Comparative chloroplast genomics and phylogenetics of Fagopyrum esculentum ssp. ancestrale -a wild ancestor of cultivated buckwheat

Abstract

Background: Chloroplast genome sequences are extremely informative about species-interrelationships owing to its non-meiotic and often uniparental inheritance over generations. The subject of our study, Fagopyrum esculentum, is a member of the family Polygonaceae belonging to the order Caryophyllales. An uncertainty remains regarding the affinity of Caryophyllales and the asterids that could be due to undersampling of the taxa. With that background, having access to the complete chloroplast genome sequence for Fagopyrum becomes quite pertinent.

Results: We report the complete chloroplast genome sequence of a wild ancestor of cultivated buckwheat, Fagopyrum esculentum ssp. ancestrale. The sequence was rapidly determined using a previously described approach that utilized a PCR-based method and employed universal primers, designed on the scaffold of multiple sequence alignment of chloroplast genomes. The gene content and order in buckwheat chloroplast genome is similar to Spinacia oleracea. However, some unique structural differences exist: the presence of an intron in the rpl2 gene, a frameshift mutation in the rpl23 gene and extension of the inverted repeat region to include the ycf1 gene. Phylogenetic analysis of 61 protein-coding gene sequences from 44 complete plastid genomes provided strong support for the sister relationships of Caryophyllales (including Polygonaceae) to asterids. Further, our analysis also provided support for Amborella as sister to all other angiosperms, but interestingly, in the bayesian phylogeny inference based on first two codon positions Amborella united with Nymphaeales.

Conclusion: Comparative genomics analyses revealed that the Fagopyrum chloroplast genome harbors the characteristic gene content and organization as has been described for several other chloroplast genomes. However, it has some unique structural features distinct from previously reported complete chloroplast genome sequences. Phylogenetic analysis of the dataset, including this new sequence from non-core Caryophyllales supports the sister relationship between Caryophyllales and asterids.

Figure 1

Gene map of the Fagopyrum esculentum chloroplast genome. The thick lines indicate the extent of the inverted repeats (IRa and IRb), which separate the genome into small (SSC) and large (LSC) single copy regions. Genes shown inside the circle are transcribed clockwise, those outside the circle are transcribed counterclockwise. Asterisk (*) indicates pseudogenes.

Figure 2

Structure of IRb/SSC and SSC/IRa in Amborella and in angiosperms with expanded IR. In Amborella IR/SSC junction occurs within ycf1; truncated copy of ycf1 is thus generated at the IRb/SSC border. Such organisation of IR/SSC junction is characteristic for most angiosperms. Lemna, Ipomoea, Jasminum and Fagopyrum represent different ways of the IR expansion. Jasminum and Fagopyrum both have included ycf1 in the IR. In Lemna not only ycf1, but also rps15 and a part of ndhH belong to the IR, and in Ipomoea overall ndhH and a part of ndhA are also duplicated.

Figure 3

Expansion of the IR region in Polygonaceae. Ethidium bromide stained 1.5% agarose gel showing PCR amplification of rps15-ycf1 and ycf1-ndhF spacers for selected Polygonaceae taxa compared to Spinacia oleracea. 1, 2 – Fagopyrum esculentum ssp. ancestrale rps15-ycf1 and ycf1-ndhF fragments respectively, 3, 4 – F. homotropicum SSC rps15-ycf1 and ycf1-ndhF, 5, 6 – F. tataricum rps15-ycf1 and ycf1-ndhF, 7, 8 – Persicaria macrophylla rps15-ycf1 and ycf1-ndhF, 9, 10 – Rheum tanguticum rps15-ycf1 and ycf1-ndhF, 11, 12 – Reynoutria japonica rps15-ycf1 and ycf1-ndhF, 13, 14 – Coccoloba uvifera rps15-ycf1 and ycf1-ndhF, 15, 16 – Spinacia oleracea rps15-ycf1 and ycf1-ndhF (ycf1-ndhF – no amplification). M is the 0.25 – 10 Kb DNA ladder (SibEnzyme Ltd, Moscow, Russia), lowermost visible lane corresponds to 0.5 Kb.

Figure 4

Maximum parsimony phylogenetic tree. This tree is based on nucleotide sequences of 61 protein-coding genes from 44 taxa. Tree length is 85896, consistency index is 0.41 and retention index is 0.48. Numbers at nodes indicate bootstrap support values; first number is for nucleotide sequence data set, second is for amino acid sequence data set. Species which differ in position according to the analysis of these two types of data are underlined. Branch lengths are proportional to the number of expected nucleotide substitutions; scale bar corresponds to 1000 changes.

Figure 5

Bayesian tree. This tree is inferred from the analysis of nucleotide data set, all codon positions are included, and each gene represents separate partition. Numbers at nodes indicate posterior probability, first number is for posterior probabilities inferred from the analysis of all codon position, second is for posterior probabilities inferred from the analysis of first two codon positions. Branch lengths are proportional to the number of expected nucleotide substitutions; scale bar corresponds to one substitution per ten sites. Species which differ in position according to the analysis of all and first two codon positions are underlined.