Genetic Diversity Analysis and Fingerprinting Construction of the Vanda species (Orchidaceae) Germplasm Resources by inter-primer binding site (iPBS) Marker

doi:10.21203/rs.3.rs-3890067/v1

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Genetic Diversity Analysis and Fingerprinting Construction of the Vanda species (Orchidaceae) Germplasm Resources by inter-primer binding site (iPBS) Marker

https://doi.org/10.21203/rs.3.rs-3890067/v1

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Plants of the genus Vanda exhibit a diverse array of leaf shapes, with the number of flowers varying from few to many, and a rich palette of colors. These characteristics render them highly valuable for ornamental purposes as tropical orchids. This study employed iPBS molecular markers to analyze the genetic diversity and phylogenetic relationships of 36 species of Vanda and 2 species of Aerides. In addition, DNA fingerprint maps were constructed. All these works will provide a reference for the identification, variety conservation, and hybrid breeding of species in Vanda. Out of 83 iPBS primers, six were selected that produced distinct amplification bands, exhibited high polymorphism, and demonstrated good repeatability. These primers were used for PCR amplification of genomic DNA from 38 samples, resulting in a total of 210 bands. Among these, all 210 bands were polymorphic, achieving a polymorphism rate of 100%. Using the POPGENE 32 software, the mean observed number of alleles (Na) for 38 samples was calculated to be 1.9936, the mean effective number of alleles (Ne) was 1.4243, the mean Nei’s genetic diversity index (He) was 0.2778, and the mean Shannon’s information diversity index (I) was 0.4404. These results indicate a rich genetic diversity among the 38 samples. The genetic similarity coefficients among the samples, calculated using NTSYS-pc 2.1 software, ranged from 0.0345 to 0.6667. Based on these coefficients, UPGMA clustering was performed, and at a similarity coefficient of 0.31, the 38 samples were classified into 7 major groups.

Vanda

iPBS molecular markers

Genetic diversity

DNA fingerprint maps

Among all the genera within the subtribe Aeridinae, the genus Vanda is the most prominent and receives the greatest attention (Gardiner et al. 2013; Liu et al. 2011; Xiang et al. 2012; Zhang et al. 2013). Currently, approximately 73 species of the genus Vanda have been discovered worldwide (Gardiner&Cribb 2013), They are primarily distributed in the tropical regions of Asia, New Guinea, and Australia (Govaerts 2012). Plants of this genus exhibit monopodial growth habits, with significant variation in stem size ranging from miniature to several meters in length. Their leaf shapes vary widely, the number of flowers ranges from few to many, and the colors are rich and diverse (Kasutjianingati et al. 2018). Plants of the genus Vanda possess not only high ornamental value but also significant medicinal importance. Various bioactive compounds and other phenolic compounds extracted from plants of the genus Vanda can be used in the treatment of diseases such as cancer, rheumatism, inflammation, and neurological disorders (Dash et al. 2008; Hossain 2011; Khan et al. 2019). Due to the impacts of global warming and increased human interference, numerous wild species within the genus Vanda are in a state of endangerment and facing extinction. To protect wild species resources, nearly all wild plants of the genus Vanda have been included in the IUCN Red List of Threatened Species. Therefore, it is urgent to undertake practical work and research in areas such as resource introduction, classification, and ex-situ conservation (Kasutjianingati et al. 2018; Khan et al. 2019).

The taxonomic history of the genus Vanda is intricate and complex. Motes (1997) referred to this genus as a 'taxonomic black hole'. Christenson (1987) opined that a 'comprehensive taxonomic revision' is necessary for this genus. Some researchers have attempted to determine the relationships between genera in the Orchidaceae family based on the characteristics of the column foot (Schlechter 1913) and pollinium numbers (Smith 1934; Dressler 1993; Holttum 1959; Senghas 1988). However, Topik et al. (2005) argued that the gynostemium and the number of pollinia in plants of the subtribe Aeridinae exhibit high diversity, and not all possess comprehensive phylogenetic information, thus they cannot be used as the sole criteria for distinguishing species within the Aeridinae subtribe. There has always been controversy regarding the relationship between certain smaller genera such as Ascocentrum Schlechter, Euanthe Schlechter, Trudelia Garay, and Christensonia Haager and the Vanda genus. Gardiner et al. (2012) consolidated the narrowly defined Vanda sensu stricto genus with several closely related genera including Ascocentrum, Euanthe, Christensonia, Neofinetia, and Trudelia, as well as Aerides flabellata. This integration established the broadly defined Vanda sensu lato genus, creating a clade comprising approximately 73 species. However, according to the latest taxonomic research on the genus Vanda, certain species have been reclassified or transferred to new taxonomic groups. With the advancement of molecular systematics, researching the intergeneric boundaries within the genus Vanda, the systematic evolution among species within the genus, and the phylogenetic relationships of closely related groups has become one of the hotspots in the taxonomic systematics of the Orchidaceae family. Currently, the taxonomic definition of the genus Vanda includes both a broadly defined (sensu lato) and a narrowly defined (sensu stricto) Vanda genus. However, the delineation and composition of these taxonomic categories lack consistent and unified support from morphological and molecular systematics evidence. Therefore, the unification of the taxonomic classification and systematic phylogenetic relationships of the genus Vanda still requires further research for substantiation.

Plants of the genus Vanda are a highly evolved group, characterized by high morphological similarity and minor differences between species, making it challenging to identify and distinguish them based solely on single morphological criteria. Only with the aid of more reliable and stable techniques can the genetic resource diversity of Vanda genus plants be effectively studied, providing optimal breeding material for the selection of superior parental lines and the cultivation of high-quality offspring. Molecular marker technology has been widely applied in various fields of biological science, such as nucleic acid sequencing and gene mapping analysis, and has also gained extensive application in the research of Vanda genus plants. Lim et al. (1999) employed RAPD molecular marker technology to study the phylogenetic relationships among the genera Vanda, Euanthe, and Ascocentrum of the genus Vanda. The clustering results indicated that the strap-leaved and terete-leaved Vanda genus plants grouped into two distinct categories. Manners et al. (2013) utilized RAPD and ISSR molecular marker techniques to analyze genetic variability in seven populations of Vanda coerulea comprising of thirty-two genotypes from Northeast India. They found that these seven populations were divided into two major groups. The clustering results indicated that there was no significant correlation between genetic differentiation among populations and geographical distance. Mukhamad et al. (2023) employed three different sets of primers (matK, rbcL, ITS2) for molecular identification of Vanda tricolor and other Vanda germplasm from various regions. The results indicated a close phylogenetic relationship between Vanda tricolor and Vanda ustii, forming a cluster with Vanda coerulea from India.

Molecular markers represent a method of marking at the DNA level, involving the fixed amplification of a specific DNA fragment and the establishment of a molecular genetic map through tracking linked markers with target trait genes. Molecular markers are characterized by their simplicity of operation, stable and repeatable results, rapid detection methods, high polymorphism, and ease of DNA extraction. They are one of the most powerful tools for variety identification and hybrid breeding, following morphological, biochemical, and cytological markers. In this study, the Inter-primer binding site (iPBS) molecular marker technique designed by Kalendar et al. (2010) was employed. This novel molecular marking technique amplifies primers designed at the conserved sites of long terminal repeat (LTR) class retrotransposons. Compared to traditional molecular marker techniques, the iPBS method is simpler and more efficient, requiring only a small amount of tissue sample. It rapidly reveals the genetic relationships between species based on genotype information.This marker has already been preliminarily applied in floral research. Duan et al. (2019) utilized iPBS markers to assess the genetic diversity of 10 wild species and 55 varieties of tree peonies. The results indicated that some tree peony varieties exhibit high genetic diversity, although there are also morphological differences.Ail et al. (2019) first utilized iPBS markers to study the genetic diversity and population structure of 131 safflower(Carthamus tinctorius L.) germplasm resources from 28 different countries. The experimental results indicated that the safflower germplasm possesses good genetic diversity. Moreover, it was determined that safflower from certain countries could be selected as superior progeny for future safflower breeding.Karik et al. (2019) first used iPBS markers to analyze the genetic diversity and population structure of 94 Turkish laurel genotypes. The results indicated that the majority of the variation in the Turkish laurel germplasm originated from within-population variation. This study contributes to a deeper understanding of the genetic relationships of Turkish laurel germplasm, providing a reference for future breeding and conservation efforts.In recent years, iPBS marker technology has been successfully applied to a variety of flowering plants for germplasm resource identification, genetic diversity analysis, and the construction of DNA fingerprint profiles. However, to date, there has been no specific research focused on Vanda. This study utilized iPBS molecular marker technology to analyze the genetic diversity of 36 Vanda spp. and 2 Aerides spp. germplasm resources and construct DNA fingerprint maps. The research explored the applicability of iPBS markers for distinguishing relationships among various subgenera within the Vanda s.l genus. It provides a theoretical foundation for the taxonomic identification, genetic mapping, and hybrid breeding of Vanda germplasm resources.

Plant Materials

In this study, a total of 38 samples were collected (Table 1), comprising 36 native species of the Vanda genus and 2 native species of the Aerides genus. The samples were numbered 1 to 38. Photographs of some of the species are presented in Fig. 1. The sample size of all tested species is 3–5 plants, which have grown for at least 3 years in the Orchidaceae Plant Germplasm Resources Garden of Flower Research Institute, Guangxi Academy of Agricultural Sciences, with no variation in the growth process. The growth conditions of the resource garden are as follows: air temperature is 25–30 ℃, relative humidity is 70–90%, and sufficient sunlight obtained through shading nets is 40%. Young and healthy leaves of the plants were selected, placed in sealed bags with proper labeling, and preserved in an icebox. They were promptly transported back to the laboratory, where they were rapidly frozen using liquid nitrogen for the extraction of genomic DNA.

Table 1

List of 38 germplasm resources
No.	Latin name of germplasm	Leaf type	Germplasm origin
1	Vanda foetide	Strap	Indonesia, Philippines
2	Vanda ampullacea	Strap	China (Yunnan), Laos, Myanmar, Thailand, etc
3	Vanda ampullaceum	Strap	China (Yunnan), Laos, Myanmar, Thailand, etc
4	Vanda bensonii	Strap	India (Assam), Myanmar, Thailand
5	Vanda bicolor	Strap	China (Xizang), Myanmar, Thailand, Vietnam
6	Vanda brunnea	Strap	China (Yunnan), Myanmar, Thailand, Vietnam
7	Vanda christensonianum	Channel	Vietnam
8	Vanda coerulea	Strap	China (Yunnan), India, Myanmar, Thailand, etc
9	Vanda coerulescens	Channel	China (Yunnan), India, Myanmar, Thailand, etc
10	Vanda concolor	Strap	China (Yunnan, Guangdong, Guangxi, Guizhou)
11	Vanda cristata	Strap	China (Yunnan, Xizang), India, Nepal, etc
12	Vanda curvifolium	Channel	Myanmar, Thailand
13	Vanda denisoniana	Strap	Laos, Myanmar, Thailand, Vietnam
14	Vanda falcata	Channel	China, Japan, South Korea
15	Vanda fuscoviridis	Strap	China, Vietnam
16	Vanda garayi	Channel	Thailand, Laos, Indonesia
17	Vanda insignis	Strap	Lesser Sunda Islands
18	Vanda lamellata	Strap	China (Taiwan), Japan, Philippines, etc
19	Vanda lilacina	Channel	China, Cambodia, Vietnam, Thailand, Laos, etc
20	Vanda limbata	Strap	Indonesia (Java), Lesser Sunda Islands
21	Vanda luzonica	Strap	Philippines
22	Vanda mariae	Strap	Philippines
23	Vanda merrillii	Strap	Philippines
24	Vanda miniatum	Channel	Laos, Myanmar, India, Malaysia
25	Vanda perplexa	Strap	Lesser Sunda Islands
26	Vanda pumila	Strap	China (Yunnan, Hainan, Guangxi), Nepal, etc
27	Vanda pusillum	Strap	Vietnam
28	Vanda roeblingiana	Strap	Philippines
29	Vanda sanderiana	Strap	Philippines
30	Vanda subconcolor	Strap	China (Yunnan, Hainan)
31	Vanda tessellata	Strap	China (Yunnan), Nepal, Myanmar, India, etc
32	Vanda testacea	Channel	Nepal, Laos, Myanmar, India, Thailand, etc
33	Vanda tricolor	Strap	Indonesia (Java), Lesser Sunda Islands
34	Vanda ustii	Strap	Philippines
35	Vanda vietnamica	Strap	Vietnam
36	Vanda teres	Terete	China, Nepal, Vietnam, Myanmar, Thailand, etc
37	Aerides flabellata	Strap	China (Yunnan), Laos, Myanmar, Thailand, etc
38	Aerides lawrenceae	Strap	Philippines

Genomic DNA Extraction and Analysis

Genomic DNA from the 38 samples was extracted using the EasyPure Genomic DNA Kit (Beijing TransGen Biotech Co., Ltd.). The integrity of the DNA was assessed with 1.0% agarose gel electrophoresis, and its concentration and purity were measured using a UV spectrophotometer. The extracted DNA was diluted to a concentration of 20 ng/µL with TE buffer and stored at -20°C for future use.

Primer Screening and iPBS PCR Amplification

The primer sequences used in the experiment were selected from the 83 iPBS primers published by Kalendar et al. (2010), and these primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. From the synthesized primers, those producing clear amplification bands, exhibiting high polymorphism, and demonstrating strong stability were selected for PCR amplification of the test samples (Table 2). The total PCR reaction volume was 20 µL, consisting of 10 µL of 2×EasyTaq® PCR SuperMix, 1 µL of template DNA, 1 µL of primer, and 8 µL of sterile water. The PCR amplification protocol was as follows: initial denaturation at 94°C for 4 min; denaturation at 94°C for 1 min, annealing at 50°C (annealing temperatures for different primers are listed in Table 2) for 45 s, extension at 72°C for 1 min, for 35 cycles; a final extension at 72°C for 7 min, with the amplification products stored at 4°C. A 10 µL sample of the PCR products was subjected to electrophoresis on a 1.0% agarose gel for detection.

Data Processing

Based on the electrophoresis patterns of the amplified products, presence of a band at the same migration position was recorded as '1', and absence as '0'. A '0, 1' matrix was constructed from the bands recorded by the amplification primers. Data analysis was performed using the NTSYS-pc 2.1 statistical software, with clustering by the UPGMA method and construction of a dendrogram. The POPGENE 32 software was used to calculate the number of alleles (Na), effective number of alleles (Ne), Nei’s genetic diversity index (He), and Shannon's information diversity index (I). Based on the clustering results, primers capable of distinguishing the test materials were selected. A DNA fingerprint map was manually constructed using the '0, 1' matrix of primer combinations.

iPBS Primer Screening and Polymorphism Analysis

From 83 iPBS primers, six were selected that produced clear amplification bands, high polymorphism, and good reproducibility. These primers were used for PCR amplification of genomic DNA from 38 germplasm resources. The amplification results (Table 2) showed that the six primers generated a total of 210 bands, all of which were polymorphic, yielding a polymorphism rate of 100%. The number of bands produced by each primer ranged from 31 to 41, with an average of 35 bands per primer. Among them, primer 2270 amplified the most bands, with 41 amplification sites, while primer 2274 amplified the least, with 31 amplification sites. On average, each primer generated 35 polymorphic bands. This indicates that the iPBS molecular marker method is highly efficient in detecting the genetic diversity of Vanda germplasm resources. The amplification results of primer 2272 on the genomic DNA of 38 germplasm resources are shown in Fig. 1. Significant differences in the amplification products among Vanda species using the same primer demonstrate the rich genetic diversity within the genus Vanda.

Table 2

Amplification results of 7 iPBS primers
Primer	Sequences(5’-3’)	Annealing temperature	Total bands	Polymorphic bands	Rate of polymorphic
2219	GAACTTATGCCGATACCA	44	33	33	100
2270	ACCTGGCGTGCCA	50	41	41	100
2272	GGCTCAGATGCCA	44	32	32	100
2274	ATGGTGGGCGCCA	51	31	31	100
2243	AGTCAGGCTCTGTTACCA	48	36	36	100
2246	ACTAGGCTCTGTATACCA	44	37	37	100
Total	-		210	210	-
Average	-		35	35	100

Genetic Diversity and Phylogenetic Relationship

Using the POPGENE 32 software, genetic diversity indices for 38 samples were calculated (Table 3). The mean observed number of alleles (Na) was 1.9936, the mean effective number of alleles (Ne) was 1.4243, the mean Nei's genetic diversity index (He) was 0.2778, and the mean Shannon's information diversity index (I) was 0.4404. These results indicate significant genetic diversity among the 38 germplasm genomes. The diversity indices confirm the abundant genetic diversity in Vanda. Additionally, using the NTSYS-pc 2.1 software, a genetic similarity coefficient matrix and clustering analysis (Fig. 3) were constructed for the 36 Vanda and 2 Aerides samples based on the sites amplified by the six primers. The genetic similarity coefficient (GS) ranged from 0.0345 to 0.6667, with a variation of 0.6322, indicating significant genetic differences among Vanda species. Within the genus Vanda, V. bensonii and V. brunnea had the highest genetic similarity coefficient of 0.6667, indicating the closest genetic distance and phylogenetic relationship between these two species. In contrast, V. foetida and V. vietnamica had the most distant relationship, with a genetic similarity coefficient of 0.0345.

Table 3

Genetic Diversity indexes of 38 Germplasm Resources
Simple size	Na	Ne	He	I
38	1.9936 ± 0.0798	1.4243 ± 0.2487	0.2778 ± 0.1179	0.4404 ± 0.1467

Cluster Analysis

The NTSYS-pc 2.1 analysis software was used to perform cluster analysis on the genetic matrix using the UPGMA method, determining the genetic relationships among the 38 germplasm resources and constructing a dendrogram (Fig. 3). The results indicate a complex genetic relationship among Vanda germplasm. At a threshold of 0.25, Vanda genus germplasm could be distinctly separated from those of other genera. At a threshold of 0.31, the 36 Vanda and 2 Aerides samples were divided into seven major groups. The first five groups comprised Vanda clusters, while the remaining two groups were clusters of closely related genera. The first major group contained 14 germplasms and could be further divided into three sub-branches. The first sub-branch included V. foetide, V. insignis, V. tricolor, V. limbata, V. mariae, and V. perplexa, with V. limbata and V. mariae being the most closely related, all originating from Indonesia. V. curvifolium and V. miniatum formed the second sub-branch, both characterized by dwarf plants and similar flower morphology and color. The third sub-branch contained V. lamellata, V. sanderiana, V. roeblingiana, V. luzonica, V. ustil, and V. merrillii, geographically originating from the Philippines. The clustering of the first major group showed some correlation with the germplasm's place of origin and plant morphology.

The second major group included four germplasms, V. ampullacea, V. ampullaceum, V. christensonianum, and V. garayi, all characterized by dwarf plants, small flowers, and bright colors. The third major group comprised only three germplasms: V. cristata, V. pumila, and V. tessellata, with V. cristata and V. pumila being more closely related and clustered into a sub-branch. The fourth major group consisted of 10 germplasms, further divided into three sub-branches. The first sub-branch included V. bensonii, V. bicolor, V. brunnea, V. concolor, V. denisoniana, V. fuscoviridis, and V. subconcolor, characterized by similar flowering periods, colors, and shapes. V. coerulea and V. coerulescens formed the second sub-branch, both featuring rare blue flowers. V. pusillum was clustered as a separate sub-branch. The fifth major group had four germplasms, with V. lilacina and V. testacea forming one sub-branch, and V. vietnamica and A. flabellata the other. V. falcata and A. lawrenceae were clustered in the sixth major group, while V. teres was alone in the seventh group, indicating these three germplasms do not belong to the genus Vanda classification. The clustering results using molecular markers were generally consistent with traditional morphological classification, demonstrating the applicability of iPBS molecular markers in the classification, identification, and phylogenetic research of Vanda germplasm resources.

Construction of DNA Fingerprint Maps

Using the six selected iPBS primers, DNA fingerprint maps were constructed for the 38 Vanda and Aerides germplasm resources. It was observed that all six primers could individually identify each of the 38 samples. In this study, primer 2272 was used to construct DNA fingerprint maps for the 38 germplasm resources (Fig. 4), where black indicates the presence of a band at a site, represented as '1' in the '0, 1' matrix, and colorless indicates the absence of a band, represented as '0'. The constructed fingerprint maps can be used for classification and identification of the 36 Vanda and 2 Aerides samples.

Although iPBS is an efficient and rapid novel molecular marker technique, there have been no reports to date on its application in the identification of Vanda varieties and analysis of genetic diversity. In this study, six primers were selected from 83 iPBS primers for their clarity in amplification bands, high polymorphism, and good reproducibility. These primers were used to amplify 38 germplasm resources, resulting in 210 detected bands, all of which were polymorphic, giving a polymorphism rate of 100%. Compared to RAPD (Lim et al. 1999; Tanee et al. 2012) and SPAR (Manners et al. 2013) molecular markers, these primers demonstrated greater efficiency, with a single primer capable of differentiating all the native Vanda species. The results of the study clearly demonstrate that the iPBS marker is a simple, efficient molecular marker with good amplification polymorphism. Its application in the identification of Vanda germplasm resources, as well as in studies of genetic diversity and phylogenetic relationships, is feasible.

The dendrogram constructed from the band patterns can distinctly differentiate between strap-leaved and terete-leaved plants within the Vanda genus. Previous studies (Lim et al. 1999; Tanee et al. 2012; Zhang et al. 2013) have all confirmed that V. teres should be classified into a separate genus, namely Papilionanthe. The clustering results obtained in this study also support this classification.

The cluster analysis diagram shows that the second major group within the genus Vanda (Vanda s.l.) was originally classified under the genus Ascocentrum. Multifaceted research findings (Lim et al. 1999; Zou et al. 2015) indicate a close relationship between the Ascocentrum and Vanda genera, and many fertile intergeneric hybrids, Ascocenda, have been produced between them. This study supports the notion of classifying Ascocentrum as a subgenus within the genus Vanda s.l..

Within the third major group, V. cristata and V. pumila are both classified under the subgenus Trudelia of the genus Vanda. Both species exhibit typical characteristics of Trudelia, with a saccate base of the labellum.

In the first and second sub-branches of the fourth major group, the germplasm, due to similar morphological features and their clustering together, aligns with the morphological and molecular classifications presented in Gardiner et al. (2013) and Zou et al. (2015). The cluster diagram shows that within the first sub-branch, the germplasms of V. concolor, V. fuscoviridis, and V. subconcolor have a closer phylogenetic relationship to each other. And the germplasms of V. bensonii, V. bicolor, V. brunnea, and V. denisoniana within this branch also exhibit a more intimate phylogenetic relationship. In the morphological classification study of Vanda by Gardiner et al. (2013), V. concolor, V. fuscoviridis, and V. subconcolor were classified under the Vanda group, while V. bensonii, V. bicolor, V. brunnea, and V. denisoniana were classified under the Obtusiloba group, and V. coerulea and V. coerulescens under the Longicalcarata group. This classification aligns with the clustering results obtained using iPBS molecular markers.

V. vietnamica was originally classified in the monotypic genus Christensonia Haager and was described as ‘a yellow A. flabellata’ (Haager 1993). In the clustering results, A. flabellata and V. vietnamica exhibited a close phylogenetic relationship and were both clustered within the genus Vanda s.l..Researchers such as Garay (1972), Seidenfaden (1973), and Christenson (1986) have advocated from a morphological perspective for the classification of A. flabellata within the genus Vanda s.l., due to its characteristic short spur and broad lip. Additionally, studies by Gardiner et al. (2013), Zhang et al. (2013), Topik et al. (2012), and the clustering results of this research consistently indicate that A. flabellata and V. vietnamica belong to the genus Vanda s.l..

In 1914, Rudolf Schlechter classified Vanda sanderiana as Euanthe, naming it Euanthe sanderiana. Only a few scholars recognized this classification, but subsequent molecular studies (Lim et al. 1999; Gardiner et al. 2013), cytological research (Tanaka & Kamemoto 1961), and results from hybrid breeding all indicate a closer phylogenetic relationship of V. sanderiana with the genus Vanda s.l.. Moreover, V. lamellata and V. sanderiana both belong to the La-mellaria section. The results of this study strongly support the classification of V. sanderiana within the genus Vanda s.l..

Although Vanda falcata was initially classified into the Vanda s.l. as the first species of the genus Neofinetia, it did not cluster within the genus Vanda s.l. in the dendrogram. Moreover, according to certain molecular studies (Fan et al. 2009; Gardiner et al. 2013; Topik et al. 2012), the clustering results indicate that the genus Neofinetia and Vanda s.l. are sister genera. Some researchers believe that V. falcata can be included in the genus Vanda s.l., adopting a broader concept of the Vanda s.l. genus to simplify the classification. This study suggests that further molecular and morphological research is needed to substantiate the classification of V. falcata within the genus Vanda s.l..

In the study results, it was found that the majority of species from the same geographical origins clustered together into a group. For example, in the first major group, species with the same geographical origin formed the Indonesian, Philippine clade, or ‘South-East Asian archipelago’ clade. This indicates that geographical factors have a significant influence on the classification of the Vanda genus.Kocyan et al. (2008) found that different species from the same geographical origin could cluster together within a sub-branch, indicating that at the sub-branch level within a genus, the geographical origin of a species seems to be more important than morphological characteristics. In addition, the classification of Vanda shows certain correlations with plant morphological traits, flowering period, flower color, and flower shape. However, some species did not exhibit a completely consistent relationship in this regard.

The classification results of this study for Vanda are mostly consistent with the findings of Lim et al. (1999), Gardiner et al. (2013), Zou et al. (2015), and Kasutjianingati & Firgiyanto (2018). However, there are some discrepancies in the classification of certain species. These differences could be attributed to the use of different research techniques and subjectivity in reading band numbers. Therefore, a unified approach to the taxonomy and phylogenetic relationships of Vanda germplasm resources should integrate evidence from molecular systematics, morphogeography, micromorphology, and other diverse studies. Currently, relying solely on a single method is insufficient to fully represent the genetic diversity of Vanda.

DNA fingerprinting is a technique that involves the amplification of specific DNA fragments from designated regions in DNA samples using particular molecular markers, resulting in a map that reflects the genetic differences between individual samples. This map can be used for individual identification, with each locus on the map having its unique significance. As DNA is less affected by external environmental factors and exhibits high polymorphism, molecular marker techniques that use DNA as a template for PCR amplification have become an effective method for the identification of Orchidaceae plant varieties. By constructing a genetic map of the genus Vanda, a foundation can be provided for the conservation, breeding, and efficient, rational utilization of its germplasm resources. There have been reports on the construction of DNA fingerprint maps for various orchids using iPBS primers. For instance, Cui et al. (2021) used two iPBS primers to construct fingerprint maps, successfully distinguishing 48 Dendrobium samples under test. Zhao et al. (2023) used two selected iPBS primers, with each primer independently capable of constructing DNA fingerprint maps. These maps successfully differentiated all 35 tested germplasm samples of Dendrobium nobile. Zeng et al. (2022) also successfully applied iPBS primers for constructing DNA fingerprint maps of Cymbidium goeringii. Until now, there have been no reports on constructing DNA fingerprint maps for the genus Vanda using this method. The six primers selected in this study all exhibited 100% polymorphism, making any one of them suitable for amplifying bands to construct fingerprint maps and identify the test materials. This study is the first to explore the genetic diversity of Vanda germplasm resources using the iPBS molecular marker method. The results indicate that iPBS molecular markers display abundant genetic diversity in 36 Vanda and 2 Aerides germplasm, providing a theoretical basis for the classification and optimization of Vanda germplasm resources. This study also introduces a new method for molecular marking in Vanda.

Author Contribution

Author ContributionY.W.Y. Designed research, conducted experiments, analyzed data, wrote the manuscript. C.X.Q., Z.Q.J., D.J.L., H.C.Y., Conducted experiments, editing manuscripts. Z.Z.B., L.J.W. Designed research, wrote the manuscript. All authors read and approved the manuscript.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (32260259, 31901092), the Natural Science Foundation of Guangxi Zhuang Autonomous Region (2021GXNSFBA075059), the Guangxi Key Research and Development Program (Guike AB21220056).

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No competing interests reported.

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Genetic Diversity Analysis and Fingerprinting Construction of the Vanda species (Orchidaceae) Germplasm Resources by inter-primer binding site (iPBS) Marker

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Abstract

Figures

Introduction

Material And Methods

Plant Materials

Genomic DNA Extraction and Analysis

Primer Screening and iPBS PCR Amplification

Data Processing

Results

iPBS Primer Screening and Polymorphism Analysis

Genetic Diversity and Phylogenetic Relationship

Cluster Analysis

Construction of DNA Fingerprint Maps

Discussion

Declarations

Author Contribution

Acknowledgments

References

Additional Declarations

Status:

Version 1