A cucurbit waterlogging tolerance related KASP molecular marker, primer group and application

Abstract

The application provides a cucurbita wet tolerance related KASP molecular marker, which is located on a cucurbita chromosome 8, and a T to C mutation occurs at the 819th nucleotide of a gene Lsi08G001510 of the cucurbita chromosome 8; a KASP primer set for amplifying the molecular marker comprises a forward primer F1, a forward primer F2 and a reverse primer R, and the nucleotide sequences are shown as SEQ ID NO. 1-3. The application also provides a cucurbita wet tolerance related KASP primer set, and application of the KASP molecular marker and the primer set in cucurbita wet tolerance identification and variety breeding. Meanwhile, the application also provides a cucurbita wet tolerance identification method. The KASP molecular marker and the corresponding KASP primer developed by the application are used for cucurbita wet tolerance identification and variety breeding, can significantly improve the breeding efficiency and accuracy, and have the characteristics of no pollution and low cost.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a KASP molecular marker, primer set, and application related to waterlogging tolerance in gourd. Background Technology

[0002] The gourd, scientifically known as *Lagenaria siceraria* (Molina) Standl., is an annual vine-like herbaceous plant belonging to the Cucurbitaceae family. Its chromosome set is 2n=2x=2245. Other names include night-blooming gourd, bottle gourd, and calabash. Gourds are now widely cultivated globally. In my country, the planting area exceeded 2 million mu (approximately 133,333 hectares) in 2024. They are mainly used as rootstock for watermelon grafting, and are also used for food, daily necessities, crafts, and ornamental purposes. Gourds possess abundant germplasm and genetic resources.

[0003] In southern my country, due to high rainfall and the impact of typhoons and torrential rains, especially during the spring plum rain season and the summer and autumn typhoon season, waterlogging disasters are highly likely to occur. Waterlogging is a significant environmental factor limiting cucurbit production in southern regions. It first affects the crop root system, leading to oxygen deficiency and anaerobic metabolism, which in turn affects the overall growth and development of the crop, ultimately impacting yield and quality. Therefore, the breeding of waterlogging-tolerant gourd varieties has become an important indicator in gourd breeding in southern China.

[0004] Currently, the breeding of waterlogging-tolerant gourd varieties mainly relies on conventional breeding techniques, including field flooding treatment and laboratory seedling identification to determine their tolerance levels, and then screening for waterlogging-tolerant materials for related variety breeding. This method is time-consuming, costly, and lacks accuracy. Although there have been preliminary studies on the genetic patterns and molecular markers of gourd root traits, molecular marker-assisted techniques that can significantly improve breeding efficiency and accuracy have not yet been developed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a KASP molecular marker, primer set and application related to waterlogging tolerance of gourd. Based on the KASP molecular marker of the present invention, waterlogging tolerance identification and variety selection of gourd can be carried out, which can significantly improve the breeding efficiency and accuracy, while reducing the cost.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0007] A KASP molecular marker related to waterlogging tolerance in gourd is disclosed. The molecular marker is located on chromosome 8 of gourd, and a T→C mutation occurs at nucleotide position 819 of the gene Lsi08G001510 on chromosome 8. The KASP primer set used to amplify the molecular marker includes forward primer F1, forward primer F2, and reverse primer R. The nucleotide sequences of forward primer F1 and forward primer F2 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the nucleotide sequence of reverse primer R is shown in SEQ ID NO.3.

[0008] As one of the preferred embodiments of the present invention, the nucleotide sequence of the gene Lsi08G001510 is shown in SEQ ID NO.4.

[0009] A KASP primer set related to waterlogging tolerance in gourd includes forward primer F1, forward primer F2, and reverse primer R; wherein the nucleotide sequences of forward primer F1 and forward primer F2 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the nucleotide sequence of reverse primer R is shown in SEQ ID NO.3.

[0010] The application of the above-mentioned KASP molecular marker, or KASP primer set, in the identification of waterlogging tolerance and variety selection of gourd.

[0011] A method for identifying the waterlogging tolerance of gourds includes the following steps:

[0012] (1) Extract genomic DNA from the gourd samples to be tested;

[0013] (2) The above KASP primer set was used to perform PCR amplification of the gourd genomic DNA using a real-time PCR instrument;

[0014] (3) Analyze the amplification results to determine the waterlogging tolerance of the gourd sample to be tested.

[0015] As one of the preferred embodiments of the present invention, in step (2), the total volume of the PCR reaction system is 5 μL, including: 2 μL of 2xTaq DNAPolymerase Mix, 1 μL of Primer Mix, and 2 μL of Cucurbita genomic DNA.

[0016] As one of the preferred embodiments of the present invention, the preparation method of the Primer Mix is ​​as follows: dilute the dry powders of forward primer F1, forward primer F2 and reverse primer R to 100 μm / mL respectively, and then mix them according to the volume ratio of "forward primer F1:forward primer F2:reverse primer R1:pure water = 1:1:2:4" to obtain the Primer Mix.

[0017] As one of the preferred embodiments of the present invention, the concentration of the gourd genomic DNA is 20 ng / μL.

[0018] As one of the preferred embodiments of the present invention, in step (2), the PCR amplification program is as follows: 94℃ pre-denaturation for 10 min, 1 cycle; 94℃ denaturation for 20 s, 61℃~55℃ annealing extension for 45 s, a total of 10 cycles, with the annealing extension temperature decreasing by 0.6℃ in each cycle; the second cycle, 94℃ denaturation for 20 s, 55℃ annealing extension for 45 s, a total of 37 cycles.

[0019] As one of the preferred embodiments of the present invention, in step (3), the PCR amplification results data are analyzed using the LGC_OMEGA genotype reading software Kluster Caller to obtain the relative fluorescence values ​​corresponding to VIC and FAM for each PCR reaction well; the samples are clustered according to the relative fluorescence values, and the genotype is determined according to the sample cluster and fluorescence type; wherein, when the sample genotype is C / C, the sample is determined to be a water-resistant or relatively water-resistant gourd material; when the sample genotype is T / T or C / T, the sample is determined to be a non-water-resistant or relatively water-resistant gourd material.

[0020] The advantages of this invention compared to the prior art are:

[0021] This invention identifies waterlogging tolerance in gourd through seedling waterlogging treatment of two parents (waterlogging-tolerant and waterlogging-intolerant) and their reciprocal crosses (F1 generation). The study reveals that waterlogging tolerance is a predominantly dominant quantitative trait. Resequencing of both parents was then performed to screen for differentially expressed loci. After waterlogging treatment identification in the F2 population, a mixed pool of extremely waterlogging-tolerant and extremely waterlogging-intolerant individuals was constructed based on their waterlogging tolerance performance. Differential loci between the parents were used to analyze the mixed pool, and waterlogging tolerance was localized using the SNP Gprime method. Combined with bioinformatics analysis, waterlogging tolerance-related mutation sites were screened. These mutation sites were then validated in a natural population. For this mutation, this invention developed a related KASP molecular marker and corresponding KASP primers.

[0022] The KASP primer set developed in this invention is used for waterlogging tolerance identification and variety selection of gourd, which can significantly improve breeding efficiency and accuracy. It also has the characteristics of being pollution-free and low-cost (rapid detection can be performed at the seedling stage without the need for large-scale field cultivation). Detailed Implementation

[0023] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0024] Example 1: Screening and identification of waterlogging tolerance-related mutation sites:

[0025] (1) Prepare a water-tolerant gourd material J03, a water-intolerant gourd material YH2, and the F1 generation of the two as parents, and the F2 population obtained by self-pollination of the F1 generation.

[0026] Among them, two parental materials are recorded in the article "Xing Nailin, Fu Yujing, Wang Yinger, et al. Identification of seedling low light tolerance of gourd rootstock germplasm resources [J]. Jiangxi Journal of Agricultural Sciences, 2019, 31(5): 11-16". The public can obtain the gourd material from the applicant within twenty years from the date of application, and use it only for repeating the relevant experiments of this invention.

[0027] (2) J03, YH2, F1 and F2 were soaked and germinated respectively, and after sowing in plug trays, they were subjected to waterlogging treatment at the 1-leaf-1-heart stage for identification. The identification method was as follows:

[0028] ① Place the burrowing tray in an open box-shaped container with dimensions larger than the length and width of the burrowing tray, and the container should be at least 4cm deep;

[0029] ② Slowly add water to the bottom of the container until the substrate is saturated with water;

[0030] ③ Continue adding water to the container until the water level is 2cm above the top surface of the seedling tray;

[0031] ④ Place the container in an artificial climate chamber. Set the daytime temperature to 25℃, the light intensity to 20,000 lux, and the relative humidity to 70%–80%. Set the nighttime temperature to 16℃ and the relative humidity to 85%–95%.

[0032] ⑤ The treatment lasted for 10 days, and the damage from waterlogging was investigated and recorded daily. The results showed that waterlogging tolerance in gourd was a predominantly dominant quantitative trait. When mortality began to appear, F2 plants that died on that day, as well as on the 2nd and 3rd days, were selected to construct a "mixed pool of plants that are extremely intolerant to waterlogging". On the 10th day, F2 plants that were least affected by waterlogging were selected to construct a "mixed pool of plants that are extremely tolerant to waterlogging".

[0033] (3) DNA was extracted from the leaves of the parents and the two extreme mixed pools of F2. The parents were resequencing to detect the differential sites between them.

[0034] Using BSA sequencing and in conjunction with the reference genome of Lagenariasiceraria var.USVL1VR-Ls published by Wu S et al. (2017) (http: / / www.cucurbitgenomics.org / organism / 13), a mutation of nucleotide T→C at position 819 on the gene Lsi08G001510 on chromosome 8 of Lagenaria (nucleotide sequence as shown in SEQ ID NO.4, used to express Protein disulfide isomerase (PDI)-like protein 3) was detected by the Gprime method.

[0035] This mutation results in a functional shift from arginine to glycine. Furthermore, homologs of this gene have been reported in some articles to potentially be associated with abiotic stresses such as waterlogging.

[0036] Therefore, this gene has been preliminarily identified as a gene related to waterlogging tolerance in gourd. Molecular markers can be developed to target this mutation.

[0037] Example 2: Development of KASP molecular markers related to waterlogging tolerance in gourd:

[0038] This embodiment develops KASP molecular markers and corresponding KASP primer sets related to waterlogging tolerance in gourd based on the mutation sites discovered in Example 1.

[0039] This embodiment of the KASP primer set includes forward primer F1, forward primer F2, and reverse primer R. Forward primer F1 is a water-resistant binding primer, with the nucleotide sequence shown in SEQ ID NO.1; forward primer F2 is a water-intolerant binding primer, with the nucleotide sequences shown in SEQ ID NO.2; and reverse primer R is a universal primer, with the nucleotide sequence shown in SEQ ID NO.3.

[0040] Example 3: Application of the KASP molecular marker of the present invention:

[0041] (1) Prepare 27 gourd germplasm resources (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, Shentongli).

[0042] The above 27 gourd germplasm resources are recorded in the article “Xing Nailin, Fu Yujing, Wang Yinger, et al. Identification of seedling low light tolerance of gourd rootstock germplasm resources [J]. Jiangxi Journal of Agricultural Sciences, 2019, 31(5): 11-16”. The public can obtain the gourd materials from the applicant within twenty years from the date of application, and use them only for repeating the relevant experiments of this invention.

[0043] (2) The 27 prepared materials were sown separately, and the leaves were sampled when they reached the 1-leaf-1-heart stage. Waterlogging tolerance was then assessed. The assessment method was as follows:

[0044] ① Place the seedling tray in an open box-shaped container with dimensions larger than the length and width of the seedling tray, and the container should be at least 4cm deep. ② Slowly add water to the bottom of the container until the substrate is saturated with water. ③ Continue adding water to the container until the water level is 2cm above the top surface of the seedling tray. ④ Place the container in an artificial climate chamber, set the daytime temperature to 25℃, the light intensity to 20000 lux, and the relative humidity to 70%–80%. Set the nighttime temperature to 16℃ and the relative humidity to 85%–95%. (5) Treat for 10 days, and investigate the waterlogging damage every 2 days.

[0045] (3) Genomic DNA was extracted from the leaves sampled in step (2), and the concentration was measured using a NanoDrop 2000. Each DNA stock solution was diluted to 20 ng / μL. The genomic DNA of the gourd was then amplified by PCR using the molecular markers described in Example 2 via a real-time PCR instrument.

[0046] The PCR reaction system (5 μL) includes: 2 μL of 2x Taq DNA Polymerase Mix, 1 μL of Primer Mix, and 2 μL of Cucurbita genomic DNA. The Primer Mix is ​​prepared as follows: dilute the dry powders of forward primer F1, forward primer F2, and reverse primer R to 100 μm / mL, then mix them according to the volume ratio of "forward primer F1:forward primer F2:reverse primer R1:pure water = 1:1:2:4".

[0047] The PCR amplification procedure is shown in Table 1.

[0048] Table 1 PCR amplification program

[0049]

[0050] (4) SNP site detection used the fluorophores FAM and VIC to distinguish between two isogenetic loci. The passive reference dye ROX was 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 was an LGC OEMGA device.

[0051] Table 2 shows the relevant excitation and emission wavelengths.

[0052] fluorescent groups Excitation light (nm) Emitted light (nm) FAM 485 520 VIC 535 556 ROX 575 610

[0053] (5) The PCR amplification results were analyzed using LGC_OMEGA's genotype reading software (Kluster Caller). The relative fluorescence values ​​of VIC and FAM for each PCR reaction well were obtained. Based on the relative fluorescence values, the samples were clustered, and the genotypes were further determined based on the sample clusters and fluorescence types.

[0054] The genotype and phenotype results of the samples measured in this embodiment are shown in Table 3. The genotype and phenotype results are generally consistent. Among the 5 waterlogging-resistant materials, the genotype is C / C; among the 7 non-waterlogging-resistant materials, 6 have the genotype T / T and 1 has the genotype C / T; among the 15 relatively waterlogging-resistant materials, 4 have the genotype C / C, 5 have the genotype C / T, and 6 have the genotype T / T.

[0055] This indicates that although other waterlogging tolerance-related genes exist in gourds besides those associated with this marker, this marker can significantly distinguish between "extremely waterlogged tolerant materials" and "waterlogged intolerant materials." For example, when the sample genotype is C / C, the sample can be identified as a waterlogged tolerant gourd material or a relatively waterlogged tolerant gourd material (i.e., definitely not a "waterlogged intolerant material"); when the sample genotype is T / T or C / T, the sample can be identified as a waterlogged intolerant gourd material or a relatively waterlogged tolerant gourd material (i.e., definitely not an "extremely waterlogged material").

[0056] Therefore, the KASP molecular marker method developed in this invention can be used for the creation of moisture-tolerant gourd germplasm resources and variety selection.

[0057] Table 3 Sample Genotypes and Phenotypes

[0058]

[0059]

[0060] 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 within the protection scope of the present invention.

Claims

1. The application of a KASP primer set in the identification of waterlogging tolerance and variety selection of gourd, characterized in that, The KASP primer set includes forward primer F1, forward primer F2, and reverse primer R; wherein the nucleotide sequences of forward primer F1 and forward primer F2 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the nucleotide sequence of reverse primer R is shown in SEQ ID NO.3; when the sample genotype is C / C, the sample is determined to be a waterlogged-tolerant or relatively waterlogged-tolerant gourd material; when the sample genotype is T / T or C / T, the sample is determined to be a waterlogged-intolerant or relatively waterlogged-tolerant gourd material.

2. A method for identifying the waterlogging tolerance of gourd, characterized in that, Includes the following steps: (1) Extract genomic DNA from the gourd sample to be tested; (2) Using the KASP primer set described in claim 1, PCR amplification of gourd genomic DNA was performed using a real-time PCR instrument; (3) Analyze the amplification results to determine the waterlogging tolerance of the gourd sample to be tested; when the sample genotype is C / C, the sample is determined to be a waterlogging tolerant gourd material or a relatively waterlogging tolerant gourd material; when the sample genotype is T / T or C / T, the sample is determined to be a waterlogging intolerant gourd material or a relatively waterlogging tolerant gourd material.

3. The method for identifying the waterlogging tolerance of gourd according to claim 2, characterized in that, In step (2), the total volume of the PCR reaction system is 5 μL, including: 2 x Taq DNA Polymerase Mix 2 μL, Primer Mix 1 μL, and Cucurbita genomic DNA 2 μL.

4. The method for identifying the waterlogging tolerance of gourd according to claim 3, characterized in that, The Primer Mix is ​​prepared as follows: dilute the dry powders of forward primer F1, forward primer F2 and reverse primer R to 100 μm / mL, and then mix them according to the volume ratio of "forward primer F1:forward primer F2:reverse primer R1:pure water = 1:1:2:4" to obtain the Primer Mix.

5. The method for identifying the waterlogging tolerance of gourd according to claim 3, characterized in that, The concentration of the gourd genomic DNA was 20 ng / μL.

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

7. The method for identifying the waterlogging tolerance of gourd according to claim 2, characterized in that, In step (3), the PCR amplification results data are analyzed using the LGC_OMEGA genotype reading software Kluster Caller to obtain the relative fluorescence values ​​of VIC and FAM for each PCR reaction well. The samples were clustered based on their relative fluorescence values, and the genotypes were determined based on the sample clusters and fluorescence types.

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