Multiple Nuclear Ortholog Next Generation Sequencing Phylogeny of Daucus.

Next generation sequencing is helping to solve the data insufficiency problem hindering well-resolved dominant gene phylogenies. We used Roche 454 technology to obtain DNA sequences from 93 nuclear orthologs, dispersed throughout all linkage groups of Daucus. Of these 93 orthologs, ten were designed to be pure exonic markers, and the remainder to have 60% or more introns. The average sequence length was about 600 nucleotides, obtained for 89 accessions of 13 species and 10 subspecies of Daucus, and an additional ten accessions of related genera (Ammi, Astrodaucus, Caucalis, Margottia, Oenanthe, Orlaya, Pseudorlaya, Torilis, and Turgenia). Phylogenetic analyses of the concatenated data matrix of 115,077 aligned nucleotides provided 100% bootstrap support for most of the external and many internal clades. Concordant with prior molecular results, Margotia gummifera and Pseudorlaya pumila were firmly embedded within Daucus. All accessions of D. capillifolius, D. carota, D. syrticus and D. sahariensis formed a 2n = 18 clade with all other Daucus (2n = 20, 22, and 44 [D. glochidiatus]) and Margotia gummifera and Pseudorlaya pumila in another clade. There was very little resolution within a clade containing all subspecies of D. carota, except a weakly supported subclade containing D. capillifolius and other accessions of D. carota from Libya and Tunisia. The best supported topologies are provided by exonic only regions of the genome, but these differ little in topology from the entire data matrix of exons and introns. Roche 454 produces well-supported (20 X coverage or more) allelic variants. An analysis of single alleles showing the best coverage vs. a single allele using ambiguity codes reconstructs essentially the same topology. We plan to use this dataset as a backbone phylogeny, but with a reduced number of markers to identify additional accessions in this clade.