USDA-ARS, Stoneville, Mississippi 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

Introduction

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

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

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

Oligonucleotide Sequences Methods Oligo Group (Hyb. Temp.) Oligonucleotide Sequences Group 1 (58C) (AC)13, (AACC)5, (AACG)5, (AAGC)5, (AAGG)5, (ATCC)5 Group 2 (52C) (AG)12, (AAC)6, (AAG)8, (ACT)12, (ATC)8 Group 3 (48C) (AAAC)6 , (AAAG)6, (AAAG)6, (AATC)6, (AATG)6, (ACAG)6, (ACCT)8, (ACTC)6, (ACTG)6 Group 4 (42C) (AAAT)8, (AACT)8, (AAGT)8, (ACAT)8, (AGAT)8

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

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

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

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

Di-nucleotide AC 9 1 AG 6 Tri-nucleotide AAG 5 AGT 3 ATA ATG 39 GTT Repeat type Repeat Sequence H. virescens H. zea Di-nucleotide AC 9 1 AG 6 Tri-nucleotide AAG 5 AGT 3 ATA ATG 39 GTT TAA 2

Tetra-nucleotide ACAG 53 14 ACAT 1 3 ACGG CAAA 9 7 CAAC 4 CATA GATG 2 Repeat type Repeat Sequence H. virescens H. zea Tetra-nucleotide ACAG 53 14 ACAT 1 3 ACGG CAAA 9 7 CAAC 4 CATA GATG 2 GTCA GTTC TAAA TTAA Penta-nucleotide CTAAC TCCTC Octa-nucleotide TTTGTCTG   Total 147 32

Amplicon size range (bp) 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). Locus (Repeat Sequence)Repeat # Amplicon size range (bp) 1 (TGA)7 169-188 2 (AACA)4 151-169 3 (CAT)7 167-177 4 (TCA)4 151-160 5 (TGA)4 165-224 6 168-202 7 (TC)10 171-191 8 (TGAC)4 159-180

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

Locus Sample Size Ho He 1 156 0.2436 0.7285 2 134 0.2687 0.4887 3 146 0.2192 0.7999 4 162 0.2716 0.5772 5 86 0.2558 0.8244 6 138 0.3043 0.9275 7 148 0.2838 0.8557 8 168 0.3095 0.7375 Mean 142 0.2696 0.7424 St. Dev   0.0303 0.1459

Amplicon size range (bp) 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). Locus (Repeat Sequence)Repeat # Amplicon size range (bp) 1 (TGA)7 249-269 2 (AGTC)4 116-129 3 (ACAT)6 156-172 4 (GTTT)4 144-232 5 (GTTT)6 386-398 6 (ATT)4 133-136 7 (TCTG)4 117-121 8 (TCTG)6 211-252 9 (ATG)6 137-144

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

Locus Sample Size Ho He 1 182 0.1099 0.397 2 192 0.4688 0.5027 3 0.3125 0.3047 4 124 0.0806 0.0774 5 0.4062 0.5188 6 186 0.1075 0.1031 7 164 0.122 0.1158 8 190 0.1684 0.3736 9 144 0.0417 0.067 Mean 174 0.2020 0.2733 St. Dev. 0.1543 0.1851

Lygus lineolaris microsatellites 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

Locus Repeat Sequence Sample size 1-15 (CA)8 358 0.2011 0.1909 1-26 Ho 1-15 (CA)8 358 0.2011 0.1909 1-26 (CA) 7 376 0.2181 0.2368 1-43 (CA) 9 368 0.8152 0.9095 2-15 (AGT) 4 374 0.5882 0.6167 2-47 (AGA) 6 370 0.6162 0.809 3-16 372 0.5914 0.7241 3-60 (GAT) 7 0.7473 0.8527 3-91 (GAT) 6 362 0.6077 0.7556 Mean 369 0.5482 0.6369 St_Dev   0.2243 0.2756

Inbreeding Coefficients for 2 Lygus populations Locus Sample Size Fis Fit Fst Nm* 1-15 358 -0.055 0.0006 388.75 1-26 376 0.0748 0.08 0.0057 43.828 1-43 368 0.0845 0.1018 0.0189 12.985 2-15 374 0.0357 0.0477 0.0124 19.842 2-47 370 0.2404 0.2417 0.0017 145.37 3-16 372 0.177 0.1827 0.007 35.677 3-60 0.1198 0.1218 0.0022 111.56 3-91 362 0.1923 0.1938 0.0018 135.83 Mean 369 0.1329 0.1391 0.0071 35.094 * Nm = Gene flow estimated from Fst = 0.25(1 - Fst)/Fst

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.

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

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