A gene for controlling long and short vine traits of watermelon, a molecular marker primer group for detecting long and short vine traits and application thereof

Abstract

The application provides a gene for controlling long and short vine traits of watermelon, a molecular marker primer group for detecting the long and short vine traits and application, and belongs to the technical field of watermelon genetic breeding. A dominant watermelon short vine mutant is found, and a F2 generation separation population is constructed by using the mutant and a long vine wild type, so that a gene for controlling vine length is located, and a gene for coding alpha-microtubule protein is found ClG42_10g0100600, Then, a molecular marker for detecting the long and short vine traits of watermelon is developed, the molecular marker is closely linked with the vine length traits of watermelon, and can be used for molecular assisted breeding of the short vine of watermelon.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of watermelon genetic breeding, and particularly relates to a gene for controlling long and short vine traits of watermelon, a molecular marker primer group for detecting the long and short vine traits and application thereof. BACKGROUND

[0002] Watermelon (Citrullus lanatus) is an annual vineous herb of Cucurbitaceae. China is the largest country in the world in terms of watermelon planting and consumption. According to the data of FAO, the planting area of watermelon in China reached 1.39 million hectares in 2022, and the total yield reached 60.54 million tons.

[0003] Internode length is one of the important plant type traits of watermelon. The main cultivars on the market have longer main vines, and the land resource utilization rate is lower due to the large area of ground cultivation. Although the hanging vine cultivation can save the planar space, the high plant increases the workload of hanging and binding vines, spraying and harvesting, and the long vine watermelon also increases the difficulty of artificial pollination in the process of hybrid breeding.

[0004] The short vine type plant has a compact plant type, does not need to be pruned, is suitable for dense planting, can fully utilize land and light, and can save labor. Therefore, creating watermelon short vine germplasm resources is the basis for breeding short vine watermelon varieties suitable for light and simple and intensive cultivation. However, if the traditional breeding method is used to breed short vine watermelon, selection needs to be performed when the plant stretches the vine, which not only needs to occupy a large plot for planting, but also has a long breeding period. If molecular marker assisted selection (MAS) is used, the short vine trait of watermelon can be selected at the seedling stage, and the breeding process can be accelerated.

[0005] At present, the short vine mutants reported on watermelon include dw-1, dw-1s, dw-2, dw-3, dw-4, SV-1 and dsh, which exhibit the traits of short internodes and short vines. However, these short vine mutants are controlled by recessive genes. At present, there is no report on short vine mutants controlled by dominant genes on watermelon. The short vine mutant controlled by the dominant gene still exhibits stable dwarfing traits even in the heterozygous state. The use of the dominant short vine mutant can accelerate the breeding of short vine watermelon varieties suitable for light and simple and intensive cultivation.

[0006] Therefore, screening of the watermelon dominant short vine mutant and further cloning of the dominant gene controlling the dwarfing trait have important significance for accelerating the breeding of watermelon short vine varieties. SUMMARY

[0007] The application aims to provide a gene for controlling the long and short vine traits of watermelons, a molecular marker primer group for detecting the long and short vine traits, and an application thereof.

[0008] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0009] The application provides a gene for controlling the long and short vine traits of watermelons.

[0010] The application further provides a KASP molecular marker primer group for detecting the long and short vine traits of watermelons, which is composed 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.

[0011] The application further provides an application of the KASP molecular marker primer group for detecting the long and short vine traits of watermelons in watermelon short vine breeding.

[0012] The application further provides a method for detecting the genotype of watermelons by using the KASP molecular marker primer group for detecting the long and short vine traits of watermelons, which comprises the following steps.

[0013] After the genomic DNA of the watermelons to be detected is extracted, the KASP molecular marker primer group for detecting the long and short vine traits of watermelons is used to perform PCR amplification on the DNA, fluorescence detection is performed, and the genotype detection result of the watermelons is obtained.

[0014] Further, the genotype of the watermelons is a homozygous long vine genotype aa, a heterozygous short vine genotype Aa, or a homozygous short vine genotype AA.

[0015] Further, the reaction system of the PCR amplification is as follows: 50 ng / μL watermelon genomic DNA 2 μL, HiGeno 2xProbe Mix 5 μL, SNP-Specific Primer 0.14 μL, ddH2O 2.86 μL, and a total of 10 μL.

[0016] Further, 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.

[0017] Further, the reaction procedure of the PCR amplification is 95℃, 10 min; 95℃, 20 s; 68℃, 40 s, 10 cycles, each cycle decreases 1℃; 95℃, 20 s; 58℃, 40 s, 40 cycles.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The present application finds a dominant short vine mutant of watermelon, and uses the F2 generation segregation population constructed by the mutant and long vine wild type to locate the gene controlling vine length, and finds a gene encoding α-tubulin ClG42_ 10g0100600 Controlling short vine trait of watermelon ， Then a molecular marker for detecting long and short vine traits of watermelon is developed, the molecular marker is closely linked to the vine length trait of watermelon, and can be used for molecular assisted breeding of short vine of watermelon. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0021] Figure 1 It is a schematic diagram of the phenotype of the short vine mutant of example 1; N+ / + represents homozygous long vine; D+ / - represents heterozygous short vine; D+ / + represents homozygous short vine;

[0022] Figure 2 It is a typing diagram of the molecular marker of example 1 in the F2 generation of the short vine mutant; the blue dot represents that the material is homozygous long vine genotype; the red dot represents that the material is homozygous short vine genotype; the green dot represents that the material is heterozygous short vine genotype;

[0023] Figure 3 It is a typing diagram of the disease-resistant short vine material in seedling stage screening of example 2; the blue dot represents long vine watermelon; the green dot represents short vine watermelon. DETAILED DESCRIPTION

[0024] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0025] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentration, intensity, and time, and the like, every intermediate value of the

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between the present specification and any incorporated document, the present specification controls.

[0027] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0028] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0029] Nucleotide sequence of the watermelon short vine trait gene ClG42_10g0100600 (SEQ ID NO. 1):

[0030]

[0031] Nucleotide sequence of the watermelon long vine trait gene (SEQ ID NO. 2):

[0032]

[0033] Example 1

[0034] The short vine trait gene of watermelon was isolated in Example 1 of the present application ClG42_10g0100600 , and a KASP molecular marker primer for detecting the long and short vine traits of watermelon was designed, and the specific method is as follows:

[0035] (1) Positioning of dominant short vine mutant candidate gene

[0036] According to the mutant library in 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, no. 8 (August 2022): 1268-84.”, a short vine mutant was found, and the short vine mutant was crossed with wild type to construct a segregation population. The segregation ratio of short vine plants and long vine plants in the segregation population was about 3:1, indicating that the short vine mutant was dominant inheritance.

[0037] According to the method shown in Figure 1 , N + / + (homozygous long vine) was used as the female parent, and D + / + (homozygous short vine mutant) was used as the male parent to obtain F1 generation plants, and the F1 generation plants were self-crossed to obtain F2 generation segregation population.

[0038] The CTAB method was used to extract the genomic DNA of the single plant of the F2 generation segregation population:

[0039] Take the tender leaves of watermelon and place them in liquid nitrogen, grind them into powder, add them to 600 μL of 2% CTAB buffer, and place them in a 65°C water bath for 30 min. After the water bath, add 600 μL of chloroform, shake well, and centrifuge at 10,000 rpm for 10 min. Repeat the chloroform extraction twice. Take the supernatant and add it to a new 1.5 mL centrifuge tube. Add an equal volume of isopropanol, mix well, and place it in a -20°C environment for 30 min. Centrifuge at 12,000 rpm for 10 min. After centrifugation, discard the supernatant, wash the precipitate with 500 μL of 75% alcohol, centrifuge at 12,000 rpm for 10 min, discard the alcohol, and dry the DNA. Add 30 μL of ddH2O to dissolve the precipitate, measure the DNA concentration with a UV spectrophotometer, dilute it to 50 ng / μL with ddH2O, and store it at 4°C for future use.

[0040] Take the DNA of 19 homozygous short vine and 23 homozygous long vine plants in the F2 separation population, perform single-plant resequencing, with a sequencing depth of 50 times the genome coverage, and use Illumina as the sequencing platform. Use G42 as the reference genome, perform whole-genome variant detection, and perform correlation analysis with the phenotype data of vine length to identify SNPs sites related to the vine length phenotype.

[0041] According to the identified SNPs sites, design KASP molecular marker primers, verify them on the F2 separation population, determine the SNP sites closely linked to the target trait, and determine the candidate gene controlling the vine length of watermelon, to obtain the watermelon dominant short vine mutant related gene ClG42_10g0100600 (SEQ ID NO. 1) and the watermelon long vine gene (SEQ ID NO. 2), both of which are 1353 bp in length and are located at Chr10: 21607895bp-21610126bp on the chromosome.

[0042] (2) Use KASP molecular markers to distinguish between watermelon short vine and long vine

[0043] According to the watermelon short vine mutant gene ClG42_10g0100600 , design KASP molecular marker primers at its SNP site, with the primer sequence being:

[0044] aa type forward primer F-FAM (SEQ ID NO. 3):

[0045] 5'-GAAGGTGACCAAGTTCATGCTTGATGTATCGTGGTGATGTTGTGC-3';

[0046] Forward primer F-HEX (SEQ ID NO. 4) of AA type:

[0047] 5'-GAAGGTCGGAGTCAACGGATTCTGATGTATCGTGGTGATGTTGTGT-3';

[0048] Reverse primer R (SEQ ID NO. 5):

[0049] 5'-TTGTACGCTTGGTCTTGATGGTG-3'.

[0050] The tender leaves of the F2 separation population were taken, and the watermelon genomic DNA was extracted by the CTAB method, and amplification was performed on a PCR instrument.

[0051] The PCR reaction system was as follows: 50 ng / μL watermelon genomic DNA 2 μL, HiGeno 2x Probe Mix 5 μL, SNP-Specific Primer (F-FAM, F-HEX, R, the final concentrations were 12 μM, 12 μM and 30 μM respectively) 0.14 μL, ddH2O 2.86 μL, a total of 10 μL.

[0052] The amplification program of PCR was as follows: 95℃, 10 min; 95℃, 20 s; 68℃ (-1℃ / cycle), 40 s, 10 cycles; 95℃, 20 s; 58℃, 40 s, 40 cycles.

[0053] After the PCR was completed, the amplification product was placed on an ABI Q5 fluorescent quantitative PCR instrument, the Genotyping experiment type was selected to read the fluorescent signal of the PCR product and to view the typing, and the results were as shown in Figure 2 .

[0054] Figure 2 In each circle, one circle represented one material to be tested, wherein a blue circle represented that the material was a homozygous long vine genotype, a red circle represented that the material was a homozygous short vine genotype, and a green circle represented that the material was a heterozygous short vine genotype.

[0055] Example 2

[0056] In the example 2 of the present application, the short vine mutant separated in the example 1 was used to transform a disease-resistant short vine material, and the molecular marker was used to screen at the seedling stage, and the specific method was as follows:

[0057] The disease-resistant material 'PKR6' was crossed with the heterozygous short vine mutant, the DNA of the F1 generation seedling leaves was extracted, and the method in the step (2) of the example 1 was used for detection.

[0058] The obtained results were as shown inFigure 3 As shown, blue dots represent long vine watermelons, and green dots represent disease-resistant short vine watermelons.

[0059] The results show that the molecular marker of the present application can be used for the assisted selection breeding of short vine watermelons and their offspring.

[0060] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

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

2.

2. A KASP molecular marker primer set for detecting the vine length and shortness trait of watermelon as described in claim 1, characterized in that, The molecular marker primer set consists of the aa-type forward primer F-FAM shown in SEQ ID NO.3, the AA-type forward primer F-HEX shown in SEQ ID NO.4, and the reverse primer R shown in SEQ ID NO.

5.

3. The application of the KASP molecular marker primer set as described in claim 2 for detecting the long and short vine traits of watermelon in the breeding of short vine watermelon.

4. A method for detecting watermelon genotype using the KASP molecular marker primer set for detecting the vine length trait of watermelon as described in claim 2, characterized in that, Includes the following steps: After extracting genomic DNA from the watermelon to be tested, the DNA was amplified by PCR using a set of KASP molecular marker primers for detecting the vine length and shortness traits of watermelon, and fluorescence detection was performed to obtain the watermelon genotype detection results. The watermelon genotype detection results include homozygous long-vine genotype aa, heterozygous short-vine genotype Aa, and homozygous short-vine genotype AA.

5. The method for detecting watermelon genotype according to claim 4, characterized in that, The PCR amplification reaction system consisted of: 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, for a total of 10 μL.

6. The method for detecting watermelon genotype according to claim 5, characterized in that, The SNP-Specific Primer consists of F-FAM at a final concentration of 12 μM, F-HEX at a final concentration of 12 μM, and R at a final concentration of 30 μM.

7. The method for detecting watermelon genotype according to claim 3, characterized in that, The PCR amplification reaction program was as follows: 95℃, 10 min; 95℃, 20 s; 68℃, 40 s, 10 cycles, decreasing by 1℃ per cycle; 95℃, 20 s; 58℃, 40 s, 40 cycles.

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