Handbook of Vanilla Science and Technology

In this analysis the V. planifolia and V. pompona sequences grouped into separate clades. Sequences from the Costa Rican hybrid were found in both the V. planifolia and V. pompona clades, suggesting both of these species were the parental species of the hybrid. For simplicity of presentation, in the tree presented in Figure 15.1, only five hybrid clone sequences are shown in each of the two clades. The phylogenetic tree produced when all the hybrid clones generated from this study were included, had the identical topology, with 6 hybrid sequences in the V. planifolia clade and 22 hybrid sequences in the V. pompona clade (not shown). Also, neighbor-joining analysis of the data generated a tree of similar topology (not shown).

In contrast to the biparental inheritance of nuclear genes such as the 5.8S rDNA genes, chloroplast genes are maternally inherited. Phylogenetic analysis of chloroplast genes can therefore be used to determine the maternal parent in an interspecific hybrid. We chose to analyze the chloroplast DNA encoded psaB gene, since sequences from other Vanilla spp. have been reported. The psaB gene encodes one of the subunits of the photosystem I protein complex (Shimada and Sugiura 1991). Maximum parsimony phylogenetic analysis of the Vanilla spp. psaB sequences is shown in Figure 15.2. The tree was based upon 1,627 total characters, of which 1,484 were constant, 88 variable characters were parsimony uninformative, and 55 variable characters were parsimony informative. Phylogenetic analysis of the psaB DNA sequences revealed the close relationship of the sequence from the Costa Rican hybrid plant with that of V. planifolia and V. tahitensis. Neighbor-joining analysis of the data generated a tree of identical topology (not shown). V. planifolia was previously determined to be the maternal parent of V. tahitensis (Lubinsky et al. 2008), and now we can conclude it was also the maternal parent of the Costa Rican hybrid plant.

Fig. 15.2 Rooted maximum parsimony phylogenetic tree of chloroplast psaB sequences. The tree length is 164 and the consistency index is 0.921.The Pogonia japonica sequence was designated as the out-group for rooting the tree. The numbers at the nodes are the bootstrap percentages based on 1,000 replications. For simplicity, the bootstrap percentages at the minor nodes are not labeled, but all were 50% or greater. Accession numbers of the sequences are given in Table 15.1.

Overall, the DNA sequence analysis of the nuclear ITS region and the chloroplast psaB gene presented here confirms that the Vanilla hybrid widely grown in Costa Rica originates from interspecific hybridization, with V. planifolia as the maternal parent and V. pompona as the paternal parent. However, it is not possible to determine from this analysis if the plant is the F1 hybrid or the progeny of a backcross.

A sample of cured beans from the hybrid and from medium-grade beans from Madagascar was extracted and the main flavor compounds analyzed by HPLC. The amounts and the ratios of the measured compounds were within the expected ranges for beans from Madagascar (Tables 15.2 and 15.3).

Table 15.2 Analysis of marker compounds from cured beans from the Costa Rican hybrid and medium-grade beans from Madagascar

Compound | Total mg | mg/mL | % Dry Wt

Costa Rican hybrid beans | ― | ― |

p-OH-Benzoic acid | 1.91 | 0.01 | 0.0076

p-OH-Benzaldehyde | 28.28 | 0.12 | 0.18

Vanillic acid | 13.70 | 0.06 | 0.08

Vanillin | 280.07 | 1.23 | 1.76

Madagascar beans | ― | ― |

p-OH-Benzoic acid | 5.84 | 0.02 | 0.03

p-OH-Benzaldehyde | 16.53 | 0.07 | 0.09

Vanillic acid | 25.76 | 0.10 | 0.15

Vanillin | 254.49 | 1.08 | 1.45

Table 15.3 Comparison of ratios of marker compounds fro grade beans from Madagascar with expected ratios | m the Costa Rican | hybrid beans and medium-grade beans from Madagascar with expected ratios

―  | Costa Rican Hybrid | Madagascar | Expected Ratios[6]

Vanillin/pHB ald | 10.25 | 15.42 | 10-20

Vanillin/Vanillic acid | 20.5 | 10.8 | 12-29

pHB acid/pHB ald | 0.08 | 0.28 | 0.15-0.35

Vanillic acid/pHB ald | 0.5 | 1.43 | 0.53-1.5