Genetic Resources of The Universal Flavor, Vanilla

Commercially cultivated vanilla ( V. planifolia ) is native to Mexico and its cultivation and breeding programmes face major bottlenecks. This study reports presence of important agronomic characters in two important and endangered species of Vanilla, V. aphylla and V. pilifera , indigenous to India. V. aphylla was tolerant to Fusarium wilt and had longer flower life than the cultivated vanilla. V. pilifera flowers were fragrant, showed signs of insect pollination and had large fruit size. The species were amenable to interspecific hybridization and successful reciprocal crosses were done. Sequence similarity studies indicated the clustering of leafy and leafless species separately.


Introduction
The genus Vanilla includes about 110 species and the species have been treated in various monographic works [1,2] including the life history of V. planifolia [3]. Vanilla planifolia (Salisb.) Ames (syn. V. fragrans Andrews.), is a tropical climbing orchid known for yielding the delicate popular flavor, vanilla [4] and is the second most expensive spice traded in the world market [5] (Spices Board 2000). The major vanilla producing countries are Madagascar, Comoro, Indonesia, Mexico and the Reunion, of which, Madagascar holds the prominent position.
Vanilla was introduced to Europe from Mexico, in about 1500 and its reputation of being an aphrodisiac followed it to countries where it was introduced. The importance of vanilla since early times in Mexico, is evident by the mention of offering vanilla as a medicinal beverage as part of a tribute during reign of Itzcóatl (Aztec Emperor) in 1427 and citing vanilla as a remedy for fatigue in Badianus manuscript in 1552 [6]. Vanilla planifolia, which yields the vanilla of commerce, is native to Mexico and parts of Central America and the history of origin of cultivated vanilla suggests that the entire stock outside Mexico may be from a single genetic source. For the last 400 years, humans have been playing important role in the dispersal and spread of vanilla in the New World.

Species of Vanilla
Studies of divergence among species of agronomic importance have been receiving greater attention. Genomics-based tools are efficient to characterize and identify genetic diversity in Vanilla and act as a significant tool for genomicsassisted plant breeding [7]. RAPD polymorphism was used to estimate the level of genetic diversity and interrelationships among few related species Vanilla planifolia, including both leafy and leafless types such as V. tahitensis, V. andamanica, V. pilifera and V. aphylla. Studies revealed that there is very limited variation within collections of V. planifolia, indicative of its narrow genetic base [8]. The British introduced Vanilla planifolia into India about 200 years ago whereas five other species are native viz., V. pilifera Holt., V. andamanica Rolfe., V. aphylla Blume., V. walkeriae Wight. and V. wightiana Lindl.
V. pilifera originally described from Malaya, recorded in Thailand is found in the Mikir hills of Northeast India. V. aphylla, an endangered species, previously known from Thailand is found in South India [9]. V. andamanica is endemic to Andaman group of Islands and is believed to be same as V. albida [10]. V. tahitensis, which is commonly exploited throughout the tropics, is indigenous to the Tahiti Islands. The presence and absence of leaves, and floral characters (colors of flower, lip, hairs on lip and ovary-pedicel etc.,), morphologically distinguish these species ( Table 1).
A preliminary analysis of the various characters of Vanilla species including the above species, showed presence and absence of leaves formed an important part in the classification of the genus which in general had the basic chromosome number x = 16. Most of the Indian species were leafless, except V. pilifera which was intermediate in character, i.e., leafless in early stages and long narrow leaves at maturity and the chromosome number in V. aphylla is 2n = 64, whereas the cultivated vanilla and V. tahitensis had a somatic chromosome number of 2n = 32 [10,11]. Differences in floral characters existed in flower color and lip characters ( Table 2). In V. pilifera vines, leaves developed as the vine grew with flowers that were narrower (2.8 x 0.8 cm) with distinct pure white ovary-pedicel (Figures 1 and 2), pale green tepals, purplish violet and longer (6 mm approx.) hairs on white lip. V. aphylla is leafless (with scales-1.8 cm) and yellowish-cream flowers (petal size 3 x 1.2 cm approx.) having tuft of hairs that are cream near tip, deep reddish inside (2-3 mm) and light green ovary-pedicel (Figures 3 and 4)

Biotechnological applications
Micropropagation and in vitro conservation techniques for the different species of Vanilla [12] and interspecific hybridization as a tool for gene flow of desirable characters from wild species into cultivated species, through pollen, have been reported [13]. Genetic interrelationships studies, using RAPD profiles [8], among different species revealed that the leafless forms of vanilla, V. aphylla and V. pilifera formed a separate sub-cluster. All the other leafy vanilla types formed a separate sub-cluster. V. pilifera, which showed an intermediate leaf character, showed only 50-56.1% similarity to V. planifolia but closely resembled V. aphylla (76.8%). Thus, the present study reveals the presence of important agronomic characters for introgression into cultivated vanilla and which can be utilized to overcome major bottlenecks in vanilla breeding. The presence of fragrance which attracts insects, coupled with signs of fruit set without hand pollination, holds V. pilifera as a potential candidate for breeding programmes, to overcome the problem of lack of natural seed set in vanilla. V. aphylla which was tolerant to Fusarium oxysporum [8] and its crossability to cultivated vanilla can be utilized as a bridging species and to help wipe out diseases arising out of monoculture. Interspecific hybridization has been reported and hence transfer of these desirable traits into cultivated vanilla,  V. planifolia, may not be hindered. The advent of biotechnological tools, offers techniques for transfer of these characters into V. planifolia, thus making the dream of transforming vanilla into a fragrant, natural seed setting, disease tolerant commercially important orchid can be turned into a reality.
The identification of a hydratase/lyase type enzyme as being a vanillin synthase offers new opportunities for the Vanilla pod-based industries. The accumulation of vanillin glucoside in the capsules of cultivated vines in response to environmental challenges may now be assessed at the molecular level. Likewise, the basis for development of genetic markers for the selection of vanilla orchid varieties with improved aromatic properties has now been laid down. Vanillin produced biologically is termed 'natural' vanillin and has a high economic value compared with chemically synthesized vanillin. Likewise, in the transition towards a bio-based economy, it is important to develop sustainable production systems to replace those currently based on fossil fuels. The demonstration that a single enzyme in the vanilla pod catalyzes the conversion of ferulic acid and ferulic acid glucoside into vanillin and vanillin glucoside provides several options for biotechnological applications [14].

Genomic DNA isolation
Genomic DNA was isolated from approximately 100 mg fresh leaves by grinding in a pestle and mortar using liquid Nitrogen and following the procedure using DNeasy® Plant Mini Kit (Qiagen, USA). The ground sample powder (100 mg) was transferred to microfuge tubes. Followed by addition of 400 μl AP1 buffer and 4 μl RNase A and mixed by vortex. The tubes were incubated at 65°C for 10 min in a water bath with intermittent mixing 2-3 times by inverting the tubes. Added 130 μl buffer P3 to the tube, mixed and incubated for 5 min on ice. The lysate was centrifuged for 5 min at 14,000 rpm. The samples were then loaded onto the QIAshredder spin columns and centrifuged at 14,000 rmp for 2 min. The flow-through was transferred to a new tube without disturbing the pellet. Added 1.5 volume of buffer AW1 and mixed by pipetting. The contents were then loaded in 650 μl fractions onto the DNeasy mini spin column and centrifuged at 8000 rmp for 1 minute. The flowthrough was discarded. The spin column was placed into a new 2 ml collection tube and added 500 μl buffer AW2, followed by centrifugation for 1 min at 8000 rpm. This last step with buffer AW2 step was repeated, with centrifugation at 14,000 rpm for 2 min. The spin columns were placed in fresh microfuge tubes and 100 μl AE buffer was added onto the membranes and incubated at room temperature for 5 min. The tubes were then centrifuged at 8000 rpm for 1 min. This step was repeated with another 100 μl of AE buffer. The eluted samples were stored at À20°C.

Measurement of purity and DNA concentration
Quality and quantity of genomic DNA was monitored by using UV/Vis. Spectrophotometry and quality was confirmed by using 0.8% Agarose Gel Electrophoresis. Each of the sample DNA was diluted to 5 ng/μL in double distilled water for use as a PCR template.

PCR amplification of DNA barcoding region and sequencing
PCR reactions were carried out using universal primers for the DNA barcode regions matK, nrDNA-ITS, rbcL and trnH-psbA. All the specific locus primers were purchased with universal M13 primer sequence at their 5 0 ends, thus enabling the direct sequencing of the PCR products using the universal M13 primers. The PCR amplification was performed in a 20 μl reaction mixture, consisting of 1X PCR buffer (2 mM Mgcl2), 200 μM each of dATP, dCTP, dGTP, dTTP; 0.5 μM of each forward and reverse primers and 1 U of Taq polymerase (TakaRa-Taq), and (5-20 ng) DNA template. DNA amplification was performed in a thermal cycler (Eppendoff, Germany). When the reaction has finished, the tubes were stored at 4°C. PCR products were separated by agarose gel electrophoresis (1.8%). The list of primers, their nucleotide sequences, annealing temperature and the specific PCR cycling conditions are shown in Table 3. A large volume PCR reaction (100 μl) per sample loci was done and PCR purification was done using Nucleospin Gel and PCR Cleanup kit (Macherey-Nagel, Germany). The purified PCR products were sequenced using M13 universal primers (M13 forward and M13 reverse primers) on ABI 3730xl  DNA sequencer at AgriGenome labs facility, Kochi, India. Each DNA barcode region was sequenced. The different universal primers used in this study for the amplification are shown in Table 4 along with the amplified product size.

Presence of important agronomic characters
Among the different species of vanilla studied Vanilla aphylla Blume and V. pilifera Holtt., flowered synchronously ( Figure 5). V. aphylla occurs naturally in South India (Figure 6) and V. pilifera (Figure 7) in Assam, Northeast India. Flowers of both the species opened sequentially and lasted for one day in V. pilifera, whereas it lasted for 2 days in V. aphylla. In the former, signs of fruit set were observed even without pollination (Figures 1 and 8) whereas V. apylla flowers did not set fruit (Figure 3), ruling out the possibility of natural fruit set in this species, which is thus similar to V. planifolia ( Table 5).   Cross sections of the ovary pedicel were observed after closing of the flowers. Persistent perianth is characteristic to the genus and also indicative of effective pollination. In flowers where pollination is not effected, the perianth is shed after the flower closes. Perianth in V. pilifera were found to persist even without pollination and the cross sections indicated initiation of seed set (Figures 9 and 10), whereas V. apyhlla did not show any indications (Figures 11 and 12). Since rostellum is present in both the species, natural pollination without an aid is ruled out. It can be suspected, that the fragrance of the V. pilifera flowers attracts insects (which were found to frequent the flowers often) to visit them and bring about effective pollination.
Pollinations both self and interspecific hybridizations between the two species were done and fruits set was observed (Figure 13).

Sequence analysis
General observations from the experiment 1. The matK sequences of VG and VP are identical.  V. planifolia Susceptible Not seen Crossable to V. aphylla < 24 Table 5. Important agronomic characters.   2. The matK sequence of V1 was different from VG/VP at 21 nucleotide positions as shown in Table 6 below.   6. The ITS sequences matching with different Vanilla sp. were downloaded and subjected to analysis using MEGA7.0 software ( Table 7).

VS matk T T T C C C T G C G G C A G C A C C T T C VG matk G A C T A T G T A C T A T T T G T T C G T VP matk G A C T A T G T A C T A T T T G T T C G T
The number of base differences per sequence from between sequences are shown. The analysis involved 14 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There was a total of 657 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Figure 14).  Table 7.

VS ITS
Estimates of evolutionary divergence between sequences.

Conclusions
The analysis involved 14 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There was a total of 657 positions in the final dataset. The analyses were conducted in MEGA7. The phylogeny analysis also revealed the separate clustering offer leafy and leafless species. Vanilla siamensis, a leafy species, indicating signs of self-pollination in its wild, in Thailand, clustered with leafless V. aphylla species. The studies further reveal the complexity of the biosynthesis of the natural vanillin synthesis. However, it is to be further analyzed whether leafy character is associated with enhanced photosynthetic products that indirectly affect the vanillin synthesis too. This reiterates the need for conservation of the genetic resources [12] of Vanilla across the continents, for implementing meaningful breeding programs, to enhance vanillin productivity in addition to disease resistance and reproductive behavior.