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Multi-locus, low copy nuclear DNA sequences have been used in plant phylogenetic studies since the late nineties ( De La Torre et al., 2006 Hughes et al., 2006 Sanderson and McMahon, 2007 Griffin et al., 2011) and, because of their bi-parental inheritance and wealth of long and independently-inherited genes ( Small et al., 2004), became the focus of plant phylogenetic reconstruction in general.
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However, when not all rDNA copies are fully homogenized as was observed for instance in tulip and peonies ( Sang et al., 1995 Booy et al., 2000 Lim et al., 2001 Alvarez and Wendel, 2003), the risk of using paralogs in phylogenetic reconstruction becomes large ( Kim and Jansen, 1994 Sang et al., 1995 Alvarez and Wendel, 2003) and hence rDNA has been disregarded as phylogenetic marker in species-level Angiosperm phylogenetics. As a special case rDNA has been used extensively in Angiosperm (and fungal) phylogenetic reconstruction, especially using the Internal Transcribed Spacer regions ( White et al., 1990 Baldwin, 1992). However, cpDNA reveals only half of the phylogenetic origin of a plant-lineage since it is uni-parentally inherited and its substitution rates are generally low compared with bi-parentally inherited nuclear DNA ( Small et al., 2004). Chloroplast DNA has the advantage of straightforward genetics: haploid, non-recombinant and highly conserved with respect to gene content and arrangement, notably among closely related species ( Olmstead and Palmer, 1992). The preponderance of data used in plant molecular phylogenetics over the last decade comes from chloroplast DNA and nuclear rDNA (notably rDNA ITS) ( APG, 2003, 2009 Chase and Reveal, 2009). We conclude that these 26 genes can be widely applied to construct a species tree for the genus Lilium. Although the resolution offered by these approaches is high, in this case there was no extra benefit in using allelic information. The three approaches generated the same topology. Twenty six gene contigs were chosen based on the presence of orthologous sequences in all cultivars, seven of which also had an orthologous sequence in Tulipa, used as out-group. We explored the potential and caveats of this strategy in four genetically distant Lilium cultivars to estimate their genetic divergence from transcriptome sequences using three approaches: POFAD (Phylogeny of Organisms from Allelic Data, uses allelic information of sequence data), RAxML (Randomized Accelerated Maximum Likelihood, tree building based on concatenated consensus sequences) and Consensus Network (constructing a network summarizing among gene tree conflicts). Next Generation Sequencing (NGS) may enable estimating relationships among genotypes using allelic variation of multiple nuclear genes simultaneously. 2Biosystematics Group, Wageningen University, Wageningen, Netherlands.1Wageningen UR Plant Breeding, Wageningen University and Research Centre, Wageningen, Netherlands.