EST-SSR molecular markers of Paphiopedilum florida and Paphiopedilum simonii, and their usage and application

Abstract

This invention discloses EST-SSR molecular markers for *Paphiopedilum florida* and *Paphiopedilum simonii*, along with their usage and applications. The nucleotide sequences of the corresponding primer sets are shown in SEQ ID NO:2-3, SEQ ID NO:5-6, SEQ ID NO:8-9, SEQ ID NO:11-12, SEQ ID NO:14-15, and SEQ ID NO:16-65. The primer sets in this invention can be widely used for typing and identifying any *Paphiopedilum* species, overcoming the shortcomings of poor primer universality in existing technologies, and achieving batch and rapid typing of *Paphiopedilum* species.

Description

Technical Field

[0001] This invention relates to the field of plant molecular identification technology, and in particular to the EST-SSR molecular markers of Paphiopedilum florida and Paphiopedilum moniliforme, as well as their usage and applications. Background Technology

[0002] Paphiopedilum spp., belonging to the genus Paphiopedilum in the family Orchidaceae, is prized for its unique flower shape and high ornamental value. With its rich phenotypic characteristics, it is considered an exotic and rare plant. As the global economy develops, Paphiopedilum is increasingly favored by orchid enthusiasts worldwide and boasts a promising market prospect.

[0003] The genus *Paphiopedilum* comprises over 80 original species, all listed in the National Key Protected Endangered Plants List, and widely distributed in Southeast Asia, South China, North India, and New Guinea. Different *Paphiopedilum* species exhibit high similarity in roots, stems, and leaves, with interspecific differences primarily identified by flower morphology. Therefore, outside of flowering season, species identification based solely on roots, stems, and leaves is difficult. This presents significant challenges for the collection and conservation of *Paphiopedilum* germplasm resources. Furthermore, the current development of the *Paphiopedilum* industry faces numerous problems, such as severe damage to orchid germplasm resources, immature conservation techniques, weak basic research related to orchids, and limited germplasm innovation methods, primarily manifested in unclear phylogenetic relationships, ambiguous evolutionary routes, and insufficient research on pollination biology. Existing *Paphiopedilum* SSR marker primers are developed using transcriptome RNA from a single *Paphiopedilum* species, exhibiting poor universality across different *Paphiopedilum* species (unable to perform PCR amplification across different *Paphiopedilum* species), thus greatly limiting their application in the identification, classification, conservation, and reintroduction into the wild of *Paphiopedilum* species.

[0004] Therefore, developing a primer that can be used across different species of the Paphiopedilum genus for germplasm resource identification is of great importance for the collection, conservation, and reintroduction of Paphiopedilum species into the wild. Summary of the Invention

[0005] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. To this end, this invention provides an EST-SSR molecular marker for *Paphiopedilum* and its usage and application. The *Paphiopedilum* SSR primers of this invention use homologous sequences co-expressed in the transcriptomes of the distantly related *Paphiopedilum variegatum* and *Paphiopedilum simonii* as templates to detect SSR sites in the homologous sequences of both species. Designed using conserved sequences at both ends of the SSR, this SSR molecular marker primer has strong universality among *Paphiopedilum* species and can be effectively used to identify and distinguish existing *Paphiopedilum* plants.

[0006] In a first aspect, the present invention provides a set of EST-SSR molecular marker primers for the genus Paphiopedilum, the primer set comprising at least 20 nucleotide sequences as shown in SEQ ID NO:2 to 61.

[0007] In some embodiments of the present invention, the primer set comprises at least 13 nucleotide sequences as shown in SEQ ID NO:2 to 61.

[0008] In some embodiments of the present invention, each nucleotide sequence in the primer set is paired up to form a primer pair (upstream primer and downstream primer), and the specific correspondence is shown in Table 1.

[0009] In some embodiments of the present invention, the primer set includes primers for SSR1, SSR6, SSR8, SSR10, SSR11, SSR12, SSR13, SSR16, SSR20, SSR23, SSR25, SSR29 and SSR30.

[0010] In some embodiments of the present invention, the primer set further includes: a nucleic acid molecule that has a sequence identity of greater than or equal to 95% with any one of the nucleotide sequences shown in SEQ ID NO:2 to 61 and has the same function as the original sequence.

[0011] In some embodiments of the present invention, each primer in the primer set may be modified by the insertion, deletion, substitution or other modification methods known to those skilled in the art of modifying one or more nucleotides, thereby improving the specificity of the primers, enabling them to be used for molecular biological research on other plants of the same genus or species, or to obtain other desired properties.

[0012] In some embodiments of the present invention, the insertion, deletion, substitution, and modification of nucleotides in the primer set all occur within the functional region sequence. In the present invention, the functional region sequence is the non-M13 sequence portion of the primer.

[0013] In some embodiments of the invention, the identity is 95%, 96%, 97%, 98%, or 99%.

[0014] In this invention, the primer set is obtained based on homologous sequences that can be co-expressed in the transcriptomes of Paphiopedilum florida and Paphiopedilum moniliforme, which are distantly related.

[0015] In this invention, to address the issue of poor universality and polymorphism of molecular markers within the *Paphiopedilum* genus, the inventors further improved upon the traditional "single-species transcriptome sequence design method" by employing a "multi-species transcriptome homologous sequence design method." Homologous sequences from the transcriptomes of the distantly related *Paphiopedilum hirsutissimum* and *Paphiopedilum concolor* were selected as templates. SSR sites in these homologous sequences were detected, and primers were designed using the conserved sequences flanking the SSRs. Because the two *Paphiopedilum* species are distantly related, and the primers underwent polymorphism comparison of homologous sequences from both species, the design exhibits strong polymorphism and universality, enabling stable amplification across all *Paphiopedilum* species.

[0016] In some embodiments of the present invention, at least one of the upstream and downstream primers in the primer set has an identifiable marker.

[0017] In some embodiments of the present invention, the upstream primer in the primer set has an identifiable marker.

[0018] In some embodiments of the present invention, the label includes fluorescent groups, specific nucleic acid molecules, nuclides, and ligands.

[0019] In some embodiments of the present invention, the marker is a specific nucleic acid molecule, specifically the M13 sequence (5'-GTAAAACGACGGCCAGT-3').

[0020] In some embodiments of the present invention, the Paphiopedilum genus includes, but is not limited to: Paphiopedilum simonii, Paphiopedilum giganteum, Paphiopedilum helenense, Paphiopedilum lancifolium, Paphiopedilum blazei, Paphiopedilum purpureus, Paphiopedilum sclerophyllum, Paphiopedilum 'King Pink', Paphiopedilum 'Colored Clouds', Paphiopedilum 'Salty Brother', Paphiopedilum 'Red Flag', Paphiopedilum 'Primula', Paphiopedilum 'Henry', Paphiopedilum 'Long Petal', Paphiopedilum 'King', Paphiopedilum purpureus, Paphiopedilum 'Red Flag', Paphiopedilum 'Leafy', Paphiopedilum 'Same Color', Paphiopedilum 'Wenshan', Paphiopedilum 'Magic Queen', or hybrids of any two of these Paphiopedilum species.

[0021] In this invention, the inventors also verified the effectiveness of the above primer set in unnamed unknown species of the genus Paphiopedilum, and found that it could also generate amplification judgment and produce typing peaks, thus demonstrating that the above primer set can be widely used in the typing of any Paphiopedilum plant.

[0022] In a second aspect, the present invention provides a Paphiopedilum species detection reagent, wherein the detection reagent comprises the Paphiopedilum EST-SSR molecular marker primer set and M13 primer described in the first aspect of the present invention.

[0023] In some embodiments of the present invention, the nucleotide sequence of the M13 primer is shown in SEQ ID NO: 1.

[0024] In this invention, the addition of the M13 primer is mainly used for labeling specific sequences, thereby making them easier to identify. Of course, those skilled in the art can also use other conventional labeling methods for labeling or identification, including but not limited to the M13 primer.

[0025] A third aspect of the present invention provides the application of the Paphiopedilum EST-SSR molecular marker primer set described in the first aspect of the present invention in the preparation of species identification and detection products for Paphiopedilum plants.

[0026] In some embodiments of the present invention, the detection product includes a detection kit and a detection chip.

[0027] In some embodiments of the present invention, the method of using the Paphiopedilum species identification and detection product is as follows:

[0028] DNA was extracted from the Paphiopedilum species to be tested, and PCR amplification was performed using the Paphiopedilum EST-SSR molecular marker primer set described in the first aspect of this invention. The amplification products were then STR-typed, and the Paphiopedilum species were determined based on the typing results.

[0029] In some embodiments of the present invention, the PCR amplification system is shown in Table 2.

[0030] In some embodiments of the present invention, the reaction procedure for the PCR amplification is shown in Table 3.

[0031] A fourth aspect of the present invention provides the application of the Paphiopedilum EST-SSR molecular marker primer set described in the first aspect of the present invention in plant species identification.

[0032] In some embodiments of the present invention, the plant is a Paphiopedilum species.

[0033] In some embodiments of the present invention, the Paphiopedilum species include, but are not limited to: Paphiopedilum simonii, Paphiopedilum giganteum, Paphiopedilum helenense, Paphiopedilum lancifolium, Paphiopedilum variegatum, Paphiopedilum purpureus, Paphiopedilum sclerophyllum, Paphiopedilum 'Pink King', Paphiopedilum 'Colored Cloud', Paphiopedilum 'Salty Brother', Paphiopedilum 'Red Flag', Paphiopedilum 'Primula', Paphiopedilum 'Henry', Paphiopedilum 'Long Petal', Paphiopedilum 'King', Paphiopedilum purpureus, Paphiopedilum 'Red Flag', Paphiopedilum 'Leafy', Paphiopedilum 'Same Color', Paphiopedilum 'Vietnamese Beauty', Paphiopedilum 'Vensan', Paphiopedilum 'Magic Queen', or hybrids of any two of these Paphiopedilum species.

[0034] The beneficial effects of this invention are:

[0035] 1. This invention is the first to develop a primer set that can be widely used for typing and identifying any Paphiopedilum species, thus effectively overcoming the shortcomings of poor primer universality in the prior art.

[0036] 2. The EST-SSR molecular marker primer set of the Paphiopedilum genus in this invention has accurate identification and characterization, and can effectively distinguish different Paphiopedilum varieties based on the typing peak, realizing the batch and rapid typing of Paphiopedilum plants. Attached Figure Description

[0037] Figure 1 The downstream primers of SSR1 to SSR16 correspond to the original SSR positions of Paphiopedilum florida and Paphiopedilum florida.

[0038] Figure 2 The downstream primers of SSR17–30 correspond to the original SSR positions of Paphiopedilum florida and Paphiopedilum simonii.

[0039] Figure 3 The diagram shows the characteristic peaks of the SSR1 primer amplification products in the DNA of *Paphiopedilum macrospottum*, *Paphiopedilum malipoense*, and *Paphiopedilum baiqiense*.

[0040] Figure 4 The diagram shows the characteristic peaks of the SSR2 primer amplification products in the DNA of *Paphiopedilum macrospottum*, *Paphiopedilum malipoense*, and *Paphiopedilum baiqiense*.

[0041] Figure 5 These are characteristic peaks of SSR1 in the DNA of some Paphiopedilum species / varieties tested.

[0042] Figure 6 The peaks of SSR6 are characteristic of DNA in some Paphiopedilum species / varieties tested.

[0043] Figure 7 This is a phylogenetic tree for plants in the genus Paphiopedilum.

[0044] Figure 8 Phylogenetic tree of orchid species.

[0045] Figure 9 The amplified bands of SSR1 in the DNA of *Lysimachia christinae*, ... and wild-type strains are shown.

[0046] Figure 10 The amplified bands of SSR6 in the DNA of *Lysimachia christinae*, ... and wild-type strains are shown.

[0047] Figure 11 The amplified bands of SSR8 in the DNA of *Lysimachia christinae*, ... and wild-type strains are shown.

[0048] Figure 12 The amplified bands of SSR10 in the DNA of *Lysimachia christinae*, ... and wild-type strains.

[0049] Figure 13 The amplified bands of SSR11 in the DNA of *Lysimachia christinae*, ... and wild-type strains.

[0050] Figure 14The amplified bands of SSR12 in the DNA of *Lysimachia christinae*, ... and wild-type strains.

[0051] Figure 15 The amplified band of SSR13 in the DNA of *Lysimachia christinae*, ... and wild-type strains.

[0052] Figure 16 The amplified band of SSR16 in the DNA of *Lysimachia christinae*, ... and wild-type strains.

[0053] Figure 17 The amplified bands of SSR20 in the DNA of *Lysimachia christinae*, ... and wild-type strains.

[0054] Figure 18 The amplified band of SSR23 in the DNA of *Lysimachia christinae*, ... and wild-type strains.

[0055] Figure 19 The amplified bands of SSR25 in the DNA of *Lysimachia christinae*, ... and wild-type strains.

[0056] Figure 20 The amplified bands of SSR29 in the DNA of *Lysimachia christinae*, ... and wild-type strains.

[0057] Figure 21 The amplified bands of SSR30 in the DNA of *Lysimachia christinae*, ... and wild-type strains. Detailed Implementation

[0058] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0059] Paphiopedilum SSR primer design

[0060] Transcriptome data for *Paphiopedilum hirsutissimum* and *P. concolor* from the *High-Density GeneticLinkage Map Construction and QTLs Identification Associated with Four Leaf-Related Traits in Lady's Slipper Orchids (Paphiopedilum concolor×Paphiopedilum hirsutissimum)* published by Li (Li, Dong-Mei, and Gen-Fa Zhu. *High-Density GeneticLinkage Map Construction and QTLs Identification Associated with Four Leaf-Related Traits in Lady's Slipper Orchids (Paphiopedilum concolor×Paphiopedilum hirsutissimum)*. *Horticulturae 2022, 8(9): 842.*) were downloaded from NCBI. Transcript raw reads were assembled into scaffold sequences using Trinity software. Bioedit software was used to search for scaffold sequences expressed in both *Paphiopedilum simonii* and *Paphiopedilum leben*. MicroSAtellite (MISA) software (http: / / pgrc.ipk-gatersleben.de / misa / ) was used to search for all SSR sites in the scaffold files, with search criteria set to at least 6 repetitions for dibasic repeats and at least 5 repetitions for tribasic, tetrabasic, pentabasic, and hexabasic repeats. SSR sites less than 100 bp apart were considered composite SSRs. Primers for SSR markers were designed using PRIMER 3 software with the following parameters: primer length 18-25 nt, annealing temperature 55-65℃, GC content 40-60%, and PCR product length 100-300 bp.

[0061] Bioedit was used to identify the polymorphism of the SSR markers mined in the above steps in the transcriptomes of Paphiopedilum florida and Paphiopedilum moniliforme. Thirty SSR markers with significant polymorphism were screened out, and the "M13" sequence (5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO: 1)) was added to the upstream primer (left primer). The primer information is shown in Table 1.

[0062] The SSR is displayed as: (repeating unit) number of repetitions. For example, (AGGTCG)5 means that AGGTCG is repeated 5 times.

[0063] Table 1 SSR primer sequence information

[0064]

[0065]

[0066]

[0067] The downstream primers all correspond to the original SSR positions of *Paphiopedilum florida* and *Paphiopedilum simonii*. The correspondence between the two is as follows: Figures 1-2 As shown.

[0068] SSR primer screening and universality test for Paphiopedilum

[0069] Young leaves were taken from plants of *P. bellatulum*, *P. malipoense*, and *P. spicerianum* as test samples. DNA was extracted using a plant genomic DNA extraction kit (Tiangen), and the extraction procedure was performed in accordance with the instruction manual.

[0070] Using the extracted DNA as a template, PCR amplification was performed using the 30 pairs of SSR primers listed in Table 1. In addition to the upstream and downstream primers, the PCR amplification system also included M13 primer (SEQ ID NO: 1) with a fluorescent label (HEX, FAM) at its 5' end. The specific PCR amplification process is shown in Table 2.

[0071] Table 2. PCR amplification system using three primers

[0072] Components concentration content Final concentration / content DNA template 50 ng / μL 1μL 50ng upstream primer 0.25μM 1μL 0.0125μM Downstream primer 5μM 1μL 0.25μM M13 fluorescent primers 3μM 1μL 0.15μM 2×Taq PCR StarMix - 10μL - <![CDATA[ddH2O]]> - 6μL - total - 20μL -

[0073] The Touchdown PCR reaction program was used (as shown in Table 3) to amplify the SSR amplification products.

[0074] Table 3. PCR amplification procedure using three primers

[0075]

[0076] The SSR amplification products were stored under light-protected frozen conditions and sent to Thermo Fisher Scientific for STR (Short Tandem Repeat) typing. The typing data were analyzed using GeneMarker (V2.4.0).

[0077] The results are shown in Table 4 and Figure 3 , Figure 4 As shown.

[0078] Table 4. Results of universality testing of 30 SSR primer pairs in the genus Paphiopedilum.

[0079]

[0080] In this context, ○ indicates that the amplification is stable and can produce a distinct STR genotyping peak.

[0081] The results above indicate that all 30 pairs of SSR primers can stably amplify *Paphiopedilum macrospotum*, *Paphiopedilum malipoense*, and *Paphiopedilum baiqiense*, and the STR typing results all produce obvious peaks. Figure 3 and Figure 4 These are characteristic peaks of SSR1 and SSR2 in the DNA of *Paphiopedilum macrospotum*, *Paphiopedilum malipoense*, and *Paphiopedilum baiqiense*, respectively.

[0082] Based on the amplification positive results obtained from the preliminary screening in Table 4 and the obtained STR peak results, 13 pairs of SSR primers with high polymorphism were selected from 30 pairs of SSR primers (SSR1, SSR6, SSR8, SSR10, SSR11, SSR12, SSR13, SSR16, SSR20, SSR23, SSR25, SSR29, and SSR30, respectively), and their identification effect in other Paphiopedilum species was tested.

[0083] Among them, the Paphiopedilum species used for testing include: Forsythia suspensa, Paphiopedilum giganteum, Paphiopedilum helenense, Paphiopedilum lancifolium, Paphiopedilum blazei, Paphiopedilum purpuratum, Paphiopedilum sclerophyllum, Paphiopedilum 'King Pink', Paphiopedilum 'Colored Clouds', Paphiopedilum 'Salty Brother', Paphiopedilum 'Red Flag', Paphiopedilum 'Primula', Paphiopedilum 'Henry', Paphiopedilum longpetal, Paphiopedilum 'King', Paphiopedilum longpetal × purple stripe hybrid, Paphiopedilum small leaf × Henry hybrid, Henry × longpetal hybrid, purple hair × red flag hybrid, Paphiopedilum with leaves, Paphiopedilum monochromatic 1, Paphiopedilum monochromatic 2, Paphiopedilum 'Wenshan', and Paphiopedilum 'Magic Queen'.

[0084] The detection method is the same as in the above embodiments.

[0085] The results are shown in Table 5 and Figures 5-6 As shown.

[0086] Table 5. Results of universality testing of 13 SSR primer pairs in the genus Paphiopedilum.

[0087]

[0088]

[0089] The results above show that all 13 SSR primer pairs were able to stably amplify in 24 Paphiopedilum species / varieties, and the STR typing results all produced obvious peaks. Figure 5 , Figure 6 (These are the characteristic peaks of SSR1 and SSR6 in the DNA of some tested species / varieties, respectively).

[0090] Based on its STR typing results, a phylogenetic tree of Paphiopedilum was constructed using the software NTsys (v2.10). Figure 7As can be seen, the 13 pairs of SSR primers can significantly distinguish the above-mentioned Paphiopedilum varieties. Among them, the hybrids can accurately be located between the two parents, indicating that the 13 pairs of SSR primers in the embodiments of the present invention can accurately determine the kinship of Paphiopedilum.

[0091] SSR primers for Paphiopedilum are universal in the genus Dendrobium of the Orchidaceae family.

[0092] There are currently over 80 species of the genus *Paphiopedilum* that have been discovered and published, and this number is constantly increasing. Due to the rarity of *Paphiopedilum* species, it is impossible to verify its universality across all *Paphiopedilum* species. Sun (Sun Yin, Zou Peishan, Jiang Nannan, Fang Yifu & Liu Guofeng (2022) Comparative Analysis of the Complete Chloroplast Genomes of Nine Paphiopedilum Species. Frontiers in Genetics, 12.) constructed a phylogenetic tree based on the chloroplast genomes of 66 species of Orchidaceae. From the phylogenetic tree ( Figure 8 As can be seen, the genera *Paphiopedilum* and *Dendrobium* belong to the subfamily Cypripedioideae and the subfamily Orchidoideae, respectively, within the tribe Epidendreae. Since *Paphiopedilum* and *Dendrobium* are relatively distantly related, if *Paphiopedilum* SSR primers can be successfully amplified by PCR in *Dendrobium* plants, it indirectly proves that the SSR primers can be amplified in all species of the *Paphiopedilum* genus.

[0093] To demonstrate the stable universality of the SSR primers developed in this embodiment of the invention within the genus Paphiopedilum, DNA was collected from three Dendrobium loddigesii plants (varieties: Hongyun, Green Jade, and wild plant) for verification.

[0094] The testing method is the same as in the above embodiments.

[0095] The test results are shown in Table 6 and Figures 7-19 As shown.

[0096] Table 6. Results of universality testing of 13 SSR primer pairs in the Dendrobium genus.

[0097]

[0098]

[0099] The results above show that all 13 SSR primer pairs were able to stably amplify in the three Dendrobium officinale varieties, and the STR typing results all produced obvious polymorphic peaks (e.g., Figure 9-21 As shown, the amplification bands of SSR1, SSR6, SSR8, SSR10, SSR11, SSR12, SSR13, SSR16, SSR20, SSR23, SSR25, SSR29, and SSR30 in the DNA of *Paphiopedilum 'Lucky'*, *Paphiopedilum 'Green Jade'*, and wild-type plants are respectively. This demonstrates that the 13 pairs of SSR primers developed and screened in the above examples can be stably amplified in neighboring species of the *Paphiopedilum* genus and exhibit polymorphism. Therefore, it can be indirectly proved that the 13 pairs of SSR primers are universal in all species of the *Paphiopedilum* genus.

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A set of EST-SSR molecular marker primers for the genus *Paphiopedilum*, characterized in that... The primer set includes nucleotide sequences as shown in SEQ ID NO:2-3, 12-13, 16-17, 20-27, 32-33, 40-41, 46-47, 50-51, and 58-61.

2. The Paphiopedilum EST-SSR molecular marker primer set according to claim 1, characterized in that, At least one of the upstream and downstream primers in the primer set has an identifiable marker.

3. The Paphiopedilum EST-SSR molecular marker primer set according to claim 2, characterized in that, The labels include fluorescent groups, specific nucleic acid molecules, nuclides, and ligands.

4. The Paphiopedilum EST-SSR molecular marker primer set according to claim 1, characterized in that, The genus *Paphiopedilum* includes: *Paphiopedilum yunnanense*, *Paphiopedilum macrocarpa*, *Paphiopedilum helenense*, *Paphiopedilum lancifolium*, *Paphiopedilum blazei*, *Paphiopedilum purpurea*, *Paphiopedilum sclerophyllum*, *Paphiopedilum 'Pink King'*, *Paphiopedilum 'Colored Cloud'*, *Paphiopedilum 'Salty Brother'*, *Paphiopedilum 'Red Flag'*, *Paphiopedilum 'Primula'*, *Paphiopedilum 'Henry'*, *Paphiopedilum longipes*, *Paphiopedilum 'King'*, *Paphiopedilum purpurea*, *Paphiopedilum 'Purpurea'*, *Paphiop 5. A reagent for detecting Paphiopedilum species, characterized in that, The detection reagent includes the Paphiopedilum EST-SSR molecular marker primer set and M13 primer as described in any one of claims 1 to 4; The nucleotide sequence of the M13 primer is shown in SEQ ID NO:

1.

6. The application of the Paphiopedilum EST-SSR molecular marker primer set according to any one of claims 1 to 4 in the preparation of Paphiopedilum species identification and detection products.

7. The application according to claim 6, characterized in that, The testing products include test kits and test chips.

8. The application of the Paphiopedilum EST-SSR molecular marker primer set according to any one of claims 1 to 4 in plant species identification; wherein the plant is a Paphiopedilum or Dendrobium plant.

Images