Cytochrome oxidase subunit 1 (COI) gene variations in mitochondrial DNA of Apis koschevnikovi in South Kalimantan
CYTOCHROME OXIDASE SUBUNIT I (COI) GENE VARIATIONS
IN MITOCHONDRIAL DNA OF Apis koschevnikovi
IN SOUTH KALIMANTAN
DZULFAQOR
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
2011
ABSTRACT
DZULFAQOR. Cytochrome Oxidase subunit I (COI) Gene Variations in Mitochondrial DNA of
Apis koschevnikovi in South Kalimantan. Supervised by RIKA RAFFIUDIN and MOCHAMAD
CHANDRA W.
Deforestation in Kalimantan forest by logging and conversion to palm oil plantation
destroyed many living organism and carbon-rich ecosystems. This condition was a serious threat
for native honeybee species from Kalimantan i.e. Apis koschevnikovi (Hymenoptera; Apidae).
Recent observations shows that the population of A. koschevnikovi has decreased persistently.
Ecological integrity of A. koschevnikovi database need to be preserved, including molecular
database. Cytochrome oxidase subunit I (COI) in mitochondrial DNA (mtDNA) can assist by
providing suitable molecular marker with DNA barcode-based technology. Currently, there are 15
haplotypes of COI gene of A. koschevnikovi has published in GeneBank database. The objective of
this research was to explore the nucleotide variations of A. koschevnikovi in South Kalimantan
based on COI gene. Five regencies were observed in South Kalimantan forest; Hulu Sungai
Selatan (HSS), Hulu Sungai Tengah (HST), Kotabaru (KB), Tanah Bumbu (TB), and Balangan
(BL). Based on alignment of 738 bp of COI A. koschevnikovi, there were 5 haplotypes of
nucleotides were found. The common haplotypes found in TB and HST samples, while others
specific in other places. None of the samples showed the same sequence with the nucleotide
sequences of GeneBank database, hence these five haplotypes were concur to be new haplotypes.
Keywords : DNA barcode, COI, A. koschevnikovi, mitochondrial DNA, haplotype.
ABSTRAK
DZULFAQOR. Variasi Gen Sitokrom Oksidase subunit I (COI) di DNA Mitokondria pada Apis
koschevnikovi di Kalimantan Selatan. Dibimbing oleh RIKA RAFFIUDIN dan MOCHAMAD
CHANDRA W.
Deforestasi di hutan Kalimantan yang berupa pembalakan dan konversi hutan menjadi
perkebunan kelapa sawit menghancurkan habitat organisme dan ekosistem. Kondisi ini merupakan
ancaman yang serius terhadap spesies asli yang berasal dari Kalimantan, termasuk Apis
koschevnikovi (Hymenoptera; Apidae). Penelitian-penelitian sebelumnya menunjukkan
kecendrungan populasi A. koschevnikovi yang terus menurun. Memelihara integritas ekologi A.
koschevnikovi sangat diperlukan, termasuk database molekulernya. Sitokrom Oksidase subunit I
(COI) di DNA mitokondria (DNAmt) dapat membantu dengan menyediakan penanda molekuler
dengan teknologi berbasis DNA barcoding. Saat ini, telah ditemukan sebanyak 15 jenis haplotipe
gen COI dari A. koschevnikovi yang telah dipublikasikan di Genbank. Tujuan dari penelitian ini
adalah untuk mengetahui variasi genetik dari A. koschevnikovi Kalimantan Selatan berdasarkan
gen COI dengan menggunakan metode sekuensing DNA. Contoh yang digunakan dalam penelitian
ini berasal dari 5 kabupaten di Kalimantan Selatan; Hulu Sungai Selatan (HSS), Hulu Sungai
Tengah (HST), Kotabaru (KB), Tanah Bumbu (TB), and Balangan (BL). Pengurutan nukleotida
dari contoh yang digunakan menghasilkan panjang nukleotida sebanyak 738 bp, dan sebanyak 5
haplotipe dari nukleotida telah ditemukan. Haplotipe umum ditemukan dari contoh yang berasal
dari Kabupaten HST dan TB. Hasil pengurutan dari contoh ini tidak menunjukkan kesamaan
dengan 15 haplotipe dari Genebank, sehingga 5 haplotipe yang ditemukan dalam penelitian ini
dapat disimpulkan sebagai haplotipe baru.
Kata kunci: DNA barkode, COI, A. koschevnikovi, DNA mitokondria, haplotipe.
CYTOCHROME OXIDASE SUBUNIT I (COI) GENE VARIATIONS IN
MITOCHONDRIAL DNA OF Apis koschevnikovi IN
SOUTH KALIMANTAN
DZULFAQOR
Intended to obtain Sarjana Sains
In Department of Biology
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
2011
Title
: Cytochrome Oxidase subunit I (COI) Gene Variations in
Mitochondrial DNA of Apis koschevnikovi in South Kalimantan
Name
: Dzulfaqor
NIM
: G34061910
Study Program : Biology
Approved by,
Dr. Ir. Rika Raffiudin
Supervisor
Drs. Mochamad Chandra W, M.Sc
Supervisor
Endorsed by
Dr. Ir. Ence Darmo Jaya Supena, M.Si.
Head of Biology Department
Tanggal Lulus :
PREFACE
All praises and thanks to Almighty Allah SWT and for the last prophet Muhammad SAW,
finally the minithesis entitled Cytochrome Oxidase subunit I (COI) Gene Variations in
Mitochondrial DNA of Apis koschevnikovi in South Kalimantan had been finished. The author
would like to send acknowledgements to Dr. Ir. Rika Raffiudin and Drs. Moch. Chandra for their
supervision and taught, and also to DIKTI that funded this research.
I am forever grateful to my parents, Lulu, Riska, and my hammies for their love and
support. Special thanks to Biology 43 crew for the great friendship, and also to members of
Zoology community especially Kak Jesi, Kak Rut, Kak Uche, Ajeng and staffs in Biology
Department for their help and support.
Bogor, February 2011
Dzulfaqor
CURRICULUM VITAE
The author was born in Bogor, July 1st 1988 from the marriage of Saiful Bahri and
Sumiati.
The author was graduated from SMA Negeri 1 Bogor in 2006 and in the same year was
accepted to study in Bogor Agricultural University. In the second year, the author entered the
Department of Biology, with Animal Bioscience as his major study. During studying in
Department of Biology, the author was active in Biology Student Association as a member of
Biosciences division (2007-2008), Chief of Committees in National Biology Competition for High
School (2008), Laboratory Assistant for General Biology, Vertebrates and Avertebrates, Animal
Physiology, and Animal Microtechnique subjects (2008-2010). The author had small research
entitled “Diversity of Medicine Herbs” in Situ Gunung Nature Reserve, Sukabumi (2008), small
research regarding Ambient Air Pollution resulting from Tire Manufacture at PT. Goodyear Tbk.,
Bogor (2009), and small research assistant for the Termites Diversity in Pangandaran Nature
Reserve, West Java (2010).
CONTENTS
Page
LIST OF TABLE ...........................................................................................................................viii
LIST OF FIGURE ..........................................................................................................................viii
LIST OF APPENDIX ....................................................................................................................viii
INTRODUCTION............................................................................................................................. 1
Background ............................................................................................................................ 1
Research Objective ................................................................................................................. 1
MATERIALS AND METHODS ...................................................................................................... 1
Time and Place ....................................................................................................................... 1
Honey bee Collection and DNA Extraction ........................................................................... 1
COI Gene Amplification ........................................................................................................ 1
Electrophoresis and DNA Visualization ................................................................................ 2
Sequencing and DNA Analysis ........................................................................................................ 2
RESULT............................................................................................................................................ 3
DNA Amplification ................................................................................................................ 3
Sequencing and DNA Analysis ........................................................................................................ 3
DISCUSSION ................................................................................................................................... 9
CONCLUSION ............................................................................................................................... 10
REFERENCES................................................................................................................................ 10
APPENDIX ..................................................................................................................................... 12
LIST OF TABLE
Page
1 Nucleotide primers for amplifying COI gene of A. koschevnikovi and primer positions based on
mitochondrial DNA of Apis mellifera (Crozier & Crozier 1993) .................................................. 2
2 A. koschevnikovi COI gene in published Genebank database (www.ncbi.nlm.nih.gov) ................ 3
3 Sample code of A. koschevnikovi used in amplification and sequencing in current study ............ 4
4 GC and AT content of A. koschevnikovi COI gene from current studies ....................................... 4
5 Haplotypes variations of A. koschevnikovi from current studies based on COI gene.................... 4
6 Position of nucleotide variations of A. koschevnikovi samples in COI gene. Common hap= Ak1
HST and Ak1 TB. Source of hap1-hap15 refer to Table 2 ............................................................ 7
7 Genetic distances of A. koschevnikovi COI gene (current study, refer to Table 3) and 15
haplotypes of COI gene from Genebank (refer to Table 2) based on Kimura-2-parameter
substitution method with 1000x bootstrap replication ................................................................... 8
LIST OF FIGURE
Page
1 Sampling location for A. koschevnikovi COI gene in South Kalimantan as shown by black square
(Raffiudin R 24 Januari 2011, personal communication). Number = colonies number (refer to
Appendix 1).. ................................................................................................................................. 2
2 DNA fragments of approximately 900 bp COI gene of Apis koschevnikovi. Lane 1-5= Ak 1BL,
Ak 1HST, Ak 8HSS, Ak 1TB, Ak 2KB. M = 100 bp DNA marker (Promega) ........................... 4
3 Nucleotides sequence of A. koschevnikovi COI region from Ak8 HSS sample and COI putative
amino acids. Number = nucleotide position. Underlined nucleotides = forward and reverse
primer. ........................................................................................................................................... 5
4 Nucleotides alignment of A. koschevnikovi COI gene resulted from this study ............................. 5
5 Putative amino acid sequences of A. koschevnikovi COI gene in this study. Number = amino
acid position,
= variation of amino acid based on six samples in this study, : = closely related
of amino acid ................................................................................................................................. 6
6 Phylogenetic tree of A. koschevnikovi from current studies and 15 haplotypes in Genebank
databases based on COI region using NJ construction with 1000x bootstrap replication.............. 9
LIST OF APPENDIX
Page
1 A. koschevnikovi honey bee collection from South Kalimantan. * = samples used for sequencing
and analysing in this study (Raffiudin R 24 Januari 2011, personal communication) ................ 13
2 Chromatogram of A. koschevnikovi cytochrome oxidase subunit 1 (COI) gene from Ak8 HSS
sample using AmF_cox1 forward primer .................................................................................... 14
3 Chromatogram of A. koschevnikovi cytochrome oxidase subunit 1 (COI) gene from Ak 8HSS
sample using AmR_cox1 reverse primer. .................................................................................... 15
INTRODUCTION
Background
Deforestation in Kalimantan due to
logging and forest conversion to palm oil
plantation destroyed many living organism
and carbon-rich ecosystems (Curran et al.
2004). This condition was a serious threat for
the species that is native in Kalimantan such
as Apis koschevnikovi, a cavity nesting honey
bee. It is classified in the order of
Hymenoptera,
suborder
of
Apocrita,
superfamily of Apoidea, and family of Apidae
(Michener 2000). The distribution of A.
koschevnikovi has establishes many new site
localities but all within the previously
confirmed limits of the tropical evergreen
forest of Sundaland (Hadisoesilo et al. 2008).
Nowadays, recent observations shows that the
population of A. koschevnikovi has decreased
persistently due to forest loss and
establishment of some clear land to make oil
palm, rubber, and coconut plantations (Otis
1991).
Preserving the ecological integrity of A.
koschevnikovi database is necessary, including
molecular database. Cytochrome oxidase
subunit I (COI) in mitochondrial DNA
(mtDNA) provide suitable molecular marker
with DNA barcode-based technology due to
their characteristic i.e no deletion and
insertion in its sequence, and slightly
variances in their sequences (Hebert et al.
2003). In animals mtDNA, COI usually
composed by over 600 bp of long, and at least
648 bp of long is required to be submitted into
Genebank database (www.ncbi.nlm.nih.gov).
Currently, there are 15 haplotypes of COI
gene of A. koschevnikovi has published in
Genebank under Accesion number AF153110
- AF153111, AY012723, AY754729 AY754732, and DQ016097 - DQ016104
(www.ncbi.nlm.nih.gov). Most of the
haplotypes occured from Sabah (haplotype 37, hap 11-15), the others are from Sarawak
(Malaysia), and West Kalimantan. However,
out of 15 haplotypes, three found in
Kalimantan. This study was aimed to explore
further to find another variations of COI
haploytpes for building the database
especially in Kalimantan forest.
Research Objective
The objective of this research was to
explore the variations of COI gene in
mitochondrial DNA of A. koschevnikovi in
South Kalimantan.
MATERIALS AND METHODS
Time and Place
The research was conducted on January
2010 up to February 2011, and it was taken
place in Division of Animal Function and
Behaviour, Department of Biology, Bogor
Agricultural University (IPB).
Honey Bee Collection and DNA Extraction
The objects of my research were DNA
from several A. koschevnikovi honey bees.
Honey bees used in this study were collection
of Dr. Rika Raffiudin (Dept. Biology IPB)
that were collected from Kabupaten Hulu
Sungai Selatan (Ak HSS), Hulu Sungai
Tengah (Ak HST), Balangan (Ak BL), Tanah
Bumbu (Ak TB), and Kotabaru (Ak KB) in
South Kalimantan (Figure 1). Several worker
honey bee from several colonies were
collected from each regencies and were
preserved in absolute ethanol.
DNA extraction has carried out by using
0.2% Cetyl Trimethyl Ammonium Bromide
(CTAB) and ethanol precipitation (Sambrook
et al. 1989).
COI Gene Amplification
TaKaRa Thermal Cycler machine was
used to amplify A. koschevnikovi DNA. COI
gene of A. koschevnikovi fragment
amplification
used AmF_cox1 forward
primer and AmR_cox1 reverse primer (Table
1) based on Apis mellifera complete sequence
(www.ncbi.nlm.nih.gov). The nucleotide
primers position were at 1915-1934 (forward)
and 2719-2739 (reverse) based Genbank
Accesion number L06178 complete A.
mellifera mitochondrial genome. COI gene
locate at nucleotide number 1794-3359 of
mitochondrial region of A. mellifera (Crozier
& Crozier 1993). The PCRs reagent were 20
µl. The PCRs reagent consists of 7.4 µl of
destilation water, 0.8 µl of 10 µM forward
primer, 0.8 µl of 10 µM reverse primer, 1 µl
DNA template collected from the extraction
(41.600 ng/µl), and 10 µl of KAPA Taq 2X
ReadyMix DNA Polymerase (containing 0.05
U/µl of KapaTaq DNA Polymerase, 25 mM of
reaction Buffer with Mg2+, 0.4 mM each
dNTP). DNA amplification in PCRs machine
occurred for 30 cycles in 5 stages i.e. 3
2
minutes (mins) at 940 C for initial denaturing,
30
P. Sebuku
P. Laut
L
t
24
Figure 1 Sampling location for A. koschevnikovi COI gene in South Kalimantan as shown
by black square (Raffiudin R 24 Januari 2011, personal communication). Number
= colonies number (refer to Appendix 1).
Table 1 Nucleotide primers for amplifying COI gene of A. koschevnikovi and primer positions
based on mitochondrial DNA of Apis mellifera (Crozier & Crozier 1993)
Primer
Forward amF_cox1
Reverse amR_cox1
Nucleotide sequence of primer
(5’-3’) (Crozier & Crozier 1993)
CCCCAGGATCATGAATTAGC
TCATTGCTGTACCAACAGGAA
minutes (mins) at 940 C for initial denaturing,
30 cycles of 1 min at 940 C (denaturing), 1
min and 30 seconds at 550 C (annealing), 2
mins at 720 C (DNA elongation) and followed
by 5 mins for final extension.
Electrophoresis and DNA Visualization
Amplified DNA was migrated using 6%
Polyacrilamid Gel Electrophoresis (PAGE)
which was submerged in 1x TBE buffer (10
mM Tris-HCl, 1 M Borat acid, and 0.1 mM
EDTA), operated for 50 minutes at 200 V.
The compositions of 6% gel acrilamide were
12 ml of aquades, 4 ml of 5xTBE, 4 ml of
acrilamide, 15 µl of TEMED, and 160 µl of
10% APS.
DNA visualization used sensitive Silver
Nitrate staining (Tegelstrom 1986). Gel
resulting from electrophoresis was washed
using CTAB solution (0.2 g/200 ml of
aquades) for 8 mins, and was flushed using
200 ml of aquades for 2 mins (2 times).
Subsequently, its gel was submerged in
NH4OH solution (2.4 ml/200 ml of aquades)
for 8 mins, and solution of mixture contained
0.32 g of AgNO3, 0.8 ml of NH4OH, 80 µl
Positions primer in mitochondrial
DNA of Apis mellifera
(Crozier & Crozier 1993)
1915-1934
2719-2739
NaOH, and 200 ml of aquades for 10 mins.
Then, the gel was flushed again using the
same condition as above, and was submerged
again in the solution of mixture contained 4 g
of NA2CO3, 100 µl of formaldehida, and 200
ml of aquades until the bands of DNA seen.
Staining process was stopped using 200 µl of
1% acetat acid in 200 ml of aquades.
Sequencing and DNA Analysis
Genetic variations in COI mitochondrial
region was analysed based on DNA
sequencing method. DNA was sequenced by
using ABI 3100 and 3730XL sequencer
machine at sequencing company. Nucleotide
sequence were edited and analysed using
Genetyx Win Version 4.0 software. Forward
and reverse sequence were aligned to obtain
contiguous sequence by using Clustal X
software (Thompson et al. 1997). All DNA
contiguous sequences was aligned together
with haplotypes databases of COI gene of A.
koschevnikovi in Genebank NCBI (National
Center for Biotechnology Information) under
Accesion number AF153110-AF153111,
3
AY012723, AY754729- AY754732, and
DQ016097-DQ016104
(www.ncbi.nlm.nih.gov) (Table 2).
Edited DNA sequences were translated
into putative amino acid using Genetyx and
were aligned by using Clustal X software.
DNA genetic distances and phylogenetic
analysis were constructed between current
samples and 15 haplotypes in Genebank as
ingroup while A. cerana with accesion
number GQ162109, and A. mellifera with
accesion number L06178 as outgroups. These
analysis was carried out by using MEGA4
software based on Kimura-2-parameter
substitution method and Neighbour Joining
(NJ) construction with 1000x bootstrap
(Tamura et al. 2007).
RESULT
DNA Amplification
There were 29 samples of A. koschevnikovi
COI gene from five regencies in South
Kalimantan were amplified using AmF_cox1
and AmR_cox1 primer (Table 3). All samples
showed the same results i.e single band with
approximately 900 bp of nucleotide (Figure
2).
Table 2 A. koschevnikovi COI gene in published Genebank database (www.ncbi.nlm.nih.gov)
No
Accesion
number
1
AF153110
2
AF153111
3
AY012723
4
AY754729
Suka, T., and Tanaka, H.
haplotipe 4
5
AY754730
Suka, T., and Tanaka, H.
haplotipe 5
6
AY754731
Suka, T., and Tanaka, H.
haplotipe 6
7
AY754732
Suka, T., and Tanaka, H.
haplotipe 7
8
DQ016097
9
DQ016098
10
DQ016099
11
DQ016100
12
DQ016101
13
DQ016102
14
DQ016103
15
DQ016104
Author
Tanaka, H. Roubik, Kato,
M., Liew, F., and Gunsalam,
Tanaka, H. Roubik, Kato,
M., Liew, F., and Gunsalam,
G.
Tanaka, H. , Suka T.,
Roubik, and Mohamed M.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Haplotypes
haplotype 1
haplotype 2
haplotype 3
haplotipe 8
Source
Brunei
Malaysia: Lamibir,
Sarawak
Malaysia: Crocker
Range Park, Sabah
Malaysia: Crocker
Range Park, Sabah
Malaysia: Crocker
Range Park, Sabah
Malaysia: Crocker
Range Park, Sabah
Malaysia: Crocker
Range Park, Sabah
Indonesia: kutai
national park, east
Kalimantan
Nucleotides
long (bp)
1041
1041
1041
1041
1041
1041
1041
1041
haplotipe 9
Indonesia: Loksad,
south Kalimantan
1041
haplotipe 10
Indonesia: field
site near from
sadap, west
Kalimantan
1041
haplotipe 11
Malaysia: Tawau,
Sabah
1041
haplotipe 12
Malaysia: Tawau,
Sabah
1041
haplotipe 13
Malaysia: Tawau,
Sabah
1041
haplotype 14
Malaysia: Tawau,
Sabah
1041
haplotype 15
Malaysia: Tawau,
Sabah
1041
4
1
2
3
4
5
M
2), while nucleotide sequence of AmF_cox1
primer found in AmR_cox1 sequence
(Appendix 3). Complete contiguous sequence
was found in Ak HSS sample colony number
8 (Ak8 HSS) i.e 826 bp (Figure 3).
Based on six contiguous of A.
koschevnikovi COI gene sequences alignment,
738 bp of nucleotide was observed. All
contiguous DNA showed rich of A-T
sequences, i.e. 75.5 % while G-C was 24.5 %
(Table 4).
There were five variations of DNA
sequences occurred from current studies
alignment. The variations respectively
occurred in nucleotide number 119, 125, 219,
254 and 272 (Figure 4). Hence, five
haplotypes were observed (Table 5). The first
haplotype was a common haplotypes was
found in Ak1 TB and Ak1 HST samples. The
second to fifth haplotypes respectively were
found in Ak2 KB, Ak8 HSS, Ak1 BL, and
Ak5
BL
sample
(Table
5).
900 bp
400 bp
Figure 2 DNA fragments of approximately
900 bp COI gene of Apis
koschevnikovi. Lane 1-5= Ak
1BL, Ak 1HST, Ak 8HSS, Ak
1TB, Ak 2KB. M = 100 bp DNA
marker (Promega).
Sequencing and DNA Analysis
Out of 29 amplified samples of A.
koschevnikovi COI gene, six samples were
sequenced (see Table 3 and Appendix 1).
Nucleotide sequence of AmR_cox1 primer
was found in AmF_cox1 sequence (Appendix
Table 3 Sample code of A. koschevnikovi used in amplification and sequencing in current study
No
1
2
3
Sample
code
Ak HSS
Ak HST
Ak BL
Regency Origin
Hulu Sungai Selatan
Hulu Sungai Tengah
Balangan
4
5
Ak KB
Ak TB
Kotabaru
Tanah Bumbu
Number of
amplified colonies
11
4
5
7
2
Samples for sequenced and
alignment analysis
Colony no.8 (Ak8 HSS)
Colony no.1 (Ak1 HST)
Colony no.1 & 5 (Ak1 BL &
Ak5 BL)
Colony no. 2 (Ak2 KB)
Colony no. 1 (Ak1 TB)
Table 4 GC and AT content of A. koschevnikovi COI gene from current studies
No
1
2
3
4
5
6
Sample Code
Ak1 HST
Ak1 TB
Ak8 HSS
Ak5 BL
Ak1 BL
Ak2 KB
A-T nucleotides (%)
75.53
75.53
75.67
75.67
75.53
75.39
G-C nucleotides (%)
24.47
24.47
24.33
24.33
24.47
24.61
Table 5 Haplotypes variations of A. koschevnikovi from current studies based on COI gene
No
Haplotype
Sample
1.
Haplotype 1
2.
Haplotype 2
Ak1 HST
Ak1 TB
Ak2 KB
3.
4.
5.
Haplotype 3
Haplotype 4
Haplotype 5
Ak8 HSS
Ak1 BL
Ak5 BL
Position of
nucleotide
alteration
Common haplotype
Nucleotide alteration
(compared with
common haplotype)
-
Substitution
no. 119 and 125
T ÆC and T ÆA
no. 219
no. 254
no. 272
G ÆA
T ÆA
C ÆT
Transition and
transversion
Transition
Transversion
Transition
-
5
CCCCAGGATCATGAATTAGCAATGATCAAATCTATAATACTATTGTCACAAGACATGCAT
P G S W I S N D Q I Y N T I V T S H A F
60
TTTTAATAATTTTTTTTATAGTTATACCATTTTTAATTGGAGGATTTGGTAATTGATTAA
L M I F F M V M P F L I G G F G N W L I
120
TTCCATTAATACTTGGATCTCCAGATATAGCTTTTCCTCGTATAAATAACATTAGATTTT
P L M L G S P D M A F P R M N N I S F W
180
GATTATTACCACCTTCATTATTAATATTATTATTAAGAAATTTATTTTACCCAAGACCAG
L L P P S L L M L L L S N L F Y P S P G
240
GAACAGGATGAACTATTTATCCTCCACTATCAGCTTATTTATATCATTCTTCACCATCAG
T G W T I Y P P L S A Y L Y H S S P S V
300
TAGATTTCGCAATTTTTTCTTTACATATATCTGGAATTTCATCAATTATAGGATCATTAA
D F A I F S L H M S G I S S I M G S L N
360
ATTTAATAGTAACAATTATAATAATAAAAAATTTTTCTTTAAATTATGATCAAATTTCAT
L M V T I M M M K N F S L N Y D Q I S L
420
TATTTCCTTGATCAGTATTTATTACAGCTATTTTATTAATTATATCATTACCTGTTTTAG
F P W S V F I T A I L L I M S L P V L A
480
CTGGAGCAATTACAATACTATTATTTGATCGAAATTTTAATACATCATTCTTTGATCCAA
G A I T M L L F D R N F N T S F F D P M
540
TAGGAGGAGGAGATCCAATTTTATATCAACATTTATTTTGATTTTTTGGTCATCCAGAAG
G G G D P I L Y Q H L F W F F G H P E V
600
TTTATATTTTAATTTTACCCGGATTTGGATTAATTTCTCATATTGTAATAAATGAAAGAG
Y I L I L P G F G L I S H I V M N E S G
660
GAAAAAAAGAAATTTTTGGAAATTTAGGAATAATTTATGCAATATTAGGAATTGGATTTC
K K E I F G N L G M I Y A M L G I G F L
720
TAGGATTTATTGTTTGAGCTCATCATATATTTACTGTTGGATTAGATGTTGATACACGAG
G F I V W A H H M F T V G L D V D T R A
780
CTTATTTTACTTCTGCAACTATAATCATTGCTGTACCAACAGGAAA
Y F T S A T M I I A V P T G *
826
Figure 3 Nucleotides sequence of A. koschevnikovi COI region from Ak8 HSS sample
and COI putative amino acid. Number = nucleotide position. Underlined
nucleotides = forward and reverse primer.
Ak1HST
Ak1TB
Ak8HSS
Ak5BL
Ak1BL
Ak2KB
TTGGTAATTGATTAATTCCATTAATACTTGGATCTCCAGATATAGCTTTTCCTCGTATAAATAACATTA
.....................................................................
.....................................................................
.....................................................................
.....................................................................
.................................................C.....A.............
[138]
[138]
[138]
[138]
[138]
[138]
Ak1HST
Ak1TB
Ak8HSS
Ak5BL
Ak1BL
Ak2KB
CAGGATGAACTGTTTATCCTCCACTATCAGCTTATTTATATCATTCTTCACCATCAGTAGATTTCGCAA
.....................................................................
...........A.........................................................
................................................................T....
..............................................A......................
.....................................................................
[276]
[276]
[276]
[276]
[276]
[276]
Figure 4 Nucleotides alignment of A. koschevnikovi COI gene in this study. Number in bracket =
nucleotide position, . = homolog nucleotide, = variations of nucleotide. Samples code
refer to Table 3.
6
Nucleotide sequence of Ak8 HSS were
translated into putative amino acid and
showed no stop codon (Figure 3). Putative
amino acid of COI gene from current studies
were aligned as well. The result showed that
there was one amino acid variation sequence
(Figure 4) i.e at nucleotide no. 219-221, codon
ATT was translated into Isoleusin (I) in Ak8
HSS sample (haplotype 3), while Valin (V)
was resulted from codon GTT in Ak1 HST
and Ak1 TB sequences (common haplotype).
Meanwhile, the other variations from
current studies alignment did not show
variations for its putative amino acid. For
examples, In Ak2 KB sample, codon TTC at
nucleotides no. 117-119 (haplotype 2) was
translated into Phenilalanin (F) and codon
CGA at nucleotides no. 123-125 was translated
into Arginin (R). At the same position in
common haplotype, codon TTT and CGT were
translated become same amino acid,
Phenilalanin and Arginin (Figure 5). In Ak1
BL sample, codon TCA at nucleotides no.
252-254 (haplotype 4) was translated become
Serin (S). At the same position in common
haplotypes, codon TCT was translated become
Serin amino acid as well. The other example
came from Ak5 BL sample, codon TTT at
nucleotides no. 270-272 (haplotype 5) was
translated into Phenilalanin (F). At the same
position in common haplotypes, codon TTC
was translated become Phenilalanin amino
acid as well.
This study performed alignment between
current sequenced samples (refer to Table 3)
and published A. koscehvnikovi COI databases
in Genebank as well. Total of 685 bp of
nucleotides was aligned. None of the current
study COI A. koschevnikovi study samples
showed the same sequence with the published
nucleotide sequences from Genebank
databases (Table 6).
Genetic distances analysis showed the
relatedness of genetic relationship between all
samples in this study, with the highest genetic
distance in Ak2 KB sample i.e 0.003-0.004
(Table 7). Phylogenetic analysis clustered all
samples in this study and published A.
koschevnikovi COI database in Genebank in
three clusters i.e cluster A, B, and C (Figure
6). All samples in this study and several
haplotypes from Sabah (Malaysia) and Brunei
were clustered in cluster A with 88 of
bootstrap value. Cluster B consisted of
haplotype 2 from Sarawak (Malaysia),
haplotype 10 from West Kalimantan, and
haplotype 11 from Sabah with 98 of bootstrap
value. However, haplotypes 3 and 4 from
Sabah was separated in cluster C having 100
of bootstrap value (Figure 6), both performed
as the basal taxon, close to A. cerana as the
outgroups.
Ak2KB
Ak8HSS
Ak1BL
Ak1HST
Ak1TB
Ak5BL
AmCrozier
AkHap13
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLFMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
*************************************:***************
60
60
60
60
60
60
60
60
Ak2KB
Ak8HSS
Ak1BL
Ak1HST
Ak1TB
Ak5BL
AmCrozier
AkHap13
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTIYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
*****:***********************************************
120
120
120
120
120
120
120
120
Figure 5
Putative amino acid sequence of A. koschevnikovi COI gene from this study. Number
= amino acid position, = variation of amino acid based on six samples in this study, :
= closely related of amino acid.
Table 6 Position of nucleotide variations of A, koschevnikovi samples in COI gene. Common hap= Ak1 HST and Ak1 TB. Source of hap1-hap15 refer to Table 2
1
2
1
5
2
1
3
3
4
3
4
5
5
1
5
4
5
7
6
3
6
6
7
2
8
1
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
2
4
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
1
2
6
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
3
8
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
1
4
1
C
.
.
.
.
T
T
T
.
.
.
.
.
.
.
.
.
.
T
T
Hap
Common hap
Ak2 KB
Ak8 HSS
Ak1 BL
Ak5 BL
Hap10_Kalbar
Hap11_Sabah
Hap2_Sarawak
Hap14_Sabah
Hap15_Sabah
Hap9_Kalsel
Hap12_Sabah
Hap1_Brunei
Hap6_Sabah
Hap7_Sabah
Hap13_Sabah
Hap8_Kaltim
Hap5_Sabah
Hap3_Sabah
Hap4_Sabah
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
T
.
.
.
.
C
C
C
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
A
.
.
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
T
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
T
.
.
.
.
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
T
C
.
.
.
C
C
C
.
.
.
.
.
.
.
.
.
.
.
.
T
A
.
.
.
A
A
A
.
.
.
.
.
.
.
.
.
.
.
.
Hap
Common hap
Ak2 KB
Ak8 HSS
Ak1 BL
Ak5 BL
Hap10_Kalbar
Hap11_Sabah
Hap2_Sarawak
Hap14_Sabah
Hap15_Sabah
Hap9_Kalsel
Hap12_Sabah
Hap1_Brunei
Hap6_Sabah
Hap7_Sabah
Hap13_Sabah
Hap8_Kaltim
Hap5_Sabah
Hap3_Sabah
Hap4_Sabah
4
6
5
T
.
.
.
.
C
C
.
.
.
.
.
.
.
.
.
.
.
C
C
4
6
8
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
4
7
7
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
C
5
3
1
T
.
.
.
.
.
.
.
.
.
.
.
.
T
A
.
.
.
T
T
5
3
4
A
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.
.
5
4
0
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
5
7
3
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
5
9
1
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
6
0
0
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
6
2
7
A
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.
6
3
1
C
.
.
.
.
T
T
T
.
.
.
.
.
.
.
.
T
.
T
T
6
3
9
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
6
4
5
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
6
5
1
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
6
6
9
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
6
7
8
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
6
8
5
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
4
7
A
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.
.
1
5
0
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
5
3
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
5
6
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
5
9
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
6
5
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
1
6
6
G
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
7
7
A
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
1
7
8
C
.
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
2
0
1
T
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
2
0
7
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
2
1
9
C
.
.
.
T
.
.
.
.
.
.
.
.
.
.
T
.
.
T
T
2
3
1
T
.
.
.
.
C
C
C
.
.
.
.
.
.
.
.
.
.
A
A
2
8
2
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
3
0
9
T
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
3
2
1
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
3
4
5
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
3
7
2
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
3
8
7
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
3
9
3
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
4
0
5
A
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
T
T
4
0
9
C
.
.
.
.
T
T
T
T
T
T
T
T
T
T
T
T
T
.
.
4
1
2
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
4
3
5
A
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
4
4
1
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
4
4
7
T
.
.
.
.
C
C
C
.
.
.
.
.
.
.
.
.
C
C
C
4
5
0
A
.
.
.
.
T
T
T
.
.
.
.
.
.
.
.
.
.
.
.
4
5
9
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
4
6
2
A
.
.
.
.
T
T
T
.
.
.
.
.
.
.
.
.
.
.
.
Table 7 Genetic distances of A. koschevnikovi COI gene (current study, refer to Table 3) and 15 haplotypes of COI gene from Genebank (refer to Table 2) based on
Kimura-2-parameter substitution method with 1000x bootstrap
[ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 ]
[ 1]
[ 2] 0.001
[ 3] 0.070 0.072
[ 4] 0.070 0.072 0.000
[ 5] 0.073 0.075 0.006 0.006
[ 6] 0.071 0.073 0.015 0.015 0.018
[ 7] 0.075 0.077 0.018 0.018 0.021 0.003
[ 8] 0.071 0.073 0.015 0.015 0.018 0.000 0.003
[ 9] 0.075 0.077 0.018 0.018 0.021 0.003 0.006 0.003
[10] 0.073 0.075 0.016 0.016 0.019 0.001 0.004 0.001 0.004
[11] 0.072 0.073 0.018 0.018 0.021 0.003 0.006 0.003 0.006 0.004
[12] 0.073 0.075 0.018 0.018 0.021 0.003 0.006 0.003 0.006 0.004 0.004
[13] 0.071 0.073 0.015 0.015 0.018 0.003 0.006 0.003 0.006 0.004 0.006 0.006
[14] 0.068 0.070 0.015 0.015 0.018 0.003 0.006 0.003 0.006 0.004 0.006 0.006 0.006
[15] 0.070 0.072 0.016 0.016 0.019 0.001 0.004 0.001 0.004 0.003 0.004 0.004 0.004 0.004
[16] 0.072 0.073 0.018 0.018 0.021 0.003 0.006 0.003 0.006 0.004 0.006 0.006 0.006 0.006 0.004
[17] 0.072 0.073 0.018 0.018 0.021 0.003 0.006 0.003 0.006 0.004 0.006 0.006 0.006 0.006 0.004 0.000
[18] 0.073 0.075 0.019 0.019 0.022 0.004 0.007 0.004 0.007 0.006 0.007 0.007 0.007 0.007 0.006 0.001 0.001
[19] 0.070 0.072 0.019 0.019 0.022 0.004 0.007 0.004 0.007 0.006 0.007 0.007 0.007 0.007 0.006 0.001 0.001 0.003
[20] 0.070 0.072 0.019 0.019 0.022 0.004 0.007 0.004 0.007 0.006 0.007 0.007 0.007 0.007 0.003 0.001 0.001 0.003 0.003
[21] 0.071 0.073 0.015 0.015 0.018 0.006 0.009 0.006 0.009 0.007 0.009 0.009 0.009 0.009 0.007 0.003 0.003 0.004 0.004 0.004
[22] 0.109 0.107 0.092 0.092 0.092 0.088 0.092 0.088 0.090 0.088 0.090 0.088 0.087 0.092 0.087 0.092 0.092 0.094 0.090 0.090 0.092
[23] 0.102 0.102 0.096 0.096 0.092 0.093 0.096 0.093 0.096 0.094 0.093 0.093 0.096 0.096 0.091 0.093 0.093 0.095 0.095 0.091 0.089 0.112
[ 1] #Ak_T_hap3_Sabah
[ 2] #Ak_T_hap4_Sabah
[ 3] #Ak_T_hap10_Kalbar
[ 4] #Ak_T_hap11_Sabah
[ 5] #Ak_T_hap2_Sarawak
[ 6] #Ak_T_hap9_Kalsel
[ 7] #Ak_T_hap15_Sabah
[ 8] #Ak_T_hap12_Sabah
[ 9] #Ak_T_hap14_Sabah
[10] #Ak_T_hap1_Brunei
[11] #Ak_T_hap6_Sabah
[12] #Ak_T_hap7_Sabah
[ 13] #Ak_T_hap8_Kaltim
[ 14] #Ak_T_hap5_Sabah
[ 15] #Ak_T_hap13_Sabah
[ 16] #Ak1HST_R_Kalsel
[ 17] #Ak1TB_R_Kalsel
[ 18] #Ak8HSS_R_Kalsel
[ 19] #Ak1BL_R_Kalsel
[ 20] #Ak5BL_R_Kalsel
[ 21] #Ak2KB_R_KAlsel
[ 22] #Ac_china
[ 23] #Am_Crozier
9
Ak T hap15 Sab ah
Ak T hap12 Sab ah
Ak T hap14 Sab ah
26
Ak T hap1 Brunei
Ak T hap9 Kalsel
19
Ak T hap6 Sab ah
65
23
Ak T hap7 Sab ah
Ak T hap8 Kaltim
28
Ak T hap5 Sab ah
Cluster A
Ak T hap13 Sab ah
34
Ak 5BL R Kalsel
Ak 1BL R Kalsel
88
37
Ak 1HST R Kalsel
49
Ak 1TB R Kalsel
46
Ak 8HSS R Kalsel
100
Ak 2KB R KAlsel
Ak T hap2 Sarawak
66
98
92
Ak T hap10 Kalb ar
Cluster B
Ak T hap11 Sab ah
Ak T hap3 Sab ah
100
Ak T hap4 Sab ah
Cluster C
Apis cerana
Apis m ellifera
0.01
Figure 6
Phylogenetic tree of A. koschevnikovi from current studies and 15 haplotypes in
Genebank databases based on COI region using NJ method with 1000x bootstrap.
Samples code refer to Table 2 and 3.
DISCUSSION
The result of A. koschevnikovi COI gene
amplification in PAGE 6% showed
approximately about 900 bp of nucleotides
long, while 801 bp of average nucleotides
long resulted from DNA sequencing toward
current samples of A. koschevnikovi. This
difference might be occur due to gel shifting
phenomenon i.e. the alteration of DNA band
movement in polyacrilamide gel.
DNA sequencing result showed different
size of nucleotides for each sample. This
condition was due to the unclear graph in
beginning of chromatogram. For alignment
purpose, all contiguous sequences must
commence and end at the same nucleotide
position.
The high number of AT nucleotides i.e
75.5% from each samples agree with insect
DNAmt that contained rich of A+T base
(Crozier et al. 1989). Longer time was needed
to amplify DNA which has a very high G-C
content (Griffiths et al. 1999).
There were two kind of nucleotide
substitution occurred from current studies
alignment i.e transition and transversion
(Table 5). Transition occurred when
nucleotide substitution did not change the
structure of nitrogene base i.e purin (A and G)
to be purin; or pirimidin (C and T) to be
pirimidin, while transversion occured when
nucleotide substitution change the structure of
nitrogene base (purin to be pirimidin; or
pirimidin to be purin) (Griffiths et al. 1999).
The nucleotide substitution does not
always resulted amino acid from transcriptiontranslation process. The alteration will effect
translated amino acid when the substitution
occurred in first or second nucleotide in codon
formation (Klug et al. 2006). For the example
in Ak8 HSS sample at nucleotide no. 219-221,
codon ATT was translated into Isoleusin (I) in
Ak8 HSS (haplotype 3), while Valin (V) was
resulted from codon GTT from Ak1 HST and
Ak1 TB sequence (common haplotype).
However, Valin and Isoleusin is still closely
related in amino acid as showed with double
dot (:) (see Figure 5). Both of them were
clustered in hydrophobic aliphatic amino acid
(Takei et al. 2006).
10
In addition, DNA variation especially third
nucleotide substitution in codon formation
does not always produce different putative
protein (Avise 1994). The examples based on
Ak2 KB sample (haplotype 2) at nucleotides
no. 117-119 and no. 123-125, Ak1 BL sample
at nucleotides no. 252-254 (haplotype 4), and
Ak5 BL sample at nucleotides no. 270-272
(haplotype 5).
The alignment between current samples
(refer to Table 3) and published A.
koschevnikovi COI databases in Genebank has
resulting 685 bp of nucleotides long (Table 5),
which fulfilled 648 nucleotides as a minimum
number of nucleotide requirement to be
submitted
to
DNA
Barcode
(www.ncbi.nlm.nih.gov). None of the current
COI A. koschevnikovi study samples showed
the same sequence with the published
nucleotide sequence from Genebank databases
(Table 5). Hence, these five haplotypes from
this research were the new haplotypes for A.
koschevnikovi COI database.
The results of this research can be used to
accomplish information about molecular
ecology studies of A. koschevnikovi as well.
Based on current studies alignment, Ak1 HST
and Ak1 TB has a same sequence, although
the distance of HST and TB regencies
approximately 150 km (Figure 1). However,
sequences variations were found in two
samples both from Balangan Regency (Ak1
BL and Ak5 BL). These results indicate that
mixed
population
occurred
in
A.
koschevnikovi distribution.
Genetic distances and phylogenetic
analysis were performed to observe genetic
relationship between current studies and
published A. koschevnikovi COI databases in
Genebank as well. Genetic distance analysis
grouped all samples in this study in one
cluster, with the highest genetic distance in
Ak2 KB sample i.e 0.003-0.004 (Table 7).
Ak2 KB sample was collected not from
Kalimantan mainland but from Sebuku island
(see sampling location number 24 in Figure
1). Hence, these highest number might be due
to geographical barrier which separate by Laut
strait and Laut island (see Figure 1).
Phylogenetic analysis grouped Ak
samples from this previous study in three
clusters (Figure 6). All samples in this study
and several haplotypes from Sabah and Brunei
were clustered in cluster A (0.000-0.009 value
of genetic distances), while haplotype 2 from
Sarawak,
haplotype
10
from
West
Kalimantan, and haplotype 11 from Sabah
were clustered in cluster B (0.000-0.022 value
of genetic distances) (Table 7). However, both
were classified in one group due to their
bootstrap value which is 100 (Figure 6).
However, Ak haplotypes 3 and 4 from
Sabah were grouped in different cluster with
100 of bootstrap value, while these samples
were collected from same region with several
haplotypes in cluster A and B. Genetic
distances of haplotypes 3 and 4 compared
with the other haplotypes showed the highest
value (0.001-0.077) (Table 7). Therefore,
these 2 haplotypes need to be reconfirmed for
the species validity.
In the case of these two anomalous
haplotypes from Genebank, therefore, one
need to be aware with the published DNA
database. In addition, further studies from the
other areas in Kalimantan is necessary to have
a complete picture of A. koschevnikovi COI
gene database.
CONCLUSION
The exploration of COI gene of A.
koschevnikovi from six samples taken from
five regencies in South Kalimantan showed
the genetic differentation. There were five
haplotypes of A. koschevnikovi COI gene were
found in this study i.e. haplotype 1, 2, 3, 4,
and 5. Haplotype 1 was a common haplotype
was found in sample Ak1 HST and Ak1 TB,
while others specific in other places. None of
the current samples showed the same
sequence with the nucleotide sequences of
Genebank database, hence these five
haplotypes were the new haplotypes of A.
koschevnikovi COI gene.
REFERENCES
Avise JC. 1994. Molecular Markers, Natural
History and Evolution. New York:
Chapman & Hall.
Crozier RH, Crozier YC, Mackinlay AG.
1989. The CO-I and CO-II region of
honeybee
mitochondrial
DNA:
evidence for variation in insect
mitochondrial evolutionary rates.
Mol Biol Evol 6: 399-411
Crozier RH, Crozier YC. 1993. The
mitochondrial genome of the
honeybee Apis mellifera: complete
sequence and genome organization,
Genetics 133: 97-117.
Curran LM et al.2004. Lowland forest loss in
protected areas of Indonesian
Borneo, Science 303: 1000-1003.
11
Griffiths A, William MG, Jeffrey HM,
Richard CL. 1999. Modern Genetic
Analysis. New York: W.H. Freeman
and Company.
Hadisoesilo et al. 2008. Morphometric
analysis and biogeography of Apis
koschevnikovi Enderlein (1906),
Apidologie 39: 495-503.
Hebert PD, Ratnasingham S, dewaard JR.
2003. Barcoding animal life:
cytochrome c oxidase subunit 1
divergences among closely related
species, Proc. R. Soc. Lond 270:
S96-S99.
Klug WS, Cummings MR, Spencer CA. 2006.
Concepts of Genetics 8th ed. New
Jersey: Pearson Education, Inc.
Michener CD. 2000. The Bees of the World.
London: The John Hopkins Univ Pr.
Otis GW. 1991. A review of the diversity of
species within Apis. In: Smith DR
(ed) Diversity in The Genus Apis.
Oxford: Westview Press. pp 29-50.
Sambrook J, Fritsch EF, Maniatis T. 1989.
Molecular Cloning: A Laboratorium
Manual Second Edition. New York:
Cold Spring Harbor Laboratory
Press.
Takei T et al. 2006. The effects of the side
chains of hydrophobic aliphatic
amino acid residues in an
amphipathic polypeptide on the
formation of a helix and its
association, J. Biochem 139: 271278.
Tamura K, Dudley J, Nei M, Kumar S. 2007.
MEGA4: molecular evolutionary
genetics analysis (MEGA) software
version 4.0, Mol Biol Evol 24: 15961599.
Tegelstrom H. 1986. Mitochondrial DNA in
natural population: an improved
routine for screening of genetic
variation based on sensitive silver
staining. Electrophoresis 7: 226-229.
Thompson JD et al. 1997. The clustal X
windows interface: flexible strategies
for multiple sequence alignment
aided by quality analysis tools. Nucl
Acid Res 24: 4876-4882.
.
APPENDIX
13
Appendix 1 A. koschevnikovi honey bee collection from South Kalimantan. * = samples used for
sequencing and analysing in this study (Raffiudin R 24 Januari 2011, personal
communication)
Southern
latitude
Longitude
east
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.49’
115 o.17’
AK 2 HSS
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.49’
115 o.17’
3.
AK 3 HSS
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.49’
115 o.17’
4.
AK 4 HSS
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.49’
115 o.17’
5.
AK 5 HSS
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.50’
115 o.18’
6.
AK 6 HSS
Kec. Losado, Kab. Hulu Sungai Selatan
02o.49’
115 o.24’
7.
AK 7 HSS
Kec. Losado, Kab. Hulu Sungai Selatan
02o.49’
115 o.25’
8.
AK 8 HSS*
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.49’
115 o.17’
9.
AK 1 BL*
Kec. Lanmpihong, Kab. Balangan
02o.20’
115 o.25’
10.
AK 2 BL
Kec. Lanmpihong, Kab. Balangan
02o.20’
115 o.25’
11.
AK 3 BL
Kec. Lanmpihong, Kab. Balangan
02o.20’
115 o.25’
12.
AK 4 BL
Kec. Lanmpihong, Kab. Balangan
02o.20’
115 o.25’
13.
AK 5 BL*
Kec. Paringin, Kab. Balangan
02o.20’
115 o.25’
14.
AK 9 HSS
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.47’
115 o.17’
15.
AK 10 HSS
Kec. Losado, Kab. Hulu Sungai Selatan
02o.49’
115 o.25’
16.
AK 11 HSS
Kec. Losado, Kab. Hulu Sungai Selatan
02o.49’
115 o.25’
17.
AK 1 HST*
Kec. Batu Benawa, Kab. Hulu Sungai Tengah
02o.38’
115 o.26’
18.
AK 2 HST
Kec. Batu Benawa, Kab. Hulu Sungai Tengah
02o.38’
115 o.25’
19.
AK 3 HST
Kec. Batu Benawa, Kab. Hulu Sungai Tengah
02o.38’
115 o.25’
20.
AK 4 HST
Kec. Alai Utara, Kab. Hulu Sungai Tengah
IN MITOCHONDRIAL DNA OF Apis koschevnikovi
IN SOUTH KALIMANTAN
DZULFAQOR
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
2011
ABSTRACT
DZULFAQOR. Cytochrome Oxidase subunit I (COI) Gene Variations in Mitochondrial DNA of
Apis koschevnikovi in South Kalimantan. Supervised by RIKA RAFFIUDIN and MOCHAMAD
CHANDRA W.
Deforestation in Kalimantan forest by logging and conversion to palm oil plantation
destroyed many living organism and carbon-rich ecosystems. This condition was a serious threat
for native honeybee species from Kalimantan i.e. Apis koschevnikovi (Hymenoptera; Apidae).
Recent observations shows that the population of A. koschevnikovi has decreased persistently.
Ecological integrity of A. koschevnikovi database need to be preserved, including molecular
database. Cytochrome oxidase subunit I (COI) in mitochondrial DNA (mtDNA) can assist by
providing suitable molecular marker with DNA barcode-based technology. Currently, there are 15
haplotypes of COI gene of A. koschevnikovi has published in GeneBank database. The objective of
this research was to explore the nucleotide variations of A. koschevnikovi in South Kalimantan
based on COI gene. Five regencies were observed in South Kalimantan forest; Hulu Sungai
Selatan (HSS), Hulu Sungai Tengah (HST), Kotabaru (KB), Tanah Bumbu (TB), and Balangan
(BL). Based on alignment of 738 bp of COI A. koschevnikovi, there were 5 haplotypes of
nucleotides were found. The common haplotypes found in TB and HST samples, while others
specific in other places. None of the samples showed the same sequence with the nucleotide
sequences of GeneBank database, hence these five haplotypes were concur to be new haplotypes.
Keywords : DNA barcode, COI, A. koschevnikovi, mitochondrial DNA, haplotype.
ABSTRAK
DZULFAQOR. Variasi Gen Sitokrom Oksidase subunit I (COI) di DNA Mitokondria pada Apis
koschevnikovi di Kalimantan Selatan. Dibimbing oleh RIKA RAFFIUDIN dan MOCHAMAD
CHANDRA W.
Deforestasi di hutan Kalimantan yang berupa pembalakan dan konversi hutan menjadi
perkebunan kelapa sawit menghancurkan habitat organisme dan ekosistem. Kondisi ini merupakan
ancaman yang serius terhadap spesies asli yang berasal dari Kalimantan, termasuk Apis
koschevnikovi (Hymenoptera; Apidae). Penelitian-penelitian sebelumnya menunjukkan
kecendrungan populasi A. koschevnikovi yang terus menurun. Memelihara integritas ekologi A.
koschevnikovi sangat diperlukan, termasuk database molekulernya. Sitokrom Oksidase subunit I
(COI) di DNA mitokondria (DNAmt) dapat membantu dengan menyediakan penanda molekuler
dengan teknologi berbasis DNA barcoding. Saat ini, telah ditemukan sebanyak 15 jenis haplotipe
gen COI dari A. koschevnikovi yang telah dipublikasikan di Genbank. Tujuan dari penelitian ini
adalah untuk mengetahui variasi genetik dari A. koschevnikovi Kalimantan Selatan berdasarkan
gen COI dengan menggunakan metode sekuensing DNA. Contoh yang digunakan dalam penelitian
ini berasal dari 5 kabupaten di Kalimantan Selatan; Hulu Sungai Selatan (HSS), Hulu Sungai
Tengah (HST), Kotabaru (KB), Tanah Bumbu (TB), and Balangan (BL). Pengurutan nukleotida
dari contoh yang digunakan menghasilkan panjang nukleotida sebanyak 738 bp, dan sebanyak 5
haplotipe dari nukleotida telah ditemukan. Haplotipe umum ditemukan dari contoh yang berasal
dari Kabupaten HST dan TB. Hasil pengurutan dari contoh ini tidak menunjukkan kesamaan
dengan 15 haplotipe dari Genebank, sehingga 5 haplotipe yang ditemukan dalam penelitian ini
dapat disimpulkan sebagai haplotipe baru.
Kata kunci: DNA barkode, COI, A. koschevnikovi, DNA mitokondria, haplotipe.
CYTOCHROME OXIDASE SUBUNIT I (COI) GENE VARIATIONS IN
MITOCHONDRIAL DNA OF Apis koschevnikovi IN
SOUTH KALIMANTAN
DZULFAQOR
Intended to obtain Sarjana Sains
In Department of Biology
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
2011
Title
: Cytochrome Oxidase subunit I (COI) Gene Variations in
Mitochondrial DNA of Apis koschevnikovi in South Kalimantan
Name
: Dzulfaqor
NIM
: G34061910
Study Program : Biology
Approved by,
Dr. Ir. Rika Raffiudin
Supervisor
Drs. Mochamad Chandra W, M.Sc
Supervisor
Endorsed by
Dr. Ir. Ence Darmo Jaya Supena, M.Si.
Head of Biology Department
Tanggal Lulus :
PREFACE
All praises and thanks to Almighty Allah SWT and for the last prophet Muhammad SAW,
finally the minithesis entitled Cytochrome Oxidase subunit I (COI) Gene Variations in
Mitochondrial DNA of Apis koschevnikovi in South Kalimantan had been finished. The author
would like to send acknowledgements to Dr. Ir. Rika Raffiudin and Drs. Moch. Chandra for their
supervision and taught, and also to DIKTI that funded this research.
I am forever grateful to my parents, Lulu, Riska, and my hammies for their love and
support. Special thanks to Biology 43 crew for the great friendship, and also to members of
Zoology community especially Kak Jesi, Kak Rut, Kak Uche, Ajeng and staffs in Biology
Department for their help and support.
Bogor, February 2011
Dzulfaqor
CURRICULUM VITAE
The author was born in Bogor, July 1st 1988 from the marriage of Saiful Bahri and
Sumiati.
The author was graduated from SMA Negeri 1 Bogor in 2006 and in the same year was
accepted to study in Bogor Agricultural University. In the second year, the author entered the
Department of Biology, with Animal Bioscience as his major study. During studying in
Department of Biology, the author was active in Biology Student Association as a member of
Biosciences division (2007-2008), Chief of Committees in National Biology Competition for High
School (2008), Laboratory Assistant for General Biology, Vertebrates and Avertebrates, Animal
Physiology, and Animal Microtechnique subjects (2008-2010). The author had small research
entitled “Diversity of Medicine Herbs” in Situ Gunung Nature Reserve, Sukabumi (2008), small
research regarding Ambient Air Pollution resulting from Tire Manufacture at PT. Goodyear Tbk.,
Bogor (2009), and small research assistant for the Termites Diversity in Pangandaran Nature
Reserve, West Java (2010).
CONTENTS
Page
LIST OF TABLE ...........................................................................................................................viii
LIST OF FIGURE ..........................................................................................................................viii
LIST OF APPENDIX ....................................................................................................................viii
INTRODUCTION............................................................................................................................. 1
Background ............................................................................................................................ 1
Research Objective ................................................................................................................. 1
MATERIALS AND METHODS ...................................................................................................... 1
Time and Place ....................................................................................................................... 1
Honey bee Collection and DNA Extraction ........................................................................... 1
COI Gene Amplification ........................................................................................................ 1
Electrophoresis and DNA Visualization ................................................................................ 2
Sequencing and DNA Analysis ........................................................................................................ 2
RESULT............................................................................................................................................ 3
DNA Amplification ................................................................................................................ 3
Sequencing and DNA Analysis ........................................................................................................ 3
DISCUSSION ................................................................................................................................... 9
CONCLUSION ............................................................................................................................... 10
REFERENCES................................................................................................................................ 10
APPENDIX ..................................................................................................................................... 12
LIST OF TABLE
Page
1 Nucleotide primers for amplifying COI gene of A. koschevnikovi and primer positions based on
mitochondrial DNA of Apis mellifera (Crozier & Crozier 1993) .................................................. 2
2 A. koschevnikovi COI gene in published Genebank database (www.ncbi.nlm.nih.gov) ................ 3
3 Sample code of A. koschevnikovi used in amplification and sequencing in current study ............ 4
4 GC and AT content of A. koschevnikovi COI gene from current studies ....................................... 4
5 Haplotypes variations of A. koschevnikovi from current studies based on COI gene.................... 4
6 Position of nucleotide variations of A. koschevnikovi samples in COI gene. Common hap= Ak1
HST and Ak1 TB. Source of hap1-hap15 refer to Table 2 ............................................................ 7
7 Genetic distances of A. koschevnikovi COI gene (current study, refer to Table 3) and 15
haplotypes of COI gene from Genebank (refer to Table 2) based on Kimura-2-parameter
substitution method with 1000x bootstrap replication ................................................................... 8
LIST OF FIGURE
Page
1 Sampling location for A. koschevnikovi COI gene in South Kalimantan as shown by black square
(Raffiudin R 24 Januari 2011, personal communication). Number = colonies number (refer to
Appendix 1).. ................................................................................................................................. 2
2 DNA fragments of approximately 900 bp COI gene of Apis koschevnikovi. Lane 1-5= Ak 1BL,
Ak 1HST, Ak 8HSS, Ak 1TB, Ak 2KB. M = 100 bp DNA marker (Promega) ........................... 4
3 Nucleotides sequence of A. koschevnikovi COI region from Ak8 HSS sample and COI putative
amino acids. Number = nucleotide position. Underlined nucleotides = forward and reverse
primer. ........................................................................................................................................... 5
4 Nucleotides alignment of A. koschevnikovi COI gene resulted from this study ............................. 5
5 Putative amino acid sequences of A. koschevnikovi COI gene in this study. Number = amino
acid position,
= variation of amino acid based on six samples in this study, : = closely related
of amino acid ................................................................................................................................. 6
6 Phylogenetic tree of A. koschevnikovi from current studies and 15 haplotypes in Genebank
databases based on COI region using NJ construction with 1000x bootstrap replication.............. 9
LIST OF APPENDIX
Page
1 A. koschevnikovi honey bee collection from South Kalimantan. * = samples used for sequencing
and analysing in this study (Raffiudin R 24 Januari 2011, personal communication) ................ 13
2 Chromatogram of A. koschevnikovi cytochrome oxidase subunit 1 (COI) gene from Ak8 HSS
sample using AmF_cox1 forward primer .................................................................................... 14
3 Chromatogram of A. koschevnikovi cytochrome oxidase subunit 1 (COI) gene from Ak 8HSS
sample using AmR_cox1 reverse primer. .................................................................................... 15
INTRODUCTION
Background
Deforestation in Kalimantan due to
logging and forest conversion to palm oil
plantation destroyed many living organism
and carbon-rich ecosystems (Curran et al.
2004). This condition was a serious threat for
the species that is native in Kalimantan such
as Apis koschevnikovi, a cavity nesting honey
bee. It is classified in the order of
Hymenoptera,
suborder
of
Apocrita,
superfamily of Apoidea, and family of Apidae
(Michener 2000). The distribution of A.
koschevnikovi has establishes many new site
localities but all within the previously
confirmed limits of the tropical evergreen
forest of Sundaland (Hadisoesilo et al. 2008).
Nowadays, recent observations shows that the
population of A. koschevnikovi has decreased
persistently due to forest loss and
establishment of some clear land to make oil
palm, rubber, and coconut plantations (Otis
1991).
Preserving the ecological integrity of A.
koschevnikovi database is necessary, including
molecular database. Cytochrome oxidase
subunit I (COI) in mitochondrial DNA
(mtDNA) provide suitable molecular marker
with DNA barcode-based technology due to
their characteristic i.e no deletion and
insertion in its sequence, and slightly
variances in their sequences (Hebert et al.
2003). In animals mtDNA, COI usually
composed by over 600 bp of long, and at least
648 bp of long is required to be submitted into
Genebank database (www.ncbi.nlm.nih.gov).
Currently, there are 15 haplotypes of COI
gene of A. koschevnikovi has published in
Genebank under Accesion number AF153110
- AF153111, AY012723, AY754729 AY754732, and DQ016097 - DQ016104
(www.ncbi.nlm.nih.gov). Most of the
haplotypes occured from Sabah (haplotype 37, hap 11-15), the others are from Sarawak
(Malaysia), and West Kalimantan. However,
out of 15 haplotypes, three found in
Kalimantan. This study was aimed to explore
further to find another variations of COI
haploytpes for building the database
especially in Kalimantan forest.
Research Objective
The objective of this research was to
explore the variations of COI gene in
mitochondrial DNA of A. koschevnikovi in
South Kalimantan.
MATERIALS AND METHODS
Time and Place
The research was conducted on January
2010 up to February 2011, and it was taken
place in Division of Animal Function and
Behaviour, Department of Biology, Bogor
Agricultural University (IPB).
Honey Bee Collection and DNA Extraction
The objects of my research were DNA
from several A. koschevnikovi honey bees.
Honey bees used in this study were collection
of Dr. Rika Raffiudin (Dept. Biology IPB)
that were collected from Kabupaten Hulu
Sungai Selatan (Ak HSS), Hulu Sungai
Tengah (Ak HST), Balangan (Ak BL), Tanah
Bumbu (Ak TB), and Kotabaru (Ak KB) in
South Kalimantan (Figure 1). Several worker
honey bee from several colonies were
collected from each regencies and were
preserved in absolute ethanol.
DNA extraction has carried out by using
0.2% Cetyl Trimethyl Ammonium Bromide
(CTAB) and ethanol precipitation (Sambrook
et al. 1989).
COI Gene Amplification
TaKaRa Thermal Cycler machine was
used to amplify A. koschevnikovi DNA. COI
gene of A. koschevnikovi fragment
amplification
used AmF_cox1 forward
primer and AmR_cox1 reverse primer (Table
1) based on Apis mellifera complete sequence
(www.ncbi.nlm.nih.gov). The nucleotide
primers position were at 1915-1934 (forward)
and 2719-2739 (reverse) based Genbank
Accesion number L06178 complete A.
mellifera mitochondrial genome. COI gene
locate at nucleotide number 1794-3359 of
mitochondrial region of A. mellifera (Crozier
& Crozier 1993). The PCRs reagent were 20
µl. The PCRs reagent consists of 7.4 µl of
destilation water, 0.8 µl of 10 µM forward
primer, 0.8 µl of 10 µM reverse primer, 1 µl
DNA template collected from the extraction
(41.600 ng/µl), and 10 µl of KAPA Taq 2X
ReadyMix DNA Polymerase (containing 0.05
U/µl of KapaTaq DNA Polymerase, 25 mM of
reaction Buffer with Mg2+, 0.4 mM each
dNTP). DNA amplification in PCRs machine
occurred for 30 cycles in 5 stages i.e. 3
2
minutes (mins) at 940 C for initial denaturing,
30
P. Sebuku
P. Laut
L
t
24
Figure 1 Sampling location for A. koschevnikovi COI gene in South Kalimantan as shown
by black square (Raffiudin R 24 Januari 2011, personal communication). Number
= colonies number (refer to Appendix 1).
Table 1 Nucleotide primers for amplifying COI gene of A. koschevnikovi and primer positions
based on mitochondrial DNA of Apis mellifera (Crozier & Crozier 1993)
Primer
Forward amF_cox1
Reverse amR_cox1
Nucleotide sequence of primer
(5’-3’) (Crozier & Crozier 1993)
CCCCAGGATCATGAATTAGC
TCATTGCTGTACCAACAGGAA
minutes (mins) at 940 C for initial denaturing,
30 cycles of 1 min at 940 C (denaturing), 1
min and 30 seconds at 550 C (annealing), 2
mins at 720 C (DNA elongation) and followed
by 5 mins for final extension.
Electrophoresis and DNA Visualization
Amplified DNA was migrated using 6%
Polyacrilamid Gel Electrophoresis (PAGE)
which was submerged in 1x TBE buffer (10
mM Tris-HCl, 1 M Borat acid, and 0.1 mM
EDTA), operated for 50 minutes at 200 V.
The compositions of 6% gel acrilamide were
12 ml of aquades, 4 ml of 5xTBE, 4 ml of
acrilamide, 15 µl of TEMED, and 160 µl of
10% APS.
DNA visualization used sensitive Silver
Nitrate staining (Tegelstrom 1986). Gel
resulting from electrophoresis was washed
using CTAB solution (0.2 g/200 ml of
aquades) for 8 mins, and was flushed using
200 ml of aquades for 2 mins (2 times).
Subsequently, its gel was submerged in
NH4OH solution (2.4 ml/200 ml of aquades)
for 8 mins, and solution of mixture contained
0.32 g of AgNO3, 0.8 ml of NH4OH, 80 µl
Positions primer in mitochondrial
DNA of Apis mellifera
(Crozier & Crozier 1993)
1915-1934
2719-2739
NaOH, and 200 ml of aquades for 10 mins.
Then, the gel was flushed again using the
same condition as above, and was submerged
again in the solution of mixture contained 4 g
of NA2CO3, 100 µl of formaldehida, and 200
ml of aquades until the bands of DNA seen.
Staining process was stopped using 200 µl of
1% acetat acid in 200 ml of aquades.
Sequencing and DNA Analysis
Genetic variations in COI mitochondrial
region was analysed based on DNA
sequencing method. DNA was sequenced by
using ABI 3100 and 3730XL sequencer
machine at sequencing company. Nucleotide
sequence were edited and analysed using
Genetyx Win Version 4.0 software. Forward
and reverse sequence were aligned to obtain
contiguous sequence by using Clustal X
software (Thompson et al. 1997). All DNA
contiguous sequences was aligned together
with haplotypes databases of COI gene of A.
koschevnikovi in Genebank NCBI (National
Center for Biotechnology Information) under
Accesion number AF153110-AF153111,
3
AY012723, AY754729- AY754732, and
DQ016097-DQ016104
(www.ncbi.nlm.nih.gov) (Table 2).
Edited DNA sequences were translated
into putative amino acid using Genetyx and
were aligned by using Clustal X software.
DNA genetic distances and phylogenetic
analysis were constructed between current
samples and 15 haplotypes in Genebank as
ingroup while A. cerana with accesion
number GQ162109, and A. mellifera with
accesion number L06178 as outgroups. These
analysis was carried out by using MEGA4
software based on Kimura-2-parameter
substitution method and Neighbour Joining
(NJ) construction with 1000x bootstrap
(Tamura et al. 2007).
RESULT
DNA Amplification
There were 29 samples of A. koschevnikovi
COI gene from five regencies in South
Kalimantan were amplified using AmF_cox1
and AmR_cox1 primer (Table 3). All samples
showed the same results i.e single band with
approximately 900 bp of nucleotide (Figure
2).
Table 2 A. koschevnikovi COI gene in published Genebank database (www.ncbi.nlm.nih.gov)
No
Accesion
number
1
AF153110
2
AF153111
3
AY012723
4
AY754729
Suka, T., and Tanaka, H.
haplotipe 4
5
AY754730
Suka, T., and Tanaka, H.
haplotipe 5
6
AY754731
Suka, T., and Tanaka, H.
haplotipe 6
7
AY754732
Suka, T., and Tanaka, H.
haplotipe 7
8
DQ016097
9
DQ016098
10
DQ016099
11
DQ016100
12
DQ016101
13
DQ016102
14
DQ016103
15
DQ016104
Author
Tanaka, H. Roubik, Kato,
M., Liew, F., and Gunsalam,
Tanaka, H. Roubik, Kato,
M., Liew, F., and Gunsalam,
G.
Tanaka, H. , Suka T.,
Roubik, and Mohamed M.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Tanaka, H. , Suka, T.,
Kahono, S., Samejima, H. ,
Maryati, M., and Roubik.
Haplotypes
haplotype 1
haplotype 2
haplotype 3
haplotipe 8
Source
Brunei
Malaysia: Lamibir,
Sarawak
Malaysia: Crocker
Range Park, Sabah
Malaysia: Crocker
Range Park, Sabah
Malaysia: Crocker
Range Park, Sabah
Malaysia: Crocker
Range Park, Sabah
Malaysia: Crocker
Range Park, Sabah
Indonesia: kutai
national park, east
Kalimantan
Nucleotides
long (bp)
1041
1041
1041
1041
1041
1041
1041
1041
haplotipe 9
Indonesia: Loksad,
south Kalimantan
1041
haplotipe 10
Indonesia: field
site near from
sadap, west
Kalimantan
1041
haplotipe 11
Malaysia: Tawau,
Sabah
1041
haplotipe 12
Malaysia: Tawau,
Sabah
1041
haplotipe 13
Malaysia: Tawau,
Sabah
1041
haplotype 14
Malaysia: Tawau,
Sabah
1041
haplotype 15
Malaysia: Tawau,
Sabah
1041
4
1
2
3
4
5
M
2), while nucleotide sequence of AmF_cox1
primer found in AmR_cox1 sequence
(Appendix 3). Complete contiguous sequence
was found in Ak HSS sample colony number
8 (Ak8 HSS) i.e 826 bp (Figure 3).
Based on six contiguous of A.
koschevnikovi COI gene sequences alignment,
738 bp of nucleotide was observed. All
contiguous DNA showed rich of A-T
sequences, i.e. 75.5 % while G-C was 24.5 %
(Table 4).
There were five variations of DNA
sequences occurred from current studies
alignment. The variations respectively
occurred in nucleotide number 119, 125, 219,
254 and 272 (Figure 4). Hence, five
haplotypes were observed (Table 5). The first
haplotype was a common haplotypes was
found in Ak1 TB and Ak1 HST samples. The
second to fifth haplotypes respectively were
found in Ak2 KB, Ak8 HSS, Ak1 BL, and
Ak5
BL
sample
(Table
5).
900 bp
400 bp
Figure 2 DNA fragments of approximately
900 bp COI gene of Apis
koschevnikovi. Lane 1-5= Ak
1BL, Ak 1HST, Ak 8HSS, Ak
1TB, Ak 2KB. M = 100 bp DNA
marker (Promega).
Sequencing and DNA Analysis
Out of 29 amplified samples of A.
koschevnikovi COI gene, six samples were
sequenced (see Table 3 and Appendix 1).
Nucleotide sequence of AmR_cox1 primer
was found in AmF_cox1 sequence (Appendix
Table 3 Sample code of A. koschevnikovi used in amplification and sequencing in current study
No
1
2
3
Sample
code
Ak HSS
Ak HST
Ak BL
Regency Origin
Hulu Sungai Selatan
Hulu Sungai Tengah
Balangan
4
5
Ak KB
Ak TB
Kotabaru
Tanah Bumbu
Number of
amplified colonies
11
4
5
7
2
Samples for sequenced and
alignment analysis
Colony no.8 (Ak8 HSS)
Colony no.1 (Ak1 HST)
Colony no.1 & 5 (Ak1 BL &
Ak5 BL)
Colony no. 2 (Ak2 KB)
Colony no. 1 (Ak1 TB)
Table 4 GC and AT content of A. koschevnikovi COI gene from current studies
No
1
2
3
4
5
6
Sample Code
Ak1 HST
Ak1 TB
Ak8 HSS
Ak5 BL
Ak1 BL
Ak2 KB
A-T nucleotides (%)
75.53
75.53
75.67
75.67
75.53
75.39
G-C nucleotides (%)
24.47
24.47
24.33
24.33
24.47
24.61
Table 5 Haplotypes variations of A. koschevnikovi from current studies based on COI gene
No
Haplotype
Sample
1.
Haplotype 1
2.
Haplotype 2
Ak1 HST
Ak1 TB
Ak2 KB
3.
4.
5.
Haplotype 3
Haplotype 4
Haplotype 5
Ak8 HSS
Ak1 BL
Ak5 BL
Position of
nucleotide
alteration
Common haplotype
Nucleotide alteration
(compared with
common haplotype)
-
Substitution
no. 119 and 125
T ÆC and T ÆA
no. 219
no. 254
no. 272
G ÆA
T ÆA
C ÆT
Transition and
transversion
Transition
Transversion
Transition
-
5
CCCCAGGATCATGAATTAGCAATGATCAAATCTATAATACTATTGTCACAAGACATGCAT
P G S W I S N D Q I Y N T I V T S H A F
60
TTTTAATAATTTTTTTTATAGTTATACCATTTTTAATTGGAGGATTTGGTAATTGATTAA
L M I F F M V M P F L I G G F G N W L I
120
TTCCATTAATACTTGGATCTCCAGATATAGCTTTTCCTCGTATAAATAACATTAGATTTT
P L M L G S P D M A F P R M N N I S F W
180
GATTATTACCACCTTCATTATTAATATTATTATTAAGAAATTTATTTTACCCAAGACCAG
L L P P S L L M L L L S N L F Y P S P G
240
GAACAGGATGAACTATTTATCCTCCACTATCAGCTTATTTATATCATTCTTCACCATCAG
T G W T I Y P P L S A Y L Y H S S P S V
300
TAGATTTCGCAATTTTTTCTTTACATATATCTGGAATTTCATCAATTATAGGATCATTAA
D F A I F S L H M S G I S S I M G S L N
360
ATTTAATAGTAACAATTATAATAATAAAAAATTTTTCTTTAAATTATGATCAAATTTCAT
L M V T I M M M K N F S L N Y D Q I S L
420
TATTTCCTTGATCAGTATTTATTACAGCTATTTTATTAATTATATCATTACCTGTTTTAG
F P W S V F I T A I L L I M S L P V L A
480
CTGGAGCAATTACAATACTATTATTTGATCGAAATTTTAATACATCATTCTTTGATCCAA
G A I T M L L F D R N F N T S F F D P M
540
TAGGAGGAGGAGATCCAATTTTATATCAACATTTATTTTGATTTTTTGGTCATCCAGAAG
G G G D P I L Y Q H L F W F F G H P E V
600
TTTATATTTTAATTTTACCCGGATTTGGATTAATTTCTCATATTGTAATAAATGAAAGAG
Y I L I L P G F G L I S H I V M N E S G
660
GAAAAAAAGAAATTTTTGGAAATTTAGGAATAATTTATGCAATATTAGGAATTGGATTTC
K K E I F G N L G M I Y A M L G I G F L
720
TAGGATTTATTGTTTGAGCTCATCATATATTTACTGTTGGATTAGATGTTGATACACGAG
G F I V W A H H M F T V G L D V D T R A
780
CTTATTTTACTTCTGCAACTATAATCATTGCTGTACCAACAGGAAA
Y F T S A T M I I A V P T G *
826
Figure 3 Nucleotides sequence of A. koschevnikovi COI region from Ak8 HSS sample
and COI putative amino acid. Number = nucleotide position. Underlined
nucleotides = forward and reverse primer.
Ak1HST
Ak1TB
Ak8HSS
Ak5BL
Ak1BL
Ak2KB
TTGGTAATTGATTAATTCCATTAATACTTGGATCTCCAGATATAGCTTTTCCTCGTATAAATAACATTA
.....................................................................
.....................................................................
.....................................................................
.....................................................................
.................................................C.....A.............
[138]
[138]
[138]
[138]
[138]
[138]
Ak1HST
Ak1TB
Ak8HSS
Ak5BL
Ak1BL
Ak2KB
CAGGATGAACTGTTTATCCTCCACTATCAGCTTATTTATATCATTCTTCACCATCAGTAGATTTCGCAA
.....................................................................
...........A.........................................................
................................................................T....
..............................................A......................
.....................................................................
[276]
[276]
[276]
[276]
[276]
[276]
Figure 4 Nucleotides alignment of A. koschevnikovi COI gene in this study. Number in bracket =
nucleotide position, . = homolog nucleotide, = variations of nucleotide. Samples code
refer to Table 3.
6
Nucleotide sequence of Ak8 HSS were
translated into putative amino acid and
showed no stop codon (Figure 3). Putative
amino acid of COI gene from current studies
were aligned as well. The result showed that
there was one amino acid variation sequence
(Figure 4) i.e at nucleotide no. 219-221, codon
ATT was translated into Isoleusin (I) in Ak8
HSS sample (haplotype 3), while Valin (V)
was resulted from codon GTT in Ak1 HST
and Ak1 TB sequences (common haplotype).
Meanwhile, the other variations from
current studies alignment did not show
variations for its putative amino acid. For
examples, In Ak2 KB sample, codon TTC at
nucleotides no. 117-119 (haplotype 2) was
translated into Phenilalanin (F) and codon
CGA at nucleotides no. 123-125 was translated
into Arginin (R). At the same position in
common haplotype, codon TTT and CGT were
translated become same amino acid,
Phenilalanin and Arginin (Figure 5). In Ak1
BL sample, codon TCA at nucleotides no.
252-254 (haplotype 4) was translated become
Serin (S). At the same position in common
haplotypes, codon TCT was translated become
Serin amino acid as well. The other example
came from Ak5 BL sample, codon TTT at
nucleotides no. 270-272 (haplotype 5) was
translated into Phenilalanin (F). At the same
position in common haplotypes, codon TTC
was translated become Phenilalanin amino
acid as well.
This study performed alignment between
current sequenced samples (refer to Table 3)
and published A. koscehvnikovi COI databases
in Genebank as well. Total of 685 bp of
nucleotides was aligned. None of the current
study COI A. koschevnikovi study samples
showed the same sequence with the published
nucleotide sequences from Genebank
databases (Table 6).
Genetic distances analysis showed the
relatedness of genetic relationship between all
samples in this study, with the highest genetic
distance in Ak2 KB sample i.e 0.003-0.004
(Table 7). Phylogenetic analysis clustered all
samples in this study and published A.
koschevnikovi COI database in Genebank in
three clusters i.e cluster A, B, and C (Figure
6). All samples in this study and several
haplotypes from Sabah (Malaysia) and Brunei
were clustered in cluster A with 88 of
bootstrap value. Cluster B consisted of
haplotype 2 from Sarawak (Malaysia),
haplotype 10 from West Kalimantan, and
haplotype 11 from Sabah with 98 of bootstrap
value. However, haplotypes 3 and 4 from
Sabah was separated in cluster C having 100
of bootstrap value (Figure 6), both performed
as the basal taxon, close to A. cerana as the
outgroups.
Ak2KB
Ak8HSS
Ak1BL
Ak1HST
Ak1TB
Ak5BL
AmCrozier
AkHap13
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLFMLLLSNLFYPSPGTG
FLIGGFGNWLIPLMLGSPDMAFPRMNNISFWLLPPSLLMLLLSNLFYPSPGTG
*************************************:***************
60
60
60
60
60
60
60
60
Ak2KB
Ak8HSS
Ak1BL
Ak1HST
Ak1TB
Ak5BL
AmCrozier
AkHap13
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTIYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
GTGWTVYPPLSAYLYHSSPSVDFAIFSLHMSGISSIMGSLNLMVTIMMMKNFS
*****:***********************************************
120
120
120
120
120
120
120
120
Figure 5
Putative amino acid sequence of A. koschevnikovi COI gene from this study. Number
= amino acid position, = variation of amino acid based on six samples in this study, :
= closely related of amino acid.
Table 6 Position of nucleotide variations of A, koschevnikovi samples in COI gene. Common hap= Ak1 HST and Ak1 TB. Source of hap1-hap15 refer to Table 2
1
2
1
5
2
1
3
3
4
3
4
5
5
1
5
4
5
7
6
3
6
6
7
2
8
1
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
2
4
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
1
2
6
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
3
8
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
1
4
1
C
.
.
.
.
T
T
T
.
.
.
.
.
.
.
.
.
.
T
T
Hap
Common hap
Ak2 KB
Ak8 HSS
Ak1 BL
Ak5 BL
Hap10_Kalbar
Hap11_Sabah
Hap2_Sarawak
Hap14_Sabah
Hap15_Sabah
Hap9_Kalsel
Hap12_Sabah
Hap1_Brunei
Hap6_Sabah
Hap7_Sabah
Hap13_Sabah
Hap8_Kaltim
Hap5_Sabah
Hap3_Sabah
Hap4_Sabah
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
T
.
.
.
.
C
C
C
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
A
.
.
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
T
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
T
.
.
.
.
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
T
C
.
.
.
C
C
C
.
.
.
.
.
.
.
.
.
.
.
.
T
A
.
.
.
A
A
A
.
.
.
.
.
.
.
.
.
.
.
.
Hap
Common hap
Ak2 KB
Ak8 HSS
Ak1 BL
Ak5 BL
Hap10_Kalbar
Hap11_Sabah
Hap2_Sarawak
Hap14_Sabah
Hap15_Sabah
Hap9_Kalsel
Hap12_Sabah
Hap1_Brunei
Hap6_Sabah
Hap7_Sabah
Hap13_Sabah
Hap8_Kaltim
Hap5_Sabah
Hap3_Sabah
Hap4_Sabah
4
6
5
T
.
.
.
.
C
C
.
.
.
.
.
.
.
.
.
.
.
C
C
4
6
8
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
4
7
7
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
C
5
3
1
T
.
.
.
.
.
.
.
.
.
.
.
.
T
A
.
.
.
T
T
5
3
4
A
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.
.
5
4
0
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
5
7
3
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
5
9
1
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
6
0
0
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
6
2
7
A
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.
6
3
1
C
.
.
.
.
T
T
T
.
.
.
.
.
.
.
.
T
.
T
T
6
3
9
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
6
4
5
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
6
5
1
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
6
6
9
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
6
7
8
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
6
8
5
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
4
7
A
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.
.
1
5
0
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
5
3
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
5
6
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
5
9
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
1
6
5
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
1
6
6
G
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
7
7
A
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
1
7
8
C
.
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
2
0
1
T
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
2
0
7
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
2
1
9
C
.
.
.
T
.
.
.
.
.
.
.
.
.
.
T
.
.
T
T
2
3
1
T
.
.
.
.
C
C
C
.
.
.
.
.
.
.
.
.
.
A
A
2
8
2
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
3
0
9
T
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
3
2
1
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
3
4
5
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
3
7
2
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
3
8
7
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
3
9
3
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
A
4
0
5
A
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
T
T
4
0
9
C
.
.
.
.
T
T
T
T
T
T
T
T
T
T
T
T
T
.
.
4
1
2
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
4
3
5
A
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
4
4
1
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
4
4
7
T
.
.
.
.
C
C
C
.
.
.
.
.
.
.
.
.
C
C
C
4
5
0
A
.
.
.
.
T
T
T
.
.
.
.
.
.
.
.
.
.
.
.
4
5
9
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
4
6
2
A
.
.
.
.
T
T
T
.
.
.
.
.
.
.
.
.
.
.
.
Table 7 Genetic distances of A. koschevnikovi COI gene (current study, refer to Table 3) and 15 haplotypes of COI gene from Genebank (refer to Table 2) based on
Kimura-2-parameter substitution method with 1000x bootstrap
[ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 ]
[ 1]
[ 2] 0.001
[ 3] 0.070 0.072
[ 4] 0.070 0.072 0.000
[ 5] 0.073 0.075 0.006 0.006
[ 6] 0.071 0.073 0.015 0.015 0.018
[ 7] 0.075 0.077 0.018 0.018 0.021 0.003
[ 8] 0.071 0.073 0.015 0.015 0.018 0.000 0.003
[ 9] 0.075 0.077 0.018 0.018 0.021 0.003 0.006 0.003
[10] 0.073 0.075 0.016 0.016 0.019 0.001 0.004 0.001 0.004
[11] 0.072 0.073 0.018 0.018 0.021 0.003 0.006 0.003 0.006 0.004
[12] 0.073 0.075 0.018 0.018 0.021 0.003 0.006 0.003 0.006 0.004 0.004
[13] 0.071 0.073 0.015 0.015 0.018 0.003 0.006 0.003 0.006 0.004 0.006 0.006
[14] 0.068 0.070 0.015 0.015 0.018 0.003 0.006 0.003 0.006 0.004 0.006 0.006 0.006
[15] 0.070 0.072 0.016 0.016 0.019 0.001 0.004 0.001 0.004 0.003 0.004 0.004 0.004 0.004
[16] 0.072 0.073 0.018 0.018 0.021 0.003 0.006 0.003 0.006 0.004 0.006 0.006 0.006 0.006 0.004
[17] 0.072 0.073 0.018 0.018 0.021 0.003 0.006 0.003 0.006 0.004 0.006 0.006 0.006 0.006 0.004 0.000
[18] 0.073 0.075 0.019 0.019 0.022 0.004 0.007 0.004 0.007 0.006 0.007 0.007 0.007 0.007 0.006 0.001 0.001
[19] 0.070 0.072 0.019 0.019 0.022 0.004 0.007 0.004 0.007 0.006 0.007 0.007 0.007 0.007 0.006 0.001 0.001 0.003
[20] 0.070 0.072 0.019 0.019 0.022 0.004 0.007 0.004 0.007 0.006 0.007 0.007 0.007 0.007 0.003 0.001 0.001 0.003 0.003
[21] 0.071 0.073 0.015 0.015 0.018 0.006 0.009 0.006 0.009 0.007 0.009 0.009 0.009 0.009 0.007 0.003 0.003 0.004 0.004 0.004
[22] 0.109 0.107 0.092 0.092 0.092 0.088 0.092 0.088 0.090 0.088 0.090 0.088 0.087 0.092 0.087 0.092 0.092 0.094 0.090 0.090 0.092
[23] 0.102 0.102 0.096 0.096 0.092 0.093 0.096 0.093 0.096 0.094 0.093 0.093 0.096 0.096 0.091 0.093 0.093 0.095 0.095 0.091 0.089 0.112
[ 1] #Ak_T_hap3_Sabah
[ 2] #Ak_T_hap4_Sabah
[ 3] #Ak_T_hap10_Kalbar
[ 4] #Ak_T_hap11_Sabah
[ 5] #Ak_T_hap2_Sarawak
[ 6] #Ak_T_hap9_Kalsel
[ 7] #Ak_T_hap15_Sabah
[ 8] #Ak_T_hap12_Sabah
[ 9] #Ak_T_hap14_Sabah
[10] #Ak_T_hap1_Brunei
[11] #Ak_T_hap6_Sabah
[12] #Ak_T_hap7_Sabah
[ 13] #Ak_T_hap8_Kaltim
[ 14] #Ak_T_hap5_Sabah
[ 15] #Ak_T_hap13_Sabah
[ 16] #Ak1HST_R_Kalsel
[ 17] #Ak1TB_R_Kalsel
[ 18] #Ak8HSS_R_Kalsel
[ 19] #Ak1BL_R_Kalsel
[ 20] #Ak5BL_R_Kalsel
[ 21] #Ak2KB_R_KAlsel
[ 22] #Ac_china
[ 23] #Am_Crozier
9
Ak T hap15 Sab ah
Ak T hap12 Sab ah
Ak T hap14 Sab ah
26
Ak T hap1 Brunei
Ak T hap9 Kalsel
19
Ak T hap6 Sab ah
65
23
Ak T hap7 Sab ah
Ak T hap8 Kaltim
28
Ak T hap5 Sab ah
Cluster A
Ak T hap13 Sab ah
34
Ak 5BL R Kalsel
Ak 1BL R Kalsel
88
37
Ak 1HST R Kalsel
49
Ak 1TB R Kalsel
46
Ak 8HSS R Kalsel
100
Ak 2KB R KAlsel
Ak T hap2 Sarawak
66
98
92
Ak T hap10 Kalb ar
Cluster B
Ak T hap11 Sab ah
Ak T hap3 Sab ah
100
Ak T hap4 Sab ah
Cluster C
Apis cerana
Apis m ellifera
0.01
Figure 6
Phylogenetic tree of A. koschevnikovi from current studies and 15 haplotypes in
Genebank databases based on COI region using NJ method with 1000x bootstrap.
Samples code refer to Table 2 and 3.
DISCUSSION
The result of A. koschevnikovi COI gene
amplification in PAGE 6% showed
approximately about 900 bp of nucleotides
long, while 801 bp of average nucleotides
long resulted from DNA sequencing toward
current samples of A. koschevnikovi. This
difference might be occur due to gel shifting
phenomenon i.e. the alteration of DNA band
movement in polyacrilamide gel.
DNA sequencing result showed different
size of nucleotides for each sample. This
condition was due to the unclear graph in
beginning of chromatogram. For alignment
purpose, all contiguous sequences must
commence and end at the same nucleotide
position.
The high number of AT nucleotides i.e
75.5% from each samples agree with insect
DNAmt that contained rich of A+T base
(Crozier et al. 1989). Longer time was needed
to amplify DNA which has a very high G-C
content (Griffiths et al. 1999).
There were two kind of nucleotide
substitution occurred from current studies
alignment i.e transition and transversion
(Table 5). Transition occurred when
nucleotide substitution did not change the
structure of nitrogene base i.e purin (A and G)
to be purin; or pirimidin (C and T) to be
pirimidin, while transversion occured when
nucleotide substitution change the structure of
nitrogene base (purin to be pirimidin; or
pirimidin to be purin) (Griffiths et al. 1999).
The nucleotide substitution does not
always resulted amino acid from transcriptiontranslation process. The alteration will effect
translated amino acid when the substitution
occurred in first or second nucleotide in codon
formation (Klug et al. 2006). For the example
in Ak8 HSS sample at nucleotide no. 219-221,
codon ATT was translated into Isoleusin (I) in
Ak8 HSS (haplotype 3), while Valin (V) was
resulted from codon GTT from Ak1 HST and
Ak1 TB sequence (common haplotype).
However, Valin and Isoleusin is still closely
related in amino acid as showed with double
dot (:) (see Figure 5). Both of them were
clustered in hydrophobic aliphatic amino acid
(Takei et al. 2006).
10
In addition, DNA variation especially third
nucleotide substitution in codon formation
does not always produce different putative
protein (Avise 1994). The examples based on
Ak2 KB sample (haplotype 2) at nucleotides
no. 117-119 and no. 123-125, Ak1 BL sample
at nucleotides no. 252-254 (haplotype 4), and
Ak5 BL sample at nucleotides no. 270-272
(haplotype 5).
The alignment between current samples
(refer to Table 3) and published A.
koschevnikovi COI databases in Genebank has
resulting 685 bp of nucleotides long (Table 5),
which fulfilled 648 nucleotides as a minimum
number of nucleotide requirement to be
submitted
to
DNA
Barcode
(www.ncbi.nlm.nih.gov). None of the current
COI A. koschevnikovi study samples showed
the same sequence with the published
nucleotide sequence from Genebank databases
(Table 5). Hence, these five haplotypes from
this research were the new haplotypes for A.
koschevnikovi COI database.
The results of this research can be used to
accomplish information about molecular
ecology studies of A. koschevnikovi as well.
Based on current studies alignment, Ak1 HST
and Ak1 TB has a same sequence, although
the distance of HST and TB regencies
approximately 150 km (Figure 1). However,
sequences variations were found in two
samples both from Balangan Regency (Ak1
BL and Ak5 BL). These results indicate that
mixed
population
occurred
in
A.
koschevnikovi distribution.
Genetic distances and phylogenetic
analysis were performed to observe genetic
relationship between current studies and
published A. koschevnikovi COI databases in
Genebank as well. Genetic distance analysis
grouped all samples in this study in one
cluster, with the highest genetic distance in
Ak2 KB sample i.e 0.003-0.004 (Table 7).
Ak2 KB sample was collected not from
Kalimantan mainland but from Sebuku island
(see sampling location number 24 in Figure
1). Hence, these highest number might be due
to geographical barrier which separate by Laut
strait and Laut island (see Figure 1).
Phylogenetic analysis grouped Ak
samples from this previous study in three
clusters (Figure 6). All samples in this study
and several haplotypes from Sabah and Brunei
were clustered in cluster A (0.000-0.009 value
of genetic distances), while haplotype 2 from
Sarawak,
haplotype
10
from
West
Kalimantan, and haplotype 11 from Sabah
were clustered in cluster B (0.000-0.022 value
of genetic distances) (Table 7). However, both
were classified in one group due to their
bootstrap value which is 100 (Figure 6).
However, Ak haplotypes 3 and 4 from
Sabah were grouped in different cluster with
100 of bootstrap value, while these samples
were collected from same region with several
haplotypes in cluster A and B. Genetic
distances of haplotypes 3 and 4 compared
with the other haplotypes showed the highest
value (0.001-0.077) (Table 7). Therefore,
these 2 haplotypes need to be reconfirmed for
the species validity.
In the case of these two anomalous
haplotypes from Genebank, therefore, one
need to be aware with the published DNA
database. In addition, further studies from the
other areas in Kalimantan is necessary to have
a complete picture of A. koschevnikovi COI
gene database.
CONCLUSION
The exploration of COI gene of A.
koschevnikovi from six samples taken from
five regencies in South Kalimantan showed
the genetic differentation. There were five
haplotypes of A. koschevnikovi COI gene were
found in this study i.e. haplotype 1, 2, 3, 4,
and 5. Haplotype 1 was a common haplotype
was found in sample Ak1 HST and Ak1 TB,
while others specific in other places. None of
the current samples showed the same
sequence with the nucleotide sequences of
Genebank database, hence these five
haplotypes were the new haplotypes of A.
koschevnikovi COI gene.
REFERENCES
Avise JC. 1994. Molecular Markers, Natural
History and Evolution. New York:
Chapman & Hall.
Crozier RH, Crozier YC, Mackinlay AG.
1989. The CO-I and CO-II region of
honeybee
mitochondrial
DNA:
evidence for variation in insect
mitochondrial evolutionary rates.
Mol Biol Evol 6: 399-411
Crozier RH, Crozier YC. 1993. The
mitochondrial genome of the
honeybee Apis mellifera: complete
sequence and genome organization,
Genetics 133: 97-117.
Curran LM et al.2004. Lowland forest loss in
protected areas of Indonesian
Borneo, Science 303: 1000-1003.
11
Griffiths A, William MG, Jeffrey HM,
Richard CL. 1999. Modern Genetic
Analysis. New York: W.H. Freeman
and Company.
Hadisoesilo et al. 2008. Morphometric
analysis and biogeography of Apis
koschevnikovi Enderlein (1906),
Apidologie 39: 495-503.
Hebert PD, Ratnasingham S, dewaard JR.
2003. Barcoding animal life:
cytochrome c oxidase subunit 1
divergences among closely related
species, Proc. R. Soc. Lond 270:
S96-S99.
Klug WS, Cummings MR, Spencer CA. 2006.
Concepts of Genetics 8th ed. New
Jersey: Pearson Education, Inc.
Michener CD. 2000. The Bees of the World.
London: The John Hopkins Univ Pr.
Otis GW. 1991. A review of the diversity of
species within Apis. In: Smith DR
(ed) Diversity in The Genus Apis.
Oxford: Westview Press. pp 29-50.
Sambrook J, Fritsch EF, Maniatis T. 1989.
Molecular Cloning: A Laboratorium
Manual Second Edition. New York:
Cold Spring Harbor Laboratory
Press.
Takei T et al. 2006. The effects of the side
chains of hydrophobic aliphatic
amino acid residues in an
amphipathic polypeptide on the
formation of a helix and its
association, J. Biochem 139: 271278.
Tamura K, Dudley J, Nei M, Kumar S. 2007.
MEGA4: molecular evolutionary
genetics analysis (MEGA) software
version 4.0, Mol Biol Evol 24: 15961599.
Tegelstrom H. 1986. Mitochondrial DNA in
natural population: an improved
routine for screening of genetic
variation based on sensitive silver
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Thompson JD et al. 1997. The clustal X
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Acid Res 24: 4876-4882.
.
APPENDIX
13
Appendix 1 A. koschevnikovi honey bee collection from South Kalimantan. * = samples used for
sequencing and analysing in this study (Raffiudin R 24 Januari 2011, personal
communication)
Southern
latitude
Longitude
east
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.49’
115 o.17’
AK 2 HSS
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.49’
115 o.17’
3.
AK 3 HSS
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.49’
115 o.17’
4.
AK 4 HSS
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.49’
115 o.17’
5.
AK 5 HSS
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.50’
115 o.18’
6.
AK 6 HSS
Kec. Losado, Kab. Hulu Sungai Selatan
02o.49’
115 o.24’
7.
AK 7 HSS
Kec. Losado, Kab. Hulu Sungai Selatan
02o.49’
115 o.25’
8.
AK 8 HSS*
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.49’
115 o.17’
9.
AK 1 BL*
Kec. Lanmpihong, Kab. Balangan
02o.20’
115 o.25’
10.
AK 2 BL
Kec. Lanmpihong, Kab. Balangan
02o.20’
115 o.25’
11.
AK 3 BL
Kec. Lanmpihong, Kab. Balangan
02o.20’
115 o.25’
12.
AK 4 BL
Kec. Lanmpihong, Kab. Balangan
02o.20’
115 o.25’
13.
AK 5 BL*
Kec. Paringin, Kab. Balangan
02o.20’
115 o.25’
14.
AK 9 HSS
Kec. Padang Batung, Kab. Hulu Sungai Selatan
02o.47’
115 o.17’
15.
AK 10 HSS
Kec. Losado, Kab. Hulu Sungai Selatan
02o.49’
115 o.25’
16.
AK 11 HSS
Kec. Losado, Kab. Hulu Sungai Selatan
02o.49’
115 o.25’
17.
AK 1 HST*
Kec. Batu Benawa, Kab. Hulu Sungai Tengah
02o.38’
115 o.26’
18.
AK 2 HST
Kec. Batu Benawa, Kab. Hulu Sungai Tengah
02o.38’
115 o.25’
19.
AK 3 HST
Kec. Batu Benawa, Kab. Hulu Sungai Tengah
02o.38’
115 o.25’
20.
AK 4 HST
Kec. Alai Utara, Kab. Hulu Sungai Tengah