Construction of molecular identity card for cultivated Dendrobium officinale in Guizhou Province
Authors: Deqiang Ren, Changya Chen, Tinglu Wang, Xia Meng, Yuan Huang, Ping Wu
Published: 2026

ABSTRACT
To establish a molecular identity card for Dendrobium officinale, providing a scientific basis for the breeding, utilization, standardized production, and quality traceability of cultivated varieties in Guizhou Province. Based on the published genome sequence of Dendrobium officinale, Simple Sequence Repeat (SSR) loci were identified using MISA (MicroSatellite identification tool) software, and SSR molecular markers were designed via Primer 3. Randomly selected primers were used for genetic analysis of cultivated Dendrobium officinale from Guizhou. Amplified fragments were digitally encoded and combined with supplementary information such as origin to construct a Quick Response (QR) molecular identity card. A total of 1,361,061 SSR markers were developed, with an average of 136 per scaffold. Mononucleotide repeats were the dominant motif (63.55%), among which (A) and (T) types accounted for 95.85%. Dinucleotide repeats constituted 11.62% of all SSR loci. Among 40 randomly selected primers, 27 were polymorphic, generating 127 polymorphic bands with a polymorphism rate of 96.27%. Genetic diversity analysis revealed rich genetic diversity in the tested materials. Cluster analysis divided the samples into four groups, based on which a molecular identity card for Guizhou cultivated Dendrobium officinale was successfully constructed. The SSR markers developed in this study exhibit high polymorphism and are suitable for genetic analysis of Dendrobium officinale. The constructed molecular identity card is of significant importance for ensuring the healthy development of the Dendrobium officinale industry in Guizhou.

Introduction

Dendrobium officinale Kimura et Migo is a precious medicinal herb in China, recognized as both a medicine and food, and ranks first among the “Nine Rare Immortal Herbs” (Huang, Wang & Nie, 2023). It is the species with the highest economic value in the genus Dendrobium, with broad market prospects in healthcare applications (Hu et al., 2024). Almost all Dendrobium officinale on the market today is artificially cultivated. Guizhou Province is a major cultivation region with the largest area of simulated wild cultivation in China. However, alongside rapid industrial development, problems such as cultivar confusion and complex origins of introduced germplasm have become prominent. Most seedlings in Guizhou are introduced from Zhejiang, Yunnan, and Fujian, alongside locally domesticated wild materials. As a special commodity, medicinal plant seedlings cannot be reliably identified by ordinary growers based on morphology alone in terms of genetic origin or geographical source. Moreover, the content of active constituents varies considerably among different cultivated varieties (Ding et al., 2005), posing challenges to medication safety.

Establishing a molecular identity card for cultivated Dendrobium officinale in Guizhou, a digital DNA profile based on molecular markers that can distinguish individuals (Wang et al., 2025), would enable rapid, accurate germplasm identification, variety labeling, and traceability, thereby supporting breeding programs and standardized cultivation (Mao et al., 2024). Among the various molecular marker techniques used in medicinal plant genetics (Wang et al., 2024), SSR (Simple Sequence Repeat) markers, also known as microsatellites, are tandemly repeated motifs of 1–6 base pairs that are highly polymorphic, codominant, and widely distributed across eukaryotic genomes. Their stability across developmental stages and environmental conditions makes them particularly advantageous over morphological or biochemical markers. Consequently, SSR markers are increasingly applied in genetic diversity analysis, genetic linkage map construction, and QTL mapping in Dendrobium officinale (Zhou et al., 2023).

Despite these advances, a critical research gap remains: existing SSR marker studies in Dendrobium officinale have predominantly relied on markers derived from transcriptomes or limited genomic regions, resulting in uneven distribution of polymorphic primers across the genome. For instance, Xie et al. (2010) developed four SSR primer pairs for germplasm purity identification, while Xu et al. (2015) identified 249 SSR loci across 36 accessions and reported high genetic diversity. However, in both studies, polymorphic primers were unevenly distributed, with fewer polymorphic markers in certain chromosomal regions—likely due to their genomic locations (e.g., telomeres, centromeres, conserved regions). This uneven distribution limits the accuracy of subsequent genetic analyses and genetic map construction. A genome-wide approach to SSR primer design is therefore essential for precise genetic characterization of this species.

To address this gap, the present study utilizes the published Dendrobium officinale genome to screen genome-wide polymorphic loci using MISA (Microsatellite Identification Tool) and to design high-throughput SSR primers via Primer 3. These markers will be used to analyze the genetic diversity of cultivated Dendrobium officinale in Guizhou Province and to construct molecular identity cards based on molecular fingerprinting. This research aims to provide a scientific basis for the breeding, utilization, and standardized cultivation of Dendrobium officinale in Guizhou.

Materials & Methods

Experimental materials

The samples used in this study were collected from cultivation sites across various counties and districts in Guizhou Province, with primary origins including Yunnan, Zhejiang, and Guizhou. All materials were authenticated as Dendrobium officinale Kimura et Migo by Shenghua Wei (Guizhou University of Traditional Chinese Medicine). The materials are deposited and maintained in the Germplasm Resource Nursery of Guizhou University of Traditional Chinese Medicine (Table 1).

10.7717/peerj.21558/table-1Table 1Germplasm resources of Dendrobium officinale used in this study.Accession IDProvenanceGeographic originCultivation method1ZJ-1Zhejiang variety, with unknown breedGuizhou Anlong Xicheng Xiushu Agriculture and Forestry Co., Ltd.Imitation wild cultivation2ZJ-2Zhejiang variety, species unknownGuizhou Anlong Xicheng Xiushu Agriculture and Forestry Co., Ltd.Facility cultivation3AL-1Locally domesticated wild species in Anlong County, Guizhou ProvinceDendrobium Garden Farmers’ Professional Cooperative, Pojiao Township, Anlong County, Guizhou ProvinceFacility cultivation4ZJ-3Zhejiang variety, species unknownXicheng Xiushu Agriculture and Forestry Co., Ltd., Anlong County, Guizhou ProvinceImitation wild cultivation5FJ-1Fujian species, variety unknownGuizhou Lvjian Shennong Organic Agriculture Co., Ltd., Dushan County, Guizhou ProvinceFacility cultivation6GZ-1Guizhou Province Guihu No. 1Guizhou Xingqian Technology Co., Ltd., Guiyang City, Guizhou ProvinceFacility cultivation7ZJ-4No. 1, Senshan, Zhejiang ProvinceGuizhou Xingqian Technology Co., Ltd., Guiyang City, Guizhou ProvinceFacility cultivation8ZJ-5Zhejiang variety, species unknownHuazhai Domestication Base, Jinping County, Guizhou ProvinceImitation wild cultivation9ZJ-6Zhejiang variety, specific variety unknownGuizhou Jinping Qianle Dendrobium officinale Planting Farmers’ Professional Cooperative, Jinping County, Guizhou ProvinceImitation wild cultivation10YN-1Dendrobium from Menghai, Yunnan ProvinceLibo Gaoli Aquaculture Production and Marketing Professional Cooperative, Libo County, Guizhou ProvinceImitation wild cultivation11YN-2Yunnan species, variety unknownHanlong Yuhu Biotechnology Co., Ltd., Luodian County, Guizhou ProvinceImitation wild cultivation12LD-1Locally domesticated wild species in Luodian County, Guizhou ProvinceProfessional Cooperative for Planting in the Stone Mountain Area under Forests, Luodian County, Guizhou ProvinceImitation wild cultivation13YN-3Soft-footed Dendrobium officinale from Guangnan County, Yunnan ProvinceGuizhou Panxian Jinzhiyuan Traditional Chinese Medicine Planting Farmers’ CooperativeFacility cultivation14ZJ-7Zhejiang variety, with unknown speciesGuizhou Wanhuyuan Ecological Agriculture Development Co., Ltd., Pingtang County, Guizhou ProvinceFacility cultivation15YN-4Lijiang County, Yunnan Province, variety unknownGuizhou Pingtang Huishengyuan Agriculture and Forestry Development Co., Ltd.Facility cultivation16YN-5Yunnan species, variety unknownGuizhou Pingtang Huishengyuan Agriculture and Forestry Development Co., Ltd.Facility cultivation17YN-6Yunnan Guangnan County Soft-leg Dendrobium officinaleGuizhou Pingtang Huishengyuan Agriculture and Forestry Development Co., Ltd.Facility cultivation18YN-7Yunnan Guangnan Soft-leg Dendrobium officinaleGuizhou Qianxi Bohui Traditional Chinese Medicine Planting Professional CooperativeFacility cultivation19ZJ-8Yandang Red, Zhejiang ProvinceGuizhou Shuicheng Lingtouyan Seed Industry Technology Development CompanyFacility cultivation20YN-8No. 1, Xilin, Yunnan ProvinceGuizhou Bailing Enterprise Group Pharmaceutical Co., Ltd. (Bailing Ecological Park), Zhenning County, Guizhou ProvinceFacility cultivation

Germplasm resources of Dendrobium officinale used in this study.

Primer design and genotyping

Genomic DNA of Dendrobium officinale was extracted and subsequently detected via gel electrophoresis following the protocol described by Pan et al. (2023). To develop genome-wide SSR markers, the published Dendrobium officinale genome (NCBI assembly accession: ASM160598v2, GCA_001605985.2. The website is https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001605985.2/) was utilized. The MISA (MicroSatellite identification tool; http://pgrc.ipk-gatersleben.de/misa/) software was employed to screen for polymorphic microsatellite loci across the genome. High-throughput primer design was performed using Primer 3 (http://primer3.ut.ee/). The synthesized primers were provided by Sangon Biotech (Shanghai) Co., Ltd. The design parameters were as follows: primer length of 18–25 bp, expected PCR product size of 100–250 bp, and GC content of 40–60%. The Misa software configuration file, Perl script, and Genome-wide SSR molecular marker developed in this research (Table S1) have been uploaded to the science database (DOI: 10.57760/sciencedb.34283).

The polymerase chain reaction (PCR) was conducted in a total volume of 10 µL, containing five µL of Taq Master Mix, one µL (approximately 50 ng) of template DNA, one µL (10 µM) of SSR primer, and three µL of ddH2O. The amplification program consisted of an initial denaturation at 95 °C for 5 min; followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 50–60 °C (temperature adjusted based on the primer’s Tm value, typically 55–60 °C) for 45 s, and extension at 72 °C for 60 s; with a final extension at 72 °C for 10 min. If initial SSR-PCR amplification results were suboptimal, adjustments to the reaction components and/or annealing temperature were made to establish the optimal protocol.

Amplification products were separated by agarose gel electrophoresis on 1.2% agarose gels using 1 × TAE as the running buffer. Electrophoresis was performed at 120 V for 30–60 min (duration adjusted as needed). The resulting DNA bands were visualized and documented using a BIO-RAD gel imaging system.

Genetic diversity analysis based on designed primers

The amplification products were scored manually. Clear or faint bands were recorded as present (1), while their absence was recorded as (0), generating a binary matrix. This matrix was analyzed using NTSYS-pc version 2.1 to construct a dendrogram based on cluster analysis. Genetic diversity parameters for the cultivated Dendrobium officinale accessions from Guizhou, including Nei’s gene diversity index (H), Shannon’s information index (I), number of polymorphic loci, and percentage of polymorphic loci (PPL), were calculated using POPGENE version 1.32.

Construction of DNA molecular identity cards

A molecular identity code for each cultivated Dendrobium officinale accession was generated by digitally encoding its molecular fingerprint data. The binary matrix (1 for presence, 0 for absence), as the core “fingerprint code”, derived from the SSR amplification products was concatenated in a fixed order of primers and fragment sizes to form a binary string. This core “fingerprint code” was supplemented with additional codes representing the germplasm’s provenance, cultivation region, and cultivation method. The combined “fingerprint code + supplementary code” formed the unique molecular identification number. This alphanumeric string was subsequently converted into a scannable Code128-A barcode using an online barcode generator (http://barcode.cnaidc.com/html/BCGcode128b.php) and into a Quick Response (QR) code using an online QR code generator (https://cli.im/), thereby producing the final DNA molecular identity card.

Results

Development of genome-wide SSR molecular markers in Dendrobium officinale

A total of 1,361,061 genome-wide SSR loci (Table S1) were identified, from which an average of 136 SSR primer pairs were designed per scaffold, indicating high genome coverage. Statistical analysis revealed seven distinct types of nucleotide SSR repeats, with significant variation in their frequency and abundance (Table 2, Fig. 1A).

10.7717/peerj.21558/table-2Table 2Distribution characteristics of different SSR motif types in the whole genome of Dendrobium officinale.TypeAnnotationNumberProportionP1Mononucleotide motif types288,31263.55%P2Dinucleotide motif types81,70518.01%P3Trinucleotide motif types33,2197.32%P4Tetranucleotide motif types2,1700.48%P5Pentanucleotide motif types4560.10%P6Hexanucleotide motif types1980.04%cCompound motif typesa46,60110.27%c*Compound motif typesb (interrupted)1,0260.23%Total453,687100% Notes. Compound motif typesaSSRs containing multiple motif types in close proximityCompound motif typesb (interrupted)Compound SSRs with intervening non-repetitive sequences between motif types

Distribution characteristics of different SSR motif types in the whole genome of Dendrobium officinale.

Notes.

Compound motif typesaSSRs containing multiple motif types in close proximityCompound motif typesb (interrupted)Compound SSRs with intervening non-repetitive sequences between motif types

SSRs containing multiple motif types in close proximity

Compound SSRs with intervening non-repetitive sequences between motif types

Statistics of different SSR repeat motif types in the whole genome of D. officinale.

Note: (A) Types and numbers of repeat motifs in the D. officinale genome. (B) Major mononucleotide motif types. (C) Major dinucleotide motif types. (D) Major trinucleotide motif types.

The predominant repeat types were mononucleotide and dinucleotide repeats, collectively accounting for 81.56% of the total SSRs identified. Mononucleotide repeats were the most frequent, constituting 63.51% of all SSRs. Among these, motifs (T) and (A) were overwhelmingly dominant, with 137,806 and 137,747 occurrences respectively, followed by (G) (6,317) and (C) (6,442) (Fig. 1B). Motifs A and T each accounted for approximately 47.77% and 47.81% of the mononucleotide repeats, respectively.

Dinucleotide repeats represented 18.01% of the total. The major dinucleotide motifs were AT/TA, CT, GA, and AG. Specifically, (AT) occurred 17,623 times (21.57% of dinucleotide repeats), (TA) 16,748 times (20.50%), (CT) 9,406 times (11.52%), (GA) 9,006 times (11.03%), and (AG) 7,812 times (9.56%), with (AT) and (TA) being the predominant types (Fig. 1C).

Trinucleotide repeats comprised 8.20% of the total. The most frequent trinucleotide motifs were (AAT) (3,863 occurrences), (ACA) (2,991), and (TTA) (2,822). Other notable motifs included (ATT) (2,064), and (CCA), (CAA), (TTG), (TAA), (ATA), (TAT) with counts ranging from 1,154 to 1,594 (Fig. 1D).

Tetranucleotide, pentanucleotide, and hexanucleotide repeats were relatively scarce, collectively constituting less than 1% of all SSRs. Tetranucleotide repeats accounted for 0.48%, with (TTTA) (319) and (TTTG) (392) as the main types. Pentanucleotide repeats (0.10%) were primarily represented by (AAAAT) (48 occurrences). Hexanucleotide repeats (0.04%) included motifs such as (GTTTAG) (six occurrences) (Fig. 2).

PCR gel electrophoresis pattern of Primer SSR35.

Genetic diversity analysis of Dendrobium officinale

Forty primer pairs were randomly selected (Table 3), of which 27 were polymorphic, 9 produced monomorphic amplification bands, and 4 failed to generate effective amplification products (Fig. 3), resulting in an effective amplification rate of 67.5%. Gel electrophoresis analysis revealed that the 27 SSR primers produced a total of 134 bands, with 127 being polymorphic, yielding a high polymorphism rate of 95%. The number of alleles per primer ranged from 2 to 8, with an average of 4.7 (Tables 4 and 5). Primers SSR15 and SSR20 exhibited the highest polymorphism, each generating 8 effective amplification bands.

10.7717/peerj.21558/table-3Table 3SSR polymorphic primers and their annealing temperatures.No.Primer nameSequence (5′ → 3′)Genomic locationAnnealing temperatures (°C)1SSR4F:5′-TGCCTTTTGAAACACTTCCC-3′scaffold10:26334-2667352R: 5′-AGCATTTCGCATGATGATTG-32SSR7F: 5′-TGAAAGCCACCTGACTTGTG-3′scaffold1000:87457-8777650R: 5′-ATGTCACATTGCAACCAAGG-3′3SSR8F: 5′-GCTATGAGAACCACCGAGGA-3′scaffold1000:118140-11847152R: 5′-ATCATGAACCATGTTGACGC-3′4SSR11F: 5′-GCCCCCTTAATGCCCTAATA-3′scaffold10008:23326-2364752R: 5′-CTTCTCGAAACTTATCGCCG-3′5SSR12F: 5′-ATGGTGAGGCTTCAAACAGG-3′scaffold10009:21788-2212952R: 5′-CCCCTCTCATTCGTTCTTCT-3′6SSR14F: 5′-TACCAAGCCACATGAAAGCA-3′scaffold1001:143018-14334750R: 5′-GCATGATCCTTTTGTTGATACG-3′7SSR15F: 5′-GTAGGCCGATTCTCCCTCTC-3′scaffold1001:162834-16318754R: 5′-CGCACCTATAGAGGCTGACC-3′8SSR16F: 5′-AGAAGCTGGGGACGAATTTT-3′scaffold10010:26091-2641254R: 5′-CCAAACCCTAGCAACCCTTT-3′9SSR17F: 5′-TCTTGGGGAGGTAGGATGTG-3′scaffold10010:35139-3545254R: 5′-TTTTTGAGGTGAAGATGGGC-310SSR18F: 5′-TTTTGCCCATGTACATAGCTT-3′scaffold10012:26594-2692950R: 5′-GTTTGGCCTCCAGGTTTACA-3′11SSR20F: 5′-AACATAAGCACGTGATGGCA-3′scaffold4138:5161-549052R: 5′-AAACCCAAGTGGGGCTTAGT-3′12SSR22F:5′-CTTTTTGTAGGGTGGGCAAA-3′scaffold1002:58637-5894958R: 5′-GCGGATCCCCATATAAGTGA-3′13SSR23F: 5′-ACCAGCTTTTCACCCATTTG-3′scaffold10029:24414-2472751R: 5′-TTAGCCGGGTTTGAGACTTG-3′14SSR24F: 5′-CCCAGCAGTGTCACCTATCA-3′caffold1003:96284-9659959R: 5′-AGGTGGTAAGCTGGGCTAGG-3′15SSR25F: 5′-CCGGCCAAATGCTTAAAAA-3′scaffold10033:13080-1347056R: 5′-AATTAAACCACCGCATGCTC-3′16SSR26F: 5′-TGAAGGCTCGCAACTCATAA-3′scaffold10042:29322-2963756R: 5′-CAACGCGAAAACAAAACACA-3′17SSR27F: 5′-CTAGCCAGCTAAGGGAGGCT-3′scaffold1005:14747-1506054R: 5′-ACCCGAATTGACACCCCTAT-3′18SSR28F: 5′-GCCCTCTTATTCACCCCTCT-3′scaffold1006:98333-9867456R: 5′-CGCACTCCGCACCTAATAAT-3′19SSR29F: 5′-CACCGAACCGAAAACCTAAA-3′scaffold1007:128676-12899854R: 5′-TTCCCCAGTGTCTCTCTTCC-3′20SSR30F: 5′-CAAACCATGCTCAATTGCAT-3′scaffold1008:80396-8074151R: 5′-GCTTACCCTTGCACTTTGGA-3′21SSR31F: 5′-CTCCAATGGGCCTAATAGCA-3′scaffold10099:5933-624858R: 5′-TCTATGTGTTTGCGTTGCGT-3′22SSR32F: 5′-TATGTTGTTCAATGGCGCTG-3′scaffold1012:142781-14309651R: 5′-AGGACCCCAAGAGGAAGTCT-3′23SSR33F: 5′-TGCAAACTGTACAGCACCAA-3′scaffold1013:121508-12182050R: 5′-TCTGATTTTGGGCAGGGTAG-3′24SSR35F: 5′-GAGGAACCTCTGCCATGAGA-3′scaffold10141:29287-2960051R: 5′-TAGGGCATCCTGTTCGTTTT-3′25SSR37F: 5′-AGCTTTTCAAAACTTGGGTTG-3′scaffold1015:139261-13961150R: 5′-AAGGGCACTGTAAGGGTGTG-3′26SSR39F: 5′-GACTCATCGGGGTAGTCCAG-3′scaffold1017:67635-6798064R: 5′-GTTCCGGGGACTTAAAATGG-3′27SSR40F: 5′-GTTGAAGGCCGCTATGATGT-3′scaffold10181:26018-2633351R: 5′-TTTAAGCTTGGCAAAATTTGAT-3′

SSR polymorphic primers and their annealing temperatures.

Statistical analysis of PCR results for 27 primer pairs.

Note: MSP denotes polymorphic amplification, MAA denotes amplifiable but monomorphic, and MSA denotes no amplification product.

10.7717/peerj.21558/table-4Table 4Effective SSR primers and their polymorphism.No.PrimerNumber of amplified bandsPolymorphic bandsPercentage of polymorphic bands (%)1SSR4331002SSR765833SSR865834SSR1176865SSR12331006SSR1454807SSR15881008SSR1665839SSR176610010SSR185510011SSR208810012SSR223310013SSR234410014SSR24215015SSR254410016SSR262210017SSR276610018SSR285510019SSR29548020SSR304410021SSR314410022SSR327710023SSR334410024SSR355510025SSR373310026SSR396610027SSR4077100

Effective SSR primers and their polymorphism.

10.7717/peerj.21558/table-5Table 5Statistics of PCR amplification bands for Dendrobium officinale SSR markers.ItemValueNumber of polymorphic primers27Number of polymorphic bands127Average number of bands per primer4.7

Statistics of PCR amplification bands for Dendrobium officinale SSR markers.

Across the 20 Dendrobium officinale accessions, a total of 129 polymorphic loci were detected, averaging 4.7 polymorphic loci per primer pair. The percentage of polymorphic loci (PPL) was 96.27%. The values for Shannon’s information index (I), Nei’s gene diversity index (H), and the average number of alleles (Na) were 0.5205, 0.3491, and 1.9627, respectively. These results indicate that the selected primers possess high polymorphism and that the tested cultivated Dendrobium officinale accessions exhibit rich genetic diversity.

The systematic cluster analysis revealed that, at a similarity coefficient threshold of 0.71, the tested Dendrobium officinale accessions could be classified into four major groups (Fig. 4). Group I comprised 13 germplasm resources: ZJ-1, ZJ-2, AL-1, ZJ-3, ZJ-6, ZJ-8, YN-1, ZJ-5, GZ-1, ZJ-4, YN-8, FJ-1, and ZJ-7. This group included eight accessions from Zhejiang, one from Fujian, two from Yunnan, and two from Guizhou. Group II consisted of 13 germplasm resources, with LD-1 representing a locally domesticated Dendrobium officinale accession from Luodian County, Guizhou Province. Group III contained two accessions introduced to Dali County (YN-4) and Guangnan County (YN-6) in Yunnan Province. Group IV included four germplasm resources, all of which were the ‘Ruanjiao’ variety of Dendrobium officinale from Guangnan County, Yunnan Province.

UPGMA dendrogram of cultivated Dendrobium officinale from Guizhou Province.

Construction of molecular identity cards for cultivated Dendrobium officinale in Guizhou

The molecular fingerprint data of the 20 cultivated Dendrobium officinale accessions were digitally encoded. Supplementary codes representing the germplasm’s geographic origin, cultivation region, and cultivation method were appended to create a composite molecular identification number following the “fingerprint code + supplementary code” format (Table 6). This alphanumeric string was subsequently converted into corresponding germplasm-specific QR codes (Fig. 5).

10.7717/peerj.21558/table-6Table 6Molecular identity cards for cultivated Dendrobium officinale accessions from Guizhou Province.Serial no.Accession IDFingerprint codeSupplementary codeOriginCultivation siteCultivation method1ZJ-11001110011100001100110100110010010100010010010110101010111010001100 1011101111000100011111111111100101111110001111111100000000000000010ZJALST2ZJ-20001110001111001100110100110001011000010000010110001000111010101100 1111111011000000011001111111111100011000010011111100010000000000010ZJALSS3AL-10011100111111101101010000100100010111010101010010101000111010001100 1011111111000101011111110111110110111000001111111110010011000000010ALALSS4ZJ-30001111111111001100010000100110010010010101010110100010111110001100 1011111111010100011111111101110111111110001111111111010011110110010ZJALST5FJ-11111110111110001101010011110110111010010111110110100001111010001100 1111111011000000010010100101110110011100001111111000010000001001010FJDSSS6GZ-10001110011110001100000000100110011110010111010000000001011111001101 1011111111010011011111111111110111111010000111111001010010000000010GZGYSS7ZJ-41001110011000001100000100100111011111010001010010000000111010001101 1111111011010010010111111100100111111010000111111101010011110011010ZJGYSS8ZJ-51001110011110001100110101100100011110010111010010110000111110001010 1111111111011000011011111111110111111110001111101101010000000110010ZJJPST9ZJ-61001110111101001100000000100000010010010101000110000000111110001101 1011111111010110011011111111110111111100001111111110010000000010010ZJJPST10YN-11001110011111001101010001100100011110010011010110000000111010001101 1011111111000000111111111111110010011000001111111110011011111010010YNLBST11YN-21001000001011101000000000100000010111010100010011100000100000010010 0011001100100000000000100001000110100000001111100100000000000000011YNLDST12LD-10101111111111001110010101110110011010010111010110101111011000001101 1001111001000100011111111111110101111100000111111011111111110000000LDLDST13YN-31001000001010101000000000100000010111010100010001100000100100100110 0011000100100000000000100001000100000001101111100100000000000000000YNPXSS14ZJ-71001110011110011100011011100111011110110111010110100001111010001100 1011111011000001010111111100100111111000000111111000011000000111000ZJPTSS15YN-41001101101101001101110100100110111010011101111000100001111110001001 1001111101000010011001111001100101110000011111000000000000001010000YNPTSS16YN-50001000001110001000000000100000011000010100010000000000101000000011 10111011001000010100001010010001001001000001111000100000000000000010YNPTSS17YN-61001111101100001100010100101001011100011000111000001000111110001001 0011111001000101000010111101000110110100011111000100000000000000010YNPTSS18YN-71011110011111101100110000100110010110010101010110000000101110000011 1111011101000000011000100001000110100000001111000100000000000000110YNQXSS19ZJ-81001100111110001100010000100110011010010111010110101000111111001000 1011111111000100011011101101110111111000001101111100010000000000010ZJSCSS20YN-81001100111000001101010000100100011000001000011110100000110110001100 0011101101000000011011111111010110111000001111111100000011110000010YNZNSS

Molecular identity cards for cultivated Dendrobium officinale accessions from Guizhou Province.

Molecular identity cards for cultivated Dendrobium officinale from Guizhou Province.

Discussion

SSR molecular markers are widely recognized for their high polymorphism, strong specificity, good reproducibility, and codominant inheritance, making them increasingly valuable for genetic diversity assessment, QTL mapping, and genetic map construction for various medicinal plants, such as Bletilla striata (Li, 2017), Paeonia lactiflora (Ji, 2013), and Melia azedarach (Cai et al., 2021). In this study, genome-wide SSR marker development based on the published genome of Dendrobium officinale enabled comprehensive coverage of the genome, providing a robust foundation for subsequent genetic analyses.

The distribution characteristics of SSR motifs in Dendrobium officinale revealed a strong AT-rich bias, with mononucleotide repeats dominated exclusively by A/T motifs and dinucleotide repeats enriched for AT/TA. This pattern aligns with observations in other monocot species (Mei et al., 2026) and contrasts with the AAG/CCT preference reported in some dicotyledonous plants (Xiao et al., 2015), reflecting potential lineage-specific genomic features. Notably, the predominance of poly (A/T) mononucleotide repeats, while consistent with the overall AT-rich composition of the genome (Zhong et al., 2025), may pose challenges for marker design, as such repeats can compromise primer specificity and amplification stability. This likely contributed to the 10% non-amplification rate observed among randomly selected primer pairs, underscoring the importance of prioritizing longer repeat motifs (e.g., di- or trinucleotides) in future marker development efforts. Nevertheless, the 67.5% polymorphism detection rate among randomly selected primers confirms that the genome-wide strategy remains efficient and reliable.

The high levels of genetic diversity detected among the cultivated Dendrobium officinale accessions from Guizhou, as reflected by the high PPL, H, and I values, are consistent with findings from previous SSR-based studies on this species (Xu et al., 2015). This pattern is likely attributable to the relatively short history of large-scale cultivation (Zheng et al., 2024) and the diverse geographical origins of the germplasm, which include wild populations and locally domesticated materials from multiple regions (Hu et al., 2024). UPGMA clustering further revealed a genetic structure characterized by broad admixture with localized differentiation. The mixed composition of Group I, which comprises accessions from several provinces, suggests historical cross-introduction and mixing of germplasm, whereas the distinct clustering of Group II (Luodian local accession) and Groups III–IV (Yunnan-specific accessions) reflects local domestication or ecological specialization. Such genetic heterogeneity has practical implications: it preserves valuable allelic diversity for future breeding efforts but also poses challenges for variety authentication and quality consistency, highlighting the need for reliable identification tools.

To address these challenges, this study developed a molecular identity card system based on the “fingerprint code + supplementary code” framework. By converting SSR-based digital fingerprints into scannable QR codes, the system enables rapid, field-deployable identification of germplasm. This approach leverages the large information capacity and low cost of QR technology (Li, Su & Yang, 2023), offering a practical solution for variety protection, traceability, and quality control in the Dendrobium officinale industry. At the same time, while the 20 accessions analyzed provide a solid proof of concept and reveal substantial genetic diversity, we recognize that this sample size is limited relative to the wide distribution and diverse cultivation sources of Dendrobium officinale in Guizhou Province.

In summary, this study established a genome-wide SSR marker set and a corresponding molecular identity card system for cultivated Dendrobium officinale in Guizhou. These tools provide a scientific basis for variety identification, traceability, and standardization, supporting the sustainable development of the Dendrobium officinale industry.

Conclusions

This study successfully developed genome-wide SSR markers for Dendrobium officinale, revealing high genetic diversity among cultivated accessions in Guizhou. Based on these polymorphic markers, a practical molecular identity card system integrating digital fingerprints with key cultivation data was constructed. This system provides a preliminary framework for germplasm identification, quality traceability, and supports the standardization and sustainable development of the Dendrobium officinale industry.

Supplemental Information

Misa software configuration file

Perl script that reads raw data, performs initial validation, and formats inputs for the main pipeline

Perl script that aggregates processed results, applies filtering criteria, and writes the final output tables