Measuring genetic variation Measuring genetic variation of tarnished plant bug, of tarnished plant bug, Lygus lineolarisLygus lineolaris, over , over

temporal and spatial scalestemporal and spatial scales

O. P. Perera, Jeff Gore, Gordon O. P. Perera, Jeff Gore, Gordon Snodgrass & Brian Scheffler & Craig Snodgrass & Brian Scheffler & Craig

AbelAbel

USDA-ARS, Stoneville, MississippiUSDA-ARS, Stoneville, Mississippi

IntroductionIntroduction

IntroductionIntroduction

Very few markers suitable for Very few markers suitable for population genetic studies population genetic studies availableavailable

No microsatellite markers No microsatellite markers for H. virescens, Helicoverpa zea, or Lygus species

IntroductionIntroduction

Simple Sequence Repeat (SSR) Simple Sequence Repeat (SSR) or microsatellite markers are or microsatellite markers are widely used in population widely used in population geneticsgenetics

Highly variable DNA regionsHighly variable DNA regionsCo-dominant markersCo-dominant markersRepeats of two to six Repeats of two to six

nucleotides are commonly usednucleotides are commonly used

MethodsMethods

Partial genomic libraries for Partial genomic libraries for each specieseach species

Enrich the library for SSR Enrich the library for SSR sequences using biotin sequences using biotin labeled repeat oligos and labeled repeat oligos and magnetic beads followed by magnetic beads followed by PCR amplificationPCR amplification

MethodsMethodsOligo GroupOligo Group

(Hyb. (Hyb. Temp.)Temp.)

Oligonucleotide SequencesOligonucleotide Sequences

Group 1Group 1

(58(58C)C)(AC)(AC)1313, (AACC), (AACC)55, (AACG), (AACG)55, , (AAGC)(AAGC)55, (AAGG), (AAGG)55, (ATCC), (ATCC)55

Group 2Group 2

(52(52C)C)(AG)(AG)1212, (AAC), (AAC)66, (AAG), (AAG)88, (ACT), (ACT)1212, , (ATC)(ATC)88

Group 3Group 3

(48(48C)C)

(AAAC)(AAAC)66 , (AAAG) , (AAAG)66, (AAAG), (AAAG)66, , (AATC)(AATC)66, (AATG), (AATG)66, (ACAG), (ACAG)66, , (ACCT)(ACCT)88, (ACTC), (ACTC)66, (ACTG), (ACTG)66

Group 4Group 4

(42(42C)C)(AAAT)(AAAT)88, (AACT), (AACT)88, (AAGT), (AAGT)88, , (ACAT)(ACAT)88, (AGAT), (AGAT)88

Genomic DNA

Digest with 4-base cutter

Ligate adapter to ends

Denature & hybridize withBiotin labeled SSR oligos

Magnetically capturerepeat sequenceshybridized to biotinlabeled SSR oligos

Perform a 2nd roundof Enrichment

PCR amplifyEnriched sequences

Clone & sequence

Validate primer pairs

Analyze sequences and design primer pairs

MethodsMethods

Genotyping with ABI3730xl Genotyping with ABI3730xl instrument and GeneMapper instrument and GeneMapper softwaresoftware

Initial screening with pooled DNA Initial screening with pooled DNA samples of laboratory insectssamples of laboratory insects

Selected primer pairs used for Selected primer pairs used for analyzing field samplesanalyzing field samples

Data analysis with PopGene v1.32Data analysis with PopGene v1.32

Results: Results: H. virescensH. virescens

DNA sequences of 192 clonesDNA sequences of 192 clones147 clones contained repeats147 clones contained repeats96 primer pairs synthesized96 primer pairs synthesized20 polymorphic primer pairs 20 polymorphic primer pairs

producing 1 or 2 peaks per producing 1 or 2 peaks per individual identifiedindividual identified

Results: Results: H. zeaH. zea

DNA sequences of 192 DNA sequences of 192 clonesclones

34 primer pairs synthesized 34 primer pairs synthesized for 34 unique clonesfor 34 unique clones

12 polymorphic primer pairs 12 polymorphic primer pairs producing 1 or 2 peaks per producing 1 or 2 peaks per individual identifiedindividual identified

Repeat typeRepeat Sequence H. virescens H. zea

Di-nucleotide AC 9 1

AG 6 0

Tri-nucleotide

AAG 5 0

AGT 3 0

ATA 1 0

ATG 39 3

GTT 1 0

TAA 1 2

Tetra-nucleotide ACAG 53 14

ACAT 1 3

ACGG 3 0

CAAA 9 7

CAAC 4 0

CATA 4 0

GATG 2 0

GTCA 1 1

GTTC 1 0

TAAA 1 0

TTAA 1 0

Penta-nucleotide

CTAAC 1 0

TCCTC 1 0

Octa-nucleotide TTTGTCTG 1 1

  Total 147 32

Repeat type Repeat Sequence H. virescens H. zea

LocusLocus (Repeat Sequence)(Repeat Sequence)Repeat #Repeat #Amplicon size range Amplicon size range

(bp)(bp)

11 (TGA)(TGA)77 169-188169-188

22 (AACA)(AACA)44 151-169151-169

33 (CAT)(CAT)77 167-177167-177

44 (TCA)(TCA)44 151-160151-160

55 (TGA)(TGA)44 165-224165-224

66 (TGA)(TGA)44 168-202168-202

77 (TC)(TC)1010 171-191171-191

88 (TGAC)(TGAC)44 159-180159-180

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).

Actual number of alleles Actual number of alleles ranged from 4 to 22ranged from 4 to 22

Effective number of alleles Effective number of alleles ranged from 3 to 12ranged from 3 to 12

Rare alleles contributed to Rare alleles contributed to reduced number of effective reduced number of effective allelesalleles

LocLocusus

SamplSample Sizee Size HHoo HHee

11 156156 0.24360.2436 0.72850.7285

22 134134 0.26870.2687 0.48870.4887

33 146146 0.21920.2192 0.79990.7999

44 162162 0.27160.2716 0.57720.5772

55 8686 0.25580.2558 0.82440.8244

66 138138 0.30430.3043 0.92750.9275

77 148148 0.28380.2838 0.85570.8557

88 168168 0.30950.3095 0.73750.7375

MeaMeann 142142 0.26960.2696 0.74240.7424

St. St. DevDev    0.03030.0303 0.14590.1459

LocuLocuss

(Repeat (Repeat Sequence)Sequence)Repeat #Repeat #

Amplicon size Amplicon size range (bp)range (bp)

11 (TGA)(TGA)77 249-269249-269

22 (AGTC)(AGTC)44 116-129116-129

33 (ACAT)(ACAT)66 156-172156-172

44 (GTTT)(GTTT)44 144-232144-232

55 (GTTT)(GTTT)66 386-398386-398

66 (ATT)(ATT)44 133-136133-136

77 (TCTG)(TCTG)44 117-121117-121

88 (TCTG)(TCTG)66 211-252211-252

99 (ATG)(ATG)66 137-144137-144

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).

Actual number of alleles Actual number of alleles ranged from 2 to 6ranged from 2 to 6

Effective number of alleles Effective number of alleles ranged from 1.3 to 4.8ranged from 1.3 to 4.8

Locus Sample Size Ho He

1 182 0.1099 0.397

2 192 0.4688 0.5027

3 192 0.3125 0.3047

4 124 0.0806 0.0774

5 192 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 lineolarisLygus lineolaris microsatellites microsatellites

Eight loci suitable for population genetics Eight loci suitable for population genetics selected so farselected so far

192 insects from 2 populations analyzed192 insects from 2 populations analyzed Number of alleles ranged from 4 to 21Number of alleles ranged from 4 to 21 Effective number of alleles ranged from 1.2 Effective number of alleles ranged from 1.2

to 11.0to 11.0

LocusRepeat Sequence Sample size HoHo HoHo

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 (CA) 7 372 0.5914 0.7241

3-60 (GAT) 7 372 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

Locus Sample Size Fis Fit Fst Nm*

1-15 358 -0.055 -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

3-60 372 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

Inbreeding Coefficients for 2 Lygus populations

* Nm = Gene flow estimated from Fst = 0.25(1 - Fst)/Fst

Other Research in Other Research in ProgressProgress

Development of microsatellites forDevelopment of microsatellites for L. L. hesperushesperus, , Sirex noctillioSirex noctillio, , Solenopsis invictaSolenopsis invicta and fungi carried by and fungi carried by SirexSirex species species ((AmylosteriumAmylosterium sp.) sp.)

Development of SNP assays for gut-specific Development of SNP assays for gut-specific genes (EcoTILLING).genes (EcoTILLING).

Sequencing of mtDNA genomes of cotton Sequencing of mtDNA genomes of cotton pest species.pest species.

Future workFuture work

Isolate and evaluate more Isolate and evaluate more SSR loci, possibly to obtain SSR loci, possibly to obtain 1-3 loci/linkage group1-3 loci/linkage group

Develop SSR based linkage Develop SSR based linkage maps maps

AcknowledgementsAcknowledgements

CollaboratorsCarlos Blanco Brian Scheffler

Technical SupportMid-South Area Genome Center

Linda Ballard Mary Duke X. Liu Sheron Simpson

SIMRUTorey Looft