Abstract Detail

Phylogenomics

Alanagreh, Lo'ai [1], Buchheim, Mark Alan [1].

Intragenomic Variation in the ITS2 rRNA: Results from Deep-Sequencing of Haematococcus.

Primary and secondary structural data from the internal transcribed spacer two (ITS2) have been used extensively for diversity studies of a myriad of eukaryotic organisms, including the green algae. Ease of amplification is due, at least in part, to the fact that ITS2 is part of the tandemly-repeated rRNA array. Nonetheless, the potential confounding influence of intragenomic variability (IGV) has yet to be addressed except in a few organisms. Moreover, none of the assessments of intragenomic variation have taken advantage of the deep-sequencing capacity of sequencing-by-synthesis (SBS) protocols. We present results from our adaptation of the 16S Metagenomics Sequencing Library Preparation (Illumina) protocol for deep sequencing of the ITS2 genes in selected isolates of the green algal genus, Haematococcus. Deep sequencing using SBS (MiSeq) yielded anywhere from more than 500,000 to just under 20,000 merged reads, easily outpacing results from recent pyrosequencing efforts. Furthermore, a conservative evaluation of these data revealed a range of three to six ITS2 variants (haplotypes) across the sampling of isolates. The relative frequency of the dominant ITS2 haplotype ranged from 0.35 to 0.96 across the isolates sampled. In all but one case, the haplotype with the greatest relative frequency corresponded to the published Sanger sequence. Although deep-sequencing generally yielded more ITS2 variants than a clone-and-sequence approach, the latter produced a few unique variants indicating that alternative priming sites for deep-sequencing will need to be explored. A survey of ITS2 variants obtained from the SAG 34-1h isolate of Haematococcus showed evidence of incomplete lineage sorting which could confound phylogenetic reconstruction. However, the same isolate also exhibited a dominant variant with the highest relative frequency. Results from phylogenetic analysis of all ITS2 variants support the conclusion that IGV is unlikely to confound analyses using ITS2 exemplars produced by standard PCR and Sanger sequencing. Our results show that SBS is reproducible and that the Illumina protocols and platforms (SBS) should be the approach of choice for assessing IGV. In addition to advancing our understanding of rRNA variation, the results of this investigation have allowed us to begin testing hypotheses regarding the maintenance of homogeneity (i.e., concerted evolution) across multi-copy genes. Comparisons between IGV in asexual lineages (e.g., Haematococcus) and sexual lineages (e.g., Chlamydomonas) suggest that the homogenizing effect of concerted evolution through unequal crossing over may be relaxed in the former.

1 - University of Tulsa, Department of Biological Science, 800 South Tucker Drive, Tulsa, Oklahoma, 74014, USA

Keywords:
ITS2
Haematococcus
Chlamydomonas
next generation sequencing
Intragenomic Variation
Concerted Evolution.

Presentation Type: Oral Paper
Session: 33, Phylogenomics II
Location: Fort Worth Ballroom 4/Omni Hotel
Date: Wednesday, June 28th, 2017
Time: 8:15 AM
Number: 33002
Abstract ID:407
Candidate for Awards:None