Use Next-Generation Sequencing Data from Corals and other Cnidaria to Illuminate the Evolution …people.bu.edu/jrf3/BI550/BI550_Marine_Genomics/Week2_files/BI550_2013... · Bioinformatics

Bioinformatics Projects

Use Next-Generation Sequencing Data from Corals and other Cnidaria to Illuminate the Evolution of Genes

and Phenotypesby John R. Finnerty

Starlet AnemoneNematostella

vectensisfree-living saltmarsh

anemone

Lined AnemoneEdwardsiella

lineataparasitic, marine,

anemone

Cauliflower coral

Pocillopora damicornis

tropical indo-pacific hard coral

Northern star coralAstrangia poculata

temperate Atlantic & Caribbean coral

Corallimorphs“mushroom corals”

solitary animals, harbor photosynthetic

algaelike corals,don’t make skeletons.

The Starlet Sea Anemone Nematostella vectensis

“Stella”

New York Times“star-haired sword carrier”

Nematostella vectensisthe starlet sea anemone

Nematostella is an estuarine species native to saltmarsh habitats along the Atlantic coast of North America.

the holey bucket

Nematostella is simple and inexpensive to culture in the laboratory.

Nematostella reproduces sexually & asexually in the lab.

Nematostella develops rapidly.

Nematostella has extensive regenerative abilities.

Nematostella boasts a sequenced genome (& thoroughly sequenced transcriptome) (1 of only 3 cnidarians with a sequenced genome at this time).

“The sea anemone genome is complex, with a gene repertoire, exon-intron structure, and

large-scale gene linkage more similar to vertebrates than to flies or nematodes, implying that the genome of the eumetazoan ancestor

was similarly complex.

While Nematostella’s genome has evolved relatively conservatively over deep time, the geographic isolation of estuarine habitats appears to have spawned ample genetic and phenotypic polymorphisms between populations.

Genetic & Phenotypic Polymorphism

Temperature Adaptation

0

1

2

3

4

5

6

7

8

13°C 21°C 29°C

Tent

acle

Reg

ener

atio

n R

ate

(tent

acle

s pe

r day

)

South Carolina

New Jersey

southern Cape Cod

Boston

Nova Scotia

Nematostella is amenable to a wide range of organismal, molecular, and reverse-genetic protocols.



anemone



anemone

Cauliflower coral








The Lined Sea Anemone Edwardsiella lineata

comb jelly

parasites

larva

larva

Ecological Impact of the host Sea walnut in the black sea Introduced in early 1980’s in ship

ballast water. By 1990, its biomass was

estimated at 1 billion tons, or roughly the same as the biomass of all fish caught in the oceans.

The anchovy catch fell from 500,000 tons to 100,000 tons from early ‘80s to 1990.

Possible biological controls: the butterfishthe lined sea anemone Edwardsiella lineataAre they specialists?

Mnemiopsis leidyi Pleurobrachia pileus Beroe ovata

Does “Ed” specialize on the sea walnut?

n = 1,865 n = 600 n = 181infected: 34% infected: 96%infected: 0%

parasites/host: 3.41 parasites/host: 13.88

How does Ed gain entry to its hosts?Mnemiopsis (sea walnut) Beroe (“shark”)

20 / 50 hosts infected Burrows through body wall. Enters through mouth.

0 / 16 hosts infected. No entry even with contact. “Immune” response?

How does Ed gain entry to its hosts?

In 6 of 7 trials, when Beroe ate an infected sea walnut, it became infected.

(a novel finding.)

Based on this transfer rate, and knowing the average # of parasites in a sea walnut, the class was able to estimate that a particular

wild-caught Beroe had eaten ~67 sea walnuts

Does its host affect Ed’s development?

Mnemiopsis

Beroe

larva

adult

0

10

20

30

40

50

60

70

% d

evel

op in

to a

dult

0 10 20 30 40 50 60days after parasite is surgically excised from hostparasite

Why is there an effect of host species?

larva

adult

parasite

Beroe

Mnemiopsis%

dev

elop

into

adu

lt

20

40

60

100

length of parasite in millimeters

80

5 10 15

y=0.404Ln(x) + 0.0056R2=0.93p<0.0001

y=0.575Ln(x) - 0.223R2=0.74p<0.006

Marine Invertebrates

comb jelly

parasites

larva

larva

How has the parasite’s unique developmental program evolved?

parasite

larva

polyp

host absenthost present

New life cycle stage.New body plan.Novel developmental program.Novel developmental regulatory gene cascade.Novel gene expression pattern.



anemone



anemone

Cauliflower coral








Mushroom Corals(aka false corals; anemone corals)

order Corallimopharia

A lineage of hexacorallian anthozoansLikely sister-group to the hard corals (Scleractinia)50 speciesSolitary polyps (not colonial)Do not build skeletons.Some species form symbioses with photosynthetic zooxanthellae.Tentacles are reduced in length or absent.Can prey on macroscopic animals by enveloping them with their oral discs and long nematocysts.

Corallimorpharia(Stephenson 1937)

Marine Genomics, class of 2011

Transcriptomic sequencing of corallimorphs subjected to variable salinities (21, 31 or 41 ppt).

Discosoma sp



anemone



anemone

Cauliflower coral








Cauliflower CoralPocillopora damicornis

The Cauliflower Coral —Pocillopora damicornis

The Cauliflower Coral —Pocillopora damicornis

a new lab model being developed at BU populations that can tolerate diverse & challenging habitats harbors substantial within-species variation. among the coral stocks we are growing, dramatic differences in the ability to tolerate acute exposure to low salinity.



anemone



anemone

Cauliflower coral








Northern Star Coral Astrangia poculata

Astrangia is facultatively symbiotic

It can survive for prolonged periods in zooxanthellate/symbiotic or azooxanthellate/aposymbiotic states.It has an extremely large latitudinal range (Caribbean to Cape Cod).It is extremely cold-tolerant.

Madracis

Stenocyathus

caryophylliids

PocilloporidaeMadrepora

caryophylliids

siderastreids

siderastreids + Leptastrea

FungiidaePlocoidafaviinids + meandriinids

caryophylliids

AnthemiphylliidaePseudosiderastrea

Thalamophyllia

Galaxea

AgariciidaeAstreopora

Alveopora

Acropora, Anacropora, Montipora

Guynia

Stephanocoenia

BathycolaSiderastrea

Porites, Goniopora, Poritipora, Stylaraea

Dendrophylliidae

Acropora digitiferaAcropora milleporaAcropora palmata


Montastrea faveolata

Porites asteroidesPorites compressa

ROBUSTA

COMPLEXA

Astrangia



anemone



anemone

Cauliflower coral








Northern Star Coral Astrangia poculata

KINDS of QUESTIONS we could ask about the transcriptome.

HOW GOOD (COMPLETE) IS THIS Astrangia TRANSCRIPTOME?

SEQUENCING & ASSEMBLING A TRANSCRIPTOME






HOW GOOD (COMPLETE) IS THIS Astrangia TRANSCRIPTOME?

Certain multi-gene families have been well characterized in other Cnidaria.Assume that these gene families are generally well conserved between cnidarians so all members of the gene family are likely to be present in Astrangia.APPROACH: Search the transcriptome to identify all members of the gene family (if present) and determine whether (i) the full sequences are present and (ii) the structure of the proteins are conserved. CANDIDATE GENE FAMILIES:

HOX genesWNT genesGiMAP genesCnidocyte specific genes: e.g., Minicollagens and/or toxins

HOX GENESTranscription factors involved in setting up different regions of the anterior-posterior axis in triploblastic animals.Organized in conserved genomic clusters.

Involved in dramatic homeotic mutations.HOX GENES

Presence of conserved Hox clusters in Cnidaria and role during development is disputed.

HOX GENES

WNT GENESSecreted factors that help to control developmental decisions during embryogenesis.A “Wnt-code” may help to set up the oral-aboral axis of the cnidarian polyp.

GiMap GENESImplicated in innate immunity in mammals but absent in Drosophila, Caenorhabditis, and Nematostella.

BMC Genomics. 2013 Jun 14;14:400. doi: 10.1186/1471-2164-14-400.The acute transcriptional response of the coral Acropora millepora to immune challenge: expression of GiMAP/IAN genes links the innate immune responses of corals with those of mammals and plants.

Cnidocyte-specific GENES

The possession of cnidocytes is the defining characteristic of Cnidaria.The origin of this venom delivery cell-type involved the origin of new structural proteins (minicollagens) and toxin proteins.

PLoS One. 2011;6(7):e22725. doi: 10.1371/journal.pone.0022725. Epub 2011 Jul 28.Morphological and molecular analysis of the Nematostella vectensis cnidom.Zenkert C, Takahashi T, Diesner MO, Özbek S.

CAN WE IDENTIFY GENES CRITICAL TO CORAL BIOLOGY?

Genes involved in CaCO3 skeletal formation.

Nature. 2011 Jul 24;476(7360):320-3. doi: 10.1038/nature10249.Using the Acropora digitifera genome to understand coral responses to environmental change.Shinzato C, Shoguchi E, Kawashima T, Hamada M, Hisata K, Tanaka M, Fujie M, Fujiwara M, Koyanagi R, Ikuta T, Fujiyama A, Miller DJ, Satoh N.

“A number of genes with putative roles in calcification were identified, and several of these are restricted to corals.”

CAN WE IDENTIFY GENES CRITICAL TO CORAL BIOLOGY?

Genes expressed by Symbiodinum.PLoS One. 2012;7(4):e35269. doi: 10.1371/journal.pone.0035269. Epub 2012 Apr 18.Symbiodinium transcriptomes: genome insights into the dinoflagellate symbionts of reef-building corals.Bayer T, Aranda M, Sunagawa S, Yum LK, Desalvo MK, Lindquist E, Coffroth MA, Voolstra CR, Medina M.

Dinoflagellates are unicellular algae that are ubiquitously abundant in aquatic environments. Species of the genus Symbiodinium form symbiotic relationships with reef-building corals and other marine invertebrates. Despite their ecologic importance, little is known about the genetics of dinoflagellates in general and Symbiodinium in particular. Here, we used 454 sequencing to generate transcriptome data from two Symbiodinium species from different clades (clade A and clade B). With more than 56,000 assembled sequences per species, these data represent the largest transcriptomic resource for dinoflagellates to date. Our results corroborate previous observations that dinoflagellates possess the complete nucleosome machinery. We found a complete set of core histones as well as several H3 variants and H2A.Z in one species. Furthermore, transcriptome analysis points toward a low number of transcription factors in Symbiodinium spp. that also differ in the distribution of DNA-binding domains relative to other eukaryotes. In particular the cold shock domain was predominant among transcription factors. Additionally, we found a high number of antioxidative genes in comparison to non-symbiotic but evolutionary related organisms. These findings might be of relevance in the context of the role that Symbiodinium spp. play as coral symbionts.Our data represent the most comprehensive dinoflagellate EST data set to date. This study provides a comprehensive resource to further analyze the genetic makeup, metabolic capacities, and gene repertoire of Symbiodinium and dinoflagellates. Overall, our findings indicate that Symbiodinium possesses some unique characteristics, in particular the transcriptional regulation in Symbiodinium may differ from the currently known mechanisms of eukaryotic gene regulation.

DOES ASTRANGIA POSSESS MOLECULAR NOVELTIES

REPORTED IN OTHER CNIDARIA?“Complex NOD-like receptor repertoire” reported in the staghorn coral Acropora digitifera. This is a group of genes implicated in innate immunity.

Mol Biol Evol. 2013 Jan;30(1):167-76. doi: 10.1093/molbev/mss213. Epub 2012 Aug 30.The complex NOD-like receptor repertoire of the coral Acropora digitifera includes novel domain combinations.Hamada M, Shoguchi E, Shinzato C, Kawashima T, Miller DJ, Satoh N.


REPORTED IN OTHER CNIDARIA?Absence of key genes involved in circadian rhythm regulation.

“Corals exhibit circadian behaviors, but little is known about the molecular mechanisms underlying the regulation of these behaviors. We surveyed the recently decoded genome of the coral, Acropora digitifera, for photoreceptor and circadian genes, using molecular phylogenetic analyses. Our search for photoreceptor genes yielded seven opsin and three cryptochrome genes. Two genes from each family likely underwent tandem duplication in the coral lineage. We also found the following A. digitifera orthologs to Drosophila and mammalian circadian clock genes: four clock, one bmal/cycle, three pdp1-like, one creb/atf, one sgg/zw3, two ck2alpha, one dco (csnk1d/cnsk1e), one slim/BTRC, and one grinl. No vrille, rev-ervα/nr1d1, bhlh2, vpac2, adcyap1, or adcyaplr1 orthologs were found. Intriguingly, in spite of an extensive survey, we also failed to find homologs of period and timeless, although we did find one timeout gene. In addition, the coral genes were compared to orthologous genes in the sea anemone, Nematostella vectensis. Thus, the coral and sea anemone genomes share a similar repertoire of circadian clock genes, although A. digitifera contains more clock genes and fewer photoreceptor genes than N. vectensis. This suggests that the circadian clock system was established in a common ancestor of corals and sea anemones, and was diversified by tandem gene duplications and the loss of paralogous genes in each lineage. It will be interesting to determine how the coral circadian clock functions without period.”

Gene. 2013 Feb 25;515(2):426-31. doi: 10.1016/j.gene.2012.12.038. Epub 2012 Dec 20.A genome-wide survey of photoreceptor and circadian genes in the coral, Acropora digitifera.Shoguchi E, Tanaka M, Shinzato C, Kawashima T, Satoh N.


REPORTED IN OTHER CNIDARIA?An expansion in the number of fluorescent proteins.

“The vivid coloration of corals depends on fluorescent proteins that include cyan (CFP), green (GFP) and red (RFP) fluorescent proteins, and a non-fluorescent blue/purple chromoprotein. We examined how many genes encoding fluorescent proteins are present in the recently sequenced genome of the coral Acropora digitifera. Based on molecular phylogenetic analysis, we found one, five, one, and three candidate genes for CFP, GFP, RFP, and chromoprotein, respectively. The CFP and GFP genes are clustered in a ~80-kb-long genomic region, suggesting that they originated from an ancestral gene by tandem duplication. Since CFP and GFP possess the same chromophore, the gene clustering may provide the first genomic evidence for a common origin of the two proteins. Comparison between the fluorescent protein genes of closely related coral species suggests an expansion of chromoprotein genes in the A. digitifera genome, and of RFP genes in the A. millepora genome. The A. digitifera fluorescent protein genes are expressed during embryonic and larval developmental stages and in adults, suggesting that the genes play a variety of roles in coral physiology.”

Zoolog Sci. 2012 Apr;29(4):260-4. doi: 10.2108/zsj.29.260.Fluorescent protein candidate genes in the coral Acropora digitifera genome.Shinzato C, Shoguchi E, Tanaka M, Satoh N.



anemone



anemone

Cauliflower coral








Stella Ed Mushroom Cauliflower Star

sequenced genome YES NO NO NO NO

sequenced transcriptome YES YES YES YES YES

access via stellabase.orgor NCBI edwardsiellabase.org skinnybastard.bu.edu

(click “edbase”) pocilloporabase.org skinnybastard.bu.edu(click “stellabase”)



anemone



anemone

Cauliflower coral








Stella Ed Mushroom Cauliflower Star

sybiosis with Symbiodinium NO NO YES

(constitutive?)YES

(constitutive)YES

(facultative)

sequenced transcriptome YES YES YES YES YES

access via stellabase.orgor NCBI edwardsiellabase.org skinnybastard.bu.edu

(click “edbase”) pocilloporabase.org skinnybastard.bu.edu(click “stellabase”)

Use Next-Generation Sequencing Data from Corals and other Cnidaria to Illuminate the Evolution …people.bu.edu/jrf3/BI550/BI550_Marine_Genomics/Week2_files/BI550_2013... · Bioinformatics

Documents