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Using transcriptomics to delineate the status of Millepora species in the Caribbean Sea. Nikolaos V. Schizas, Dannise Ruiz Ramos, Ingrid Ortiz Gonzalez, Audrey Majeske , Ernesto Weil University of Puerto Rico, Mayagüez Department of Marine Sciences. Background.

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slide1

Using transcriptomics to delineate the status of Millepora species in the Caribbean Sea

Nikolaos V. Schizas, Dannise Ruiz Ramos, Ingrid Ortiz Gonzalez,

Audrey Majeske, Ernesto Weil

University of Puerto Rico, Mayagüez

Department of Marine Sciences

background
Background

The hydrocoral genus Millepora consists of 19 species distributed in warm waters around the globe

12 are found in the Indo-Pacific, 7 in the Atlantic/Caribbean

slide3

Caribbean species of Milleporaspp.

Clockwise from the upper left: M. alcicornis, M. squarrosa, M. striata, and M. complanata

background1
Background
  • Millepora is an important reef framework builder in some reefs
  • Millepora is a voracious plankton feeder, consuming up to 8 prey cm-2 day-1
  • Milleporidsare superior space competitors over gorgonians
  • Millepora are susceptible to bleaching
  • Despite their abundance, geographical distribution and geological importance, the milleporids have seldom received attention in coral reef studies
slide5

Millepora visually dominate some reefs

Milleporaovergrowing gorgonians

(Photo:CCMA/NOAA)

slide6

Questions

1) Are there reliable morphological characters that separate the species?

2) Do the different species/morphotypes of Caribbean Millepora represent genetically distinct taxa?

Photo credit- E. Weil

slide7

M. alcicornisbranching vs. encrusting vs. M. complanata

M. alcicornisbranching

M. alcicornisencrusting

M. complanata

MORPHOLOGY

Ruiz Ramos, Weil, Schizas (2014). Zoological Studies.

slide8

Morphological Measurements

g = Gastropore, d = Dactylopore,

1. Diameter of gastropore,

2. Diameter of dactylopore,

3. Distance among dactylopores,

4. Distances from gastroporeto nearest dactylopore,

5. Distances among

gastropores.

Ruiz Ramos et al. (2014). Zoological Studies

slide9

GOOD

BAD

Ruiz Ramos et al. 2014. Zoological Studies

slide10

BAD

GOOD

GOOD

Ruiz Ramos et al. 2014. Zoological Studies

slide11

Molecular Methods

Samples from 7 localities across the Caribbean were added for genetic analysis: Bocas del Toro (Panama), Grand Cayman, Mona (Puerto Rico - PR), Parguera (PR), Vieques (PR), Guadeloupe and Curaçao.

One gene approach: mtCOI a variable marker in hydrozoans

slide12

Unresolved Gene genealogy – COI gene

M. squarrosa

RED = M. complanata

BLUE = M. alcicornis(branching)

GREEN = M. alcicornis (encrusting)

Circle = M. striata

Diamonds: haplotypes shared among the morphotypes.

We cannot distinguish M. complanata from

M. alcicornis and M. striata.

Milleporasquarrosais sufficiently different, genetically.

slide13

Comparative transcriptomics

1) Presence/absence of transcripts

2) Differential expression

3) Sequence comparison

slide14

Study Design

Ion Proton (P1 chip) – One transcriptome per chip

2 transcriptomes M. alcicornis vs. 1 transcriptomeM. complanata

Test for consistency - 2 independent runs of M. alcicornis

M. alcicornisbranching

M. complanata

slide16

Pre-processing pipeline

Contamination-free sequences

RNA-seq data is aligned

to reference genomes

Symbiodinium and

Human genomes

slide17

Assembly pipeline

M. alcicornisRNA-seq data 1

M. alcicornis RNA-seq data 2

slide18

Post-Assembly Analysis Pipeline

  • Bowtie (Aligner) – Aligns raw RNA-seq reads against assembled transcripts.
      • Can be used to count the number of reads that are well represented in the assembled transcriptome.
      • Can be used for visualization of reads alignment, information about read depth.
  • RSEM – Also uses Bowtie to estimates gene and isoform expression levels from RNA-Seq data for each sample).
  • Merge - Create a matrix using the expected fragment count data produced by RSEM across samples used for differential expression analysis.
  • EdgeR- Estimates differential gene expression across samples (pairwise comparison).
slide19

Differential Expression between replicated data sets –M. alcicornis

Data from 253 putatively identified gene components suggests consistency between runs in Ion Proton

29 (11%)

224 (89%)

conclusions
Conclusions
  • Consistency between the two Ion Proton runs on M. alcicornis
  • Despite the high number of reads the resulting data sets were relatively small
  • Lack of a reference genome/transcriptome
  • We cannot explain the high contamination of rRNA(RiboMinus Kit–removes 12S and 18S rRNAs instead of the micropoly(A) Purist Kit which selects for mRNAs.
continuing work
Continuing work…
  • We are adding paired-end Illumina data for M. alcicornis and M. complanata.
  • Generate high quality transcriptomes of M. striata and M. squarrosa.
  • Perform transplant experiments and examine gene expression patterns in “native” vs. transplanted M. alcicornis colonies.
slide24

Acknowledgments

We thank the Seven Bridges Genomics: Sebastian Wernicke, Lu Zhang NemanjaIlic, and JelenaRadenkovic, for the bioinformatics analysis and guidance in this project.

We thank the NIH Cancer Genome Facilities for allowing to use the Ion Proton and their facilities.

The school of Arts and Sciences, University of Puerto Rico supported partially the travelling costs.

Puerto Rico Sea Grant for providing funds for additional Illumina runs and training of students in NGS techniques