MOLECULAR DIVERSITY IN HONEY BEES USING SIMPLE SEQUENCE REPEATS (SSR) , CYTOCHROME OXIDASE–I (CO-I) AND CYTOCHROME OXIDASE –II (CO-II)

By

Khalid Ali Khan (Ph.D. Student) 432108568

Supervisor : Dr. Ahmad bin Abdullah Al Ghamdi

Seminar II (PLPT-695)

Department of Plant Protection , King Saud University -Riyadh

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://www.padil.gov.au/pests-and-diseases/Pest/Main/135533/8764#

http://tyanto.wordpress.com/apiary/bee_comparison500/

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 locationon a chromosome ( Kumar,2009).

*http://scienceaid.co.uk/biology/genetics/inheritance.html

The DNA sequence in individuals differs(Polymorphism) and individuals are classified on the basis of these variations in the sequence of DNA.

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/

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 phenotypiceffects.

Mononucleotide SSR (A)11: AAAAAAAAAAA

Dinucleotide SSR (GT)6: GTGTGTGTGTGT

Trinucleotide SSR (CTG)4: CTGCTGCTGCTG

*http://www.coursework4you.co.uk/essays-anddissertations/sample69.php

http://en.wikipedia.org/wiki/File:Mitochondrial_DNA_en.svg

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 characterize mtDNA in colony

Morphometery has long been the only method usedto study the variation among the honey bee populations.

Ruttner(1988) published areport on the taxonomy ofthis 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

Diversity in Honey Bees(Apis mellifera)

http://presstige.cz/ztresnaku/honey-bee-life-cycle&page=5

Lineage A

Lineage M

Linea

ge Y

A-Lineage (Africa )M-Lineage (Western Europe)

C- Lineage(South Eastern Europe)

O-LineageNear East and Middle East  Y-Lineage(Ethopia)

(Ruttner, 1988;Ferreira et al.2008)(Frank et al.2000)

Lineage - O

Lineage- C

Distribution of Apis mellifera lineage

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 honeybee population from the ibernian Peninsula and Sicily.

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)

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).

Molecular Diversity of Apis mellifera in United States of America

 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

PURPOSE OF STUDY

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.

MATERIALS AND METHODS

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).

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.

mt DNA CO-I , CO-II intergenic region sequencing

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

Microsatellites/ SSR markers

Estoup et al. 1995

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 .

 The phylogenetic relationship among the different honey bee lineage in Kingdom of Saudi Arabia will beconstructed .

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

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.

References:

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. NucleicAcids 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 thehoneybee 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.

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.

ACKNOWLEDGEMENT

Dr. Ahmad Al-Ghamdi (My Supervisor )

Dr. Yehya Al-Zakki Dr. Javed Ansari

thanks