A ssr core primer set, kit, identification method and application for durian variety identification

Abstract

The application discloses a kind of for durian variety identification SSR core primer group, kit, identification method and application, belong to plant variety identification technical field.This core primer group contains 7 pairs of SSR primer, nucleotide sequence is sequentially as shown in SEQ ID NO:1 to SEQ ID NO:14, can be matched with 14 pairs of supporting SSR primer and form 21 pairs of core primer combination, average polymorphism information index reaches 0.7125.The application also provides detection kit containing the primer group, and corresponding variety identification method: extracting the genomic DNA of sample to be measured, obtains genotype spectrum by multiplex PCR amplification and capillary electrophoresis detection, compares with SSR fingerprint database, determines the variety of sample to be measured.The application is short in detection cycle, determination standard is unified, suitable for durian germplasm identification, seedling true and false identification.

Description

Technical Field

[0001] This invention relates to the field of plant variety identification technology, and in particular to an SSR core primer set, kit, identification method and application for durian variety identification. Background Technology

[0002] durian( Durio zibethinus Durian is a prized tropical fruit tree with extremely high economic value. With the rapid expansion of my country's durian consumption market, the problems of numerous varieties, a chaotic seedling market, and difficulty in distinguishing genuine from counterfeit varieties have become increasingly prominent. Traditional morphological identification is greatly affected by environment and developmental stage, making it difficult to accurately differentiate similar varieties.

[0003] Currently, durian variety identification mainly involves two technical approaches: morphological identification and molecular marker identification. Morphological identification distinguishes varieties by observing phenotypic traits such as leaves, flowers, and fruits. However, its results are greatly affected by environmental conditions and plant development stages, and the identification cycle is long, failing to meet the industry's demand for rapid identification. Among molecular marker identification methods, SSR markers have been widely used in variety identification and fingerprinting of various crops such as apples, citrus, and rice due to their advantages such as co-dominant inheritance, high polymorphism, good repeatability, and stable results. In the field of durian, several studies have attempted to use SSR markers for germplasm resource genetic diversity analysis and variety identification, but significant technical shortcomings still exist.

[0004] In domestic research, Deng Huidong et al. used fluorescent SSR technology to analyze the polymorphism and genetic diversity of 54 durian germplasm accessions from Hainan, screening out 19 pairs of polymorphic SSR primers. The average polymorphism information index (PIC) was 0.643, indicating only moderate polymorphism and limited ability to distinguish varieties. Internationally, Lin Xinge et al. developed 79,178 SSR markers based on whole-genome sequencing, providing a vast resource of markers for durian genetic research. However, this study did not systematically screen and validate these markers in practical applications, and standardized marker combinations that can be directly used for variety identification have not yet been established. Early international studies used only 3-7 pairs of SSR primers to distinguish a small number of durian varieties, resulting in a limited number of markers and inconsistent polymorphism levels, which could not support accurate identification of germplasm resources.

[0005] In summary, existing durian SSR identification technologies suffer from four core problems: First, there is a lack of systematically screened core primer combinations with excellent polymorphism; existing markers generally exhibit low polymorphism, resulting in insufficient resolution for variety identification. Second, standardized testing procedures and multiplex PCR detection systems have not been established, leading to low testing efficiency and high costs. Third, a unified database of standard genotypes for reference varieties has not been established, making it difficult to recognize test results from different laboratories and batches. Therefore, developing a set of highly polymorphic and highly discriminative core SSR primers and establishing standardized identification methods are of significant practical importance for regulating durian variety management and ensuring the healthy development of the industry. Summary of the Invention

[0006] This invention provides an SSR core primer set, kit, identification method, and application for durian variety identification, in order to overcome the above-mentioned problems.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention provides an SSR core primer set for durian variety identification, the SSR core primer set comprising 7 pairs of SSR primers: LL28, LL51, LL57, LL61, LL63, LL87, and LL105; The nucleotide sequences of the forward and reverse primers of the seven SSR primer pairs are as follows: Primer pair LL28: SEQ ID NO:1 and SEQ ID NO:2; Primer pair LL51: SEQ ID NO:3 and SEQ ID NO:4; Primer pair LL57: SEQ ID NO:5 and SEQ ID NO:6; Primer pair LL61: SEQ ID NO:7 and SEQ ID NO:8; Primer pair LL63: SEQ ID NO:9 and SEQ ID NO:10; Primer pair LL87: SEQ ID NO:11 and SEQ ID NO:12; Primer pair LL105: SEQ ID NO:13 and SEQ ID NO:14; The durian varieties mentioned are Musang King, Golden Pillow, Black Thorn, Squirrel King, Long Stem, Calamari, Kan Yao, and Indonesian Red.

[0008] Furthermore, the 5' ends of the forward primers of the seven SSR primer pairs are all connected to fluorescent staining groups, namely ROX, FAM, or VIC; wherein, the 5' ends of the forward primers of primer pair LL28, LL51, LL57, LL87, and LL105 are connected to ROX, the 5' ends of the forward primers of primer pair LL63 are connected to FAM, and the 5' ends of the forward primers of primer pair LL61 are connected to VIC.

[0009] Another aspect of the present invention provides the application of the aforementioned SSR core primer set in durian variety identification, determining the genetic diversity of durian germplasm resources, constructing durian variety linkage maps, or determining the kinship between durian varieties.

[0010] Another aspect of the present invention provides a kit containing the aforementioned SSR core primer set.

[0011] Another aspect of the present invention provides a method for identifying durian varieties, comprising the following steps: S1: Extract genomic DNA from the durian sample to be tested; S2: Using the obtained DNA as a template, multiplex fluorescent PCR amplification was performed using the fluorescently modified SSR core primer set and its 14 pairs of fluorescent SSR primers. S3: Perform capillary electrophoresis fluorescence fragment analysis on the amplification products to obtain the allele size of each SSR locus and construct a multi-site genotype profile of the sample to be tested. S4: Compare the genotype profile with the reference variety SSR fingerprint database to determine the variety of the durian sample to be tested; The nucleotide sequences of the forward and reverse primers of the 14 pairs of fluorescent SSR primers are as follows: Primer pair LLW2: SEQ ID NO:15 and SEQ ID NO:16; Primer pair LLW3: SEQ ID NO:17 and SEQ ID NO:18; Primer pair LLW4: SEQ ID NO:19 and SEQ ID NO:20; Primer pair LLW5: SEQ ID NO:21 and SEQ ID NO:22; Primer pair LLW7: SEQ ID NO:23 and SEQ ID NO:24; Primer pair LLW8: SEQ ID NO:25 and SEQ ID NO:26; Primer pair LLW9: SEQ ID NO:27 and SEQ ID NO:28; Primer pair LLW10: SEQ ID NO:29 and SEQ ID NO:30; Primer pair LLW11: SEQ ID NO:31 and SEQ ID NO:32; Primer pair LLW12: SEQ ID NO:33 and SEQ ID NO:34; Primer pair LLW13: SEQ ID NO:35 and SEQ ID NO:36; Primer pair LLW15: SEQ ID NO:37 and SEQ ID NO:38; Primer pair LLW16: SEQ ID NO:39 and SEQ ID NO:40; Primer pair LLW19: SEQ ID NO:41 and SEQ ID NO:42.

[0012] Furthermore, in the 14 pairs of fluorescent SSR primer pairs, the 5' end of the forward primer is connected to a fluorescent group of ROX, FAM, VIC, or TAMRA; wherein, the 5' end of the forward primer of primer pair LLW12 is connected to ROX; the 5' end of the forward primers of primer pairs LLW5, LLW7, LLW10, LLW15, and LLW16 is connected to ROX; The 5' end is connected to TAMRA; the 5' ends of the forward primers of primer pair LLW2, LLW4, LLW11, and LLW19 are connected to VIC; the 5' ends of the forward primers of primer pair LLW3, LLW8, LLW9, and LLW13 are connected to FAM.

[0013] Further, in step S4, the reference variety SSR fingerprint database is a pre-established database containing standard allele size data for each locus corresponding to the 7 pairs of SSR primers and the 14 pairs of fluorescent SSR primers for 8 representative durian varieties.

[0014] The beneficial effects of this invention are: 1. This invention is based on the aril transcriptome data of three durian varieties: Musang King, Tomani, and Golden Pillow. Through SSR locus mining, primer design, and multiple rounds of screening, seven pairs of self-developed core SSR primers were obtained, which, together with 14 pairs of validated matching SSR primers, form a core primer set of 21 pairs. Using this primer set, 40 durian germplasm accessions were analyzed, detecting a total of 154 alleles with an average polymorphism information index (PIC) of 0.7125. Among them, 19 primer pairs had PIC values ​​higher than 0.5, indicating highly polymorphic loci. Compared with the existing technology with an average PIC of 0.643, the polymorphism level is improved by approximately 10.8%, enabling high-resolution differentiation of durian varieties.

[0015] 2. Based on the primer fluorescent labeling type and amplicon fragment range, this invention constructs five sets of multiplex PCR reaction systems. A single reaction well can simultaneously amplify four SSR sites, and all 21 sites require only five reactions to complete the amplification. This represents a 75% improvement in detection efficiency compared to single-primer PCR, significantly reducing detection costs and reagent consumption. Combined with capillary electrophoresis fluorescence detection technology, the identification cycle of traditional morphological identification (1-2 growing seasons) can be shortened to 2-3 days, meeting the industry's needs for durian germplasm resource screening and rapid identification of seedling authenticity.

[0016] 3. This invention uses 8 durian germplasm samples as experimental materials and accurately determines the allele fragment size of each variety by capillary electrophoresis. It establishes an SSR fingerprint database, i.e. a standard genotype database, covering 8 main durian varieties, to provide a unified comparison benchmark for the test results of different laboratories. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Figure A shows the results of chromosome fluorescence staining and telomere fluorescence in situ hybridization of *Durian simonii*, while Figure B shows the results of chromosome fluorescence staining karyotype detection and telomere fluorescence in situ hybridization detection. Figure 2 The following are examples of flow cytometry ploidy analysis results for two varieties, Golden Pillow and Squirrel King. Figure A shows the flow cytometry ploidy analysis results for Golden Pillow and Figure B shows the flow cytometry ploidy analysis results for Squirrel King. Figure 3 DNA integrity testing of 40 durian samples ( Figure 3The varieties corresponding to lanes 1-40 are, in order: Chun Chun, Lim Fong Jiao, D600, Hulu, Zhujiao, Wugonghuang, Jiapili, Yen Thong, Lobster, Sultan King, KK5, TEMBAGA, Dream Lover, Red Shrimp, Jawi Super, Keladi Gold, Manik Urai, Pilo King, Yellow Ginger, Tok Megat, Angbak, Tok L1 Tok, Kunyu No. 7, Green Skin, Golden Bun, Golden Phoenix, Red Meat, MDUR 88, Long Stem, Musang King, Golden Pillow, Salika, Ouyuege, Squirrel King, Indonesian Red, Big Fruit, Jialun, Malaysian Red, Black Thorn, Ganyao; Lane M is Marker). Figure 4 The agarose gel electrophoresis screening results of the 7 pairs of SSR primers provided for this invention ( Figure 4 Lanes 1, 3, 5, 7, 9, 11, and 13 in the middle lane correspond to the Musang King breed; lanes 2, 4, 6, 8, 10, 12, and 14 correspond to the Golden Pillow breed; lane M is Marker. Figure 5 The results of polyacrylamide gel electrophoresis detection of some samples on the 7 pairs of SSR primers provided in this invention are as follows: Figure 5 The varieties corresponding to the lanes in each group from left to right are Musang King, Golden Pillow, Blackthorn, Squirrel King, Longstalk, Caucasian, Gan Yao, and Indonesian Red; lane M is the marker). Figure 6 The results of capillary electrophoresis detection of 7 pairs of fluorescent primers for Musang King; Figure 7 The results of capillary electrophoresis detection of 7 pairs of fluorescent primers for *Pholiota chinensis* are shown. Figure 8 The results of UPGMA cluster analysis of the eight durian samples are used to verify the findings of this invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: Durian Sample Collection and Plumpness Analysis 1. Durian sample collection Forty durian germplasm resources were collected from the durian resource nursery in Sanya, Hainan. For each germplasm, two types of experimental materials were collected simultaneously: healthy mature leaves and tender shoot tips. The leaf samples were used for subsequent primer screening and construction of a standard genotype database, while the shoot samples were used for ploidy level analysis experiments.

[0021] The specific varieties of the 40 durian germplasm resources are shown in Table 1 below: Table 1. Variety names of 40 durian germplasm resources

[0022] 2. Analysis of the polyploidy level of durian resources First, fluorescence in situ hybridization chromosome karyotype analysis was performed on the Golden Pillow variety. The results are as follows: Figure 1 As shown, the results of chromosome fluorescence staining karyotype detection for durian are as follows: Figure 1 As shown in Figure A, the results of telomere fluorescence in situ hybridization detection are as follows: Figure 1 As shown in Figure B, chromosome counting of mitotic cells revealed that the somatic chromosome number of this variety was 56. Combined with the diploid report of the "KanYao" variety (2n=56) in the literature (Jietal., 2025), the Golden Pillow variety was identified as a diploid control. Subsequently, 40 durian shoots were collected and analyzed for ploidy level using flow cytometry, with Golden Pillow as the control variety. The results showed that the ploidy of 39 resources was consistent with that of Golden Pillow, all being diploid and suitable for subsequent SSR molecular marker analysis. Figure 2 Example of flow cytometry ploidy analysis results for two varieties, Golden Pillow and Squirrel King ( Figure 2 Image A shows a golden pillow. Figure 2 Image B in the middle is of the Squirrel King.

[0023] Example 2: Development and Screening of SSR Primer Sets 1. SSR locus discovery and primer design Transcriptome data of the aril of three durian varieties—Musang King, Tomanee, and Golden Pillow—were obtained from public databases. SSR loci were detected in the Unigene sequence using MISA 1.0 (https: / / webblast.ipk-gatersleben.de / misa / ), yielding a total of 5577 SSR loci. Primers were designed for these 5577 SSR loci using Primer 3 software, with three sets of primers designed for each SSR locus. Additionally, 30 pairs of SSR primers with discriminative capabilities were collected from the literature and included in the initial screening.

[0024] 2. Primer initial screening DNA from two durian germplasm materials, Musang King and Golden Pillow, collected in Example 1, was randomly selected and PCR amplified using primer pairs with 5577 SSR loci designed above. The amplified products were detected by 1% agarose gel electrophoresis. Based on the quality of the electrophoretic bands, 175 primer pairs capable of producing clear and bright bands were selected. Among them, the agarose gel electrophoresis screening results of the 7 core SSR primer pairs finally determined in this invention (primer pair LL28, primer pair LL51, primer pair LL57, primer pair LL61, primer pair LL63, primer pair LL87, and primer pair LL105) are as follows: Figure 4 As shown.

[0025] 3. Genomic DNA extraction Leaf tissues were collected from the above 40 durian germplasm samples, with three biological replicates for each sample. Genomic DNA was extracted using a modified CTAB method, and the specific steps are as follows: Weigh 100 mg of leaf sample into a 2.0 mL centrifuge tube, freeze in liquid nitrogen, and grind thoroughly into powder. Add 800 µL of CTAB extraction buffer preheated to 65 °C to each tube, mix thoroughly, and incubate in a 65 °C water bath for 45-60 min. During incubation, gently invert the centrifuge tube every 15 min to mix. After the water bath, centrifuge at 12000 rpm for 10 min. Transfer the supernatant to a new centrifuge tube, add an equal volume of a mixture of chloroform and isoamyl alcohol (chloroform to isoamyl alcohol volume ratio 24:1) to each tube, mix gently, and let stand at room temperature for 10 min, then centrifuge at 12000 rpm for 10 min. Transfer the supernatant to a new centrifuge tube again, add an equal volume of pre-chilled isopropanol, gently invert to mix, and incubate at -20 °C for 30 min to precipitate the DNA. After DNA precipitation, centrifuge at 12000 rpm for 10 min. Discard the supernatant, wash the DNA precipitate twice with 70% ethanol solution, discard the ethanol, air dry the precipitate at room temperature, add 100 µL of double-distilled water or TE buffer to fully dissolve the DNA, and finally use an ultra-micro UV spectrophotometer to detect the DNA concentration and purity. Store qualified DNA samples at -20℃ for later use.

[0026] 4. DNA integrity detection using agarose gel electrophoresis To verify the quality of the extracted DNA, this experiment used 1% agarose gel electrophoresis to detect the DNA integrity of each sample. The procedure is as follows: (1) Gel preparation: Weigh 1 g of agarose and add 100 mL of 1× TAE buffer to prepare a 1% (w / v) agarose gel. Heat in a microwave oven until the agarose is completely dissolved, then cool to about 50-60℃, add 3 μL of nucleic acid dye, and mix gently. Pour the gel solution into the gel casting tank with the comb already placed in it, avoiding the formation of air bubbles. Let it stand at room temperature for 20-30 min until the gel is completely solidified, then carefully remove the comb. Transfer the gel to the electrophoresis tank and add 1× TAE buffer until the liquid level is 1-2 mm above the gel surface.

[0027] (2) Sample preparation and loading: Take 1 μL of DNA sample and mix it with 1 μL of 6× loading buffer and 4 μL of ddH2O, for a total volume of 6 μL; carefully add the mixture to the gel wells with a pipette and record the sample order; add 5 μL of DNA molecular weight standard to the side loading wells.

[0028] (3) Electrophoresis detection: Cover the electrophoresis tank, turn on the power, and set the voltage to 120V for constant voltage electrophoresis; electrophoresis for 20~30 minutes, and stop electrophoresis when the bromophenol blue indicator migrates to 2 / 3 of the gel position.

[0029] (4) Observation and photography: Place the gel in a gel imaging system, observe the DNA bands and take photos for storage.

[0030] DNA integrity test results of 40 durian samples are as follows: Figure 3 As shown in the figure, the results indicate that the DNA bands of each sample are clear and the main band is bright, indicating that the extracted DNA is of good integrity and can be used for subsequent PCR amplification and labeling analysis.

[0031] 5. Screening of polymorphic primers Using DNA from eight durian samples with significant phenotypic differences out of the 40 samples in Example 1 as material, the DNA was mixed in equal amounts and used as amplification templates. PCR amplification was performed using the 175 pairs of SSR primers pre-screened and the 30 pairs of SSR primers collected from the literature, and the amplification products were detected by 6% polyacrylamide gel electrophoresis. The eight durian samples with significant phenotypic differences were: Musang King, Golden Pillow, Black Thorn, Squirrel King, Long Stem, Jialun, Ganyao, and Indonesian Red.

[0032] 5.1 PCR Amplification The total volume of the PCR amplification reaction system is 20 μL. The concentration and amount of each component are prepared according to Table 2. The system can be fine-tuned according to the actual conditions during the experiment.

[0033] Table 2 PCR amplification reaction system

[0034] The PCR reaction program was set as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; after the cycle, 72℃ extension for 10 min, and the amplification products were stored at 4℃.

[0035] 5.2 Polyacrylamide gel electrophoresis detection (1) Gel preparation (taking 6% non-denaturing PAGE as an example): Assemble the vertical electrophoresis glass plate and secure it with clips to ensure a seal. Prepare a 6% separating gel (20 mL system): 4 mL of 30% acrylamide solution, 4 mL of 5×TBE, 11.8 mL of ddH2O, 160 μL of 10% APS, and 16 μL of TEMED, for a total volume of 20 mL. Finally, add TEMED, mix immediately, and pour the gel along one side of the glass plate using a pipette, avoiding the formation of air bubbles. Immediately insert the comb and polymerize at room temperature for 30 min. After the gel has completely solidified, carefully remove the comb and rinse the sample wells with deionized water to remove unpolymerized acrylamide.

[0036] (2) Sample preparation: Take 2 μL of PCR amplification product, mix it with an equal volume of 6×PAGE loading buffer, and centrifuge briefly before use.

[0037] (3) Electrophoresis detection: Fix the gel plate in the electrophoresis tank and add 1×TBE buffer to the inner and outer tanks; use a flat-tipped pipette to add the sample to the gel wells, with a sample volume of 2 μL per well, and add molecular weight standard to the side wells at the same time; cover the electrophoresis tank, connect the power supply, set the constant voltage to 150 V, and the electrophoresis time is about 1.5 h.

[0038] (4) Silver staining: After electrophoresis, carefully pry open the glass plate, peel the gel into a plastic box, add 0.2% AgNO3 solution (freshly prepared), and stain in the dark for 7 min. Discard the AgNO3, rinse the gel twice quickly with deionized water; add 3% Na2CO3 (containing 0.02% formaldehyde) staining solution, and gently shake until the bands are clearly visible. Finally, place the gel on a transmission light box or scanner, take a picture and save it.

[0039] Based on the electrophoresis results, primer pairs with polymorphism were initially screened out. Subsequently, the polymorphic primers were retested using DNA from eight individual durian samples with significant phenotypic differences as templates to assess the stability of their identification ability. After two rounds of screening, a total of 21 pairs of core SSR primers with stable polymorphism and excellent identification ability were obtained, of which 7 pairs were independently developed and 14 pairs were from the literature. The names and nucleotide sequences (forward and reverse primers) of the 7 independently developed core SSR primer pairs are shown in Table 3. Table 3. Sequences of 7 pairs of self-developed core SSR primers

[0040] The names and nucleotide sequences (forward and reverse primers) of the 14 pairs of matching SSR primers are shown in Table 4. Table 4. Sequences of 14 pairs of matching SSR primers

[0041] Subsequently, fluorescently labeled versions of the 21 primer pairs listed in Tables 3 and 4 were synthesized, and fingerprint data were collected from the eight durian samples gathered in Example 1. The polyacrylamide gel electrophoresis results of the seven self-developed core SSR primer pairs in Table 3 in the aforementioned eight durian samples are as follows: Figure 5 As shown, Figure 5 The results show that the seven pairs of self-developed core SSR primers provided by this invention can amplify clear and stable bands in the above eight durian samples. Moreover, the band size and number among different samples show obvious polymorphism, indicating that the selected primers have good variety identification ability and can be used for the subsequent fluorescent labeling fingerprint analysis of eight durian materials.

[0042] 6. Capillary electrophoretic fingerprint acquisition Fluorescently labeled versions of the 21 pairs of core SSR primers listed in Tables 3 and 4 were synthesized. The 5' ends of the forward primers were labeled with four fluorescent dyes: ROX, TAMRA, VIC, and FAM. The primer grouping and fluorescent labeling are detailed in Table 5. Based on the fluorescence color and amplicon fragment range, five sets of primers were constructed according to the primer grouping scheme in Table 5 and Table 6, respectively, for multiplex PCR amplification of eight durian varieties: Musang King, Golden Pillow, Black Thorn, Squirrel King, Long Stem, Jialun, Ganyao, and Indonesian Red. Each well could simultaneously amplify four or more SSR loci. Using this scheme, amplification of all 21 loci in a single sample requires only five wells, improving detection efficiency by 75% compared to traditional single-primer single-well PCR.

[0043] Table 5 Primer grouping

[0044] Note: The values ​​in parentheses represent the allelic variation ranges for each primer. Amplification products from primers within the same group can be subjected to multiplex electrophoresis. The grouping can be adjusted by changing the fluorescent label based on the primer allelic variation ranges.

[0045] Table 6. Multiplex fluorescent PCR system with 5 primer sets

[0046] Note: In each system, add 0.5 μL of each primer, and the final concentration of each primer is 0.25 μmol / L.

[0047] After amplification, capillary electrophoresis is performed. The procedure is as follows: The PCR amplification products from the five reaction wells corresponding to a single sample were diluted 50-fold and then mixed in equal volumes. One μL of the mixture was then added to 0.1 μL of the molecular weight internal standard (GeneScan). TM 500 LIZ ® Prepare a 10 μL sample loading mixture by adding 8.9 μL of deionized formamide and 10 μL of 5 μL of PCR reagent. Denature the mixture at 95°C for 5 min in a PCR instrument, then immediately cool it on ice for 10 min. After brief centrifugation, load the sample onto an ABI 3730xl DNA analyzer. Use a POP-7... TM Separating gels and 1×Buffer were used for electrophoresis separation according to the instrument's preset program. Raw electrophoresis data were analyzed using GeneMarker V2.2.0 software. Allelic fragment sizes were calculated based on molecular weight internal standards. Alleles for each sample on each primer pair were recorded: homozygous sites were denoted as X / X, heterozygous sites as X / Y, and missing data sites as 0 / 0. All genotype data were finally exported to an Excel spreadsheet for subsequent statistical analysis.

[0048] The capillary electrophoresis peak diagrams of the Musang King and Golden Pillow varieties under 7 pairs of fluorescent primers are as follows: Figure 6 , Figure 7 As shown.

[0049] The fingerprint data of Musang King, Golden Pillow, Blackthorn, Squirrel King, Long-stalked, Gynostemma pentaphyllum, Gan Yao, and Indonesian Red under the seven primer pairs LL28, LL51, LL57, LL61, LL63, LL87, and LL105 are as follows: Musang King fingerprint data: 210 / 210, 265 / 265, 280 / 288, 226 / 254, 186 / 204, 269 / 281, 202 / 207; Jinzhen fingerprint data: 204 / 208, 265 / 265, 280 / 280, 229 / 254, 192 / 192, 269 / 269, 207 / 207; Blackthorn fingerprint data: 204 / 204, 262 / 265, 280 / 284, 254 / 261, 186 / 192, 269 / 269, 202 / 207; Squirrel King fingerprint data: 204 / 210, 265 / 265, 280 / 288, 254 / 254, 192 / 196, 269 / 275, 207 / 207; Long-handled fingerprint data: 204 / 210, 265 / 265, 280 / 280, 254 / 261, 186 / 202, 269 / 269, 202 / 207; Jia Lun's fingerprint data: 204 / 210, 265 / 265, 280 / 280, 252 / 254, 186 / 192, 269 / 269, 207 / 207; Gan Yao's fingerprint data: 204 / 210, 265 / 265, 280 / 280, 254 / 254, 192 / 192, 269 / 275, 207 / 207; Indonesian red fingerprint data: 208 / 210, 265 / 265, 288 / 288, 254 / 254, 186 / 186, 263 / 269, 202 / 207.

[0050] Example 3: Evaluation of Polymorphism and Variety Identification Ability of Core SSR Markers Using 40 durian germplasms collected in Example 1 as materials, 21 pairs of SSR primers screened in Example 2 were used for capillary electrophoresis typing. The obtained fingerprint data were statistically analyzed to evaluate the polymorphism and variety identification ability of the primers. The results showed that a total of 154 alleles were detected by the 21 primer pairs, with a maximum of 14 alleles detected by a single primer and an average of 7.3 alleles detected per primer. The polymorphism information of the 21 pairs of SSR primers is shown in Table 7.

[0051] Table 7 Polymorphism information of 21 pairs of SSR primers

[0052] Specifically, among the 7 pairs of self-developed core SSR primers, the allelic variation range of the first primer pair (LL28) is 202-210 bp, and the 4 effective alleles are 202 / 204 / 208 / 210 bp respectively; The allele variation range of the second primer pair (LL51) is 262-265 bp, and the two effective alleles are 262 / 265 bp. The allele variation range of the third primer pair (LL57) is 170 / 288 bp, and the nine valid alleles are: 170 / 173 / 185 / 273 / 275 / 279 / 280 / 284 / 288 bp; The allele variation range of the fourth primer pair (LL61) is 226-263 bp, and the six effective alleles are 226 / 229 / 252 / 254 / 261 / 263 bp. The allele variation range of the fifth primer pair (LL63) is 184-213 bp, and the 10 valid alleles are: 184 / 186 / 192 / 194 / 196 / 198 / 200 / 202 / 204 / 213 bp; The allele variation range of the sixth primer pair (LL87) is 263-281 bp, and the four valid alleles are 263 / 269 / 275 / 281 bp.

[0053] The allele variation range of the 7th primer pair (LL105) is 193-213 bp, and the 5 valid alleles are 193 / 202 / 204 / 207 / 213 bp.

[0054] The polymorphism information content (PIC) of the 21 primer pairs ranged from 0.3372 to 0.8786, with an average PIC value of 0.7125. Among the 21 SSR primer pairs, 19 pairs had PIC values ​​higher than 0.5, indicating highly polymorphic sites, meeting the standard requirements for high polymorphism of marker sites. (Note: The correlation between PIC value and gene polymorphism is as follows: PIC value greater than 0.50 indicates highly polymorphic sites; PIC value between 0.25 and 0.50 indicates moderately polymorphic sites; PIC value less than 0.25 indicates low polymorphic sites, indicating that the gene is relatively conserved.)

[0055] The allelic fragment sizes of the seven pairs of self-developed core SSR primers (LL28, LL51, LL57, LL61, LL63, LL87, and LL105 in Table 3) in the above eight varieties were summarized to establish a durian SSR fingerprint database, i.e., a standard genotype database, providing a unified basis for comparison of test results from different laboratories. The allelic variation range of the seven pairs of self-developed core SSR primers and the genotype data of the eight reference varieties are shown in Table 8.

[0056] Table 8. Allelic variations of 7 pairs of SSR primers and information on 8 reference varieties.

[0057] Note: Table 8 only lists the genotype data of mainstream commercial varieties and does not cover all the germplasm corresponding to the variations.

[0058] Example 4: Determination of Differential Loci for Identification of 8 Durian Varieties 1. Determination of differential loci Based on the specific genotype data of the eight durian varieties on the seven pairs of self-developed core SSR primers given in Table 7 of Example 3, the eight durian varieties were compared pairwise, resulting in a total of 28 pairs of data (the calculation formula is 8×7÷2=28, that is, each variety is paired with the other seven, but each pair will be counted repeatedly, so it is divided by 2). The results are shown in Table 9. The difference sites in the comparison data of all varieties are not completely the same. Therefore, the technical solution of the present invention can directly distinguish the eight main durian varieties through molecular biology.

[0059] Table 9. Statistical analysis of pairwise differences among eight durian varieties.

[0060] 2. Cluster analysis Based on genotypic data from 8 durian samples at 7 SSR loci, Nei's genetic distance was calculated, and cluster analysis was performed using UPGMA (unweighted group average). The clustering results are as follows: Figure 8 As shown, Figure 8 The results showed that Gan Yao and Squirrel King clustered in the same branch, indicating a close phylogenetic relationship. This clustering result is consistent with the pairwise comparison results based on the number of differential loci in Table 8, where Gan Yao and Squirrel King had two differential loci. Indonesian Red and Musang King formed separate branches independently of all other tested varieties in the cluster analysis.

[0061] Based on the above cluster analysis and differential site comparison results, it is shown that the 7 pairs of SSR core primers screened in this invention can effectively distinguish among the 8 tested durian varieties, and the difference in the number of differential sites is highly consistent with the conclusion of the cluster analysis based on genetic distance, further verifying the variety identification effectiveness of the primer set set in this invention.

[0062] In summary, this invention, using eight durian germplasm resources as experimental materials, systematically completed a full set of experimental work, including germplasm ploidy level verification, multiple rounds of SSR primer screening, optimization of multiplex PCR amplification system, establishment of capillary electrophoresis detection process, construction of standard genotype database, and verification of variety identification methods. Through a two-stage screening strategy of mixed template initial screening and single-sample secondary screening, 21 pairs of polymorphic stable core SSR primers with excellent variety identification capabilities were finally obtained, including 7 pairs of self-developed primers and 14 pairs of literature-derived primers. Based on the above 7 pairs of self-developed core SSR primers, a standard genotype database covering eight representative durian germplasm resources was established. The identification method provided by this invention is simple to operate, has a short detection cycle, and provides objective and unified judgment standards. It can be stably applied to scenarios such as durian variety authenticity identification, precise germplasm resource identification, and variety rights protection, providing reliable molecular detection technology support for seedling traceability and standardized management of germplasm resources in the durian industry.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An SSR core primer set for durian variety identification, characterized in that, The SSR core primer set includes 7 pairs of SSR primers: LL28, LL51, LL57, LL61, LL63, LL87, and LL105. The nucleotide sequences of the forward and reverse primers of the seven SSR primer pairs are as follows: Primer pair LL28: SEQ ID NO:1 and SEQ ID NO:2; Primer pair LL51: SEQ ID NO:3 and SEQ ID NO:4; Primer pair LL57: SEQ ID NO:5 and SEQ ID NO:6; Primer pair LL61: SEQ ID NO:7 and SEQ ID NO:8; Primer pair LL63: SEQ ID NO:9 and SEQ ID NO:10; Primer pair LL87: SEQ ID NO:11 and SEQ ID NO:12; Primer pair LL105: SEQ ID NO:13 and SEQ ID NO:14; The durian varieties mentioned are Musang King, Golden Pillow, Black Thorn, Squirrel King, Long Stem, Calamari, Kan Yao, and Indonesian Red.

2. The SSR core primer set according to claim 1, characterized in that, The 5' ends of the forward primers of all seven SSR primer pairs are connected to fluorescent staining groups, namely ROX, FAM, or VIC. Specifically, the 5' ends of the forward primers of primer pair LL28, LL51, LL57, LL87, and LL105 are connected to ROX; the 5' ends of the forward primers of primer pair LL63 are connected to FAM; and the 5' ends of the forward primers of primer pair LL61 are connected to VIC.

3. The application of the SSR core primer set described in claim 1 in durian variety identification, determining the genetic diversity of durian germplasm resources, constructing durian variety linkage maps, or determining the phylogenetic relationships among durian varieties, characterized in that, The durian varieties mentioned are Musang King, Golden Pillow, Black Thorn, Squirrel King, Long Stem, Calamari, Kan Yao, and Indonesian Red.

4. A reagent kit, characterized in that, It contains the SSR core primer set as described in claim 1.

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