for identifying pepper mosaic and its primers are obtained; using this marker and primers can identify the occurrence of pepper mosaic in pepper germplasm in advance, screen healthy pepper plants with normal leaves, and can genotype different pepper leaf types of the selected pepper germplasm; at the same time, the present invention provides a method for typing pepper mosaic or selecting pepper plants with normal leaves. Different leaf type pepper plants can be identified and distinguished through conventional laboratory detection and amplification. The method is convenient, easy to operate, can be used in advance for the screening and identification of pepper mosaic occurrence, shorten the breeding screening time, and also provide more excellent pepper germplasm resources, providing technical support for pepper germplasm identification and strain breeding. Description of the Drawings

[0030] Figure 1 It is the leaf morphology genetic map of the 59×Z81 hybrid combination (a is the hybridization map of the normal leaf "59" (female parent) and the mosaic leaf "Z81" (male parent), b is the hybridization phenotype map of different leaf type peppers).

[0031] Figure 2 Distribution map of Gvalue in the genome (a is the distribution map of Δ(SNP-index), b is the linkage candidate interval of the mosaic trait on chromosome 12).

[0032] Figure 3 Genetic linkage map of pepper leaf color mutation.

[0033] Figure 4 Genotyping results of the ILITYHIA gene in parental and F1 (a) and F2 (b) populations. Specific implementation manners

[0034] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0036] The parental "59" (female parent P1) and "Z81" (male parent P2) and the progeny F1 and F2 pepper materials used in the present invention are all planted in South China Agricultural University.

[0037] Example 1 Phenotypic analysis of pepper mosaic and construction of genetic linkage map

[0038] 1. Phenotypic analysis

[0039] Through field statistics, it was found that the pepper leaves of the F1 generation plants obtained by bagging and crossing the normal-leaved "59" (female parent) and mosaic-leaved "Z81" (male parent) peppers all showed normal leaves, indicating that normal leaves of peppers are dominant traits ( Figure 1 a). In the F2 generation segregation population (466 plants) obtained by selfing the F1 generation plants in bags, 362 pepper leaves showed normal leaves and 104 pepper leaves showed mosaic leaves ( Figure 1 b). After chi-square test, it was found that the segregation ratio basically followed normal leaves: mosaic leaves = 3:1, and the P value was 0.32 > 0.05, which conformed to the genetic law controlled by a single gene.

[0040] 2. Genome resequencing

[0041] Genome resequencing was performed on two parental pools of pepper No. 59 (sequencing completed) and pepper Z81, as well as two extremes of the F2 generation with "mottled leaves" and "normal leaves". The sequencing depth of the parents was 20x, and the sequencing depth of the extreme pooled F2 generation was 50x (about 10% of the F2 generation population size). Based on the MGI-seq 2000 high-throughput sequencing platform (produced by BGI Genomics Co., Ltd., Shenzhen), a total of 392.79 Gb of data was generated, with an average Q20 of 97.1% and an average Q30 of 90.6%. The GC content was normal, as shown in Table 1 below, indicating that the amount of data obtained by sequencing was sufficient and the data quality was very high, which could be used for subsequent experimental analysis.

[0042] Table 1 Summary of Genome Resequencing Data

[0043]

[0044] 3. Initial Mapping Results of BSA-seq for Pepper Leaf Color

[0045] Using the "CA59" pepper genome of this research group as the reference genome, the resequencing data of the two parents and the two extreme leaf color pooled genomes in the F2 generation were used to detect the single nucleotide polymorphism (SNP) and short fragment insertion-deletion (InDel) variation information present. The SNP-index value and Gprime value were calculated using the high-quality SNPs of the pooled "mottled leaves" and "normal leaves" of pepper leaves. Subsequently, the Δ(SNP-index) value was obtained by calculating the difference, and the Δ(SNP-index) distribution map and the distribution map of Gprime on the chromosome were drawn based on the results. According to Δ(SNP-index), with a 95% confidence level as the screening threshold, it was found that there was an obvious peak on chromosome 12 ( Figure 2 a); located at the position of 7.29 Mb - 256.84 Mb, with a length of 251.2 Mb. This peak was used as the candidate interval for the gene controlling leaf color mutation in this population for subsequent research.

[0046] 4. Linkage Genetic Map of Pepper Mottled Leaf Trait

[0047] According to the initial mapping results of the above pepper mottled leaf gene, the linkage candidate interval of the mottled leaf trait was at the position of 7.29 Mb - 256.84 Mb (251.2 Mb) on chromosome 12 ( Figure 2 b). To further narrow down the candidate interval, the DNA of P1, P2, F1 generations and 466 F2 generation plants was used as templates for molecular marker development, and according to the genotyping of polymorphic InDels in the F2 generation population, the QTL IicMapping software was used to construct its linkage genetic map.

[0048] Twenty-three polymorphic markers were screened in the candidate interval of chromosome 12 for the construction of a linkage genetic map. The results showed that the interval linked to the pepper mosaic trait was between KASP 2 and KASP 3( Figure 3 ), the physical positions of the markers were 122,883,926 bp - 122,949,130 bp, the physical distance was 65.204 Kb, the genetic distance was 1.21 cM, the LOD value was 40.15, and the contribution rate was 29.55%.

[0049] Example 2 Analysis of candidate genes within the genetic candidate interval for pepper mosaic

[0050] 1. Sequence analysis and functional annotation of candidate genes

[0051] Seven candidate genes were located in the screened candidate interval of Example 1 using Diamond and BLAST software, and the CDS sequences of these seven candidate genes were aligned to the NT database, NR database, and Swissprot database for functional annotation. The results showed that all seven genes in the candidate interval were annotated, as shown in Table 2. By searching for genes related to pepper mosaic, it was found that within the interval mapped in the present invention, there was an ILITYHIA (Capann_ca59V2g38035), which might be an important candidate gene for the pepper mosaic trait in this population.

[0052] Table 2 Gene functional annotation within the pepper mosaic candidate interval

[0053]

[0054] 2. Expression analysis of candidate genes

[0055] To determine the strongest candidate gene controlling the pepper mosaic trait, the CDS sequences of all candidate genes within the candidate interval were extracted, specific primers were set respectively, and the expression levels of different candidate genes in the two parents were detected. Using the ubiquitin extension protein gene CA12g20490 of pepper as an internal reference gene, fluorescence quantitative PCR was performed using ChamQUniversal SYBR qPCR Master Mix from Nanjing Novoprotein Biotechnology Co., Ltd.

[0056] Through the fluorescence quantitative PCR experiment, it was found that only the expression levels of ILITYHIA (Capann_ca59V2g38035) in the two parents were significantly different, and it was speculated that ILITYHIA was the key candidate gene controlling the pepper mosaic trait in this population.

[0057] Example 3 Development and verification of KASP molecular markers for identifying the occurrence of pepper mosaic

[0058] The analysis of Example 2 shows that the ILITYHIA gene is considered to be the key gene controlling the mosaic trait of pepper in this population, located at 122862093bp-122885329bp on chromosome 12. According to the resequencing of the parental genome, there are 5 SNPs in the full length of the ILITYHIA gene. Through the genome sequence and mutation information, the sequence of 200bp before and after the mutation site was extracted, and the sequence was submitted to SNPWay (http: / / www.snpway.com / ) to design primers, and BLAST was performed on SolGenomics Network (https: / / solgenomics.net / tools / blast / ) to detect the uniqueness of the sequence in the pepper genome. It was found that only the 122883926 site KASP2 was unique.

[0059] Subsequently, by designing and synthesizing primers for the site (Table 3), fluorescence quantitative PCR was performed in the parents and F1 to detect its polymorphism; the fluorescence quantitative PCR reaction system is shown in Table 4 below, and the fluorescence quantitative PCR reaction program is: 94°C, 15 min; 94°C, 20 s; 78°C, 10 s; 62°C, 1 min; 10 cycles; 94°C, 20 s; 57°C, 1 min; 30 cycles; 25°C, 1 min; and typing analysis was performed using the BioRad CFXmanager software that comes with the fluorescence quantitative PCR instrument.

[0060] Table 3 Primer information of KASP marker

[0061]

[0062] Table 4 Fluorescence quantitative PCR reaction system

[0063]

[0064] Using BioRad CFXmanager software to check the fluorescence quantitative typing results, it was found that the KASP2 primers were polymorphic in both parents and the F1 generation. Figure 4 As shown in a, Z81 (P2) is clustered with blue squares (Allele 2) near the Y axis, and the pepper shows mosaic leaves; F1 is clustered with green triangles (Heterozygote) near the diagonal of the X and Y axes (in the middle of the horizontal and vertical axes), and the pepper shows normal leaves; No. 59 (P1) is clustered with orange dots (Allele 1) near the X axis, and the pepper shows normal leaves, which can be used for subsequent typing detection of the F2 generation population.

[0065] Further typing analysis was performed on 384 F2 strains using the same method as above. The results are as follows: Figure 4As shown in b, it was found that 82 F2 individual plants had the same genotype as P1; 93 had the same genotype as P2; and 197 had the same genotype as F1. Combining with the field phenotypes, among the 384 F2 populations, 380 plants had known phenotypes, and 320 plants had consistent genotypes and phenotypes, with a consistency rate of 84.21%. It can be used for detecting, screening, and identifying pepper mosaic, typing pepper mosaic and plants with different leaf types, and obtaining excellent pepper germplasm resources.

[0066] In summary, the present invention constructs a linkage genetic map of pepper mosaic traits based on different pepper leaf type (normal leaf, mosaic leaf) segregation populations, locates the strongest candidate genes controlling pepper mosaic traits, develops KASP molecular markers for identifying the occurrence of pepper mosaic, and provides a method for pepper molecular marker-assisted breeding to type pepper mosaic or select pepper plants with normal leaves; detecting with KASP markers and primers can type plants with different pepper leaf types, and can identify pepper mosaic and normal leaf plants in both parental and offspring populations. Combining with field phenotype analysis, the consistency rate reaches 84.21%, which can screen and identify the occurrence of pepper mosaic in advance, shorten the breeding screening time, and at the same time provide more excellent pepper germplasm resources, providing technical support for pepper germplasm identification and strain selection.

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A KASP molecular marker for identifying pepper mosaic, characterized in that, The KASP molecular marker is located at the 122883926 bp locus on chromosome 12 of pepper. The base at this locus is T in normal leaves and A in variegated leaves.

2. A specific primer set for detecting the KASP molecular marker according to claim 1, characterized in that, Containing primer 1: 5-'GAAGGTGACCAAGTTCATGCTACATCGCCTCGTTTTACCACAT-3', primer 2: 5-'GAAGGTCGGAGTCAACGGATTACATCGCCTCGTTTTACCACAA-3'.

3. The primer set according to claim 2, characterized in that, The primer set further includes: primer 3: 5-'TGGGAATGATTGAGGAAACTAGCA-3'.

4. The application of the KASP molecular marker according to claim 1, or the primer set according to claim 2 or 3, in detecting different leaf types of pepper, screening or identifying pepper mosaic, and pepper mosaic typing.

5. The application of the KASP molecular marker according to claim 1, or the primer set according to claim 2 or 3, in breeding pepper plants with normal leaves.

6. The application of the KASP molecular marker according to claim 1, or the primer set according to claim 2 or 3, in preparing a kit for detecting different leaf types of pepper.

7. A kit for detecting different leaf types of pepper, characterized in that, The kit contains the specific primer set described in claim 2 or 3.

8. A method for screening or identifying pepper mosaic, characterized in that, Detect the 122883926 bp locus on chromosome 12 of the plant to be tested. The plant with base A at this locus is variegated leaf pepper, and the plant with base T at this locus is normal leaf pepper.

9. A method for pepper mosaic typing or breeding pepper plants with normal leaves, characterized in that, Including the following steps: S1. Extract the DNA of the sample to be tested; S2. Using the sample extracted in step S1 as a template, perform qPCR detection with the specific primer set described in claim 2 or 3; S3. Analyze the detection results of step S2 with qPCR genotyping software; if the genotyping result is close to the abscissa, mark it as X; If the genotyping result is in the middle position between the abscissa and the ordinate, mark it as XY; If the genotyping result is close to the ordinate, mark it as Y.

10. The method according to claim 9, characterized in that, In step S3, the plant identified as Y is variegated leaf pepper plant, and the plants identified as XY and X are normal leaf pepper plants.

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