2. MOLECULAR DIVERSITY IN HONEY BEES USING SIMPLE
SEQUENCE REPEATS (SSR) , CYTOCHROME OXIDASE–I
(CO-I) AND CYTOCHROME OXIDASE –II (CO-II)
Seminar II (PLPT-695)
By
Khalid Ali Khan Supervisor : Dr. Ahmad bin Abdullah
(Ph.D. Student) 432108568 Al Ghamdi
Department of Plant Protection ,
King Saud University -Riyadh

3. A lot of variations among the organisms is found. The
organisms are classified on the basis of these variations.
There are different parameters/markers to measure the
diversity in the organisms. Some important markers are:
1-Morphological Markers
Organisms are selected on the base of appearance .re.g.
pigmentation etc.
Maarkers
http://tyanto.wordpress.com/apiary/ http://www.padil.gov.au/pests-and-
bee_comparison500/ diseases/Pest/Main/135533/8764#

4. 2-Chemical Markers
Organisms are grouped/classified on the base of biochemical
properties. e.g. Different humans have different blood groups.
3-Molecular Markers
A molecular marker is a
DNA sequence with a known location
on a chromosome ( Kumar,2009).
The DNA sequence in individuals differs
(Polymorphism) and individuals are
classified on the basis of these
variations in the sequence of DNA.
*http://scienceaid.co.uk/biology/genetics/inheritance.html

5. Some Important Molecular Markers
a) Restriction Fragment Length
Polymorphism(RFLP)
b) Randomly Amplified Polymorphic
DNA(RAPD)
c) Simple Sequence Repeats (SSR)/
Microsatellites.
d) Amplified Fragment Length
Polymorphism(AFLP)
e) Single Nucleotide Polymorphism (SNP)
http://cnx.org/content/m26561/latest/

6. Microsatellites/SSR
 Short segments of DNA
consisting of repeated
sequences, sandwiched
between flanking sequences.
 Present in the non – coding
region of DNA so they do not
have any apparent phenotypic
effects.
Mononucleotide SSR (A)11:
AAAAAAAAAAA
Dinucleotide SSR (GT)6:
GTGTGTGTGTGT
Trinucleotide SSR (CTG)4: CTGCTGCTGCTG
*http://www.coursework4you.co.uk/essays-anddissertations/sample69.php

7. Mitochondrial DNA
 It is located in mitochondria,( an
organelle in eukaryotic cells that
convert the chemical energy from
food into adenosine triphosphate
(ATP).
 mtDNA CO I – CO-II gene
region of Apis mellifera
exhibit high degree of
genetic variability within
and among the A.mellifera
lineage.
(Collet et al.2006; Ozdil et al.2009)
 mtDNA is inherited solely from the
mother(Avise,2004). Only one worker
bee is required to genetically http://en.wikipedia.org/wiki/File:Mit
characterize mtDNA in colony ochondrial_DNA_en.svg

8. Diversity in Honey Bees(Apis mellifera)
Morphometery has long
been the only method used
to study the variation among
the honey bee populations.
Ruttner(1988) published a
report on the taxonomy of
this species , based on the
analysis of morphological
characters .
Morphometery has shown that Apis mellifera has
differentiated into 26 subspecies.
On the basis of specific behavior and ecological
characteristics these subspecies are distributed into
five lineages
http://presstige.cz/ztresnaku/honey-bee-life-cycle&page=5

9. Distribution of Apis mellifera lineage
A-Lineage (Africa )
M-Lineage (Western Europe)
C- Lineage
(South Eastern Europe)
Lineage- C
O-Lineage
Near East and Middle East Lineage - O
Y-Lineage(Ethopia)
(Ruttner, 1988;Ferreira et al.2008)
(Frank et al.2000)

10. Molecular Diversity of Apis mellifera in Africa
Frank et al.(2001) analyses 738 honey bee colonies from 64
localities in 21 African countries by using Dra-I RFLP of CO I
– CO-II mitochondrial region .
mtDNA of African honey bee
appeared to be composed of
highly divergent lineage.
The African lineage “A” was present
in all the localities except
NORTH EASTERN AFRICA.
In North eastern Africa two newly described
lineage “O” and “Y” were observed in high
proportion.
Lineage “A” was present in high proportion in honey
bee population from the ibernian Peninsula and Sicily.

11. Range of Divergence percentage within and
between lineage
Table : Range of divergence within and between lineage using 40
complete DNA sequence of COI-CO II inter-genic region.
(Frank, et al.2001)

12. Molecular Diversity of Apis mellifera in
United States of America
 Delaney ( 2009) analyzed the genetic diversity of honey bees in
two regions of United States.
 The western commercial breeding population (WCBP) and the
southeastern commercial breeding population (SCBP) were
sampled.
 Genetic diversity was analyzed by using DraI restriction
fragment length polymorphism of the COI - COII mitochondrial
region and 10 polymorphic microsatellite loci.
 The C-lineage honey population was dominant in both regions.
 The frequency of M- lineage was low .
 The A- lineage was also found in the southeastern commercial
breeding population (SCBP).

14. PURPOSE OF STUDY
The studies on genetic variation of Apis mellifera using SSR
and DNA sequence have not been extensively conducted in
Kingdome of Saudi Arabia.
Therefore this study will focus on genetic diversity in honey
bees (Apis mellifera) by using
1- Sequencing of mtDNA CO-I and CO-II intergenic region
2-Simple Sequence Repeats/SSR Markers

15. MATERIALS AND METHODS
Collection of bee samples
Honey bee samples will be collected from different
localities in Kingdom of Saudi Arabia .
About 100-200 worker bees will be collected from each
location and these bees will be put in a sample kit
(containing vials with 95% ethanol) until the extraction of
DNA.
DNA Extraction
DNA will be extracted from honey bee worker thoracic
region by using the Puregene DNA Isolation Kit D-5000
A(Qiagen Valencia, CA).

16. mt DNA CO-I , CO-II intergenic region sequencing
 The mt DNA CO-I , CO-II intergenic region will be amplified by using
E2 (5’- GGC AGA ATA AGT GCA TTG-3’) and
H(5’-CAA TAT CAT CAT TGA TGA CC-3’) PCR primer
(Garney et al.1993)
 PCR will be conducted with 2 µl of the extracted DNA for total
volume of 50 µl.
 Amplicons will be separated by gel electrophoresis in 1 % agarose .
 The amplified DNA will be purified using Nanosep Centrifugal
devices (Pall life Science Ann, Arbor, MI ) and sent to the laboratory
for DNA sequencing.
 Mitotypes will be assigned and compared by conducting Basic
Local Alignment Search Tool ( BLAST) of DNA sequence available
on Gene Bank.

17. Microsatellites/ SSR markers
The diversity in honey bees will be studied through microsatellites/
SSR markers . Twelve microsatellites 12 loci out of 75 (A7, A28, A1
13, B124, A43, A24 , A88, A14, A76, A107, A29 and A35) will be
chosen to perform the study . (Estoup et al. 1993).These loci have
shown good proxies for assessing genetic variations in A.mellifera
Estoup et al. 1995

18.  PCR amplification of above mentioned loci will be carried out
as per Estoup et al.(1995). The sequences of primers and
optimal PCR conditions are given for each locus in previous
table.
 Amplicons will be separated by gel electrophoresis in 1 %
agarose .
 The microsatellites fragment size will be scored using
GeneMapper software .

19. The phylogenetic relationship among the
different honey bee lineage in
Kingdom of Saudi Arabia will be
constructed .
The molecular diversity in honey bee through
SSR will also be measured in Kingdom of Saudi
Arabia .
http://www.extension.org/pages/58650
/proceedings-of-the-american-bee-
research-conference-2011

20. References:
Avise, J.C.2004.Molecular markers,natural history and
evolution(2nd.ed.) Sinauer Assosiates, Inc.Sunderland,MA.
Collet, T., K. M. Ferreira, M. C. Arias, A.E.E. Soares, and M. A. Del
Lama. 2006. Genetic structure of Africanized honeybee
populations (Apis mellifera L.) from Brazil and Uruguay
viewed through mitochondrial DNA COI-COII patterns.
Heredity 97: 329-335.
Delaney, D. A.,M.D. Meixner, N.M. Schiff, and W.S. Sheppard.2009.
Genetic Characterization of Commercial Honey Bee
(Hymenoptera: Apidae) Populations in the United States by
UsingMitochondrial and Microsatellite Markers. Ann. Entomol.
Soc. Am. 102(4): 666-673.

21. Estoup, A.,L. Garnery, M. Solignac, and J. M.Cornuett .1995.
Microsatellite Variation in Honey Bee (Apis mellifera L.)
Populations: Hierarchical Genetic Structure and Test of the
Infinite Allele and Stepwise Mutation Models. Genetics
140:679-695.
Estoup,A., M. Solignac, M.Harry, and J. M.Cornuet .19 93
Characterization of (GT), and (CT), microsatellites in two
insect species: Apis mllijma and Bombus terrestris. Nucleic
Acids Res. 21: 1427-1431.
Franck, P., L. Garnery, A. Loiseau, B. P. Oldroyd, H. R.Hepburn, M.
Solignac, and J. M. Cornuet. 2001. Genetic diversity of the
honeybee in Africa: microsatellite and mitochondrial data.
Heredity 86: 420-430.
Garnery, L., M. Solignac, G. Celebrano, and J. M. Cornuet. 1993. A
simple test using restricted PCR-amplifed mitochondrial-
DNA to study the genetic-structure of Apis mellifera L.
Experientia 49: 1016-1021.

22. Ozdil,F.A.,A.Y.Mehmet,A.Yildiz,and H.G. Hall.2009.Molecular
characterization of Turkish honey bee populations(Apis
mellifera ) inferred from mitochondrial DNA RFLP and
sequence results.Apidologie 40: 570-576.
RUTTNER, F. 1988. Biogeography and Taxonomy of Bees.
Springer-Verlag, Berlin.

23. ACKNOWLEDGEMENT
 Dr. Ahmad Al-Ghamdi (My Supervisor )
 Dr. Yehya Al-Zakki
 Dr. Javed Ansari