Molecular diversity in honey bees using simple sequence
MOLECULAR DIVERSITY IN HONEY BEES USING SIMPLESEQUENCE REPEATS (SSR) , CYTOCHROME OXIDASE–I(CO-I) AND CYTOCHROME OXIDASE –II (CO-II) Seminar II (PLPT-695)ByKhalid Ali Khan Supervisor : Dr. Ahmad bin Abdullah(Ph.D. Student) 432108568 Al GhamdiDepartment of Plant Protection ,King Saud University -Riyadh
A lot of variations among the organisms is found. Theorganisms are classified on the basis of these variations.There are different parameters/markers to measure thediversity in the organisms. Some important markers are:1-Morphological MarkersOrganisms are selected on the base of appearance .re.g.pigmentation etc.Maarkershttp://tyanto.wordpress.com/apiary/ http://www.padil.gov.au/pests-and-bee_comparison500/ diseases/Pest/Main/135533/8764#
2-Chemical MarkersOrganisms are grouped/classified on the base of biochemicalproperties. 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
Some Important Molecular Markersa) 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 DNAconsisting of repeatedsequences, sandwichedbetween flanking sequences. Present in the non – codingregion of DNA so they do nothave any apparent phenotypiceffects.Mononucleotide SSR (A)11:AAAAAAAAAAADinucleotide SSR (GT)6:GTGTGTGTGTGTTrinucleotide SSR (CTG)4: CTGCTGCTGCTG*http://www.coursework4you.co.uk/essays-anddissertations/sample69.php
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
Diversity in Honey Bees(Apis mellifera)Morphometery has longbeen the only method usedto study the variation amongthe honey bee populations.Ruttner(1988) published areport on the taxonomy ofthis species , based on theanalysis of morphologicalcharacters .Morphometery has shown that Apis mellifera hasdifferentiated into 26 subspecies.On the basis of specific behavior and ecologicalcharacteristics these subspecies are distributed intofive lineages http://presstige.cz/ztresnaku/honey-bee-life-cycle&page=5
Distribution of Apis mellifera lineageA-Lineage (Africa )M-Lineage (Western Europe)C- Lineage(South Eastern Europe) Lineage- CO-LineageNear East and Middle East Lineage - OY-Lineage(Ethopia)(Ruttner, 1988;Ferreira et al.2008)(Frank et al.2000)
Molecular Diversity of Apis mellifera in AfricaFrank et al.(2001) analyses 738 honey bee colonies from 64localities in 21 African countries by using Dra-I RFLP of CO I– CO-II mitochondrial region .mtDNA of African honey beeappeared to be composed ofhighly divergent lineage.The African lineage “A” was presentin all the localities exceptNORTH EASTERN AFRICA.In North eastern Africa two newly describedlineage “O” and “Y” were observed in highproportion.Lineage “A” was present in high proportion in honeybee population from the ibernian Peninsula and Sicily.
Range of Divergence percentage within and between lineageTable : Range of divergence within and between lineage using 40complete DNA sequence of COI-CO II inter-genic region.(Frank, et al.2001)
Molecular Diversity of Apis mellifera inUnited 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).
PURPOSE OF STUDYThe studies on genetic variation of Apis mellifera using SSRand DNA sequence have not been extensively conducted inKingdome of Saudi Arabia.Therefore this study will focus on genetic diversity in honeybees (Apis mellifera) by using1- Sequencing of mtDNA CO-I and CO-II intergenic region2-Simple Sequence Repeats/SSR Markers
MATERIALS AND METHODSCollection of bee samplesHoney bee samples will be collected from differentlocalities in Kingdom of Saudi Arabia .About 100-200 worker bees will be collected from eachlocation and these bees will be put in a sample kit(containing vials with 95% ethanol) until the extraction ofDNA. 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).
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.
Microsatellites/ SSR markersThe diversity in honey bees will be studied through microsatellites/SSR markers . Twelve microsatellites 12 loci out of 75 (A7, A28, A113, B124, A43, A24 , A88, A14, A76, A107, A29 and A35) will bechosen to perform the study . (Estoup et al. 1993).These loci haveshown good proxies for assessing genetic variations in A.mellifera 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 thedifferent honey bee lineage inKingdom of Saudi Arabia will beconstructed .The molecular diversity in honey bee throughSSR will also be measured in Kingdom of SaudiArabia . http://www.extension.org/pages/58650 /proceedings-of-the-american-bee- research-conference-2011
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.
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.
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