ChloroMitoSSRDB: Open Source Repository of Perfect and Imperfect Repeats in Organelle Genomes for Evolutionary Genomics.

0.31 MB·English

by  Sablok, Gaurav

DNA RESEARCH 20, 127–133, (2013) doi:10.1093/dnares/dss038
Advance Access publication on 2 January 2013
ChloroMitoSSRDB: Open Source Repository of Perfect and Imperfect
Repeats in Organelle Genomes for Evolutionary Genomics
GAURAV Sablok1,*, SURESH B. Mudunuri2, SUJAN Patnana3, MARTINA Popova4, MARIO A. Fares5,6,
and NICOLA LA Porta1
Sustainable Agro-ecosystems and Bioresources Department, IASMA Research and Innovation Centre, Fondazione
Edmund Mach, Via E. Mach 1, San Michele all‘Adige, Trentino 38010, Italy1; Department of Computer Science &
Engineering, Vishnu Institute of Technology, Vishnupur, Bhimavaram, Andhra Pradesh, India2; TalentSprint Edu.
Services, International Institute of Information Technology (IIIT) Campus, Gachibowli, Hyderabad, Andhra Pradesh,
India3; Department of Plant Physiology and Molecular Biology, University of Plovdiv, 24 Tsar Assen St., Plovdiv
4000, Bulgaria4; Department of Genetics, Trinity College Dublin, Universityof Dublin, Dublin 2, Dublin, Ireland5
and Department of Abiotic Stress, Instituto de Biolog´ıa Molecular y Celular de Plantas, CSIC-UPV, Ingeniero Fausto
Elio s/n, Val`encia 46022, Spain6
*To whom correspondence should be addressed. Tel. þ39 3270484732. Email: [email protected]
Edited by Prof. Kenta Nakai
(Received 3September2012;accepted 26November 2012)
Abstract
Microsatellitesorsimplesequencerepeats(SSRs)arerepetitivestretchesofnucleotides(A,T,G,C)that
aredistributedeitherassinglebasepairstretchesorasacombinationoftwo-tosix-nucleotidesunitsthat
arenon-randomly distributed withincodingand innon-codingregionsofthe genome.ChloroMitoSSRDB
is a complete curated web-oriented relational database of perfect and imperfect repeats in organelle
genomes. The present version of the database contains perfect and imperfect SSRs of 2161 organelle
genomes (1982 mitochondrial and 179 chloroplast genomes). We detected a total of 5838 chloroplast
perfectSSRs,37297chloroplastimperfectSSRs,5898mitochondrialperfectSSRsand50355mitochon-
drial imperfect SSRs across these genomes. The repeats have been further hyperlinked to the annotated
gene regions (coding or non-coding) and a link to the corresponding gene record in National Center for
Biotechnology Information(www.ncbi.nlm.nih.gov/) to identify and understand the positional relation-
shipoftherepetitivetracts.ChloroMitoSSRDBisconnectedtoauser-friendly webinterfacethatprovides
useful information associated with the location of the repeats (coding and non-coding), size of repeat,
motifandlengthpolymorphism,etc.ChloroMitoSSRDBwillserveasarepositoryfordevelopingfunction-
al markers for molecular phylogenetics, estimating molecular variation across species. Database URL:
ChloroMitoSSRDB can be accessed as an open source repository at www.mcr.org.in/chloromitossrdb.
Key words: chloroplast; database; mitochondria; microsatellites; simple sequence repeats; web interface
1. Introduction japonica, it has been demonstrated that the distribu-
tion of microsatellites is also influenced by the motif
Microsatellites, or simple sequence repeats (SSRs), sequence and the sequence characteristics of the
are repetitive stretches of a tandemly repeated motif adjoining regions possessing the microsatellites, in
of one to six base pairs, which has evolved and addition to the replication slippage and point muta-
expanded owing to the replication slippage mechan- tion model.2 These repetitive stretches may occur in
ism that is supposed to be the cause of their high coding and in non-coding regions of the genome.
polymorphic rates.1 Recently, using a genome-wide SSRs have been potentially designated as a class of
alignment of two Orzya species var. indica and co-dominant markers for evaluating germplasm,
#TheAuthor2013.PublishedbyOxfordUniversityPressonbehalfofKazusaDNAResearchInstitute.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.
org/licenses/by-nc/3.0/),whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginal
workisproperlycited.
128 Organelle Repeat Database [Vol. 20,
establishing phylogenetic and evolutionary relation- and structural data.17 A database of universally pub-
ships.Ithasbeenobservedthatclustersofmicrosatel- lished primer sequences of chloroplast genomes has
lite motifs with moderate GC are abundant on been developed, providing a platform for studying
chromosome number 2 in the model plant molecular variations and evolution in chloroplasts.18
Arabidopsis thaliana, which suggests that repetitive Theseorganelle genomeshavebeenexploitedfurther
stretches may be biased towards the accumulation fortheminingofgenes,exons,introns,geneproducts,
in a certain regions.3 Microsatellites have been asso- taxonomy, RNA editing sites, SNPs and haplotype
ciated with various functional roles such astheir pos- information, all of which are displayed as curated
sibleroleintheregulationofpromoters,transcription informationinGOBASE.19Acomprehensiverepository
and translation, and these sequence repeats have of unique proteins expressed in chloroplast proteome
been credited with evolutionary importance.4–6 The using liquid chromatography-mass spectrometry/
positioning of microsatellites in the genome seems mass spectrometry has been developed (AT_
to play an important role in their regulatory activity; CHLORO), serving as a knowledge base to explore the
hence, studying the distribution and understanding envelope proteins.20 However, a complete curated
the possible reasons of microsatellites expansions web-oriented integrated repository of repeat pattern
across genomes have currently been the focus of is still lacking. This has motivated us to undertake a
current intense research. genome-wide study and to develop a web-enabled
Organelle genomes, plant chloroplast and animal interface to analyse the perfect and the imperfect
mitochondrial genomes have been referred to as repeatsinorganellegenomes.
natural counterparts.7,8 Features such as conserved We propose ChloroMitoSSRDB that offers a wide
gene order, lack of heteroplasmy (occurrence of visualization of perfect and imperfect repeats across
more than one type of organelle genome), low re- the chloroplast and mitochondrial genomes with
combination rates and their relative small size are corresponding genomic coordinates. The aim of
making these organelle genomes the widely used ChloroMitoSSRDBistoconstituteaplatformtoaccess
tools for phylogenetic studies. However, lack of het- theutilityofSSRsasmarkersforphylogeneticclassifica-
eroplasmy has not been universally observed in all tion across species. To our knowledge, this is the first
the mitochondrial genomes and has been earlier po- updatedintegratedrepositoryofthegenomicrepeats
tentially reviewed with the occurrence and factors in chloroplast and mitochondrial genomes accessible
affecting the stoichiometry of heteroplasmy in mito- viawebinterface.
chondrial genomes of plants and animals.9 The uni-
parental inheritance of the organelle markers
provides a means to elucidate the genetic flow and 2. Material and methods
genetic structure of the population and the organelle
markers have been widely used in population studies 2.1. Genome data retrieval and pattern search
(fora review seeProvan etal.).8 Insilico development Allthestudiedchloroplast (179)andmitochondrial
of SSRs of organelle genomes has brought them up (1982) genomes were retrieved from the National
as potential markers for transferability among the Center for Biotechnology Information (NCBI) RefSeq
species, ease of development and as key players in database (www.ncbi.nlm.nih.gov/). The required files
genome length variation. They have been widely such as gbk, fna, faa, gff and ptt were downloaded for
demonstrated as potential markers for establishing the studied chloroplast and mitochondrial genomes
molecular evolutionary histories, demographic diver- and were stored as flat files sorted for each genome.
sity and resolving phylogeny in a wide variety of For the identification of the perfect and imperfect
species from Pinus (forest species) to Oryza sativa repeats, the software tool Imperfect Microsatellite
(Monocots).10–12 There have been recent reports on Extractor (IMEx)21 has been used, which uses a sliding
the identification of perfect repeats in organelle windowalgorithmtoidentifytheregionswitharepeti-
genomes of various organisms.11–16 However, previ- tive stretch of a particular nucleotide motif either
ous studies have only been focused on a relatively stretched perfectlyor with levels of imperfection.
small number of genomes and only perfect repeats Thealgorithmallowstheusertospecifytheminimal
have been identified. A proper characterization length of the consecutive nucleotide stretch and
system that would allow researchers to search for reportstheSSRmotif,motifrepeatcounts,coordinates
the association of these repeats with the coding or oftheSSRstractinthegenomeanditslocationrelative
non-codingregions has been lacking in these reports. to coding and non-coding regions. The association of
Inthepastfewyears,systematiccuratedwebreposi- the repeats in coding and intercoding regions was
torieshavebeendevelopedfortheorganellegenomes, determined based on the sequence annotation infor-
whichincludesFUGOIDthatdisplaysthecurateddistri- mation available in GenBank database (NCBI, www.
butionofintronsinorganellegenomeswithfunctional ncbi.nlm.nih.gov). We applied the following length
No. 2] G. Sablok et al. 129
Figure1. SchematicillustrationshowingtheflowoftheorganizationofthedatainChloroMitoSSRDB.
criteria(Mono-,12;Di-,6;Tri-,4;andforTetra-toHexa
Table 1. Structure of the table ‘chloromitometa’ that stores the
repeats,aminimumstretchofthreeminimumrepeti- meta-information of all the mitochondrial and chloroplast
tions) to define each SSRs as a true repeat. In case of genomes
imperfect repeats, the parameter for imperfection
Information Field Datatype Key Example
percentage (p%) is set to 10% indicating the level of
Accession acc_no int(11) 5881414,
imperfectionallowedineachrepeattract.
number 110189662
SequenceID seq_id varchar(11) PRI NC_000834,
AC_000022
3. Results and discussions
Sequence seq_name varchar(500) Rattusnorvegicus
name strainWistar
3.1. Structure of ChloroMitoSSRDB database
mitochondrion,
ChloroMitoSSRDBishostedona32-bitLinuxserver Porphyra
pre-installed with MySQL (http://www.mysql.com/), purpurea
chloroplast
Apache (http://www.apache.org/) and PHP (http://
www.php.net/) commonly called as LAMP. A flow Sequence seq_type varchar(50) Complete
type genome,
chart explaining the organization and the work flow
complete
of the ChloroMitoSSRDB has been presented (Fig. 1). sequence
ChloroMitoSSRDBisbasedonasimplecomprehensive
Sequence seq_length int(11) 16613bp,
relationaldatabasemanagementsystem,MySQL,that length 7686bp
is sufficient for organizing, storing and retrieving the Nucleotide a_per Float 33.06%
data with a single query. The details of the relational composition
MySQL tables used in the construction of the ofA
ChloroMitoSSRDB database are explained in Tables 1 Nucleotide t_per Float 41.87%
composition
and 2. Table 1 shows the metadata for each genome,
ofT
whereas the structure of the MySQL relational tables
Nucleotide g_per Float 13.58%
depicting the repeat information stored for the
composition
coding and the non-coding regions is given in ofG
Table 2. Each query has been split into hierarchical
Nucleotide c_per Float 11.49%
levels of information that displays information on composition
each Genome (e.g. accession, sequence length and ofC
nucleotide composition) (Table 1). Organelle organelle Char(1) M(for
The information for the genome composition (A-, type Mitochondrion),
C(Chloroplast)
T-, G- and C- counts, etc.) has been computed from
TaxonID taxon Int 263995
the flat files obtained from the NCBI RefSeq database
(Table 1). The complete repeat information of the
database is stored in two different tables (refer repeat information includes the details of individual
Table 2), storing the perfect and imperfect repeats repeatssuchasthesequenceID,startandendcoordi-
of all chloroplast and mitochondrial genomes. The nates of the repeat, the repeating motif, number of
130 Organelle Repeat Database [Vol. 20,
Table 2. Structure of the tables ‘chloromitoperfectmicrosatellite’ and ‘chloromitoimperfectmicrosatellite’ that store the repeat
informationofallperfectandimperfectmicrosatellitesofmitochondrialandchloroplastgenomes
Information Field Datatype Key Example
SequenceID index_no varchar(11) PRI NC_000834,AC_000022
Startingco-ordinateofSSR start int(11) PRI 172,12843
Endingco-ordinateofSSR end int(11) PRI 182,12885
motif(repeatingunit) motif varchar(10) AT,G,CAAC
Numberofrepetitions iterations int(5) 3,7
Lengthofrepeattract tract_length int(11) 12bp,18bp
NucleotidecompositionofA a_per Float 50.00%
NucleotidecompositionofT t_per Float 0.00%
NucleotidecompositionofG g_per Float 33.33%
NucleotidecompositionofC c_per Float 16.67%
Repeatpositioninformation coding_info varchar(50) Coding(ifrepeatisinthecodingregion)orNull
(ifoutside)
ProteinID(ifrepeatincodingregion) protein_id int(11) 110189664(ifrepeatisinthecodingregion)or
0(ifnon-coding)
aImperfectionpercentageofthetract imperfection Float 9%,0%
aAlignmentline1 alignment_line1 Text TTAA-TAATTAA
aAlignmentline2 alignment_line2 Text ***********
aAlignmentline3 alignment_line3 Text TTAATTAATTAA
aThelastfourcolumns(imperfection,alignment_line1,alignment_line2andalignment_line3)arepresentonlyinthetable
thatstoresimperfectmicrosatellites(chloromitoimperfectmicrosatellite).
iterations, total tract length, nucleotide composition and report page: (i) the first search pattern is accord-
of the repeat, protein information of coding repeats. ingtotheorganelleclassificationandithasbeenclas-
In addition, the table displaying the imperfect sified into chloroplast and mitochondrial genomes,
repeats also stores the imperfection percentage and (ii) the second search pattern has been classified
alignment information that can be used to study accordingtothetypeofrepeatpattern(perfectorim-
the evolution of these repeats. perfect) and (iii). the last search pattern allows the
usertoselecttherepeatsize.Withtheappropriatese-
lectionpattern,theuserwillbedirectedtotheorgan-
3.2. Web visualization of ChloroMitoSSRDB elle-specific page (chloroplast and mitochondrial)
The front end of the database is integrated viaweb containing the list of the organism for which the
accessible PHP scripts. The web interface allows SSRs have been identified, which are further linked
various patternsof searchfor therepeats in organelle to the organism-specific repeat pages for further in-
genomes. The complete browsing outlay of the formation on the distribution of the repetitive tracts.
ChloroMitoSSRDBisdisplayed(Fig.2).Thecuratedin- To ease the access of the database and to enhance
formation is organized into several search patterns, the user functionality, we also provide chloroplast
and proper navigation pageshave been provided. The andmitochondrialrepeat-specificpagesalphabetically
curatedinformationfromtheIMExhasbeenprocessed orderedaccordingtotheorganismname.Anadvanced
furtheraccordingtogeneIDs,organismname,andthe search option has been provided to filter the repeats
SSRsweresortedaccordingtothecodingornon-coding based on the user-specific criteria allowing the user
regions. The position of the coding regions has been to search for a repeat region of a specific length. An
determined using the annotated ptt files of each optiontoexportthesearchresultsandtherepeatinfor-
chloroplastandmitochondrialgenomeasdownloaded mationinexcelformathasbeenprovided,sothatthe
fromtheNCBIRefseqdatabase. user can save and analyse the repeats, design primers
ChloroMitoSSRDBinterfaceprovidesinformationon andcanutilizetheinformationforfurtherdownstream
several repeat statistics, including the distribution of processingoftheobservedrepeats.
the repeat types, length of the motifs and their posi- A query page for every organism is directed to a
tions (coding or non-coding repeats). The querying ChloroMitoSSRDB repeat summary page for organ-
of ChloroMitoSSRDB through the web interface is ism-specific summary page that gives a detailed illus-
organized into three search patterns that accomplish tration of the distribution of the perfect and the
all interface functionalities: query page, result page imperfect repeats distribution and the genome
N
o
.
2
]
G
.
S
a
b
lo
k
e
t
a
l.
Figure2. Howtobrowse:schematicbrowsingofChloroMitoSSRDB.
1
3
1
132 Organelle Repeat Database [Vol. 20,
composition using bar and pie charts. The genome platformtounderstandtherepetitivenatureoforgan-
composition and the repeat occurrence graphs were ellegenomes.
generated dynamically based on the repeat informa-
tion using Libchart, a PHP chart drawing library (http Acknowledgements: G.S. thanks Ivan Milev for
://naku.dohcrew.com/libchart/). The repeat pattern reading the manuscript. S.M. gratefully acknowledges
summarydisplayedontheorganismspecificpageare the support of Vishnu Educational Society in
clickable links, which redirects and give further providing necessary infrastructure and resources to
informationonthestartandendoftheSSRrepeatcon- carry out the database development.
tainingtract,Motifandtheoccurrenceoftherespective
repeatpatternacrossthegenomes.
Funding
Mutations in the SSR stretches prevailing in the
codingregionmayaffectthesubsequenttranscription
This work was supported by BIOMASFOR
and translation of the gene harbouring the repetitive
stretches of SSRs.22 Mutations in chloroplast SSRs (Z0912003I, Italy) and EC FP7 (BIOSUPPORT,
(mutation rates at cpSSR loci as between 3.2(cid:2)1025 Bulgaria). M.A.F. was supported by a grant from the
and 7.9(cid:2)1025) have been described as low when Spanish Ministerio de Ciencia e Inovacio´n (BFU2009-
compared with substitution rates.23 Recently, it was 12022).
observed that the plant mitochondrial substitution
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