A two-tier bioinformatic pipeline to develop probes for target capture of nuclear loci with applications in Melastomataceae.

Johanna R Jantzen,Fabian A Michelangeli,Marcelo Reginato,Ryan A Folk,Nico Cellinese,Pamela S Soltis,Prabha Amarasinghe,Douglas E Soltis

doi:10.1002/aps3.11345

Abstract

PremisePutatively single‐copy nuclear (SCN) loci, which are identified using genomic resources of closely related species, are ideal for phylogenomic inference. However, suitable genomic resources are not available for many clades, including Melastomataceae. We introduce a versatile approach to identify SCN loci for clades with few genomic resources and use it to develop probes for target enrichment in the distantly related Memecylon and Tibouchina (Melastomataceae).MethodsWe present a two‐tiered pipeline. First, we identified putatively SCN loci using MarkerMiner and transcriptomes from distantly related species in Melastomataceae. Published loci and genes of functional significance were then added (384 total loci). Second, using HybPiper, we retrieved 689 homologous template sequences for these loci using genome‐skimming data from within the focal clades.ResultsWe sequenced 193 loci common to Memecylon and Tibouchina. Probes designed from 56 template sequences successfully targeted sequences in both clades. Probes designed from genome‐skimming data within a focal clade were more successful than probes designed from other sources.DiscussionOur pipeline successfully identified and targeted SCN loci in Memecylon and Tibouchina, enabling phylogenomic studies in both clades and potentially across Melastomataceae. This pipeline could be easily applied to other clades with few genomic resources.

Highlights

PREMISE: Putatively single-copy nuclear (SCN) loci, which are identified using genomic resources of closely related species, are ideal for phylogenomic inference
We introduce a versatile approach to identify SCN loci for clades with few genomic resources and use it to develop probes for target enrichment in the distantly related Memecylon and Tibouchina (Melastomataceae)
With a minimum gene length of 500 bp enforced, we identified 948 putatively SCN loci based on the A. thaliana genome and 1045 loci based on the T. cacao genome

Summary

Methods

We identified putatively SCN loci using MarkerMiner and transcriptomes from distantly related species in Melastomataceae. Published loci and genes of functional significance were added (384 total loci). Using HybPiper, we retrieved 689 homologous template sequences for these loci using genome-skimming data from within the focal clades. The detailed workflows of our two-tiered approach (Fig. 1) are described below. Genome-skimming data sets were constructed for two species each of Olisbeoideae For the Olisbeoideae samples, total genomic DNA was extracted from herbarium samples (M. afzelii: van der Burgt et al 947 [MO], M. torricellense: Takeuchi and Ama 16241 [MO]) following Doyle and Doyle (1987). Raw FASTQ reads were trimmed and adapters removed using Trimmomatic 0.33 (Bolger et al, 2014) with a sliding window of 20 bp and quality score of Q20 or greater, and the final quality of reads was assessed using FastQC (Andrews, 2010)

Results

Discussion

Conclusion