Orchid Functional Genomics Database Special Issue - Database Orchidstra

41 Pages·2013·2.37 MB·English

Orchidstra: An Integrated Orchid Functional
Genomics Database
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Chun-lin Su1,3, Ya-Ting Chao1,3, Shao-Hua Yen1, Chun-Yi Chen1, Wan-Chieh Chen1, c
i
Yao-Chien Alex Chang2 and Ming-Che Shih1,* a
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1AgriculturalBiotechnologyResearchCenter,AcademiaSinica,Taipei11529,Taiwan F
o
2DepartmentofHorticultureandLandscapeArchitecture,NationalTaiwanUniversity,Taipei10617,Taiwan. c
3Theseauthorscontributedequallytothiswork. u
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*Correspondingauthor:E-mail:[email protected];Fax,[phone].
(Received November 9, 2012; Accepted January 5, 2013) Is
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A specialized orchid database, named Orchidstra (URL: Abbreviations: BLAST, basic local alignment search tool; enlo
http://orchidstra.abrc.sinica.edu.tw), has been constructed CAM, crassulacean acid metabolism; EIF5A, eukaryotic trans- –ad
e
toorchciodllefcutn,ctainonnoatlagteenaonmdicsshsatruedigeesn.oTmheicOinrcfohridmaacteiaoenisfoar lGateinoen Oinnittiaotloiogny;faKcEtoGrG5,AK;yEoStTo, eExnpcryecslsoepdedseiaquoefncGeentaegs; GanOd, Dad from
largefamilyofAngiospermsthatexhibitsextraordinarybio- Genomes; miRNA, microRNA; NGS, next-generation sequen- tab http
diversity in terms of both the number of species and their cing; SRA, sequence read archive; TSA, transcriptome s
distributionworldwide.Orchidsexhibitmanyuniquebiolo- shotgun assembly. ase://aca
gical features; however, investigation of these traits is cur- sde
m
rentlyconstrainedduetothelimitedavailabilityofgenomic ic
information.Transcriptomeinformationforfiveorchidspe- Introduction .o
u
p
cies and one commercial hybrid has been included in the .c
Orchidaceae, the orchid family, diverged from the Liliaceae o
Orchidstra database. Altogether, these comprise >380,000 m
andAmaryllidaceae,isthelargestfamilyofAngiosperms,with /p
non-redundant orchid transcript sequences, of which c
>110,000 are protein-coding genes. Sequences from the >800generaand>25,000species.Continuousidentificationof p/a
teritahnesrcrfirpotmomoeutpshuottrgeuandsafsrsoemmbnleyxt-(gTeSnAe)ratwioenreseqoubetanicniendg ngeenwosmpeeciaensdanrdepmeotilteicvuelasremquaernkecress)(,frocommbbointhedthweicthhloarolrpealadsyt rticle-a
complex morphological variations, means that systematic bs
technologies assembled into contigs, or from conventional orchid classification is a never-ending pursuit that continues tra
cDNA library approaches. An annotation pipeline using tochangeasnewcriteriaandevidenceemerge(Dressler1993, ct/5
Gene Ontology, KEGG and Pfam was built to assign gene Pridgeon et al. 1999, Chase et al. 2005, Pridgeon et al. 2005). 4/2
descriptions and functional annotation to protein-coding /e
Orchid family genomes are generally large, and vary 168-fold 11
genes. Deep sequencing of small RNA was also performed (1C=0.33–55.4pg)overall,indicatinggreatevolutionarydiver- /18
for Phalaenopsis aphrodite to search for microRNAs 7
sity(Leitchetal.2009).Thelargesizeandcomplexityofmost 7
(miRNAs), extending the information archived for this 43
orchidgenomestendtohampergenomicapproachestoorchid 9
species to miRNA annotation, precursors and putative b
research. y
target genes. The P. aphrodite transcriptome information g
The broad range of biodiversity seen among orchids pro- ue
was further used to design probes for an oligonucleotide s
microarray, and expression profiling analysis was carried vides a great opportunity for exploration of the unique and t on
intriguing features that evolved during the adaptation of 1
out. The intensities of hybridized probes derived from 3
the family to various environments that are not represented F
microarray assays of various tissues were incorporated into e
bymodelorganismssuchasArabidopsisandrice.Suchfeatures b
the database as part of the functional evidence. In the ru
includeflowerpatternformation,crassulaceanacidmetabolism a
future, the content of the Orchidstra database will be ry
(CAM) photosynthesis to assimilate carbon at night, epi- 2
expanded with transcriptome data and genomic informa- 0
phytic habitat with high water and nutrient usage efficiency, 19
tion from more orchidspecies.
unique seed development, symbiosis with mycorrhizae, and
Keywords: Annotation (cid:2) Expression profile (cid:2) many others. Besides their biological novelty, orchids are
Next-generation sequencing (cid:2) Orchidaceae (cid:2) Transcriptome also of great commercial interest. Taiwan is one of the
shotgun assembly. majororchid-producingandexportingcountriesintheworld.
PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004,availableonlineatwww.pcp.oxfordjournals.org
!TheAuthor2013.PublishedbyOxfordUniversityPressonbehalfofJapaneseSocietyofPlantPhysiologists.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense
(http://creativecommons.org/licenses/by-nc/3.0/),whichpermitsunrestrictednon-commercialuse,distribution,
andreproductioninanymedium,providedtheoriginalworkisproperlycited.
PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004 !TheAuthor2013. 1
C.-l.Su.etal.
Together with other countries such as Japan, the USA, associated with the assembled contigs or expressed se-
China, The Netherlands and countries in Southeast Asia, they quence tags (ESTs) accessible to the public. We hope to pro-
sharealargemarketoforchidtradingandhavebuiltanindus- mote orchid functional research with rich sequence
try of orchid nurseries maintained by advanced greenhouse information in association with functional annotation. Since
facilities. genomicresearchoftenconsumesalargeamountofresources,
Despitebeinganimportantfamily inthePlantae,genomic focusing on a model orchid species is important for in-depth
information about orchids has been relatively scarce until research. Our research team applied NGS technologies to
recently. Rapid advances in DNA sequencing technology obtain transcriptome shotgun sequence and developed a
known as next-generation sequencing (NGS or massively par- streamlined process for de novo assembly followed by the
allelsequencing)inrecentyearshaveledtowideandpopular annotation of a potential orchid model species, P. aphrodite
applications in genomic research generating a large volume (Suetal.2011).
D
of sequence information and causing a drop in per-base cost The original Orchidstra database was constructed mainly o
w
(Wall et al. 2009, Metzker 2010). Abundant orchid sequences based on the transcriptomic information including sequences n
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includingourownresearchresultshavebeendepositedinthe andannotationsofP.aphroditederivedfromapreviousstudy a
d
e
GenBankTSA/SRAdatabase(transcriptomeshotgunassembly/ (Suetal.2011).Afterthedevelopmentofmethodologyforde d
sequencereadarchive)sincethetechnologyhasbeenavailable. novo assembly and annotation of transcriptome information, fro
m
Anotherimportantmilestoneinrecentgenomicresearchisthe weexpandtheapplicationstothetranscriptomicinformation h
developmentofbioinformaticprocesses,especiallyfordenovo collectedfromvariousorchidspecies.Moregenomicdatasuch ttp
s
assembly when the reference genome is unavailable. Various as miRNA information and expression profiles of P. aphrodite ://a
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strategies, algorithms and software have been developed, havebeencontinuouslygeneratedbyuseversince.Withmore ad
e
resulting in rapid accumulation of sequence data of many sequence information available including those of multiple m
ic
non-model organisms in the databases (Surget-Groba and orchid species retrieved from the internet database, small .o
u
Montoya-Burgos 2010, Su et al. 2011, Zhang et al. 2011). NGS RNA data as well as expression profiles, there is a need to p
.c
techniquesarealsoappliedtotheidentificationanddetection update the Orchidstra database for the purposes of studies om
of small functional RNA, especially microRNA (miRNA) of comparative genomics and functional genomics. The /p
c
p
(Gustafson etal.2005, Johnsonetal.2007, Simonetal.2009). Orchidstra database is now expanded and reconstructed to /a
In addition, high-throughput sequencing techniques provide integratecomplexgenomicinformation. rtic
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an alternative tool for gene expression profiling known as In order to enrich useful transcriptome resources for -a
b
RNaseq(MartinandWang2011,Tariqetal.2011).Withdevel- researchers interested in orchid functional studies and com- s
tra
opments of the technique, computation and application, parativegenomics,wedownloadedtheTSA/ESTdataofseveral c
NGS has revolutionized modern genomic research with rich orchid species from GenBank to carry out further analysis. t/54
/2
information,highpaceandlowcost. Altogether, transcriptomic information from six orchids was /e
1
Severaldatabasesspecializedtocertainorchidspecieshave collected in the database we built (for illustration, see 1
/1
beenpreviouslyestablishedwithadditionalfunctionalannota- Supplementary Fig. S1). All of these orchids belong to the 8
7
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tion. OrchidBase (URL: http://http://orchidbase.itps.ncku.edu. Epidendroideae, one of the five subfamilies of Orchidaceae. 4
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tw/est/home2012.aspx) stores sequences of expressed tran- Epidendroideae is the largest subfamily, with >500 genera 9 b
y
scriptsfromthreePhalaenopsisspecies,P.aphrodite,P.equestris and >20,000 species. Most members of this subfamily are g
u
and P. bellina, obtained using a combination of conventional tropical epiphytes, some with pseudobulbs (Dressler 1990). e
s
Sanger sequencing and the high-throughput sequencing plat- Many genera in this subfamily such as Phalaenopsis, Cattleya, t o
n
forms, Roche 454 and Illumina Solexa (Fu et al. 2011). This Oncidium,DendrobiumandCymbidiumareofmajorcommer- 1
3
database offers 8,501 assembled contigs and 76,116 from cial value in the world floral market. However, not all tran- F
e
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cDNA libraries built from various tissues, resulting in 84,617 scriptome sequences of orchid species in GenBank are ru
a
non-redundant transcribed sequences. The sources of the abundant enough or their library sources cover insufficient ry
cDNA library covered 11 tissues from the three species representative plant tissues to reach the level of depth for a 20
1
(Fu et al. 2011). Another database, OncidiumOrchidGenome faircomparison.GenomicinformationofP.aphroditeisstillthe 9
Base (URL: http://predictor.nchu.edu.tw/oogb/) was built most abundant that is available to carry out a thorough
specifically for the transcriptome sequences of Oncidium analysis.
Gower Ramsey (Chang et al. 2011), a commercial hybrid. The ThemainpurposeofconstructingtheOrchidstradatabase
authors applied Roche 454 pyrosequencing techniques to istosharerichgenomicinformationwithresearchersinorder
obtain sequence data from six tissues, including many flower tofacilitatemolecularbiologicalresearch,includingfunctional
stages, and generated reads for assembly. This database offers studiesofgenes,interactingnetworksandregulatorymechan-
50,908assembledcontigsand120,219singletonsthatledtothe isms of orchid biology. The moth orchid, P. aphrodite, with
discoveryofflowering-associatedgenes. abundant genomic information in our database, may serve
Although sequence information is growing in public as a research model system for exploring many interesting
databases, proper and sufficient annotation is often not anduniquebiologicalfeaturesinorchids.
2 PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004 !TheAuthor2013.
Orchidfunctionalgenomicsdatabase
Database Contents Erycina pusilla. Erycina is closely related to the genus,
Oncidium,andalsobelongstotheTribeCymbidieae,Subtribe
Orchid species and library source
Oncidiinae.Erycinahasadifferentmorphologyandphysiology
Currently, five species and one hybrid of orchids are included fromPhalaenopsisorchids.Erycinaismainlydistributedintrop-
in the Orchidstra database (Supplementary Fig. S1). These ical America and has a diploid (2n=2x=10) genome of 1C=
speciesrepresenttheorchidspeciesforwhichthemostabun- 1.5pg(Chaseetal.2005).TranscriptomeinformationforErycina
dantsequenceinformationisdepositedinGenBank.Theyare wasbuiltinthesamewayaswebuiltitforP.aphrodite.Erycina
P.aphrodite,P.equestris,P.bellina,Erycinapusilla,Dendrobium has the advantage of small plant size, short life cycle
nobile and Oncidium Gower Ramsey. We generated NGS data andyear-roundblooming,suggestingitssuitabilityforuseasa
forthetwoofthem,P.aphroditeandE.pusilla.Sequencesofthe modelorchid.
otherfour,P.equestris,P.bellina,D.nobileandOncidiumGower
Ramsey, were downloaded from GenBank in NCBI. A detailed Dendrobium nobile. The genus Dendrobium belongs to the Do
w
descriptionofthelibrariesandsequenceinformationisgivenin Tribe Podocjilaeae, Subtribe Dendrobiinae. There are about n
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theMaterialsandMethods. 1,200speciesinthegenusDendrobium,distributedthroughout ad
most of Southeast Asia and the Southwest Pacific islands. ed
Phalaenopsisaphrodite. ThegenusPhalaenopsis(alsoknown Dendrobium has a genome size of 1C=0.75–5.85pg (Jones fro
m
as moth orchids) belonging to tribe Vandeae contains >60 et al. 1998). The Dendrobium sequence was obtained from h
species and is an important source of breeding parents for NCBI (Liang et al. 2012). A collection of 15,017 ESTs from the ttps
commercial hybrids. TheTaiwan native P. aphrodite is diploid vernalizedaxillarybudsandvegetativetissuesofD.nobilewere ://a
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(2n=2x=38) with a genome size estimated as about assembled for 9,616 unique gene clusters (Liang et al. 2012). ad
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1C=1.4pg (Lin et al. 2001). The transcriptome sequence of Dendrobium spp. is known to botanists for both its value in m
this Phalaenopsis species was obtained by assembly of reads theornamentalflowermarketanditsuseintraditionalherbal ic.o
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from high-throughput sequencing techniques, namely Roche medicine. p.c
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454andIlluminaSolexa(Suetal.2011).Phalaenopsisaphrodite m
Data processing and contents
is an epiphyte with a CAM photosynthesis pattern and has /pc
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large white flowers that are of interest to orchid breeders. The outline of the analysis process pipeline is shown in a /a
Phalaenopsis is indigenous throughout most of Southeast flow chart (Fig. 1A). TSAs of contigs or singletons >200bp rtic
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AsiaandAustralia. were first searched by BlastX against the NCBI nr database -a
b
for potential open reading frames and similarity to currently s
Phalaenopsis equestris. Also native to Taiwan, P. equestris is known genes. Altogether, 381,918 non-redundant TSAs were trac
closely related to P. aphrodite, with a similar geological distri- stored in the Orchidstra database and divided into 114,933 t/54
bution.ThediploidP.equestris(2n=2x=38)hasagenomesize protein-coding and 266,985 non-coding TSAs (Table 1). /2/e
1
estimatedtobe1C=1.69pg(Linetal.2001).Thecharacteristic The protein-coding TSAs are annotated with terms identified 1
/1
large number of small flowers on the stalk of P. equestris is inGeneOntology(GO;GeneOntologyConsortium2013),Pfam 8
7
also of interest to breeders and it is often used as a breeding (Finnetal.2010)andKEGG(KyotoEncyclopediaofGenesand 74
3
parent. The transcript sequences of P. equestris and P. bellina Genomes; Tanabe and Kanehisa 2012) (Table 2). Annotation 9 b
have been published (Tsai et al. 2006), and were downloaded procedures were performed as described previously (Su et al. y g
u
fromNCBIandannotatedin-house. 2011).CorrespondinghomologstoArabidopsisandricearealso e
s
listed,withE-valuesandidentitytoindicatesequencesimilarity. t o
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Phalaenopsis bellina. Phalaenopsis bellina (2n=2x=38) is ThedatabasewebuiltwasnamedOrchidstra(URL:http:// 1
3
one of few Phalaenopsis with fragrance and has a genome orchidstra.abrc.sinica.edu.tw), a combination of the words F
e
sizeof1C=7.52pg(Linetal.2001). ‘orchid’and‘orchestra’,torepresenttheharmoniousinterplay bru
among collections of genes to bring about the beauty of ary
Oncidium Gower Ramsey. Oncidium is a genus with about orchids. Orchidstra is a web-based open-access database with 20
1
330 species under Tribe Cymbidieae, Subtribe Oncidiinae. value-addedannotationsincludinggeneexpressionprofilingas 9
Oncidium has a genome size of 1C=0.6–5.73pg (L. Hanson, functional evidence. TSA sequences, gene descriptions and
I.J. Leitch and M.D. Bennett, unpublished data from the functional annotation such as GO, KEGG and Pfam were
Jodrell Laboratory, Royal Botanic Gardens, Kew, 1999). assignedtoprotein-codingTSAs.
Oncidium spp. and hybrids are important in the flower Structural RNAs including rRNA, tRNA, small nuclear RNA
market mostly as cut flowers. Oncidium Gower Ramsey is a (snRNA) and small nucleolar RNA (snoRNA) were identified
popular commercial hybrid with a complex breeding back- and separated from mRNA with Rfam (http://rfam.sanger.ac.
ground. Sequence information of this hybrid was mainly con- uk/) and the Silva database (http://www.arb-silva.de/).
tributed by the group building the OncidiumOrchidGenome Phalaenopsisaphroditelongnon-codingRNAs(lncRNAs)that
Basedatabase(Changetal.2011)andsomeESTs contributed hadahighdegreeofnucleotidesequencehomologywithmany
byotherresearchers. other plant species were identified by multiple sequence
PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004 !TheAuthor2013. 3
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Fig. 1 Overview of the information process pipeline for next-generation sequencing data. (A) Flow chart of the sequence data process
pipeline (for details see the Materials and Methods). (B) Data content of Phalaenopsis aphrodite in the Orchidstra. Expressed
transcripts (TSA for transcriptome shotgun assembly) were cross-linked to miRNA (SR for small RNA) by the target genes and precursors
identification.
4 PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004 !TheAuthor2013.
Orchidfunctionalgenomicsdatabase
Table 1 Statistics of transcriptome shotgun assemblies (TSAs) in the Orchidstra database
Orchid species No. of No. of No. of Average N50c Tissue sourced Data Expression
coding nc TSAsa total lengthb (bp) sourcee profiling
TSAs TSAs (bp)
Phalaenopsis aphrodite 42,573 191,233 233,806 875 405 R, L, S, F, PE, ST, FB, IN, PC TSA Yes
Erycina pusilla 31,515 51,550 83,065 783 533 R, L, F, PE TSA NA
Oncidium Gower Ramsey 26,786 20,900 47,686 668 619 L, F, PB, IN, FB TSA, cDNA NA
Dendrobium nobile 10,515 3,302 13,817 639 669 L, S, AB cDNA NA
Phalaenopsis equestris 2,401 0 2,401 631 684 FB cDNA NA
Phalaenopsis bellina 1,143 0 1,143 686 740 FB cDNA NA
Total 114,933 266,985 381,918 773 494 D
o
ancTSA,non-coding TSA. w
bAverage lengthofthenucleotide sequence ofprotein-coding TSAs. nlo
c N50oftotal assembledTSAs. ad
d Code for tissue sources: R, root; L, leaf; S, stem; F, open flower; PE, pedicel; ST, stalk; IN, inflorescence; FB, flower bud; PB, pseudobulb; AB, auxiliary bud; ed
PeCD,aptarostooucorcrem:.TSA,transcriptome shotgun assembly sequence;cDNA,reverse transcriptase-mediated cDNA library. from
h
ttp
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Table 2 Statistics of functional affiliates in the Orchidstra database ad
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Orchid species No. of No. of TSAs with m
ic
coding TSAs Pfam GO KEGG Rice homolog At homolog .o
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Phalaenopsis aphrodite 42,573 24,084 16,701 15,216 23,002 24,205 .c
o
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Erycina pusilla 31,515 20,731 16,229 15,932 22,686 21,833 /p
c
Oncidium Gower Ramsey 26,786 12,189 24,283 12,562 16,671 18,579 p
/a
Dendrobium nobile 10,515 7,429 7,706 1,388 7,981 7,761 rtic
Phalaenopsis equestris 2,401 1,667 1,335 1,675 1,852 1,805 le-a
b
Phalaenopsis bellina 1,143 839 746 1,093 949 934 s
Total 114,933 66,939 67,000 47,866 73,141 75,117 trac
t/5
RicehomologswereobtainedfromBlastagainstMSURiceGenomeAnnotationProjectRelease7,andAt(Arabidopsisthaliana)homologswerefromtheArabidopsis 4
TAIR10 release. /2
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1
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alignment and were grouped together in the database. in the Orhcidstra database. The resulting expression profiles 74
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Precursors of miRNA were also discovered in the non-coding generated from hybridization of probes labeled by RNA ex- 9
b
TSAsusingasmallRNAanalysispipeline. tractedfromvarioustissueswereshowninaheatmappattern y g
In addition to long expressed transcripts, analysis of deep to indicate their relative expression level with a color-coded ue
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sequencing results of small RNA for P. aphrodite provides gradient. This information should be useful for determining t o
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miRNA annotation, precursors and putative target genes for tissuesforamplificationofgenesofinterestbyPCR. 1
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this species. Small RNA was isolated from leaf, root, flower F
and germinating seeds (protocorm) of P. aphrodite and sub- Accessory tools for analysis ebru
jectedtoSolexaforsequencing.Weidentified3,251P.aphrodite Inordertocreateauser-friendlyenvironment,usefulaccessory ary
miRNAsequencesfor88publiclyknownplantmiRNAfamilies functions are provided on the front page or under the Tool 20
1
gathered from GenBank SRA050114. Each miRNA has its bar (Supplementary Fig. S1). Users have options to initiate 9
own page to display annotation together with the expression the search for a gene of interest. First, a search box on the
level in various tissues, precursors and predicted target genes; home page provides a quick choice of species, type of tran-
if applicable, the relevant internal links to corresponding TSA scripts and keyword. Secondly, an advanced search in the
data(includingprecursorsandtargetgenes)arealsoprovided. ‘Tools’ provides more detailed criteria for finding the TSA.
Sequences from TSA and small RNA are cross-linked by the Thirdly, one can use Blast, also in the ‘Tools’, to find the TSA
identificationofprecursorandtargetgenes(Fig.1B). if thehomologoussequenceisavailable. Orchidstra integrates
theBLASTutilitybyprovidingaweb-basedinterfacetosearch
Gene expression profiling
againstthelocalsequencedatabasesofallspeciesinOrchidstra.
To enrich functional annotation for P. aphrodite, we included Thisfunctioncaneasilybeexecutedandnavigated.Inaddition,
theresultsofmicroarrayanalysisinthetranscriptinformation an advanced search function provides improved data search
PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004 !TheAuthor2013. 5
C.-l.Su.etal.
accuracy with input keywords and related information. Users available in graphics (Fig. 2A). Pfam domains can be directly
canalsobrowseGOtermsorKEGGpathways(ECorKnumber linked to the Sanger Institute Pfam database (http://pfam.
fromtheKEGGdatabase,http://www.genome.jp/kegg/)inthe sanger.ac.uk/) by the corresponding protein family (PF)
database. GO analysis charts and KEGG pathways are also numberforamoredetaileddescription.
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Fig.2 ExamplesoffunctionalannotationintheOrchidstradatabase.(A)KEGGpathwayingraphicview.Steroidbiosynthesisisdemonstrated
here.TheECnumberwithinacoloredboxindicatesgeneswithP.aphroditeidentity.(B)Comparisonofexpressionprofilesofmultiplegenes
showstissueexpressionpatterns.
6 PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004 !TheAuthor2013.
Orchidfunctionalgenomicsdatabase
Besidesafunctionalannotationsearch,Orchidstraprovides
avisualdisplayofmicroarraydatausingacolorcharttoshow
relative intensities of signals among tissues. Users can use the
functionof‘Expressionprofile’underthe‘Tools’toinputmul-
tiplesequencecontigIDstogenerateanintegratedexpression
profile(Fig.2B).
Cross-species comparison
Manyofthegenomicdatabaseswerecomprisedofinformation
on a single species as a model organism for research. The
Arabidopsis 1001 genome project was intended for whole-
genome sequencing of 1,001 Arabidopsis ecotypes (Cao et al. D
o
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2011, Schneeberger et al. 2011) and to build a database for n
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comparative purposes (http://www.1001genomes.org/index. a
d
html). Another example is the Sol Genomics Network ed
(http://solgenomics.net/) which consists of genomic informa- fro
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tionontomato,tobacco,potato,petuniaandpepper,allwithin h
theSolanaceaefamily.Informationincludingsequencesofgen- ttp
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omes and transcriptomes, mutant phenotypes and available ://a
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lines, quantitiative loci and markers, and many features are a
d
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presentintheSolGenomicsNetworkforcomprehensiveana- m
lysis(Bombarelyetal.2011). ic.o
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Orchidstra was designed to integrate information across p
.c
orchid species and to serve as a general reference source in Fig. 3 Comparison of Arabidopsis and rice homologs of expressed om
support oforchid functional genomics research. Since orchids TSAs among four orchid species in the Orchidstra database. (A) /p
c
adapt to widely distributed habitats and evolve into a large Number of Arabidopsis homologs. (B) Number of rice homologs. p/a
number of species, it is rational to believe frequent events of The number in brackets indicates the total number of homologs rtic
gene variation and natural selection may have occurred. found in the species. Pa, Phalaenopsis aphrodite; Ep, Erycina pusilla; le-a
Og,OncidiumGowerRamsey;andDn,Dendrobiumnobile. b
Incorporationoftranscriptomedatafrommultipleorchidspe- s
cies can broaden the information base available for compara- trac
tive analysis within and between species, especially in the t/54
absenceofareferencegenomeformostorchids. sequencing depth and tissue sources of transcriptome, and /2/e1
Due to incomplete transcriptome information of the cur- 1
genomevariationsbetweenspecies. /1
rentdatabasecollectionincludinganunevendepthofsequen- 8
cingeffortsandvarioustissuesfromwhereRNAsampleswere We selected the eukaryotic translation initiation factor 5A 774
(EIF5A) gene as an example to demonstrate the usage of 3
etixntgruacisthedg,eitneissdoifffitchuoltsetocmomakmeoannlyovsheraarleldcoammpoanrgisopnlatnotsdiosr- the Orchidstra database for phylogeny analysis. The amino 9 by g
acid identities of EIF5A between plants we selected are high, u
uniquelyownedbycertainspecies(Table1). e
A Venn diagram was plotted for comparing genes in ranging from 84% to 97% (Fig. 4A). Phylogeny analysis st o
indicated that these EIF5A orthologs are highly conserved n
common among orchid species. The number of homologous 1
TSA sequences derived from sequence alignment to among plant species and that sequence diversity exists 3 F
Arabidopsis and rice wascompared (Fig. 3A,B). Phalaenopsis amongorchids(Fig.4B). ebru
equestrisandP.bellinawereexcludedfromthisanalysisbecause ary
of their small sample sizes in TSA number. The number of Conclusions and Future Implementation 201
homologs from D. nobile isalso low forcomparison, although 9
notexcluded.HomologscommonlysharedamongP.aphrodite, Orchidbiologyresearchhasgainedmomentuminrecentyears
E. pusilla and Oncidium Gower Ramsey (Fig. 3; 2,496 due to significant value of the commercial market and the
Arabidopsis homologs and 2,422 for rice) make up approxi- unique biological features exhibited by orchids attracting
mately20%ofthetotalcodingTSAs.TheseTSAsareprobably researcher’sinterest.Technologicaldevelopmentssuchasmo-
responsibleforthefundamentalphysiologyfoundacrosshigher leculartoolsandavailabilityofgenomicsequenceinformation
plantspecies.From12%to16%ofthehomologsinP.aphrodite have also contributed to the research progress. Whether to
areunique.Thismayreflectrichsequenceinformationinclud- facilitate biotechnological development for horticultural pur-
ing the number and length of the assembled contigs derived posesorforfundamentalresearchpurposes,easyaccesstorich
from P. aphrodite. The interspecies variation in number genomic information about the orchid family will facilitate
of homologs may reflect a combination of differences in in-depth research on the molecular function and regulation
PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004 !TheAuthor2013. 7
C.-l.Su.etal.
D
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h
ttp
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ic
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Fig.4 SequencecomparisonsofEIF5Agenes.(A)MultiplesequencealignmentrevealshighaminoacididentityofEIF5Abetweenplantspecies. 1
1
(B)PhylogeneticanalysisofEIF5Afromvariousspecies.ContigidisusedforEIF5AoforchidspeciesthatcanbefoundintheOrchidstradatabase, /1
8
whileotherspeciesaregivenaGenBankid. 77
4
3
9
b
y
g
u
e
s
ofgenesaswellasareasofevolutionaryandecologicalinterest. orchidgenomicinformationdatabaseshouldfacilitatemolecu- t o
n
NGS technology has successfully reduced the cost and effort lar research on gene functions and regulatory mechanisms 1
3
required for fast accumulation of sequence information. ofthemanyinterestingbiologicalfeaturesoforchids. F
e
Here, we built an informatics data processing pipeline to b
ru
assemble reads into contigs and functionally annotate a
ry
genes from de novo organisms effectively and constructed a Materials and Methods 2
0
1
database, Orchidstra, which can serve as a gateway to access 9
Library construction and data sources
informationabouttheabundanceofgenesexpressedinorchid
species. Phalaenopsis aphrodite.
In the future, we intend to generate and collect more Tissuesource:vegetative(roots,stem,leaf);reproductive(stalk,
sequence information from other orchid species especially in flowerbuds, younginflorescences, flowers offull blossom and
different subfamilies. Since there are a great number of spe- senescence); germinating seed (protocorm formation, proto-
cies in the orchid family, it will be intriguing to understand cormdevelopmentandseedlingformation)
the phylogeny of gene families during the course of their Libraryconstructionanddatasource:Suetal.(2011)
evolution. Providing more transcript sequences from species Sequencingtechniques:IllumiaSolexaandRoche454platform
indifferentorchidsubfamilies shouldbehelpfulinpromoting NCBI accession numbers: SRA030409, SRA050114; TSA:
orchid comparative genomic studies, and a comprehensive JI626343–JI831113
8 PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004 !TheAuthor2013.
Orchidfunctionalgenomicsdatabase
Sequences obtained: 246,242 contigs (from SRA030409) and FJ237035, FJ237036, FJ237037, FJ237038, FJ237040, FJ348573,
22,829,317uniquereads(fromSRA050114) FJ618566, FJ618567, FJ859988, FJ859989, FJ859990, FJ859991,
FJ859993, FJ859994, FJ859995, FJ859996, HM140840,
HM140841, HM140842, HM140843, HM140844, HM140845,
Phalaenopsis equestris.
HM140846, HM140847, HM146076, HM146077, HQ585983,
Tissuesource:flowerbud HQ585984,HQ591455
Libraryconstructionanddatasource:Tsaietal.(2006) Sequencesobtained:48,380contigs
Sequencingtechnique:Sangersequencing
Data processing—sequence analysis and
NCBI accession numbers: CK855526–CK857579, CB031751–
functional annotation
CB035289,BU744268–BU744277,CK901119
Sequencesobtained:2,455ESTs(afterdatacleanup) Raw data were obtained from three sources: local NGS data
D
for whole transcriptomes and small RNA transcriptomes, o
w
GenBank ESTs (Benson et al. 2012) and GenBank TSAs (for n
Phalaenopsis bellina. lo
the process pipeline, see Fig. 1A). After assembly, sequences a
d
Tissuesource:flowerbud with high similarity to sequences of potential ‘contaminants’ ed
Libraryconstructionanddatasource:Tsaietal.(2006) suchasbacteria,virusesandchloroplastswereremovedpriorto fro
m
Sequencingtechnique:Sangersequencing annotation. Blast2GO was incorporated into the autoannota- h
NCBI accession numbers: CK857580–CK859399, CO742089– tion pipeline for functional annotation and possible pathway ttps
CO742627 analysis (Gotz et al. 2008). After annotation procedures, ://a
c
Sequenceobtained:1,208ESTs(afterdatacleanup) description of the best BLAST (Altschul et al. 1990) hit, the ad
e
GO terms (Gene Ontology Consortium 2013), Pfam domains m
Erycina pusilla. ic
(http://pfam.sanger.ac.uk/) (Finn et al. 2010), enzyme codes .o
u
Tissuesource:leaf,root,flower,pedicel and corresponding KEGG pathway (http://www.genome.jp/ p.c
Libraryconstructionanddatasource:unpublisheddata kegg/) (Tanabe and Kanehisa 2012) were assigned to every om
Sequencingtechnique:IllumiaSolexaandRoche454platform protein-coding TSA that meets the specified threshold. /pc
p
NCBIaccessionnumber:SRA037585.1 Non-coding RNAs were annotated according to the similarity /a
Sequencesobtained:88,203contigs searchofthepublicdatabasefornon-codingRNAfamiliesand rtic
le
structured RNA elements including Rfam (http://rfam.sanger. -a
b
Dendrobium nobile. ac.uk/), Silva (http://www.arb-silva.de/) and miRBase (http:// s
tra
www.mirbase.org/) (Griffiths-Jones et al. 2008). Non-coding c
Tissue source: vegetative (auxiliary bud) and seedling RNAs showing a high degree of similarity between various t/54
(leafandstem) /2
Libraryconstructionanddatasource:Liangetal.(2012) specieswereidentifiedusingUniGenedata.Thesequencean- /e1
notation information and data from microarray experiments 1
Sequencingtechnique:Sangersequencing /1
were included in the database. The protein-coding TSAs and 8
NCBI accession numbers: HO189246–HO204626, JQ063042, 77
non-codingRNAsinOrchidstracanbeaccessedandanalyzed 4
JQ063043, JQ063457, JQ063458, JQ063459, JQ063460, 3
AY608889, DQ462460, DQ462469, EF535598, EF535599, byvariousonlinetoolsandservices,includingavarietyofkey- 9 by
wordsearchorbrowseoptions,visualizationofmicroarraypro- g
GR410230, GR410231, GU357498, GU382674, GU382675, u
filingofvarioustissuesandaBLASTserverfordatabasesearch. e
HQ388352 Thesequencesandannotationscanberetrieveddirectlyfrom st o
Sequencesobtained:15,398ESTs n
thedatabase. 1
3
F
Oncidium Gower Ramsey. Database construction e
b
ru
Tissuesource:leaf,pseudobulbs,younginflorescences,inflores- Nucleotidesequencesinfastaformat,blastresults,annotations ary
cences,flowerbuds,matureflowers includingGOresults,KEGGresults,Pfamandhomologsearch 2
0
1
Libraryconstructionanddatasource:Changetal.(2011) results were stored. To access these genomic resources in a 9
Sequencingtechnique:Roche454andSangersequencing user-friendlyandaccurateway,acustomizeddatabasewasde-
NCBIaccessionnumbers:HS521830–HS524732andJL898334– signed and built, Orchidstra. Orchidstra is a web application
JL943742; AF276233, AF276234, AF276235, AF276236, builtusingthree-tierarchitecture.ItrunswiththeApacheWeb
AF276237, AY196350, AY496865, AY940147, AY940148, server and MySQL database in Linux OS. PHP and JavaScript
AY953937, AY953938, AY953939, AY973631, AY973632, scripts were used to create the user interface coupled with
AY973633, AY973634, AY974325, AY974326, AY974327, MySQL, a relational database management system. The URL
DQ289592, DQ289593, DQ289594, DQ289595, DQ302727, address of the Orchidstra database is http://orchidstra.abrc.
EF570111, EF570112, EF570113, EF570114, EF570115, sinica.edu.tw. Current features include resource browsing and
EF570116, EU130454, EU130455, EU130456, EU130457, online tools such as searching, blasting and linking to corres-
EU130458, EU130459, EU130460, EU583501, EU583502, pondingPfam,GOandKEGG.
PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004 !TheAuthor2013. 9
C.-l.Su.etal.
Expression profiling sequencingdataarecleanedup.Forexample,PASRrepresents
smallRNAidinP.aphrodite.
Expression profiles of P. aphrodite were analyzed using a
We defined the protein-coding TSAs as the transcript
custom-madeorchidmicroarray.Thismicroarraywasdesigned
assemblies which matched against the NCBI nr database with
based on the transcriptome sequences we obtained from
anE-valueof(cid:4)1e-10,aswellasthenon-codingtranscriptswith
high-throughput sequencing and printed on glass slides by
anE-valueof>1e-10.RelativedegreesofhomologyfromBlast
Agilent eArray and the SurePrint platform (Agilent). The first
results were applied to assign gene identity for orchid TSAs
version of the orchid biochip featured 67,038 probes from
using terms such as homolog, similar to, weakly similar to
43,662 annotated genes and two popular orchid viruses,
andputativeprotein(Suetal.2011).
Ondontoglossum ringspot virus (ORSV) and Cymbidium
Homologs to Arabidopsis and rice were defined when the
mosaic virus (CymMV). RNAs isolated from root, leaf,
TSAs were submitted to BlastX against the protein databases
full-bloom flowers, flower buds, stalk, young inflorescences
D
with scale leaves, and small buds were labeled and hybridized of Arabidopsis TAIR10 (Lamesch et al. 2012) and MSU Rice ow
Genome Annotation Project Release 7 (Ouyang et al. 2007) n
to the microarray. Signals detected were globally normalized lo
withanE-value(cid:4)1e-20. a
andcomparedusingGeneSpringGX7.3software(Agilent).The d
e
d
intensitiesofthesignalsweredisplayedonacolor-codedchart fro
fortheconvenienceofviewers. Supplementary data m
h
ttp
Phylogenetic analysis SupplementarydataareavailableatPCPonline. s
://a
Theaminoacid sequenceoftheEIF5A geneusedinthephyl- ca
d
ogenyanalysis wasdownloaded fromtheOrchidstradatabase Disclaimer em
or NCBI GenBank. The sequence was aligned by Mega5 ic
(Tamura et al. 2011) with the Neighbor–Joining method Theauthorsacceptnoliabilityfortheaccuracyofthesequence .oup
(Saitou and Nei 1987). The result of the phylogeny tree was or expression information. Validation by users is strongly .co
encouraged. m
evaluated by bootstrap resampling of 1,000 replicates /p
c
(Felsenstein 1985) and the evolutionary distances were com- p
/a
putedusingthePoissoncorrectionmethod. Funding rtic
le
System requirements -a
This work was supported by Academia Sinica [under the b
s
The Orchidstra database is supported by the latest versions Development Program of Industrialization for Agricultural tra
c
of Microsoft Internet Explorer, Mozilla Firefox and Apple Biotechnology (http://dpiab.sinica.edu.tw/index_en.php), t/5
4
Safari. In order to experience the Orchidstra website fully, (grantNo.098S0311)];theNationalScienceCouncil. /2
/e
we suggest that you upgrade to a recent browser such as 11
Microsoft Internet Explorer 8 or later, Mozilla Firefox 9 and /18
Acknowledgements 7
Apple Safari 5. We recommend that the best resolution 7
4
3
for browsing is 1,024(cid:3)768. In addition, JavaScript is used TheauthorswouldliketoexpresstheirgratitudetoDr.Tsai-Mu 9
b
on this website, thus enabling Javascript 1.2 or later is Shen of the National Chia-Yi University, for providing plant y g
recommended. material of native Phalaenopsis aphrodite collected from ue
s
Da-Wu Mountain in Taiwan, and Dr. Chang Chen from the t o
Definition of terms n
National Chung HsingUniversity and Dr. Choun-SeaLinfrom 1
3
Inthismanuscript,TSAisusedtodescribeexpressedtranscript Academia Sinica for providing the Erycina pusilla materials. F
e
sequences from in-house-generated or downloaded reads High-throughput sequencing work was performed by the bru
including ESTs and NGS reads (SRA) that were assembled NGS core facility in Academia Sinica led by Dr. Mei-Yeh Lu. ary
intouniqueandcontiguoussequences.IntheOrchidstradata- Dr. Tzyy-Jen Chiou and Dr. Ho-Ming Chen, both from 2
0
1
base,aTC(fortranscriptcontig)isinitialedandplacedinfront Academia Sinica, provided precious advice related to small 9
of the identification number. The authors wish to clarify that RNA(especiallymiRNAandtasiRNA).
theTCheredoesnotstandfor‘tentativeconsensus’asusually
recognized for the clustering procedure of UniGene or Gene
indexformation. Methodsused in ourde novo assembly pro- References
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Altschul,S.F.,Gish,W.,Miller,W.,Myers,E.W.andLipman,D.J.(1990)
from clustering as described previously in the Materials and
Basiclocalalignmentsearchtool.J.Mol.Biol.215:403–410.
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Benson,D.A.,Karsch-Mizrachi,I.,Clark,K.,Lipman,D.J.,Ostell,J.and
prefixtheidnumberofassembledtranscriptsand,inE.pucilla,
Sayers,E.W.(2012)GenBank.NucleicAcidsRes.40:D48–D53.
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sequences derived from small RNA deep sequencing after Fischer-York, T. et al. (2011) The Sol Genomics Network
10 PlantCellPhysiol.54(2):e11(1–11) (2013) doi:10.1093/pcp/pct004 !TheAuthor2013.