1. 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

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. 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

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

11. 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

12. 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

13. 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 2
(AACA)4 3
(CAT)7 4
(TCA)4 5
(TGA)4 6 7
(TC)10 8
(TGAC)4

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

15. 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

16. 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 2
(AGTC)4 3
(ACAT)6 4
(GTTT)4 5
(GTTT)6 6
(ATT)4 7
(TCTG)4 8
(TCTG)6 9
(ATG)6

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

18. 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

19. 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

20. Locus Repeat Sequence Sample size 1-15 (CA)8 358 0.2011 0.1909 1-26Ho
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

21. 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

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. 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