SNP SNP ho hone ney be y bee di diagno gnostic pa c pane nel - PowerPoint PPT Presentation

SNP SNP ho hone ney be y bee di diagno gnostic pa c pane nel Tugrul Giray, Ph.D. Department of Biology University of Puerto Rico San Juan, PR

Introduction • The population of honey bees on Puerto Rico (PR) is less aggressive than Africanized Honey bees (AHB). • PR bees are exposed to fewer infectious agents than those on the U.S. mainland. • Aim is to develop a certification program for the exportation of the honey bees of PR. • One component is to develop a diagnostic single nucleotide polymorphism (SNP) assay that can unambiguously identify PR honey bees.

SNP genotype dataset 1 • Avalos et al. 2017 – 2.8 Million SNPs Population Num. samples European honey bees (EHB), Hawaii 30 Africanized honey bees (AHB), Mexico 30 Gentle Africanized honey bees (gAHB), PR 30

SNP genotype dataset 2 • Wallberg et al. 2014 – 8 Million SNPs Population Num. samples Adansonii 10 Anatoliaca 10 Capensis 10 Carnica 10 EU domestic 20 Iberiensis 10 Ligustica 10 Mellifera Norway 10 Mellifera Sweden 10 Scutellata 10 Syriaca 10 US domestic 10

SNP selection process Step 1: All SNPS (10.8 Million SNPs) Total number of SNPs from Avalos et al. 2017 & Wallberg et al. 2014 Step 2: Shared (183,000 SNPs) Present in both datasets Present in 220 honey bee samples from both datasets Step 3: Assay Criteria (7,086 SNPs) No neighboring SNP in 16 flanking bases on either side of chosen SNP Must meet requirement for Agena assay design

Methods used for SNP Selection • Principle Component Analysis ( PCA) Variable reduction technique that determines the most important variables for understanding the difference among a set of observations. The important variables are combined together to produce a single representative value for each observation.

Methods for SNP Selection • Discriminant Analysis of Principle Components ( DAPC) Discriminant Analysis determines whether pre-defined groups can be distinguished from each other. We use the previously established principal components to determine whether the groups can be separated and identify which SNP's are important for each group. Combination of Principle Component Analysis (PCA) and Discriminant Analysis. DAPC allows us to determine whether different groups of bees can be separated and identify diagnostic SNPs for each group.

Methods for Selection from SNP Pool 7,086 SNPs DAPC was run using all 7,086 SNPs ( From step 3 ) to identify clusters of closely related samples

Specific SNP Selection • Subset of fewer (42) SNPs with high Linear Discriminant (LD) values generated 42 SNPs similar clusters (as using all 7,086 SNPs) • gAHB was separated from the rest using 42 SNPs • DAPC was repeated with different subset of high LD value SNPs to identify SNPs that separate individual populations Identified 134 SNPs that differentiated all the populations in the two datasets

Agena SNP panel – genotyping results Num. samples Percentage Num. samples Sample Country matched matched tested prediction prediction Puerto Rico 163 150 92.02 Argentina 54 43 79.63 Bolivia 70 70 100.00 Costa Rica 11 11 100.00 France 26 22 84.62 Israel 28 28 100.00 Italy 51 39 76.47 Kenya 59 59 100.00 Madagascar 14 14 100.00 Malta 11 4 36.36 Mexico 54 54 100.00 Morocco 25 25 100.00 Panama 37 36 97.30 Portugal 12 12 100.00 Georgia 29 29 100.00 Seychelles 19 19 100.00 South Africa 68 68 100.00 Spain 8 8 100.00 Tunisia 15 15 100.00 Turkey 85 74 87.06 U.S. 109 101 92.66 Zambia 49 37 75.51 Total 997 918 92.09

Conclusion • Identified a set of 134 SNPs that differentiates 15 populations in the reference datasets. • A diagnostic Agena SNP assay was developed using these 134 SNPs. • Agena assay was run on 834 samples from 21 countries as well as 163 samples from Puerto Rico. • Overall 92% of samples matched their predicted assignation.

Acknowlegements (I) Tugrul Giray Dept. of Biology, University of Puerto Rico, San Juan, Puerto Rico Rosanna Giordano Know Your Bee, Inc., San Juan, Puerto Rico Ravikiran Donthu " " Jose Marcelino Dept. of Biology, University of Puerto Rico, San Juan, Puerto Rico Jose Agosto Rivera " " Aixa Ramirez Lluch " " Yarira Ortiz-Alvarado " " Catalina P. Rodriguez Alemany " " Charles A. Cuff " " Carlos Ortiz " " FanFan Noel " " Cesar Ramirez " " Shirley Cruz " " Ísada C Cordero-Ford " " Stephanie F Cardona " " Janpierre Aleman-Rios " " Sebastian A. Silva Echeandia " " Tilden Aponte " " Alfredo Ghezzi " " Jose Acadio " " Ricardo Pappa " " Silvia Planas " " Yadira Ortiz-Ruiz " " Valence Washington " " Madhavi Kuchibhotla " " Rita Frontera Facultad Ciencias Naturales, Univ. Puerto Rico, Río Piedras Campus Carlos Pereira " " Shu-Ching Chen School of Computing and Information Sciences, Florida International University, Miami, FL Hector Cen " " Maria Presa Reyes " "

Acknowlegements (II) Everett Weber Michigan State University Arian Avalos Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana IL Felipe Soto-Adames Florida Dept. of Agriculture and Consumer Services, Division of Plant Industry, Gainesville, FL Paul Scalley " " David Westervelt " " Carmen Fraccica (Collector Florida II, Tampa) " " Robert Horsburgh (Collector Florida I, St. Augustine) " " Julieta Brambila USDA, Plant Protection and Quarantine (PPQ), Gainesville, FL Robyn Rose USDA, APHIS Tony & Becky Hogg (Collectors Florida III, Jefferson County) Florida State Beekeepers Association Tallahassee, FL 32311 Jay Evans USDA-ARS, Beltsville, MD, 20705 Rodrigo A. Velarde M. Instituto Apícola Boliviano, PROMIEL-SEDEM, La Paz, Bolivia Nabor H. Mendizabal Chavez " " Mike Allsopp Agricultural Research Council, Stellenbosch, South Africa Elliud Mulli (Kenya, Madagascar & Seychelles) International Centre of Insect Physiology and Ecology, Nairobi, Kenya Ernesto Guzman-Novoa Honey Bee Research Centre, School of Environmental Sciences, University of Guelph, Canada Adriana Correa Benitez Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México Gaetana Mazzeo Dipartimento di Agricoltura, Alimentazione e Ambiente, Università degli Studi di Catania, Italia Victoria Soroke Agricultural Research Organization ARO, Bet Dagan, Israel Jose Antonio Ruiz University of Cordoba, Spain Adrian Bugeja Douglas Institute of Earth Systems, Rural Sciences Farmhouse, University of Malta, Msida, Malta Faten Ben Abdelkader Laboratoire d’Apiculture et de Zoologie. INAT, Université de Carthage, Tunis-Mahrajène, Tunisia Daniel Ball Forest Fruits Ltd., Lusaka, Zambia Alberto Galindo Fundación Miguel Lillo, Tucumán, Argentina Alejandra A. Scannapieco Instituto de Genética ‘Ewald A. Favret’, INTA & CICT, Buenos Aires, Argentina Eduardo Virla Fundación Miguel Lillo, Tucumán, Argentina Hermogenes Fernandez-Marin Centro de Biodiversidad y Descubrimiento de Drogas, INDICASAT AIP, Cuidad del Saber, Panama Cesar Guillen Sanchez CORBANA, Corporacion Bananera Nacional, Pococí, Limón, Costa Rica Zachary Wang Michigan State University, Dept. of Entomology, MSU Apiculture Lab, East Lansing, MI Jennifer Holmes (Collector Florida IV, Stuart) Hani Honey Company Stuart, FL