Intergeneric Hybridization between Phalaenopsis 2166 and Vanda ‘saint valentine’: Characterization of Parents Using ndhE cpDNA Partial Sequence

ABSTRACT An intergeneric cross between Phalaenopsis 2166 and Vanda ‘saint valentine’ has successfully produced protocorms that will be grown further to form seedlings. The present study aims to genetically characterize both parents by using ndhE partial gene as its sequence is shown polymorphic among five orchid genera of the subtribe Oncidiinae. The results reveal that the ndhE partial sequences of Phalaenopsis 2166 and Vanda ‘saint valentine’ are considerably homologous with those of Oncidium. However, alignment of ndhE partial sequences between both parents shows only 58% similarity, leading to the conclusion that a relatively high genetic difference between them may occur.

Parent selection is an important initial step that determines the success of hybridization in orchids. This must involve knowledge of parental traits, particularly those of economic values such as flower size and shape of the hybrids will be produced (Widyastoety et al. 2010). For successful hybridization, maternal parents of strong and long-lasting flower buds with short gynostemium enabling pollen tubes to reach embryo sacs more readily should be selected (Hartati 2010).
Intergeneric hybridization between genus Phalaenopsis and Vanda has been reported to successfully produce seedlings with a number of characteristics seemingly showing maternal inheritance (Hartati 2010). Several species of Phalaenopsis and Vanda are commercially exploited to produce hybrids of high economic and aesthetic values (Sharma et al. 2013). Phalaenopsis is an epiphytic monopodial orchid with long leaves and various flower patterns, while Vanda is also epiphytic monopodial with flowers of sharp colour. Some Phalaenopsis species show long-lasting flowers with a blooming period of up to three months. On the other hand, Vanda flowers blossom commonly for only three weeks.
Monopodial orchids have stems of continual apical growth with little or no axillary sprouting (Johnson & Kane 2007). From an evolutionary point of view, monopodials are the most diverse developing orchids by means of anagenesis. The members of this group show potentials in horticultural practices because they have vigorous stems and long-lasting flowers blooming for 15 -30 days (Sharma et al. 2013).
More specifically, a cross between Phalaenopsis 2166 of pink flower with red spots as the female parent and Vanda 'saint valentine' of plain red as the male parent has also been performed resulting in hybrid fruits brought in Phalaenopsis. The seeds of this intergeneric cross have been grown in vitro and successfully produced protocorms or zygotic embryos. Then, these intergeneric protocorms will be grown to form seedlings using several in vitro culture media.
To predict whether the phenotypical traits of the hybrid seedlings are prone to correspond to those of Phalaenopsis 2166 as their female parent or not, characterization of both Phalaenopsis 2166 and Vanda 'saint valentine' genotypes using a particular marker from cpDNA is required. The ndhE gene is one of the sequences in the cpDNA suitable to be used as a molecular marker in orchids since the sequence is polymorphic among five orchid genera of the subtribe Oncidiinae (Wu et al. 2010). Hence, it is assumed that the sequence also shows polymorphism between Phalaenopsis 2166 and Vanda 'saint valentine', so that both genera can be genetically characterized using ndhE as a molecular marker. This study aims to characterize Phalaenopsis 2166 and Vanda 'saint valentine' as parents of the intergeneric hybridization using ndhE partial sequence.
In our study genomic DNAs were isolated from the completely developed second leaves of Phalaenopsis 2166 and Vanda 'saint valentine' following the CTAB method (Doyle & Doyle 1990). We used Phalaenopsis 2166 of four years old and Vanda 'saint valentine' of six years old. The quality and quantity of the isolated DNAs were measured using a genequant. Amplification of ndhE partial sequences was performed using universal primers, i.e. 5' -GCTAGCCCAATAGCTGCTTC -3' as forward primer and 5' -TCGAAGCATGGTTAGAGCAC -3' as reverse primer. This pair of primers have been designed using Primer 3 on the basis of ndhE conserved areas of three Oncidium hybrid cultivars available at the NCBI database, i.e. Grower Ramsey (acc. no. GU175400.1), Grower Ramsey 'Sunkist' (acc. no. GU175389.1) and Sweet Sugar 'Million Coin' (acc. no. GU175397.1). A total volume of 10 µl PCR mixture containing 2.5 µl template DNA, 5 µl Gotaq green, 2.25 µl nuclease-free water (NFW), and 0.25 µl of individual primer was subjected to PCR condition as follows: predenaturation at 94°C for 3 mins, proceeded by 35 cycles of denaturation at 94°C for 30 secs, primer annealing at 50°C for 30 secs, primer elongation at 72°C for 90 secs, and terminated by final elongation at 72°C for 10 mins prior to storage at 4°C. The PCR products were visualized in a 1.5% agarose gel using TBE buffer. These were then sent to Firstbase Malaysia for sequencing after (Sanger & Nicklen 1977) automated with terminator labelling.
Data of sequences were edited using Bioedit version 7.0.4.1 (Hall 1999) and were checked manually. The edited sequences were then examined for similarity with ndhE sequences available at the NCBI database using BLAST analysis. Sequence alignment was carried out with ClustalW (Thompson et al. 1994), which was also implemented in Bioedit version 7.0.4.1 (Hall 1999).
An amplicon resulting from Vanda 'saint valentine' sample is well visualized as an electrophoretic band of approximately 160 bp. BLAST analysis on the sequence of the amplicon shows 91% similarity with that of cpDNA of an orchid species, i.e., Neofinetia falcata (acc. no. KT726909.1). In addition, they also show 90% similarity with ndhE gene sequences of three Oncidium hybrid cultivars, i.e., Grower Ramsey, Grower Ramsey 'Sunkist' and Sweet Sugar 'Million Coin'. This certainly makes sense, because the ndhE sequences of the Oncidium hybrid cultivars are from which the PCR primers used in this study have been designed. Thus, the amplicon obtained from Vanda 'saint valentine' is undoubtedly a ndhE partial sequence.
As in the case of Vanda 'saint valentine', the amplicon obtained from Phalaenopsis 2166 has also about 160 bp in length. Its sequence shows 92% similarity with ndhE gene sequences of the three Oncidium hybrid cultivars. The similarity to these sequences is even slightly higher than that of Vanda 'saint valentine' (i.e., 90% similarity). Thus, the amplicon of Phalaenopsis 2166 sample is also obviously a ndhE partial sequence like that of Vanda 'saint valentine'.
The partial ndhE sequences of both Phalaenopsis 2166 and Vanda 'saint valentine' have now available at the NCBI database with accession numbers of MH646649 and MH646650 respectively. The alignment of both sequences, however, shows only 58% similarity (Figure 1).
Intergeneric hybridization in plants, either naturally or artificially, are very important to evolution and speciation. It has been proven to provide beneficial significance including increased genetic diversity, improved environmental adaptation, and reproductive isolation breaking. On the other hand, it has also some detrimental effects such as genetic domination which may result in the potential extinction of original species. To assess intergeneric hybridization in plants, molecular markers could be employed along with morphological and chemical properties (Akita et al. 2021).
CpDNA constitutes a source of molecular markers commonly used to assess intergeneric hybridization in plants. Some of them are microsatellites or simple sequence repeats (SSR), the application of which in the pine family has been reviewed revealing their potentials as molecular markers in evolutionary biology of the plant family (Filiz & Koc 2014;Niu et al. 2017). Another marker widely used is ndhE gene, which is a member of ndh genes. The sequences have been proven to be polymorphic among five orchid genera of the family Oncidiinae, i.e. Oncidium, Beallara, Odontoglossum, Odontocidium, and Zelenkocidium (Wu et al. 2010).
The ndh genes are assumed to increase photosynthetic performance in fluctuating terrestrial conditions (Peredo et al. 2013). As many as 11 subunits of ndh genes, i.e., from ndhA to ndhK, are encoded in cpDNA of higher plants. In addition, three cyanobacterial nuclear-encoded subunits genes, i.e., ndhM, ndhN, and ndhO, are found in cpDNA. This indicates that nuclear ndh genes originated in cyanobacteria and were transferred from cpDNA to the nuclear genome during evolution. In Oncidium hybrid cultivar Grower Ramsey, however, all the 11 subunits of ndh genes are encoded in cpDNA, and it is merely ndhE that is translated into a functional protein. Genes that do not encode functional protein have usually a high evolution rate causing high polymorphism. This is also the case in ndh genes of Phalaenopsis aphrodite (Wu et al. 2010;Kim et al. 2020). It is reported that ndhE genes in P. aphrodite and P. equestris are not in complete condition, while ndhA, ndhE, and ndhI genes in Erycina and two varieties of Oncidium have the same pattern (Luo et al. 2014;Smidt et al. 2020).
Complex ndh genes are responsible for encoding NADH dehydrogenase, which serves as transferring electrons from NADH to plastoquinone in the cyclic electron cycle. NADH should be reduced into NADPH in the non-cyclic electron cycle in photosystem I, but when NADPH is not needed, electron is not caught by NADH. In this case, cyclic electron cycle occurs, where electron from photosystem I is caught by pheredoxin, and then is passed to cytochrome B6 complex, cytochrome f, and back to photosystem I. From this cyclic electron cycle, only ATP is produced. All the other photosynthetic elements are encoded by cpDNA genes (Wu et al. 2010;Peredo et al. 2013;Wang et al. 2013;Kim & Chase 2017;Dong et al. 2018;Zhu et al. 2019). The loss of ndh genes or the presence of pseudogenes has been elucidated in the genome of monocotyl plants. Degenerative genes in cpDNA of photosynthetic orchids result from the change in ndh gene structure. Non-functional ndh genes controlled by cpDNA are observed in C3 and CAM plants (Pan et al. 2012;Kim et al. 2020). The loss of genes encoded in cpDNA has no effect on the plant life cycle (Luo et al. 2014).
The absence of ndh genes is found in monocotyledon plants other than orchid species of Najas flexilis and Petrosavia stellaris. This loss of ndh genes is caused by adaptation to submerge environments. In orchids, particular types of ndh genes sometimes disappear, but ndhE can be found in some orchid species of Oncidium, Cymbidium, and Cattleya. Specifically, in Dendrobium, ndhE gene is found but with a deletion of 21 bp (Kim et al. 2015).
In Phalaenopsis aphrodite ndhA, ndhF, and ndhH genes are not found in the cpDNA, while ndhB, ndhC, ndhD, ndhE, ndhG, ndh I, ndhJ, and ndhK genes show variation (Wu et al. 2010;Sui et al. 2018;Kim et al. 2020). Then, in P. aphrodite, which is the ancestor of all orchids, a change in ndh gene control into the nuclear genome occurs. It is confirmed that ndhE gene is observed in P. aphrodite (Kim et al. 2015). In Oncidium, ndhE gene is subjected to truncation and becomes pseudogene, while in P. equestris, the loss of ndhA, ndhE, ndhF, and ndhH genes is reported (Jheng et al. 2012;Kim et al. 2020).
Vanda and Phalaenopsis are of two different genera despite the same subtribe, i.e. Aeridinae. Vanda is, however, known capable of hybridization with other monopodial genera, e.g. Rhyncostylis, Aerides, Ascocentrum, and Phalaenopsis (Sharma et al. 2013;Johnson & Kane 2007). Nevertheless, some intergeneric hybridizations show reciprocally different results. For instance, the relative success of intergeneric hybridization between Renanthera imschootiana as the female parent and V. coerulea as the male parent is reported, while the reciprocal crosses remain failed although both genera are monopodials (Kishor & Sharma 2009). The use of V. testacea as the female parent instead of V. coerulea shows better results (Kishor & Sharma 2008). Similarly, the percentage of pods ready to harvest is relatively higher when Phalaenopsis sp. are used as male parents in the intergeneric crosses of Phalaenopsis x Vanda rather than in the case of the reciprocal combinations (Hartati 2010). On the contrary, we found in our study that no pod at all was produced when Vanda 'saint valentine' was used as the female parent and Phalaenopsis 2166 served as the male parent. The occurrence of maternal inheritance was demonstrated in the case between Phalaenopsis and Vanda previously reported (Hartati 2010) so that it will be very interesting to further study the ndhE partial sequences of the intergeneric hybrid seedlings that will be produced.
The relatively low similarity between ndhE partial sequences of Vanda 'saint valentine' and Phalaenopsis 2166 leads to the conclusion that a relatively long genetic distance between both genera may exist. This supports their taxonomical status as belonging to two different genera, implying the possibility of a heterosis phenomenon in the intergeneric hybrids that will be produced.

AUTHORS CONTRIBUTION
M.D. designed, analysed the research data, and supervised all the processes, A.H.S. collected the data and wrote the manuscript.