Complete chloroplast genome of Oncidium Gower Ramsey and evaluation of molecular markers for identification and breeding in Oncidiinae

Abstract

Background: Oncidium spp. produce commercially important orchid cut flowers. However, they are amenable to intergeneric and inter-specific crossing making phylogenetic identification very difficult. Molecular markers derived from the chloroplast genome can provide useful tools for phylogenetic resolution.

Results: The complete chloroplast genome of the economically important Oncidium variety Onc. Gower Ramsey (Accession no. GQ324949) was determined using a polymerase chain reaction (PCR) and Sanger based ABI sequencing. The length of the Oncidium chloroplast genome is 146,484 bp. Genome structure, gene order and orientation are similar to Phalaenopsis, but differ from typical Poaceae, other monocots for which there are several published chloroplast (cp) genome. The Onc. Gower Ramsey chloroplast-encoded NADH dehydrogenase (ndh) genes, except ndhE, lack apparent functions. Deletion and other types of mutations were also found in the ndh genes of 15 other economically important Oncidiinae varieties, except ndhE in some species. The positions of some species in the evolution and taxonomy of Oncidiinae are difficult to identify. To identify the relationships between the 15 Oncidiinae hybrids, eight regions of the Onc. Gower Ramsey chloroplast genome were amplified by PCR for phylogenetic analysis. A total of 7042 bp derived from the eight regions could identify the relationships at the species level, which were supported by high bootstrap values. One particular 1846 bp region, derived from two PCR products (trnHGUG -psbA and trnFGAA-ndhJ) was adequate for correct phylogenetic placement of 13 of the 15 varieties (with the exception of Degarmoara Flying High and Odontoglossum Violetta von Holm). Thus the chloroplast genome provides a useful molecular marker for species identifications.

Conclusion: In this report, we used Phalaenopsis. aphrodite as a prototype for primer design to complete the Onc. Gower Ramsey genome sequence. Gene annotation showed that most of the ndh genes inOncidiinae, with the exception of ndhE, are non-functional. This phenomenon was observed in all of the Oncidiinae species tested. The genes and chloroplast DNA regions that would be the most useful for phylogenetic analysis were determined to be the trnHGUG-psbA and the trnFGAA-ndhJ regions. We conclude that complete chloroplast genome information is useful for plant phylogenetic and evolutionary studies in Oncidium with applications for breeding and variety identification.

Figure 1

Parents of 15 varieties of Oncidiinae. ¹Yellow background: Oncidium; white: Beallara; blue: Odontoglossum; purple: Odontocidium; green: Degarmoara; red: Zelenkocidium. ²Onc. Aloha = Onc. Goldiana × Onc. Star Wars. ³Mtssa Charles = Brassia verrucosa × Milt. spectabilis.

Figure 2

Primers for Oncidiinae ndh gene and phylogenetic analysis. ¹Primer sequences, annealing site of the forward primer in Onc. Gower Ramsey and the anticipated amplicon size (bp) are presented. ²Different background colors indicate different experiments; gray: ndh gene identification; yellow: phylogenetic analysis.

Figure 3

Gene map of Onc. Gower Ramsey chloroplast genome. The thick lines indicate the extent of the IRa and IRb, which separate the genome into SSC and LSC regions. Genes on the outside of the map are transcribed clockwise and genes on the inside of the map are transcribed counterclockwise.

Figure 4

Maximum likelihood phylogram for 61 conserved protein-coding genes. All nodes have 100% ML bootstrap support unless otherwise indicated. Horizontal branch lengths are proportional to the number of inferred substitutions/site along that branch. One node, marked "nr," was not resolved in the ML bootstrap consensus tree. The position of Oncidium in a clade of Asparagales is indicated with an arrow.

Figure 5

Summary of ndh gene patterns in Oncidiinae. ¹Different background colors indicate different genera; yellow: Oncidium, white: Beallara, blue: Odontoglossum, pink: Odontocidium, purple: Colmanara, green: Degarmoara, red:Zelenkocidium. ²GR: Gower Ramsey, Sunkiss: Gower Ramsey 'Sunkiss', L. H.: Lemon heart, M. C.: Sweet sugar 'Million Coin', E. star: Eurostar, M. J.: Peggy Ruth Carpenter 'Morning Joy', H. D.: Marfitch 'Howard Dream', S. S.: Tahoma Glacier 'Sugar Sweet', S. E.: Smile Eri, M. H.: Margarete Holm, V. v. H.: Violetta. von Holm, G.G.: Golden Gate, W.G.: Wildcat 'Garfield', Dgmra: Dgmra. Flying High, L. A.: Little Angel. ³'black star': absent genes (no sequence exists). 'white star': stop codon (There is no change in gene size but there are stop codons within coding sequences). 'black triangle': truncated genes (only partial coding sequences are observed). 'white triangle': frame shift (reading frame shifted or nucleotides deleted). 'white circle': functional protein., -: no PCR product obtained using the primers in Figure 2.

Figure 6

Structure of ndh genes are different in Oncidiinae varieties. Numbers indicate the positions in the chloroplast genome. The angled dashed lines indicate the gaps. Different colors indicate different ndh genes, a color key is shown at the bottom of each part of the Figure. Detailed information is shown in Figure 5. ¹ including most of the Oncidiinae except Zelenkocidium Little Angel. ²including three Oncidium Gower Ramsey varieties, Bllra. Tahoma Glacier 'Sugar Sweet', Odm. Violetta, von Holm, Odcdm. Wild cat 'Garfield', and Zelenkoncidium Little Angel. ³including four Oncidium varieties, two Odontoglossum varieties, one Odontocidium, Dgmra Fly High, and Zelenkoncidium Little Angel. ⁴ including Bllra. Eurostar, Bllra. Marfitch 'Howard Dream', and Bllra. Smile Eri. ⁵including four Oncidium varieties, two Odontoglossum varieties, two Odontocidium varieties, Dgmra. Fly High. ⁶including Bllra. Eurostar, Bllra. Peggy Ruth Carpenter, and Bllra. Marfitch 'Howard Dream'. ⁷including three Oncidium varieties. ⁸including Bllra. Eurostar, Bllra. Tahoma Glacier 'Sugar Sweet', Bllra Peggy Ruth Carpenter and Bllra. Smile Eri.

Figure 7

Maximum parsimony phylogenetic trees using different cpDNA regions of 15 varieties of Oncidiinae. These trees are based on the nucleotide sequences of (A) matK (B) trnH^GUG-psbA+matK (C) trnH^GUG-psbA+trnF^GAA-ndhJ (D) from all eight cpDNA regions. The numbers at the nodes indicate bootstrap support values. The scale bar indicates a branch length corresponding to 100 character-state changes.