EST-SSR molecular marker primer set developed based on Cherry Blossom transcriptome sequence and its application

Abstract

This invention relates to the field of plant molecular marker technology, specifically to an EST-SSR molecular marker primer set developed based on the transcriptome sequence of *Prunus campanulata* and its applications. The EST-SSR molecular marker primer set contains 19 pairs of primers, with sequences shown in SEQ ID Nos. 1–38. The EST-SSR molecular marker primers developed based on *Prunus campanulata* transcriptome data exhibit greater accuracy, stability, and effectiveness compared to traditional molecular markers. Furthermore, the EST-SSR markers are mostly distributed in regions related to gene coding regions or regulatory sequences, providing a theoretical basis and important technical means for *Prunus campanulata* germplasm resource identification, genetic diversity analysis, molecular-assisted breeding, and fingerprinting, and have promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of plant molecular marker technology, specifically to an EST-SSR molecular marker primer set developed based on the transcriptome sequence of Prunus campanulata and its application. Background Technology

[0002] The campanulata cherry (Cerasus campanulata) is a high-quality cherry blossom germplasm resource in my country and one of the four major ornamental cherry blossom species. It boasts high ornamental value, wide adaptability, and strong resistance to adverse conditions, making it highly valuable for development and utilization. The campanulata exhibits a relatively large coefficient of variation in various traits among individuals, indicating potential for selective breeding. In recent years, domestic research on the breeding and horticultural application of the campanulata cherry has developed rapidly, with many new varieties being authorized. Therefore, the identification of numerous campanulata cherry varieties is particularly important.

[0003] DNA fingerprinting, a marker-based identification technology, offers advantages such as accuracy, simplicity, speed, and ease of automation. Rapid variety identification through DNA fingerprinting is a crucial technique for variety identification and conservation. SSR molecular markers possess advantages including abundant quantity, genome-wide coverage, high polymorphism, co-dominance, and ease of operation.

[0004] The publication number CN117925898A, entitled "An InDel Marker Primer Combination for Cherry Blossom Germplasm Resources and Its Application," discloses an InDel marker combination consisting of five primer pairs that can be used to identify Cherry Blossom germplasm from seven different geographical sources and construct DNA fingerprinting. However, this method only targets a limited number of germplasm resources from different geographical sources, and the sample size and genetic diversity covered are significantly insufficient. Furthermore, the paper does not provide core polymorphism metrics, such as allele count (Na), effective allele count (Ne), and polymorphism information content (PIC) analysis, making it impossible to objectively evaluate the actual polymorphism level and practical value of the primer set. In addition, it lacks population genetic tests such as Hardy-Weinberg equilibrium (HWE) testing, making it difficult to assess whether the loci meet population genetic expectations, among other potential problems. Compared with SSR markers, this InDel marker combination has inherent limitations in its identification ability. SSR markers typically possess multi-site, high polymorphism, and multiple allele characteristics, resulting in stronger discriminative ability. In contrast, InDel markers are mostly biallelic with lower polymorphism, limiting their accuracy in distinguishing closely related or different varieties from the same origin. Although the selected InDel marker primer set can identify seven geographical germplasm samples of *Prunus campanulata*, its reliability in identification efficiency, breadth of application, and stability of primer polymorphism remain significant shortcomings, thus limiting the efficiency and accuracy of *Prunus campanulata* germplasm identification and genetic diversity analysis. Summary of the Invention

[0005] This invention addresses the problems existing in the prior art by providing an EST-SSR molecular marker primer set developed based on the transcriptome sequence of *Prunus campanulata*, which can be used to construct a fingerprint map of *Prunus campanulata* and analyze the genetic diversity of germplasm resources.

[0006] To achieve the above objectives, the technical solution adopted by this invention is: an EST-SSR molecular marker primer set developed based on the transcriptome sequence of *Prunus campanulata*, the EST-SSR molecular marker primer set containing 19 primer pairs, including: CCFSSR036, CCFSSR056, CCFSSR093, CCFSSR195, CCFSSR215, CCFSSR217, CCFSSR241, CCFSSR267, CCFSSR275, CCFSSR293, CCFSSR296, CCFSSR315, CCFSSR316, CCFSSR317, CCFSSR342, CCFSSR365, CCFSSR381, CCFSSR390, and CCFSSR398;

[0007] The CCRSSR036 primer set sequence is shown in SEQ ID No. 1-2;

[0008] The CCRSSR056 primer set sequence is shown in SEQ ID No. 3-4;

[0009] The CCRSSR093 primer set sequence is shown in SEQ ID No. 5-6;

[0010] The CCRSSR195 primer set sequence is shown in SEQ ID No. 7-8;

[0011] The CCRSSR215 primer set sequence is shown in SEQ ID No. 9-10;

[0012] The CCRSSR217 primer set sequence is shown in SEQ ID No. 11-12;

[0013] The CCRSSR241 primer set sequence is shown in SEQ ID No. 13-14;

[0014] The CCRSSR267 primer set sequence is shown in SEQ ID No. 14-15;

[0015] The CCRSSR275 primer set sequence is shown in SEQ ID No. 17-18;

[0016] The CCRSSR293 primer set sequence is shown in SEQ ID No. 19-20;

[0017] The CCRSSR296 primer set sequence is shown in SEQ ID No. 21-22;

[0018] The CCRSSR315 primer set sequence is shown in SEQ ID No. 23-24;

[0019] The CCRSSR316 primer set sequence is shown in SEQ ID No. 25-26;

[0020] The CCRSSR317 primer set sequence is shown in SEQ ID No. 27-28;

[0021] The CCRSSR342 primer set sequence is shown in SEQ ID No. 29-30;

[0022] The CCRSSR365 primer set sequence is shown in SEQ ID No. 31-32;

[0023] The CCRSSR381 primer set sequence is shown in SEQ ID No. 33-34;

[0024] The CCRSSR390 primer set sequence is shown in SEQ ID No. 35-36;

[0025] The CCRSSR398 primer set sequence is shown in SEQ ID No. 37-38.

[0026] A second objective of this invention is to provide a kit for analyzing the genetic diversity of *Chlorophytum comosum* germplasm resources or constructing fingerprint profiles, the kit containing the primer sets described in SEQ ID No. 1 to 38.

[0027] A third objective of this invention is to provide the application of sequences such as SEQ ID No. 1-38 or the above-described kits in the genetic diversity analysis of *Chlorophytum comosum* germplasm resources.

[0028] A fourth objective of this invention is to provide the application of sequences such as SEQ ID No. 1 to 38 or the above-described kit in the construction of *Chlorophytum comosum* fingerprints.

[0029] The beneficial technical effects of this invention are as follows: The EST-SSR molecular marker primers developed based on *Prunus campanulata* transcriptome data possess accuracy, stability, and effectiveness compared to traditional molecular markers. Furthermore, EST-SSR markers are mostly distributed in regions related to gene coding regions or regulatory sequences, providing a theoretical basis and important technical means for *Prunus campanulata* germplasm resource identification, genetic diversity analysis, molecular-assisted breeding, and fingerprinting construction, and have promising application prospects. This invention is characterized by high efficiency and low cost, with numerous and comprehensive molecular marker sites developed, and it is universally applicable among *Prunus campanulata* strains. Attached Figure Description

[0030] 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 only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is an agarose gel electrophoresis image of the PCR products from the eight cherry blossom germplasm materials in Example 1, amplified using the CFSSR315-CCFSSR320 molecular marker primers. In the image, SSR 315, SSR 316, SSR 317, SSR 318, SSR 319, and SSR320 represent primer set numbers; C20, C22, C25, C29, C31, C37, F91, and F101 represent *Prunus campanulata* sample numbers; and M stands for Marker.

[0032] Figure 2 This is a capillary fluorescence detection image of the PCR products after amplification using CCFSSR275 primers from the eight cherry blossom germplasm materials in Example 1.

[0033] Figure 3 The UPGMA clustering results are shown for the 47 Prunus campanulata germplasm materials in Example 2. Detailed Implementation

[0034] 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, and 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.

[0035] Example 1

[0036] Development and screening of polymorphic primers for Prunus campanulata

[0037] 1. Transcriptome sequencing

[0038] The *Prunus campanulata* samples were collected from healthy young leaves in Xiamen, Fujian Province, for RNA extraction. High-throughput transcriptome sequencing yielded 75,582 unigenes for SSR locus searching, with an average length of 1.69 Kb.

[0039] 2. SSR locus identification

[0040] MISA was used to detect unigenes and obtain EST-SSR locus information. A total of 30,654 SSR loci were detected in 21,217 unigenes.

[0041] 3. Primer design

[0042] EST-SSR primers were designed using Primer 3 software, resulting in a batch design of 16,692 primer pairs. The main parameters set during primer design were: primer length between 20-23 bp; annealing temperature around 60℃; base repeat number ≤ 4; and no two consecutive A / T bases at the 5' and 3' ends. The designed primers were evaluated using Oligo 7.0 software. Finally, 400 pairs were selected for marker development and validation.

[0043] 4. Genomic DNA Extraction and Detection

[0044] Healthy young leaves from eight cherry blossom germplasm materials with significant differences in ornamental traits (Table 1) were selected, and genomic DNA was extracted using the TSINGKE Plant DNA Extraction Kit (universal type). After PCR amplification, the DNA concentration and purity were detected by 1% agarose gel electrophoresis and an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). After passing the tests, the DNA extract was diluted to 25 ng / μl and stored at -20°C for later use.

[0045] Table 1. Eight cherry blossom germplasm materials tested.

[0046]

[0047] 5. PCR amplification reaction

[0048] The PCR amplification system and amplification procedure are as follows (Table 2 and Table 3).

[0049] Table 2 PCR amplification reaction system

[0050]

[0051] Table 3 PCR amplification program

[0052]

[0053] Note: The first amplification was annealed at 62℃, and the second amplification was annealed at 55℃ with primers containing fluorescently modified adapters.

[0054] 6. Polymorphic primer screening

[0055] The amplified PCR products were detected by 1% agarose gel electrophoresis (2 μl sample + 6 μl bromophenol blue) to obtain identification gel images. When using 'double flag' as the DNA template, 303 primer pairs were successfully amplified, resulting in 303 sets of valid image data, all of which showed high consistency. Figure 1 The most representative CCFSSR315-CCFSSR320 groups were selected for demonstration. Template concentration was then determined using gel electrophoresis, diluted with water, and further screened using capillary electrophoresis. Results showed that 186 primer pairs were successfully amplified in all 8 samples. Figure 2 As shown in the figure (the image is a partial representative case of CCFSSR275 capillary electrophoresis analysis), it accounted for 46.5% of the synthesized primers. After further analysis, 169 primer pairs with high polymorphism were finally screened out in all 8 cherry blossom varieties.

[0056] Using these 169 pairs of EST-SSR polymorphic primers of Prunus campanulata as candidate primers, through analysis, 19 pairs (Table 4) of primers with high polymorphic information and stable amplification bands were finally selected as core primers for genetic diversity analysis and fingerprinting of Prunus campanulata.

[0057] Table 4. Information on 19 EST-SSR core primer pairs screened from the *Prunus campanulata* transcriptome.

[0058]

[0059] Example 2: Application of EST-SSR molecular marker primer set to genetic diversity analysis of Prunus campanulata.

[0060] 1. Sources of germplasm resources

[0061] Of the 47 tested materials (Table 5), 38 were of the *Cephalotaxus fortunei* (also known as *Cephalotaxus fortunei* var. *fujianensis*), 3 were of the *Cephalotaxus dielsiana* var. *dielsiana*, 2 were of the *Cephalotaxus speciosa* var. *spirosa*, 2 were of the *Cephalotaxus yedoensis* var. *yonnanensis*, 1 was of the *Cephalotaxus yunnanensis* var. *cerasoides*.

[0062] Table 5. Basic information on the 47 cherry blossom germplasm resources tested.

[0063]

[0064] 2. Genomic DNA extraction

[0065] DNA was extracted from the 47 germplasm resources listed in Table 5 using the TSINGKE Plant DNA Extraction Kit (General Type). After extraction, DNA concentration and purity were determined by 1% agarose gel electrophoresis and an ND-1000 spectrophotometer. Once the DNA samples passed the tests, the extract was diluted to 25 ng / μl and stored at -20°C for later use.

[0066] 3. PCR amplification reaction

[0067] Using the DNA described above as a template, PCR amplification was performed using the 19 pairs of polymorphic primers selected in Table 4. The PCR reaction system and procedure were the same as those in Tables 2 and 3.

[0068] 4. Genetic diversity analysis

[0069] PCR amplification products were detected and analyzed using capillary electrophoresis, and the results were read and analyzed. Genetic diversity of *Prunus campanulata* was analyzed by comparing amplified polymorphic bands. Specifically, using PopGen 32 and GenALEx version 6.501 software, various genetic diversity indices for SSR loci and populations were calculated, including observed alleles (Na), effective alleles (Ne), Shannon index (I), polymorphism information index (PIC), observed heterozygosity (Ho), expected heterozygosity (He), major allele frequency (MP), and inbreeding coefficient (Fis).

[0070] Table 6 Polymorphism of 19 EST-SSR primers

[0071]

[0072] Note: Na: observed alleles, Ne: effective alleles, I: Shannon index, PIC: polymorphism information index, Ho: observed heterozygosity, He: expected heterozygosity, PHWE: Hardy-Weinberg equilibrium. *: P<0.05, **: P<0.01, ***: P<0.001, ns: P>0.05.

[0073] The results showed that 139 allele loci were detected in 47 samples using 19 primer pairs (Table 6), with the number of alleles ranging from 4 to 15, averaging 7.3 alleles per locus. The total number of effective alleles was 49.7590, ranging from 1.3867 to 7.6568, with an average of 2.6189 effective alleles per locus. The Shannon index (I) ranged from 0.5554 to 2.2745, with an average of 1.1335. The polymorphism information content (PIC) ranged from 0.2579 to 0.8563, with an average of 0.4968. Therefore, the 19 EST-SSR primer pairs exhibited high polymorphism. The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.1277 to 0.7234 and 0.2819 to 0.8787, respectively, with means of 0.3577 and 0.5416. The mean inbreeding coefficient was 0.3324. The Hardy-Weinberg equilibrium test revealed that 18 of the 19 SSR loci deviated from Hardy-Weinberg equilibrium at P < 0.01, possibly due to locus over-heterozygosity.

[0074] 5. Cluster analysis

[0075] Based on the Nei genetic distance, a UPGMA tree was constructed in POPULATIONS 1.2.30 software (with 1000 replicates) for cluster analysis of 47 tested materials. The cluster tree was formatted and edited using FigTree version 1.4.2 software. Clustering results ( Figure 3 The results show that C36 high-bowl cherry stands alone, while the three tail-leaf cherry varieties C2, C5, and C22 cluster together, and C25 Showa cherry is also separated from the other varieties. Within the bell-shaped cherry lineage, varieties with close phenotypic and phylogenetic relationships, such as the red double-petaled 'Peony Cherry', 'Jiangshan Beauty', 'Wangdi', 'Fugui', and 'Xiamen Beauty', cluster together. Meanwhile, pink varieties with spreading petals, such as 'Red Pink Beauty', 'Guangzhou Cherry', and 'Fuzhou Feihan', cluster together. Therefore, the EST-SSR clustering results can effectively reflect the phylogenetic relationships among the tested cherry blossom varieties and provide a reference for pedigree tracing among varieties.

[0076] Example 3: Application of EST-SSR molecular marker primer set in the construction of DNA fingerprinting of different germplasm of Prunus campanulata.

[0077] The amplification bands of the above 19 pairs of EST-SSR primers in 47 germplasm materials were detected by capillary electrophoresis. Based on the detection results, fingerprint analysis and genotyping were performed. The allelic marker configuration of each sample at each SSR locus was recorded, and the 19 pairs of SSR primers were encoded sequentially. The encoded information was combined to form the SSR fingerprint map of the *Prunus campanulata* samples.

[0078] No single primer pair could completely distinguish the 47 materials. Based on the polymorphism information index of the primers, CCRSSR342 and CCRSSR316 were selected first for variety identification. Based on the results, another primer pair was added sequentially for combined identification. When CCRSSR342, CCRSSR316, and CCRSSR317 were selected for combined identification, C11, C15, C17, C18, C20, and C28 remained indistinguishable. Analysis of the remaining primers revealed that CCRSSR215 could distinguish C17, C20, and C28, while CCRSSR267 and CCRSSR036 could distinguish C11, C15, and C18. Therefore, six primer pairs, CCRSSR342, CCRSSR316, CCFSSR317, CCRSSR267, CCRSSR036 and CCRSSR215 (Table 7), were finally selected to construct DNA fingerprint profiles for 47 cherry blossom germplasm materials (Table 8).

[0079] Table 7. Six pairs of EST-SSR primers used for fingerprinting.

[0080]

[0081] Table 8. EST-SSR fingerprint codes of 47 tested cherry blossom varieties

[0082]

[0083] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An EST-SSR molecular marker primer set developed based on the transcriptome sequence of Prunus subhirtella, characterized in that: the EST-SSR molecular marker primer set comprises 19 primer sets, including CCRSSR036, CCRSSR056, CCRSSR093, CCRSSR195, CCRSSR215, CCRSSR217, CCRSSR241, CCRSSR267, CCRSSR275, CCRSSR293, CCRSSR296, CCRSSR315, CCRSSR316, CCRSSR317, CCRSSR342, CCRSSR365, CCRSSR381, CCRSSR390 and CCRSSR398; the sequence of the CCRSSR036 primer set is shown as SEQ ID No. 1-2; the sequence of the CCRSSR056 primer set is shown as SEQ ID No. 3-4; the sequence of the CCRSSR093 primer set is shown as SEQ ID No. 5-6; the sequence of the CCRSSR195 primer set is shown as SEQ ID No. 7-8; the sequence of the CCRSSR215 primer set is shown as SEQ ID No. 9-10; the sequence of the CCRSSR217 primer set is shown as SEQ ID No. 11-12; the sequence of the CCRSSR241 primer set is shown as SEQ ID No. 13-14; the sequence of the CCRSSR267 primer set is shown as SEQ ID No. 14-15; the sequence of the CCRSSR275 primer set is shown as SEQ ID No. 17-18; the sequence of the CCRSSR293 primer set is shown as SEQ ID No. 19-20; the sequence of the CCRSSR296 primer set is shown as SEQ ID No. 21-22; the sequence of the CCRSSR315 primer set is shown as SEQ ID No. 23-24; the sequence of the CCRSSR316 primer set is shown as SEQ ID No. 25-26; the sequence of the CCRSSR317 primer set is shown as SEQ ID No. 27-28; the sequence of the CCRSSR342 primer set is shown as SEQ ID No. 29-30; the sequence of the CCRSSR365 primer set is shown as SEQ ID No. 31-32; the sequence of the CCRSSR381 primer set is shown as SEQ ID No. 33-34; the sequence of the CCRSSR390 primer set is shown as SEQ ID No. 35-36; the sequence of the CCRSSR398 primer set is shown as SEQ ID No. 37-38.

2. A kit for analyzing genetic diversity or constructing a fingerprint of a Prunus yedoensis germplasm resource, characterized by, the kit contains the EST-SSR molecular marker primer set of claim 1.

3. The use of the EST-SSR molecular marker primer set of claim 1 or the kit of claim 2 in the genetic diversity analysis of Prunus serrulata germplasm resources.

4. The use of the EST-SSR molecular marker primer set according to claim 1 or the kit according to claim 2 in the construction of a fingerprint of Prunus yedoensis.

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