Molecular Methods for Distinguishing Between Wild and Commercial Cotton Variants of Anthonomus grandis. Raul Ruiz-Arce

Transcription

1 Molecular Methods for Distinguishing Between Wild and Commercial Cotton Variants of Anthonomus grandis Raul Ruiz-Arce

2 USDA APHIS PPQ Mission Lab (ML) Molecular methods development at ML TW/BW diagnostics at ML Description & performance of three methods Summary for captures Recent advancements and future work

3 Overview of Facilities, Expertise, and Diagnostics at USDA APHIS Mission Lab - Edinburg, TX

4 4 Molecular Scientists 3 Entomologists 1 GIS Specialist 3 Biologists 1 Plant Pathologist 18 Technicians 3 Office Administration 1 IT specialist 2 Interns

5 Citrus health response programs Detection and ID citrus pests Citrus greening biocontrol Mexican fruit fly SIT support Strain development & QC Colony health Molecular diagnostics Conventional/NGS* *Fruit flies ID, Source estimation BW/TW variant ID Helicoverpa ID methods Old/New World Bollworm

6 Organelles Membranes DNAs Their importance to molecular work

7 DNA molecule Sample preservation Contaminants Harvesting DNA

8 Why: Pest$ take a big bite We re a small world Quality food is important What do they tell us: Confirm ID Differences among species Where pests originate History and movement Speciation

9 3 temperature Denaturing Annealing Extension

10 Indigenous to C America/S Mexico Found in Cuba & Haiti in the 1800s First reported in Texas 1892 S America ( s)

11 Morphological - Burke (1968) and Burke et al (1986) Molecular Roehrdanz (2001) ML RAPD-PCR 1990s 2000s Southeastern boll weevil (BW) Host commercial cotton (Gossypium hirsutum) Thurberia weevil (TW) Host wild cotton (G. thurberi), occurs in N Mexico & SW US

12 Captures Trapped weevils initiate response Challenge in using older methods Morphology (Burke, 1968) Molecular (Roehrdanz, 2001) Opportunity for Improving management ID source of infestation Helps in decision for management Reduction to resources

13 Molecular Methods (Barr et al., 2013) Sequencing COI Barcode (Folmer et al 1994) SCAR (seq characterized amplified region) Classical Taxonomy (perf by C. Reuter) Morphometrics (Burke, 1968)

14 n=158 weevils 30 sample sites 10 Wild cotton 20 Comm cotton

15 503 base pair fragment Reference database (n=158), 31 unique seqs N=84, 14 sites in 7 MX states N=74, 16 sites in 4 US States

16 The list of variable nucleotide positions for the 31 COI haplotypes recovered from the Anthonomus spp. specimens. The diagnostic positions distinguishing Thurberia and boll weevil highlighted. AN2 T C A A C G C G T C A C A C T G G A A A A G T T G T A A A A A C G C A A A T A C T T A T A A A A AN G.... A A G G G A AN4 AN6.. G.. A.... G A TW * Sonora, Chihuahua, Coahuila, Durango, AZ AN7.. G G G C... T... AN8.. G G A.. G AN14.. G G G A G AN G G A C AN16.. G. T..... G SNPs consistent with boll weevil AN17.. G G A AN18.. G G... C A.. G AN19.. G G AN G AN21.. G G A AN G AN23.. G.... A.. G A AN A AN26. T G G... C A.. G AN5 C T G... T. C T... T C C A T T AN1 G.. G. A... T G A... A A A C. C.. G T. C.... G G. AN9 G.. G. A... T. A... A.... G... A C G C. G G T... G. C T. C.. C. G.. AN10 G.. G. A... T G A... A... G.... A C G C G. G T. C.... G G. AN11 G.. G. C... T. A... A A C G C.. G.... G.. T. C.. C. G.. AN12 G.. G. A BW... T. A... A A C G C.. G.... G.. T. C.. C. G.. AN13 A.. G. A... T G A... A A C G C G. G T. C.. C. G G. AN25 G.. G. A... T. A... A A C. C.. G.... G.. T. C.. C. G.. AN27 G.. G. A... T. A... A.... G... A C G C. G G T A.. G. C T. C.. C. G.. AN28 G.... A... T. A G.. A A C G C G T. C. G C. G G. AN29 G.. G. A... T G A... A A C G C G. G T. C.... G G. AN30 G.. G. A... T G A... A... G. A.. A C G C G. G T. C.... G G. AN31 G.. G. A... T G A... A A C. C G. G T. C.... G G.

17 Neg C Developed from excised band fixed to BW - = not BW + = BW

18 Femur Ratio (length:width) (Burke, 1968) TW BW

19 Identification results for n=158 with different methods (Barr et al 2013). ID Morphometrics SCAR COI profile Correct* 89.4% 82.9% 94.3% *considered correct when the results agreed w/host

20 C. Reuter (Phoenix, Az) Morp ID then, Shipped to ML Day 1 Intake DNA Isolation minimally invasive Day 2 PCR/Electrophoresis Day 3 Send off for sequencing Day 5+ Interpret data/complete report

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22 Capture Date n Collection Site Reported ID March Ojinaga, Chihuahua March Delicias, Chihuahua 10 May Sonoyta, Sonora 1 Oct Caborca, Sonora 22 Oct Caborca, Sonora 7 July Delicias, Chihuahua 16 July Nuevo San Lucas, Chi 17 July Delicias, Chihuahua BW BW TW TW TW NOT A. grandis NOT A. grandis NOT A. grandis

23 Provides early warning Results to managers within ~5-7days Helps improve management strategies Identifies hot spots/feedback Reduction to resources TW ID = no spraying, trade is not impacts, etc BW ID = target only problematic areas

24 1. Sequence and assemble the CBW genome 2. Resolve the population genomic structure of A. grandis in northern Mexico 3. Infer movement/dispersal of CBW in northern Mexico and southern Texas 4. Identify source populations of potential reintroductions in southern Texas 5. Additional marker development

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26 * Mediterranean fruit fly (Ceratitis capitata) Frequent US infestations (captures) Identified informative markers (conserved & fine scale) Informative reference database Results: Effective tool for determining the source of introductions Improve pest management strategies Minimize future infestations Identify hot-spots *Photograph by: Scott Bauer, USDA

27 = GEO DIST DNA Marker set A = Source = Regional-level DNA marker set B = Source = Below Regional-level