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The popular hybrid orchid Vanda Miss Joaquim was made Singapore's national flower in 1981. It was originally described in the Gardeners’ Chronicle in 1893, as a cross between Vanda hookeriana and Vanda teres. However, no record had been kept as to which parent contributed the pollen. This study was conducted using DNA barcoding techniques to determine the pod parent of V. Miss Joaquim, thereby inferring the pollen parent of the hybrid by exclusion.
Two chloroplast genes, matK and rbcL, from five related taxa, V. hookeriana, V. teres var. alba, V. teres var. andersonii, V. teres var. aurorea and V. Miss Joaquim ‘Agnes’, were sequenced. The matK gene from herbarium specimens of V. teres and V. Miss Joaquim, both collected in 1893, was also sequenced.
No sequence variation was found in the 600-bp region of rbcL sequenced. Sequence variation was found in the matK gene of V. hookeriana, V. teres var. alba, V. teres var. aurorea and V. Miss Joaquim ‘Agnes’. Complete sequence identity was established between V. teres var. andersonii and V. Miss Joaquim ‘Agnes’. The matK sequences obtained from the herbarium specimens of V. teres and V. Miss Joaquim were completely identical to the sequences obtained from the fresh samples of V. teres var. andersonii and V. Miss Joaquim ‘Agnes’.
The pod parent of V. Miss Joaquim ‘Agnes’ is V. teres var. andersonii and, by exclusion, the pollen parent is V. hookeriana. The herbarium and fresh samples of V. teres var. andersonii and V. Miss Joaquim share the same inferred maternity. The matK gene was more informative than rbcL and facilitated differentiation of varieties of V. teres.
In angiosperms, plastid genes are generally transmitted through the maternal line (Birky 1994; Mogensen 1996). By sequencing plastid genes, maternal identification is possible. DNA barcoding is an initiative aimed at giving a genetic identity to all organisms, and is at present most commonly applied to animals and plants. For animals, the mitochondrial gene, cytochrome c oxidase I (COI), has proven to be sufficiently informative (Hebert et al. 2003). However, due to the lower mitochondrial substitution rate in plants (cited from Newmaster et al. 2006), other plastid gene regions have had to be evaluated for use—these include atpF-atpH spacer, matK, rbcL, rpoB, rpoC1, psbK-psbI spacer and trnK-psbA spacer (CBOL Plant Working Group 2009). The Consortium for the Barcode of Life (CBOL) has recommended a combination of rbcL and matK for DNA barcoding of plants (CBOL Plant Working Group 2009). It is important to note that DNA barcoding is not equivalent to molecular taxonomy (Newmaster et al. 2006). The purpose of DNA barcoding is primarily for the expedient identification of new species (Chase et al. 2005; Newmaster et al. 2006). Even so, the chloroplast genes rbcL and matK have been used in many phylogenetic studies across plant families and in the Orchidaceae (Cameron et al. 1999; Goldman et al. 2001; Hidayat et al. 2005; Barrett and Freudenstein 2008; Freudenstein and Senyo 2008; Cameron 2009; CBOL Plant Working Group 2009).
The chloroplast gene rbcL encodes ribulose-1,5-bisphosphate carboxylase oxygenase. It is ~1.4 kb in length. Another chloroplast gene, matK, is located within the trnK intron. This 1.5-kb gene encodes a maturase required for chloroplast activities such as photosynthesis (Barthet and Hilu 2007). The matK region has been shown to have a significantly faster substitution rate than rbcL—up to 3-fold faster at the nucleotide level and 6-fold at the amino acid level (Johnson and Soltis 1994; Olmstead and Palmer 1994; Xiang et al. 1998; Muller et al. 2006). matK is considered to be one of the most informative loci for determining phylogenetic relationships (Hilu et al. 2003).
The precise identification of an orchid hybrid's parentage is of utmost importance to botanists, breeders and enthusiasts alike. The parental characteristics give an indication of a hybrid's growth requirements, flowering habit and other phenotypic aspects. There are occasional instances when the parentage of a particular orchid hybrid is disputed. Some examples include Vanda Tan Chay Yan, Vanda Mimi Palmer (Chew 2008) and the more exalted Vanda Miss Joaquim, Singapore's national flower. We have chosen V. Miss Joaquim as our case study to evaluate the feasibility of using DNA barcoding for the identification of the maternal parent of an orchid hybrid. Although some Vanda species have been assigned to the genus Papilionanthe (Tay 2004), we have chosen to use Vanda for ease of reference.
The hybrid V. Miss Joaquim arose in the garden of Miss Agnes Joaquim, an Armenian lady residing in Singapore during the late 19th to early 20th century. Vanda Miss Joaquim is the result of a cross between Vanda hookeriana and Vanda teres. Unfortunately, there was no record of which plant contributed the pollen (Ridley 1893). Nevertheless, H. N. Ridley, then Director of the Singapore Botanic Gardens, described the hybrid in the Gardeners’ Chronicle in 1893—the Royal Horticultural Society records V. hookeriana as the seed parent and V. teres as the pollen parent. In herbarium records at the Singapore Botanic Gardens, the V. Miss Joaquim specimen, filed by Ridley in 1893, is recorded as Vanda Agnes Joaquim and its parentage is described as (V. teres×V. hookeriana).
In the ensuing years, culture of the plant spread beyond the shores of Singapore, first to Malaysia, then to Hawaii (Yeoh 1963b; Alphonso 1981). Vanda Miss Joaquim was selected as Singapore's national flower on 15 April 1981 (Alphonso 1981). It is the only hybrid orchid designated as a national flower. Vanda Miss Joaquim is also the first orchid hybrid from Singapore to be registered and the first hybrid Vanda in the world to have been registered with the Royal Horticultural Society (Chew 2007).
Vanda teres is native to Burma (Myanmar) and its distribution extends to northeastern India and the Himalayas (Veitch and Sons 1893; Holttum 1953). In its natural habitat, it is ‘found in the hot plains and valleys scrambling over the branches of the largest trees and exposed to the full glare of the sun’ (Veitch and Sons 1893). In his Orchids of Malaya, Holttum (1953) described four varieties of V. teres: var. gigantea, var. aurorea, var. andersoni (also spelt andersonii) and var. alba. In this study, we used three varieties of V. teres: var. aurorea, var. andersonii and var. alba. It is not known which variety of V. teres gave rise to V. Miss Joaquim (Alphonso 1981); however, V. teres var. aurorea and V. teres var. andersonii are more common (Yam 1999).
Vanda hookeriana is a Malaysian species found in wet or swampy sites in Johore and the Kinta Valley in Malaysia (Yeoh 1963a; Dourado 1979). It is also known as the Kinta Weed (Yeoh 1963a; Dourado 1979). Vanda hookeriana requires more shade and moisture than other similar species and, in its native habitat, can be found scrambling between small shrubs and bushes (Yeoh 1963a). There are two varieties of V. hookeriana: the coloured variety and the alba type. We were unable to obtain the alba variety for this study as it is extremely rare in the wild (Laycock 1931; Dourado 1979) and may have well disappeared from cultivation (Tay 2004).
Vanda Miss Joaquim is a vigorous grower and flowers freely year round. Its leaves have inherited the characteristic kink from V. hookeriana but are, on the whole, intermediate between V. hookeriana and V. teres (Ridley 1893). Additional varieties of V. Miss Joaquim include ‘Rose Marie’, ‘Douglas’ (Teoh 1981) and ‘Singapore Botanic Gardens’ (Teoh 1981). In this article, we deal specifically with the parentage of the ‘Agnes’ variety, which is the varietal name retrospectively given to the original V. Miss Joaquim (Alphonso 1981; Teoh 1981). It has been speculated that, based on flower morphology, the variety of V. teres that gave rise to V. Miss Joaquim is var. aurorea (Yam 1999). The coloured variety of V. hookeriana has been suggested as the other parent of V. Miss Joaquim (Yam 1999).
In this study, we set out to resolve the issue of parentage by tracing the maternal line using DNA sequencing methods. We report here the results obtained by sequencing the rbcL and matK genes from V. hookeriana, V. teres and V. Miss Joaquim. We also sequenced the matK gene of the original herbarium specimens of V. Miss Joaquim collected by H. N. Ridley in 1893 and V. teres collected by Miss Joaquim in 1893.
Fresh samples were collected from the Singapore Botanic Gardens and the National Institute of Education: V. Miss Joaquim ‘Agnes’, V. hookeriana, V. teres var. alba, V. teres var. andersonii and V. teres possibly var. aurorea. Only the alba variety was in flower when we collected the specimens, and therefore our identification of the other two varieties is based first on the fact that the V. teres var. andersonii was so labelled at the Singapore Botanic Gardens and, second, on Holttum's description (Holttum 1953) that the leaves of V. teres var. aurorea are ‘green, not spotted’ and the leaves of V. teres var. andersonii are ‘green, curved upwards towards stem’. Additional fresh samples were obtained from J. B. Tay—these comprise two subvarieties of V. teres var. alba, two subvarieties of V. teres var. andersonii and one specimen of V. Miss Joaquim ‘Agnes’.
Herbarium samples were obtained from the SING Herbarium at the Singapore Botanic Gardens: V. Miss Joaquim from 1893, collector H. N. Ridley, V. teres from 1893, collector Miss Joaqium, V. hookeriana collected from Kampar Swamp, Perak, Malaysia, in 1895 by Charles Curtis, and that collected from Kinta Valley, Malaysia, in 1889 by E. Brewster.
All plant samples were deposited at the SING Herbarium and given specimen voucher numbers SING 2010-760 (V. teres var. andersonii), SING 2011-014 (V. Miss Joaquim ‘Agnes’), 2011-015 (V. teres var. aurorea), SING 2011-016 (V. teres var. alba) and 2011-017 (V. hookeriana).
The Finnzymes Phire Plant Direct PCR kit was used for all polymerase chain reactions (PCR). Plant samples were crushed in the dilution buffer provided with the Phire Plant Direct PCR kit. For fresh plant samples, PCR conditions were as follows: denaturation temperature 98 °C, annealing temperature 55 °C for rbcL1F/rbcLaR, matK56F/matK1027R and matK56F/matK1520R primer pairs, and extension at 72 °C, for a total of 40 cycles. For herbarium specimens, the nested PCR technique was employed, as previously described (Sambrook and Russell 2001). Primers matK-19F and matK1520R were used for the first-round nested PCR with denaturation and extension temperatures as described above. Primer annealing was done at 45 °C and the PCR was cycled 50 times. Second-round nested PCR was done using primers matK56F and matK1027R and cycling conditions were as described above for the primer pair. Primers used for amplification of rbcL: rbcL1F 5′-ATG TCA CCA CAA ACA GAA ACT AAA GC-3′ (Molvray et al. 2000) and rbcLaR 5′-CTT CTG CTA CAA ATA AGA ATC GAT CTC-3′ (Kress et al. 2005; Kress and Erickson 2007; Fazekas et al. 2008) (Fig. 1). Primers used for amplification of matK: matK-19F 5′-CGT TCT GAC CAT ATT GCA CTA TG-3′ (Molvray et al. 2000; Goldman et al. 2001; Pridgeon et al. 2001; Salazar et al. 2003; Whitten et al. 2007; van den Berg et al. 2009), matK56F 5′-GGC AAC AAA ACT TCC TAT ATC C-3′, matK743F 5′-CTT CTG GAG TCT TTC TTG AGC-3′, matK1520R 5′-CGG ATA ATG TCC AAA TAC CAA ATA-3′ (Whitten et al. 2000; Williams et al. 2001; Chase et al. 2009; Neubig et al. 2009), matK1027R 5′-CCA GAA AAG AGA AGG AAT AAT TGG-3′ and matK336R 5′-GAC TCC AAA ACC TTC TGA TAC C-3′ (Fig. 1). PCR products were purified using Wizard® PCR and Gel Clean-up System (Promega Corporation, Madison, WI, USA), according to the manufacturer's recommendations.
Purified PCR products were sent to AITBiotech Pte Ltd, Singapore, for sequencing. All samples were sequenced bi-directionally. Sequencing for rbcL was done in both directions using the rbcL1F and rbcLaR primers. Sequencing for matK was also done bi-directionally using primers matK56F and matK1027R. To obtain complete sequence coverage of the matK amplification product, the primers matK743F and matK336R were designed and used for sequencing. The larger matK fragment amplified using the primers matK56F and matK1520R was sequenced using primers matK56F, matK1027R and matK1520R.
Sequence data were analysed using Lasergene SeqMan software from DNASTAR, Inc., Madison, WI, USA. Nucleotide and amino acid sequence alignments were performed using the ClustalW algorithm (Thompson et al. 1994) provided by the Lasergene MegAlign software from DNASTAR, Inc., Madison, WI, USA. DNA bases showing similarity or identity were highlighted using the BoxShade software available on http://mobyle.pasteur.fr/cgi-bin/portal.py?form=boxshade.
We amplified and sequenced the rbcL gene from all five fresh plant samples (see Materials and methods). Polymerase chain reaction for each sample was done in duplicate and each PCR product was sequenced in both directions using primers rbcL1F and rbcLaR. As the amplification product was ~600-bp, no internal primers were necessary to obtain a full sequence in either direction. Replicate sequences for each sample were aligned and a consensus sequence was obtained. An alignment of consensus nucleotide sequences from all samples was done and no nucleotide variation was observed (Fig. 2). As no base variation was observed, a third replicate for PCR and sequencing was not necessary. This was also the reason why PCR of the rbcL gene from the herbarium specimens was not attempted.
Amplification of the matK gene from the five fresh plant samples was done in triplicate. For the herbarium specimens (Fig. 3), the nested PCR technique was employed and replicate amplification experiments were attempted. Repeated attempts at amplifying matK from all herbarium material for V. hookeriana failed. Only one successful nested PCR product was obtained for V. Miss Joaquim. For V. teres, two separate PCR reactions using primers matK-19F and matK1520R were successful and these were re-amplified using the internal nested primers matK56F and matK1027R. A total of three amplification products were finally obtained. All amplification products were sequenced in both directions using primers matK56F and matK1027R. As the amplification product was 1-kb long, internal primers matK743F and matK336R were used to obtain complete sequence coverage of the products. Replicate sequences from each sample were aligned to obtain a consensus nucleotide sequence, and these consensus sequences were used in the final alignment (Fig. 4). The nucleotide sequence alignment (Fig. 4) reveals base variations at 10 sites within the 1-kb sequence length aligned. The alignment was trimmed to a 720-bp region (Fig. 4) within which the 10 base variations are clustered. Codon positions of the base variants are summarized in Table 1. Translations of the nucleotide sequences were performed in silico and the predicted amino acid sequences were aligned (Fig. 5). The amino acid sequence alignment indicates that the variable nucleotides translate into sequence substitutions at the amino acid level (Fig. 5). From the nucleotide and protein sequence alignments, it is apparent that the sequence of V. teres var. andersonii is identical to the sequence of V. Miss Joaquim. In addition, the matK sequence of the fresh sample of V. Miss Joaquim is identical to that of the herbarium specimens for V. Miss Joaquim and V. teres. The maximum number of nucleotide base variations between samples is seven base substitutions between V. hookeriana and V. teres var. andersonii.
To obtain a better sampling of specimens, additional fresh samples of V. teres var. alba, V. teres var. andersonii and V. Miss Joaquim ‘Agnes’, along with our existing samples of V. hookeriana, V. teres var. alba and V. teres var. aurora, were amplified over an ~1.5-kb extended region demarcated by primers matK56F (this study) and matK1520R (Whitten et al. 2000; Williams et al. 2001; Chase et al. 2009; Neubig et al. 2009). This region encompasses the standard barcode regions defined by the 1R_KIM and 3F_KIM primers (Fazekas et al. 2008), and the matK390F and matK1326R primers (Cuénoud et al. 2002). Amplification products were sequenced once using the primers matK56F, matK1027R and matK1520R (Fig. 1). The three sequences obtained for each amplification product were assembled to form contigs. These contigs were then aligned to give an interspecific matK alignment (Fig. 6). Additional variation was observed only for V. hookeriana and V. teres var. alba in this extended amplification product. The results obtained confirm our earlier finding that the matK sequence of V. teres var. andersonii is identical to that of V. Miss Joaquim ‘Agnes’.
During this study, we only observed flowering of the V. Miss Joaquim (Fig. 7E) and V. teres var. alba (Fig. 7B) plants. Vanda hookeriana, V. teres var. andersonii and V. teres var. aurorea did not flower. In order to identify the plants, we relied heavily on leaf morphology (Fig. 7) and the plant labelling of V. hookeriana and V. teres var. andersonii at the Singapore Botanic Gardens. Identification of V. hookeriana (Fig. 7A) was straightforward as it unmistakably possessed the characteristic leaf kink. Vanda hookeriana stem and leaves were also clearly less stout than in V. teres, a growth form that seems less vigorous. Our V. teres var. andersonii (Fig. 7C) has leaves that are curved towards the stem, matching Holttum's description (Holttum 1953). In addition, it possesses purple spots, which although not mentioned by Holttum to be present in V. teres var. andersonii, is specifically mentioned as being absent from the variety aurorea (Holttum 1953). Thus, our identification of V. teres var. aurorea (Fig. 7D) relies on the absence of spots; the slight curvature of the leaves towards the stem is less pronounced than that in V. teres var. andersonii; and it is the other most common variety besides V. teres var. andersonii. Other V. teres varieties are unlikely as V. teres var. gigantea was mentioned by Holttum as having ‘straight purple-spotted leaves’ (Holttum 1953).
We were unable to find any sequence variation in the 600-bp region of the rbcL gene sequenced. This finding is in accordance with many other studies that have shown that rbcL has a slower rate of nucleotide substitution compared with other loci used for phylogenetic analyses. It is possible that sequencing a larger fragment of rbcL may reveal some sequence variations. We found matK to be a very useful locus for delineating the relationships between the plants studied. Sequence information obtained from matK alone facilitated the identification of the pod parent of V. Miss Joaquim. The matK sequences alone were sufficient to differentiate V. teres to the variety level. It is interesting to note that V. teres var. alba has a sequence that is more similar to V. hookeriana than any of the other V. teres varieties (Fig. 4 and Table 1). Vanda teres var. alba differs from V. hookeriana by three bases and from the other two V. teres varieties by seven bases. Only one base substitution distinguishes V. teres var. aurorea from V. teres var. andersonii (Fig. 4 and Table 1). We found that the nucleotide substitution rate throughout all three codon positions in matK was fairly consistent, with a frequency of 0.4 for codon 1 and 0.3 for positions 2 and 3 (Table 1). Despite our small data set, our finding is in agreement with other reports that the substitution rate is comparable between all codon positions (Johnson and Soltis 1994; Hilu and Liang 1997; Xiang et al. 1998; Hilu et al. 2003; Barthet and Hilu 2008). The high rate of amino acid substitution in matK seen in our study has also been noted in previous studies (Olmstead and Palmer 1994; Barthet and Hilu 2008).
The sample size of this study was restricted by the difficulty in finding plants in bloom or that were clearly labelled. In particular, while it was easy to find plants labelled V. teres, only two plants were found labelled to the level of variety—these were both labelled V. teres var. andersonii. Additional samples of V. hookeriana, V. teres var. alba, V. teres var. andersonii and V. Miss Joaquim ‘Agnes’ were obtained to address sampling effects. Amplification and sequencing of the extended 1.5-kb fragment of the matK gene, encompassing the standard barcode regions, from these additional plant samples and our existing specimens confirmed our earlier conclusions derived using the matK56F and matK1027R primers. We therefore conclude that the sequences obtained using the matK56F and matK1027R primers were indicative of the variation observed within the standard barcode regions defined by the 1R_KIM, 3R_KIM, matK390F and matK1326R primers.
The herbarium specimens served to confirm that V. teres was the pod parent of the original V. Miss Joaquim described in 1893. We have shown that the fresh sample of V. teres var. andersonii is identical in sequence to the herbarium specimen. In addition, the fresh sample of V. Miss Joaquim ‘Agnes’ is also identical in sequence to the herbarium specimen of V. Miss Joaquim from 1893. This suggests that the contemporary and herbarium samples of V. Miss Joaquim ‘Agnes’ share the same inferred maternal parent. The flowers of the present-day V. Miss Joaquim remain morphologically similar to that described by Ridley (1893) and depicted by the Royal Horticultural Society in 1897 (Royal Horticultural Society Orchid Committee 1897; Royal Horticultural Society 2010) (Fig. 8).
Identification of plants can be challenging, if not impossible, when labels are lost or destroyed. We have shown that matK can be used to identify and distinguish terete Vanda species and varieties. In orchid hybridization, records of parentage are occasionally lost, ambiguous or erroneous. Due to the complexity of many orchid hybrids, sequencing nuclear genes could potentially be a long and difficult process. We have shown that DNA barcoding using the chloroplast gene matK is a useful technique for identification and/or confirmation of parentage, particularly when the maternal parent is disputed. We have shown that the direct PCR technique can be used to obtain amplification products from herbarium specimens. Due to the lack of high-molecular-weight DNA, the nested PCR technique may be employed to produce a higher yield, yet specific amplification product.
In conclusion, we have answered a 118-year-old question of the pod parent of Singapore's national flower, V. Miss Joaquim. We have shown through DNA amplification and sequencing of the original 1893 herbarium specimens and present-day plants that the pod parent of V. Miss Joaquim is V. teres var. andersonii while the pollen parent, by exclusion, is V. hookeriana. In the course of our investigation, it was shown that V. teres var. andersonii and V. teres var. aurorea differed in their matK sequence by one nucleotide. The rbcL sequence of the various Vanda specimens did not show any sequence variation. This result concurs with observations in Leguminosae (Lavin et al. 2005) that the matK gene is suitable for orchid barcoding and parentage determination as it gives a much greater resolution at the infrafamilial level.
Further work can be done to confirm the role of V. hookeriana as the pollen parent of V. Miss Joaquim by using random amplification of polymorphic DNA. Additional specimens of V. Miss Joaquim and the various V. teres varieties can also be sequenced to minimize the probability of biased results due to sampling effects. The option of sequencing the entire chloroplast genome (Nock et al. 2011) for better resolution can also be considered, now that next-generation sequencing methods have made whole-genome sequencing much more cost effective.
All sequences obtained from fresh plant specimens have been deposited in the GenBank database under accession numbers HQ439013–HQ439022.
This work was supported by the Singapore Botanic Gardens, National Parks Board, Singapore.
Both authors conceptualized the project and experimental strategy. G.S.K. performed the experiments and prepared the manuscript. T.F.C. edited the manuscript.
We thank the Keeper of the Singapore Herbarium, Paul Leong, Serena Lee and J.B. Tay for their assistance in providing fresh and herbarium specimens used in this study. David Lim assisted in identifying some of the plant specimens used. We thank Khoon Meng Wong for comments on the manuscript.