Handbook of Vanilla Science and Technology

F1 hybrids and recent backcross progenies are expected to have ITS copies from both parental species, and sequence analysis of cloned ITS fragments can be used to confirm the hybrid status of the plants. Such an analysis was used to determine the parental species of a recently formed natural Citrus hybrid (Xu et al. 2006). We used analysis of ITS sequences to confirm the hybrid nature of a Vanilla widely cultivated in Costa Rica. The ITS region of V. planifolia, V. pompona, and the presumed hybrid, was PCR amplified, cloned into a bacterial plasmid, and sequences of 6 to 28 clones per plant sample were obtained as described in the Methods Section. Maximum parsimony phylogenetic analysis of the Vanilla spp. ITS sequences is shown in Figure 15.1. The tree was based upon 686 total characters, of which 342 were constant, 163 variable characters were parsimony uninformative, and 181 characters were parsimony informative. Figure 15.1 includes previously reported ITS sequences, which were generated by direct sequencing of PCR products, and the sequences of the cloned ITS PCR products from V. planifolia, V. pompona, and the hybrid plant generated in this study. The accession numbers of the sequences used in the analysis presented in Figure 15.1 are listed in Table 15.1. The sequence from Clematepistephium smilacifolium, a species also in the tribe Vanilleae, was also included in the analysis. The Pogonia japonica sequence was chosen to root the tree, since the tribe Pogonieae is sister to the tribe Vanilleae (Cameron 2009).

Fig. 15.1 Rooted maximum parsimony phylogenetic tree of nuclear ITS sequences. The tree length is 872 and the consistency index is 0.727. The Pogonia japonica sequence was designated as the outgroup 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 60% or greater. Accession numbers of the sequences are given in Table 15.1.

Table 15.1 Accession 15.1 and 15.2 numbers of DNA sequences used in the phylogenetic analyses presented in Figures

Sequence name | Accession number | Source

ITS Sequences

C. smilacifolium | FJ425838 | Cameron 2009

P. japonica | AF151011 | Cameron and Chase 1999

V. africana | FJ425834 | Cameron 2009

V. aphylla | AF151006 | Cameron and Chase 1999

V. bahiana | EU498163 | Pansarin et al. 2008

V. barbellata | FJ425835 | Cameron 2009

V. edwalii | EU498165 | Pansarin et al. 2008

V. hirsuta | AF391786 | Clements et al. 2002

V. imperialis | FJ425830 | Cameron 2009

V. planifolia 1 | AF391786 | Clements et al. 2002

V. planifolia 11 | GQ867239 | This study

V. planifolia 12 | GQ867240 | This study

V. planifolia 13 | GQ8672241 | This study

V. planifolia 14 | GQ867242 | This study

V. planifolia 15 | GQ867243 | This study

V. planifolia 17 | GQ867244 | This study

V. planifolia 18 | GQ867245 | This study

V. planifolia 19 | GQ867246 | This study

V. pompona 1 | EU498164 | Pansarin et al. 2008

V. pompona 5 | GQ867234 | This study

V. pompona 6 | GQ867235 | This study

V. pompona 7 | GQ867236 | This study

V. pompona 8 | GQ867237 | This study

V. pompona 9 | GQ867238 | This study

V. roscheri | FJ425840 | Cameron 2009

Vanilla hybrid 1 | GQ867248 | This study

Vanilla hybrid 4 | GQ867271 | This study

Vanilla hybrid 5 | GQ867273 | This study

Vanilla hybrid 6 | GQ867265 | This study

Vanilla hybrid 7 | GQ867266 | This study

Vanilla hybrid 8 | GQ867261 | This study

Vanilla hybrid 9 | GQ867257 | This study

Vanilla hybrid 15 | GQ867253 | This study

Vanilla hybrid 17 | GQ867254 | This study

Vanilla hybrid 18 | GQ867247 | This study

psaB Sequences

C. smilacifolium | AY380964 | Cameron 2004

P. japonica | AY381061 | Cameron 2004

V. africana | AY381089 | Cameron 2004

V. aphylla | AY381088 | Cameron 2004

V. barbellata | AY381090 | Cameron 2004

V. edwallii | EU498093 | Pansarin et al. 2008

V. imperialis | AY381091 | Cameron 2004

V. inodora | AY381092 | Cameron 2004

V. palmarum 1 | AY381093 | Cameron 2004

V. palmarum 2 | EU498092 | Pansarin et al. 2008

V. planifolia | GQ888507 | This study

V. pompona | GQ888509 | This study

V. roscheri | AY381095 | Cameron, 2004

V. tahitensis | GQ888508 | This study

Vanilla hybrid | GQ888510 | This study