Genetic Relatedness among Dendrobium (Orchidaceae) Species and Hybrids Using Morphological and AFLP Markers

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  • 1 School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, University Kebangsaan Malaysia, 43600 Bangi, Malaysia
  • 2 Horticulture Research Centre, MARDI Headquarters, P.O. Box 12301, 50774 Kuala Lumpur, Malaysia
  • 3 School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, University Kebangsaan Malaysia, 43600 Bangi, Malaysia

Dendrobium is one of the largest genera in the Orchidaceae family. Information on the genetic diversity and relationships among species and hybrids is important for breeding purposes and species conservation. The objectives of this study were to assess genetic relatedness and to determine whether morphological, molecular, or combined analysis can discriminate among Dendrobium species, commercial hybrids, and interspecific hybrids. A total of 81 Dendrobium accessions were characterized with 12 amplified fragment length polymorphism (AFLP) primer pairs and 21 morphological characters. Mean genetic relatedness for morphological characters, AFLP analysis, and combined analysis were 0.61, 0.37, and 0.43, respectively. Dendrograms were generated using an unweighted pair group method with arithmetic averages (UPGMA); the analysis was performed on a Jaccard similarity coefficient matrix. The data from morphological characters revealed that the Jaccard similarity coefficient ranged from 0.20 to 1.0, where the tested 81 Dendrobium accessions could be grouped into four clusters. For the AFLP analysis, the number of polymorphic fragments for each primer varied from 80 to 284 with 78% average percentage of polymorphic loci and the similarity coefficient ranging from 0.125 to 1.0 with Dendrobium accessions grouped into three clusters. The similarity coefficients estimated through a combined analysis of morphological and AFLP data ranged from 0.21 to 1.0 and the Dendrobium accessions appeared clustered into two groups. The results revealed some similarities among the three data sets. The combined data set was the most useful in discriminating Dendrobium accessions based on species sections and relationship among species and their hybrids. The correlation between the AFLP data and the combined data was highly significant (r = 0.98, P > 0.001), indicating the usefulness of AFLP data for species discrimination and hybrid identity in the absence of floral morphological characters.

Abstract

Dendrobium is one of the largest genera in the Orchidaceae family. Information on the genetic diversity and relationships among species and hybrids is important for breeding purposes and species conservation. The objectives of this study were to assess genetic relatedness and to determine whether morphological, molecular, or combined analysis can discriminate among Dendrobium species, commercial hybrids, and interspecific hybrids. A total of 81 Dendrobium accessions were characterized with 12 amplified fragment length polymorphism (AFLP) primer pairs and 21 morphological characters. Mean genetic relatedness for morphological characters, AFLP analysis, and combined analysis were 0.61, 0.37, and 0.43, respectively. Dendrograms were generated using an unweighted pair group method with arithmetic averages (UPGMA); the analysis was performed on a Jaccard similarity coefficient matrix. The data from morphological characters revealed that the Jaccard similarity coefficient ranged from 0.20 to 1.0, where the tested 81 Dendrobium accessions could be grouped into four clusters. For the AFLP analysis, the number of polymorphic fragments for each primer varied from 80 to 284 with 78% average percentage of polymorphic loci and the similarity coefficient ranging from 0.125 to 1.0 with Dendrobium accessions grouped into three clusters. The similarity coefficients estimated through a combined analysis of morphological and AFLP data ranged from 0.21 to 1.0 and the Dendrobium accessions appeared clustered into two groups. The results revealed some similarities among the three data sets. The combined data set was the most useful in discriminating Dendrobium accessions based on species sections and relationship among species and their hybrids. The correlation between the AFLP data and the combined data was highly significant (r = 0.98, P > 0.001), indicating the usefulness of AFLP data for species discrimination and hybrid identity in the absence of floral morphological characters.

Orchids are commercially important in Malaysia. Export value is expected to increase further because of a wider product range, longer shelf life, and ability to export flowers year-round. The orchid family is probably one of the most important plant families from a horticultural point of view. Orchidaceae is one of the largest families among angiosperms. The family includes 800 genera and 25,000 species (Stewart and Griffiths, 1995). Orchids are distributed in all regions of the world except Antarctica and are found growing in many different habitats and elevation gradients (Pridgeon, 2000). They are well known for their economic importance and widely cultivated for ornamental purposes and cosmopolitan in distribution (Bechtel et al., 1992).

The genus Dendrobium is the third largest in the family Orchidaceae, comprising ≈1184 species worldwide (Leitch et al., 2009). Dendrobium plants are among the most prevalent orchids for commercial production of cut flowers and potted plants (Chen and Tsi, 2000) and the most popular genus in horticultural industries. The genetic diversity of the Dendrobium genus is not well known (Wang et al., 2009).

Orchid classification has been poorly understood because of the lack of fossil records, the large number of species, and a historical emphasis on characters related to floral morphology. Circumscription of genera, subtribes, tribes, and subfamilies and the relationships among them were unclear because of homoplasy in morphological and anatomical features (Chase, 2006). There has been no explicit morphological cladistic analysis of Orchidaceae (Freudenstein and Rasmussen, 1999). Phylogenetic research using DNA sequences is now unraveling the complex picture at every taxonomic level. Assessment of genetic variability is important for the use of genetic resources and for determining the uniqueness of genotypes to exploit heterosis (Franco et al., 2001). Genetic diversity can be inferred from genetic similarity or distance estimates determined using phenotypic variation and/or molecular markers.

Molecular markers are not subject to environmental influence, and because of that, they are considered superior to morphological markers (Máric et al., 2004). Several types of molecular markers are available and have been used in assessment of orchid genetic diversity; ribosomal DNA internal transcribed spacer (ITS) region-based analysis was used to ascertain the phylogenetic relationship among 20 Dendrobium species (Chiang et al., 2012), whereas a phylogenetic tree from ribosomal-ITS2 sequences was constructed for 18 species of the genus Dendrobium (Chaudhary et al., 2012; Wang et al., 2009) used the intersimple sequence repeat technique to evaluate genetic diversity and phylogenetic relationship among 31 Dendrobium species and Shengnan et al. (2011) determined the genetic diversity among 31 species of Dendrobium species using random amplified polymorphic DNA markers.

AFLP markers are dominant markers. The AFLP technique can generate hundreds of highly replicable markers, allowing for high-resolution genotyping (Mueller and Wolfenbarger, 1999). Approximately 90% of the AFLP polymorphisms reflect point mutations and most AFLP fragments are independent loci (Buntjer et al., 2002). The loss of a restriction site removes the fragment from the profile rather than changing its size (Albertson et al., 1999), which means there is no need for prior DNA sequence information. When compared with other DNA-based markers (Vos et al., 1995), many markers can be analyzed in a short time, and only a small amount of DNA is needed. AFLP markers are highly reproducible and reliable (Bagley et al., 2001; Jones et al., 1997), high-throughput, cost-effective, and have wide genome coverage (Hansen et al., 1999; Prashanth et al., 2002). AFLP has proven to be an extremely effective tool for distinguishing closely related genotypes (Xiang et al., 2003).

AFLP markers have been used in the assessment of genetic relationships in many plant species such as rice (Zhu et al., 1998), Arabidopsis thaliana (L.) (Breyne et al., 1999), bluegrass (Renganayaki et al., 2001), Carica papaya (Kim et al., 2002), Hibiscus tiliaceus (Tang et al., 2003), and potato (Esfahani et al., 2009).

Molecular and morphological data matrices are very informative tools for the estimation of genetic distances (Vieira et al., 2007). Few orchid studies have analyzed at the same time both molecular data and morphological characters for the same set of taxa, but these have demonstrated that both types of characters are complementary and the structural characters often mark clades recovered in the combined analyses (Albert, 1994; Figueroa et al., 2008). Different studies found that the distribution of morphological character states provides additional support to the groupings recovered in molecular phylogenetic trees (Figueroa et al., 2008; Salazar, 2009; Salazar et al., 2009).

The objectives of this study were to 1) determine genetic relatedness among Dendrobium accessions from different species and hybrids; and 2) determine whether morphological, molecular, or combined analysis can discriminate Dendrobium species, Dendrobium commercial hybrids, and Dendrobium interspecific hybrids.

Materials and Methods

Plant materials.

Eighty-one Dendrobium accessions were used in this study from 13 Dendrobium species, eight commercial hybrids, and 17 interspecific hybrids. All plant materials were ≈2 years old. Dendrobium species were propagated by seeds or cuttings, whereas the commercial hybrids and interspecific hybrids were vegetatively propagated. Leaf samples of accessions were collected from the Malaysian Agricultural Research and Development Institute orchid greenhouse. A list of the Dendrobium accessions, their corresponding accession numbers, and classification is provided in Table 1. The geographical origin of the Dendrobium species in Malaysia is given in Nor Hazlina et al. (2013).

Table 1.

List of Dendrobium accessions, their corresponding accession numbers, and classification.

Table 1.

Morphological data analysis.

All plants were assessed for 21 morphological traits, all of which were referred by Sheehan and Sheehan (1994): plant height, root type, leaf type, leaf height, leaf width, pseudobulb habit, pseudobulb character, pseudobulb structure of cross-section, pseudobulb inter node, flower arrangement, flower structure, sepal structure, petal structure, flower color arrangement, hairy flowers, sepal color, sepal with more than one color, petal color, petal with more than one color, labellum color (upper side), and labellum color (inner side). All measurements were made using a digital caliper and flower color was standardized using the Royal Horticultural Society Color Chart.

Average values were applied for each quantitative character. All morphological data were standardized using the Transform option in Past Version 1.92 (Hammer et al., 2001) to reduce the influence of different scales. Morphological character data were used to estimate similarity coefficients, according to Jaccard (1908) and to construct dendrograms using the UPGMA in PAST Version 1.92 software (Hammer et al., 2001).

Amplified fragment length polymorphism analysis.

Genomic DNA was extracted from fresh leaf tissues using the modified 2× cetyltrimethyl ammonium bromide protocol based on Doyle and Doyle (1990). Some samples had very limited leaf tissues; in this case, a DNeasy Plant Mini Kit (QIAGEN, Germany) was used.

AFLP analysis was carried out according to Vos et al. (1995). Briefly, 250 ng of genomic DNA was digested with restriction enzymes EcoRI and MseI. The digested DNA fragments were ligated to EcoRI and MseI adaptors with T4 DNA ligase. Sixty-four primer combinations were used for the selective amplification. The products were separated on 5% denaturing polyacrylamide gels, and the DNA bands were visualized using silver staining (Fritz et al., 1999). Of these, 12 primer pairs that generated well-defined patterns were chosen for this study (Table 2).

Table 2.

Total number of amplified fragment length polymorphism loci examined using 12 primer combinations, percentage of polymorphic loci per primer combination among 81 Dendrobium accessions, and hybrids.

Table 2.

For the selected 12 primer combinations, the genomic DNA of 81 Dendrobium accessions were further amplified using selected EcoRI and MseI primers. The EcoRI-based primer was labeled with fluorescent dye (FAM and NED) at the 5′ end. After selective amplification, amplified fragments were separated on polyacrylamide gel with an ABI Genetic Analyser 3100 (Applied Biosystems, USA) after being mixed with DNA size standards labeled with fluorescent dye ROX (red). Data of fragments were processed with GeneMapper 4.0 (Applied Biosystems, USA) for size and intensity of each band.

Only clear and unambiguous DNA bands were included in the analysis. DNA bands were scored for presence (1) and absence (0). A pairwise comparisons of accessions, based on the presence of unique and shared polymorphic products, were used to estimate similarity coefficients, according to Jaccard (1908), and while constructing dendrograms using the UPGMA, the Mantel test was performed to test the correlation between the morphological data and AFLP data with PAST Version 1.92 software (Hammer et al., 2001).

Combined data analysis.

All 81 Dendrobium accessions with morphological and molecular data were included in a third data set to compute the similarity coefficient of Jaccard (1908). This data set was used as input for cluster analysis using the UPGMA to generate a dendrogram using PAST Version 1.92 software (Hammer et al., 2001). Correlation coefficients between the combined data with morphological data and between the combined data with AFLP data were inferred from Mantel tests (Hammer et al., 2001).

Results

Morphological characterization.

The data from morphological characters (Supplemental Table 1) revealed that the similarity coefficient, ranging from 0.20 to 1.0 with an 0.61 average (data not provided), imply a broad genetic base for the Dendrobium accessions investigated. Figure 1 represents the dendrogram for the Dendrobium accessions generated by UPGMA analysis of the 81 Dendrobium accessions with the 21 morphological characters. Dendrobium accessions were grouped into four well-defined clusters; the first cluster, which consisted of 51 accessions, was further divided to two subclusters: subcluster Ia contained 46 accessions (Fig. 1). In this subcluster (Ia), the coefficient similarity was 1 between (Bigi1, Bigi2, Bigi3, Bigi4, Bigi5) and (DS1, DS2, DS3), which is plausible because Dendrobium bigibbum is one of the parents for the interspecific hybrid DS (data not provided). On the other hand, the coefficient similarity was also 1 between (Bigi1, Bigi2, Bigi3, Bigi4, Bigi5) and (TF3, TF7), although Dendrobium bigibbum was not a parent for the interspecific hybrid TF (data not provided) and subcluster Ib with five hybrids (Soni1, Soni2, Caes1, Caes2, and Caes3). These findings imply that these hybrids are closely related because Dendrobium Ceasar ‘Giant’ is an ancestor of Dendrobium Sonia. The second cluster consisted of 10 accessions and was further divided into two subclusters (Fig. 1): subcluster IIa with one species (Anos2, Anos3, Anos5, and Anos6) belonging to section Dendrobium and subcluster IIb, which contained three hybrids (Chao1, Chao2, and Chao3) belonging to Dendrobium Chao Praya Gem and three interspecific hybrids (TF1, TF2, and TF5) belonging to Dendrobium Tuanku Fauziah. This result is acceptable because the interspecific hybrid TF is a progeny from Dendrobium Chao Praya Gem. The third cluster comprised eight accessions, all belonging to Dendrobium crumenatum and Dendrobium setifolium, which are in section Rhopalanthe. The fourth cluster comprised 12 accessions from Dendrobium leonis, Dendrobium grande, and Dendrobium aloifoliums, all belonging to section Aporum.

Fig. 1.
Fig. 1.

Dendrogram for the Dendrobium accessions generated by unweighted pair group method with arithmetic averages (UPGMA) analysis of the 81 Dendrobium accessions with the 21 morphological characters.

Citation: HortScience horts 49, 5; 10.21273/HORTSCI.49.5.524

AFLP analysis.

A total of 2701 fragments ranging in size from 70 bp to 500 bp were scored using 12 primer combinations. The number of polymorphic fragments for each primer varied from 80 [E-AGC/M-CAT (NED)] to 284 [E-AGC/M-CAA (NED), (E-AGC/M-CTC (NED)]. The average percentage of polymorphic loci (polymorphic loci/total loci) was 78%, ranging from 28% for primer combination E-AGC/M-CAT (NED) to 100% for primer combinations E-AGC/M-CAA (NED), E-AAC/M-CAC (FAM), and E-AGC/M-CTC (NED). The number of bands and the degree of polymorphism revealed by each primer combination is given in Table 2.

The similarity coefficient ranged from 0.125 to 1.0 with an average of 0.37 (data not provided), indicating wide genetic distance among the accessions. Figure 2 represents the dendrogram for the 81 Dendrobium accessions generated by a UPGMA analysis of the AFLP data. Dendrobium accessions appeared clustered into three well-defined clusters; the first cluster consisted of 42 accessions and was further divided into two subclusters: subcluster Ia contained all commercial hybrids, all interspecific hybrids, and two species (Bigi1, Bigi2, Bigi3, Bigi4, Bigi5) belonging to section Phalaenthe and (Croc1, Croc2, Croc3, and Croc4) belonging to section Pedilonum. Subcluster Ib had eight accessions (Lamel1, Lamel2, Lamel3) from section Pedilonum (Farm1, Farm2, Farm3, Farm4, and Farm5) from section Callista. The second cluster comprised 24 accessions and was divided into two subclusters: subcluster IIa contained 12 accessions, all belonging to section Aporum, and subcluster IIb contained 12 accessions all belonging to section Rhopalanthe. The third cluster comprised 15 accessions and was further divided into two subclusters: subcluster IIIa contained Anos1, Anos4, Anos2, Anos3, Anos5, and Anos6 from section Dendrobium; Aggr1, Aggr2, Aggr3, and Aggr4 from section Callista; and subcluster IIIb contained five accessions (Pier1, Pier2, Pier3, Pier4, and Pier5) from section Dendrobium.

Fig. 2.
Fig. 2.

Dendrogram for the 81 Dendrobium accessions generated by a unweighted pair group method with arithmetic averages (UPGMA) analysis of the amplified fragment length polymorphism (AFLP) data.

Citation: HortScience horts 49, 5; 10.21273/HORTSCI.49.5.524

The correlation between the morphological data and AFLP data was highly significant (r = 0.55) with P > 0.001.

Combined analysis.

The similarity coefficient estimated through a combined analysis of morphological and AFLP data ranged from 0.21 to 1.0 with an average of 0.43 (data not provided), indicating broad genetic distance among the accessions. Figure 3 represents the dendrogram for 81 Dendrobium accessions generated by UPGMA analysis for the combined analysis of the AFLP and morphological data. Dendrobium accessions appeared clustered into two well-defined groups; the first group consisted of 39 accessions and was divided into four subclusters: subcluster Ia contained 11 accessions all belonging to section Dendrobium, and subcluster Ib contained four accessions (Aggr1, Aggr2, Aggr3, and Aggr4) and they belonged to section Callista, whereas subcluster Ic contained 12 accessions (Hend1, Hend2, Hend3, Hend4, Seti1, Seti2, Seti3, Seti4, Seti5, Crum1, Crum2, and Crum3); all 12 accessions belonged to the Rhopalanthe section. Subcluster Id comprised 12 accessions (Leon1, Leon2, Leon3, Leon4, Leon5, Grand1, Grand2, Grand3, Alio1, Alio2, Alio3, and Alio4), all belonging to section Aporum. The second cluster consisted of 42 accessions and was further divided into two subclusters; subcluster IIa contained 12 accessions from Callista and Pedilonum sections, whereas subcluster IIb contained 30 accessions; five accessions (Bigi1, Bigi2, Bigi3, Bigi4, and Bigi5) belonged to the Dendrobium bigibbum species from section Phalaenanthe and 25 accessions were all commercial and interspecific hybrids. The interspecific hybrids (TF1, TF2, TF3, TF4, TF5, TF6, and TF7) were in the same cluster with (Chao1 and Chao2) and the similarity coefficient between them ranged from 0.81 to 0.86 because Dendrobium Chao Praya Gem is a parent for the interspecific hybrid Dendrobium Tuanku Fauziah. The interspecific hybrids (TN1, TN2, TN4, and TN5) were in the same cluster with (Caes1 and Caes2) with the similarity coefficient of 0.90 (data not provided) because Dendrobium Caeser ‘Giant’ is a parent for the interspecific hybrid Dendrobium Tuanku Najihah.

Fig. 3.
Fig. 3.

Dendrogram for 81 Dendrobium accessions generated by unweighted pair group method with arithmetic averages (UPGMA) analysis for the combined analysis of the amplified fragment length polymorphism (AFLP) and morphological data.

Citation: HortScience horts 49, 5; 10.21273/HORTSCI.49.5.524

The correlation coefficients between both the morphological and AFLP matrices were positive and significant (r = 0.69** and r = 0.98,** respectively) with the combined analysis matrix.

Discussion

Genetic diversity in plants is essential for successful breeding and creation of new cultivars. Evaluation of the relationship of Dendrobium accessions (species, commercial hybrids, and interspecific hybrids) with morphological and AFLP data analysis can help breeders choose parental plants for breeding. However, phylogenetic relationships and the genetic diversity information of the Dendrobium genus are very limited (Burke et al., 2008; Gu et al., 2007; Wang et al., 2009; Yue et al., 2006).

The cluster analysis in this study for the 81 Dendrobium accessions based on 21 morphological characters revealed four clusters (Fig. 1). An earlier examination using 16 different morphological characters revealed only three clusters (Nor Hazlina et al., 2013). Wang et al. (2009) reported that using a Euclidian distance matrix for 31 Dendrobium species based on 28 morphological traits revealed six clusters belonging to six sections. Our results indicated that the morphological data used in the cluster analysis were efficient in differentiating among the hybrids from their parents and according to the species section with some inconsistencies as reported by Chen and Tsi (2000) and Tsi et al. (1999).

AFLP fingerprinting data have the potential to be an integral part of the new plant varieties protection system (Xiang et al., 2003). In this study, 78% of the AFLP loci were polymorphic among the 81 Dendrobium accessions. Our results are in agreement with other studies on Dendrobium species, where 70.6% of the AFLP loci were polymorphic among 30 commonly grown Dendrobium cultivars (Quan et al., 2012). Xiang et al. (2003) found that 83% of the AFLP loci were polymorphic among 43 Dendrobium hybrids. Other studies reported higher polymorphism; Yin et al. (2007) reported 99.7% polymorphism among 38 Dendrobium species using AFLP techniques with eight pairs of primer combinations, and Zhu and Li (2011) found 99.8% polymorphism among 63 hybrid cultivars and 37 native species of Asian Dendrobium with the use of eight primer AFLP combinations.

The moderate association (r = 0.55) between genetic relatedness using morphological and AFLP analysis may be the result of several reasons; molecular analysis provides a wider genome sampling than the morphological analysis, and molecular markers are not subject to either natural or artificial selection, whereas the morphological characters are subject to both natural and artificial selection (Vieira et al., 2007).

Our results strongly indicated that AFLP markers were effective in distinguishing between the Dendrobium accessions. Compared with morphological characters, DNA fingerprints reflect directly the variability for plants at the genetic level with reliable and tremendous data sets for reproducible estimation of genetic relatedness for investigations on plant systematics (Wang et al., 2009).

The analysis for the combined morphological and AFLP data revealed a dendrogram somewhat similar to the dendrograms generated using the morphological and molecular data. The combined analysis concluded in the subcluster Id with the Aporum section (Leon, Grand, and Aloi) (Fig. 3), which was similar to the results seen in the cluster analysis of the morphological data (Fig. 1) for the fourth cluster, from the molecular data cluster analysis (Fig. 2) for subcluster IIa; also subcluster Ic in the combined analysis (Fig. 3), which was similar to subcluster IIb in the AFLP data analysis (Fig. 2); and the second cluster for the combined data analysis (Fig. 3) similar to the first cluster for the AFLP data analysis (Fig. 2) with respect for some differences in the similarity coefficient between the accessions.

The correlation coefficient for the AFLP data with the combined analysis (r = 0.98) was higher than the correlation coefficient between the morphological characters and combined analysis (r = 0.69), which could be the result of the different number of data points for AFLP markers and morphological characters (Vieira et al., 2007). The cluster analysis of the combined data widely resembles that of the AFLP data but with reliable assignment of species to sections. Crosses between species, which are closely related (in the same cluster), are expected to yield a high seed set with good viability.

Conclusions

In this study we combined morphological and AFLP data to evaluate genetic relatedness among Dendrobium accessions. The combined data set coincides best to species sections and relationship between species and their hybrids. The integration of morphological and AFLP markers can be a powerful tool for distinguishing among closely related germplasm materials and can be used in recognizing unknown origins of germplasm materials, plant variety preservation, and/or cultivar identification.

Literature Cited

  • Albert, V.A. 1994 Cladistic relationships of the slipper orchids (Cypripedioideae: Orchidaceae) from congruent morphological and molecular data Lindleyana 9 115 132

    • Search Google Scholar
    • Export Citation
  • Albertson, R.C., Markert, J.A., Danley, P.D. & Kocher, T.D. 1999 Phylogeny of a rapidly evolving clade: The cichid fishes of lake Malawi, East Africa Proc. Natl. Acad. Sci. USA 96 5107 5110

    • Search Google Scholar
    • Export Citation
  • Bagley, M.J., Anderson, S.L. & May, B. 2001 Choice of methodology for assessing genetic impact of environmental stressors: Polymorphism and reproducibility of RAPD and AFLP fingerprints Ecotoxicol. 10 239 244

    • Search Google Scholar
    • Export Citation
  • Bechtel, H., Cribb, P. & Launert, E. 1992 The manual of cultivated orchid species. 3rd Ed. Blandford Press, London, UK

  • Breyne, P., Rombaut, D., Van Gysel, A., Van Mantagu, M. & Gerats, T. 1999 AFLP analysis of genetic diversity within and between Arabidopsis thaliana ecotypes Mol. Gen. Genet. 26 627 634

    • Search Google Scholar
    • Export Citation
  • Buntjer, J.B., Osten, M., Nijman, I.J., Kuiper, M.T. & Lenstra, J.A. 2002 Phylogeny of bovine species based on AFLP fingerprinting Heredity 88 46 51

  • Burke, J.M., Bayly, M.J., Peter, B., Adams, P.B. & Ladiges, P.Y. 2008 Molecular phylogenetic analysis of Dendrobium (Orchidaceae), with emphasis on the Australian section Dendrocoryne, and implications for generic classification Aust. Syst. Bot. 21 1 14

    • Search Google Scholar
    • Export Citation
  • Chase, M. 2006 Royal Botanic Gardens, Kew Science & Horticulture. 11 Jan. 2013. <http://www.Orchidaceae.htm/>

  • Chaudhary, B., Chattopadhyay, P., Verma, N. & Banerjeem, N. 2012 Understanding the phylomorphological implications of Pollinia from Dendrobium (Orchidaceae) Amer. J. Plant Sci. 3 816 828

    • Search Google Scholar
    • Export Citation
  • Chen, S.C. & Tsi, Z.H. 2000 The orchards of China. 2nd Ed. The Chinese Forestry Press, Beijing, China

  • Chiang, C.H., Yu, T.A., Lo, S.F., Kuo, C.L., Peng, W.H. & Tsay, H.S. 2012 Molecular authentication of Dendrobium species by multiplex polymerase chain reaction and amplification refractory mutation system analysis J. Amer. Soc. Hort. Sci. 137 438 444

    • Search Google Scholar
    • Export Citation
  • Doyle, J.J. & Doyle, J.L. 1990 Isolation of plant DNA from fresh tissue Focus 12 13 15

  • Esfahani, S.T., Shiran, B. & Balali, G. 2009 AFLP markers for the assessment of genetic diversity in European and North American potato varieties cultivated in Iran CBAB 9 75 86

    • Search Google Scholar
    • Export Citation
  • Figueroa, C., Salazar, G.A., Zavaleta, A. & Engleman, M. 2008 Root character evolution and systematics in Cranichidinae, Prescottiinae and Spiranthinae (Orchidaceae, Cranichideae) Ann. Bot. (Lond.) 101 509 520

    • Search Google Scholar
    • Export Citation
  • Franco, J., Crossa, J., Ribaut, J.M., Betran, J., Warburton, M.L. & Khairallah, M. 2001 A method for combining molecular markers and phenotypic attributes for classifying plant genotypes Theor. Appl. Genet. 103 944 952

    • Search Google Scholar
    • Export Citation
  • Freudenstein, J.V. & Rasmussen, F.N. 1999 What does morphology tell us about orchid relationships?—A cladistic analysis Amer. J. Bot. 86 225 248

  • Fritz, A.K., Caldwell, S. & Worall, W.D. 1999 Molecular mapping of Russian wheat aphid resistance from triticale accession PI 386156 Crop Sci. 39 1707 1710

    • Search Google Scholar
    • Export Citation
  • Gu, S., Ding, X.Y., Wang, Y., Zhou, Q., Ding, G., Li, X.X. & Qian, L. 2007 Isolation and characterization of microsatellite markers in Dendrobium officinale, an endangered herb endemic to China Mol. Ecol. Notes 7 1166 1168

    • Search Google Scholar
    • Export Citation
  • Hammer, O., Harper, D.A.T. & Ryan, P.D. 2001 PAST: Paleontological statistics software package for education and data analysis Palaeontol. Electronica 4 9

    • Search Google Scholar
    • Export Citation
  • Hansen, M., Kraft, T., Christiansson, M. & Nilsson, N.O. 1999 Evaluation of AFLP in Beta Theor. Appl. Genet. 98 845 852

  • Jaccard, P. 1908 Nouvelles recherches sur la distribution florale Bull. Soc. Vaud. Sci. Nat. 44 223 270

  • Jones, C.J., Edward, K.J., Castaglione, S., Winfield, M.O., Sala, F., Van de Wiel, C., Bredemeijer, G., Vosman, B., Matthes, M., Daly, A., Brettschneider, R., Bettini, P., Buiatti, M., Maestri, E., Malcevschi, A., Marmiroli, N., Aert, R., Volckaert, G., Rueda, J., Linacero, R., Vazquez, A. & Karp, A. 1997 Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories Mol. Breed. 3 381 390

    • Search Google Scholar
    • Export Citation
  • Kim, M.S., Moore, P.H., Zee, F., Fitch, M.M., Steiger, D.L., Manshardt, R.M., Paull, R.E., Drew, R.A., Sekioka, T. & Ming, R. 2002 Genetic diversity of Carica papaya as revealed by AFLP markers Genome 45 503 512

    • Search Google Scholar
    • Export Citation
  • Leitch, I.J., Kahandawala, I., Suda, J., Hanson, L., Ingrouille, M.J., Chase, M.W. & Fay, M.F. 2009 Genome size diversity in orchids: Consequences and evolution Ann. Bot. (Lond.) 104 469 481

    • Search Google Scholar
    • Export Citation
  • Máric, S., Laríc, S., Artincic, J., Pejíc, I. & Kozumplink, V. 2004 Genetic diversity of hexaploid wheat cultivars estimated by RAPD markers, morphological traits and coefficients of parentage Plant Breed. 123 366 369

    • Search Google Scholar
    • Export Citation
  • Mueller, U.G. & Wolfenbarger, L.L. 1999 AFLP genotyping and fingerprinting Trends Ecol. Evol. 14 389 394

  • Nor Hazlina, M.S., Wahba, L.E., Fadelah, A. & Wickneswari, R. 2013 Genetic relationships among 81 Dendrobium accessions from Malaysia Malays. Appl. Biol. 42 35 40

    • Search Google Scholar
    • Export Citation
  • Prashanth, S.R., Parani, M., Mohanty, B.P., Talame, V., Tuberosa, R. & Parida, A. 2002 Genetic diversity in cultivars and landraces of Oryza sativa subsp. indica as revealed by AFLP markers Genome 45 451 459

    • Search Google Scholar
    • Export Citation
  • Pridgeon, A. 2000 The illustrated encyclopedia of orchids. Timber Press, Portland, OR

  • Quan, Z., Yongping, Z., Weiming, G., Weijun, L. & Guangdong, W. 2012 Assessment of genetic relationships among Spring Dendrobium cultivars and varietal materials using amplified fragment length polymorphism (AFLP) analysis Afr. J. Biotechnol. 11 14777 14785

    • Search Google Scholar
    • Export Citation
  • Renganayaki, K., Read, J.C. & Fritz, A.K. 2001 Genetic diversity among Texas bluegrass genotypes (Poa arachnifera Torr.) revealed by AFLP and RAPD markers Theor. Appl. Genet. 102 1037 1045

    • Search Google Scholar
    • Export Citation
  • Salazar, G.A. 2009 DNA, morphology, and systematics of Galeoglossum (Orchidaceae, Cranichidinae), p. 161–172. In: Pridgeon, A.M. and J.P. Suarez (eds.). Proc. of the Second Scientific Conference on Andean Orchids. Universidad Técnica Particular de Loja, Loja, Ecuador

  • Salazar, G.A., Cabrera, L.I., Santiago Madriñán, S. & Chase, M.W. 2009 Phylogenetic relationships of Cranichidinae and Prescottiinae (Orchidaceae, Cranichideae) inferred from plastid and nuclear DNA sequences Ann. Bot. (Lond.) 104 403 416

    • Search Google Scholar
    • Export Citation
  • Sheehan, T. & Sheehan, M. 1994 An illustrated survey of orchid genera. Timber Press, Inc., Portland, OR.

  • Shengnan, Z., Zhenhua, Z., Shangguo, F., Shang, W., Nana, S. & Huizhong, W. 2011 Analysis on the genetic diversity among 31 species of Dendrobium based on RAPD marker Journal of Hangzhou Normal University (Natural Science Edition) 4 333 339

    • Search Google Scholar
    • Export Citation
  • Stewart, J. & Griffiths, M. 1995 Manual of orchids. Timber Press, Portland, OR

  • Tang, T., Jian, Z.S. & Shi, S. 2003 Genetic diversity of Hibiscus tiliaceus (Malvaceae) in China assessed using AFLP markers Ann. Bot. (Lond.) 92 409 414

    • Search Google Scholar
    • Export Citation
  • Tsi, Z.H., Chen, S.C., Luo, Y.B. & Zhu, G.H. 1999 Orchidaceae (3). In: Tsi, Z.H. (ed.). Angiospermae, Monocotyledoneae, Flora Reipublicae Popularis Sinicae. Vol. 19. Science Press, Beijing, China

  • Vieira, E.A., Félix de Carvalho, F.I., Bertan, I., Kopp, M.M., Zimmer, P.D., Benin, G., Gonzalez da Silva, J.A., Hartwig, I., Malone, G. & Costa de Oliveira, A. 2007 Association between genetic distances in wheat (Triticum aestivum L.) as estimated by AFLP and morphological markers Genet. Mol. Biol. 30 392 399

    • Search Google Scholar
    • Export Citation
  • Vos, P., Hogers, R., Bleeker, M., Reijans, M., Vandelee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. & Zabeau, M. 1995 AFLP: A new technique for DNA fingerprinting Nucleic Acids Res. 23 4407 4414

    • Search Google Scholar
    • Export Citation
  • Wang, H.Z., Feng, S.G., Lu, J.J., Shi, N.N. & Liu, J.J. 2009 Phylogenetic study and molecular identification of 31 Dendrobium species using inter-simple sequence repeat (ISSR) markers Sci. Hort. 122 440 447

    • Search Google Scholar
    • Export Citation
  • Xiang, N., Hong, Y. & Lam-Chan, L.T. 2003 Genetic analysis of tropical orchid hybrids (Dendrobium) with fluorescence amplified fragment length polymorphism (AFLP) J. Amer. Soc. Hort. Sci. 128 731 735

    • Search Google Scholar
    • Export Citation
  • Yin, B., Ying-hua, B., Wen-quan, W., Li-li, J. & Yu-ning, Y. 2007 Analysis of the phylogenetic relationship of Dendrobium in China by AFLP technique Acta Horticulturae Sinica. 34 1569 1574

    • Search Google Scholar
    • Export Citation
  • Yue, G.H., Lam-Chan, L.T. & Hong, Y. 2006 Development of simple sequence repeat (SSR) markers and their use in identification of Dendrobium varieties Mol. Ecol. Notes 6 832 834

    • Search Google Scholar
    • Export Citation
  • Zhu, G.F. & Li, D.M. 2011 Genetic relationships among native species and hybrid cultivars of Asian Dendrobium (Orchidaceae) using amplified fragment length polymorphism markers HortScience 46 192 196

    • Search Google Scholar
    • Export Citation
  • Zhu, J., Gale, M.D., Quarrie, S., Jackson, M.T. & Bryan, G.J. 1998 AFLP markers for the study of rice biodiversity Theor. Appl. Genet. 96 602 611

Supplemental Table 1.

Morphological Character Data for 81 Dendrobium accessions based on 21 traits.

Supplemental Table 1.Supplemental Table 1.Supplemental Table 1.

If the inline PDF is not rendering correctly, you can download the PDF file here.

Contributor Notes

To whom reprint requests should be addressed; e-mail wicki@ukm.my.

  • View in gallery

    Dendrogram for the Dendrobium accessions generated by unweighted pair group method with arithmetic averages (UPGMA) analysis of the 81 Dendrobium accessions with the 21 morphological characters.

  • View in gallery

    Dendrogram for the 81 Dendrobium accessions generated by a unweighted pair group method with arithmetic averages (UPGMA) analysis of the amplified fragment length polymorphism (AFLP) data.

  • View in gallery

    Dendrogram for 81 Dendrobium accessions generated by unweighted pair group method with arithmetic averages (UPGMA) analysis for the combined analysis of the amplified fragment length polymorphism (AFLP) and morphological data.

  • Albert, V.A. 1994 Cladistic relationships of the slipper orchids (Cypripedioideae: Orchidaceae) from congruent morphological and molecular data Lindleyana 9 115 132

    • Search Google Scholar
    • Export Citation
  • Albertson, R.C., Markert, J.A., Danley, P.D. & Kocher, T.D. 1999 Phylogeny of a rapidly evolving clade: The cichid fishes of lake Malawi, East Africa Proc. Natl. Acad. Sci. USA 96 5107 5110

    • Search Google Scholar
    • Export Citation
  • Bagley, M.J., Anderson, S.L. & May, B. 2001 Choice of methodology for assessing genetic impact of environmental stressors: Polymorphism and reproducibility of RAPD and AFLP fingerprints Ecotoxicol. 10 239 244

    • Search Google Scholar
    • Export Citation
  • Bechtel, H., Cribb, P. & Launert, E. 1992 The manual of cultivated orchid species. 3rd Ed. Blandford Press, London, UK

  • Breyne, P., Rombaut, D., Van Gysel, A., Van Mantagu, M. & Gerats, T. 1999 AFLP analysis of genetic diversity within and between Arabidopsis thaliana ecotypes Mol. Gen. Genet. 26 627 634

    • Search Google Scholar
    • Export Citation
  • Buntjer, J.B., Osten, M., Nijman, I.J., Kuiper, M.T. & Lenstra, J.A. 2002 Phylogeny of bovine species based on AFLP fingerprinting Heredity 88 46 51

  • Burke, J.M., Bayly, M.J., Peter, B., Adams, P.B. & Ladiges, P.Y. 2008 Molecular phylogenetic analysis of Dendrobium (Orchidaceae), with emphasis on the Australian section Dendrocoryne, and implications for generic classification Aust. Syst. Bot. 21 1 14

    • Search Google Scholar
    • Export Citation
  • Chase, M. 2006 Royal Botanic Gardens, Kew Science & Horticulture. 11 Jan. 2013. <http://www.Orchidaceae.htm/>

  • Chaudhary, B., Chattopadhyay, P., Verma, N. & Banerjeem, N. 2012 Understanding the phylomorphological implications of Pollinia from Dendrobium (Orchidaceae) Amer. J. Plant Sci. 3 816 828

    • Search Google Scholar
    • Export Citation
  • Chen, S.C. & Tsi, Z.H. 2000 The orchards of China. 2nd Ed. The Chinese Forestry Press, Beijing, China

  • Chiang, C.H., Yu, T.A., Lo, S.F., Kuo, C.L., Peng, W.H. & Tsay, H.S. 2012 Molecular authentication of Dendrobium species by multiplex polymerase chain reaction and amplification refractory mutation system analysis J. Amer. Soc. Hort. Sci. 137 438 444

    • Search Google Scholar
    • Export Citation
  • Doyle, J.J. & Doyle, J.L. 1990 Isolation of plant DNA from fresh tissue Focus 12 13 15

  • Esfahani, S.T., Shiran, B. & Balali, G. 2009 AFLP markers for the assessment of genetic diversity in European and North American potato varieties cultivated in Iran CBAB 9 75 86

    • Search Google Scholar
    • Export Citation
  • Figueroa, C., Salazar, G.A., Zavaleta, A. & Engleman, M. 2008 Root character evolution and systematics in Cranichidinae, Prescottiinae and Spiranthinae (Orchidaceae, Cranichideae) Ann. Bot. (Lond.) 101 509 520

    • Search Google Scholar
    • Export Citation
  • Franco, J., Crossa, J., Ribaut, J.M., Betran, J., Warburton, M.L. & Khairallah, M. 2001 A method for combining molecular markers and phenotypic attributes for classifying plant genotypes Theor. Appl. Genet. 103 944 952

    • Search Google Scholar
    • Export Citation
  • Freudenstein, J.V. & Rasmussen, F.N. 1999 What does morphology tell us about orchid relationships?—A cladistic analysis Amer. J. Bot. 86 225 248

  • Fritz, A.K., Caldwell, S. & Worall, W.D. 1999 Molecular mapping of Russian wheat aphid resistance from triticale accession PI 386156 Crop Sci. 39 1707 1710

    • Search Google Scholar
    • Export Citation
  • Gu, S., Ding, X.Y., Wang, Y., Zhou, Q., Ding, G., Li, X.X. & Qian, L. 2007 Isolation and characterization of microsatellite markers in Dendrobium officinale, an endangered herb endemic to China Mol. Ecol. Notes 7 1166 1168

    • Search Google Scholar
    • Export Citation
  • Hammer, O., Harper, D.A.T. & Ryan, P.D. 2001 PAST: Paleontological statistics software package for education and data analysis Palaeontol. Electronica 4 9

    • Search Google Scholar
    • Export Citation
  • Hansen, M., Kraft, T., Christiansson, M. & Nilsson, N.O. 1999 Evaluation of AFLP in Beta Theor. Appl. Genet. 98 845 852

  • Jaccard, P. 1908 Nouvelles recherches sur la distribution florale Bull. Soc. Vaud. Sci. Nat. 44 223 270

  • Jones, C.J., Edward, K.J., Castaglione, S., Winfield, M.O., Sala, F., Van de Wiel, C., Bredemeijer, G., Vosman, B., Matthes, M., Daly, A., Brettschneider, R., Bettini, P., Buiatti, M., Maestri, E., Malcevschi, A., Marmiroli, N., Aert, R., Volckaert, G., Rueda, J., Linacero, R., Vazquez, A. & Karp, A. 1997 Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories Mol. Breed. 3 381 390

    • Search Google Scholar
    • Export Citation
  • Kim, M.S., Moore, P.H., Zee, F., Fitch, M.M., Steiger, D.L., Manshardt, R.M., Paull, R.E., Drew, R.A., Sekioka, T. & Ming, R. 2002 Genetic diversity of Carica papaya as revealed by AFLP markers Genome 45 503 512

    • Search Google Scholar
    • Export Citation
  • Leitch, I.J., Kahandawala, I., Suda, J., Hanson, L., Ingrouille, M.J., Chase, M.W. & Fay, M.F. 2009 Genome size diversity in orchids: Consequences and evolution Ann. Bot. (Lond.) 104 469 481

    • Search Google Scholar
    • Export Citation
  • Máric, S., Laríc, S., Artincic, J., Pejíc, I. & Kozumplink, V. 2004 Genetic diversity of hexaploid wheat cultivars estimated by RAPD markers, morphological traits and coefficients of parentage Plant Breed. 123 366 369

    • Search Google Scholar
    • Export Citation
  • Mueller, U.G. & Wolfenbarger, L.L. 1999 AFLP genotyping and fingerprinting Trends Ecol. Evol. 14 389 394

  • Nor Hazlina, M.S., Wahba, L.E., Fadelah, A. & Wickneswari, R. 2013 Genetic relationships among 81 Dendrobium accessions from Malaysia Malays. Appl. Biol. 42 35 40

    • Search Google Scholar
    • Export Citation
  • Prashanth, S.R., Parani, M., Mohanty, B.P., Talame, V., Tuberosa, R. & Parida, A. 2002 Genetic diversity in cultivars and landraces of Oryza sativa subsp. indica as revealed by AFLP markers Genome 45 451 459

    • Search Google Scholar
    • Export Citation
  • Pridgeon, A. 2000 The illustrated encyclopedia of orchids. Timber Press, Portland, OR

  • Quan, Z., Yongping, Z., Weiming, G., Weijun, L. & Guangdong, W. 2012 Assessment of genetic relationships among Spring Dendrobium cultivars and varietal materials using amplified fragment length polymorphism (AFLP) analysis Afr. J. Biotechnol. 11 14777 14785

    • Search Google Scholar
    • Export Citation
  • Renganayaki, K., Read, J.C. & Fritz, A.K. 2001 Genetic diversity among Texas bluegrass genotypes (Poa arachnifera Torr.) revealed by AFLP and RAPD markers Theor. Appl. Genet. 102 1037 1045

    • Search Google Scholar
    • Export Citation
  • Salazar, G.A. 2009 DNA, morphology, and systematics of Galeoglossum (Orchidaceae, Cranichidinae), p. 161–172. In: Pridgeon, A.M. and J.P. Suarez (eds.). Proc. of the Second Scientific Conference on Andean Orchids. Universidad Técnica Particular de Loja, Loja, Ecuador

  • Salazar, G.A., Cabrera, L.I., Santiago Madriñán, S. & Chase, M.W. 2009 Phylogenetic relationships of Cranichidinae and Prescottiinae (Orchidaceae, Cranichideae) inferred from plastid and nuclear DNA sequences Ann. Bot. (Lond.) 104 403 416

    • Search Google Scholar
    • Export Citation
  • Sheehan, T. & Sheehan, M. 1994 An illustrated survey of orchid genera. Timber Press, Inc., Portland, OR.

  • Shengnan, Z., Zhenhua, Z., Shangguo, F., Shang, W., Nana, S. & Huizhong, W. 2011 Analysis on the genetic diversity among 31 species of Dendrobium based on RAPD marker Journal of Hangzhou Normal University (Natural Science Edition) 4 333 339

    • Search Google Scholar
    • Export Citation
  • Stewart, J. & Griffiths, M. 1995 Manual of orchids. Timber Press, Portland, OR

  • Tang, T., Jian, Z.S. & Shi, S. 2003 Genetic diversity of Hibiscus tiliaceus (Malvaceae) in China assessed using AFLP markers Ann. Bot. (Lond.) 92 409 414

    • Search Google Scholar
    • Export Citation
  • Tsi, Z.H., Chen, S.C., Luo, Y.B. & Zhu, G.H. 1999 Orchidaceae (3). In: Tsi, Z.H. (ed.). Angiospermae, Monocotyledoneae, Flora Reipublicae Popularis Sinicae. Vol. 19. Science Press, Beijing, China

  • Vieira, E.A., Félix de Carvalho, F.I., Bertan, I., Kopp, M.M., Zimmer, P.D., Benin, G., Gonzalez da Silva, J.A., Hartwig, I., Malone, G. & Costa de Oliveira, A. 2007 Association between genetic distances in wheat (Triticum aestivum L.) as estimated by AFLP and morphological markers Genet. Mol. Biol. 30 392 399

    • Search Google Scholar
    • Export Citation
  • Vos, P., Hogers, R., Bleeker, M., Reijans, M., Vandelee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. & Zabeau, M. 1995 AFLP: A new technique for DNA fingerprinting Nucleic Acids Res. 23 4407 4414

    • Search Google Scholar
    • Export Citation
  • Wang, H.Z., Feng, S.G., Lu, J.J., Shi, N.N. & Liu, J.J. 2009 Phylogenetic study and molecular identification of 31 Dendrobium species using inter-simple sequence repeat (ISSR) markers Sci. Hort. 122 440 447

    • Search Google Scholar
    • Export Citation
  • Xiang, N., Hong, Y. & Lam-Chan, L.T. 2003 Genetic analysis of tropical orchid hybrids (Dendrobium) with fluorescence amplified fragment length polymorphism (AFLP) J. Amer. Soc. Hort. Sci. 128 731 735

    • Search Google Scholar
    • Export Citation
  • Yin, B., Ying-hua, B., Wen-quan, W., Li-li, J. & Yu-ning, Y. 2007 Analysis of the phylogenetic relationship of Dendrobium in China by AFLP technique Acta Horticulturae Sinica. 34 1569 1574

    • Search Google Scholar
    • Export Citation
  • Yue, G.H., Lam-Chan, L.T. & Hong, Y. 2006 Development of simple sequence repeat (SSR) markers and their use in identification of Dendrobium varieties Mol. Ecol. Notes 6 832 834

    • Search Google Scholar
    • Export Citation
  • Zhu, G.F. & Li, D.M. 2011 Genetic relationships among native species and hybrid cultivars of Asian Dendrobium (Orchidaceae) using amplified fragment length polymorphism markers HortScience 46 192 196

    • Search Google Scholar
    • Export Citation
  • Zhu, J., Gale, M.D., Quarrie, S., Jackson, M.T. & Bryan, G.J. 1998 AFLP markers for the study of rice biodiversity Theor. Appl. Genet. 96 602 611

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