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Thummajitsakul, S., Silprasit, K., Klinbunga, S., Sittipraneed, S.The partial mitochondrial sequence of the Old World stingless bee, Tetragonula pagdeni(2013) Journal of Genetics, pp. 1-5. Article in Press.
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The partial mitochondrial sequence of the Old World stinglessbee, Tetragonula pagdeni
By: Thummajitsakul, S (Thummajitsakul, Sirikul)[ 1,2 ] ; Silprasit, K (Silprasit, Kun)[ 3 ] ; Klinbunga, S
(Klinbunga, Sirawut)[ 4 ] ; Sittipraneed, S (Sittipraneed, Siriporn)[ 2 ]
JOURNAL OF GENETICS
Volume: 92 Issue: 2 Pages: 299-303
DOI: 10.1007/s12041-013-0243-1
Published: AUG 2013
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Keywords
Author Keywords: stingless bees; mitochondrial DNA gene order; long PCR; Tetragonula; Tetragonula
pagdeni
KeyWords Plus: APIS-MELLIFERA; MOLECULAR PHYLOGENY; GENE ORGANIZATION; DNA;
APIDAE; EVOLUTIONARY; HYMENOPTERA; MELIPONINI; ORIGIN; GENOME
Author Information
Reprint Address: Thummajitsakul, S (reprint author)
Srinakharinwirot Univ Ongkharak, Fac Hlth Sci, Nakhon Nayok 26120, Thailand.
Addresses:
[ 1 ] Srinakharinwirot Univ Ongkharak, Fac Hlth Sci, Nakhon Nayok 26120, Thailand
[ 2 ] Chulalongkorn Univ, Dept Biochem, Fac Sci, Bangkok 10330, Thailand
[ 3 ] Srinakharinwirot Univ, Fac Environm Culture & Ecotourism, Nakhon Nayok 26120, Thailand
[ 4 ] Natl Sci Ctr Genet Engn & Biotechnol NSTDA, Klongluang 12120, Pathum Thani, Thailand
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INDIAN ACAD SCIENCES, C V RAMAN AVENUE, SADASHIVANAGAR, P B #8005, BANGALORE
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Language: English
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ISSN: 0022-1333
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RESEARCH NOTE
The partial mitochondrial sequence of the Old World stingless bee,Tetragonula pagdeni
SIRIKUL THUMMAJITSAKUL1,2∗, KUN SILPRASIT3, SIRAWUT KLINBUNGA4 and SIRIPORN SITTIPRANEED2
1Faculty of Health Science, Srinakharinwirot University Ongkharak, Nakhon-Nayok 26120, Thailand2Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
3Faculty of Environmental Culture and Ecotourism, Srinakharinwirot University, Nakhon-Nayok 26120, Thailand4Aquatic Molecular Genetics and Biotechnology, National Science Center for Genetic Engineering and Biotechnology
(NSTDA), 113 Paholyothin Road, Klong 1, Klong Luang, Pathum Thani 12120, Thailand
[Thummajitsakul S., Silprasit K., Klinbunga S. and Sittipraneed S. 2013 The partial mitochondrial sequence of the Old World stingless bee,Tetragonula pagdeni. J. Genet. 92, xx–xx]
Introduction
The partial mitochondrial sequence of Tetragonula pagdeniwas examined using long PCR technique and subsequentlycloned, and sequenced using primer walking strategy. Theobjective of this study was to characterize T. pagdeni mito-chondrial DNA sequence, focussing on gene order. Thepartial mitochondrial sequence of T. pagdeni obtained inthis study represents approximately 69.2% of the MeliponinimtDNA. The protein-coding genes have a bias towardsAT-rich codons. Codon usage and amino acid sequences donot deviate substantially from those reported for other sting-less bees or honeybees. Similar to other Meliponini species(Arias et al. 2006), the mitochondrial genome of T. pagdenilacks the COI–COII intergenic region.
In past, mitochondrial DNA was widely employed toresolve population structure, phylogeography and phyloge-netic relationships at various taxonomic levels (Kmiec et al.2006). Generally, animal mtDNA exhibits a high evolutionrate compared to that of the nuclear DNA and very conservedgene order comprising two rRNA genes, 22 tRNA genes, 13protein-coding genes, as well as a noncoding control region(D-loop) (Wolstenholme 1992). The mtDNA has been exten-sively used in honeybee, Apis mellifera, to investigate natu-ral range origin, and genetic polymorphisms based on PCR-RFLP method (Smith and Brown 1990). For stingless bees,the mitochondrial genome of several Meliponine species hasbeen determined through RFLP analysis (Francisco et al.
∗For correspondence. E-mail: [email protected].
2001). Besides, the nearly complete mtDNA sequence hasbeen recently determined in stingless bee, Melipona bicolor(Silvestre et al. 2008). However, the information of mtDNAsequences from Tetragonula species is very limited, onlyfew mitochondrial genes such as 16S rRNA gene reportedin genetic variability study (Costa et al. 2003; Rasmussenand Cameron 2007; Thummajitsakul et al. 2011). T. pagdeniSchwarz is widely distributed in Indo-Malayan/Australasianand Neotropical regions (Michener 2000) and is one ofthe most common indigenous stingless bees in Thailand(Sakagami 1978).
Materials and methods
Sample and DNA extraction
Individuals of T. pagdeni collected from a single colony werepreserved in 95% ethanol and stored at 4◦C until required.Total genomic DNA was extracted from each entire bee usingphenol–chloroform extraction and ethanol precipitation fol-lowing Thummajitsakul et al. (2011).
PCR and sequencing
In our previous studies, the mtDNA regions for cytb, COI,16S rRNA and ATP(6, 8) + COIII genes of T. pagdeniwere amplified by using primer pairs; cyt-b-F/R, COI-F/R,LR13107-F/LR12647-R and ATPS6-F/tRNA-ASP-R, res-pectively (table 1). On the basis of each sequence por-tion, three sets of primer pairs LR12647-R + COI2494,LR12677 + cytb10729 and COIII9821 + cytb5031 were
Keywords. stingless bees; mitochondrial DNA gene order; long PCR; Tetragonula; Tetragonula pagdeni.
Journal of Genetics, Vol. 92, No. 2, August 2013
Sirikul Thummajitsakul et al.
Table 1. Sequences of primers used in PCR reactions of T. pagdeni mtDNA.
mtDNA genes Primer Sequence
ATPase (6,8) ATPS6-F 5′-AAG ATA TAT GGA AAT AAG CT-3′tRNA-ASP-R 5′-ATA AAA TAA CGT CAA AAT GTC A-3′
COI COI-F 5′- ATA ATT ATT GTT GCT GAT GTA-3′COI-R 5′-CTA TTC ATA TAA CTG GAA TTT C-3′
Cytb cyt b-F 5′-TTC ACT ATA TTA TAA AAG ATG TAA GTT C-3′cyt b-R 5′-GGC AAA AAG AAA ATA TCA TTC AGG-3′
16S rRNA LR13107-F 5′-TGG CTG CAG TAT AAC TGA CTG TAC AAA GG-3′LR12647-R 5′-GAA ACC AAT CTG ACT TAC GTC GAT TTG A-3′
COI, COII, ATPase8, ATPase6, COIII, LR12647-R 5′-GAAACCAATCTGACTTACGTCGATTTGA-3′ND3, 12S, 16S rRNA COI2494 5′-CGAGCATATTTTACATCAGCAACAAT-3′
Cytb, ND6, ND5, ND4, ND4L LR12677 5′-GTTCAAATCGACGTAAGTCAGATTGGTTTC-3′cytb10729 5′-AGCTCCTCAAAATGATATTTGTCCTCATGG-3′
COIII, ND3, 12S, 16S rRNA, ND1, cytb COIII9821 5′-TTAACGATAGAGTTTACGGGTCAAT-3′cytb5031 5′-AGCTACAGCATTTCTTGGGTATGTAC-3′
designed on each gene for the 16S/COI, 16S/cytb andcytb/COIII regions, respectively (table 1).
PCR was carried out with 30 cycles of a 25 μL reac-tion volume containing 4.75 μL of sterile distilled H2O,2.5 μL of 10× LA PCR buffer II (Takara Bio, Shiga, Japan),4.0 μL of dNTP (2.5 mM), 5.0 μL of each primer (0.4 μM),0.25 μL of 1.25 unit Takara LA TaqTM (Takara), and 1.0 μLof template containing approximately 5 ng DNA. The PCRcondition was used with denaturation at 98◦C for 10 s andannealing and extension combined at the same tempera-ture (60◦C) for 10 min. The PCR products were verifiedon a 1.0% agarose gel and visualized by ethidium bro-mide staining via ultraviolet transillumination. Each purifiedPCR product was cloned into pGEM R©-T easy vectors(Promega, Madison, USA) and subsequently sequenced byprimer walking using BigDye terminator cycling condi-tions on a 3730xl automatic sequencer (Sequencing Service,Macrogen, Seoul, Korea). All sequences were analysed andcompared by the homology search to assure the correct frag-ments using BLASTN (nucleotide similarity) available athttp://www.ncbi.nlm.nih.gov.
Sequence analysis
Each coding region was identified by searching for openreading frames, including start and stop codons. The com-parisons of nucleotide or amino acid sequences of T. pagdeni
Figure 1. Phylogenetic analysis of cyt-b mtDNA genes amongthe bees was conducted using neighbour-joining method, usingTriatoma dimidiata as outgroup (AF301594).
were performed by alignment with those of M. bicolor andA. mellifera using the BLASTX algorithm (NCBI). TransferRNA and ribosomal RNA sequences were identified by eyecomparison with homologues of A. mellifera, M. bicolor andBombus ignites. Phylogenetic analysis using cyt-b mtDNAsequence among these bees was shown by neighbour-joiningmethod in MEGA ver. 5.10 (Tamura et al. 2011) (figure 1).
Results and discussion
The total size of stingless bees mtDNA has been estimatedin M. bicolor species by RFLP analysis and sequencingas approximately 18,500 bp and 14,422 bp (Silvestreet al. 2008), respectively. In our study, the partial mtDNAsequence of T. pagdeni obtained was 12,802 bp or 69.2% ofthe total mtDNA genome of Meliponini species. The incom-plete mtDNA sequence contained 11 complete genes, a par-tial COI gene, both rRNA genes and 12 tRNA genes. Eachprotein-coding gene in T. pagdeni was similar in lengthand nucleotides to their counterpart genes in other sting-less bees (M. bicolor) and honey bees (A. melliferra) (table2). The four overlapping regions, ND4/ND5, cytb/tRNA-Ser(S2), ND1/tRNA-Leu (L1) and ATP8/ATP6, were observed(figure 2). Some of the overlapped genes have been reportedfor M. bicolor mtDNA by Silvestre et al. (2008) such as ND1and tRNA-Leu (L1) shared six nucleotides; ATP6 and ATP8shared 10 nucleotides. Moreover, 14 noncoding regions wereobserved with total intergenic region of 419 bp. In honeybeeA. mellifera, the number of noncoding nucleotides, exclud-ing the COI–COII intergenic and control region is 618 bp(Crozier and Crozier 1993).
The noncoding region found in mtDNA sequences ofT. pagdeni showed no significant similarities with anyregions of the mtDNA of M. bicolor. The intergenic regionbetween COI and COII genes of A. mellifera mtDNA isknown as hypervariable region. This intergenic region hasbeen widely studied in A. mellifera, and size polymorphism
Journal of Genetics, Vol. 92, No. 2, August 2013
Partial mitochondrial sequence of Tetragonula pagdeni
Table 2. Size, start codon and stop codon comparisons of protein-coding genes between T. pagdeni (Tp), M. bicolor (Mb) and A. mellifera(Am).
% Nucleotide similarityLength (bp) Start codon Stop codon between two species
Gene Tp Mb Am Tp Mb Am Tp Mb Am Tp and Mb Tp and Am
ND4L 300 279 264 ATA ATA ATT TAA TAA TAA 69% 68%ND4 1392 1323 1344 ATG ATT ATA TAG TAA TAA 78% 71%ND5 1665 1647 1665 ATC ATT ATT TAA TAA TAA 76% 70%ND6 522 540 504 ATT ATT ATT TAA TAA TAA 71% 69%cytb 1089 1050 1152 ATT ATT ATG TAA TAA TAA 79% 74%ND1 933 930 918 ATA ATA ATT TAA TAA TAA 76% 72%ND3 351 354 354 ATA ATA ATA TAA TAA TAA 71% 0%COIII 780 780 777 ATG ATG ATG TAG TAA TAA 70% 67%ATPase6 687 684 681 ATG ATG ATG TAG TAA TAA 69% 66%ATPase8 168 168 159 ATT ATT ATT TAA TAA TAA 72% 73%COII 627 678 676 ATT ATT ATT TAG TAA T 76% 71%
has been reported (from 200 to 650 bp) among subspecies(Garnery et al. 1992, Franck et al. 1998). It has also beenreferred as a possible second origin of mtDNA replicationand transcription (Cornuet et al. 1991). Our results reveal thatthe COI–COII intergenic region was absent in T. pagdeni, ashas been previously reported in the case of M. bicolor and atleast 16 other Meliponini species (Arias et al. 2006).
The A + T contents of each protein-coding gene inT. pagdeni were high, ranging from 66 to 87% (table 3), sim-ilar to M. bicolor (87%, Silvestre et al. 2008) and A. mel-lifera (85%, Crozier and Crozier 1993). The highly biasedA + T content has been known as a major characteristicof the mitochondrial genome of M. bicolor and A. mellif-era (Crozier and Crozier 1993; Silvestre et al. 2008). The
advantage of AT bias could be explained by one hypoth-esis that the DNA polymerase could use those bases in amore efficient way during mtDNA replication (Clary andWolstenholme 1985) because the energetic cost to breakA–T links is lower compared to G–C links. The A + T-richregion of A. melifera is located between the 12S rRNAand tRNA-Glu genes (Crozier and Crozier 1993). It con-tains several short repeating sequences (6–13 bp) with vary-ing copy numbers (two-to-four copies), scattered throughthe whole region and a polythymidine stretch. This poly-thymidine stretch is reported to be a transcription control orthe initiation of replication (Zhang and Hewitt 1997). Forstingless bee M. bicolor, the A + T-rich region couldnot be sequenced because of difficulties in amplification
Figure 2. Overlapping the three sequenced fragments (5855, 4879 and 5089 bp, respectively) obtained from longPCR amplification with LR12647-R + COI2494, COIII9821 + cytb5031 and LR12677 + cytb10729, respectively andsequenced by primer walking.
Journal of Genetics, Vol. 92, No. 2, August 2013
Sirikul Thummajitsakul et al.
Table 3. Presentation of base compositions (%) in each gene ofT. pagdeni mtDNA.
Gene Length A + T content (%)
ND4L 300 87ND4 1392 83ND5 1665 83ND6 522 86cytb 1089 77ND1 933 83ND3 351 66COIII 780 68ATPase6 687 66ATPase8 168 80COII 627 6916S rRNA 1351 7712S rRNA 762 76tRNA-Pro 67 84tRNA-Phe 67 85tRNA-Asn 68 82tRNA-Thr 67 79tRNA-Ser(2) 67 85tRNA-Leu(1) 69 83tRNA-Val 68 88tRNA-Gln 68 79tRNA-Arg 61 85tRNA-Glu 65 71tRNA-Asp 81 78tRNA-Leu(2) 65 75
(Silvestre et al. 2008), but it was described by its sizeusing RFLP analysis. It was estimated in size approxi-mately 3300 bp, about 2.5-kb longer than in A. mellifera(Crozier and Crozier 1993). In this study, because we werenot able to amplify the entire mtDNA of T. pagdeni, theA + T-rich region, ND2 and 10 tRNA genes were not foundon the sequenced mtDNA fragment. The DNA amplification
failure can be explained by the existence of tandem repeats,heteroplasmy and great length variation at intraspecific andinterspecific levels (Zhang and Hewitt 1997), including par-tial duplications that commonly occur in insect mtDNA. Allprotein-coding genes of T. pagdeni indicated slight differ-ences when compared to M. bicolor and A. mellifera. Thenucleotide sequence of T. pagdeni is more similar to M.bicolor than A. mellifera.
The 12S and 16S rRNA genes of T. pagdeni mtDNA wereestimated to be 762 and 1351 bp, respectively, and showed83% and 78% similarity compared to the partial 12S (437 bp)complete 16S rRNA (1354 bp) nucleotide sequence genesof M. bicolor, respectively. The srRNA and lrRNA genes ofT. pagdeni were located between the tRNA-Leu (1) andtRNA-Val gene and between the tRNA-Val and tRNA-Glngenes, respectively (figure 3). The order of 12S rRNA, 16SrRNA, ND1, cytb and ND6 genes of T. pagdeni mtDNA wasdifferent from those of M. bicolor (Silvestre et al. 2008).All tRNA sequences had 61–81 nucleotides with 12 to 29%G + C and 70.77 to 88% A + T (data not shown). The anti-codon nucleotides were identical to those commonly foundfor the corresponding tRNA genes in A. mellifera, M. bicolorand B. ignitus mtDNA. However, tRNA-Gln found in T. pag-deni mtDNA resembled those in A. mellifera and B. ignitusmtDNA, but not seen in M. bicolor mtDNA (Crozier andCrozier 1993; Silvestre et al. 2008). The tRNA-Gln gene waslocated between 12S and tRNA-Arg (figure 2).
In conclusion, the nearly complete mitochondrial genomeof T. pagdeni may provide critical information for futuremtDNA analyses on biology, ecology and evolution atintraspecific and interspecific levels of other Tetragonulaspecies. Studies of mtDNA gene organization illustrate anexpedited rate in hymenopterans (Dowton et al. 2002).The mitochondrial gene order could be explained byseveral mechanisms. One of the most extensively-accepted
A.
B.
C.
Figure 3. Gene organization of the mtDNA from T. pagdeni (A), M. bicolor (B) (Silvestre et al. 2008) and A. melliferra (Crozier andCrozier 1993). All tRNA genes were indicated by the amino acid they encode: I (tRNAIle), A (tRNAAla), K (tRNALys), M (tRNAMet), W(tRNATrp), Y (tRNATyr), L2 (tRNALeu(UUR)), D (tRNAAsp), G (tRNAGly), R (tRNAArg), N (tRNAAsn), E (tRNAGlu), F (tRNAPhe),H (tRNAHis), T (tRNAThr), P (tRNAPro), S2 (tRNASer(UCN)), L1 (tRNALeu(CUN)) and V (tRNAVal).
Journal of Genetics, Vol. 92, No. 2, August 2013
Partial mitochondrial sequence of Tetragonula pagdeni
mechanisms is tandem duplication of gene regions as a resultof slipped-strand mispairing followed by gene deletions(Levinson and Gutman 1987; Macey et al. 1998).
Acknowledgements
We thank Department of Biochemistry, Faculty of science,Chulalongkorn University and Aquatic Molecular Genetics andBiotechnology Laboratory, National Center for Genetic Engineer-ing and Biotechnology (BIOTEC), NSTDA, for providing facilitiesrequired for the experiments.
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Received 6 November 2012, in final revised form 8 February 2013; accepted 22 February 2013Published on the Web: 26 June 2013
Journal of Genetics, Vol. 92, No. 2, August 2013
