Molecular marker primer of winter melon fruit type major gene BhFS2.1 as well as marking method and application of molecular marker primer

Abstract

The invention relates to the technical field of molecular genetics, in particular to a molecular marker primer of a winter melon fruit type major gene BhFS2.1 as well as a marking method and application of the molecular marker primer. According to the molecular marker primer, an F2 genetic population is obtained by hybridizing and selfing a long and thin white gourd material and a thick and short white gourd material, and then QTL detection is performed by combining the genotype and phenotype of the F2 genetic population; a major gene for controlling the fruit type of the winter melon is successfully identified and named as BhFS2.1, a molecular marker closely linked with the gene is developed, two pairs of simple and easy-to-use functional markers 2M57.86 and 2M59.91 are obtained, and experimental verification shows that the two molecular markers can accurately judge whether a breeding intermediate material is a slender fruit type wax gourd or a thick and short fruit type wax gourd in the seedling stage, so that the breeding intermediate material can be quickly and accurately selected. The method can be used for quickly screening required fruit type strains (species) in the seedling stage, and the detection is convenient, quick and convenient and is not influenced by the environment.

Description

[Technical field]

[0001] The present invention relates to the technical field of molecular genetics, and in particular to a molecular genetics primer of a wax gourd fruit type major gene BhFS2.1 and a labeling method and application thereof. [Background technology]

[0002] Winter melon (Benincasa hispida Cogn.) is an annual vine herbaceous plant of the genus Wax Gourd in the Cucurbitaceae family. It has high nutritional value and therapeutic effects. It is an important vegetable crop and food processing raw material for my country's southern vegetables to the north. At present, the fruit shape of winter melons on the market in my country is relatively single, mainly cylindrical, and the variety is single, which limits the development of winter melon fruit shape diversity and provides consumers with limited choice. Exploring the main effect genes that control the fruit shape of winter melon and using molecular markers to assist in the cultivation of new fruit type varieties are green and economical effective measures that are in line with the market and can achieve results in a short period of time.

[0003] At present, there are not many studies on wax gourd fruit shape, and even fewer related patents. Guangxi University used the nearly spherical wax gourd inbred line MY-1 (fruit shape index = 1.06) and the long cylindrical wax gourd inbred line GX-71 (fruit shape index = 4.56) as parents to construct 4 generations of genetic populations (P1, P2, F1, F2), and used the plant quantitative trait major gene + polygene mixed genetic model analysis method to study the genetic law of fruit shape; cluster segregation analysis (BSA-seq) combined with KASP analysis to locate the candidate gene that controls wax gourd fruit shape (fruit shape index). It was found that there was a very significant correlation between the fruit longitudinal diameter, fruit transverse diameter and fruit shape index, among which the fruit shape index was controlled by 2 pairs of additive-dominant-epistatic major genes + additive-dominant-epistatic polygenes, and the major gene inheritance rate was 97.03%. BSA-seq was initially located on chromosome 2 and finely located within the interval of 19,571bp in physical distance. Bch02G01683 (a member of the IQD protein family) was identified as the target gene, which regulates wax gourd fruit shape by affecting the number and size of cells. The Vegetable Research Institute of Guangdong Academy of Agricultural Sciences has developed an Indel molecular marker that is closely linked to wax gourd fruit size. The marker primers are located for the genetic population constructed with large-fruit wax gourd B227 and small-fruit wax gourd HF3 as parents. A fruit type major gene BhFS was located within the interval of 16.80-16.90Mb on chromosome 10. After designing primers, a large-fruit specific marker BhFS168 of 168bp and a small-fruit specific marker BhFS173 of 173bp were obtained. The markers have good repeatability and strong specificity. They can be used to identify wax gourd fruit size phenotypes in the seedling stage, and can accurately, quickly and effectively identify wax gourd fruit size.

[0004] Due to the complex genetics of winter melon, long breeding cycle, cumbersome operation and other factors, the breeding process of winter melon is slow. The use of molecular assisted selection (Marker-assisted selection, MAS) technology can effectively solve this problem and improve the efficiency of breeding. Therefore, it is urgent to carry out more research and marker development related to winter melon fruit types, simplify breeding steps and procedures, and improve breeding efficiency. [Summary of the invention]

[0005] In view of the above, it is necessary to provide a molecular marker primer for the major effect gene BhFS2.1 of wax gourd fruit type and its marking method and application. This molecular marker can distinguish the fruit type of wax gourd in the seedling stage, providing a new idea for the future genetic breeding of wax gourd, and can greatly shorten the cultivation cycle for breeding of wax gourd fruit type.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The invention discloses an application of a primer set for detecting a molecular marker of the major gene BhFS2.1 for fruit type of wax gourd in identifying wax gourd fruit type lines or varieties at the seedling stage, wherein the primer set comprises a primer pair 2M57.86 and / or a primer pair 2M59.91; the nucleic acid sequence of the primer pair 2M57.86 is as shown in SEQ ID NO.1-2 (5'-AGTTGTATTGGGAGGGTATG-3'; the downstream primer sequence is: 5'-GGGGCATCAACAATCTTTCT-3'); the nucleic acid sequence of the primer pair 2M59.91 is as shown in SEQ ID NO.3-4 (5'-GAGGAGGGAGCTAAGATGAA-3'; the downstream primer sequence is: 5'-CAAAATCATCTGGCAACAAG-3'); the wax gourd materials are a short and thick fruit type parent 2020-mdL and a long and thin fruit type parent 2019-ss-4-1.

[0008] The present invention also includes a method for breeding wax gourd fruit-type strains or varieties at the seedling stage using the primer set, the method comprising:

[0009] Primer pair 2M57.86 was used to amplify the genomic DNA of the wax gourd sample to be tested. If a target fragment of 111 bp could be amplified, it means that the wax gourd sample is a slender fruit type wax gourd; if a target fragment of 76 bp could be amplified, it means that the wax gourd sample is a thick and short fruit type wax gourd; if two bands could be amplified, it means that the wax gourd sample is a medium fruit type wax gourd;

[0010] Or use primer pair 2M59.91 to amplify the genomic DNA of the wax gourd sample to be tested. If a 133bp target fragment can be amplified, it means that the wax gourd sample is a slender fruit type wax gourd; if a 103bp target fragment can be amplified, it means that the wax gourd sample is a thick and short fruit type wax gourd; if two bands can be amplified, it means that the wax gourd sample is an intermediate fruit type wax gourd.

[0011] The present invention has the following beneficial effects:

[0012] 1. The present invention newly detected a wax gourd fruit type major gene on chromosome 2 of wax gourd and developed molecular markers that are closely linked to and co-segregated with the gene. The invention then carried out in-depth design and modification on several pairs of molecular markers to obtain two pairs of simple and easy-to-use functional markers 2M57.86 and 2M59.91. With the help of MAS technology, the breeding of fruit type positive single plants or lines (species) can be effectively carried out, thereby shortening the breeding cycle of wax gourd fruit type varieties. Experimental verification shows that the two molecular markers of the present invention can accurately determine whether the intermediate breeding material is a slender fruit type wax gourd, a thick and short fruit type wax gourd, or an intermediate fruit type wax gourd with the characteristics of both at the seedling stage, thereby quickly screening the desired fruit type lines (species). The detection is convenient, fast and easy, and is not affected by the environment.

[0013] 2. The auxiliary breeding selection has clear goals and saves costs. In the traditional breeding method, it is first necessary to collect materials with different fruit types, create intermediate materials through a series of approaches such as hybridization, backcrossing and self-pollination, and identify and select the longitudinal diameter, transverse diameter and fruit type index of wax gourd. The operation is cumbersome and complicated, and is easily affected by the environment. At the same time, the fruit type identification cycle is long, and statistical analysis can only be performed after the fruit is mature and stable, which takes a lot of time. Therefore, the traditional wax gourd breeding cycle is long, difficult, and costly; and the method of the present invention uses molecular markers to detect the main effect gene loci of fruit type, which is used for the genotype detection of strains (species) in wax gourd breeding. Positive individual plants carrying the required fruit type can be identified in the wax gourd seedling stage, and other unnecessary plants are eliminated, which greatly improves the screening efficiency of wax gourd fruit type cultivation, saves production costs, and promotes the breeding process of new fruit type strains (species) of wax gourd.

Brief Description of the Drawings

[0014] Figure 1 This is a phenotypic identification diagram of the fruit types of winter melon 2019-ss-4-1, 2020-mdL and F1 plants.

[0015] Figure 2 This is the phenotypic distribution map of the fruit type index of the winter melon 2020-mdL / 2019-ss-4-1F2 population.

[0016] Figure 3 This is the location map of the wax gourd fruit type major gene BhFS2.1 on chromosome 2.

[0017] Figure 4 This is the agarose gel electrophoresis detection diagram of the amplified products of molecular marker 2M57.86 in different wax gourd materials.

[0018] Figure 5 This is the agarose gel electrophoresis detection diagram of the amplified products of molecular marker 2M59.91 in different wax gourd materials.

[0019] Figure 4-Figure 5 In the figure, M is BM2000 DNA marker, and the band sizes from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp, respectively; R is 2019-ss-4-1, S is 2020-mdL, lanes 1-4 are 4 negative wax gourd materials (all with short and thick fruit type), and lanes 5-12 are part of 2020-mdL / 2019-ss-4-1F 2:3 The arrow points to the target band of R elongated fruit material 2019-ss-4-1. In the positive samples, there is a target band on the same horizontal line as R (111bp, 2M57.86, Figure 4 ; or 133bp, 2M59.91, Figure 5 ), while this band was not present in negative samples. [Specific implementation method]

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0021] The parents used in the embodiments of the present application: the slender variety 2019-ss-4-1 (longitudinal diameter = 112.0 cm, horizontal diameter = 10.2 cm, fruit type index = 10.99) was collected by the applicant from the wild in Baise City, Guangxi Zhuang Autonomous Region, China, and the short and thick variety 2020-mdL (longitudinal diameter = 58.3 cm, horizontal diameter = 20.9 cm, fruit type index = 2.79) was collected by the applicant from the wild in Guilin City, Guangxi Zhuang Autonomous Region, China; they are now stored in the germplasm resource bank of this institute, and the applicant promises that any unit and individual can obtain the above varieties from our unit.

[0022] Example 1

[0023] This embodiment is a scheme for obtaining molecular markers, which is as follows:

[0024] Acquisition of molecular markers

[0025] 1. Construction of six-generation materials and phenotypic identification

[0026] (1) Construction of six generations (P1, P2, F1, F2, BC1P1, BC1P2) of materials. The early fruit type identification experiment showed that the wax gourd material 2019-ss-4-1 was an elongated fruit type with a fruit type index of 9.03, while 2020-mdL was a short and thick fruit type with a fruit type index of 2.92. In order to locate and clone the fruit type locus, the present invention used the short and thick wax gourd material 2020-mdL as the female parent and the elongated material 2019-ss-4-1 as the male parent to hybridize to obtain F1, and F1 was backcrossed with the parents to obtain BC1P1 and BC1P2. I P2, F1 self-pollination generation obtained 2020-mdL / 2019-ss-4-1F2 genetic segregation population, six generations of materials were used for wax gourd fruit type index genetic law and phenotypic identification; F2 self-pollination generation obtained the corresponding F 2:3 Family, used for screening of recombinant individual strains.

[0027] (2) Statistics of fruit type index phenotype. The two parents (2019-ss-4-1 and 2020-mdL) and a total of 201 F2 plants were planted in the field. Self-pollination was performed during the flowering period. One upper fruit was kept on each plant, and the other fruits were thinned out and routine production management was performed. When the fruit matured, the agronomic traits such as the length and diameter of the fruit were counted, and the fruit type index was calculated.

[0028] 2. Molecular marker analysis of the 2020-mdL / 2019-ss-4-1F2 population

[0029] (1) According to the average fruit type index of the F2 population, 30 extremely slender plants and 30 extremely short and thick plants were selected, and their leaf genomic DNA was extracted using the CTAB method (Murray et al. 1980). Equal amounts of DNA were mixed to construct slender and short DNA pools, which were sent to the company for BSA-seq sequencing to obtain basic information for preliminary positioning. In view of the large number of positioning intervals and the large range, insertion-deletion (InDel) markers were developed in the target region for verification based on the resequencing data of both parents.

[0030] (2) Extract leaf genomic DNA from parents 2019-ss-4-1 and 2020-mdL and each individual plant in the F2 population. The two parents were screened for polymorphism using the verification markers obtained in step (1), and the PCR reaction was performed on a BIOER amplification instrument. PCR reaction system: 0.5 μL DNA template, 0.3 μL 10 μM primer, 0.2 μL 10 mM dNTP, 1 μL 10×PCR Buffer (containing Mg2+), 0.1 μL TaqDNApolymerase (5U / μL), and 10 μL with ddH2O. The amplification product length of the InDel primers used in this experiment is generally between 100-300 bp, and the PCR reaction program used is: 94°C 4 min, 94°C 30 sec, 60°C 30 sec, 72°C 45 sec, 34 cycles, 72°C 10 min. The different conditions used by different primers are mainly reflected in the annealing temperature, which is slightly adjusted individually.

[0031] The amplified products were separated by 3% agarose gel, and the amplified DNA bands were observed and recorded by gel imaging. Polymorphic primers between the parents were used for amplification analysis in the F2 population to obtain the genotype information of the F2 population.

[0032] (3) According to the linkage markers and the genotype information of the F2 segregating population, the JoinMap3.0 software was used to construct a partial genetic map of wax gourd and obtain the genetic distance of each molecular marker based on the linkage exchange law. Finally, combined with the molecular marker genotype information of the individual plants in the F2 population and the corresponding fruit shape index, the composite interval mapping method of MapQTL 5 software was used to scan the QTL loci of the target chromosome.

[0033] 3. Results and analysis

[0034] Phenotypic identification of fruit type index of winter melon 2020-mdL, 2019-ss-4-1 and F1 plants Figure 1 As shown in the figure, it can be seen that the winter melon fruit type identification results show that 2019-ss-4-1 is slender (vertical diameter = 112.0cm, horizontal diameter = 10.2cm, fruit type index = 10.99), while 2020-mdL is short and thick (vertical diameter = 58.3cm, horizontal diameter = 20.9cm, fruit type index = 2.79), and F1 is intermediate (vertical diameter = 92.1cm, horizontal diameter = 16.7cm, fruit type index = 5.51), indicating that F1 is co-dominant inheritance, in which long and thick are dominant. Among them, the fruit type index calculation formula is: fruit type index = vertical diameter / horizontal diameter.

[0035] The phenotypic distribution of the fruit type index of winter melon 2020-mdL / 2019-ss-4-1F2 population is shown in the figure Figure 2As shown in the figure, the distribution of fruit type index of individual plants in 201 F2 populations is continuous, with a minimum value of 2.45 and a maximum value of 10.99, which conforms to the normal distribution. According to the experimental results, we divide the fruit type index ≤3.0 into short and thick fruit type winter melon, 3<fruit type index ≤6.0 into medium fruit type winter melon, and 6.0<fruit type index into slender fruit type index winter melon. Combined with the phenotyping results of the sixth generation of 2019-ss-4-1 and 2020-mdL, the ratio of short and thick fruit type (fruit type index ≤5.51):slender fruit type (fruit type index>5.51) in the F2 population is 145:56 (χ 2 =0.88<χ 2 0.01,1 =3.84), and the chi-square test showed that it conformed to the separation law of a pair of dominant genes.

[0036] MapQTL 5 software was used to analyze the genotype and fruit type index phenotype of individual plants in the F2 population. After QTL scanning, the location map of the major effect gene of wax gourd fruit type on chromosome 2 was obtained. Figure 3 As shown in the figure, the vertical value on the horizontal axis represents the genetic distance (cM) between markers, and the vertical axis represents the LOD value. The results show that there is a significant QTL locus between the 2M57.86 and 2M59.91 markers on chromosome 2, with an LOD value of 28.8 and a contribution rate of 78.9%. The gene was named BhFS2.1. Then, the molecular markers 2M57.86 and 2M59.91 were used to screen recombinant single plants and to screen positive single plants in backcross breeding.

[0037] Therefore, the use of the above molecular markers to identify the presence of the BhFS2.1 gene has a very high efficiency, which greatly improves the breeding progress of wax gourd fruit type lines (species). The primer pairs and their sequences developed using the method of this embodiment are: the upstream primer sequence of the primer pair 2M57.86 is: 5'-AGTTGTATTGGGAGGGTATG-3'; the downstream primer sequence is: 5'-GGGGCATCAACAATCTTTCT-3' (SEQ ID NO.1-2). The upstream primer sequence of the primer pair 2M59.91 is: 5'-GAGGAGGGAGCTAAGATGAA-3'; the downstream primer sequence is: 5'-CAAAATCATCTGGCAACAAG-3' (SEQ ID NO.3-4).

[0038] Example 2

[0039] The molecular markers obtained in Example 1 were verified, and the specific verification method was as follows:

[0040] 1. Materials and Methods

[0041] (1) Materials: 1 slender fruit type wax gourd material 2019-ss-4-1, 1 short and thick fruit type wax gourd material 2020-mdL, 4 negative wax gourd materials (all short and thick fruit type), 9 2020-mdL×2019-ss-4-1F 2:3 Pedigree of the population. Molecular marker primers: 2M57.86 and 2M59.91.

[0042] (2) Method: The genomic DNA of the leaves of the sample to be tested was extracted by CTAB extraction method, and the sample DNA was amplified using primer pair 2M57.86 and 2M59.91. The amplified products were separated by 3% agarose gel, and the amplified DNA bands were observed and recorded by gel imaging (the amplification method was the same as in Example 1).

[0043] 2. Results:

[0044] Using the above method, PCR amplification was performed on the genomic DNA of 14 different samples of wax gourd materials, including 2019-ss-4-1 and 2020-mdL. The results are as follows Figure 4-Figure 5 As shown. The results show that in the positive samples, primers 2M57.86 (or 2M59.91) can be used to amplify a fragment of 111bp (or 133bp), indicating that the wax gourd sample contains the high fruit type index slender gene in the 2019-ss-4-1 material; while in the negative samples, these fragments cannot be amplified, and 76bp (or 103bp) fragments are amplified, respectively, indicating that the low fruit type index short gene in the 2020-mdL material of the wax gourd sample; if two bands can be amplified, it means that the wax gourd sample contains both genes and is an intermediate fruit type. This shows that the molecular marker method provided by the present invention can accurately screen out positive samples containing the desired fruit type, thereby greatly improving the efficiency of positive single plant selection in wax gourd breeding.

[0045] Figure 4 and Figure 5 Agarose gel electrophoresis detection of band types of the amplified products of molecular markers 2M57.86 and 2M59.91 in different wax gourd materials, where M is BM2000 DNA marker, and the band sizes from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp, respectively; R is 2019-ss-4-1, S is 2020-mdL, lanes 1-4 are 4 negative wax gourd materials (all with short and thick fruit types), and lanes 5-12 are part of 2020-mdL / 2019-ss-4-1F 2:3 Family; the arrow points to the target band of the parent of R material 2019-ss-4-1. In the positive samples, there is a target band (111bp, 2M57.86, Figure 4; or 133bp, 2M59.91, Figure 5 ), while this band was not present in negative samples.

[0046] In summary, the molecular markers 2M57.86 and 2M59.91 developed in the present application are closely linked and co-segregated with the wax gourd fruit type major gene BhFS2.1. The above molecular markers can be used to effectively distinguish whether the wax gourd seedlings are slender fruit type, thick and short fruit type, or intermediate type, that is, the fruit type of the wax gourd at maturity can be accurately predicted. The detection is convenient, fast and easy, not affected by the environment, and can quickly screen breeding intermediate materials, accelerate the breeding of fruit type varieties (seeds), assist in clarifying breeding goals, and save costs.

[0047] Example 3

[0048] Phenotypic verification: From the electrophoresis results of Example 2, the wax gourd materials in lane R (positive material 2019-ss-4-1), lane S (negative material 2020-mdL) and lanes 1 to 12 were selected for planting to perform phenotypic verification of the fruit type.

[0049] The specific method is as follows:

[0050] A total of 16 samples of plants to be verified were planted in the field, and self-pollination was performed during the flowering period. One melon was left on each plant, and the rest were thinned out and managed in a conventional manner. When the fruit matured, the agronomic traits of the fruit, such as the length and diameter, were counted, and the fruit shape index was calculated.

[0051] The results are shown in Table 1:

[0052] Table 1 Fruit type index of materials in different lanes

[0053] Lanes Longitudinal diameter / cm Horizontal diameter / cm Fruit type index Lanes Longitudinal diameter / cm Horizontal diameter / cm Fruit type index R 106.1 11.6 9.15 7 84.1 16.2 5.18 S 34.2 13.9 2.46 8 92.8 17.9 5.18 1 46.2 18.5 2.50 9 106.7 12.0 8.89 2 57.1 22.1 2.58 10 49.5 18.8 2.64 3 53.2 20.4 2.61 11 81.7 15.3 5.34 4 58.2 22.3 2.61 12 79.1 14.8 5.35 5 73.9 14.4 5.13 R 107.5 12.2 8.79 6 86.1 16.8 5.15 S 47.8 17.8 2.68

[0054] As can be seen from Table 1, when the electrophoresis band is consistent with lane R (2019-ss-4-1) (lane 9), their fruit shape is all slender, and the fruit shape index is relatively high (close to 10.99); when the electrophoresis band is consistent with lane S (2020-mdL) (lane 9, lanes 1-4, lane 10), their fruit shape is basically short and thick, and the fruit shape index is relatively low (close to 2.79); and when there are two bands in the electrophoresis band (the band types of 2019-ss-4-1 and 2020-mdL exist) (lanes 5-8, lanes 11-12), their fruit shape is intermediate, and the fruit shape index is in the middle (close to 5.51).

[0055] Therefore, combined with Example 2, in summary, molecular markers 2M57.86 and 2M59.91 can be used to breed wax gourd fruit type lines (species), and the specific method is as follows:

[0056] Primer pair 2M57.86 was used to amplify the genomic DNA of the winter melon sample to be tested. If a target fragment of 111bp could be amplified, it means that the winter melon sample contains the high fruit type index gene in the 2019-ss-4-1 material, and is a slender fruit type winter melon; if a target fragment of 76bp could be amplified, it means that the winter melon sample contains the low fruit type index gene in the 2020-mdL material, and is a short and thick fruit type winter melon; if two bands could be amplified, it means that the winter melon sample contains both genes, and is an intermediate fruit type winter melon.

[0057] Or use primer pair 2M59.91 to amplify the genomic DNA of the winter melon sample to be tested. If a 133bp target fragment can be amplified, it means that the winter melon sample contains the high fruit type index gene in the 2019-ss-4-1 material, and it is a slender fruit type winter melon; if a 103bp target fragment can be amplified, it means that the low fruit type index gene in the 2020-mdL material of the winter melon sample is a thick and short fruit type winter melon; if two bands can be amplified, it means that the winter melon sample contains both genes and is an intermediate fruit type winter melon.

[0058] In summary, the main effect gene position of the molecular marker detection developed by the present application is clear and easy to identify. By detecting the molecular markers closely linked to the gene, the fruit type of the wax gourd plant can be accurately distinguished, and its detection is convenient, fast and convenient, and is not affected by the environment. The positive individual plants carrying the elongated fruit type gene can be identified at the wax gourd seedling stage, and other plants that do not carry the gene are eliminated, which greatly improves the screening efficiency of the wax gourd fruit type, saves production costs, and promotes the breeding process of the wax gourd fruit type strain (kind).

[0059] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. Application of a primer set for detecting the molecular marker of wax gourd fruit type major gene BhFS2.1 in identifying wax gourd fruit type strains or varieties at the seedling stage, characterized in that: The primer set includes primer pair 2M57.86 and / or primer pair 2M59.91; the nucleic acid sequence of the primer pair 2M57.86 is shown in SEQ ID NO.1-2; the nucleic acid sequence of the primer pair 2M59.91 is shown in SEQ ID NO.3-4; the wax gourd material is the short and thick fruit type parent 2020-mdL and the slender fruit type parent 2019-ss-4-1.

2. A method for breeding wax gourd fruit type strains or varieties at the seedling stage using the primer set as claimed in claim 1, characterized in that: The method is: Primer pair 2M57.86 was used to amplify the genomic DNA of the wax gourd sample to be tested. If a target fragment of 111 bp could be amplified, it indicated that the wax gourd sample was of the slender fruit type; if a target fragment of 76 bp could be amplified, it indicated that the wax gourd sample was of the thick and short fruit type; if two bands could be amplified, it indicated that the wax gourd sample was of the intermediate fruit type; Or use primer pair 2M59.91 to amplify the genomic DNA of the wax gourd sample to be tested. If a 133bp target fragment can be amplified, it means that the wax gourd sample is a slender fruit type wax gourd; if a 103bp target fragment can be amplified, it means that the wax gourd sample is a thick and short fruit type wax gourd; if two bands can be amplified, it means that the wax gourd sample is an intermediate fruit type wax gourd.

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