KASP marker primer combination for detecting watermelon red flesh characters and application of KASP marker primer combination

Abstract

The invention discloses a KASP marker primer combination for detecting watermelon red flesh characters and application of the KASP marker primer combination, and belongs to the technical field of molecular biology breeding. According to the invention, a KASP labeled primer combination is designed according to an SNP15442987 site of a watermelon, and the KASP labeled primer combination comprises an upstream primer 1 of which the nucleotide sequence is shown as SEQ ID NO.1, an upstream primer 2 of which the nucleotide sequence is shown as SEQ ID NO.2 and a downstream common primer of which the nucleotide sequence is shown as SEQ ID NO.3. The primer combination is applied to detection of watermelon pulp color traits, the accuracy rate is 100%, observation can be carried out without being carried out in the watermelon fruit ripening period, operation is easy and convenient, and the workload of screening and identification through naked eyes is greatly reduced; the detection time is short, only 1-2 hours are needed, and the method is suitable for batch detection of large-group multiple samples. Meanwhile, by using the KASP marker, a material with stable characters can be obtained in only three generations, so that the breeding period is greatly shortened, and the breeding efficiency is improved.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biological breeding, and in particular to a KASP marker primer combination for detecting a red flesh trait of a watermelon and an application thereof. Background Art

[0002] Watermelon (Citrullus lanantus) is an important horticultural crop in the world. my country ranks first in the world in terms of total annual watermelon production and planting area, playing an important role in the development of my country's agricultural industry. With the improvement of people's living standards, the requirements for watermelon quality are getting higher and higher. The flesh color is an important indicator for measuring watermelon quality. At present, the common watermelon flesh color on the market is red, which is also the most popular and widely accepted flesh color by consumers.

[0003] In addition to red, there are also watermelon germplasm resources with orange, yellow, green and white flesh colors. The nutritional value of watermelons of different flesh colors is also different. For example, yellow flesh watermelons are rich in carotenoids and have antioxidant effects; orange flesh watermelons are rich in β-carotene and lutein, which have vision care effects; green flesh watermelons have the effects of promoting blood circulation and preventing anemia; white flesh watermelons are low in sugar and rich in amino acid mineral salts, which have anti-cancer and anti-aging effects. In addition to nutritional value, some other flesh-colored watermelon resources are also disease-resistant and stress-resistant. It is an important breeding goal for breeders to select functional and highly resistant red flesh watermelon varieties with different nutritional components through breeding technology.

[0004] In the research on genetic localization and marker development of the red flesh trait of watermelon, Li et al. used a hybrid population of scarlet flesh watermelon × coral red flesh watermelon to locate the scarlet flesh trait within the 40kb interval of chromosome 6, developed two InDel molecular markers based on sequence variation in the localized interval, and verified the markers in F2, BC1P2 populations and 56 scarlet flesh watermelon samples (Li et al., Discovery of the genomic region and candidate genes of the scarlet red flesh color (Yscr) locus in watermelon (Citrullus lanatus L.). Frontiers in Plant Science, 2020, 11: article 116). This study developed molecular markers based on specific population materials and has material specificity. Red-fleshed watermelons are rich in lycopene. Fall et al. located the QTL for the lycopene content of red-fleshed watermelons and pink-fleshed watermelons, hoping to find sites associated with flesh color through differences in lycopene content, but no stable QTL sites were found (Fall et al., QTLs associated with flesh quality traits in an elite×elite watermelon population. Euphytica, 2019, 215:30). In summary, the current identification of the red flesh trait of watermelons mainly relies on observations of the fruit maturity period. The existing markers related to flesh color are population-specific and have limitations in the identification of different hybrid populations or natural watermelon population materials. Therefore, it is necessary to provide a molecular identification technology for the red flesh trait of watermelons that can be quickly identified and has universality. Summary of the invention

[0005] The purpose of the present invention is to provide a KASP marker primer combination for detecting the red flesh trait of watermelon and an application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention designs a KASP marker primer combination according to the watermelon SNP_15442987 site, which is applied to identify the color of watermelon flesh with an accuracy rate of 100%. The operation is simple and the detection time is short, which greatly reduces the workload of screening and identification by naked eye.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a KASP marker primer combination for detecting the red flesh trait of watermelon, comprising an upstream primer 1 with a nucleotide sequence as shown in SEQ ID NO.1, an upstream primer 2 as shown in SEQ ID NO.2, and a downstream common primer as shown in SEQ ID NO.3;

[0008] The SNP site detected by the KASP marker primer combination is located at the 15442987th base of chromosome 4 of watermelon, and the upstream and downstream sequences are shown in SEQ ID NO.4. There is a C / A mutation at the 501st base of the sequence.

[0009] Preferably, the molar ratio of the upstream primer 1, the upstream primer 2 and the downstream common primer is 1:1:3.

[0010] The invention provides an application of the KASP marker primer combination in preparing a product for detecting the red flesh trait of watermelon.

[0011] Preferably, the product is a kit.

[0012] The present invention also provides a kit for detecting the red flesh trait of watermelon, comprising the above-mentioned KASP marker primer combination.

[0013] The present invention also provides the use of the KASP labeled primer combination or the kit in any of the following:

[0014] (1) Identify the color characteristics of watermelon flesh;

[0015] (2) Screening high-purity parents of red-fleshed watermelon;

[0016] (3) Cultivate red-fleshed watermelon varieties.

[0017] The present invention also provides a method for identifying the color properties of watermelon flesh, comprising the following steps:

[0018] Extracting genomic DNA of the watermelon to be tested;

[0019] Using the genomic DNA as a template, the above-mentioned KASP labeled primer combination is used to perform a PCR reaction to obtain a PCR amplification product;

[0020] According to the fluorescence signal value of the PCR amplification product, the genotyping result is obtained to identify the flesh color trait of the watermelon to be tested.

[0021] Preferably, if the result of the genotyping is a homozygous genotype C:C, the flesh color trait of the watermelon to be tested is identified as red flesh;

[0022] If the result of the genotyping is the homozygous genotype A:A, the flesh color trait of the tested watermelon is identified as non-red flesh.

[0023] Optionally, the PCR reaction system includes: 2.5 μL of 2×KASPmaster mix, 1.25 μL of primer combination and 1.25 μL of genomic DNA.

[0024] Optionally, the PCR reaction program is: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20 s, annealing and extension at 61-55°C for 60 s, decreasing by 0.6°C in each cycle, for 10 cycles; denaturation at 95°C for 20 s, annealing and extension at 55°C for 60 s, for 27 cycles.

[0025] The present invention discloses the following technical effects:

[0026] The present invention uses the GWAS method to locate a significant association site SNP_15442987 that controls the red flesh trait of watermelon, and according to the variable site, a KASP molecular marker associated with the red flesh trait of watermelon is developed. The KASP marker primer combination designed by the present invention includes an upstream primer 1 with a nucleotide sequence as shown in SEQ ID NO.1, an upstream primer 2 as shown in SEQ ID NO.2, and a downstream common primer as shown in SEQ ID NO.3. The primer combination is applied to the detection of watermelon flesh color traits, with an accuracy rate of 100%, and it is not necessary to observe the watermelon fruit in the ripening period, the operation is simple, and the workload of screening and identification by naked eyes is greatly reduced; the detection time is short, only 1-2 hours are needed, and it is suitable for batch detection of large groups and multiple samples. At the same time, using the KASP marker, materials with stable traits can be obtained in only 3 generations, which greatly shortens the breeding cycle and improves the breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 Manhattan plot for genomic association analysis of watermelon red flesh traits;

[0029] Figure 2 The figure is a genotyping result diagram of the Cla_15442987 marker of the present invention in 94 natural population samples of watermelon; wherein, the blue dots represent the C:C genotype fluorescence signal corresponding to the primer Cla_15442987, which is a homozygous individual plant with red watermelon flesh color; the red dots represent the A:A genotype fluorescence signal corresponding to the primer Cla_15442987, which is a homozygous individual plant with non-red watermelon flesh color; and the black squares are negative controls. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1 Development and application of KASP molecular marker for watermelon red flesh trait

[0036] 1. Genome-wide association analysis locates the red flesh trait of watermelon

[0037] The whole genome (Genome version: Watermelon Genome v2.5) of 143 natural watermelon population materials was resequenced using Illumina sequencing technology, with an average sequencing depth of 15×. After filtering with the condition of minimum allele frequency (MAF) ≥ 5% and missing data rate ≤ 20%, a total of 2,923,709 SNPs were detected, providing rich genetic variation for watermelon breeding. The red flesh trait of 143 watermelon materials was analyzed, and the FaST-LMM (Factored Spectrally Transformed Linear Mixed Models) model was used to perform a genome-wide association analysis between the watermelon flesh color trait and all SNP variant sites. The interval 14172730-15856268 on chromosome 4 was significantly associated with the red flesh trait of watermelon. The Manhattan plot of the FaST-LMM model is shown in the figure. Figure 1 As shown in the figure, there are 40 SNP sites in the association interval. Further analysis of the locations of the 40 SNP sites showed that SNP_15442987 was located in the exon region of the Cla97C04G070940 gene. This site produced a base mutation from C to A, resulting in a change in the encoded amino acid, which in turn caused differences in the flesh color trait. Therefore, SNP site 15442987 is the SNP site that causes the variation of the red flesh trait of watermelon.

[0038] 2. Development of KASP marker for detecting red flesh trait of watermelon

[0039] By comparing the genomic sequences of site 15442987, which was significantly associated with the red flesh trait of watermelon, a 1bp variation was identified at the genomic level between red-fleshed and non-red-fleshed watermelons. This variation caused a mutation from C to A, resulting in differences in the red flesh trait.

[0040] According to the variation of this site, a primer combination was designed in the 500bp region upstream and downstream of the variation site (1001bp in total). The specific sequence is as follows:

[0041] FAM fluorescently labeled upstream primer 1 (SEQ ID NO.1):

[0042] Cla_15442987-F1:

[0043] 5'-GAAGGTGACCAAGTTCATGCTTGGACAAGGCATCGAGAGAA-3';

[0044] VIC fluorescently labeled upstream primer 2 (SEQ ID NO.2):

[0045] Cla_15442987-F2:

[0046] 5'-GAAGGTCGGAGTCAACGGATTTGGACAAGGCATCGAGAGAC-3';

[0047] Downstream common primer (SEQ ID NO.3):

[0048] Cla_15442987-R: 5'-GCCATTGTCTTGATGCCAC-3'.

[0049] The above primers were synthesized by Nanjing Jisi Huiyuan Biotechnology Co., Ltd.

[0050] Upstream and downstream regions of the variant site (SEQ ID NO.4):

[0051]

[0052] The SNP_15442987 site is located at the 501st base of the sequence shown in SEQ ID NO.4 and has been marked in the sequence.

[0053] The Cla_15442987 primer was used to detect the red flesh trait of watermelon. If it was a C:C genotype, it was determined to be a homozygous individual plant with red flesh color; if it was an A:A genotype, it was determined to be a homozygous individual plant with non-red flesh color.

[0054] 3. Use Cla_15442987 marker primer combination to detect the red flesh trait of watermelon

[0055] (1) Genomic DNA extraction

[0056] The total DNA of leaves of 94 natural watermelon population samples was extracted using FlaPure Plant DNA Extraction Kit (Beijing Jinsha Biotechnology Co., Ltd.). The flesh color traits of the 94 watermelon samples are shown in Table 1.

[0057] Table 194 watermelon natural population materials and flesh color phenotypes

[0058]

[0059]

[0060] (2) PCR reaction system and procedure: The watermelon DNA extracted in step (1) was used as a template and the Cla_15442987 primer was used for PCR amplification.

[0061] The PCR reaction system was 5 μL in total and consisted of the following components: 2.5 μL 2×KASPmaster mix, 1.25 μL Primer mix, and 1.25 μL DNA template. The Primer mix was a mixture of Cla_15442987-F1:Cla_15442987-F2:Cla_15442987-R in a ratio of 1:1:3.

[0062] The PCR reaction program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20 s, annealing and extension at 61-55°C for 60 s, decreasing by 0.6°C in each cycle, for 10 cycles; denaturation at 95°C for 20 s, annealing and extension at 55°C for 60 s, for 27 cycles.

[0063] (3) Reading of fluorescence signal value of PCR amplification product

[0064] After the PCR amplification cycle is completed, the fluorescence signal value is read using a fluorescence quantitative PCR instrument, and the genotyping results of the watermelon samples are obtained based on the fluorescence differences. If it is a homozygous genotype C:C, it is judged to be a watermelon sample with red flesh color, represented by a blue dot in the genotyping diagram; if it is a homozygous genotype A:A, it is judged to be a watermelon sample with non-red flesh color, represented by a red dot in the genotyping diagram.

[0065] 94 different watermelon samples were tested using the Cla_15442987 primer to obtain the genotyping diagram, as shown in Figure 2 The KASP test results of 94 watermelon samples are shown in Table 2.

[0066] Table 2 KASP test results of 94 watermelon samples

[0067]

[0068]

[0069]

[0070] The results in Table 2 show that, through the identification and screening of the Cla_15442987 primer combination, the material in which the C:C fluorescence signal corresponding to the primer Cla_15442987 is detected is the homozygous material of the watermelon with red flesh color. The material in which the A:A fluorescence signal corresponding to the primer Cla_15442987 is detected is the homozygous material of the watermelon with non-red flesh color. Therefore, the genotyping result of the Cla_15442987 primer combination of the present invention is completely consistent with the flesh color phenotype, and the detection accuracy is 100%, which can be applied to the rapid identification of the red flesh trait of watermelon. Through the screening of molecular markers, the workload of screening and identification by naked eye after the fruit matures is greatly reduced, the identification accuracy is improved, and the breeding process is accelerated.

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A KASP marker primer combination for detecting the red flesh trait of watermelon, characterized in that: It comprises an upstream primer 1 having a nucleotide sequence as shown in SEQ ID NO.1, an upstream primer 2 as shown in SEQ ID NO.2, and a downstream common primer as shown in SEQ ID NO.3; The SNP site detected by the KASP marker primer combination is located at the 15442987th base of chromosome 4 of watermelon, and the upstream and downstream sequences are shown in SEQ ID NO.

4. There is a C / A mutation at the 501st base of the sequence.

2. The KASP marker primer combination according to claim 1, characterized in that: The molar ratio of the upstream primer 1, the upstream primer 2 and the downstream common primer is 1:1:

3.

3. Use of the KASP marker primer combination as claimed in claim 1 or 2 in the preparation of a product for detecting the red flesh trait of watermelon.

4. The use according to claim 3, characterized in that The product is a test kit.

5. A kit for detecting the red flesh trait of watermelon, characterized in that: The method comprises the KASP marker primer combination according to claim 1 or 2.

6. Use of the KASP labeling primer combination according to claim 1 or 2 or the kit according to claim 5 in any of the following: (1) Identify the color characteristics of watermelon flesh; (2) Screening high-purity parents of red-fleshed watermelon; (3) Cultivate red-fleshed watermelon varieties.

7. A method for identifying the color properties of watermelon flesh, characterized in that: The following steps are involved: Extracting genomic DNA of the watermelon to be tested; Using the genomic DNA as a template, and using the KASP labeled primer combination described in claim 1 or 2 to perform a PCR reaction to obtain a PCR amplification product; According to the fluorescence signal value of the PCR amplification product, the genotyping result is obtained to identify the flesh color trait of the watermelon to be tested.

8. The method according to claim 7, characterized in that If the result of the genotyping is a homozygous genotype C:C, the flesh color trait of the tested watermelon is identified as red flesh; If the result of the genotyping is the homozygous genotype A:A, the flesh color trait of the tested watermelon is identified as non-red flesh.

9. The method according to claim 7, characterized in that The PCR reaction system includes: 2×KASPmastermix 2.5 μL, primer combination 1.25 μL and genomic DNA 1.25 μL.

10. The method according to claim 7, characterized in that The PCR reaction program is: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20 s, annealing and extension at 61-55°C for 60 s, decreasing 0.6°C in each cycle, 10 cycles; denaturation at 95°C for 20 s, annealing and extension at 55°C for 60 s, 27 cycles.

Images