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Apr 09, 2023 114 likes •272 views

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

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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

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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.

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SNP genotype dataset 1

Population

  • Num. samples

European honey bees (EHB), Hawaii 30 Africanized honey bees (AHB), Mexico 30 Gentle Africanized honey bees (gAHB), PR 30

  • Avalos et al. 2017 – 2.8 Million SNPs
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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

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SNP selection process

Step 2: Shared (183,000 SNPs)

Present in both datasets Present in 220 honey bee samples from both datasets

Step 1: All SNPS (10.8 Million SNPs)

Total number of SNPs from Avalos et al. 2017 & Wallberg et al. 2014

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

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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.

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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

  • f bees can be separated and identify diagnostic SNPs for each group.
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Methods for Selection from SNP Pool

DAPC was run using all 7,086 SNPs (From step 3) to identify clusters

  • f

closely related samples

7,086 SNPs

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Specific SNP Selection

  • Subset of fewer (42) SNPs with high

Linear Discriminant (LD) values generated similar clusters (as using all 7,086 SNPs)

  • gAHB was separated from the rest using 42

SNPs

  • DAPC was repeated with different subset
  • f high LD value SNPs to identify SNPs

that separate individual populations

42 SNPs

Identified 134 SNPs that differentiated all the populations in the two datasets

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Agena SNP panel – genotyping results

Sample Country

  • Num. samples

tested

  • Num. samples

matched prediction Percentage matched 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

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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.
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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 " "

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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

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SLIDE 14

Acknowlegements (III)

David Baez Cuevas (Collector Florida V, Oviedo)

Amateur beekeeper

Yves Le Conte

INRA, Bees and Environment Research Unit, Avignon, France

Mert Kükrer

Middle East Technical University, METU, Dept. of Biological Sciences, Ankara, Turkey

Amina Chakir

University Cadi Ayyad, Semlalia Faculty of Sciences, Marrakech, Morocco

Val Dolcini Pollinator Partnership Tom Van Arsdall

" "

Jacki Sands Qiagen Bi Linton

" "

Manuel O. Mendez

" "

Don Vendrone

" "

Rosalie Frechette Genome Quebec Andrew Bradford Agena Veronique Petrucci Enrique Adames-Soto

Alejandro Echevaria Puerto Rico Beekeeper Antonio Ayala " " Carlos Erminio " " Cristino Morreno " " Daniel Capucio " " Edgardo Lugo " " Emanuel Rosario " " Israel Diaz " " Jaime Soto " " Miguel Carbonel " " Pablo Ramirez Ruiz " " Raul Torres " " Roberto Martinez " " Roberto Mercado " " Ruben Parilla " " Sonia y Rafael Sanchez Sola " " Victor Agion Acosta " " Wilfredo A. Mezquita " " Willy Burgos " "