Method for identifying mitochondrial DNA fingerprint of melon cultivar and hybrid purity and primer combination thereof

Abstract

The application discloses a method for identifying melon mitochondrial DNA fingerprint and hybrid seed purity and a primer combination thereof. Specifically disclosed are a mitochondrial SNP molecular marker combination comprising 24 SNP sites on melon mitochondrial DNA, and primer groups 01 to 24 for detecting the mitochondrial SNP molecular marker combination. The developed molecular marker combination and SNP primer combination can be used for identifying melon mitochondrial DNA fingerprint and first-generation hybrid seed purity, and have important application values in the process of agricultural production and variety breeding for clearly determining the pedigree of melons and the purity of commodity seeds. The method provided by the application has the advantages of high throughput, accuracy, low cost, simple operation, saving of manpower and material resources, and the like, and has a very broad application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a method for identifying mitochondrial DNA fingerprints of melon varieties and purity of hybrid seeds and a primer combination thereof. BACKGROUND

[0002] Mitochondria are the main site of plant aerobic respiration and have a semi-autonomous organelle genome. Among the more than 600 known angiosperms, the mitochondria of most plants are strictly maternally inherited, while the mitochondria of melon (Cucumis melo L.) exhibit rare paternal inheritance. That is, the mitochondrial genes of melon hybrid offspring all come from the paternal parent, and the gene recombination rate is much lower than that of the nuclear genome. With the continuous development of sequencing technology, the mitochondrial genome sequence of melon has been assembled, and its genome includes one large circle and two small circles. The large circle genome is about 2.71 Mb, and the two small circles have genomes of 0.15 Mb and 0.047 Mb, respectively.

[0003] China is the country with the largest melon planting area and the highest consumption in the world. At present, more than 2000 melon varieties have been applied for registration and protection, and 98% of the market varieties are one-generation hybrids. This also brings new challenges for melon variety tracing, seed purity quality detection and market supervision. The heteromorphous seed of hybrid seed is mainly the seed produced by selfing of the maternal parent, and the paternal inheritance characteristics of melon mitochondria can be used to detect the purity of hybrid varieties. In addition, the mitochondrial genes of melon are not only related to the respiratory metabolic pathway, but also related to important traits such as sex differentiation and low temperature tolerance. Therefore, the development of melon mitochondrial DNA molecular markers is of great significance for melon pedigree tracing, variety purity identification, functional gene analysis, etc.

[0004] At present, the study of melon germplasm resource variation group provides the possibility for mining melon mitochondrial SNP variation based on big data. As the third generation of DNA molecular markers, SNP is genetically stable and easy to detect automatically. KASP (Kompetitive Allele Specific PCR), i.e. competitive allele-specific PCR technology, is a commonly used method for detecting SNP typing, and has the characteristics of high stability, accuracy and low cost, and has been widely used in high-throughput molecular assisted breeding and variety identification. SUMMARY

[0005] One of the purposes of the present application is to provide a mitochondrial SNP molecular marker combination and its detection primer set for identifying mitochondrial DNA fingerprints of melon varieties, identifying purity of melon hybrid seeds, tracing melon varieties and / or analyzing melon genetic relationships. The technical problems to be solved are not limited to the technical topics described, and other technical topics not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0006] To achieve the above object, the present application first provides a mitochondrial SNP molecular marker combination, which can comprise the following 24 SNP sites on the mitochondrial DNA of melon: G25469C, C26825G, A43442C, T55919C, C84914G, G289473C, G327258C, G388559C, T544861G, C732705A, G782469T, G1015555T, T1240089G, C1264778G, T1300424G, C1552239G, G1883266C, C1948365G, C2024981A, T2114515G, G2419091A, G2501159C, G2573569C and C2651373A.

[0007] The reference genome sequence of the melon mitochondrial DNA has a GeneBank ID number of MG947207.

[0008] The present application also provides a primer set for amplifying (or detecting) the mitochondrial SNP molecular marker combination.

[0009] Further, the primer set can comprise primer set 01 to primer set 24 for amplifying the mitochondrial DNA fragment containing the SNP site, wherein:

[0010] Primer set 01 comprises forward primers with nucleotide sequences of SEQ ID NO: 1 at positions 22-47 and SEQ ID NO: 2 at positions 22-47, and a reverse primer with a nucleotide sequence of SEQ ID NO: 3;

[0011] Primer set 02 comprises forward primers with nucleotide sequences of SEQ ID NO: 4 at positions 22-45 and SEQ ID NO: 5 at positions 22-45, and a reverse primer with a nucleotide sequence of SEQ ID NO: 6;

[0012] Primer set 03 comprises forward primers with nucleotide sequences of SEQ ID NO: 7 at positions 22-48 and SEQ ID NO: 8 at positions 22-47, and a reverse primer with a nucleotide sequence of SEQ ID NO: 9;

[0013] Primer set 04 comprises forward primers with nucleotide sequences of SEQ ID NO: 10 at positions 22-46 and SEQ ID NO: 11 at positions 22-45, and a reverse primer with a nucleotide sequence of SEQ ID NO: 12;

[0014] Primer set 05 includes a forward primer with nucleotide sequences of positions 22-44 of SEQ ID NO: 13 and positions 22-44 of SEQ ID NO: 14, and a reverse primer with nucleotide sequence of SEQ ID NO: 15;

[0015] Primer set 06 includes a forward primer with nucleotide sequences of positions 22-46 of SEQ ID NO: 16 and positions 22-46 of SEQ ID NO: 17, and a reverse primer with nucleotide sequence of SEQ ID NO: 18;

[0016] Primer set 07 includes a forward primer with nucleotide sequences of positions 22-44 of SEQ ID NO: 19 and positions 22-44 of SEQ ID NO: 20, and a reverse primer with nucleotide sequence of SEQ ID NO: 21;

[0017] Primer set 08 includes a forward primer with nucleotide sequences of positions 22-44 of SEQ ID NO: 22 and positions 22-44 of SEQ ID NO: 23, and a reverse primer with nucleotide sequence of SEQ ID NO: 24;

[0018] Primer set 09 includes a forward primer with nucleotide sequences of positions 22-46 of SEQ ID NO: 25 and positions 22-44 of SEQ ID NO: 26, and a reverse primer with nucleotide sequence of SEQ ID NO: 27;

[0019] Primer set 10 includes a forward primer with nucleotide sequences of positions 22-49 of SEQ ID NO: 28 and positions 22-50 of SEQ ID NO: 29, and a reverse primer with nucleotide sequence of SEQ ID NO: 30;

[0020] Primer set 11 includes a forward primer with nucleotide sequences of positions 22-52 of SEQ ID NO: 31 and positions 22-54 of SEQ ID NO: 32, and a reverse primer with nucleotide sequence of SEQ ID NO: 33;

[0021] Primer set 12 includes a forward primer with nucleotide sequences of positions 22-47 of SEQ ID NO: 34 and positions 22-48 of SEQ ID NO: 35, and a reverse primer with nucleotide sequence of SEQ ID NO: 36;

[0022] Primer set 13 includes a forward primer with nucleotide sequences of positions 22-44 of SEQ ID NO: 37 and positions 22-42 of SEQ ID NO: 38, and a reverse primer with nucleotide sequence of SEQ ID NO: 39;

[0023] Primer set 14 includes a forward primer with nucleotide sequences of positions 22-46 of SEQ ID NO: 40 and positions 22-46 of SEQ ID NO: 41, and a reverse primer with nucleotide sequence of SEQ ID NO: 42;

[0024] Primer set 15 includes a forward primer with nucleotide sequences of positions 22-51 of SEQ ID NO: 43 and positions 22-50 of SEQ ID NO: 44, and a reverse primer with nucleotide sequence of SEQ ID NO: 45;

[0025] Primer set 16 includes a forward primer with nucleotide sequences of positions 22-47 of SEQ ID NO: 46 and positions 22-47 of SEQ ID NO: 47, and a reverse primer with nucleotide sequence of SEQ ID NO: 48;

[0026] Primer set 17 includes a forward primer with nucleotide sequences of positions 22-44 of SEQ ID NO: 49 and positions 22-44 of SEQ ID NO: 50, and a reverse primer with nucleotide sequence of SEQ ID NO: 51;

[0027] Primer set 18 includes a forward primer with nucleotide sequences of positions 22-44 of SEQ ID NO: 52 and positions 22-44 of SEQ ID NO: 53, and a reverse primer with nucleotide sequence of SEQ ID NO: 54;

[0028] Primer set 19 includes a forward primer with nucleotide sequences of positions 22-44 of SEQ ID NO: 55 and positions 22-45 of SEQ ID NO: 56, and a reverse primer with nucleotide sequence of SEQ ID NO: 57;

[0029] Primer set 20 includes a forward primer with nucleotide sequences of positions 22-52 of SEQ ID NO: 58 and positions 22-51 of SEQ ID NO: 59, and a reverse primer with nucleotide sequence of SEQ ID NO: 60;

[0030] Primer set 21 includes a forward primer with nucleotide sequences of positions 22-44 of SEQ ID NO: 61 and positions 22-46 of SEQ ID NO: 62, and a reverse primer with nucleotide sequence of SEQ ID NO: 63;

[0031] Primer set 22 includes a forward primer with nucleotide sequences of positions 22-43 of SEQ ID NO: 64 and positions 22-43 of SEQ ID NO: 65, and a reverse primer with nucleotide sequence of SEQ ID NO: 66;

[0032] Primer set 23 includes a forward primer with nucleotide sequences of positions 22-39 of SEQ ID NO: 67 and positions 22-39 of SEQ ID NO: 68, and a reverse primer with nucleotide sequence of SEQ ID NO: 69;

[0033] Primer set 24 includes a forward primer with nucleotide sequences of positions 22-46 of SEQ ID NO: 70 and positions 22-47 of SEQ ID NO: 71, and a reverse primer with nucleotide sequence of SEQ ID NO: 72.

[0034] Furthermore, primer set 01 was used to amplify the mitochondrial DNA fragment containing SNP site G25469C; primer set 02 was used to amplify the mitochondrial DNA fragment containing SNP site C26825G; primer set 03 was used to amplify the mitochondrial DNA fragment containing SNP site A43442C; primer set 04 was used to amplify the mitochondrial DNA fragment containing SNP site T55919C; primer set 05 was used to amplify the mitochondrial DNA fragment containing SNP site C84914G; primer set 06 was used to amplify the mitochondrial DNA fragment containing SNP site G289473C; primer set... Primer set 07 is used to amplify the mitochondrial DNA fragment containing SNP site G327258C; primer set 08 is used to amplify the mitochondrial DNA fragment containing SNP site G388559C; primer set 09 is used to amplify the mitochondrial DNA fragment containing SNP site T544861G; primer set 10 is used to amplify the mitochondrial DNA fragment containing SNP site C732705A; primer set 11 is used to amplify the mitochondrial DNA fragment containing SNP site G782469T; primer set 12 is used to amplify the mitochondrial DNA fragment containing SNP site G1015555T; primer set 13... Primer set 14 is used to amplify mitochondrial DNA fragments containing SNP site T1240089G; primer set 15 is used to amplify mitochondrial DNA fragments containing SNP site C1264778G; primer set 16 is used to amplify mitochondrial DNA fragments containing SNP site T1300424G; primer set 17 is used to amplify mitochondrial DNA fragments containing SNP site C1552239G; primer set 18 is used to amplify mitochondrial DNA fragments containing SNP site G1883266C; primer set 1948365G; primers Primer set 19 was used to amplify the mitochondrial DNA fragment containing SNP site C2024981A; primer set 20 was used to amplify the mitochondrial DNA fragment containing SNP site T2114515G; primer set 21 was used to amplify the mitochondrial DNA fragment containing SNP site G2419091A; primer set 22 was used to amplify the mitochondrial DNA fragment containing SNP site G2501159C; primer set 23 was used to amplify the mitochondrial DNA fragment containing SNP site G2573569C; and primer set 24 was used to amplify the mitochondrial DNA fragment containing SNP site C2651373A.

[0035] Furthermore, in primer sets 01 to 24, the 5' ends of the two forward primers in each set are connected to different fluorescent tag sequences.

[0036] Furthermore, in primer sets 01 to 24, the 5' end of the first forward primer in each set is connected to a FAM fluorescent tag sequence, and the 5' end of the second forward primer is connected to a HEX fluorescent tag sequence.

[0037] The FAM fluorescent tag sequence is 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO: 73); the HEX fluorescent tag sequence is 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO: 74).

[0038] The primers described in this article may be KASP primers.

[0039] Furthermore, the primer sets described in this paper may include primer sets 01 to 24, wherein:

[0040] Primer set 01 may include forward primer 01F1 (SEQ ID NO: 1), forward primer 01F2 (SEQ ID NO: 2), and reverse primer 01R (SEQ ID NO: 3);

[0041] Primer set 02 may include forward primer 02F1 (SEQ ID NO: 4), forward primer 02F2 (SEQ ID NO: 5), and reverse primer 02R (SEQ ID NO: 6);

[0042] Primer set 03 may include forward primer 03F1 (SEQ ID NO: 7), forward primer 03F2 (SEQ ID NO: 8), and reverse primer 03R (SEQ ID NO: 9);

[0043] Primer set 04 may include forward primer 04F1 (SEQ ID NO: 10), forward primer 04F2 (SEQ ID NO: 11), and reverse primer 04R (SEQ ID NO: 12);

[0044] Primer set 05 may include forward primer 05F1 (SEQ ID NO: 13), forward primer 05F2 (SEQ ID NO: 14), and reverse primer 05R (SEQ ID NO: 15);

[0045] Primer set 06 may include forward primer 06F1 (SEQ ID NO: 16), forward primer 06F2 (SEQ ID NO: 17), and reverse primer 06R (SEQ ID NO: 18);

[0046] Primer set 07 may include forward primer 07F1 (SEQ ID NO: 19), forward primer 07F2 (SEQ ID NO: 20), and reverse primer 07R (SEQ ID NO: 21);

[0047] Primer set 08 may include forward primer 08F1 (SEQ ID NO: 22), forward primer 08F2 (SEQ ID NO: 23), and reverse primer 08R (SEQ ID NO: 24);

[0048] Primer set 09 may include forward primer 09F1 (SEQ ID NO: 25), forward primer 09F2 (SEQ ID NO: 26), and reverse primer 09R (SEQ ID NO: 27);

[0049] Primer set 10 may include forward primer 10F1 (SEQ ID NO: 28), forward primer 10F2 (SEQ ID NO: 29), and reverse primer 10R (SEQ ID NO: 30);

[0050] Primer set 11 may include forward primer 11F1 (SEQ ID NO: 31), forward primer 11F2 (SEQ ID NO: 32), and reverse primer 11R (SEQ ID NO: 33);

[0051] Primer set 12 may include forward primer 12F1 (SEQ ID NO: 34), forward primer 12F2 (SEQ ID NO: 35), and reverse primer 12R (SEQ ID NO: 36);

[0052] Primer set 13 may include forward primer 13F1 (SEQ ID NO: 37), forward primer 13F2 (SEQ ID NO: 38), and reverse primer 13R (SEQ ID NO: 39);

[0053] Primer set 14 may include forward primer 14F1 (SEQ ID NO: 40), forward primer 14F2 (SEQ ID NO: 41), and reverse primer 14R (SEQ ID NO: 42);

[0054] Primer set 15 may include forward primer 15F1 (SEQ ID NO: 43), forward primer 15F2 (SEQ ID NO: 44), and reverse primer 15R (SEQ ID NO: 45);

[0055] Primer set 16 may include forward primer 16F1 (SEQ ID NO: 46), forward primer 16F2 (SEQ ID NO: 47), and reverse primer 16R (SEQ ID NO: 48);

[0056] Primer set 17 may include forward primer 17F1 (SEQ ID NO: 49), forward primer 17F2 (SEQ ID NO: 50), and reverse primer 17R (SEQ ID NO: 51);

[0057] Primer set 18 may include forward primer 18F1 (SEQ ID NO: 52), forward primer 18F2 (SEQ ID NO: 53), and reverse primer 18R (SEQ ID NO: 54);

[0058] Primer set 19 may include forward primer 19F1 (SEQ ID NO: 55), forward primer 19F2 (SEQ ID NO: 56), and reverse primer 19R (SEQ ID NO: 57);

[0059] Primer set 20 may include forward primer 20F1 (SEQ ID NO: 58), forward primer 20F2 (SEQ ID NO: 59), and reverse primer 20R (SEQ ID NO: 60);

[0060] Primer set 21 may include forward primer 21F1 (SEQ ID NO: 61), forward primer 21F2 (SEQ ID NO: 62), and reverse primer 21R (SEQ ID NO: 63);

[0061] Primer set 22 may include forward primer 22F1 (SEQ ID NO: 64), forward primer 22F2 (SEQ ID NO: 65), and reverse primer 22R (SEQ ID NO: 66);

[0062] Primer set 23 may include forward primer 23F1 (SEQ ID NO: 67), forward primer 23F2 (SEQ ID NO: 68), and reverse primer 23R (SEQ ID NO: 69);

[0063] Primer set 24 may include forward primer 24F1 (SEQ ID NO: 70), forward primer 24F2 (SEQ ID NO: 71), and reverse primer 24R (SEQ ID NO: 72).

[0064] The present invention also provides a kit or DNA chip that may contain any of the primer sets described herein.

[0065] The kit or DNA chip has at least one of the following uses:

[0066] C1) is used to identify the mitochondrial DNA fingerprint of melon varieties;

[0067] C2) is used to identify the purity of melon hybrids;

[0068] C3) is used for the identification of the authenticity of melon varieties;

[0069] C4) is used for tracing the origins of melon varieties;

[0070] C5) was used for kinship analysis of melons.

[0071] This invention also provides any of the following applications of the mitochondrial SNP molecular marker combination, any of the primer sets described herein, or the kit or DNA chip:

[0072] A1) Application in identifying mitochondrial DNA fingerprints of melon varieties;

[0073] A2) Application in identifying the purity of melon hybrids;

[0074] A3) Application in the identification of the authenticity of melon varieties;

[0075] A4) Application in the tracing of melon varieties;

[0076] Application of A5 in the analysis of kinship in melons.

[0077] This invention also provides the use of any of the primer sets described herein in the preparation of products for detecting the mitochondrial SNP molecular marker combinations.

[0078] The product may be a reagent, reagent kit, chip, or test strip.

[0079] The present invention also provides a method for identifying the mitochondrial DNA fingerprint of a melon variety or the purity of a melon hybrid, the method comprising using any of the primer sets described herein or the kits or DNA chips to identify the mitochondrial DNA fingerprint of a melon variety or the purity of a melon hybrid.

[0080] The above method may include the following steps:

[0081] B1) Extract genomic DNA from the melon to be tested;

[0082] B2) Using the genomic DNA as a template, perform PCR amplification using any of the primer sets described herein, and collect fluorescence signals;

[0083] B3) Determine the genotype of the melon to be tested based on the fluorescence signal.

[0084] Furthermore, in the primer sets, the molar ratio of the two forward primers to the reverse primers in each primer set 01 to primer set 24 is 2:2:5.

[0085] Further, B3) can specifically be: if a tested melon variety (the melon to be tested) shows a blue fluorescent signal based on a certain SNP site, then the genotype of the tested melon variety based on that SNP site is homozygous for "the complementary base of the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F1""; if a tested melon variety shows a red fluorescent signal based on a certain SNP site, then the genotype of the tested melon variety based on that SNP site is homozygous for "the complementary base of the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F1""; The genotype of a tested melon variety is homozygous if it shows a green fluorescent signal based on a certain SNP site. One base is the complementary base of the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F1", and the other base is the complementary base of the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F2".

[0086] Furthermore, the method also includes further identifying the mitochondrial DNA fingerprint of the melon variety or the purity of the melon hybrid based on the genotype of the melon to be tested. For example, cluster analysis can be performed on the melon variety to be tested based on the genotype of the melon to be tested (based on the genotype of 24 mitochondrial SNP loci) to construct a mitochondrial DNA fingerprint map of the melon variety, etc., to identify the mitochondrial DNA fingerprint of the melon variety, identify the authenticity of the melon variety, distinguish different melon varieties, and identify the kinship of melon resources.

[0087] Furthermore, the number of homozygous genotypes and the number of missing genotypes can be counted based on the genotypes of the melons to be tested (based on genotypes at 24 mitochondrial SNP loci), and then the seed purity of the melon hybrid to be tested can be calculated. The formula for calculating the seed purity of the melon hybrid to be tested is: Hybrid seed purity = Number of homozygous genotypes of the father / (96 - Number of missing genotypes) × %.

[0088] This invention also provides a method for identifying the authenticity of melon varieties, classifying melon varieties, or analyzing the phylogenetic relationships of melons. The method may include using any of the primer sets, kits, or DNA chips described herein to identify the authenticity of melon varieties, classify melon varieties, or analyze the phylogenetic relationships of melons.

[0089] With the development of genomics technology, the mitochondrial genome of melon has been assembled, and whole-genome resequencing data of 149 melon resources have been published, providing marker resources for screening mitochondrial genetic loci in melon. This invention utilizes large-scale melon variant genome data and a mitochondrial reference genome to develop polymorphic SNP primer combinations in melon. The SNP primer combinations provided by this invention can be used to identify the mitochondrial DNA fingerprint of melon and the purity of first-generation hybrids, which has significant application value in clarifying the pedigree and commercial purity of melons during agricultural production and variety breeding. The method provided by this invention has advantages such as high throughput, accuracy, low cost, simple operation, and saving manpower and resources, and has a very broad application prospect. Attached Figure Description

[0090] Figure 1 The SNP genotyping effect of 24 primer sets in some tested melon varieties was studied.

[0091] Figure 2 Cluster diagram of 120 tested melon varieties based on 24 SNP primer sets.

[0092] Figure 3 A graph showing the relationship between the number of SNP markers (i.e., the number of SNP loci) and the differentiation of 120 tested melon varieties.

[0093] Figure 4 The results of SNP analysis show the purity of the melon hybrid to be tested.

[0094] Figure 5 The field phenotypes of normal and heteromorphic plants of the melon variety Jingyugu No. 2 are shown. Detailed Implementation

[0095] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0096] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0097] Example 1: SNP primer combinations for identifying mitochondrial DNA fingerprints and hybrid purity in melon varieties.

[0098] I. Discovery of Mitochondrial SNP Sites in Melon

[0099] In this study, SNP loci were identified based on resequencing data from 149 representative melon accessions, which represent a wide range of genetic diversity in melons. The data were compared with the melon mitochondrial reference genome to screen for perfect SNP loci that exhibited no other variations within 30 bp flanking the SNP, high polymorphism, were non-population-specific, and were mitochondrial genome-specific. Ultimately, 24 SNP loci were identified.

[0100] Specifically, the screening criteria for SNP sites are as follows: First, SNP sites with MAF>0.3, heterozygosity less than 0.05, deletion rate less than 0.1, polymorphism in both thin-skinned and thick-skinned melon populations, and conserved 30bp sequences on both wings (no InDel, no SSR, no other SNPs) were selected in the mitochondrial genome, resulting in 83 perfect SNPs in melon mitochondria; then, KASP primers were designed and SNP genotyping was performed, and 24 SNP primer sets were successfully genotyped.

[0101] Basic information on 24 SNP sites in the melon mitochondrial genome is detailed in Table 1. The locations of the SNP sites in the mitochondria were determined based on the alignment of the melon mitochondrial reference genome sequence, version number MG947207 (downloadable from: https: / / www.ncbi.nlm.nih.gov / nuccore / MG947207.1 / ).

[0102] Table 1. Basic information on 24 SNP loci in the mitochondrial genome of melon

[0103]

[0104]

[0105] II. Obtaining SNP primer combinations for identifying mitochondrial DNA fingerprints and hybrid purity in melon varieties

[0106] Based on the 24 SNP sites discovered in step one, the inventors of this invention designed, screened, and synthesized 24 primer combinations suitable for identifying the mitochondrial DNA fingerprint and hybrid purity of melon varieties using the allele competitive specific PCR (KASP) method. Each primer combination consists of 24 primer sets. The name of each primer set is shown in column 2 of Table 2. Each primer set consists of 3 primer sequences used to amplify one SNP site. The nucleotide sequences of each primer in the 24 primer sets are shown in column 4 of Table 2.

[0107] Table 2. 24 SNP primer combinations for identifying mitochondrial DNA fingerprinting and hybrid purity in melons.

[0108]

[0109]

[0110]

[0111]

[0112] Note: A single underscore indicates a FAM fluorescent tag sequence, and a double underscore indicates a HEX fluorescent tag sequence.

[0113] Example 2: Validation of SNP primer combinations

[0114] The basic information of the 120 tested melon varieties in this embodiment is shown in Table 3. These 120 commercial melon varieties were bred by more than 20 breeding units in China and are all common market varieties, including 87 thick-skinned melons and 33 thin-skinned melons. They exhibit rich genetic variation in terms of growth period, rind color, flesh color, fruit weight, and sugar content, and are representative of the varieties.

[0115] Table 3. Basic Information of 120 Tested Melon Varieties

[0116]

[0117]

[0118] 1. Obtaining genomic DNA from the tested melon varieties

[0119] Genomic DNA was extracted from the young roots (mixed with the young roots of 10 seeds) of 120 tested melon varieties using the CTAB method to obtain the genomic DNA of the tested melon varieties.

[0120] The quality and concentration of genomic DNA from the tested melon varieties must meet the requirements for PCR. The standards are as follows: agarose gel electrophoresis shows a single DNA band without obvious diffusion; the A260 / A280 ratio detected by a Nanodrop2000 (Thermo) UV spectrophotometer is around 1.8, and the A260 / A230 ratio is greater than 1.6; the concentration of genomic DNA from the tested melon varieties is 30-50 ng / μL.

[0121] 2. Using genomic DNA from 120 tested melon varieties as templates, PCR amplification was performed using 24 primer sets to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2", and primers containing "R" was 2:2:5.

[0122] The PCR reaction system and reagents are determined based on the microplate used, as detailed in Table 4 below.

[0123] Table 4. PCR reaction system for KASP marker detection

[0124]

[0125]

[0126] The PCR reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touch down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, and continued amplification for 26 cycles.

[0127] 3. After completing step 2, when the temperature of each PCR amplification product drops below 40℃, the fluorescence value is read by scanning with the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is read at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is read at an excitation wavelength of 528nm and an emission wavelength of 560nm). The genotype of each SNP locus of the 120 tested melon varieties is determined based on the fluorescence signal color. The specific judgment principles are as follows: If a tested melon variety shows a blue fluorescent signal based on a certain SNP site, then the genotype of the tested melon variety based on that SNP site is homozygous, consisting of the complementary base of the first 3' terminal base of the primer that amplifies the SNP site and whose name contains "F1"; if a tested melon variety shows a red fluorescent signal based on a certain SNP site, then the genotype of the tested melon variety based on that SNP site is homozygous, consisting of the complementary base of the first 3' terminal base of the primer that amplifies the SNP site and whose name contains "F2"; if a tested melon variety shows a green fluorescent signal based on a certain SNP site, then the genotype of the tested melon variety based on that SNP site is heterozygous, with one base being the complementary base of the first 3' terminal base of the primer that amplifies the SNP site and whose name contains "F1", and the other base being the complementary base of the first 3' terminal base of the primer that amplifies the SNP site and whose name contains "F2".

[0128] It should be noted that if the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20s, 55℃ annealing and extension for 1min, 5 cycles) until the results are satisfactory.

[0129] SNP typing results of 24 primer sets are shown below Figure 1 .

[0130] The results showed that all 24 primer sets could achieve good typing results in 120 tested melon varieties.

[0131] 4. Cluster analysis

[0132] Based on the genotypes of 120 tested melon varieties at 24 mitochondrial SNP loci, cluster analysis was performed on the 120 tested melon varieties using MiniMarker and MEGA7 software.

[0133] Cluster diagram of 120 tested melon varieties based on 24 primer sets is shown below. Figure 2 As shown in the figure. The results showed that the 24 primer sets could distinguish the 120 tested melon varieties in Table 3. Therefore, the primer sets developed in Example 1 can be applied to the identification of mitochondrial DNA fingerprints in melon varieties, distinguishing different melon varieties and effectively identifying the phylogenetic relationships of melon resources.

[0134] 5. Efficiency Evaluation

[0135] Species authenticity identification can be reduced by using sequential analysis. The inventors of this invention compared the relationship between the number of SNP markers (i.e., the number of SNP loci) and the discrimination rate of 120 tested melon varieties.

[0136] Based on the genotypes of 120 melon varieties at 24 mitochondrial SNP loci, Excel was used to count the number of pairwise differing loci between the 120 melon varieties. The following judgments were then made: If two melon varieties have one or more differing loci, they are considered different varieties with a distant pedigree; the more differing loci, the more distant the genetic relationship. If two melon varieties have zero differing loci, they are considered to be, or possibly are, the same melon variety, sharing the same pedigree.

[0137] Experimental results show that ( Figure 3 The 24 primer sets (i.e., 24 mitochondrial SNPs) achieved a discrimination rate of 98.5% in 120 melon varieties. A total of 7140 combinations were obtained through pairwise comparisons among the 120 melon varieties. Of these, 108 combinations had 0 mitochondrial SNP differential sites, 221 combinations had 1 mitochondrial SNP differential site, and 362 combinations had 2 mitochondrial SNP differential sites. This demonstrates that the 24 SNP markers developed in this invention have high discrimination ability.

[0138] Example 3: Identification of the purity of the Jingyugu No. 2 melon hybrid using the SNP primer combination developed in Example 1.

[0139] 1. The purity of the Jingyugu No. 2 melon hybrid was identified using the SNP primer combination developed in Example 1.

[0140] Jingyugu No. 2 is a common market variety and can be purchased commercially. The specific identification steps are as follows:

[0141] (1) 96 seeds of the Jingyugu No. 2 hybrid melon and its parent were planted to obtain melon seedlings to be tested; leaves or roots of the melon seedlings to be tested were taken and genomic DNA was extracted by CTAB method to obtain the genomic DNA of the melon variety to be tested.

[0142] (2) Using the genomic DNA of the melon variety to be tested as a template, PCR amplification was performed using 24 mitochondrial SNP primer sets to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2", and primers containing "R" in their names was 2:2:5.

[0143] The PCR reaction system and reagents are determined based on the microplate used, as detailed in Table 4.

[0144] The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touch down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, and continued amplification for 26 cycles.

[0145] (3) After completing step (2), when the temperature of each PCR amplification product drops below 40℃, the fluorescence value is read by scanning with the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is read at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is read at an excitation wavelength of 528nm and an emission wavelength of 560nm). The fluorescence signal color is obtained, and the following judgment is made: If the melon variety to be tested shows a blue fluorescence signal based on a certain SNP site, then the genotype of the melon variety to be tested based on that SNP site is homozygous for "the complementary base of the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F1". If the tested melon variety shows a red fluorescent signal based on a certain SNP site, then the genotype of the tested melon variety based on that SNP site is homozygous, consisting of "the complementary base of the first 3' terminal base of the primer that amplifies the SNP site and whose name contains "F2""; if the tested melon variety shows a green fluorescent signal based on a certain SNP site, then the genotype of the tested melon variety based on that SNP site is heterozygous, with one base being "the complementary base of the first 3' terminal base of the primer that amplifies the SNP site and whose name contains "F1"" and the other base being "the complementary base of the first 3' terminal base of the primer that amplifies the SNP site and whose name contains "F2"".

[0146] It should be noted that if the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20s, 55℃ annealing and extension for 1min, 5 cycles) until satisfactory results are obtained. SNP sites showing homozygous differences between parents were selected, and the primer set of these SNPs was used to perform SNP typing on the DNA of 96 melon hybrid seeds.

[0147] (4) Using the screened mitochondrial SNP primers, SNP typing was performed on 96 Jingyugu No. 2 melon hybrids. The results were analyzed based on the PCR product signals, such as... Figure 4 The genotypes of 96 seeds were determined, and the number of homozygous genotypes and the number of missing genotypes were counted. The seed purity of this tested melon hybrid was calculated using the following formula:

[0148] Hybrid seed purity = Number of homozygous genotypes in the father / (96 - Number of deletion genotypes) × %

[0149] Based on the SNP typing results, the purity of the tested melon hybrid is 92 / (96-0)=95.83%. Therefore, the purity of the tested melon hybrid is 95.83%.

[0150] 2. Plant 96 seeds of the Jingyugu No. 2 melon variety. Determine the purity of the 96 melon varieties based on their field phenotypes.

[0151] On July 20, 2023, 96 seeds of the Jingyugu No. 2 melon variety were planted in the greenhouse of the Vegetable Research Institute of Beijing Academy of Agricultural and Forestry Sciences. The spacing between plants in the field was 35 cm and the row spacing was 60 cm, with other management practices the same as in general field management. Thirty days after planting, during the fruiting period, field phenotypes were investigated. The hybridization rate of the variety was checked based on its characteristics, and the purity was calculated as the percentage of plants of this variety out of the 96 melon plants tested.

[0152] Phenotypic results showed that two heterotypic plants were found in the field. Figure 5 The identification results of the SNP primer combination developed in Example 1 were completely consistent with the phenotypic identification results.

[0153] Therefore, it can be seen that the SNP primer combination developed in Example 1 can be used to identify the purity of melon hybrids.

[0154] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A primer set, characterized in that, The primer set consists of primer set 01 to primer set 24 for amplifying the mitochondrial DNA fragment containing the SNP site, wherein: The primer set 01 comprises forward primers with nucleotide sequences of SEQ ID NO: 1 22-47, SEQ ID NO: 2 22-47, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 3; The primer set 02 comprises forward primers with nucleotide sequences of SEQ ID NO: 4 22-45, SEQ ID NO: 5 22-45, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 6; The primer set 03 comprises forward primers with nucleotide sequences of SEQ ID NO: 7 22-48, SEQ ID NO: 8 22-47, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 9; The primer set 04 comprises forward primers with nucleotide sequences of SEQ ID NO: 10 22-46, SEQ ID NO: 11 22-45, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 12; The primer set 05 comprises forward primers with nucleotide sequences of SEQ ID NO: 13 22-44, SEQ ID NO: 14 22-44, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 15; The primer set 06 comprises forward primers with nucleotide sequences of SEQ ID NO: 16 22-46, SEQ ID NO: 17 22-46, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 18; The primer set 07 comprises forward primers with nucleotide sequences of SEQ ID NO: 19 22-44, SEQ ID NO: 20 22-44, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 21; The primer set 08 comprises forward primers with nucleotide sequences of SEQ ID NO: 22 22-44, SEQ ID NO: 23 22-44, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 24; The primer set 09 comprises forward primers with nucleotide sequences of SEQ ID NO: 25 22-46, SEQ ID NO: 26 22-44, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 27; The primer set 10 comprises forward primers with nucleotide sequences of SEQ ID NO: 28 22-49, SEQ ID NO: 29 22-50, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 30; The primer set 11 comprises forward primers with nucleotide sequences of SEQ ID NO: 31 22-52, SEQ ID NO: 32 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 33; The primer set 12 comprises forward primers with nucleotide sequences of SEQ ID NO: 34 22-52, SEQ ID NO: 35 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 36; The primer set 13 comprises forward primers with nucleotide sequences of SEQ ID NO: 37 22-52, SEQ ID NO: 38 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 39; The primer set 14 comprises forward primers with nucleotide sequences of SEQ ID NO: 40 22-52, SEQ ID NO: 41 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 42; The primer set 15 comprises forward primers with nucleotide sequences of SEQ ID NO: 43 22-52, SEQ ID NO: 44 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 45; The primer set 16 comprises forward primers with nucleotide sequences of SEQ ID NO: 46 22-52, SEQ ID NO: 47 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 48; The primer set 17 comprises forward primers with nucleotide sequences of SEQ ID NO: 49 22-52, SEQ ID NO: 50 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 51; The primer set 18 comprises forward primers with nucleotide sequences of SEQ ID NO: 52 22-52, SEQ ID NO: 53 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 54; The primer set 19 comprises forward primers with nucleotide sequences of SEQ ID NO: 55 22-52, SEQ ID NO: 56 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 57; The primer set 20 comprises forward primers with nucleotide sequences of SEQ ID NO: 58 22-52, SEQ ID NO: 59 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 60; The primer set 21 comprises forward primers with nucleotide sequences of SEQ ID NO: 61 22-52, SEQ ID NO: 62 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 63; The primer set 22 comprises forward primers with nucleotide sequences of SEQ ID NO: 64 22-52, SEQ ID NO: 65 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 66; The primer set 23 comprises forward primers with nucleotide sequences of SEQ ID NO: 67 22-52, SEQ ID NO: 68 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO: 69; The primer set 24 comprises forward primers with nucleotide sequences of SEQ ID NO: 70 22-52, SEQ ID NO: 71 22-54, respectively, and a reverse primer with a nucleotide sequence of SEQ ID NO:

72. The primer set 12 comprises forward primers with nucleotide sequences of SEQ ID NO: 34 (22-47), SEQ ID NO: 35 (22-48), and reverse primers with nucleotide sequences of SEQ ID NO: 36; The primer set 13 comprises forward primers with nucleotide sequences of SEQ ID NO: 37 (22-44), SEQ ID NO: 38 (22-42), and reverse primers with nucleotide sequences of SEQ ID NO: 39; The primer set 14 comprises forward primers with nucleotide sequences of SEQ ID NO: 40 (22-46), SEQ ID NO: 41 (22-46), and reverse primers with nucleotide sequences of SEQ ID NO: 42; The primer set 15 comprises forward primers with nucleotide sequences of SEQ ID NO: 43 (22-51), SEQ ID NO: 44 (22-50), and reverse primers with nucleotide sequences of SEQ ID NO: 45; The primer set 16 comprises forward primers with nucleotide sequences of SEQ ID NO: 46 (22-47), SEQ ID NO: 47 (22-47), and reverse primers with nucleotide sequences of SEQ ID NO: 48; The primer set 17 comprises forward primers with nucleotide sequences of SEQ ID NO: 49 (22-44), SEQ ID NO: 50 (22-44), and reverse primers with nucleotide sequences of SEQ ID NO: 51; The primer set 18 comprises forward primers with nucleotide sequences of SEQ ID NO: 52 (22-44), SEQ ID NO: 53 (22-44), and reverse primers with nucleotide sequences of SEQ ID NO: 54; The primer set 19 comprises forward primers with nucleotide sequences of SEQ ID NO: 55 (22-44), SEQ ID NO: 56 (22-45), and reverse primers with nucleotide sequences of SEQ ID NO: 57; The primer set 20 comprises forward primers with nucleotide sequences of SEQ ID NO: 58 (22-52), SEQ ID NO: 59 (22-51), and reverse primers with nucleotide sequences of SEQ ID NO: 60; The primer set 21 comprises forward primers with nucleotide sequences of SEQ ID NO: 61 (22-44), SEQ ID NO: 62 (22-46), and reverse primers with nucleotide sequences of SEQ ID NO: 63; The primer set 22 comprises forward primers with nucleotide sequences of SEQ ID NO: 64 (22-43), SEQ ID NO: 65 (22-43), and reverse primers with nucleotide sequences of SEQ ID NO: 66; The primer set 23 comprises forward primers with nucleotide sequences of SEQ ID NO: 67 (positions 22-39) and SEQ ID NO: 68 (positions 22-39), and a reverse primer with a nucleotide sequence of SEQ ID NO:

69. The primer set 24 comprises forward primers with nucleotide sequences of SEQ ID NO: 70 (positions 22-46) and SEQ ID NO: 71 (positions 22-47), and a reverse primer with a nucleotide sequence of SEQ ID NO:

72.

2. The primer set according to claim 1, characterized in that, In each of the primer sets 01-24, the 5' ends of the two forward primers are connected with different fluorescent label sequences.

3. The primer set according to claim 1 or 2, characterized in that, In each of the primer sets 01-24, the 5' end of the first forward primer is connected with a FAM fluorescent label sequence, and the 5' end of the second forward primer is connected with a HEX fluorescent label sequence.

4. A kit or DNA chip, characterized in that, The kit or DNA chip comprises any one of the primer sets according to claims 1-3.

5. Use of any one of the primer sets according to claims 1-3 or the kit or DNA chip according to claim 4 in: A1) identifying mitochondrial DNA fingerprints of melon cultivars; A2) identifying purity of melon hybrid seeds; A3) identifying authenticity of melon cultivars; A4) tracing origins of melon cultivars; A5) analyzing genetic relationships of melon cultivars.

6. A method for identifying the mitochondrial DNA fingerprint of a melon cultivar or the purity of a melon hybrid, characterized in that, The method comprises using any one of the primer sets according to claims 1-3 or the kit or DNA chip according to claim 4 to identify mitochondrial DNA fingerprints of melon cultivars or purity of melon hybrid seeds.

7. The method of claim 6, wherein, The method comprises the following steps: B1) extracting genomic DNA of melon to be tested; B2) using the genomic DNA as a template, performing PCR amplification with any one of the primer sets according to claims 1-3, and collecting fluorescent signals; B3) determining the genotype of the melon to be tested according to the fluorescent signals.

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