Washington Tree Fruit Research Commission

Research Reports

Development of Genetic Markers for Differentiation of Intercepted Immature Lepidoptera Pests in Deciduous Fruits (2002)

FINAL PROJECT REPORT
WTFRC Project #
YEAR 0/0
Organization Project #
Title:Development of Genetic Markers for Differentiation of Intercepted Immature Lepidoptera Pests in Deciduous Fruits
PI: L. G. Neven
Organization:USDA-ARS
 PDF version of report

Co-PIs

T. R. Unruh, Research Entomologist, USDA-ARS

Accomplishments

1.                    Extracted DNA from 6 species of Lepidoptera, codling moth, lesser apple worm, oblique-banded leafroller, omnivorous leafroller, Lacanobia fruitworm, oriental fruit moth.
2.                    Tested, using polymerase chain reactions, 13 primer sets designed to amplify mitochondrial DNA (7 for COI and 6 for COII) and 2 primer sets to amplify genomic DNA (ITS region).
3.                    Differences in banding patterns were noted for 4 of the COI primer sets, all 6 of the COII primer sets, and both of the ITS primer sets.
4.                    Sequencing of the bands obtained from C and I primer sets of COI were performed..
5.                    Comparisons of the banding patterns of 5 populations of codling moth using primer sets N and O of the ITS region were made.  These are in the process of sequencing.

Results

We have perfected a DNA extraction method for Lepidopteran larvae, and have obtained good quality DNA from the extraction method.  The DNA was used in amplification reactions using primers designed to amplify specific regions of mitochondrial DNA (primer sets A-J for the COI region and primer sets D-M for the COII regions).  Sequence analysis of bands obtained from primer sets C and I of the COI region indicate 2 to 3 areas of polymorphism (differences) which may be useful in species identification.  Additional bands obtained from primer sets G, F, and K are currently being sequenced.

 

Primer set N of the ITS region was used to amplify DNA from 12 to 13 individual codling moths obtained from 5 different populations in Washington State.  A number of differences in the banding pattern among individuals were noted, as well as differences between populations.  The 1200 bp band was the most common amplification product.  Population #5 yielded the most different sizes of amplified bands among individuals.  It appears that the number and size differences in bans obtained from primer set N may not be as useful in population identification as other primer sets.  Primer set O is currently half-way through this process, and appears to have a more consistent banding pattern among individuals in populations.

Once the sequences are complete for the above mentioned primer sets, DNA analysis will be performed on various populations of these species.  Also, searches will be made on GenBank to compare our sequences against published sequences for other insect species.  Once a unique sequence is identified for a specific species, a primer set will be made specifically to that region, and then tested against various populations of that species and against other species.

 


Figure 1.  1.8% Agarose gel electrophoretic pattern of moth mitochondrial DNA amplified with SR-J-14233 and T1-N-24 mitochondrial primers.  LAW-Lesser Apple Worm, OLR-Omnivorous Leafroller; OBLR-Oblique-banded Leafroller; OFM-Oriental Fruit moth; CM-Codling moth; LS-Lacanobia Fruitworm (from 1st report).

 

 

 

 

 

 

 

 

 

 

 

 


Table 1.  Description of samples and band molecular weights resulting from DNA amplification using SR-J-14233 and T1-N-24 mitochondrial primers.  LAW-Lesser Apple Worm, OLR-Omnivorous Leafroller; OBLR-Oblique-banded Leafroller; OFM-Oriental Fruit moth; CM-Codling moth; LS-Lacanobia Fruitworm (from 1st report).

 

 

LANE

INSECT

LENGTH

1

LAW

790 bp

2

LAW

790 bp

3

OLR

995 bp

4

MW marker

Marker

5

OBLR

564 bp

6

OFM

800 bp

7

OBLR

564 bp

8

OBLR

564 bp

9

OFM

800 bp

10

MW marker

Marker

11

CM

1360 bp

12

CM

1360 bp

13

CM

1360 bp

14

CM

1360 bp

15

LS

940 bp + 520 bp

16

LS

940 bp + 520 bp

17

LS

940 bp + 520 bp

18

LS

940 bp + 520 bp

 

 

 


Table 2.  Description of primer sets used for DNA amplification of mitochondrial and genomic sequences.  Primer sequences are given in Simon et al. (1994).

 

Region Amplified

Primer Set

Primer 1

Primer 2

Cytochrome Oxidase I

A

C1-J-1751

C1-N-2191

 

B

C1-J-1752

TL2-3014

 

C

C1-J-2183

TL2-3014

Cytochrome Oxidase II

D

C2-J-3400

C2-N-3661

 

E

C2-J-3279

C2-N-3389

 

F

C2-J-3400

TK-N-3785

Cytochrome Oxidase I

G

C1-J-1859

C1-N-2191

 

H

C1-J-1859

L2-N-3014

 

I

C1-J-2195

C1-N-2329

 

J

C1-J-2195

L2-N-3014

Cytochrome Oxidase II

K

C2-J-3138

C2-N-3389

 

L

C2-J-3571

C2-N-3661

 

M

C2-J-3571

TK-N-3785

ITS

N

ITS 4

ITS 5

ITS

O

ITS 2-5'

ITS 2-3'

 

 

Simons, C., F. Frati, A. Beckenbach, B. Crespi, H. Liu, P. Flook.  1994.  Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a complication of conserved polymerase chain reaction primers.  Ann. Entomol. Soc. Am.  87(6): 651-701.

 


Table 3.  List of the number and size of amplified bands obtained from the DNA of codling moth (CM), Oriental Fruit moth (OFM), Lesser Apple Worm (LAW), Oblique banded Leafroller (OBLR), Pandemis Leafroller (PLR), Cabbage looper (T.ni), Omnivorous leafroller (OLR), and Lacanobia Fruitworm (LFW), using various primer sets for COI, COII, and ITS (as listed in Table 2).  Green indicated bands which have been sequenced and yellow are bands in the process of being sequenced.

 

 

 

 

 

 

 

 

 

Figure 2.  Number of bands from amplification of DNA from codling moth (CM), Oriental Fruit moth (OFM), Lesser Apple Worm (LAW), Oblique banded Leafroller (OBLR), Pandemis Leafroller (PLR), Cabbage looper (T.ni), Omnivorous leafroller (OLR), and Lacanobia Fruitworm (LFW), using various primer sets for COI, COII, and ITS. 


Figure 3.  Size and number of bands amplified from individual codling moths obtained from 5 different populations in Washington State.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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