KASP molecular marker related to waterlogging resistance of cucurbit, primer group and application

Abstract

The invention provides a KASP molecular marker related to waterlogging resistance of cucurbit, which is positioned on a chromosome 8 of the cucurbit, and the nucleotide at the 819th site of the gene Lsi08G001510 of the chromosome 8 of the cucurbit is subjected to T-C mutation; the KASP primer group for amplifying the molecular marker comprises a forward primer F1, a forward primer F2 and a reverse primer R, and nucleotide sequences of the forward primer F1, the forward primer F2 and the reverse primer R are respectively shown as SEQ ID NO.1-3. The invention also provides a KASP primer group related to the waterlogging resistance of the cucurbit, and application of the KASP molecular marker and the primer group in waterlogging resistance identification and variety breeding of the cucurbit. Meanwhile, the invention further provides a method for identifying the waterlogging resistance of the cucurbit. The KASP molecular marker and the corresponding KASP primer developed by the invention are used for identifying the waterlogging tolerance of the cucurbit and breeding varieties, the breeding efficiency and accuracy can be remarkably improved, and meanwhile, the KASP molecular marker has the characteristics of no pollution and low cost.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a KASP molecular marker related to waterlogging tolerance of gourd, a primer set and an application thereof. Background Art

[0002] Gourd is an annual vine herbaceous plant of the Cucurbitaceae family, with a scientific name of Lagenaria siceraria (Molina) Standl., a chromosome set of 2n=2x=2245, and aliases such as night-blooming gourd, gourd, and gourd. Gourd is now a widely cultivated crop in the world. In my country, the planting area exceeded 2 million mu in 2024. It is mainly used as a rootstock for watermelon grafting, for food, daily use, and for cultural and artistic craft processing and ornamental purposes, with rich germplasm and gene resources.

[0003] In southern my country, due to heavy rainfall and the influence of typhoons and rainstorms, especially in the spring rainy season and the summer and autumn typhoon rainy season, waterlogging disasters are very likely to occur. Waterlogging is an important environmental factor that restricts the production of melons in southern China. It first affects the root system of crops, causing them to lack oxygen, causing crops to undergo anaerobic metabolism, and then affecting the overall growth and development of crops, and ultimately affecting crop yield and quality. Therefore, the selection and breeding of waterlogging-resistant gourd varieties has become an important indicator for gourd breeding in southern China.

[0004] However, at present, the breeding of waterlogging-resistant varieties of gourds mainly relies on the application of conventional breeding technology, relying on field flooding treatment and laboratory seedling identification to identify their tolerance level, and then screen waterlogging-resistant materials for relevant variety breeding. This method has a long cycle, high cost, and low accuracy. Although there have been preliminary studies on the genetic laws and molecular markers of the root traits of gourds, molecular marker-assisted technologies 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 related to waterlogging tolerance of gourd, a primer set and application. Based on the KASP molecular marker of the present invention, waterlogging tolerance identification and variety breeding of gourd can be carried out, which can significantly improve breeding efficiency and accuracy while reducing costs.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A KASP molecular marker related to waterlogging tolerance of gourd, wherein the molecular marker is located on chromosome 8 of gourd, and a T→C mutation occurs at the 819th nucleotide of gene Lsi08G001510 on chromosome 8 of gourd; a KASP primer set for amplifying the molecular marker comprises a forward primer F1, a forward primer F2 and a reverse primer R; wherein the nucleotide sequences of the forward primer F1 and the forward primer F2 are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively, and the nucleotide sequence of the reverse primer R is shown as 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 of cucurbit includes a forward primer F1, a forward primer F2 and a reverse primer R; wherein the nucleotide sequences of the forward primer F1 and the forward primer F2 are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively, and the nucleotide sequence of the reverse primer R is shown as SEQ ID NO.3.

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

[0011] A method for identifying waterlogging tolerance of gourd comprises the following steps:

[0012] (1) extracting genomic DNA of the gourd sample to be tested;

[0013] (2) using the KASP primer set to perform PCR amplification reaction on the cucurbit genomic DNA using a fluorescent quantitative 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 DNA Polymerase Mix, 1 μL of Primer Mix, and 2 μL of cucurbit genomic DNA.

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

[0017] As one of the preferred embodiments of the present invention, the concentration of the cucurbit 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: 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; in the second round of cycles, denaturation at 94°C for 20 s, annealing and extension at 55°C for 45 s, for a total of 37 cycles.

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

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

[0021] The present invention identifies two parents (waterlogging tolerant and waterlogging intolerant) and their reciprocal cross F1 during seedling stage waterlogging treatment, and finds that waterlogging tolerance of gourd is quantitatively dominant, and then resequencing the two parents to screen the differential sites. After the F2 population is identified by waterlogging treatment during seedling stage, a mixed pool of extreme waterlogging tolerance and extreme waterlogging intolerant is constructed according to the waterlogging tolerance performance, and the mixed pool is tested using the differential sites between the parents, and the waterlogging tolerance is located using the SNP Gprime method, combined with bioinformatics analysis, and the waterlogging tolerance-related mutation sites are screened. Through natural population verification, the mutation sites related to waterlogging tolerance of gourd are obtained. In response to this mutation, the present invention develops relevant KASP molecular markers and corresponding KASP primers.

[0022] The KASP primer set developed by the present invention is used for identification of waterlogging tolerance and variety breeding of cucurbits, which can significantly improve breeding efficiency and accuracy, and 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 DESCRIPTION

[0023] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the experimental methods used in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0024] Example 1, Screening and determination of mutation sites related to waterlogging tolerance:

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

[0026] Among them, the two parental materials are recorded in the article "Xing Nailin, Fu Yujing, Wang Yinger, et al. Identification of weak light tolerance of gourd rootstock germplasm resources in the seedling stage [J]. Journal of Jiangxi 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, only for repeating experiments related to the present invention.

[0027] (2) Seeds of J03, YH2, F1 and F2 were soaked and germinated respectively. After sowing in plug trays, they were flooded at the one-leaf and one-heart stage. The treatment and identification methods were as follows:

[0028] ① Place the plug tray in an open box-shaped container with a length and width larger than that of the plug tray, and the depth of the container should be more than 4 cm;

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

[0030] ③Continue to add water into the container until the water level is 2 cm higher than the upper surface of the plug tray;

[0031] ④ Place the container in an artificial climate chamber, set the daytime temperature at 25°C, the light at 20,000 lux, and the relative humidity at 70% to 80%; set the nighttime temperature at 16°C and the relative humidity at 85% to 95%;

[0032] ⑤ Treat for 10 days, and investigate and record the waterlogging damage every day. The results showed that waterlogging tolerance of gourd is quantitatively dominant. When death began to occur, the F2 single plant materials that died on that day and on the 2nd and 3rd days were selected to construct the "extremely intolerant to waterlogging mixed pool", and on the 10th day, the F2 single plant materials that were least affected by waterlogging were selected to construct the "extremely tolerant to waterlogging mixed pool".

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

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

[0035] This mutation leads to a functional change from arginine to glycine. At the same time, the homologous gene of this gene has been reported in related articles to be related to adverse stress such as waterlogging.

[0036] Therefore, the gene was preliminarily identified as a gene related to waterlogging tolerance in cucurbit. Related molecular markers can be developed for this mutation.

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

[0038] This example develops KASP molecular markers related to waterlogging tolerance of cucurbit based on the mutation sites found in Example 1 and the corresponding KASP primer sets.

[0039] The KASP primer set of this embodiment includes a forward primer F1, a forward primer F2 and a reverse primer R. Among them, the forward primer F1 is a waterlogging-tolerant site binding primer, and the nucleotide sequence is shown in SEQ ID NO.1; the forward primer F2 is a waterlogging-intolerant site binding primer, and the nucleotide sequence is shown in SEQ ID NO.2; the reverse primer R is a universal primer, and the nucleotide sequence is shown in SEQ ID NO.3.

[0040] Example 3: Application of KASP molecular markers 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 weak light tolerance of gourd rootstock germplasm resources in the seedling stage [J]. Journal of Jiangxi Agricultural Sciences, 2019, 31(5): 11-16". The public can obtain the gourd materials from the applicant within 20 years from the date of application, only for repeating experiments related to the present invention.

[0043] (2) The 27 prepared materials were sown separately and grown to the 1-leaf 1-heart stage. Leaf samples were taken and then waterlogging tolerance treatment was identified. The treatment identification method was:

[0044] ① Place the plug tray in an open box-shaped container with a length and width larger than that of the plug tray, and the container should be more than 4 cm deep; ② Slowly add water to the bottom of the container until the substrate is saturated with water; ③ Continue to add water to the container until the water level is 2 cm above the upper surface of the plug tray; ④ Place the container in an artificial climate chamber, set the daytime temperature at 25°C, the light at 20,000 lux, and the relative humidity at 70% to 80%; set the nighttime temperature at 16°C and the relative humidity at 85% to 95%; (5) Treat for 10 days and investigate the damage caused by waterlogging every 2 days.

[0045] (3) Extract genomic DNA from the leaves sampled in step (2), measure the concentration using NanoDrop 2000, and dilute each DNA stock solution to 20 ng / μL. Then, use the molecular markers described in Example 2 to perform PCR amplification of the gourd genomic DNA using a fluorescent quantitative 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 cucurbit genomic DNA. The Primer Mix is ​​prepared as follows: the forward primer F1, forward primer F2, and reverse primer R dry powder are diluted to 100 μm / mL respectively, and then diluted and mixed 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 program is shown in Table 1.

[0048] Table 1 PCR amplification program

[0049]

[0050] (4) SNP site detection uses the fluorophores FAM and VIC to distinguish two isogenic sites. Passive reference dye ROX is used to correct the signal difference between wells due to reaction volume errors. The relevant excitation and emission wavelengths are shown in Table 2. The reading software is the OEM equipment of LGC.

[0051] Table 2 Related excitation and emission wavelengths

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

[0053] (5) Analyze the PCR amplification result 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.

[0054] The genotypes and phenotypes of the samples tested in this example are shown in Table 3. The genotypes and phenotypes of the results are consistent as a whole. The genotypes of the 5 waterlogging-tolerant materials are all C / C; among the 7 waterlogging-intolerant materials, the genotypes of 6 are T / T and 1 is C / T; among the 15 waterlogging-tolerant materials, the genotypes of 4 are C / C, 5 are C / T, and 6 are T / T.

[0055] This indicates that, although there are other waterlogging tolerance-related genes in the gourd besides the genes associated with this marker, the marker can be used to significantly distinguish "extreme waterlogging tolerance materials" from "waterlogging intolerant materials". For example, when the sample genotype is C / C, it can be determined that the sample is a waterlogging tolerant gourd material or a waterlogging tolerant gourd material (i.e., it is definitely not a waterlogging intolerant material); when the sample genotype is T / T or C / T, it can be determined that the sample is a waterlogging intolerant gourd material or a waterlogging tolerant gourd material (i.e., it is definitely not a waterlogging intolerant material).

[0056] Therefore, the KASP molecular marker method developed in the present invention can be used for the creation of moisture and waterlogging resistant gourd germplasm resources and variety breeding.

[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 in the protection scope of the present invention.

Claims

1. A KASP molecular marker related to waterlogging tolerance of gourd, characterized in that: The molecular marker is located on chromosome 8 of calabash, and a T→C mutation occurs at the 819th nucleotide of gene Lsi08G001510 on chromosome 8 of calabash; the KASP primer set used to amplify the molecular marker includes a forward primer F1, a forward primer F2 and a reverse primer R; wherein the nucleotide sequences of the forward primer F1 and the forward primer F2 are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively, and the nucleotide sequence of the reverse primer R is shown as SEQ ID NO.

3.

2. The KASP molecular marker related to waterlogging tolerance of cucurbit according to claim 1, characterized in that: The nucleotide sequence of the gene Lsi08G001510 is shown in SEQ ID NO.

4.

3. A KASP primer set related to waterlogging tolerance of gourd, characterized in that: It comprises a forward primer F1, a forward primer F2 and a reverse primer R; wherein the nucleotide sequences of the forward primer F1 and the forward primer F2 are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively, and the nucleotide sequence of the reverse 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 the identification of waterlogging tolerance and variety breeding of cucurbits.

5. A method for identifying waterlogging tolerance of gourd, characterized in that: The steps include: (1) extracting genomic DNA of the gourd sample to be tested; (2) using the KASP primer set described in claim 3 to perform PCR amplification reaction on the genomic DNA of the gourd using a fluorescent quantitative PCR instrument; (3) Analyze the amplification results to determine the waterlogging tolerance of the gourd sample to be tested.

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

7. The method for identifying waterlogging tolerance of bottle gourd according to claim 6, characterized in that: The preparation method of the Primer Mix is ​​as follows: dilute the forward primer F1, forward primer F2 and reverse primer R dry powder to 100 μm / mL respectively, and then dilute and 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.

8. The method for identifying waterlogging tolerance of bottle gourd according to claim 6, characterized in that: The concentration of the cucurbit genomic DNA is 20 ng / μL.

9. The method for identifying waterlogging tolerance of bottle gourd according to claim 5, characterized in that: In the step (2), the PCR amplification program is: pre-denaturation at 94°C for 10 min, 1 cycle; denaturation at 94°C for 20 s, annealing and extension at 61°C-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; in 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.

10. The method for identifying waterlogging tolerance of bottle gourd according to claim 5, characterized in that: In the step (3), the PCR amplification result data is analyzed using the genotype reading software Kluster Caller of LGC_OMEGA to obtain the relative fluorescence values ​​corresponding to VIC and FAM of each PCR reaction well; According to the relative fluorescence values, the samples were clustered and the genotypes were determined according to the sample clusters and fluorescence types. When the sample genotype was C / C, the sample was determined to be a moisture-resistant gourd material or a relatively moisture-resistant gourd material. When the sample genotype was T / T or C / T, the sample was determined to be an intolerant gourd material or a relatively moisture-resistant gourd material.

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