RPL2 gene-based KASP molecular marker related to waterlogging resistance of cucurbit, primer group and application of RPL2 gene-based KASP molecular marker and primer group

Abstract

The invention provides a KASP molecular marker related to waterlogging resistance of cucurbit based on an RPL2 gene, and the molecular marker corresponds to a mutation site of an exon region of a gene Lsi09G006060 on a cucurbit 9 chromosome, specifically, the mutation of G-A of 6589121st nucleotide of the cucurbit 9 chromosome. The invention also provides a KASP primer group for detecting the molecular marker, and also provides application of the molecular marker and the primer group in detection of waterlogging resistance of cucurbit, screening of waterlogging-resistant materials of cucurbit and breeding of waterlogging-resistant varieties of cucurbit, and corresponding methods. The molecular marker and the primer group can quickly judge the genotype of the cucurbit through fluorescent quantitative PCR, are used for detecting the waterlogging resistance of the cucurbit, screening waterlogging-resistant materials and breeding varieties, solve the problems of long period, high cost and low precision of conventional breeding, and are pollution-free in detection.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and plant resistance breeding, and in particular to a KASP molecular marker related to waterlogging tolerance of cucurbit based on an RPL2 gene, a primer set and an application thereof. Background Art

[0002] Lagenaria siceraria (Molina) Standl., also known as night-blooming gourd, gourd, and ground cattail, originated in Africa and is an annual climbing herb in the Cucurbitaceae family. It is widely cultivated worldwide, with over 2 million mu (approximately 1.5 acres) of planted area in my country in 2024. It is primarily used as a rootstock for watermelon grafting, but also has other uses, including food, daily use, cultural relics, and ornamental purposes. It boasts a rich germplasm and genetic resources.

[0003] Southern my country is prone to waterlogging due to heavy rainfall, typhoons, and downpours, particularly during the spring rainy season and the typhoon-prone summer and autumn rainy seasons. The occasional, short-term downpours in northern China during summer and autumn can also cause devastating damage to melon production. Waterlogging, a key environmental factor limiting melon production in rainy and torrential regions, primarily affects crop roots, leading to hypoxia and a shift to anaerobic metabolism. This in turn affects overall crop growth and development, ultimately reducing yield and quality. Therefore, the selection and breeding of waterlogging-tolerant gourd varieties has become a key focus in gourd breeding in flood-prone areas.

[0004] At present, the selection and breeding of waterlogging-resistant varieties of gourds is still mainly based on conventional breeding technology, that is, through field flooding treatment combined with laboratory seedling identification, the tolerance level of the material is determined, and then the waterlogging-resistant materials are screened and variety breeding is carried out based on this. This process has problems such as long cycle, high cost, and insufficient accuracy. In the existing technology, although there are reports on the genetic laws of root traits related to waterlogging resistance of gourds and a small number of molecular marker screenings, molecular marker-assisted breeding technologies that can significantly improve breeding efficiency and accuracy are still relatively scarce. Therefore, it is of great practical significance to develop more molecular markers related to waterlogging tolerance of gourds for the creation of waterlogging-resistant gourd germplasm resources and the selection and application of highly water-resistant varieties. Summary of the Invention

[0005] The present invention aims to provide a KASP molecular marker, primer set and application related to waterlogging tolerance of cucurbit based on the RPL2 gene, so as to solve the problems of long cycle, high cost and low accuracy in the existing identification and variety selection of waterlogging tolerance of cucurbit, and insufficient related efficient molecular marker-assisted breeding technology, thereby improving the efficiency and accuracy of the creation of waterlogging-resistant cucurbit germplasm resources and the breeding of highly waterlogging-resistant varieties, and providing technical support for cucurbit production in flood-prone areas.

[0006] The present invention adopts the following technical solutions to solve the above technical problems: A KASP molecular marker related to waterlogging tolerance in cucurbit based on the RPL2 gene, the molecular marker corresponding to a mutation site in the exon region of the gene Lsi09G006060 on chromosome 9 of cucurbit; the gene Lsi09G006060 encodes 50Sribosomal protein L2, i.e., RPL2 protein; the mutation site is a G→A mutation at nucleotide position 6589121 on chromosome 9 of cucurbit (G corresponds to a waterlogging-tolerant phenotype, and A corresponds to a waterlogging-intolerant phenotype).

[0007] In the present invention, the genome of chromosome 9 of Cucurbita cucurbita is a public sequence, and its original genome sequencing data and transcriptome sequence read data have been deposited in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) of the United States, with the corresponding access numbers NHZF00000000 (genome) and SRP107894 (transcriptome). The complete genome sequence and its annotation information can also be obtained through the Cucurbit Genomics Database (accessed at: http: / / cucurbitgenomics.org).

[0008] As one of the preferred embodiments of the present invention, the mutation site is specifically located in the 9th exon region of the gene Lsi09G006060 on Chromosome 9 of Cucurbita cucurbita. The sequence of the gene Lsi09G006060 before mutation is shown in SEQ ID NO. 4, and the sequence after mutation is shown in SEQ ID NO. 5.

[0009] A KASP primer set for detecting the above-mentioned molecular marker comprises an upstream primer F1, an upstream primer F2 and a downstream primer R; wherein the nucleotide sequences of the upstream primer F1 and the upstream primer F2 are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively, and the nucleotide sequence of the downstream primer R is shown as SEQ ID NO.3.

[0010] An application of the KASP molecular marker or KASP primer set in detecting waterlogging tolerance of gourd, screening waterlogging-resistant gourd materials, and breeding waterlogging-resistant gourd varieties.

[0011] A method for detecting waterlogging tolerance of a calabash, comprising the following steps: (1) Extracting genomic DNA from the gourd material to be tested; (2) using the genomic DNA as a template and performing fluorescent quantitative PCR amplification using the KASP primer set described in claim 3; (3) Determine the genotype based on the fluorescence signal of the PCR product: If the genotype is G / G homozygous, the material is a "waterlogging-tolerant material" or "relatively waterlogging-tolerant material" (most of them are "waterlogging-tolerant materials", and a few of them are "relatively waterlogging-tolerant" due to the regulation of other waterlogging-tolerant genes); If the genotype is A / A homozygous, the material is a "waterlogging-intolerant material" or "relatively waterlogging-tolerant material" (most of them are "waterlogging-intolerant materials", and a few of them are "relatively waterlogging-tolerant" due to the regulation of other genes); If the genotype is A / G heterozygous, the material is a "waterlogging-intolerant material" or "relatively waterlogging-tolerant material" (no waterlogging-tolerant performance). Among them, the waterlogging tolerance of "relatively waterlogging-tolerant materials" is between that of "waterlogging-tolerant materials" and "waterlogging-intolerant materials", that is, waterlogging tolerance of "waterlogging-intolerant materials" < waterlogging tolerance of "waterlogging-tolerant materials" < waterlogging tolerance of "waterlogging-tolerant materials".

[0012] As one of the preferred embodiments of the present invention, in step (2), the PCR reaction system is 5 μL, including: 2 μL of 2 x TaqDNA Polymerase Mix, 1 μL of 4 x SNP Primer Mix, and 2 μL of 20 ng / μL genomic DNA.

[0013] As one of the preferred embodiments of the present invention, in step (2), the PCR amplification program is as follows: pre-denaturation at 94°C for 10 min, 1 cycle; denaturation at 94°C for 20 s, annealing and extension at 61°C to 55°C for 45 s, for a total of 10 cycles, with the annealing and extension temperature decreasing by 0.6°C in each cycle; the second round of denaturation at 94°C for 20 s, annealing and extension at 55°C for 45 s, for a total of 37 cycles.

[0014] As one of the preferred embodiments of the present invention, in step (3), the fluorescence signal is analyzed using Kluster Caller software of LGC_OMEGA, and the genotype is determined by the relative fluorescence values ​​of VIC and FAM.

[0015] A method for screening gourd waterlogging-resistant materials comprises the following steps: using the method for detecting waterlogging tolerance of gourd to detect the genotype of the gourd material, and screening G / G homozygous materials as waterlogging-resistant candidate materials.

[0016] A method for breeding gourd varieties resistant to waterlogging is characterized in that the genotype of the gourd material is detected by the above-mentioned method for detecting waterlogging tolerance of the gourd, and G / G homozygous materials are screened as waterlogging-resistant candidate materials; field waterlogging stress identification is performed on the screened waterlogging-resistant candidate materials, and materials with the lowest degree of waterlogging damage and normal growth and development are selected to cultivate the waterlogging-resistant gourd varieties.

[0017] The advantages of the present invention over the prior art are: The application finds that the wet and waterlogging tolerance of cucurbit is a partial dominant quantitative inheritance by identifying two parents (wet and waterlogging tolerance and not wet and waterlogging tolerance) and their reciprocal F1 through seedling stage wet and waterlogging treatment, then resequencing the two parents, screening the difference sites, analyzing the genotype of the difference sites of two extreme separation pools of F2 generation, screening the functional mutation sites by combining bioinformatics analysis, obtaining the wet and waterlogging tolerance related gene sites through natural population verification, and developing the related KASP molecular marker and the corresponding KASP primer for the sites.

[0018] The KASP molecular marker developed in the application can quickly detect the genotype of cucurbit by fluorescence quantitative PCR, directly correlate with wet and waterlogging tolerance, greatly shorten the identification and variety breeding cycle, and reduce the cost. The KASP molecular marker of the application is designed for the specific mutation site (Lsi09G006060 gene 9th exon G→A mutation) on the 9th chromosome of cucurbit directly related to wet and waterlogging tolerance, and the genotype is consistent with the phenotype as a whole after verification of 27 germplasm resources, which can significantly distinguish extreme wet and waterlogging tolerance and not wet and waterlogging tolerance materials, and improve the identification accuracy. The KASP primer of the application can not only be used for wet and waterlogging tolerance identification of cucurbit, but also be directly applied to wet and waterlogging tolerance material screening, variety breeding and germplasm resource creation; at the same time, the KASP molecular marker detection process does not need complex experimental operation, has the characteristics of no pollution, and meets the efficient and environmental protection needs of modern breeding technology. DETAILED DESCRIPTION

[0019] The embodiments of the application are described in detail below, which are implemented on the premise of the technical scheme of the application, and detailed implementation modes and specific operation processes are given, but the protection scope of the application is not limited to the following embodiments. At the same time, the experimental methods used in the following are conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

[0020] Example 1, screening and determination of wet and waterlogging tolerance related mutation sites of cucurbit: (1) Test material preparation Select 1 wet and waterlogging tolerance cucurbit material "J03", 1 not wet and waterlogging tolerance cucurbit material "YH2", and the F1 generation prepared by the two as parents and the F2 separation population obtained by selfing of F1. Among them, "J03" and "YH2" materials are recorded in "Xing Nailing, Fu Yujing, Wang Ying'er, et al. Cucurbit stock resources seedling stage light tolerance identification [J]. Jiangxi Journal of Agricultural Sciences, 2019, 31(5): 11-16" (the material name is completely consistent with the literature), the public can obtain the above-mentioned materials from the applicant for twenty years from the application date, and only for repeating the related experiments of the application.

[0021] (2) Waterlogging treatment at the seedling stage and analysis of genetic patterns Seeds of "J03", "YH2", reciprocal cross F1 and F2 populations were soaked and germinated before being sown in plug trays. When the seedlings grew to the one-leaf, one-heart stage, the F2 plants were numbered and leaf samples were collected (for subsequent DNA extraction), followed by waterlogging stress identification.

[0022] The method for identifying waterlogging stress is as follows: place the plug tray in an open, box-shaped container (depth ≥ 4 cm) that is larger than the tray in length and width. Slowly add water to the container until the substrate is saturated with water. Continue adding water until the water level is 2 cm above the top surface of the tray. Place the container in an artificial climate chamber set at a daytime temperature of 25°C, 20,000 lux, and a relative humidity of 70%-80%, and a nighttime temperature of 16°C and a relative humidity of 85%-95%. This treatment continues for 10 days, with waterlogging damage to the plants recorded daily. Results indicate that waterlogging tolerance in cucurbits exhibits a partially dominant quantitative inheritance pattern.

[0023] According to the waterlogging tolerance performance of the F2 population, “single plants that died 3 days before death” were screened to construct an extremely waterlogging-intolerant mixed pool, and “single plants that were least affected by waterlogging 10 days after treatment” were screened to construct an extremely waterlogging-tolerant mixed pool.

[0024] (3) DNA extraction and mutation site screening The genomic DNA of leaves from the two extreme mixed pools of "J03", "YH2" and F2 was extracted using the conventional CTAB method. The purity and concentration of DNA were detected by agarose gel electrophoresis and NanoDrop 2000, respectively, to ensure that the DNA quality met the requirements of subsequent experiments.

[0025] Whole-genome resequencing was performed on the two parents, "J03" and "YH2", and the differential SNP sites between the parents were screened through bioinformatics analysis; combined with BSA (cluster segregation analysis) sequencing technology, the Gprime association algorithm was used to perform genotyping analysis on the differential sites in the F2 extreme mixed pool.

[0026] The results showed that there was a functional mutation site in the 9th exon region of gene Lsi09G006060 (encoding 50S ribosomal protein L2, i.e. RPL2) on chromosome 9 of cucurbita lappa, specifically a G→A mutation at nucleotide position 6589121 on chromosome 9 of cucurbita lappa (the sequences of gene Lsi09G006060 before and after mutation are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively).

[0027] Bioinformatics analysis revealed that the G→A mutation causes premature termination of protein synthesis encoded by the RPL2 gene (G corresponds to a waterlogging-tolerant phenotype, and A corresponds to a waterlogging-intolerant phenotype). Studies have shown that homologous genes of this gene are involved in reactive oxygen species metabolism, a crucial physiological pathway for plant responses to adverse stress. Under waterlogging conditions, hypoxia in plant roots triggers intracellular accumulation of reactive oxygen species. Disruption of reactive oxygen metabolism can lead to a range of damage, including cell membrane damage and protein degradation. It is hypothesized that in cucurbits, the RPL2 gene may regulate the synthesis of proteins involved in reactive oxygen species metabolism (as ribosomal proteins involved in protein biosynthesis), thereby affecting the plant's ability to scavenge reactive oxygen species under waterlogging stress and, in turn, altering waterlogging tolerance. The G→A mutation in this gene impairs the synthesis of the encoded protein, potentially disrupting the balance of reactive oxygen species metabolism and resulting in differences in waterlogging tolerance. Therefore, this mutation is preliminarily identified as a key functional site associated with waterlogging tolerance in cucurbits.

[0028] Example 2: Development of KASP molecular markers and primers related to waterlogging tolerance in cucurbits: Based on the "G→A mutation site 6589121 on chromosome 9 of gourd" screened in Example 1, this example designed and developed a KASP molecular marker and a matching primer set for detecting this site.

[0029] The KASP primer set of this embodiment includes an upstream primer F1, an upstream primer F2, and a downstream primer R. The upstream primer F1 is a primer binding to the waterlogging-tolerant site, with a nucleotide sequence as shown in SEQ ID NO. 1; the upstream primer F2 is a primer binding to the waterlogging-intolerant site, with a nucleotide sequence as shown in SEQ ID NO. 2; and the downstream primer R is a universal primer, with a nucleotide sequence as shown in SEQ ID NO. 3.

[0030] Example 3: Application and verification of the KASP molecular marker of the present invention: This example uses 27 cucurbit germplasm resources to verify the effectiveness of the KASP molecular marker developed in Example 2 in identifying waterlogging tolerance and screening cucurbit materials. The specific steps are as follows: (1) Prepare 27 gourd germplasm resources, with sample numbers as “Z063”, “JL082”, “Z077”, “SD0810”, “J081”, “C2002”, “DP-1”, “FH1”, “HGZ”, “HN1”, “J03”, “JTT”, “JZS”, “KZ”, “T2002”, “Y4”, “Y1”, “Z062”, “Z0618”, “Z078”, “Z0620”, “YH2”, “JTK01”, “JX0812”, “JX0815”, “TB10”, and “Shentongli”. The 27 materials are all recorded in the article "Xing Nailin, Fu Yujing, Wang Yinger, et al. Identification of weak light tolerance of gourd rootstock germplasm resources at the seedling stage [J]. Journal of Jiangxi Agricultural Sciences, 2019, 31(5):11-16" (the sample numbers of the 27 materials are consistent with the names in the article). The public can obtain the materials from the applicant within 20 years from the date of this application, only for repeating experiments related to the present invention.

[0031] (2) The seeds of the 27 materials were soaked and germinated before being sown in plug trays. When the seedlings grew to the one-leaf and one-heart stage, leaf samples were collected (for DNA extraction) and then treated according to the "Waterlogging Stress Identification Method" in Example 1 (the artificial climate chamber conditions, flooding depth, and treatment time were all the same). The treatment lasted for 10 days, and the waterlogging damage of the plants was investigated every 2 days. The plants were divided into three levels according to their waterlogging resistance: Tolerance to waterlogging: After 10 days of treatment, the plants showed no obvious wilting and grew normally; Relatively resistant to waterlogging: the plants wilt slightly after 10 days of treatment and can grow normally after water supply is restored; Intolerant to waterlogging: plants will wilt severely or die within 10 days of treatment and will not be able to resume growth.

[0032] (3) The genomic DNA of the leaves of the above 27 materials was extracted by CTAB method, and the DNA concentration was uniformly diluted to 20 ng / μL using NanoDrop 2000, which was used as the template for subsequent fluorescence quantitative PCR.

[0033] (4) Fluorescence quantitative PCR amplification detection was performed on the extracted genomic DNA using the molecular markers described in Example 2. The PCR reaction system configuration (total system 5 μL): 2×Taq DNA Polymerase Mix 2 μL, 4×SNP Primer Mix 1 μL, 20 ng / μL genomic DNA 2 μL; wherein, the preparation method of 4×SNP Primer Mix is ​​as follows: upstream primer F1, upstream primer F2 and downstream primer R dry powder were diluted to 100 μm / mL respectively, and then diluted and mixed according to the volume ratio of "upstream primer F1: upstream primer F2: downstream primer R: pure water = 1:1:2:4".

[0034] The PCR amplification program is shown in Table 1.

[0035] Table 1 PCR amplification procedure

[0036] (5) SNP detection uses the fluorophores FAM and VIC to distinguish two isogenic loci. The passive reference dye ROX is used to correct for signal differences between wells due to reaction volume errors. The relevant excitation and emission wavelengths are shown in Table 2. The reading software is LGC's OMEGA equipment.

[0037] Table 2 Related excitation and emission wavelengths

[0038] (6) Analyze the PCR amplification data using LGC_OMEGA's genotype reading software (Kluster Caller). Obtain the relative fluorescence values ​​corresponding to VIC and FAM for each PCR reaction well. Cluster the samples based on the relative fluorescence values, and further determine the genotype based on the sample clusters and fluorescence types.

[0039] The genotypes and waterlogging-tolerant phenotypes of the samples tested in this example are shown in Table 3 (tolerance levels: tolerant, relatively tolerant, and intolerant). The results showed that the genotypes and phenotypes were generally consistent: all five waterlogging-tolerant materials had a G / G genotype; among the seven waterlogging-intolerant materials, five had an A / A genotype and two had an A / G genotype; and among the 15 relatively waterlogging-tolerant materials, six had an A / A genotype, three had an A / G genotype, and six had a G / G genotype.

[0040] Table 3 Genotypes and phenotypes of samples

[0041] These results indicate that, although other genes associated with waterlogging tolerance exist in cucurbits besides the gene associated with this marker, this marker can significantly distinguish between extremely waterlogging-tolerant (G / G homozygous) and waterlogging-intolerant (mainly A / A homozygous) materials. Therefore, this method can be used to detect the genotypes of cucurbit materials and screen G / G homozygous materials as waterlogging-tolerant candidates. These selected waterlogging-tolerant candidates can then be tested under field waterlogging stress, with the goal of selecting materials with the least waterlogging damage and normal growth and development to cultivate waterlogging-tolerant cucurbit varieties.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A KASP molecular marker related to waterlogging tolerance in cucurbit based on the RPL2 gene, characterized in that: The molecular marker corresponds to a mutation site in the exon region of gene Lsi09G006060 on chromosome 9 of cucurbita; the gene Lsi09G006060 encodes 50S ribosomal protein L2; the mutation site is a G→A mutation at nucleotide position 6589121 on chromosome 9 of cucurbita.

2. The molecular marker according to claim 1, characterized in that The mutation site is specifically located in the 9th exon region of gene Lsi09G006060 on chromosome 9 of gourd.

3. A KASP primer set for detecting the molecular marker according to claim 1 or 2, characterized in that: It includes an upstream primer F1, an upstream primer F2 and a downstream primer R; wherein the nucleotide sequences of the upstream primer F1 and the upstream primer F2 are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively, and the nucleotide sequence of the downstream primer R is shown as SEQ ID NO.

3.

4. Use of the KASP molecular marker according to claim 1 or 2, or the KASP primer set according to claim 3, in detecting waterlogging tolerance of cucurbit, screening waterlogging-resistant cucurbit materials, and breeding waterlogging-resistant cucurbit varieties.

5. A method for detecting waterlogging tolerance of a gourd, characterized in that: The following steps are involved: (1) Extracting genomic DNA from the gourd material to be tested; (2) using the genomic DNA as a template and performing fluorescent quantitative PCR amplification using the KASP primer set described in claim 3; (3) Determine the genotype based on the fluorescence signal of the PCR product: if the genotype is G / G homozygous, the material is resistant to waterlogging or relatively resistant to waterlogging; if the genotype is A / A homozygous, the material is not resistant to waterlogging or relatively resistant to waterlogging; if the genotype is A / G heterozygous, the material is not resistant to waterlogging or relatively resistant to waterlogging.

6. The method according to claim 5, characterized in that In step (2), the PCR reaction system is 5 μL, including: 2 μL of 2 x Taq DNA Polymerase Mix, 1 μL of 4 x SNP Primer Mix, and 2 μL of 20 ng / μL genomic DNA.

7. The method according to claim 5, characterized in that In step (2), the PCR amplification program is as follows: pre-denaturation at 94°C for 10 min, 1 cycle; denaturation at 94°C for 20 s, annealing and extension at 61°C to 55°C for 45 s, for a total of 10 cycles, with the annealing and extension temperature decreasing by 0.6°C in each cycle; the second cycle, denaturation at 94°C for 20 s, annealing and extension at 55°C for 45 s, for a total of 37 cycles.

8. The method according to claim 5, characterized in that In step (3), the fluorescence signal is analyzed using KlusterCaller software of LGC_OMEGA, and the genotype is determined by the relative fluorescence values ​​of VIC and FAM.

9. A method for screening gourd waterlogging-resistant materials, characterized in that: The method described in any one of claims 5 to 8 is used to detect the genotype of the gourd material, and the G / G homozygous material is screened as a candidate material for waterlogging resistance.

10. A method for breeding gourd varieties resistant to waterlogging, characterized in that: The method described in any one of claims 5 to 8 is used to detect the genotype of the gourd material, and G / G homozygous materials are screened as candidate materials for waterlogging resistance; the screened candidate materials for waterlogging resistance are subjected to field waterlogging stress identification, and materials with the lowest degree of waterlogging damage and normal growth and development are selected to cultivate gourd varieties that are waterlogging-resistant.

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