intragenomic_rrna_variability_in_plants

initial reasoning

So I had this notion that intra-genomic variation in the ITS region of the rRNA cluster in plant genomes has to do with genome size, and that ITS in the rRNA should not be used as a marker for phylogenetic analyses. The former is known and published although in pieces (alvarez 2003 & prokopowhich 2003 & plenty more I’m sure). The latter is also commonly accepted, yet the ITS is commonly used in plant phylogenetic studies. What is missing is a bigger integrated study of plant rRNA cistrons including big plant genomes and an assessment of how this realistically can effect phylogenetic studies. I seem to remember I assessed this as a novel thing when a student report pointed me to the intragenomic variability of the ITS, but a quick search through literature indicates this is not the case. My guess is that the student at the time missed out on this quite simple literature search or I missed out on his literature research in his report. The initial students observations were (1) there is a big intra-genome variability in the Azolla ITS region (2) the variability has some pattern (3) this pattern is consistent among species of Azolla. Here I plan an open systematic look into this matter.

Build-up

Meta-analysis part:

1. Hypothesis: rRNA copy nr increases with genome size.
   • Alternative hypothesis: phylogenetic pattern
   • Yes, Prokopowich (2003) showed this for 68 plants and 94 animals in 2003.
     • How many more plant genomes and rRNA copy nrs are available these days, and is it worth redoing this 15 years after date.
     • Prokopowhich: “It might be expected that the need for ribosomes would increase as genome size increases if the relative proportion of protein-coding genes remains constant. However, the number of protein-coding genes does not increase proportionately (reviewed in Cavalier-Smith 1985).”
2. Do ITS lengths (and possibly functional content) relate to genome size
   • Alternative hypothesis: phylogenetic pattern
   • Or is it all over the place: Rogers 1987
   • Really, we should be talking about a length distribution per genome

Assembly analysis part:

3. Technical: How many rRNA copies are preserved in draft genomes
   • Sub: differentiate among sequencing strategies
   • Sub/comeback: are these rRNA clusters (or single rRNA cistrons) a consensus, identical, or do they diverge. (per assembly strategy) * How do rRNA clusters end up in genomes (metagenomes) and how do these compare to sanger sequenced results.
   • Sub: do these differentiate from rRNA PCR fragments sequenced in databases

Reads backmapping to rRNA fragments part:

4. Intra genome variation of
   • rRNA genes -> should be practically zero * this seems not to be published
   • ITS regions
     * This is published at least in non-plants
5. Certain pattern in ITS variation
   • Is there functional regions in ITS regions
   • Similar in individuals of the same species
   • Similar in the same genes
   • Family…

Combine:

6. Overall ITS variation as a function of genome size
   • Alternative: phylogenetic
7. Realistic worst case scenario’s when mis-base-calling such fragments.

Data to be generated collected

List of plants with

• Plant

  • Name
  • Origin
  • Specimen

• Genome size

  • Size
  • Genome size as [C-values]
  • Technique of size determination

• known rRNA copy nr

  • Technique

• raw sequencing data available

  • accession nr

• annotated genomes

  • accession nr
  • fasta
  • gff (including rRNA repeats)
  • assembly technique

Novelties to be found:

Scripted redo of Prokopowhich with more genomes and more modern data, not truly novel but I will have to do this to get started anyhow.

How do rRNA clusters end up in genomes (metagenomes) and how do these compare to sanger sequenced results. <- implications for placing sequenced results in a tree.

Is there an (explainable pattern) of intragenomic variability in plant ITS regions.

Simulate how bad variable base calls due to intragenomic variability can mess up a phylogeny.

What I miss… a more evolution central experiment/explanation of the data like Ganley 2007

Conclusion… ITS is not really trustworthy. Use single copy genomic genes. But that’s not a new advise is it. Perhaps we can give a more nuanced advise, how bad is it really in simulations and is it genome size (~copy nr) dependent.

Questions I still have

Has the ITS some folding function in the self-assembly of the ribosome

Can we do phylogeny and alignment on a variable sequence; multiple sequence alignments of seqlogos/hmms… or is this black magic.

#Literature alvarez 2003 Ribosomal ITS sequences and plant phylogenetic inference Ganley 2007 Highly efficient concerted evolution in the ribosomal DNA repeats: Total rDNA repeat variation revealed by whole-genome shotgun sequence data Prokopowich 2003 The correlation between rDNA copy number and genome size in eukaryotes Rogers 1987 Ribosomal RNA genes in plants: variability in copy number and in the intergenic spacer