The complete plastid genomes of Ophrys iricolor and O. sphegodes (Orchidaceae) and comparative analyses with other orchids

Abstract

Sexually deceptive orchids of the genus Ophrys may rapidly evolve by adaptation to pollinators. However, understanding of the genetic basis of potential changes and patterns of relationships is hampered by a lack of genomic information. We report the complete plastid genome sequences of Ophrys iricolor and O. sphegodes, representing the two most species-rich lineages of the genus Ophrys. Both plastomes are circular DNA molecules (146754 bp for O. sphegodes and 150177 bp for O. iricolor) with the typical quadripartite structure of plastid genomes and within the average size of photosynthetic orchids. 213 Simple Sequence Repeats (SSRs) (31.5% polymorphic between O. iricolor and O. sphegodes) were identified, with homopolymers and dipolymers as the most common repeat types. SSRs were mainly located in intergenic regions but SSRs located in coding regions were also found, mainly in ycf1 and rpoC2 genes. The Ophrys plastome is predicted to encode 107 distinct genes, 17 of which are completely duplicated in the Inverted Repeat regions. 83 and 87 putative RNA editing sites were detected in 25 plastid genes of the two Ophrys species, all occurring in the first or second codon position. Comparing the rate of nonsynonymous (dN) and synonymous (dS) substitutions, 24 genes (including rbcL and ycf1) display signature consistent with positive selection. When compared with other members of the orchid family, the Ophrys plastome has a complete set of 11 functional ndh plastid genes, with the exception of O. sphegodes that has a truncated ndhF gene. Comparative analysis showed a large co-linearity with other related Orchidinae. However, in contrast to O. iricolor and other Orchidinae, O. sphegodes has a shift of the junction between the Inverted Repeat and Small Single Copy regions associated with the loss of the partial duplicated gene ycf1 and the truncation of the ndhF gene. Data on relative genomic coverage and validation by PCR indicate the presence, with a different ratio, of the two plastome types (i.e. with and without ndhF deletion) in both Ophrys species, with a predominance of the deleted type in O. sphegodes. A search for this deleted plastid region in O. sphegodes nuclear genome shows that the deleted region is inserted in a retrotransposon nuclear sequence. The present study provides useful genomic tools for studying conservation and patterns of relationships of this rapidly radiating orchid genus.

Fig 1

Gene map of Ophrys sphegodes (a) and Ophrys iricolor (b) plastid genomes. Genes drawn inside the circle are transcribed in the clockwise direction, and genes drawn outside are transcribed in the counter-clockwise direction. Different functional groups of genes are colour-coded. The darker grey in the inner circle corresponds to G/C content, and the lighter grey corresponds to A/T content. LSC, Large Single Copy; SSC, Small Single Copy; IRA/B, Inverted Repeat A/B. The enlargement shows that the loss of the partial duplicated gene of ycf1 and the truncation of ndhF gene in O. sphegodes are correlated with the shift of the junction between the IR and SSC.

Fig 2

In silico validation of ndhF deletion (using software CNView) comparing O. sphegodes plastid reads against reference genome of O. iricolor (a) and O. iricolor plastid reads against reference genome of O. sphegodes (b). Y-axis represents normalized coverage values.

Fig 3. Results of BLASTX search of scaffold1075174 (length 5,436 bp): Putative domain hits are indicated by the colored arrows.