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SRX9635182: ddRADseq Barleria
1 ILLUMINA (NextSeq 500) run: 7.3M spots, 1G bases, 447.8Mb downloads

Design: Quantity and concentration of DNA were assessed using a BioTek Instruments (Winooski, VT) Synergy H1 Hybrid Multi-Mode Microplate Reader, with a target concentration of 20-40 ng/l. If DNA extractions were low quality or had low concentration, the samples were re-extracted with the precipitation time increased from overnight to as long as two-weeks. The 192 samples were prepared for double-digest RADseq (Baird et al., 2008; Davey and Blaxter, 2010) following the protocol developed by Parchman et al. (2012) and modified by Tripp et al. (2017) in which genomic DNA was fragmented using restriction enzymes EcoRI and MseI. Our only modification to the Parchman-Tripp protocol was to use 6 l of DNA in each well (20-40 ng/l) for 120-240 ng total. Unique barcodes developed by Tripp et al. (2017) were ordered from IDT (Coralville, IA, USA). ddRADseq protocols were trialed and modified by using samples of freshly-extracted DNA at concentrations of 50 ng/l and 100 ng/l, as well as increasing the primer concentration from 5M to 10M. After filtering, DNA samples at 20-40 ng/l were most successful for library preparation. Once all of the sample libraries were prepared in this manner, the resulting PCR amplicons were combined and sent to the University of California, Riverside Institute for Integrative Genome Biology Genomics Core Facility. There, 200-500 base fragments were size-selected using a Sage Scientific Blue Pippin and 96 samples were multiplexed and sequenced on two lanes of an Illumina HiSeq 2500 (San Diego, CA) using Single End 100 b kits.
Submitted by: Claremont Graduate University
Study: A RADseq Phylogeny of Barleria (Acanthaceae) Resolves Fine-Scale Relationships
show Abstracthide Abstract
Barleria is a genus of approximately 300 species of herbs and shrubs with a wide distribution across the paleotropics; the genus is especially diverse in Angola, Tanzania, and Madagascar. Among Acanthaceae, Barleria can be recognized by a combination of characters: four sepals composed of a small inner pair and larger outer pair; double cystoliths in epidermal cells; an abaxial pair of stamens with filaments that twist through 180 degrees and cross just distal to the synstapetal zone; and globose, honeycombed pollen. Historically, natural groupings in the genus have proven elusive due to rich morphological variation among species of Barleria, including of traits that are homogeneous at the genus level in other Acanthaceae. Previous morphological studies classified Barleria into two subgenera and seven sections. More recently, a molecular study that sampled 53 Barleria species for four chloroplast loci and the nuclear locus nrITS supported the subgenera and four of the seven recognized sections. The aim of our study was to estimate relationships in the genus through a phylogenetic analysis that samples 190 accessions representing 184 taxa, including varieties and subspecies of 167 species of Barleria (56% of total species diversity of ~300). This sample represents all sections and encompasses the geographic range of Barleria. We also included ten outgroups sampled from related genera (i.e., Andrographis, Crabbea, Lepidagathis, Justicia, Neuracanthus, Whitfieldia). Single nucleotide polymorphism data were generated using double-digest restriction-site associated DNA sequencing. The RADseq phylogeny corroborated the topology estimated from the chloroplast and nrITS data, but with greatly increased resolution and support of fine-scale relationships. Importantly, the RADseq phylogeny found six major lineages in subg. Barleria and resolved a polytomy that included the type species of the genus, B. cristata. The topology suggests at least four independent dispersal events to Madagascar with subsequent radiation. Our results are a step towards a stable subgeneric classification and broadly inform our understanding of diversity and evolution in one of the largest genera of Acanthaceae.
SAMN16965131 • SRS7835191 • All experiments • All runs
Name: 212 Barleria carruthersiana
Instrument: NextSeq 500
Strategy: RAD-Seq
Selection: PCR
Layout: SINGLE
Runs: 1 run, 7.3M spots, 1G bases, 447.8Mb
Run# of Spots# of BasesSizePublished


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