Method for constructing muskmelon variety molecular identity card based on InDel marker and application thereof

Abstract

The invention particularly relates to a method for constructing a muskmelon variety molecular identity card based on an InDel marker and application of the method. The invention provides application of an InDel molecular marker combination in identification or auxiliary identification of muskmelon germplasm and preparation of a product for identification or auxiliary identification of muskmelon germplasm, the InDel molecular marker combination comprises nine InDel molecular marker sites, and the nine InDel molecular marker sites respectively provide unique molecular identity cards for 40 thick-peel muskmelon varieties to be tested, namely MInd3, MInd62, MInd68, MInd102, MInd130, MInd135, MInd141, MInd171 and MInd192. The identification card can be used for quickly, efficiently and accurately distinguishing all 40 thick-skin muskmelon varieties, and a scientific basis is provided for standardizing a muskmelon seed market, solving muskmelon variety property right disputes and intellectual property protection.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and crop breeding, and specifically relates to a method for constructing a molecular identity card of melon varieties based on InDel markers and its application. Background Art

[0002] Melon (Cucumis melo L.) belongs to the genus Cucumis in the Cucurbitaceae family. It is rich in nutrients and has a sweet taste, and is deeply loved by consumers. It occupies an important position in the fruit consumption of Chinese residents. Due to the advantages of high output efficiency, high land utilization rate and multiple cropping index in melon cultivation, it has become one of the important cash crops in China. According to the statistics of the Food and Agriculture Organization of the United Nations (http: / / www.fao.org / ), the planting area of melons in China in 2023 was 395,800 hm 2 , accounting for 36.23% of the global total. China is the country with the largest cultivation area, the most varieties and the highest yield of melons in the world. With the rapid development of the melon industry, the competition in the melon seed market has become increasingly fierce. Every year, a large number of new melon varieties flood into the market, with a wide variety. Among them, disputes caused by counterfeit and shoddy seeds occur from time to time. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to quickly, efficiently and accurately identify some melon varieties. For this purpose, the present invention provides the application of an InDel molecular marker combination, and the combination contains 9 InDel molecular marker loci, namely MInd3, MInd62, MInd68, MInd102, MInd130, MInd135, MInd141, MInd171, MInd192;

[0004] The above application can be any one of the following:

[0005] A1) Identifying or assisting in identifying melon germplasm;

[0006] A2) Preparing a product for identifying or assisting in identifying melon germplasm.

[0007] In the above application, the germplasm can be species, varieties, plants, seeds, cells and / or DNA fragments, etc.

[0008] The present invention also provides the application of a substance for detecting InDel molecular markers. The InDel molecular marker can be the above 9 InDel molecular marker loci, and the application can be the application of the substance in any one of the following:

[0009] B1) Identifying or assisting in identifying melon germplasm;

[0010] B2) Preparing a product for identifying or assisting in identifying melon germplasm.

[0011] In the above application, the substance contains PCR primers for amplifying a genomic DNA fragment of melon containing the InDel molecular marker.

[0012] In the above application, the PCR primers are a composition, and the composition may be composed of primer pairs MI3, MI62, MI68, MI102, MI130, MI135, MI141, MI171, and MI192;

[0013] The amplification product of the primer pair MI3 may be the MInd3; the amplification product of the primer pair MI62 may be the Mind62; the amplification product of the primer pair MI68 may be the Mind68; the amplification product of the primer pair MI102 may be the Mind102; the amplification product of the primer pair MI130 may be the Mind130; the amplification product of the primer pair MI135 may be the Mind135; the amplification product of the primer pair MI141 may be the Mind141; the amplification product of the primer pair MI171 may be the Mind171; the amplification product of the primer pair MI192 may be the Mind192.

[0014] In the above application, the nucleotide sequence of the forward primer of the MI3 primer pair may be SEQ ID NO: 1, and the nucleotide sequence of the reverse primer of the MI3 primer pair may be SEQ ID NO: 2; the nucleotide sequence of the forward primer of the MI62 primer pair may be SEQ ID NO: 3, and the nucleotide sequence of the reverse primer of the MI62 primer pair may be SEQ ID NO: 4; the nucleotide sequence of the forward primer of the MI68 primer pair may be SEQ ID NO: 5, and the nucleotide sequence of the reverse primer of the MI68 primer pair may be SEQ ID NO: 6; the nucleotide sequence of the forward primer of the MI102 primer pair may be SEQ ID NO: 7, and the nucleotide sequence of the reverse primer of the MI102 primer pair may be SEQ ID NO: 8; the nucleotide sequence of the forward primer of the MI130 primer pair may be SEQ ID NO: 9, and the nucleotide sequence of the reverse primer of the MI130 primer pair may be SEQ ID NO: 10; the nucleotide sequence of the forward primer of the MI135 primer pair may be SEQ ID NO: 11, and the nucleotide sequence of the reverse primer of the MI135 primer pair may be SEQ ID NO: 12; the nucleotide sequence of the forward primer of the MI141 primer pair may be SEQ ID NO: 13, and the nucleotide sequence of the reverse primer of the MI141 primer pair may be SEQ ID NO: 14; the nucleotide sequence of the forward primer of the MI171 primer pair may be SEQ ID NO: 15, and the nucleotide sequence of the reverse primer of the MI171 primer pair may be SEQ ID NO: 16; the nucleotide sequence of the forward primer of the MI192 primer pair may be SEQ ID NO: 17, and the nucleotide sequence of the reverse primer of the MI192 primer pair may be SEQ ID NO: 18.

[0015] The present invention also provides a method for identifying or assisting in the identification of melon germplasm. The method includes using the genomic DNA of the melon germplasm to be identified as a template, performing PCR amplification with the PCR primers to obtain a PCR product, and determining the polymorphism of the above InDel molecular marker combination in the melon germplasm to be identified according to the PCR product to identify the melon germplasm to be identified.

[0016] In the above method, determining the polymorphism of the above InDel molecular marker combination in the melon germplasm to be identified according to the PCR product may include the following steps:

[0017] S1) Detecting the PCR product by agarose gel electrophoresis;

[0018] S2) Arranging the alleles obtained by each pair of primers in ascending order of molecular weight according to the electrophoresis results, and assigning values starting from 1 in sequence;

[0019] S3) According to the fixed order of the InDel molecular marker combination, encode the alleles obtained for each melon variety with assigned numerical codes, and then a unique string, namely the unique molecular identification number, can be obtained for each tested melon material.

[0020] S4) If the molecular identification number of the melon germplasm to be identified is consistent with the existing identification number, then determine the variety of the melon germplasm to be identified.

[0021] In the above application or method, the electrophoresis pattern of MInd3 has three band types, namely 256 / 292bp, 256bp, and 292bp. Among them, 256 / 292bp indicates that there are two DNA fragments with lengths of 256bp and 292bp respectively in MInd3; 256bp indicates that there is only a DNA fragment with a length of 256bp in MInd3; 292bp indicates that there is only a DNA fragment with a length of 292bp in MInd3. The nucleotide sequence of 256bp is obtained after the deletion of the base sequence from the 196th to the 231st position of the 292bp, and the 292bp is SEQ ID NO: 19 in the sequence listing.

[0022] The electrophoresis pattern of MInd62 has three band types, namely 259 / 275bp, 259bp, and 275bp. Among them, 259 / 275bp indicates that there are two DNA fragments with lengths of 259bp and 275bp respectively in the PCR product MInd62 obtained by using the MI62 primer pair; 259bp indicates that there is only a DNA band with a length of 259bp in MInd62; 275bp indicates that there is only a DNA band with a length of 275bp in MInd62. The nucleotide sequence of 259bp is obtained after the deletion from the 187th to the 202nd position of the 275bp, and the 275bp is SEQ ID NO: 20 in the sequence listing.

[0023] The electrophoresis pattern of MInd68 has three band types, namely 228 / 244bp, 228bp, and 244bp. Among them, 228 / 244bp indicates that the PCR product MInd68 obtained by using the MI68 primer pair is two DNA fragments with lengths of 228bp and 244bp respectively; 228bp indicates that there is only a DNA fragment with a length of 228bp in MInd68; 244bp indicates that there is only a DNA fragment with a length of 244bp in MInd68. The nucleotide sequence of 228bp is obtained after the deletion from the 122nd to the 137th position of the 244bp, and the 244bp is SEQ ID NO: 21 in the sequence listing.

[0024] The electrophoresis pattern of Mind102 has three bands, namely 251 / 278 bp, 251 bp, and 278 bp. Among them, 251 / 278 bp indicates that Mind102 is two DNA fragments with lengths of 251 bp and 278 bp respectively; 251 bp indicates that Mind102 only has a DNA fragment with a length of 251 bp; 278 bp indicates that Mind102 only has a DNA fragment with a length of 278 bp. The nucleotide sequence of 251 bp is obtained by deleting the base sequence from the 96th to the 122nd position of the 278 bp, and the 278 bp is sequence 22 (SEQ ID NO: 22) in the sequence listing.

[0025] The electrophoresis pattern of Mind130 has three bands, namely 242 / 269 bp, 242 bp, and 269 bp. Among them, 242 / 269 bp indicates that Mind130 is two DNA fragments with lengths of 242 bp and 269 bp respectively; 242 bp indicates that Mind130 only has a DNA fragment with a length of 242 bp; 269 bp indicates that Mind130 only has a DNA fragment with a length of 269 bp. The nucleotide sequence of 242 bp is obtained by deleting the base sequence from the 157th to the 183rd position of the 269 bp, and the 269 bp is sequence 23 (SEQ ID NO: 23) in the sequence listing.

[0026] The electrophoresis pattern of Mind135 has three bands, namely 279 / 306 bp, 279 bp, and 306 bp. Among them, 279 / 306 bp indicates that Mind135 is two DNA fragments with lengths of 279 bp and 306 bp respectively; 279 bp indicates that Mind135 only has a DNA fragment with a length of 279 bp; 306 bp indicates that Mind135 only has a DNA fragment with a length of 306 bp. The nucleotide sequence of 279 bp is obtained by deleting the base sequence from the 138th to the 164th position of the 306 bp, and the 279 bp is sequence 24 (SEQ ID NO: 24) in the sequence listing.

[0027] The electrophoresis pattern of Mind141 has three bands, namely 245 / 272 bp, 245 bp, and 272 bp. Among them, 245 / 272 bp indicates that Mind141 is two DNA fragments with lengths of 245 bp and 272 bp respectively; 245 bp indicates that Mind141 only has a DNA fragment with a length of 245 bp; 272 bp indicates that Mind141 only has a DNA fragment with a length of 272 bp. The nucleotide sequence of 245 bp is obtained by deleting the base sequence from the 124th to the 150th position of the 272 bp, and the 272 bp is sequence 25 (SEQ ID NO: 25) in the sequence listing.

[0028] The electrophoresis spectrum of Mind171 has three band types, namely 263 / 290bp, 263bp and 290bp. Among them, 263 / 290bp means that MInd171 has two DNA fragments of 263bp and 290bp in length respectively; 263bp means that there is only a DNA fragment of 263bp in length in MInd171; 290bp means that there is only a DNA fragment of 290bp in length in MInd171. The nucleotide sequence of 263bp is obtained after the base sequence of the 160th to 186th position of 290bp is deleted, and the 290bp is sequence 26 in the sequence list (SEQ ID NO: 26).

[0029] The electrophoresis spectrum of Mind192 has three band types, namely 267 / 294bp, 267bp and 294bp. Among them, 267 / 294bp means that MInd192 is two DNA fragments of 267bp and 294bp in length; 267bp means that there is only a DNA fragment of 267bp in length in MInd192; 294bp means that there is only a DNA fragment of 294bp in length in MInd192. The nucleotide sequence of 267bp is obtained after the base sequence of the 195th to 221st position of the 294bp is deleted, and the 294bp is sequence 27 (SEQ ID NO: 27) in the sequence list.

[0030] The present invention also provides an application of the above method in identifying or assisting in identifying the germplasm purity or authenticity of melon germplasm. In the application, the germplasm purity may be seed purity.

[0031] The present invention also provides a primer composition for identifying or assisting in identifying melon varieties, and the primer composition can be the PCR primer.

[0032] The present invention also provides a reagent or a kit for identifying or assisting in identifying melon varieties, and the reagent or the kit may contain the primer composition.

[0033] In the present application, the melon germplasm can be at least one of the following 40 kinds of melons: Dongfang Mi No. 4, Dongfang Mi No. 1, Dongfang Mi No. 3, Dongfang Mi No. 2, DM4, Jinshuai, Huanghua, Jinmiyue, Jinmilang, Jiutai No. 1, Dongfang Mi No. 5, Sunshine 86 King, Dongfang Mi No. 6, Du Mi No. 5, Xizhou Mi No. 25, Xizhou Mi No. 17, Du Mi Green 25, Dongfang Mi No. 7, Sutian Jasper, Bachu Liuxiang Melon, Dongfang Mi No. 8, Pearl No. 7, Sanxiong No. 5, Pearl No. 3, Yugu, Pearl No. 4, Chinese Crisp Pear, Pearl No. 5, Sutian No. 4, Xiboluoto No. 2, Qingmi No. 1, Zhengtai Net Pattern No. 5, Cuitian, Berg, Mario, Xinniu Meilong No. 3, Jintian No. 1, Pearl No. 2, Pearl No. 6 and Huozhou Mi 186.

[0034] Determining the polymorphisms of the 9 InDel molecular markers of the melon germplasm to be identified based on the PCR products to identify the melon germplasm to be identified can be the corresponding relationship in Table 3 of the specification, that is, if the polymorphisms of the 9 InDel molecular markers of the melon germplasm to be identified are consistent with any one of the 40 melon germplasms, then the melon germplasm to be identified is or is a candidate for the melon germplasm that is consistent with it; if there is at least one polymorphism of the 9 InDel molecular markers of the melon germplasm to be identified that is inconsistent with a specific melon germplasm among the 40 melon germplasms, then the melon germplasm to be identified is or is a candidate for not being the specific melon germplasm.

[0035] In one embodiment, if the melon germplasm to be identified meets the following 9 conditions F1)-F9), then the melon germplasm to be identified is or is a candidate for 'Dongfangmi No. 4'; if the melon germplasm to be identified does not meet at least one of the 9 conditions, then the melon germplasm to be identified is or is a candidate for not being 'Dongfangmi No. 4'; the 9 conditions are:

[0036] F1) There are two bands with different lengths after gel electrophoresis of MInd3, and the band pattern is 256 / 292 bp;

[0037] F2) There are two bands with different lengths after gel electrophoresis of MInd62, and the band pattern is 259 / 275 bp;

[0038] F3) There are two bands with different lengths after gel electrophoresis of MInd68, and the band pattern is 228 / 244 bp;

[0039] F4) There are two bands with different lengths after gel electrophoresis of MInd102, and the band pattern is 251 / 278 bp;

[0040] F5) There are two bands with different lengths after gel electrophoresis of MInd130, and the band pattern is 242 / 269 bp;

[0041] F6) There are two bands with different lengths after gel electrophoresis of MInd135, and the band pattern is 279 / 306 bp;

[0042] F7) There are two bands with different lengths after gel electrophoresis of MInd141, and the band pattern is 245 / 272 bp;

[0043] F8) There are two bands with different lengths after gel electrophoresis of MInd171, and the band pattern is 263 / 290 bp;

[0044] F9) There are two bands with different lengths after gel electrophoresis of MInd192, and the band pattern is 267 / 294 bp.

[0045] After meeting the above conditions, the molecular ID number of the melon to be identified is 333333333, and it can be identified as 'Dongfangmi No. 4'.

[0046] The molecular ID of melon varieties constructed by the present invention using InDel marker technology provides a unique molecular ID for 40 tested thick-skinned melon varieties. Using this ID, the 40 thick-skinned melon varieties can be quickly, efficiently, and accurately distinguished, providing a scientific basis for standardizing the melon seed market, resolving melon variety property right disputes, and intellectual property protection. Specific Embodiments

[0047] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0048] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0049] Example 1: Mining of InDel molecular marker primers related to melon variety identification and establishment of detection methods

[0050] 1 Materials and Methods

[0051] 1.1 Test Materials

[0052] A total of 40 common thick-skinned melon varieties on the market were used in this study, including 22 types of Hami melons, 12 types of Western-style melons, and 6 types of netted melons. The detailed information is shown in Table 1.

[0053] Table 1 Information table of 40 melon varieties

[0054]

[0055]

[0056] 1.2 Test Methods

[0057] 1.2.1 Genomic DNA Extraction

[0058] (1) Take about 1 cm 2 of melon leaves and put them into a 2 mL centrifuge tube. Add 3 steel beads with a diameter of 3 mm and 900 μL of CTAB extraction solution, and then use a shaking grinder to grind the sample at 65 Hz for 3 min to crush it;

[0059] (2) Place a 2 mL centrifuge tube in a 65 °C water bath for 1 h, and invert and mix it every 15 min;

[0060] (3) After the water bath, add 900 μL of chloroform, shake vigorously to mix, and centrifuge at 12,000 rpm for 10 min;

[0061] (4) Pipette 750 μL of the supernatant into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol solution, mix well and place at -20 °C overnight;

[0062] (5) After centrifuging at 12,000 rpm for 10 min, pour off the supernatant, add 500 μL of 75% ethanol to wash the precipitate, then centrifuge at 12,000 rpm for 10 min, and pour off the supernatant;

[0063] (6) Place the 1.5 mL centrifuge tube in a fume hood to dry, add 30 μL of sterile water, flick the tube wall several times to ensure that all the DNA is dissolved, and place at -20 °C for later use.

[0064] The CTAB (1 L) formulation is as follows:

[0065] Reagents used Weight / volume Cetyltrimethylammonium bromide (CTAB) 20g Sodium chloride (NaCl) 81.82g 0.5M EDTA 40 mL 1M Tris-HCl 100 mL <![CDATA[ddH 2 O]]> Make up to 1 L

[0066] 1.2.2 Whole-genome resequencing and InDel marker mining

[0067] Take the seedling leaves of the hybrid F1 melon variety 'Dongfangmi 4' and its male parent M15-3 and female parent M06-1-3 respectively, quickly freeze them in liquid nitrogen, and use these as samples to send to Shanghai Ling'en Biotechnology Co., Ltd. for whole-genome resequencing. After data quality control, clean data are obtained. Align the sequencing reads with the gene sequences of the melon whole-genome melon DHL92 genome (http: / / cucurbitgenomics.org / organism / 3) using the BWA (http: / / bio-bwa.sourceforge.net / ) software, and screen for InDel sites that are different between the male and female parents. Design primers in the conserved regions on both sides of the different sites using the Primer Primer 5.0 software. The primer fragment length ranges from 20 to 25 bp, the PCR amplification product length ranges from 180 to 300 bp, and the annealing temperature ranges from 55 to 61 °C. A total of 200 pairs of InDel primers are designed and commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0068] 1.2.3 PCR amplification system and amplification program

[0069] The PCR reaction system is shown in the following table. The reaction procedure is pre-denaturation at 94°C for 3 min, then denaturation at 94°C for 30 s, annealing at 55 - 61°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles, and finally extension reaction at 72°C for 10 min. The PCR products were detected by electrophoresis on a 4% agarose gel (preparation method: 2 g of agarose, 50 ml of TAE buffer), electrophoresed at a constant voltage of 100 V for 80 min, and photographed with an ultraviolet gel imager. The PCR reaction system is as follows:

[0070]

[0071]

[0072] 2 Results and Analysis

[0073] 2.1 InDel Marker Screening

[0074] InDel loci with differences between the male parent M15-3 and the female parent M06-1-3 of 'Dongfangmi No. 4' were screened and 200 pairs of InDel primers were synthesized. These primers were used to perform PCR amplification on the genomic DNA of 40 tested melon varieties (Table 1). The amplification products were detected by electrophoresis on a 4% agarose gel. After strict screening, 9 pairs of InDel primers were finally determined. The amplified maps of these primers had the characteristics of clear bands, strong stability, and significant polymorphism. The specific sequences of these 9 pairs of InDel primers are shown in Table 2.

[0075] Table 2 Specific InDel Primer Sequences

[0076]

[0077] After amplification according to the reaction conditions of 1.2.3 and detection of the PCR products by electrophoresis on a 4% agarose gel, the allelic information of 40 tested melon varieties at 9 InDel loci is shown in Table 3.

[0078] The PCR products were detected by electrophoresis on an agarose gel. The electrophoretic map bands of 40 tested melon varieties are as follows:

[0079] 1) The electrophoresis pattern of the PCR product MInd3 obtained using the MI3 primer pair has three band types, namely 256 / 292 bp, 256 bp, and 292 bp. Among them, 256 / 292 bp indicates that there are two DNA bands with lengths of 256 bp and 292 bp respectively in the PCR product MInd3 obtained using the MI3 primer pair; 256 bp indicates that there is only one DNA band with a length of 256 bp in the PCR product MInd3 obtained using the MI3 primer pair; 292 bp indicates that there is only one DNA band with a length of 292 bp in the PCR product MInd3 obtained using the MI3 primer pair. The nucleotide sequence of the 256 bp is obtained after the deletion of the base sequence from position 196 to position 231 of the 292 bp, and the 292 bp is sequence 19 (SEQ ID NO: 19) in the sequence listing.

[0080] 2) The electrophoresis pattern of the PCR product MInd62 obtained using the MI62 primer pair has three band types, namely 259 / 275 bp, 259 bp, and 275 bp. Among them, 259 / 275 bp indicates that there are two DNA bands with lengths of 259 bp and 275 bp respectively in the PCR product MInd62 obtained using the MI62 primer pair; 259 bp indicates that there is only one DNA band with a length of 259 bp in the PCR product MInd62 obtained using the MI62 primer pair; 275 bp indicates that there is only one DNA fragment with a length of 275 bp in the PCR product MInd62 obtained using the MI62 primer pair. The nucleotide sequence of the 259 bp is obtained after the deletion from position 187 to position 202 of the 275 bp, and the 275 bp is sequence 20 (SEQ ID NO: 20) in the sequence listing.

[0081] 3) The electrophoresis pattern of the PCR product MInd68 obtained using the MI68 primer pair has three band types, namely 228 / 244 bp, 228 bp, and 244 bp. Among them, 228 / 244 bp indicates that the PCR product MInd68 obtained using the MI68 primer pair is two DNA fragments with lengths of 228 bp and 244 bp respectively; 228 bp indicates that there is only one DNA fragment with a length of 228 bp in the PCR product MInd68 obtained using the MI68 primer pair; 244 bp indicates that there is only one DNA fragment with a length of 244 bp in the PCR product MInd68 obtained using the MI68 primer pair. The nucleotide sequence of the 228 bp is obtained after the deletion from position 122 to position 137 of the 244 bp, and the 244 bp is sequence 21 (SEQ ID NO: 21) in the sequence listing.

[0082] 4) The electrophoresis pattern of the PCR product MInd102 obtained using the MI102 primer pair has three band types, namely 251 / 278 bp, 251 bp, and 278 bp. Among them, 251 / 278 bp indicates that the PCR product MInd102 obtained using the MI102 primer pair is two DNA fragments with lengths of 251 bp and 278 bp respectively; 251 bp indicates that there is only one DNA fragment with a length of 251 bp in the PCR product MInd102 obtained using the MI102 primer pair; 278 bp indicates that there is only one DNA fragment with a length of 278 bp in the PCR product MInd102 obtained using the MI102 primer pair. The nucleotide sequence of the 251 bp is obtained after the deletion of the base sequence from position 96 to position 122 of the 278 bp, and the 278 bp is sequence 22 (SEQ ID NO: 22) in the sequence listing.

[0083] 5) The electrophoresis pattern of the PCR product MInd130 obtained using the MI130 primer pair has three band types, namely 242 / 269 bp, 242 bp, and 269 bp. Among them, 242 / 269 bp indicates that the PCR product MInd130 obtained using the MI130 primer pair is two DNA fragments with lengths of 242 bp and 269 bp respectively; 242 bp indicates that there is only one DNA fragment with a length of 242 bp in the PCR product MInd130 obtained using the MI130 primer pair; 269 bp indicates that there is only one DNA fragment with a length of 269 bp in the PCR product MInd130 obtained using the MI130 primer pair. The nucleotide sequence of the 242 bp is obtained after the deletion of the base sequence from position 157 to position 183 of the 269 bp, and the 269 bp is sequence 23 (SEQ ID NO: 23) in the sequence listing.

[0084] 6) The electrophoresis pattern of the PCR product MInd135 obtained using the MI135 primer pair has three band types, namely 279 / 306 bp, 279 bp, and 306 bp. Among them, 279 / 306 bp indicates that the PCR product MInd135 obtained using the MI135 primer pair is two DNA fragments with lengths of 279 bp and 306 bp respectively; 279 bp indicates that there is only one DNA fragment with a length of 279 bp in the PCR product MInd135 obtained using the MI135 primer pair; 306 bp indicates that there is only one DNA fragment with a length of 306 bp in the PCR product MInd135 obtained using the MI135 primer pair. The nucleotide sequence of the 279 bp is obtained after the deletion of the base sequence from position 138 to position 164 of the 306 bp, and the 279 bp is sequence 24 (SEQ ID NO: 24) in the sequence listing.

[0085] 7) The electrophoresis pattern of the PCR product MInd141 obtained using the MI141 primer pair has three band types, namely 245 / 272, 245, and 272. Among them, 245 / 272 indicates that the PCR product MInd141 obtained using the MI141 primer pair is two DNA fragments with lengths of 245 bp and 272 bp respectively; 245 indicates that there is only one DNA fragment with a length of 245 bp in the PCR product MInd141 obtained using the MI141 primer pair; 272 indicates that there is only one DNA fragment with a length of 272 bp in the PCR product MInd141 obtained using the MI141 primer pair. The nucleotide sequence of the 245 bp is obtained after the deletion of the base sequence from the 124th to the 150th position of the 272 bp, and the 272 bp is sequence 25 (SEQ ID NO: 25) in the sequence listing.

[0086] 8) The electrophoresis pattern of the PCR product MInd171 obtained using the MI171 primer pair has three band types, namely 263 / 290, 263, and 290. Among them, 263 / 290 indicates that the PCR product MInd171 obtained using the MI171 primer pair is two DNA fragments with lengths of 263 bp and 290 bp respectively; 263 indicates that there is only one DNA fragment with a length of 263 bp in the PCR product MInd171 obtained using the MI171 primer pair; 290 indicates that there is only one DNA fragment with a length of 290 bp in the PCR product MInd171 obtained using the MI171 primer pair. The nucleotide sequence of the 263 bp is obtained after the deletion of the base sequence from the 160th to the 186th position of the 290 bp, and the 290 bp is sequence 26 (SEQ ID NO: 26) in the sequence listing.

[0087] 9) The electrophoresis pattern of the PCR product MInd192 obtained using the MI192 primer pair has three band types, namely 267 / 294, 267, and 294. Among them, 267 / 294 indicates that the PCR product MInd192 obtained using the MI192 primer pair is two DNA fragments with lengths of 267 bp and 294 bp respectively; 267 indicates that there is only one DNA fragment with a length of 267 bp in the PCR product MInd192 obtained using the MI192 primer pair; 294 indicates that there is only one DNA fragment with a length of 294 bp in the PCR product MInd192 obtained using the MI192 primer pair. The nucleotide sequence of the 267 bp is obtained after the deletion of the base sequence from the 195th to the 221st position of the 294 bp, and the 294 bp is sequence 27 (SEQ ID NO: 27) in the sequence listing.

[0088] Table 3 Allele information table of 40 tested melon varieties at 9 InDel loci

[0089]

[0090]

[0091]

[0092] 2.2 Construction of Molecular Identification Cards for Melon Varieties

[0093] According to the detection results of 4% agarose gel electrophoresis, record the banding patterns amplified by each pair of InDel markers in 40 melon varieties, and arrange the alleles obtained by each pair of primers in ascending order, and assign values starting from 1 with Arabic numerals (see Table 4 for details).

[0094] Table 4 Allele Assignment Criteria for 9 InDel Loci

[0095]

[0096] According to the fixed order of 9 InDel loci, code the alleles obtained for each melon variety according to the assigned numbers, and the unique string of each tested melon material, that is, the unique molecular identification card number, can be obtained (see Table 5 for details).

[0097] Table 5 Molecular Identification Card Information of 40 Tested Melon Varieties

[0098]

[0099]

[0100] The above experimental results show that the 9 pairs of InDel marker primers in Table 2 can distinguish the 40 melon germplasms in Table 1, and conduct authenticity and seed purity identification within the corresponding germplasm range. The 40 melon germplasms in Table 1 are hereinafter referred to as 40 melon varieties for short.

[0101] 2.3 Identification of Authenticity within the Range of 40 Melon Varieties

[0102] The method for identifying authenticity within the range of 40 melon varieties includes: using the genomic DNA of the melon to be tested as a template, performing PCR amplification with the 9 pairs of InDel marker primers in Table 2 according to 1.2.3, detecting the PCR products of the melon to be tested by agarose gel electrophoresis, reading the different band types corresponding to the 9 primers according to the electrophoresis pattern, and assigning values to different types of bands respectively according to the method in Table 4. According to the fixed order of 9 InDel loci, code the assigned numbers obtained for the melon variety to be tested, and the molecular identification card number of the melon variety to be tested can be obtained. Compare this identification card number with that in Table 5, and if they are consistent, it is the corresponding melon variety.

[0103] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art.

Claims

1. Application of InDel molecular marker combination, characterized in that: The combination contains 9 InDel molecular marker sites, namely MInd3, MInd62, MInd68, MInd102, MInd130, MInd135, MInd141, MInd171, and MInd192; The application is any of the following: A1) Identify or assist in identifying melon germplasm; A2) Preparation of products for identification or assistance in identification of melon germplasm.

2. Application of a substance labeled with an InDel molecule, characterized in that: The InDel molecular markers are the 9 InDel molecular marker sites described in claim 1, and the application is the application of the substance in any of the following: B1) Identify or assist in identifying melon germplasm; B2) Preparation of products for identification or assistance in identification of melon germplasm.

3. The use according to claim 2, characterized in that: The material contains PCR primers for amplifying a melon genomic DNA fragment containing the InDel molecular marker.

4. The use according to claim 3, characterized in that: The PCR primers are a composition, which consists of a MI3 primer pair, a MI62 primer pair, a MI68 primer pair, a MI102 primer pair, a MI130 primer pair, a MI135 primer pair, a MI141 primer pair, a MI171 primer pair and a MI192 primer pair; The amplification product of the MI3 primer pair is the MInd3 described in claim 1; the amplification product of the MI62 primer pair is the Mind62 described in claim 1; the amplification product of the MI68 primer pair is the Mind68 described in claim 1; the amplification product of the MI102 primer pair is the Mind102 described in claim 1; the amplification product of the MI130 primer pair is the Mind130 described in claim 1; the amplification product of the MI135 primer pair is the Mind135 described in claim 1; the amplification product of the MI141 primer pair is the Mind141 described in claim 1; the amplification product of the MI171 primer pair is the Mind171 described in claim 1; the amplification product of the MI192 primer pair is the Mind192 described in claim 1.

5. The use according to claim 4, characterized in that: The nucleotide sequence of the forward primer of the MI3 primer pair is SEQ ID NO: 1, and the nucleotide sequence of the reverse primer of the MI3 primer pair is SEQ ID NO: 2; the nucleotide sequence of the forward primer of the MI62 primer pair is SEQ ID NO: 3, and the nucleotide sequence of the reverse primer of the MI62 primer pair is SEQ ID NO: 4; the nucleotide sequence of the forward primer of the MI68 primer pair is SEQ ID NO: 5, and the nucleotide sequence of the reverse primer of the MI68 primer pair is SEQ ID NO: 6; the nucleotide sequence of the forward primer of the MI102 primer pair is SEQ ID NO: 7, and the nucleotide sequence of the reverse primer of the MI102 primer pair is SEQ ID NO: 8; the nucleotide sequence of the forward primer of the MI130 primer pair is SEQ ID NO: 9, and the nucleotide sequence of the reverse primer of the MI130 primer pair is SEQ ID NO: 10; the nucleotide sequence of the forward primer of the MI135 primer pair is SEQ ID NO: NO: 11, the nucleotide sequence of the reverse primer of the MI135 primer pair is SEQ ID NO: 12; the nucleotide sequence of the forward primer of the MI141 primer pair is SEQ ID NO: 13, and the nucleotide sequence of the reverse primer of the MI141 primer pair is SEQ ID NO: 14; the nucleotide sequence of the forward primer of the MI171 primer pair is SEQ ID NO: 15, and the nucleotide sequence of the reverse primer of the MI171 primer pair is SEQ ID NO: 16; the nucleotide sequence of the forward primer of the MI192 primer pair is SEQ ID NO: 17, and the nucleotide sequence of the reverse primer of the MI192 primer pair is SEQ ID NO:

18.

6. A method for identifying or assisting in identifying melon germplasm, characterized in that: The method comprises using the genomic DNA of the melon germplasm to be identified as a template, performing PCR amplification using the PCR primers described in claim 3 or 4 to obtain a PCR product, and determining the polymorphism of the InDel molecular marker combination described in claim 1 of the melon germplasm to be identified based on the PCR product to identify the melon germplasm to be identified.

7. The method according to claim 6, characterized in that The specific steps of determining the polymorphism of the InDel molecular marker combination described in claim 1 for the melon germplasm to be identified based on the PCR product are as follows: S1) detecting the PCR product by agarose gel electrophoresis; S2) arranging the alleles obtained by each pair of primers in order of molecular weight from small to large according to the electrophoresis results, and assigning values ​​starting from 1; S3) According to the fixed order of the InDel molecular marker combination, the alleles obtained from each melon variety are coded according to the assigned digital number, so that a unique string of characters for each tested melon material, i.e., a unique molecular ID number, can be obtained; S4) If the molecular ID number of the melon germplasm to be identified is consistent with the existing ID number, the variety of the melon germplasm to be identified is determined.

8. Use of the method of claim 6 or 7 in identifying or assisting in identifying the authenticity of melon germplasm.

9. A primer composition for identifying or assisting in identifying melon varieties, characterized in that: The primer composition contains the PCR primer according to claim 3 or 4.

10. A reagent or kit for identifying or assisting in identifying melon varieties, characterized in that: The reagent or kit contains the primer composition according to claim 9.

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