Preliminary Characterization of Mitochondrial Genome of ... · ResearchArticle Preliminary Characterization of Mitochondrial Genome of Melipona scutellaris, a Brazilian Stingless

Research ArticlePreliminary Characterization of Mitochondrial Genome ofMelipona scutellaris a Brazilian Stingless Bee

Manuella Souza Silverio1 Viniacutecius de Rezende Rodovalho1

Ana Maria Bonetti1 Guilherme Correcirca de Oliveira2 Sara Cuadros-Orellana2

Carlos Ueira-Vieira1 and Anderson Rodrigues dos Santos3

1 Universidade Federal de Uberlandia Instituto de Genetica e Bioquımica Avenida Para 1720 Campus UmuaramaBloco 2E Sala 246 2∘ Piso 38400-902 Uberlandia MG Brazil

2 Rene Rachou Research Center-Fiocruz Molecular and Cellular Pathology Laboratory Avenida Augusto de Lima 1715Barro Preto 30190-002 Belo Horizonte MG Brazil

3 Universidade Federal de Uberlandia Faculdade de Computacao Avenida Joao Naves de Avila 2121 Campus Santa MonicaBloco 1B Sala 148 38400-902 Uberlandia MG Brazil

Correspondence should be addressed to Anderson Rodrigues dos Santos arsantosfcufubr

Received 28 February 2014 Accepted 28 May 2014 Published 16 June 2014

Academic Editor Sankar Subramanian

Copyright copy 2014 Manuella Souza Silverio et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Bees aremanufacturers of relevant economical products and have a pollinator role fundamental to ecosystems Traditionally studiesfocused on the genusMelipona have beenmostly based on behavioral and social organization and ecological aspects Only recentlythe evolutionary history of this genus has been assessed usingmolecularmarkers includingmitochondrial genes Even though thesestudies have shed light on the evolutionary history of the Melipona genus a more accurate picture may emerge when full nuclearand mitochondrial genomes ofMelipona species become available Here we present the assembly annotation and characterizationof a draft mitochondrial genome of the Brazilian stingless beeMelipona scutellaris usingMelipona bicolor as a reference organismUsing Illumina MiSeq data we achieved the annotation of all protein coding genes as well as the genes for the two ribosomalsubunits (16S and 12S) and transfer RNA genes as well Using the COI sequence as a DNA barcode we found thatM cramptoni isthe closest species toM scutellaris

1 Introduction

Melipona scutellaris popularly known as urucu bee is a stin-gless bee species profusely found from Bahia to PernambucoBrazilian states They are present in urban and rural environ-ments and their pollinator role is pivotal to the ecosystems inwhich they live [1 2]

The genus Melipona has long been target of ecologicalgenetic and especially behavioral and pollination studiesExisting molecular phylogenetic studies have shown that theMelipona genus clusters with other neotropical MeliponiniFurthermore Melipona spp form a well-supported mono-phyletic group as determined by recent studies Stinglessbees especially compose an ancient group distributedworldwide around tropics making them important species

for phylogenetic relationships studies Thus the elucidationof social behavioral evolution relies on a better understandingof phylogenetic relationships for eusocial insects [3 4] Forthis reason there is an increasing interest on getting moreaccurate phylogenetic reconstructions based on molecularaspects

Phylogenetic studies have explored nuclear ribosomalgenes Since 18S and 28S subunits are short genes they aretherefore easily amplified and sequenced Besides such genesare located in very informative regions where conservedor variable sequences may enlighten phylogenetic relationsbetween species [5 6] The mitochondrial genome has alsobeen included in such studies since its potential for pro-viding evolutionary information was recognized Metazoanmitogenomes are about 16 kb long and contain 37 genes 22

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 927546 6 pageshttpdxdoiorg1011552014927546

2 BioMed Research International

tRNAs 2 rRNAs (16S and 12S subunits) and 13 oxidativephosphorylation proteinsmdash7 from complex I (ND1 ND2ND3 ND4 ND4L ND5 and ND6) one from complex III(cytochrome B) three from complex IV (COI COII andCOIII) and two from complexV (ATPase6 andATPase8) [7]

The tRNA genes are embedded in variable regionsThrough evolution these regions underwent rearrangementsmore often than protein coding regions Therefore tRNAorder is a tool for comparative phylogenetic analysis betweenspecies [8] Additionally the high copy number per cell lowrecombination rate high mutational rate and dominantlymaternal heritance have made the mitochondrial genome apowerful tool for evolutionary studies

DNA barcoding or taxon identification using a stan-dardized genomic region was initially used in the studyof animal specimens [9] and later for a wider diversityof organisms DNA barcoding based on the cytochromec oxidase subunit 1 (COI) gene has since then become awidely accepted molecular marker for species identificationThis 650 bp long sequence is a simple and reliable tool formetazoan species identification indicating their moleculardivergences or similarities [4] Being such a short sequenceit is useful for robust phylogeny analysis and important fortracking and measuring ecosystems biodiversity what haslately been referred to asmetabarcoding [10]

Other genes may be employed for DNA barcoding butespecially among Arthropoda the interest on COI over othergenes is mainly due to its well conserved and single copysequence which makes the amplification by PCR reactioneasier allowing the usage of a small set of primers StillCOI sequence presents faster substitution rates than nucleargenes and its variations are remarkably more interspecificthan intraspecific [10 11]

DNA barcoding applications for insects have been verysuccessful [11] This technique allows species identificationin different life stages (eggs larvae nymphs and pupae)when morphologic characteristics are not easily identified[12 13] Besides it makes species identification possible withonly tissues or fragmented parts of the insect [14] Furtherthe importance of phylogenetic relationship analysis relies onunderstanding ecosystems biodiversity taking into accountthat insects are important at pollination decomposition pestcontrol and even disease vectors [10]

The present study characterized the draft ofM scutellarismitogenome annotation according to its gene order geneconservation and taxonomic characterization by DNA bar-coding

2 Material and Methods

21 Biological Material Total DNA was extracted from apool of five male individuals from the Meliponary UFU atUniversidade Federal de Uberlandia campus Umuarama (S180 551015840W 450 171015840) DNA extraction was performed withCTAB buffer which consists of 2 (wv) CTAB diluted in100mM Tris-HCl 20mM EDTA and 14M NaCl Immedi-ately before maceration 02 (vv) 120573-mercaptoethanol wasadded 150 120583L of CTAB buffer was added and maceration

was performed manually with a pestle Then 350120583L ofCTAB buffer and 5 120583L of RNase solution (100mgmL) wereadded following incubation at 37∘C for 1 hour Later 5120583L ofproteinase K solution (20mgmL) was added with additionalincubation at 50∘C for 1 hour For homogenate extractionaddition of 240120583L of PhenolChloroformIsoamyl alcohol(25 24 1) and then centrifugation at 12000timesg for 10 min-utes Supernatant was transferred for a new tube and DNAwas precipitated with 500120583L of absolute ethanol followingcentrifugation at 12000timesg for 15 minutes Ethanol 70was used for pellet washing with a volume of 500120583L andcentrifugation at 12000timesg for 3 minutes This step wasrepeated [15] Pellet was dried at room temperature overnightand eluted into 100 120583L of MiliQ water

22 Mitochondrial Genome Sequencing The total genomesequencing of M scutellaris was performed at the ReneRachou Research Center Fiocruz Minas (Belo HorizonteMG) using an Illumina platform (MiSeq) and a paired-endstrategy The library was constructed with the Nextera XTDNA Sample Preparation Kit following the manufacturerrsquosinstructions Fragments of 404 bp long were carried out forsequencing The average read length was 250 bp and the finalthroughput was 84Gbp

23 Genome Assembly In order to achieve more reliableand accurate results two kinds of assembly software wereemployed SOAPdenovo2 [16] and Velvet [17] The first Mscutellaris mitogenome created in this work was made usingcontigs generated by the SOAPdenovo2 softwarewith varyingkmer parameter between 23 and 127

The assembly by Velvet generated assemblies for differentkmer values 31 41 51 61 71 81 91 or 99 The MuMmerPackage 30 [18] aligned the contigs from each pair of assem-blies with the reference sequence and show-tiling was used tocomplete the scaffolding of the set of contigs Combinationsof the following set of parameters were used in show-tiling-i 50 70 or 90 -V 0 5 or 10 -v 10 50 or 90 -c included ornot

The generated sequences were filtered according to thefollowing conditions (i) sequence size between 14000 and17000 base pairs (ii) AT content greater than or equal to 84(iii) maximum gap tolerance of 100 nucleotides

The remaining sequences were submitted to MITOS [19]for functional annotation Steps for Velvet assembly andsubsequent annotation with MITOS are schemed in Figure 1The referencemitogenomeofM bicolorwas also submitted inorder to provide amore reliable basis for further analysisThedata from annotations were used for analysis of gene orderand similarity to reference sequence

The genes from the reference genome of M bicolor arelisted in the first columnof Table 1 Relatively to each gene theadjacent genes were analyzed (upstream and downstream)according to their frequencies (occurrence ldquoOCRrdquo inTable 1)The annotated genes fromM scutellaris were locally alignedagainst the reference mitogenome highlighting identity andE-value Transfer RNAs secondary structures were predictedusing tRNASCAN-SE software [20]

BioMed Research International 3

VelvetAssembly for each

kmer value

MITOSSequence annotation

CombinationsAssemblies were

combined in pairs

FilteringGenomes were filtered by

Sequence size 14ndash17kbpGap tolerance 100 bp

Repetitions were removed

NucmerAlignment with thereference sequence

Show-tilingScaffolding with combinations

of the set of parameters

minusc rarr included or not

64

assembliesassemblies

3456

genomes genomes

8

35

AT content ge 84minusi rarr 50 70 or 90minusV rarr 0 5 or 10minus rarr 10 50 or 90

Figure 1 Velvet genome assembly parameters adopted and MITOS annotation

3 Results and Discussion

In total 36 assemblies were performed generating 36 anno-tations which allowed inferring that all protein-coding genesand tRNA genes were annotated Using all annotations thegene order analysis revealed synteny between M scutellarismitogenome and the reference genome Ribosomal genesidentities were also analyzed (Table 1) as well as the conser-vation of secondary structure for tRNA

31 Organization andPartial Characterization ofM scutellarisMitogenome In Table 1 the ldquoreference generdquo column corre-sponds to the mitochondrial gene order of M bicolor Con-sidering the genes that are found in the reference genomecolumn ldquoOCRrdquo (second column) shows howmany times eachgene is annotated forM scutellaris within the 36 annotationsldquoUpstream generdquo and ldquodownstream generdquo columns refer towhich genes are found in those respective positions relativeto the reference gene Finally ldquoOCRrdquo columns (fifth and sixthcolumns) mean how many times a gene is found in suchposition relative to another gene also considering the totalof 36 annotations

M scutellarismitogenome shows an overall high syntenywhen compared to M bicolor mitogenome Figure 2 is anillustrative scheme which gathers the annotated genes underthe most frequent order among all 36 assemblies Althoughall genes were found they are not all present in the followingscheme as long as some few onesmust yet have their positionvalidated

Regarding tRNA genes aligning the annotated genesagainst the reference genome did not generate considerableidentity values for all cases However submitting the Mscutellaris sequences to tRNAScan-SE it was possible to inferthat the tRNA secondary structures ofM scutellaris are viableand well conserved An example is displayed in Figure 3

In Table 1 it is possible to notice that in some cases thereis more than one gene in the upstream andor downstream

position providing different possibilities of gene organi-zation Such genes are tRNAA tRNAK tRNAG tRNARtRNAT and tRNAL1

The coding-protein genes for M scutellaris are syntenicto their homologous in M bicolor mitogenome Some tRNAgenes are also syntenic namely tRNAM tRNAW tRNAYtRNAL2 tRNAD tRNAF tRNAP and tRNAS2 However it isalso possible to infer that some tRNA genes have underwentrearrangement which are tRNAV tRNAS1 tRNAN andtRNAA Some other tRNA genes must yet have their positionvalidated since their occurrence (OCR) is very low comparedto the average such as tRNAI tRNAG tRNAE and tRNAL1(not shown in Figure 2) It is known that tRNAs order isa particular feature of each insect species [8] and tRNAsdistribution copy number and codon usage patterns areespecially important in evolutionary studies [21]

Our results provided evidences of possible duplication oftwo genes inM scutellarismitogenome in comparison toMbicolor In Table 1 it is possible to see the occurrences fortRNAQ downstream to ND5 and 12S genes 3536 and 2936respectively Still tRNAA has also high occurrences for beingupstream to 12S (3036) composing the following cluster16S tRNAN tRNAA 12S But there is also a high occurrencefor being downstream to tRNAT (2736) whose position isnot yet clear although it is already unlikely to be part of thecluster mentioned above

A single gene found in M bicolor mitogenome was notfound inM scutellaris which is tRNAH On the other handgene tRNAX was annotated suggesting a tRNA that is notidentified requiring further validation

Moreover submitting the mitogenome sequence of Mbicolor to MITOS as a support for further analysis a genethat is not found in the currently available sequence wasannotated namely tRNAC This gene was also annotatedin M scutellaris mitogenome in the same position Suchsynteny suggests that tRNAC gene may be present in bothgenomes and this information provided byMITOSmay havebeen omitted by other kinds of annotation software


Table 1 Annotated genes inM scutellarismitogenome upstreamdownstream relations and similarity analysis

Reference gene Upstream gene Downstream gene Similarity to reference sequenceName OCRlowast Name OCRlowast Name OCRlowast Blast algorithm Best identity E-valuetrnI 2 trnA 2 trnQ 2 blastn mdash mdash

trnA 34 trnTtrnN

2725 rrnS 30 blastn 9206 200E minus 021

trnK 16 trnQlowastlowast 5 trnEtrnM

64 blastn 9362 200E minus 016

trnM 28 trnXlowastlowast 22 nad2 27 blastn 9571 200E minus 028nad2 36 trnM 27 trnC 32 tblastx 9643 200E minus 018trnC 32 nad2 32 trnW 30 blastn mdash mdashtrnW 32 trnC 30 trnY 25 blastn 9692 600E minus 028trnY 26 trnW 25 cox1 25 blastn mdash mdashcox1 36 trnY 25 trnL2 20 tblastx 9833 200E minus 099trnL2 20 cox1 20 cox2 20 blastn mdash mdashcox2 36 tnrL2 20 tnrD 32 tblastx 9655 300E minus 063trnD 32 cox2 32 atp8 31 blastn mdash mdashatp8 35 trnD 31 atp6 34 tblastx 9375 200E minus 005atp6 36 atp8 34 cox3 36 tblastx 9444 100E minus 009cox3 36 atp6 36 trnV 17 tblastx 9412 400E minus 027

trnS1 27 trnRnad3

1210 trnF 19 blastn 9688 200E minus 027

trnG 3 cox3 3 nad3atp8

21 blastn mdash mdash

nad3 36 trnVcox3

1714 trnR 24 tblastx 9500 700E minus 006

trnR 24 nad3 24 trnS1trnN


trnN 34 rrnL 25 trnA 25 blastn 9672 900E minus 026trnE 12 trnK 6 trnF 7 blastn mdash mdashtrnF 28 trnS1 19 nad5 15 blastn mdash mdashnad5 36 trnF 15 trnQlowastlowast 35 tblastx 9583 100E minus 013trnH 0 mdash mdashnad4 36 trnQlowastlowast 35 nad4l 28 tblastx 9500 100E minus 028nad4l 33 nad4 28 trnP 26 tblastx 9474 013

trnT 32 nad4l 9 trnA 27 blastn mdash mdashtrnQlowastlowast 7

trnP 35 nad4l 26 nad6 35 blastn mdash mdashnad6 35 trnP 36 cob 35 tblastx 9412 400E minus 022cob 36 nad6 35 trnS2 35 tblastx 9565 100E minus 011trnS2 35 cob 35 nad1 35 blastn 10000 700E minus 032nad1 36 trnS2 35 rrnL 34 tblastx 9286 500E minus 021

trnL1 2 trnYnad1

11

rrnLtrnL2


rrnL 36 nad1 34 trnN 25 blastn 9526 300E minus 095trnV 23 cox3 17 nad3 17 blastn mdash mdashrrnS 36 trnA 30 tnrQ 29 blastn 9508 400E minus 024lowastOCR occurrences in different assemblieslowastlowastGenes not found inM bicolor mitogenome


I A

A

K M

M

ND2

ND2

C W Y COI L2

L2

COII D

ATP8

ATP6 COIII S1

S1 S1C W Y COI COII D

ATP8

ATP6 COIII

G

Q Q

ND3

ND3G

R N

N

E F

F

ND5

ND5

H ND4

ND4

ND4L

ND4L

T P

P

ND6

ND6

CytB S2

S2

ND1

ND1CytB

L1 V16S

16S

12S

12S

M bicolor

M scutellaris

Figure 2 Comparison betweenM bicolorrsquos mitogenome and gene organization achieved forM scutellaris Genes whose position is not yetvalidated were omitted (in gray)

U

U

U U

UU

U U

U

U U U UU

U

U

U

U

U

U

U

U

U

U

UUA

A

AA

AAAA

A

AAA

A

A

A

AA

A

AA

A

A

A

A

A

A

A

AAA

AC

C

C

G

G

G

G

G

G

(a)

U

UUUUU

U

UU

U

U

U

U

U

U

U

U

U

UU

U

UU

UU

U

UA

A

A

A

A A

A

A

A

A

A

AA

A

A

AA

A

A

A

A

A

A

AA

AA

AAA

C

CC

G

G

G

G

GG

(b)

Figure 3 Comparison of secondary structure predictions for tRNAS2 genes ofMelipona scutellaris (a) andMelipona bicolor (b)

Concerning the protein-coding regions they are probablynot yet complete Genesrsquo lengths are not all as the expectedpreventing a conclusion about the codon usage in M scutel-larisrsquo mitogenome

Finally comparing both assemblies N50 values adoptedwere 343 for SOAPdenovo2 and 722-362 for Velvet Thelonger scaffolds achieved had close lengths which are 15580and 15206 for SOAPdenovo2 and Velvet respectively Ingeneral all assemblies generated scaffolds around 14000ndash15580 bp long However despite the good quality of the data(phred gt 30) it was not possible to achieve any significantscaffold in a first approach For this reason the presentmethodology was adopted in order to reach the evidencesof genesrsquo presence As discussed above all protein-codingsequences were annotated as well as transfer RNA-codingand ribosomal subunits genes It is assumed that using asingle library of inserts 400 bp long may have influences overthe results under the expected requiring a complementarylibrary for subsequent analyses

32 DNA Barcoding for Taxonomic Identification The cyto-chrome c oxidase subunit I gene (COI) sequence annotated

in this study was submitted to BOLD Systems database [22]for taxonomic analysis It was validated as belonging to theMelipona genus with 100 probability

Melipona cramptoni had the highest identity (9899) toM scutellarisCOI sequence Other closest hits wereMeliponarufiventris (9843) andMelipona eburnea (9840)The ref-erence organismM bicolor was ranked at 97th position with952 identity This observation highlights the limitations ofusingM bicolor as a reference for assembly

Ahigher identity betweenM scutellaris andM rufiventriswas already expected Rocha et al [23] have demonstratedby cytogenetic studies that amongMelipona species there aredifferent heterochromatin content and distribution patternsTwo groupsmay be distinguished for such characteristics thatmay be considered for evolutionary analysis as well

Rocha et al [23] established M bicolor as belonging toGroup I which comprises species with less than 50 ofheterochromatin Species belonging to such group displayheterochroma-tin content concentrated in one portion ofthe chromosome On the other hand Group II is composedof species with heterochromatin content higher than 50spread along the chromosome extension By cytogenetic


studies M scutellaris and M rufiventris were classified asbeing part of Group II Therefore DNA barcoding resultrevealing a closer relation between M scutellaris and Mrufiventris rather than M bicolor confirms cytogenetic stud-ies However the close relation betweenM scutellaris andMcramptoni has not yet been investigated

4 Conclusion

The results generated to the mitogenome of M scutellaris inthis study suggest a conserved character in comparison toMbicolor Protein coding genes order especially appears to bewell conserved Some tRNAgenes underwent rearrangementbut a conservation pattern could also be analyzed as well

Through taxonomic identification search on BOLD Sys-tems database it was possible to assume that M cramptoniis the closest species to M scutellaris (9899) although Mbicolor is also found among closely related species (952identity) The second closest species to M scutellaris foundat BOLD database is M rufiventris what is confirmed bycytogenetic studies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] A Pianaro Ecologia quımica de abelhas brasileiras Meliponarufiventris Melipona scutellaris Plabeia droryana Nannotrig-ona testaceicornis Tetragonisca angustula e Centris trigonoides[MS thesis] UNICAMP Chemistry Institute 2007

[2] W E Kerr G A Carvalho and V A Nascimento A AbelhaUrucu Biologia Manejo e Conservacao Liber Liber 1996

[3] S R Ramırez J C Nieh T B Quental D W Roubik V LImperatriz-Fonseca and N E Pierce ldquoA molecular phylogenyof the stingless bee genus Melipona (Hymenoptera Apidae)rdquoMolecular Phylogenetics and Evolution vol 56 no 2 pp 519ndash525 2010

[4] C Rasmussen and S A Cameron ldquoGlobal stingless bee phylog-eny supports ancient divergence vicariance and long distancedispersalrdquo Biological Journal of the Linnean Society vol 99 no1 pp 206ndash232 2010

[5] J M Henriques Identificacao molecular (DNA barcode) dospeixes da Bacia do Rio Ribeira de Iguape e dos Rios Costeirosdo Estado de Sao Paulo [Doctorate thesis] UNESP BiosciencesInstitute 2010

[6] M S Caterino S Cho and F A H Sperling ldquoThe currentstate of insect molecular systematics a thriving tower of babelrdquoAnnual Review of Entomology vol 45 pp 1ndash54 2000

[7] W S Sheffler Mitochondria John Wiley amp Sons 2nd edition2008

[8] C M Rodovalho Caracterizacao do transcriptoma e genomamitocondrial da formiga cortadeira Atta laevigata (FORMICI-DAE ATTINI) [Doctorate thesis] UNESP Biosciences Institute2011

[9] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergences

among closely related speciesrdquo Proceedings of the Royal SocietyB Biological Sciences vol 270 no 1 pp S96ndashS99 2003

[10] DW Yu Y Ji and B C Emerson ldquoBiodiversity soup metabar-coding of arthropods for rapid biodiversity assessment andbiomonitoringrdquo Methods in Ecology and Evolution vol 3 pp613ndash623 2012

[11] M Ander K Troell and J Chirico ldquobarcoding of biting midgesin the genus Culicoides a tool for species determinationrdquoMedical and Veterinary Entomology vol 27 no 3 pp 323ndash3312013

[12] M Virgilio T Backeljau B Nevado and M De Meyer ldquoCom-parative performances of DNA barcoding across insect ordersrdquoBMC Bioinformatics vol 11 article 206 2010

[13] D-S Park R Foottit E Maw and P D N Hebert ldquoBarcodingbugs DNA-based identification of the true bugs (insectaHemiptera Heteroptera)rdquo PLoS ONE vol 6 no 4 Article IDe18749 2011

[14] M A Smith C Bertrand K Crosby et al ldquoWolbachia andDNA barcoding insects patterns potential and problemsrdquoPLoS ONE vol 7 no 5 Article ID e36514 2012

[15] H ChenM Rangasamy S Y Tan HWang and B D SiegfriedldquoEvaluation of five methods for total DNA extraction fromwestern corn rootworm beetlesrdquo PLoSONE vol 5 no 8 ArticleID e11963 2010

[16] R Luo B Liu Y Xie et al ldquoSOAPdenovo2 an empiricallyimproved memory-efficient short-read de novo assemblerrdquoGigaScience vol 1 no 1 pp 18ndash23

[17] D R Zerbino and E Birney ldquoVelvet algorithms for de novoshort read assembly using de Bruijn graphsrdquo Genome Researchvol 18 no 5 pp 821ndash829 2008

[18] S Kurtz A Phillippy A L Delcher et al ldquoVersatile and opensoftware for comparing large genomesrdquo Genome Biology vol 5no 2 article R12 2004

[19] M Bernt A Donath F Juhling et al ldquoMITOS improved denovo metazoan mitochondrial genome annotationrdquo MolecularPhylogenetics and Evolution vol 69 no 2 pp 313ndash319 2013

[20] P Schattner A N Brooks and T M Lowe ldquoThe tRNAscan-SE snoscan and snoGPS web servers for the detection of tRNAsand snoRNAsrdquoNucleic Acids Research vol 33 no 2 ppW686ndashW689 2005

[21] S K Behura M Stanke C A Desjardins J H Werren and DW Severson ldquoComparative analysis of nuclear tRNA genes ofNasonia vitripennis and other arthropods and relationships tocodon usage biasrdquo InsectMolecular Biology vol 19 no 1 pp 49ndash58 2010

[22] S Ratnasingham and P D N Hebert ldquoBOLD the barcode oflife data system barcodingrdquoMolecular Ecology Notes vol 7 no3 pp 355ndash364 2007

[23] M P Rocha S G Pompolo A Fernandes and L A O CamposldquoMeliponamdashsix decades of cytogeneticrdquo Journal of Biosciencesvol 23 no 1 pp 111ndash117 2007

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tRNAs 2 rRNAs (16S and 12S subunits) and 13 oxidativephosphorylation proteinsmdash7 from complex I (ND1 ND2ND3 ND4 ND4L ND5 and ND6) one from complex III(cytochrome B) three from complex IV (COI COII andCOIII) and two from complexV (ATPase6 andATPase8) [7]

The tRNA genes are embedded in variable regionsThrough evolution these regions underwent rearrangementsmore often than protein coding regions Therefore tRNAorder is a tool for comparative phylogenetic analysis betweenspecies [8] Additionally the high copy number per cell lowrecombination rate high mutational rate and dominantlymaternal heritance have made the mitochondrial genome apowerful tool for evolutionary studies

DNA barcoding or taxon identification using a stan-dardized genomic region was initially used in the studyof animal specimens [9] and later for a wider diversityof organisms DNA barcoding based on the cytochromec oxidase subunit 1 (COI) gene has since then become awidely accepted molecular marker for species identificationThis 650 bp long sequence is a simple and reliable tool formetazoan species identification indicating their moleculardivergences or similarities [4] Being such a short sequenceit is useful for robust phylogeny analysis and important fortracking and measuring ecosystems biodiversity what haslately been referred to asmetabarcoding [10]

Other genes may be employed for DNA barcoding butespecially among Arthropoda the interest on COI over othergenes is mainly due to its well conserved and single copysequence which makes the amplification by PCR reactioneasier allowing the usage of a small set of primers StillCOI sequence presents faster substitution rates than nucleargenes and its variations are remarkably more interspecificthan intraspecific [10 11]

DNA barcoding applications for insects have been verysuccessful [11] This technique allows species identificationin different life stages (eggs larvae nymphs and pupae)when morphologic characteristics are not easily identified[12 13] Besides it makes species identification possible withonly tissues or fragmented parts of the insect [14] Furtherthe importance of phylogenetic relationship analysis relies onunderstanding ecosystems biodiversity taking into accountthat insects are important at pollination decomposition pestcontrol and even disease vectors [10]

The present study characterized the draft ofM scutellarismitogenome annotation according to its gene order geneconservation and taxonomic characterization by DNA bar-coding

2 Material and Methods

21 Biological Material Total DNA was extracted from apool of five male individuals from the Meliponary UFU atUniversidade Federal de Uberlandia campus Umuarama (S180 551015840W 450 171015840) DNA extraction was performed withCTAB buffer which consists of 2 (wv) CTAB diluted in100mM Tris-HCl 20mM EDTA and 14M NaCl Immedi-ately before maceration 02 (vv) 120573-mercaptoethanol wasadded 150 120583L of CTAB buffer was added and maceration

was performed manually with a pestle Then 350120583L ofCTAB buffer and 5 120583L of RNase solution (100mgmL) wereadded following incubation at 37∘C for 1 hour Later 5120583L ofproteinase K solution (20mgmL) was added with additionalincubation at 50∘C for 1 hour For homogenate extractionaddition of 240120583L of PhenolChloroformIsoamyl alcohol(25 24 1) and then centrifugation at 12000timesg for 10 min-utes Supernatant was transferred for a new tube and DNAwas precipitated with 500120583L of absolute ethanol followingcentrifugation at 12000timesg for 15 minutes Ethanol 70was used for pellet washing with a volume of 500120583L andcentrifugation at 12000timesg for 3 minutes This step wasrepeated [15] Pellet was dried at room temperature overnightand eluted into 100 120583L of MiliQ water

22 Mitochondrial Genome Sequencing The total genomesequencing of M scutellaris was performed at the ReneRachou Research Center Fiocruz Minas (Belo HorizonteMG) using an Illumina platform (MiSeq) and a paired-endstrategy The library was constructed with the Nextera XTDNA Sample Preparation Kit following the manufacturerrsquosinstructions Fragments of 404 bp long were carried out forsequencing The average read length was 250 bp and the finalthroughput was 84Gbp

23 Genome Assembly In order to achieve more reliableand accurate results two kinds of assembly software wereemployed SOAPdenovo2 [16] and Velvet [17] The first Mscutellaris mitogenome created in this work was made usingcontigs generated by the SOAPdenovo2 softwarewith varyingkmer parameter between 23 and 127

The assembly by Velvet generated assemblies for differentkmer values 31 41 51 61 71 81 91 or 99 The MuMmerPackage 30 [18] aligned the contigs from each pair of assem-blies with the reference sequence and show-tiling was used tocomplete the scaffolding of the set of contigs Combinationsof the following set of parameters were used in show-tiling-i 50 70 or 90 -V 0 5 or 10 -v 10 50 or 90 -c included ornot

The generated sequences were filtered according to thefollowing conditions (i) sequence size between 14000 and17000 base pairs (ii) AT content greater than or equal to 84(iii) maximum gap tolerance of 100 nucleotides

The remaining sequences were submitted to MITOS [19]for functional annotation Steps for Velvet assembly andsubsequent annotation with MITOS are schemed in Figure 1The referencemitogenomeofM bicolorwas also submitted inorder to provide amore reliable basis for further analysisThedata from annotations were used for analysis of gene orderand similarity to reference sequence

The genes from the reference genome of M bicolor arelisted in the first columnof Table 1 Relatively to each gene theadjacent genes were analyzed (upstream and downstream)according to their frequencies (occurrence ldquoOCRrdquo inTable 1)The annotated genes fromM scutellaris were locally alignedagainst the reference mitogenome highlighting identity andE-value Transfer RNAs secondary structures were predictedusing tRNASCAN-SE software [20]



kmer value



combined in pairs








64


3456

genomes genomes

8

35

















trnA 34 trnTtrnN





trnS1 27 trnRnad3




nad3 36 trnVcox3







trnL1 2 trnYnad1

11

rrnLtrnL2




I A

A

K M

M

ND2

ND2

C W Y COI L2

L2

COII D

ATP8

ATP6 COIII S1


ATP8

ATP6 COIII

G

Q Q

ND3

ND3G

R N

N

E F

F

ND5

ND5

H ND4

ND4

ND4L

ND4L

T P

P

ND6

ND6

CytB S2

S2

ND1

ND1CytB

L1 V16S

16S

12S

12S

M bicolor

M scutellaris


U

U

U U

UU

U U

U

U U U UU

U

U

U

U

U

U

U

U

U

U

UUA

A

AA

AAAA

A

AAA

A

A

A

AA

A

AA

A

A

A

A

A

A

A

AAA

AC

C

C

G

G

G

G

G

G

(a)

U

UUUUU

U

UU

U

U

U

U

U

U

U

U

U

UU

U

UU

UU

U

UA

A

A

A

A A

A

A

A

A

A

AA

A

A

AA

A

A

A

A

A

A

AA

AA

AAA

C

CC

G

G

G

G

GG

(b)











4 Conclusion





References
































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



kmer value



combined in pairs








64


3456

genomes genomes

8

35

















trnA 34 trnTtrnN





trnS1 27 trnRnad3




nad3 36 trnVcox3







trnL1 2 trnYnad1

11

rrnLtrnL2




I A

A

K M

M

ND2

ND2

C W Y COI L2

L2

COII D

ATP8

ATP6 COIII S1


ATP8

ATP6 COIII

G

Q Q

ND3

ND3G

R N

N

E F

F

ND5

ND5

H ND4

ND4

ND4L

ND4L

T P

P

ND6

ND6

CytB S2

S2

ND1

ND1CytB

L1 V16S

16S

12S

12S

M bicolor

M scutellaris


U

U

U U

UU

U U

U

U U U UU

U

U

U

U

U

U

U

U

U

U

UUA

A

AA

AAAA

A

AAA

A

A

A

AA

A

AA

A

A

A

A

A

A

A

AAA

AC

C

C

G

G

G

G

G

G

(a)

U

UUUUU

U

UU

U

U

U

U

U

U

U

U

U

UU

U

UU

UU

U

UA

A

A

A

A A

A

A

A

A

A

AA

A

A

AA

A

A

A

A

A

A

AA

AA

AAA

C

CC

G

G

G

G

GG

(b)











4 Conclusion





References
































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




trnA 34 trnTtrnN





trnS1 27 trnRnad3




nad3 36 trnVcox3







trnL1 2 trnYnad1

11

rrnLtrnL2




I A

A

K M

M

ND2

ND2

C W Y COI L2

L2

COII D

ATP8

ATP6 COIII S1


ATP8

ATP6 COIII

G

Q Q

ND3

ND3G

R N

N

E F

F

ND5

ND5

H ND4

ND4

ND4L

ND4L

T P

P

ND6

ND6

CytB S2

S2

ND1

ND1CytB

L1 V16S

16S

12S

12S

M bicolor

M scutellaris


U

U

U U

UU

U U

U

U U U UU

U

U

U

U

U

U

U

U

U

U

UUA

A

AA

AAAA

A

AAA

A

A

A

AA

A

AA

A

A

A

A

A

A

A

AAA

AC

C

C

G

G

G

G

G

G

(a)

U

UUUUU

U

UU

U

U

U

U

U

U

U

U

U

UU

U

UU

UU

U

UA

A

A

A

A A

A

A

A

A

A

AA

A

A

AA

A

A

A

A

A

A

AA

AA

AAA

C

CC

G

G

G

G

GG

(b)











4 Conclusion





References
































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


I A

A

K M

M

ND2

ND2

C W Y COI L2

L2

COII D

ATP8

ATP6 COIII S1


ATP8

ATP6 COIII

G

Q Q

ND3

ND3G

R N

N

E F

F

ND5

ND5

H ND4

ND4

ND4L

ND4L

T P

P

ND6

ND6

CytB S2

S2

ND1

ND1CytB

L1 V16S

16S

12S

12S

M bicolor

M scutellaris


U

U

U U

UU

U U

U

U U U UU

U

U

U

U

U

U

U

U

U

U

UUA

A

AA

AAAA

A

AAA

A

A

A

AA

A

AA

A

A

A

A

A

A

A

AAA

AC

C

C

G

G

G

G

G

G

(a)

U

UUUUU

U

UU

U

U

U

U

U

U

U

U

U

UU

U

UU

UU

U

UA

A

A

A

A A

A

A

A

A

A

AA

A

A

AA

A

A

A

A

A

A

AA

AA

AAA

C

CC

G

G

G

G

GG

(b)











4 Conclusion





References
































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Conclusion





References
































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Preliminary Characterization of Mitochondrial Genome of ... · ResearchArticle Preliminary Characterization of Mitochondrial Genome of Melipona scutellaris, a Brazilian Stingless