Gene for controlling long and short vine characters of watermelons, molecular marker primer group for detecting long and short vine characters and application

Abstract

The invention provides a gene for controlling long and short vine characters of watermelons, a molecular marker primer group for detecting the long and short vine characters and application, and belongs to the technical field of genetic breeding of watermelons. According to the invention, a dominant watermelon short-vine mutant is found, an F2-generation segregation population constructed by the mutant and a long-vine wild type is utilized to locate a gene for controlling the vine length, a gene ClG4210g0100600 for coding alpha-tubulin is found, and then a molecular marker for detecting the long-vine and short-vine traits of watermelons is developed. The molecular marker is closely linked with the watermelon vine length character and can be used for molecular assisted breeding of watermelon short vines.

Description

Technical Field

[0001] The invention relates to the technical field of watermelon genetic breeding, and in particular to a gene for controlling the long and short vine traits of watermelon, a molecular marker primer set for detecting the long and short vine traits, and applications thereof. Background Art

[0002] Watermelon (Citrullus lanatus) is an annual vine herbaceous plant of the Cucurbitaceae family. my country is the world's largest watermelon grower and consumer. According to FAO statistics, my country's watermelon planting area reached 1.39 million hectares in 2022, with a total output of 60.54 million tons.

[0003] Internode length is one of the important plant traits of watermelon. The main varieties currently grown on the market have long main vines, so creeping cultivation occupies a large area and has a low utilization rate of land resources. Although hanging vine cultivation can save flat space, too high plants will increase the workload of hanging and tying vines, spraying pesticides, harvesting, etc. At the same time, long-vine watermelon also increases the difficulty of artificial pollination during hybrid breeding.

[0004] The short-vine type plants are compact, do not require pruning, are suitable for dense planting, can make full use of land and light, and can save labor. Therefore, the creation of short-vine watermelon germplasm resources is the basis for cultivating short-vine watermelon varieties suitable for simplified and intensive cultivation. However, if traditional breeding methods are used to select short-vine watermelons, it is necessary to select when the plants are extending their vines. Not only does it require a large plot of land for planting, but the breeding cycle is also long. If molecular marker-assisted selection breeding (MAS, Molecular-maker Assisted Selection) is used, the short-vine trait of watermelons can be selected at the seedling stage, which speeds up the breeding process.

[0005] At present, the short vine mutants reported in watermelon include dw-1, dw-1s, dw-2, dw-3, dw-4, SV-1, and dsh, which show short internodes and shortened vine length, but these short vine mutants are controlled by recessive genes. At present, short vine mutants controlled by dominant genes have not been reported in watermelon. Short vine mutants controlled by dominant genes will still show stable dwarfing traits even in the heterozygous state of genotype. The use of dominant short vine mutants can accelerate the breeding of short vine watermelon varieties suitable for light simplification and intensive cultivation.

[0006] Therefore, screening dominant short-vine mutants of watermelon and further cloning the dominant genes controlling dwarfing traits are of great significance for accelerating the breeding of short-vine watermelon varieties. Summary of the invention

[0007] The purpose of the present invention is to provide a gene controlling the long and short vine traits of watermelon, a molecular marker primer set for detecting the long and short vine traits and an application thereof. The present invention screens the dominant gene controlling the short vine trait of watermelon, and designs the KASP molecular marker primers for detecting the long and short vine traits of watermelon, laying a foundation for the breeding of high-quality short-vine watermelon varieties.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a gene for controlling the long and short vine traits of watermelons. The gene for controlling the short vine trait of the watermelon is a dominant gene, and the nucleotide sequence of the gene for controlling the short vine trait of the watermelon is shown as SEQ ID NO.1; the gene for controlling the long vine trait of the watermelon is a recessive gene, and the nucleotide sequence of the gene for controlling the long vine trait of the watermelon is shown as SEQ ID NO.2.

[0009] The present invention also provides a KASP molecular marker primer set for detecting the long and short vine traits of watermelon, wherein the molecular marker primer set consists of an aa type forward primer F-FAM shown in SEQ ID NO.3, an AA type forward primer F-HEX shown in SEQ ID NO.4, and a reverse primer R shown in SEQ ID NO.5.

[0010] The present invention also provides an application of a KASP molecular marker primer set for detecting the long and short vine traits of watermelon in watermelon short vine breeding.

[0011] The present invention also provides a method for detecting watermelon genotype using a KASP molecular marker primer set for detecting the long and short vine traits of watermelon, comprising the following steps: After extracting the genomic DNA of the watermelon to be tested, the DNA was amplified by PCR using the KASP molecular marker primer set for detecting the long and short vine traits of watermelon, and fluorescence detection was performed to obtain the watermelon genotype detection result.

[0012] Furthermore, the watermelon genotype is homozygous long vine genotype aa, heterozygous short vine genotype Aa, and homozygous short vine genotype AA.

[0013] Furthermore, the reaction system of the PCR amplification is: 2 μL of 50 ng / μL watermelon genomic DNA, 5 μL of HiGeno 2xProbe Mix, 0.14 μL of SNP-Specific Primer, and 2.86 μL of ddH2O, a total of 10 μL.

[0014] Furthermore, the SNP-Specific Primer consists of F-FAM with a final concentration of 12 μM, F-HEX with a final concentration of 12 μM, and R with a final concentration of 30 μM.

[0015] Furthermore, the reaction program of the PCR amplification is 95°C, 10 min; 95°C, 20 s; 68°C, 40 s, 10 cycles, minus 1°C for each cycle; 95°C, 20 s; 58°C, 40 s, 40 cycles.

[0016] The beneficial effects of the present invention compared with the prior art are: The present invention discovered a dominant short-vine mutant of watermelon, and used the mutant and the long-vine wild type to construct an F2 generation segregating population to locate the gene that controls vine length, and found a gene encoding α-tubulin ClG42_ 10g0100600 Can control the short vine characteristics of watermelon ， Then, a molecular marker for detecting the long and short vine traits of watermelon was developed. This molecular marker is closely linked to the watermelon vine length trait and can be used for molecular-assisted breeding of short vines in watermelon. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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 labor.

[0018] Figure 1 The phenotype diagram of the short vine mutant in Example 1; N+ / + indicates homozygous long vines; D+ / - indicates heterozygous short vines; D+ / + indicates homozygous short vines; Figure 2 This is the typing diagram of the molecular markers in the F2 generation of the short vine mutant of Example 1; blue dots indicate that the material is a homozygous long vine genotype; red dots indicate that the material is a homozygous short vine genotype; green dots indicate that the material is a heterozygous short vine genotype; Figure 3 This is a phenotyping diagram for screening disease-resistant short-vine materials at the seedling stage in Example 2; blue dots represent long-vine watermelons; green dots represent short-vine watermelons. DETAILED DESCRIPTION

[0019] 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.

[0020] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0021] 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.

[0022] 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 the skilled artisan. The present invention description and examples are exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Gene controlling short vine trait in watermelon ClG42_10g0100600 Nucleotide sequence (SEQ ID NO.1): Nucleotide sequence of the gene controlling the vine length trait of watermelon (SEQ ID NO.2):

[0025] Example 1 Example 1 of the present invention isolates a gene controlling the short vine trait of watermelon ClG42_10g0100600 , and designed KASP molecular marker primers that can detect the long and short vine traits of watermelon. The specific method is as follows: (1) Location of candidate genes for dominant short-vine mutants According to the prior art "Deng, Yun, Shoucheng Liu, Yilin Zhang, Jingsheng Tan, Xiaopeng Li, Xiao Chu, Binghua Xu, et al. "A Telomere-to-Telomere Gap-Free Reference Genome of Watermelon and Its Mutation Library Provide Important Resources for Gene Discovery and Breeding". Molecular Plant 15, Issue 8 (August 2022): 1268–84.", a short vine mutant was found in the mutant library. The short vine mutant was hybridized with the wild type to construct an isolated population. The separation ratio of short vine plants and long vine regular plants in the isolated population was about 3:1, indicating that the short vine mutant was dominant.

[0026] according to Figure 1 As shown, N + / + (homozygous long creeper) is the female parent, D + / + (Homozygous short vine mutant) The male parents were crossed to obtain F1 generation plants, and the F1 generation plants were self-pollinated to obtain the F2 generation segregating population.

[0027] Extraction of genomic DNA from leaves of individual plants in the F2 generation segregation population using the CTAB method: Take young watermelon leaves and place them in liquid nitrogen, grind them into powder, add them to 600 μL 2% CTAB buffer, and place them in a 65℃ water bath for 30 min; after the water bath, add 600 μL chloroform, shake and mix, centrifuge at 10,000 rpm in a centrifuge for 10 min, and extract chloroform twice; take the supernatant and add it to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, invert and mix, place at -20℃, let stand for 30 min, and centrifuge at 12,000 rpm in a centrifuge for 10 min; after centrifugation, pour out the supernatant, wash the precipitate with 500 μL 75% alcohol, centrifuge at 12,000 rpm in a centrifuge for 10 min, pour out the alcohol, absorb the excess alcohol and dry the DNA; add 30 μL ddH2O to dissolve the precipitate, measure the DNA concentration with a UV spectrophotometer, and dilute to 50 with ddH2O ng / μL, stored at 4℃ for later use.

[0028] DNA from 19 homozygous short vines and 23 homozygous long vines in the F2 generation segregation population was collected for single plant resequencing, with a sequencing depth of 50 times the genome coverage, and the sequencing platform was Illumina. Using G42 as the reference genome, whole genome variation detection and association analysis with the phenotype data of vine length were performed to identify SNPs sites associated with the vine length phenotype.

[0029] Based on the identified SNPs, KASP molecular marker primers were designed. The SNPs closely linked to the target trait were confirmed by verification on the F2 segregation population. The candidate genes controlling the vine length of watermelon were also identified, and the genes related to the dominant short vine mutant of watermelon were obtained. ClG42_10g0100600 (shown in SEQ ID NO.1), and watermelon vine growth gene (shown in SEQ ID NO.2), both genes are 1353 bp in length, and their specific locations on the chromosome are: Chr10: 21607895bp-21610126bp.

[0030] (2) Using KASP molecular markers to distinguish short and long vines in watermelon Watermelon short vine mutant gene ClG42_10g0100600 , KASP molecular marker primers were designed at its SNP site, and the primer sequences were: aa type forward primer F-FAM (SEQ ID NO.3): 5'-GAAGGTGACCAAGTTCATGCTTGATGTATCGTGGGTGATGTTGTGC-3'; AA type forward primer F-HEX (SEQ ID NO.4): 5'-GAAGGTCGGAGTCAACGGATTCTGATGTATCGTGGTGATGTTGTGT-3'; Reverse primer R (SEQ ID NO.5): 5'-TTGTACGCTTGGTCTTGATGGTG-3'.

[0031] Young and tender leaves of watermelon from the F2 segregation population were taken, and the watermelon genomic DNA was extracted using the CTAB method and amplified on a PCR instrument.

[0032] The PCR reaction system was: 2 μL of 50 ng / μL watermelon genomic DNA, 5 μL of HiGeno 2x Probe Mix, 0.14 μL of SNP-Specific Primer (the final concentrations of F-FAM, F-HEX, and R were 12 μM, 12 μM, and 30 μM, respectively), and 2.86 μL of ddH2O, for a total of 10 μL.

[0033] The PCR amplification program was 95°C, 10 min; 95°C, 20 s; 68°C (-1°C / cycle), 40 s, 10 cycles; 95°C, 20 s; 58°C, 40 s, 40 cycles.

[0034] After PCR, place the amplified product on the ABI Q5 fluorescent quantitative PCR instrument, select the Genotyping experiment type to read the fluorescent signal of the PCR product and check the typing. The results are as follows Figure 2 shown.

[0035] Figure 2 In the figure, each dot represents a material to be tested, where a blue dot indicates that the material is a homozygous long-vine genotype, a red dot indicates that the material is a homozygous short-vine genotype, and a green dot indicates that the material is a heterozygous short-vine genotype.

[0036] Example 2 Example 2 of the present invention utilizes the short vine mutant isolated from Example 1 to breed disease-resistant short vine materials, and uses molecular markers to screen them at the seedling stage. The specific method is as follows: The disease-resistant material 'PKR6' was hybridized with the heterozygous short-vine mutant, and DNA was extracted from leaves of the F1 generation seedlings, and the DNA was detected using the method of step (2) of Example 1.

[0037] The results are as follows Figure 3 As shown, the blue dots represent long-vine watermelons, and the green dots represent disease-resistant short-vine watermelons.

[0038] The obtained results indicate that the molecular markers of the present invention can be used for assisted selection breeding of short-vine watermelon and its progeny.

[0039] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene for controlling the long and short vine traits of watermelon, characterized in that: The gene controlling the short vine trait of the watermelon is a dominant gene, and the nucleotide sequence of the gene controlling the short vine trait of the watermelon is shown in SEQ ID NO.1; the gene controlling the long vine trait of the watermelon is a recessive gene, and the nucleotide sequence of the gene controlling the long vine trait of the watermelon is shown in SEQ ID NO.

2.

2. A KASP molecular marker primer set for detecting the long and short vine traits of watermelon as claimed in claim 1, characterized in that: The molecular marker primer set consists of an aa-type forward primer F-FAM shown in SEQ ID NO.3, an AA-type forward primer F-HEX shown in SEQ ID NO.4, and a reverse primer R shown in SEQ ID NO.

5.

3. Use of the KASP molecular marker primer set for detecting the long and short vine traits of watermelon according to claim 2 in watermelon short vine breeding.

4. A method for detecting watermelon genotype using the KASP molecular marker primer set for detecting watermelon vine length traits according to claim 2, characterized in that: The steps include: After extracting the genomic DNA of the watermelon to be tested, the KASP molecular marker primer set for detecting the long and short vine traits of watermelon was used to perform PCR amplification on the DNA, and fluorescence detection was performed to obtain the watermelon genotype detection result.

5. The method for detecting watermelon genotype according to claim 3, characterized in that: The watermelon genotype detection results include homozygous long vine genotype aa, heterozygous short vine genotype Aa, and homozygous short vine genotype AA.

6. The method for detecting watermelon genotype according to claim 3, characterized in that: The reaction system of the PCR amplification is: 2 μL of 50 ng / μL watermelon genomic DNA, 5 μL of HiGeno 2x Probe Mix, 0.14 μL of SNP-Specific Primer, and 2.86 μL of ddH2O, a total of 10 μL.

7. The method for detecting watermelon genotype according to claim 5, characterized in that: The SNP-Specific Primer is composed of F-FAM with a final concentration of 12 μM, F-HEX with a final concentration of 12 μM, and R with a final concentration of 30 μM.

8. The method for detecting watermelon genotype according to claim 3, characterized in that: The reaction program of the PCR amplification was 95°C, 10 min; 95°C, 20 s; 68°C, 40 s, 10 cycles, with 1°C minus for each cycle; 95°C, 20 s; 58°C, 40 s, 40 cycles.

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