The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron

Abstract

The complete sequence of the chloroplast genome of cassava (Manihot esculenta, Euphorbiaceae) has been determined. The genome is 161,453 bp in length and includes a pair of inverted repeats (IR) of 26,954 bp. The genome includes 128 genes; 96 are single copy and 16 are duplicated in the IR. There are four rRNA genes and 30 distinct tRNAs, seven of which are duplicated in the IR. The infA gene is absent; expansion of IRb has duplicated 62 amino acids at the 3' end of rps19 and a number of coding regions have large insertions or deletions, including insertions within the 23S rRNA gene. There are 17 intron-containing genes in cassava, 15 of which have a single intron while two (clpP, ycf3) have two introns. The usually conserved atpF group II intron is absent and this is the first report of its loss from land plant chloroplast genomes. The phylogenetic distribution of the atpF intron loss was determined by a PCR survey of 251 taxa representing 34 families of Malpighiales and 16 taxa from closely related rosids. The atpF intron is not only missing in cassava but also from closely related Euphorbiaceae and other Malpighiales, suggesting that there have been at least seven independent losses. In cassava and all other sequenced Malphigiales, atpF gene sequences showed a strong association between C-to-T substitutions at nucleotide position 92 and the loss of the intron, suggesting that recombination between an edited mRNA and the atpF gene may be a possible mechanism for the intron loss.

Fig. 1

Map of the M. esculenta plastid genome represented in its circular monomeric form. Large and small single copy regions (LSC, SSC) are separated by the inverted repeats (IRa and IRb, 26,954 bp, respectively). Genes illustrated inside the circle are transcribed in the clockwise direction and genes illustrated outside the circle are transcribed in the counter-clockwise direction. Split genes or genes with introns are marked with asterisks

Fig. 2

Alignment of atpF genes from cassava and Populus alba showing that there is 94.1% sequence identity in the exon regions and the precise loss of the intron in cassava. The nucleotide position 92 is highlighted in cassava, where C-U editing occurs. Primers used for DNA sequencing are also highlighted

Fig. 3

Ethidium bromide stained 1.5% agarose gel showing amplification products for selected Malpighiales taxa screened for the presence/absence of the atpF intron. Intron present, lanes 1 Putranjiva roxburghii, 2 Hybanthus concolor, 3 Bischofia javanica, 4 Ricinus communis, 5 Suregada glomerulata, 6 Tetrorchidium cf. macrophyllum. Intron absent, 7 Elateriospermum tapos, 8 Manihot grahamii, 9 Micrandra inundata, 10 Savia bahamensis, 11 Lophopyxis maingayi 12 Malesherbia weberbaueri. L Invitrogen 1 Kb Plus DNA Ladder. Underlined taxa have been sequenced from these PCR products. See Appendix for sample details

Fig. 4

Phylogenetic tree of the 76-taxon atpF dataset. Tree shown is from a ML analysis with −lnL = 8741.21059. Number series above or below branches indicate support from BI, ML, and MP analyses, respectively; single numbers indicate all 3 values are identical. Asterisk indicates <50% support in that analysis. Intron absence is indicated with an open square; all other taxa possess the atpF intron. A filled gray square indicates the presence of a thymine at nucleotide 92; all other taxa possess a cytosine at that site, which is potentially RNA edited as a C-U conversion. GenBank numbers are indicated for published data, all other taxa are newly generated (see Appendix)