Phylogenetic relationships of selected commercial Dendrobium hybrids in Sri Lanka

Abstract

Nuclear rDNA-ITS regions and chloroplast matK genes are useful in delineating plant species. In this study, genetic relatedness of eight commercial Dendrobium hybrids (A-H) with a range of attractive flower colours was studied using nuclear rDNA-ITS and chloroplast matK sequences. Genomic DNA was extracted from fresh, young leaves using a modified cetyltrimethylammonium bromide based protocol. rDNA-ITS and matK were amplified using PCR in 25 μl reactions containing 1X PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 1.25 U Taq DNA polymerase, 0.4 μM forward and reverse primers and 1.00 μl of genomic DNA. The optimized thermal cycling conditions were initial denaturation at 95 oC for 5 minutes, 35 (rDNA-ITS) and 40 (matK) cycles of denaturation at 95 oC for 40 seconds, annealing at 55 oC (rDNA-ITS) and 48 oC (matK) for 40 seconds, extension at 72 oC for 40 seconds and final extension at 72 oC for 10 minutes. rDNA-ITS and matK PCR products were subjected to Sanger sequencing. Sequences were manually edited using BioEdit 7.0.5.3. and ContigExpress software. Sequences were aligned to the nucleotide database in the National Center for Biotechnology Information using mega BLAST program. Forty-three related sequences were obtained from GenBank and the sequences were aligned using ClustalW implemented in MEGA 7.0.26 software. Phylogenetic analysis was performed by generating trees of ITS, matK and concatenated sequences of ITS and matK. The phylogenetic relationships were analyzed using Maximum Likelihood analysis with 1000 bootstrap replications. Phalaenopsis aphrodite, Liparis kumokiri and Malaxis spicata were used as outgroups. Combined gene-tree was estimated using RAxML-HPC BlackBox tool in CIPRES Science Gateway platform. Resulting trees were viewed using Figtree v1.4.3. In the combined gene tree, selected hybrids were clustered into two distinct groups. Dendrobium hybrids A, B, C, E and F were clustered with Dendrobium bigibbum var bigibbum and Dendrobium phalaenopsis (72% bootstrap). Hybrids G, H and D were clustered with Dendrobium nindii and Dendrobium taurinum (79% bootstrap). In matK gene tree, all the selected hybrids were clustered together with Dendrobium kingianum (90% bootstrap). In rDNA-ITS gene tree, hybrids A, B, C, E and F were clustered with Dendrobium bigibbum var bigibbum and Dendrobium phalaenopsis while hybrids D, G and H were clustered with Dendrobium taurinum and Dendrobium nindii (81% bootstrap). Therefore, though high variation in floral morphology is observed among the selected imported commercial hybrids, they were represented from a narrow genetic background. This is an indicative of genetic bottleneck most likely due to selective breeding and it is important to incorporate more diverse varieties in future breeding programs to maintain a diverse genetic background