A set of ssr core primers for identification of passion fruit varieties and application thereof

Abstract

The application discloses a kind of SSR core primer group for passion fruit variety identification and application thereof, belong to molecular biology and agricultural biotechnology field.The SSR core primer group is composed of 6 pairs of high polymorphism, good stability, clear amplification band SSR primer, its nucleotide sequence is as shown in SEQ ID NO:1-SEQ ID NO:12.The application also provides a method for identifying passion fruit varieties using the above primer group, including extracting the genomic DNA of the sample to be tested, performing PCR amplification with the primer group, detecting the amplified product to obtain the genotype using fluorescence-labeled capillary electrophoresis, comparing with the fingerprint database and counting the number of difference sites to obtain the identification result.The application can quickly and accurately identify passion fruit varieties, effectively distinguish varieties with similar genetic backgrounds, and the identification period only needs 2-3 days, which greatly shortens the 18-24 months required by traditional DUS test, and is suitable for passion fruit variety authenticity identification, variety purity detection, germplasm resource evaluation and other fields.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and agricultural biotechnology, specifically to an SSR core primer set for passion fruit variety identification and its application. Background Technology

[0002] Passionflower ( Passiflora edulis Passion fruit is one of the important economic fruit trees in tropical regions of my country. Statistics show that the planting area of ​​passion fruit in my country has exceeded 1 million mu (approximately 66,667 hectares), with major varieties including Tainong, Mantianxing, the Golden Series, and Xiangfei, among dozens of others. With the continuous expansion of planting scale and the rapid increase in the number of varieties, problems such as mixed varieties, homonymous varieties, and homonymous varieties are becoming increasingly prominent in production. For example, "Da Huangjin" and "Xiao Huangjin" are similar in appearance but have significantly different commercial values; "Mantianxing" and "Miyu" are difficult to distinguish in terms of seedling morphology. These problems seriously restrict the standardized development of the passion fruit industry and the efficient utilization of germplasm resources.

[0003] Currently, passion fruit variety identification mainly relies on field morphological observation and DUS (specificity, uniformity, and stability) testing. This method requires at least two growth cycles (approximately 18-24 months), consuming significant land resources. Furthermore, plant phenotypes are easily affected by environmental factors such as climate and soil, making it difficult to guarantee the accuracy, stability, and comparability of the identification results. Therefore, establishing an efficient, accurate, and environmentally independent variety identification technology system has become a critical issue that the passion fruit industry urgently needs to address.

[0004] SSR (simple repeat sequence) markers, due to their advantages such as codominant inheritance, rich polymorphism, good reproducibility, ease of operation, and low cost, have been incorporated into national standards (e.g., GB / T 39917-2021, NY / T 2594-2014) for variety identification in major crops such as rice, maize, and soybean, and have been recommended by the International Union for the Protection of New Varieties of Plants (UPOV) as the preferred molecular marker for variety identification. In passion fruit, several studies have reported on the development and application of SSR markers: Cerqueira-Silva et al. (2014) developed 15 pairs of SSR primers from the passion fruit genome and used them for genetic diversity analysis; Ocampo et al. (2016) obtained more than 200 SSR loci using transcriptome sequencing; and domestic scholars have also developed thousands of SSR loci through transcriptome sequencing and used them for germplasm resource evaluation.

[0005] However, existing SSR marker technology for passion fruit still has significant shortcomings in practical variety identification applications. Firstly, the polymorphism levels of reported SSR primers vary widely; many primers exhibit unclear amplification bands, poor repeatability, or lack of polymorphism in actual validation. Primers that have not undergone systematic screening and large-scale germplasm validation are difficult to use directly for accurate variety identification. Secondly, existing markers are mostly randomly selected, without optimizing core primer combinations for major cultivated passion fruit varieties. In conclusion, developing a set of core SSR primers with high polymorphism, good stability, strong identification ability, and systematic validation is of great significance for standardizing passion fruit variety management, protecting breeders' intellectual property rights, and promoting the healthy and orderly development of the industry. Summary of the Invention

[0006] To overcome the above-mentioned technical problems, this invention provides a rapid identification method for passion fruit varieties using SSR molecular markers and a dedicated primer combination, which is applicable to passion fruit germplasm resource identification, variety rights protection, seedling quality supervision, and breeding material authenticity testing.

[0007] This invention provides an SSR core primer set for passion fruit variety identification, which consists of the following 6 pairs of primers: The forward and reverse nucleotide sequences of the first pair of primers are shown in SEQ ID NO:1 and SEQ ID NO:2; The forward and reverse nucleotide sequences of the second pair of primers are shown in SEQ ID NO:3 and SEQ ID NO:4; The forward and reverse nucleotide sequences of the third pair of primers are shown in SEQ ID NO:5 and SEQ ID NO:6; The forward and reverse nucleotide sequences of the fourth pair of primers are shown in SEQ ID NO:7 and SEQ ID NO:8; The forward and reverse nucleotide sequences of the fifth pair of primers are shown in SEQ ID NO:9 and SEQ ID NO:10; The forward and reverse nucleotide sequences of the sixth primer pair are shown in SEQ ID NO:11 and SEQ ID NO:12.

[0008] Furthermore, the amplification fragment ranges of the six primer pairs are as follows: 177-201 bp, 170-190 bp, 198-268 bp, 154-180 bp, 129-189 bp, and 191-250 bp.

[0009] This invention also provides a method for identifying passion fruit varieties using the SSR core primer set, comprising the following steps: S1: Extract genomic DNA from the passion fruit sample to be tested; S2: Using the DNA extracted in step S1 as a template, perform PCR amplification using the 6 pairs of primers respectively; S3: Capillary electrophoresis was used to detect the PCR amplification products obtained in step S2 to obtain the allelic genotypes of the sample at 6 SSR loci. S4: Compare the alleles obtained in step S3 with the SSR fingerprint database of known passion fruit varieties to determine the variety of passion fruit.

[0010] Further, the PCR in step S2 is fluorescent PCR: the 5' end of the forward primer of the third pair of primers is labeled with ROX, the 5' end of the forward primer of the second pair of primers and the 5' end of the forward primer of the sixth pair of primers are labeled with TAMRA, the 5' end of the forward primer of the first pair of primers and the fifth pair of primers is labeled with VIC, and the 5' end of the forward primer of the fourth pair of primers is labeled with FAM; the labeled first to fourth pairs of primers form one group, and the labeled fifth and sixth pairs of primers form another group, which are then subjected to PCR amplification, and the PCR amplification products within the same group are detected by capillary electrophoresis in step S3.

[0011] Furthermore, in step S4, the SSR fingerprint database uses the allele fragments of standard varieties as a reference for calibration, and alleles with fragment size differences within ±1 bp in different laboratory tests are determined to be the same allele.

[0012] The present invention also provides a passion fruit variety identification kit, comprising the SSR core primer set, PCR reaction buffer, dNTPs, Taq DNA polymerase, molecular weight standard, and standard variety DNA control.

[0013] The present invention also provides the use of the SSR core primer set or the kit in the identification of passion fruit variety authenticity, detection of passion fruit variety purity, evaluation of genetic diversity of passion fruit germplasm resources, passion fruit assisted DUS test, and parent selection or identification of hybrid offspring in passion fruit breeding.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: I. The six pairs of core SSR primers provided by this invention, combined with fluorescently labeled capillary electrophoresis technology, can complete the entire identification process from DNA extraction to data analysis within 2-3 days, significantly shortening the identification cycle of 18-24 months required by traditional DUS testing, and significantly improving identification efficiency. It is suitable for application scenarios such as authenticity identification of passion fruit varieties, purity detection of varieties, and large-scale germplasm resource surveys.

[0015] Second, the six primer pairs of this invention detected 49 alleles in 40 germplasm accessions, with an average of 6.8 alleles detected per primer pair. The average polymorphism information content (PIC) was 0.6553. The PIC values ​​of the six markers were all higher than 0.5, indicating that they were all highly polymorphic sites. This effectively distinguished the eight passion fruit germplasm accessions tested, making up for the shortcomings of existing markers in terms of identification ability.

[0016] Third, this invention establishes an SSR fingerprint database calibrated with allele fragments of 8 standard varieties as references, and designs a 6-pair primer combination containing four fluorescent labels: ROX, TAMRA, VIC, and FAM, as well as a dual PCR-capillary electrophoresis detection system. A single injection can detect 4 sites, and all 6 labels can be detected in only 2 reaction wells, which greatly improves the detection efficiency. 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 This is an electrophoresis image of 40 passion fruit samples for DNA integrity testing in this invention (Note: Wells 1-40 are DNA sampling wells of 40 passion fruit varieties, corresponding to the following varieties in order: Xiangfei, Tainong, Da Huangjin, Mantianxing, Qinmi, Huangjin No.1, Dongmi, Miyu, Zhuangmi 07, Jindu No.3, Huangjin No.2, Hangkong Tainong, Huangjin No.3, Hangkong Da Huangjin, Renongzhen No.1, Renongzhen Z053, Huangguo No.1, Daguo, Longzhuguo No.1, Margaret, Ruixiang, Huangguo No.5, Zhuangmi 06, Huangguo No.2, Huangguo No.3, Huangguo No.4, Honghua, Longzhuguo No.2, Huangguo No.6, Guava Xiao Huangjin, Nanqu Youxuan, Chunhuang, Fujian No.3, Huangguo No.7, Yunnan Pu'er, Tianyuan, Qingpi 1, Apple 5, Jiaguo No.1, Wujin; Well M is the marker).

[0019] Figure 2 The agarose gel electrophoresis results are for the verification of the 6 pairs of SSR primers of this invention (Note: Wells 1, 5, 7, 9 and 11 correspond to the variety Xiangfei; Wells 2, 4, 6, 8, 10 and 12 correspond to the variety Dahuangjin; Well M is Marker).

[0020] Figure 3 The results of polyacrylamide gel electrophoresis are used to verify the polymorphism of some primer pairs in the mixed sample method of this invention (Note: M well is the marker).

[0021] Figure 4The results of polyacrylamide gel electrophoresis verification of the 6 pairs of polymorphic, stable, and highly distinguishable core SSR primers in this invention are shown (Note: Wells 1-8, 9-16, 17-24, 25-32, 33-40, and 41-48 are divided into 6 groups, and the varieties corresponding to each group in order are Xiangfei, Tainong, Huangjin No. 1, Da Huangjin, Mantianxing, Qinmi, Zhuangmi 06, and Zhuangmi 07, and well M is the marker).

[0022] Figure 5 The results of capillary electrophoresis detection of the six pairs of SSR core primers in this invention are shown.

[0023] Figure 6 The results of UPGMA cluster analysis of the eight passion fruit samples of this invention are used to verify the results. Detailed Implementation

[0024] 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.

[0025] Example 1 Development and screening of SSR core primers for passion fruit: 1.1 Plant materials Forty passion fruit germplasm resources were collected, including the following varieties: Xiangfei, Tainong, Da Huangjin, Mantianxing, Qinmi, Huangjin No.1, Dongmi, Miyu, Zhuangmi 07, Jindu No.3, Huangjin No.2, Hangkong Tainong, Huangjin No.3, Hangkong Da Huangjin, Renongzhen No.1, Renongzhen Z053, Huangguo No.1, Daguo, Longzhuguo No.1, Margaret, Ruixiang, Huangguo No.5, Zhuangmi 06, Huangguo No.2, Huangguo No.3, Huangguo No.4, Honghua, Longzhuguo No.2, Huangguo No.6, Guava Xiao Huangjin, Nanqu Youxuan, Chunhuang, Fujian No.3, Huangguo No.7, Yunnan Pu'er, Tianyuan, Qingpi 1, Apple No.5, Jiaguo No.1, Wujin.

[0026] All samples were collected from the Passionflower Germplasm Resource Nursery in Hainan Province. Each sample consisted of young leaves, which were flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0027] 1.2 Transcriptome Sequencing and SSR Locus Mining Flower tissues from *Passiflora purpurea* (Tainong) and *Passiflora yunnanensis* (Guava Little Golden) were selected, and total RNA was extracted for transcriptome sequencing. SSR loci were detected in the Unigene sequence using MISA 1.0 software (https: / / webblast.ipk-gatersleben.de / misa / ), with parameters set as follows: ≥10 single nucleotide repeats, ≥6 dinucleotide repeats, ≥5 trinucleotide repeats, ≥5 tetranucleotide repeats, ≥5 pentanucleotide repeats, and ≥5 hexanucleotide repeats. A total of 37,323 SSR loci were obtained.

[0028] 1.3 Primer Design Three sets of primers were designed for each SSR locus using Primer3 software. The design parameters were: primer length 18-27 bp, GC content 40%-60%, annealing temperature (Tm) 52-60℃, expected product length 100-350 bp, and the Tm difference between the upstream and downstream primers not exceeding 5℃. Preliminary screening of the specificity of each primer pair was performed using e-PCR, resulting in over 18,000 SSR loci suitable for primer design. From these, 1000 target loci were selected for primer synthesis and preliminary screening.

[0029] 1.4 DNA Extraction and Integrity Detection Leaves from 40 passionflower samples were collected, with three biological replicates for each sample. Genomic DNA was extracted using a modified CTAB method: 0.2 g of leaves were ground into powder in liquid nitrogen, and 1 mL of CTAB extraction buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0, 0.2% β-mercaptoethanol) preheated at 65°C was added. The mixture was inverted 2-3 times during the incubation period. Extraction was performed twice with an equal volume of chloroform:isoamyl alcohol (24:1). DNA was precipitated by adding 2 / 3 volume of isopropanol. The sample was washed twice with 70% ethanol and dried before being dissolved in TE buffer.

[0030] DNA concentration and purity (OD) were determined using an ultra-micro UV spectrophotometer. 260 / 280 The ratio is between 1.8 and 2.0. Take 1 μL of DNA for 1% agarose gel electrophoresis to check DNA integrity. Results are as follows... Figure 1 As shown, wells 1-40 are for 40 passion fruit samples with DNA spotting, all displaying clear main bands without tailing or degradation; well M is the DNA molecular weight standard (DL2000 DNA Marker). This indicates that the extracted DNA is of good integrity and meets the requirements for subsequent PCR amplification.

[0031] 1.5 Preliminary amplification and verification of primers Two samples of passion fruit DNA (Xiangfei and Dahuangjin) were randomly selected and PCR amplified using the primers synthesized in step 1.3.

[0032] PCR products were subjected to agarose gel electrophoresis. The amplification effect of the primers was initially screened based on the electrophoretic pattern. The screening criteria were: the amplified product showed a single, clear band with sufficient brightness (indicating high amplification efficiency), no visible non-specific amplification bands, no primer dimer dispersion or bands, and the amplified fragment size was basically consistent with the target region expected by the primer design. Primer pairs meeting all the above conditions were deemed qualified for the initial screening and proceeded to the subsequent polymorphism verification stage. This included the six target primer pairs from the technical solution of this invention, and the results are as follows: Figure 2 As shown, wells 1-12 contain the PCR products of the six primer pairs, all of which amplified clear and bright single bands without nonspecific amplification or primer dimers; well M contains the DNA molecular weight standard (DL2000). This indicates that the six primer pairs of the present invention can effectively amplify the target fragment.

[0033] 1.6 Polymorphic primer screening To efficiently screen primers with polymorphism, a mixed template method was employed. Eight passion fruit samples with significant phenotypic differences (namely Xiangfei, Tainong, Golden No. 1, Da Huangjin, Mantianxing, Qinmi, Zhuangmi 06, and Zhuangmi 07) were used, and equal amounts of DNA were mixed as templates. PCR amplification was performed using the primers synthesized in step 1.3. The products were then subjected to 6% non-denaturing polyacrylamide gel electrophoresis (crosslinking degree 19:1, 1×TBE buffer, 150 V constant voltage electrophoresis for 3 h) followed by silver staining.

[0034] Silver staining procedure: After electrophoresis, fix the gel in 10% ethanol for 10 min, sensitize it with 1% nitric acid for 5 min, rinse twice with deionized water, stain with 0.2% silver nitrate solution for 15 min, rinse quickly with deionized water, and develop with 3% sodium carbonate solution (containing 0.02% formaldehyde) until the bands are clear. Finally, terminate the reaction with 10% glacial acetic acid.

[0035] Electrophoresis results of selected primers are as follows Figure 3 As shown in the figure, the primers indicated by the arrows amplified multiple clear bands of different sizes in the mixed template, indicating that the primers of the present invention exhibit polymorphism in 8 materials with significant phenotypic differences, and all 6 pairs of core primers in the technical solution of the present invention are included therein.

[0036] 1.7 Stability Verification of Core Primers The six pairs of polymorphic core primers were selected to perform PCR amplification and 6% polyacrylamide gel electrophoresis on the DNA of eight passion fruit samples (Xiangfei, Tainong, Huangjin No. 1, Da Huangjin, Mantianxing, Qinmi, Zhuangmi 06, and Zhuangmi 07) to evaluate their polymorphic stability and variety differentiation ability. The PCR amplification reaction system is shown in Table 1. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 45 s, 30 cycles; 72℃ final extension for 7 min. The evaluation criteria were: (1) clear amplified bands and good reproducibility; (2) at least multiple alleles were detected in the eight materials; (3) the amplified fragment size range was convenient for capillary electrophoresis detection (100-350 bp).

[0037] Table 1 PCR amplification reaction system

[0038] Figure 4 The polyacrylamide gel electrophoresis screening results of the 6 pairs of core primers are shown. The images of each group of 8 wells show the genotypic electrophoresis performance of Xiangfei, Tainong, Huangjin No.1, Da Huangjin, Mantianxing, Qinmi, Zhuangmi06, and Zhuangmi07 on the XFL-F1370, XFL-F2259, XFL-F5342, XFL-F5939, XFL-F7699, and XFL-F8329 markers, respectively. It can be seen that each pair of primers produces clear and distinguishable polymorphic bands in 8 samples, and the number of alleles is abundant.

[0039] The specific sequences and numbers of the six primer pairs confirmed in this invention are shown in Table 2.

[0040] Table 2. Six pairs of SSR core primer sequences

[0041] 1.8 Fluorescent labeling and capillary electrophoresis detection To improve detection throughput, six primer pairs were divided into two groups based on the amplification fragment range and fluorescent labeling wavelength of each primer. Multiplex fluorescent PCR amplification was performed using eight DNA samples extracted from passionflower as templates. The labeled primers and groupings are shown in Table 3, and the PCR amplification reaction system is shown in Table 4. The fluorescent labeling position of all primer pairs was at the 5' end of the forward primer.

[0042] Table 3 Primer grouping and fluorescent labeling

[0043] Table 4 Multiplex fluorescent PCR amplification reaction system

[0044] Note: In each system, add 0.5 μL of each primer, and the final concentration of each primer is 0.25 μmol / L.

[0045] The PCR amplification programs for both groups were identical: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 45 s, for 30 cycles; and 72℃ final extension for 7 min.

[0046] Subsequently, capillary electrophoresis fingerprinting was performed on eight passionflower samples. For the two groups of fluorescent PCR amplifications, 1 μL of the product and 0.5 μL of GeneScan were taken respectively. TM 500 LIZ ® The internal standard and 8.5 μL of Hi-Di formamide were mixed, denatured at 95°C for 5 min, cooled in an ice bath, and then subjected to electrophoresis on a gene analyzer. Allele fragment sizes were analyzed using GeneMapper software, accurate to ±0.5 bp.

[0047] The following are the precise genotypes (in bp) of alleles from eight major passion fruit varieties: Fragrant Concubine: 201 / 201, 234 / 250, 158 / 158, 170 / 181, 207 / 207, 184 / 190; Taiwan Farmers: 193 / 201, 197 / 250, 155 / 158, 170 / 185, 198 / 198, 170 / 178; Gold No. 1: 201 / 201, 234 / 236, 158 / 158, 170 / 181, 207 / 207, 170 / 184; Large gold: 201 / 201, 236 / 236, 158 / 158, 170 / 170, 207 / 207, 190 / 190; Starry Sky: 201 / 201, 250 / 250, 158 / 158, 170 / 170, 207 / 207, 190 / 190; Qinmi: 201 / 201, 234 / 236, 158 / 158, 170 / 170, 207 / 207, 184 / 184; Zhuangmi 06: 193 / 201, 236 / 250, 158 / 158, 170 / 170, 207 / 207, 184 / 190; Zhuangmi 07: 193 / 201, 197 / 236, 155 / 158, 170 / 181, 207 / 207, 184 / 190.

[0048] Figure 5The capillary electrophoresis results of the "Xiangfei" variety at 6 core SSR marker sites are shown. Each site produces 1-2 peaks (homozygous or heterozygous). (Special note:) Figure 5 The first two short peaks in the XFL-F7699 group are impurity peaks. The peak positions correspond to the allele fragment sizes, and the peak heights reflect the fluorescence signal intensity.

[0049] In this protocol, the amplification products of each primer group are staggered in size to ensure they occupy different detection windows in capillary electrophoresis, avoiding peak overlap caused by fragments of similar length. Different primers within the group are labeled with different fluorescent dyes, and the emission spectra of each dye are separated, preventing fluorescence signal overlap and interference in capillary electrophoresis. The repeatability and stability of the multiplex detection system were verified using DNA samples from different passionflower germplasm resources, confirming that the amplified bands at each site were clear, with good peak shapes and no cross-interference.

[0050] Compared with conventional gel electrophoresis, this method has significant advantages: First, it uses six pairs of primers in two groups for multiplex amplification, and two capillary electrophoresis cycles simultaneously detect six sites, increasing throughput by three times; second, the combination of capillary electrophoresis and fluorescent internal standard achieves fragment size resolution accuracy of 1 bp, providing objective and automated results that avoid errors and subjectivity associated with manual plate reading; and third, multiplex PCR and two injection cycles significantly reduce reagents and consumables, resulting in a marked reduction in the cost of detecting a single site.

[0051] Example 2 Assessment of polymorphism of core SSR markers and their ability to distinguish varieties: 2.1 Allele Statistics The capillary electrophoresis fingerprint data of 40 passion fruit samples collected in Example 1 were analyzed. The results are shown in Table 5. A total of 49 alleles were detected in 6 pairs of core SSR markers, with an average of 8.2 alleles detected per primer pair, indicating that the SSR markers screened in this invention have high polymorphism.

[0052] Table 5 Polymorphism information of core SSR marker sites

[0053] Specifically: The allele variation range of the first primer pair (XFL-F1370) is 177-201 bp, and the nine valid alleles are: 177 / 178 / 181 / 182 / 189 / 190 / 193 / 197 / 201 bp. The allele variation range of the second primer pair (XFL-F8329) is 170-190 bp, and the five effective alleles are 170 / 178 / 182 / 184 / 190 bp. The allele variation range of the third primer pair (XFL-F7699) is 198-268 bp, and the eight valid alleles are: 198 / 207 / 229 / 231 / 233 / 235 / 242 / 268 bp; The allele variation range of the fourth primer pair (XFL-F5342) is 154-180 bp, and the nine effective alleles are: 154 / 155 / 156 / 157 / 158 / 159 / 161 / 162 / 280 bp; The allele variation range of the fifth primer pair (XFL-F5939) is 129-189 bp, and the 11 valid alleles are: 129 / 142 / 167 / 170 / 178 / 180 / 181 / 183 / 185 / 187 / 189 bp; The allele variation range of the sixth primer pair (XFL-F2259) is 191-250 bp, and the seven valid alleles are 191 / 197 / 199 / 204 / 234 / 236 / 250 bp.

[0054] 2.2 Analysis of Polymorphic Information Content According to the classification criteria of Cho et al. (1996): PIC > 0.50 indicates a high polymorphism site, 0.25 < PIC ≤ 0.50 indicates a moderate polymorphism site, and PIC ≤ 0.25 indicates a low polymorphism site. The PIC values ​​of the six core SSR primer pairs in this invention range from 0.5659 to 0.7358, with an average PIC value of 0.6553, all belonging to high polymorphism sites. Among them, primer 2 (XFL-F8329) has the highest PIC value (0.7358), and primer 4 (XFL-F5342) has the lowest PIC value (0.5659), but both are still above the high polymorphism threshold. The above results indicate that the SSR markers screened in this invention have excellent potential for variety differentiation.

[0055] 2.3 Allelic variation information The allele fragment sizes of the 6 primer pairs in the above 8 varieties were summarized to establish a passion fruit SSR fingerprint database, which facilitates data comparison between different laboratories. That is, the allelic variation range of the 6 primer pairs and some reference variety data are shown in Table 6.

[0056] Table 6. Allelic variations of the 6 SSR primer pairs and information on 8 reference varieties.

[0057] Example 3 Identification of 8 different sites for passion fruit varieties: 3.1 Determination of Differential Sites Based on the specific genotype data of the eight varieties given in Table 6, pairwise comparisons were performed on the eight varieties, resulting in 28 comparison data sets, as shown in Table 7. The comparison data for all varieties showed no identical differences at any one site. Therefore, the technical solution of this invention can directly distinguish the eight main passion fruit varieties using molecular biology methods.

[0058] Table 7. Statistical analysis of pairwise differences among eight passion fruit varieties.

[0059] 3.2 Cluster Analysis Based on genotypic data from 8 passion fruit samples at 6 SSR loci, Nei's genetic distance was calculated, and cluster analysis was performed using UPGMA (unweighted group average). Clustering results are as follows: Figure 6 As shown, the results indicate that *Mantianxing* and *Dahuangjin* clustered in the same branch, indicating a close kinship; *Qinmi* and *Huangjin 1* clustered in another branch, also showing a close genetic relationship. These clustering results are consistent with the paired results based on the number of differential loci (see Table 7), where *Mantianxing* and *Dahuangjin* had one differential locus, and *Qinmi* and *Huangjin 1* had two differential loci. The Tainong variety formed a separate branch independently of all other tested varieties in the cluster analysis, with at least four differential loci between it and the other varieties.

[0060] Based on the above cluster analysis and differential site comparison results, it is shown that the six pairs of SSR core primers screened in this invention can effectively distinguish eight tested passion fruit varieties, and the difference in the number of differential sites is highly consistent with the cluster analysis conclusion based on genetic distance, further verifying the variety identification effectiveness of the primer set set in this invention.

[0061] In summary, this invention addresses the issues of mixed passion fruit varieties and the lack of standardized procedures in existing SSR marker technologies by providing a core primer set consisting of six pairs of highly polymorphic SSR primers, along with a corresponding variety identification method and kit, for identifying eight major passion fruit varieties. This technical solution requires only 2-3 days for identification, significantly shortening the time compared to traditional DUS testing. It offers advantages such as accurate identification, good repeatability, strong discriminative ability, and low cost, providing an efficient and reliable molecular detection method for passion fruit variety authenticity identification, germplasm resource evaluation, and auxiliary DUS testing.

[0062] 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 passion fruit variety identification, characterized in that, The primer set consists of the following 6 pairs of primers: The forward and reverse nucleotide sequences of the first pair of primers are shown in SEQ ID NO:1 and SEQ ID NO:2; The forward and reverse nucleotide sequences of the second pair of primers are shown in SEQ ID NO:3 and SEQ ID NO:4; The forward and reverse nucleotide sequences of the third pair of primers are shown in SEQ ID NO:5 and SEQ ID NO:6; The forward and reverse nucleotide sequences of the fourth pair of primers are shown in SEQ ID NO:7 and SEQ ID NO:8; The forward and reverse nucleotide sequences of the fifth pair of primers are shown in SEQ ID NO:9 and SEQ ID NO:10; The forward and reverse nucleotide sequences of the sixth primer pair are shown in SEQ ID NO:11 and SEQ ID NO:

12.

2. The SSR core primer set according to claim 1, characterized in that, The amplification fragment ranges of the six primer pairs are as follows: 177-201 bp, 170-190 bp, 198-268 bp, 154-180 bp, 129-189 bp, and 191-250 bp.

3. A method for identifying passion fruit varieties using the SSR core primer set described in claim 1, characterized in that, Includes the following steps: S1: Extract genomic DNA from the passion fruit sample to be tested; S2: Using the DNA extracted in step S1 as a template, perform PCR amplification using the 6 pairs of primers respectively; S3: Capillary electrophoresis was used to detect the PCR amplification products obtained in step S2 to obtain the allelic genotypes of the sample at 6 SSR loci. S4: Compare the alleles obtained in step S3 with the SSR fingerprint database of known passion fruit varieties to determine the variety of passion fruit.

4. The method according to claim 3, characterized in that, The PCR in step S2 is fluorescent PCR: the 5' end of the forward primer of the third pair of primers is labeled with ROX, the 5' end of the forward primer of the second pair of primers and the 5' end of the forward primer of the sixth pair of primers are labeled with TAMRA, the 5' end of the forward primer of the first pair of primers and the fifth pair of primers is labeled with VIC, and the 5' end of the forward primer of the fourth pair of primers is labeled with FAM; the labeled first to fourth pairs of primers are grouped together, and the labeled fifth and sixth pairs of primers are grouped together, and PCR amplification is performed separately. The PCR amplification products within the same group are detected by capillary electrophoresis in step S3.

5. The method according to claim 3, characterized in that, In step S4, the SSR fingerprint database uses the allele fragments of standard varieties as a reference for calibration, and alleles with fragment size differences within ±1 bp in tests from different laboratories are identified as the same allele.

6. A passion fruit variety identification kit, characterized in that, The kit contains the SSR core primer set as described in claim 1, PCR reaction buffer, dNTPs, Taq DNA polymerase, molecular weight standards, and standard DNA controls.

7. The use of the SSR core primer set of claim 1 or the kit of claim 6 in the identification of passion fruit variety authenticity, detection of passion fruit variety purity, evaluation of genetic diversity of passion fruit germplasm resources, passion fruit assisted DUS test, and parent selection or identification of hybrid offspring in passion fruit breeding.

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