Measuring genetic variation of tarnished plant bug, Lygus lineolaris , over temporal and spatial scales

1. Measuring genetic variation of tarnished plant bug, Lygus lineolaris, over temporal and spatial scales O. P. Perera, Jeff Gore, Gordon Snodgrass & Brian Scheffler & Craig Abel USDA-ARS, Stoneville, Mississippi

2. Introduction

3. Introduction • Very few markers suitable for population genetic studies available • No microsatellite markers for H. virescens, Helicoverpa zea, or Lygus species

4. Introduction • Simple Sequence Repeat (SSR) or microsatellite markers are widely used in population genetics • Highly variable DNA regions • Co-dominant markers • Repeats of two to six nucleotides are commonly used

5. Methods • Partial genomic libraries for each species • Enrich the library for SSR sequences using biotin labeled repeat oligos and magnetic beads followed by PCR amplification

6. Methods

7. Genomic DNA Validate primer pairs Digest with 4-base cutter Analyze sequences and design primer pairs Ligate adapter to ends Clone & sequence Denature & hybridize with Biotin labeled SSR oligos Perform a 2nd round of Enrichment Magnetically capture repeat sequences hybridized to biotin labeled SSR oligos PCR amplify Enriched sequences

8. Methods • Genotyping with ABI3730xl instrument and GeneMapper software • Initial screening with pooled DNA samples of laboratory insects • Selected primer pairs used for analyzing field samples • Data analysis with PopGene v1.32

9. Results: H. virescens • DNA sequences of 192 clones • 147 clones contained repeats • 96 primer pairs synthesized • 20 polymorphic primer pairs producing 1 or 2 peaks per individual identified

10. Results: H. zea • DNA sequences of 192 clones • 34 primer pairs synthesized for 34 unique clones • 12 polymorphic primer pairs producing 1 or 2 peaks per individual identified

13. The sequence and the number of repeat units at each polymorphic locus and the amplicon size range observed in the test population of H. virescens (data for 8 loci shown).

14. Actual number of alleles ranged from 4 to 22 • Effective number of alleles ranged from 3 to 12 • Rare alleles contributed to reduced number of effective alleles

16. The sequence and the number of repeat units at each polymorphic locus and the amplicon size range observed in the test population of H. zea (data for 9 loci shown).

17. Actual number of alleles ranged from 2 to 6 • Effective number of alleles ranged from 1.3 to 4.8

19. Eight loci suitable for population genetics selected so far • 192 insects from 2 populations analyzed • Number of alleles ranged from 4 to 21 • Effective number of alleles ranged from 1.2 to 11.0 Lygus lineolaris microsatellites

21. Inbreeding Coefficients for 2 Lygus populations * Nm = Gene flow estimated from Fst = 0.25(1 - Fst)/Fst

22. Other Research in Progress • Development of microsatellites for L. hesperus, Sirex noctillio, Solenopsis invicta and fungi carried by Sirex species (Amylosterium sp.) • Development of SNP assays for gut-specific genes (EcoTILLING). • Sequencing of mtDNA genomes of cotton pest species.

23. Future work • Isolate and evaluate more SSR loci, possibly to obtain 1-3 loci/linkage group • Develop SSR based linkage maps

24. Collaborators Carlos Blanco Brian Scheffler Technical Support Mid-South Area Genome Center Linda Ballard Mary Duke X. Liu Sheron Simpson SIMRU Torey Looft Acknowledgements