A dCAPS marker for red trait in late development of Chinese pepper fruit and its application

Abstract

The present invention provides a new SNP site associated with the color of Chinese pepper fruit in the late development stage, which is located at position 239864112 on chromosome 6 (reference genome version: ASM227189v2); for the above SNP site, the present invention also provides a corresponding dCAPS molecular marker, a specific primer and a method for identifying the fruit color of Chinese pepper in the late development stage at the seedling stage by using the dCAPS molecular marker. The method is based on the characteristics of the SNP site and the application of the mismatch base enzyme cleavage site, combined with PCR amplification and enzyme cleavage methods, greatly improving the identification efficiency of the fruit color of Chinese pepper. The development of the marker is of great significance to the breeding of Chinese peppers with different fruit colors.

Description

Technical Field

[0001] The invention relates to the fields of pepper genetic breeding and molecular biology, and in particular to a dCAPS marker for red traits in the late development stage of Chinese pepper fruits and an application thereof. Background Art

[0002] Chinese pepper (Capsicum chinense) is one of the five cultivated species of the genus Capsicum. China has rich pepper germplasm resources, and some resources have fruits with prominent fragrance and strong spicy taste, and the fruits are red in the late development stage. The color of pepper fruit is an important breeding goal. Genetic studies have shown that when yellow pepper fruits are hybridized with red pepper fruits, the F1 representative is red, indicating that red is a dominant trait and yellow is a recessive trait. Therefore, through traditional phenotypic identification methods, plants with red pepper fruits may have red and yellow pepper fruits in their offspring, which makes the color of pepper fruits unstable in the late development stage, profoundly affecting breeding efficiency.

[0003] Molecular marker breeding is an important means of breeding. By developing molecular markers that are closely linked to the target traits, stable, and easy to detect, the genotype can be directly selected, and the target traits can be selected in the early stages of plant growth and development. At the same time, co-dominant molecular markers can also distinguish between homozygous and heterozygous genotypes, which can greatly improve the accuracy and efficiency of selection in breeding and significantly shorten the breeding cycle. Therefore, in the process of breeding new varieties with Chinese pepper as one of the parents, it is very urgent to identify whether the plant to be tested contains the fruit red gene in Chinese pepper in the early stages of plant growth and development, and at the same time determine whether it is a homozygous genotype or a heterozygous genotype. Molecular marker breeding is very urgent. Summary of the invention

[0004] The first aspect of the present invention aims to provide a SNP site associated with the color of Chinese pepper fruit in the late development stage, wherein the SNP site is located at position 239864112 on chromosome 6 of Chinese pepper (reference genome version: ASM227189v2).

[0005] The second aspect of the present invention aims to provide an application of the SNP site in identifying or screening the color of Chinese pepper fruits in the late development stage.

[0006] Specifically, the present invention determines whether the Chinese pepper is red in the late stage of development according to the genotype of the SNP site.

[0007] In one or more embodiments, if the genotype of the SNP site is CC, the fruit color of the tested Chinese pepper in the late development stage is red.

[0008] In one or more embodiments, if the genotype of the SNP site is CH (H is one of the bases T, A, and G), the fruit color of the Chinese pepper to be tested in the late stage of development is red.

[0009] In one or more embodiments, if the genotype of the SNP site is HH (H is one of the bases T, A, and G), the fruit color of the Chinese pepper to be tested in the late stage of development is non-red.

[0010] The third aspect of the present invention aims to provide a specific dCAPS molecular marker associated with the color of Chinese pepper fruit in the late development stage, the nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1, position 239864112 of the sequence (reference genome version: ASM227189v2).

[0011] The fourth aspect of the present invention aims to provide a primer pair for detecting the specific dCAPS molecular marker of the present invention, wherein the primer pair consists of a primer F and a primer R;

[0012] The primer F is as follows a1) or a2):

[0013] a1) the sequence shown in SEQ ID NO: 2;

[0014] a2) a single-stranded DNA molecule having the same function as the sequence shown in SEQ ID NO: 2 after one or more nucleotides are substituted and / or deleted and / or added;

[0015] The primer R is as follows b1) or b2):

[0016] b1) the sequence shown in SEQ ID NO: 3;

[0017] b2) A single-stranded DNA molecule having the same function as the sequence shown in SEQ ID NO: 3 after one or more nucleotides are substituted and / or deleted and / or added.

[0018] In one or more embodiments, the primer pair consists of a primer F and a primer R, wherein the primer F is a1), and the primer R is b1).

[0019] In one or more embodiments, the primer pair consists of a primer F and a primer R, wherein the primer F is a1), and the primer R is b2).

[0020] In one or more embodiments, the primer pair consists of a primer F and a primer R, wherein the primer F is a2) and the primer R is b1).

[0021] In one or more embodiments, the primer pair consists of primer F and primer R, wherein primer F is a2) and primer R is b2).

[0022] It is well known to those skilled in the art that the design of primers must comply with certain principles, such as controlling the length of the primers, the GC content of the primers, the Tm value of the primers, the number of mismatched bases, etc., and can be designed using conventional software in the art, or can be designed directly on the relevant website. As long as the primers comply with the above design principles, they may be used for the detection of the present application. Preferably, the primer pair described in the present application includes the sequence shown in SEQ ID NO:2 and the sequence shown in SEQ ID NO:3.

[0023] The fifth aspect of the present invention aims to provide the use of the specific dCAPS molecular marker in identifying or screening whether Chinese pepper fruits are red or non-red in the late development stage.

[0024] The sixth aspect of the present invention aims to provide a method for identifying or screening whether Chinese pepper fruits are red or non-red in the late development stage based on dCAPS molecular marker technology, comprising the following steps:

[0025] S1. Extract genomic DNA from pepper parents and offspring;

[0026] S2. performing PCR amplification on the extracted genomic DNA using the primer pair of the present invention;

[0027] S3. The amplified PCR product was digested with EcoRI and then subjected to electrophoresis;

[0028] S4. Result judgment: If the enzyme cleavage product electrophoresis obtains an electrophoresis band of 100bp, the genotype of the SNP site of the Chinese pepper sample is HH (H is one of the bases T, A, and G), and the fruit of the sample is non-red in the late stage of development; if the enzyme cleavage product electrophoresis obtains an electrophoresis band of 79bp, the genotype of the pepper sample is CC, and the fruit of the sample is red in the late stage of development; if the enzyme cleavage product electrophoresis obtains electrophoresis bands of 100bp and 79bp, the genotype of the SNP site of the pepper sample is CH (H is one of the bases T, A, and G), and the fruit of the sample is red in the late stage of development.

[0029] In one or more embodiments, the PCR amplification system (25 μl) in step S2 is: 5 μl genomic DNA, 12.5 μl 2x Rapid Taq Master Mix, 2 μl 10 μM upstream primer, 2 μl 10 μM downstream primer, and ddH2O to make up to 25 μl.

[0030] In one or more embodiments, the conditions for PCR amplification in step S2 are:

[0031] Step 1: pre-denaturation at 94°C for 5 min; Step 2: denaturation at 94°C for 30 s;

[0032] Step 3: annealing at 55℃ for 30s; Step 4: extension at 72℃ for 60s;

[0033] Step 5: Repeat Step 2-4 for 35 cycles; Step 6: Extend at 72°C for 7 min.

[0034] In one or more embodiments, the enzyme digestion system (25 μl) in step S3 is: 1 μl of restriction endonuclease, 5 μl of restriction endonuclease buffer, 10 μl of PCR product, and ddH2O to make up to 25 μl.

[0035] In one or more embodiments, the conditions for the enzyme cleavage in step S3 are: Step 1: incubate at 37° C. for 15 min; Step 2: inactivate at 65° C. for 20 min.

[0036] The seventh aspect of the present invention aims to provide a product for identifying or screening whether Chinese pepper fruits are red or non-red in the late development stage, the product comprising any of the above primer pairs and EcoRI enzyme, preferably, the product also comprises a DNA extraction reagent.

[0037] In one or more embodiments, the method for using the product for identifying or screening Chinese peppers for red or non-red varieties in the late stage of fruit development of the present invention is as follows: extracting the genome of the pepper to be tested by the DNA extraction reagent provided by the product, then performing PCR amplification using the primer pair provided by the product, digesting the amplified product with the EcoRI enzyme provided by the product and then performing electrophoresis, and judging the fruit color of the Chinese pepper according to the electrophoresis band; if the electrophoresis of the digestion product obtains an electrophoresis band of 100bp, the genotype of the SNP site of the pepper sample is HH (H is one of the bases T, A, and G), and the fruit of the sample is non-red in the late stage of fruit development; if the electrophoresis of the digestion product obtains an electrophoresis band of 79bp, the genotype of the pepper sample is CC, and the sample is a red variety; if the electrophoresis of the digestion product obtains electrophoresis bands of 100bp and 79bp, the genotype of the SNP site of the pepper sample is CH (H is one of the bases T, A, and G), and the sample is a red variety.

[0038] The present invention provides a new SNP site related to the red fruit trait in the late development stage of Chinese pepper, which is located at position 239864112 on chromosome 6, and has a polymorphism of C / H (H is one of the bases T, A, and G). By identifying the site in the seedling stage, it is possible to accurately identify whether an unknown Chinese pepper sample is red, and batch identification can be performed, which greatly improves the identification efficiency and accuracy.

[0039] For the above-mentioned SNP sites, the present invention also provides corresponding dCAPS molecular markers, specific primers, and a method for quickly identifying the fruit color of Chinese peppers in the late development stage using dCAPS molecular markers in the seedling stage. The method is based on the characteristics of the SNP site and the application of the mismatch base enzyme cleavage site, combined with PCR amplification and enzyme cleavage methods, which greatly improves the identification efficiency of the fruit color of Chinese peppers. The development of this marker is of great significance to the breeding of different fruit colors of Chinese peppers. The dCAPS marker of the red fruit trait of the late development of Chinese peppers described in the present invention can be used for molecular marker-assisted breeding, greatly shortening the breeding cycle, and can also identify whether the Chinese pepper sample to be tested is a homozygous genotype or a heterozygous genotype, which has important theoretical and practical significance for the breeding of new varieties of Chinese pepper fruit colors. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Illustration of the design of 100bp sequences upstream and downstream of the SNP and CCS_dCAPS primers.

[0041] Figure 2 Validation diagram of the developed dCAPS molecular markers in natural populations and progeny.

[0042] M is D2000 Marker; double bands represent heterozygous genotypes, and single band represents homozygous genotypes. DETAILED DESCRIPTION

[0043] The following is a detailed description of the present invention. The following description of the technical features is based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted that:

[0044] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0045] In the present specification, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0046] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0047] In this specification, the use of "optional" or "optional" indicates that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0048] In this specification, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used indicates weight or mass percentage.

[0049] In this specification, unless otherwise stated, "plurality" means that there are two or more.

[0050] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0053] Example 1 Obtaining SNP loci associated with fruit color in late development of Chinese pepper

[0054] Using Chinese peppers with red fruits in the late stage of fruit development and Chinese peppers with yellow fruits in the late stage of fruit development as materials, based on the simplified genome sequencing (GBS) technology, GATK and other software were used to detect population SNPs:

[0055] (1) Select reads that map to unique positions on the genome for subsequent analysis;

[0056] (2) GATK (-type UnifiedGenotyper) was used to detect population SNPs in the filtered bam files;

[0057] (3) To reduce false positive SNPs caused by sequencing errors, the SNP base support number for parents and offspring is required to be no less than 4.

[0058] (4) Statistical SNP-related information: number of heterozygous SNPs, number of homozygous SNPs, and heterozygous SNP ratio.

[0059] By analyzing the genotype test results of the two parents, polymorphic markers between the parents were developed. The sites that were homozygous for both parents and polymorphic between the parents were screened. Through the F2 segregation population, a significant bin marker related to the red fruit trait in the late development of Chinese pepper was obtained. The CCS gene was discovered by annotating and analyzing the 1Mb upstream and downstream of the obtained marker, and a non-synonymous mutation SNP was found in the gene. The SNP site was at position 239864112 on chromosome 6 of Chinese pepper (reference genome version: ASM227189v2), the base of the red fruit sample was C, and the base of the yellow fruit sample was A.

[0060] Example 2 Development of dCAPS molecular markers related to the color of Chinese pepper fruit in late development (named CCS_dCAPS in the present invention)

[0061] 1. Primer design:

[0062] The dCAPS primers were designed according to the SNP sites obtained in Example 1, and the specific steps were as follows:

[0063] Using the online software dCAPS Finder 2.0, the SNP mutation site was selected, the mismatch base (TC→AA) was introduced, and the appropriate restriction endonuclease (EcoRI) was selected to design one of the primers; the other primer was designed using Snapgene viewer, the primer was designed to be 20-25bp, and the GC content was 30-60%; the annealing temperature was 55-60°C, and the annealing temperature and length of the upstream and downstream primers were kept consistent as much as possible.

[0064] The final primer sequences are as follows:

[0065]

[0066] like Figure 1 The figure shows the upstream and downstream 100 bp sequences of the SNP of the present invention and the CCS_dCAPS primer design diagram, wherein [C / A] is the SNP mutation site, the arrows underline the upstream and downstream primer positions, and the arrows are the restriction sites.

[0067] The genome of Chinese peppers with red or yellow fruits in the late stage of fruit development was used as a template and PCR amplification was performed using the above primers. The resulting product was 100 bp, and its sequence is shown in SEQ ID NO: 1 in the sequence listing. The 101st base of the sequence is C / A, which is a polymorphic site.

[0068] 2. Extraction of sample genomic DNA

[0069] The genomic DNA was extracted from the young leaves of pepper samples in the parent or F2 segregation population in Example 1 using a DNA extraction kit ( Plant DNA Isolation Mini Kit DC104) (Novizan Biotech Co., Ltd., Nanjing):

[0070] (1) Take 100 mg of fresh plant tissue and grind the plant tissue using a grinder;

[0071] (2) Immediately add 400 μl buffer A1 and 4 μl RNase A to the ground sample and vortex to fully lyse.

[0072] (3) 65°C water bath for 10 min. During the water bath, invert the centrifuge tube 2-3 times to mix the sample.

[0073] (4) Add 130 μl of Buffer A2 to the mixture, mix thoroughly, place on ice for 5 min, and then centrifuge at 14,000 rpm (18,400 × g) for 10 min. Pipette the supernatant into a new centrifuge tube.

[0074] (5) Take 1.5 times the volume of the supernatant, Buffer A3, and mix immediately by pipetting;

[0075] (6) The mixture in (5) was transferred to an adsorption column, centrifuged at 12000 rpm (13400 × g) for 1 min, and the filtrate was discarded;

[0076] (7) Add 600 μl of Buffer AW, centrifuge at 12000 rpm (13400 × g) for 1 min, discard the filtrate, and repeat the process;

[0077] (8) Place the adsorption column in a new 1.5 ml centrifuge tube, add 50 μl of preheated Elution Buffer to the center of the adsorption column, leave at room temperature for 5 min, centrifuge at 12000 rpm (13400 × g) for 1 min, discard the adsorption column, and store at -20°C for future use.

[0078] 3. PCR amplification

[0079] Using the genomic DNA obtained above as a template, the following amplification system was constructed:

[0080] (1) The PCR amplification system is (25 μl):

[0081] 2x Rapid Taq Master Mix: 12.5μl

[0082] Upstream primer: 2 μl (concentration 10 μM)

[0083] Upstream primer: 2 μl (concentration 10 μM)

[0084] Genomic DNA: 5 μl

[0085] ddH2O: make up to 25μl.

[0086] (2) The PCR amplification procedure is as follows:

[0087] Step 1: Pre-denaturation at 94°C for 5 min;

[0088] Step 2: denaturation at 94°C for 30 seconds;

[0089] Step 3: anneal at 55℃ for 30s;

[0090] Step 4: 72℃ extension for 60s;

[0091] Step 5: Repeat Step 2-4 for 35 cycles;

[0092] Step 6: Extend at 72℃ for 7 minutes.

[0093] 4. Enzyme Digestion

[0094] The PCR product obtained in the above step was digested with restriction endonuclease EcoRI (NEB)

[0095] (1) Enzyme digestion system (25 μl) is as follows:

[0096] Restriction enzyme: 1 μl

[0097] Restriction enzyme buffer: 5 μl

[0098] PCR product: 10 μl

[0099] ddH2O: make up to 25 μl

[0100] (2) Enzyme digestion reaction conditions are as follows:

[0101] Step 1: Incubate at 37°C for 15 min;

[0102] Step 2: Inactivate at 65℃ for 20 minutes.

[0103] 5. Electrophoresis

[0104] The above enzyme cleavage products were subjected to agarose gel electrophoresis and photographed using a gel imager. The electrophoresis conditions were: 3.5% agarose gel, voltage: 150 V, time: 35 min.

[0105] 6. Result judgment: If the enzyme cleavage product electrophoresis obtains an electrophoresis band of 100bp, the genotype of the SNP site of the Chinese pepper sample is AA, and the fruit of the sample is yellow in the late stage of development; if the enzyme cleavage product electrophoresis obtains an electrophoresis band of 79bp, the genotype of the Chinese pepper sample is CC, and the fruit of the sample is red in the late stage of development; if the enzyme cleavage product electrophoresis obtains electrophoresis bands of 100bp and 79bp, the genotype of the SNP site of the Chinese pepper sample is CA, and the fruit of the sample is red in the late stage of development.

[0106] Example 3 Application of dCAPS molecular marker (named CCS_dCAPS in the present invention)

[0107] Hainan yellow lantern (Capsicumchinense), a local variety collected from Lingshui, Hainan, with yellow fruit in the late stage of development, is used as the male parent, numbered 'HNUCC16', and Chinese pepper, which is red in the late stage of development, is collected from the College of Horticulture of Hainan University and numbered 'HNUCC22' as the female parent. The F1 generation is obtained by hybridization, and then the F2 generation population is obtained by self-pollination. The present invention randomly selects 39 pepper seedlings from 200 F2 generation populations as samples to be tested, and uses the method described in the present invention to identify or predict the color of the late stage of fruit development of each sample; then the pepper F2 generation population seedlings are transplanted to the test field, and watering and fertilization are carried out during the period to ensure that each sample grows normally until mature fruits grow; the color of the late stage of fruit development of each sample identified by the method described in the present invention at the seedling stage is compared with the actual color of the late stage of fruit development of Chinese pepper, and the accuracy of the detection method described in the present invention is judged.

[0108] 1. Select young leaves of each sample at the seedling stage for genomic DNA extraction

[0109] 2. Using the genomic DNA obtained above as a template, perform PCR amplification respectively:

[0110] The PCR amplification system was (25 μl): 2x Rapid Taq Master Mix 12.5 μl, upstream primer (SEQ ID NO: 2) 2 μl (concentration 10 μM), downstream primer (SEQ ID NO: 3) 2 μl (concentration 10 μM), genomic DNA 5 μl, and ddH2O was added to make up to 25 μl.

[0111] The PCR amplification procedure is:

[0112] Step 1: pre-denaturation at 94°C for 5 min; Step 2: denaturation at 94°C for 30 s;

[0113] Step 3: annealing at 55℃ for 30s; Step 4: extension at 72℃ for 60s;

[0114] Step 5: Repeat Step 2-4 for 35 cycles; Step 6: Extend at 72°C for 7 min.

[0115] 3. The amplified products obtained above were digested with restriction endonuclease EcoRI (NEB):

[0116] The enzyme digestion system (25 μl) is as follows: 1 μl restriction endonuclease, 5 μl restriction endonuclease buffer, 10 μl PCR product, and ddH2O to

[0117] The enzyme digestion reaction conditions are as follows: Step 1: incubate at 37°C for 15 min; Step 2: inactivate at 65°C for 20 min.

[0118] 4. The enzyme-digested products were subjected to agarose gel electrophoresis and photographed using a gel imager. The electrophoresis conditions were: 3.5% agarose gel, voltage: 150 V, time: 35 min.

[0119] 5. Identification or prediction is performed according to the following standards: if the enzyme cleavage product electrophoresis obtains an electrophoresis band of 100bp, the genotype of the SNP site of the Chinese pepper sample is AA, and the fruit of the sample is yellow in the late stage of development; if the enzyme cleavage product electrophoresis obtains an electrophoresis band of 79bp, the genotype of the Chinese pepper sample is CC, and the fruit of the sample is red in the late stage of development; if the enzyme cleavage product electrophoresis obtains electrophoresis bands of 100bp and 79bp, the genotype of the SNP site of the Chinese pepper sample is CA, and the fruit of the sample is red in the late stage of development.

[0120] 6. The color of the late fruit development of each sample identified or predicted by the above method was compared with the actual color of the late fruit development of each sample grown in the field. The results are as follows:

[0121]

[0122]

[0123] The above test shows that the result of identifying or predicting the color of the late fruit development of Chinese pepper at the seedling stage by the method of the present invention is completely consistent with the actual color of the late fruit development, and the accuracy rate is 100%. Therefore, the molecular marker of the present invention has strong applicability, and can be used to quickly identify and screen the color traits of the late fruit development of Chinese pepper at the seedling stage in practical breeding applications.

[0124] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. An application of a SNP locus associated with the red trait of Chinese pepper fruit in the late development period in identifying or screening the red trait of Chinese pepper fruit in the late development period, characterized in that: The SNP site is at position 239864112 on chromosome 6 of Chinese pepper, the base is C / A, and the reference genome version is ASM227189v2. It can be used to determine whether the fruit is red in the late stage of development in Chinese pepper during the seedling stage.

2. A primer pair of specific dCAPS molecular markers related to the red trait in the late development of Chinese pepper fruit developed based on the SNP locus of claim 1, characterized in that: The primer pair consists of primer F and primer R; The primer F is the sequence shown in SEQ ID NO: 2; The primer R is the sequence shown in SEQ ID NO:

3.

3. Use of the primer pair of the specific dCAPS molecular marker according to claim 2 in identifying or screening the red trait of Chinese pepper fruit in the late development stage and whether it is homozygous.

4. A method for identifying or screening red or non-red varieties of Chinese peppers in the late development stage of fruit based on dCAPS molecular marker technology, characterized in that: The following steps are involved: S1. Extract genomic DNA from Chinese pepper parent and offspring plants; S2. performing PCR amplification on the extracted genomic DNA using the primer pair according to claim 2; S3. The amplified PCR product was digested with EcoRI and then subjected to electrophoresis; S4. Result judgment: If the enzyme cleavage product electrophoresis obtains an electrophoresis band of 100bp, the genotype of the SNP site of the Chinese pepper sample is HH, H is one of the bases T, A, and G, and the fruit of the sample is non-red in the late stage of development; if the enzyme cleavage product electrophoresis obtains an electrophoresis band of 79bp, the genotype of the pepper sample is CC, and the fruit of the sample is red in the late stage of development; if the enzyme cleavage product electrophoresis obtains electrophoresis bands of 100bp and 79bp, the genotype of the SNP site of the pepper sample is CH, H is one of the bases T, A, and G, and the fruit of the sample is red in the late stage of development.

5. A product for identifying or screening whether Chinese pepper fruits are red varieties in the late development stage, characterized in that: The product comprises the primer pair according to claim 2 and EcoRI enzyme.

6. The product according to claim 5, characterized in that Also contains DNA extraction reagents.

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