Assessing Symbiodinium diversity in scleractinian corals via next-generation sequencing-based genotyping of the ITS2 rDNA region.
Bottom Line: A genetic distance cut-off of 0.03 collapsed intragenomic ITS2 variants of isoclonal cultures into single OTUs.When applied to the analysis of field-collected coral samples, our analyses confirm that much of the commonly observed Symbiodinium ITS2 diversity can be attributed to intragenomic variation.We conclude that by analysing Symbiodinium populations in an OTU-based framework, we can improve objectivity, comparability and simplicity when assessing ITS2 diversity in field-based studies.
Affiliation: Red Sea Research Center, King Abdullah University of Science and Technology (KAUST), 23955, Thuwal, Saudi Arabia.Show MeSH
Mentions: Depending on the taxon investigated, we identified between 102 and 331 distinct ITS2 sequence variants, including cultured strains and field-collected specimens (mean = 219.67). Taking only isoclonal culture samples into account, we identified on average 230.86 ITS2 sequence variants per culture, indicating that there is a substantial number of distinct ITS2 sequence variants found within Symbiodinium genomes (Table1). Despite the high number of distinct ITS2 copies, read counts for the different ITS2 copies showed a highly uneven distribution (Fig.1A). When sorting ITS2 variants of isoclonal cultures by sequence read abundance, the most abundant ITS2 copies were on average ∼20 times more prevalent than the second most common ITS2 copy (all clades: 21.84-fold, clade A's: 8.25-fold, clade B's: 39.88-fold, clade C's: 8.49-fold). Further, the five most abundant ITS2 copies from any culture made up >80% of associated reads, indicating that only few distinct ITS2 genes make up the majority of genomic gene copies. This was substantiated by a rarefaction analysis, which indicated that most of the numerous ITS2 copies were captured at very low abundance in each genome (Fig.1B). For instance, subsampling of isoclonal cultures to 2000 reads yielded on average less than half of the distinct ITS2 copies we were able to recover taking all sequence reads into account. Additionally, the rarefaction curves at this sampling depth did not approach saturation. This effectively illustrates that intragenomic diversity lies in low-abundant genomic ITS2 copies, which also seems to far exceed what could be captured by ‘traditional’ sequencing methods. Comparison of pyrosequencing data of culture CCMP2467 (type A1) with 25 sequences generated by cloning and sequencing showed that both techniques identified the same most dominant sequence, but numerical ranking of the next most common sequences did not particularly match up well between cloning and 454 data (not shown). Comparing pyrosequencing data to DGGE fingerprinting of isoclonal Symbiodinium cultures, DGGE yielded a single dominant band accompanied by few, faint, background bands. The dominant ITS2 variants produced by 454 pyrosequencing (numerically abundant) and DGGE (brightest band) were identical in sequence and were representative sequences of the Symbiodinium type analysed (Table1, Fig.2). Accordingly, 454 pyrosequencing, cloning and sequencing and DGGE produced uniform results in regard to identifying the ITS2 variants most representative of the genome.
Affiliation: Red Sea Research Center, King Abdullah University of Science and Technology (KAUST), 23955, Thuwal, Saudi Arabia.