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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPMENT OF ADVANCED TECHNOLOGIES FOR THE SCREWWORM ERADICATION PROGRAM

Location: Screwworm Research

Title: Lyophilization of Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae yields high-quality DNA for use in AFLP genetic studies

Authors
item Clark, Pete -
item Isenhour, David -
item Skoda, Steven
item Molina-Ocha, Jaime -
item Gianni, Claudia -
item Foster, John -

Submitted to: International Journal of Tropical Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 11, 2009
Publication Date: July 28, 2009
Citation: Clark, P.L., Isenhour, D.J., Skoda, S.R., Molina-Ocha, J., Gianni, C., Foster, J.E. 2009. Lyophilization of Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae yields high-quality DNA for use in AFLP genetic studies. International Journal of Tropical Insect Science. 29(2):79-85.

Interpretive Summary: Agricultural research in the 21st century has become a collaborative effort. Research on crop pests such as the fall armyworm (FAW) can involve international collaboration because it is a pest not only in the Southern United States but also Latin and South America. Our interest to study the genetic variation of twenty four sub-populations of FAW from the Southern United States, Mexico, Puerto Rico, Brazil, and Argentina made necessary that we investigate insect collection procedures that preserve the integrity of DNA for molecular genetic analysis. The samples were collected primarily from maize (corn), but also included outliers collected from pigweed, Royal Paulonia, lemon tree, and bermudagrass. A common insect preservation technique is to place individual insects in 95% ethyl alcohol (ETOH). However, various regulations for shipping and large size of this insect often prevent large numbers of samples stored in ETOH from being imported. Genomic DNA was extracted from samples preserved in 95% ETOH, lyophilized (i.e. freeze-dried), and fresh insects and then evaluated using DNA quantification and PCR-AFLP (polymerase chain reaction -amplified fragment length polymorphism; a molecular genetic technique). All three treatments yielded high quality/ high molecular weight (approx. 70 to 150 ~g) DNA. No differences in quality of genomic DNA for AFLP analysis were observed. Lyophilization was thus used to preserve the FAW samples for genetic analysis in this study. This storage technique should prove useful not only in other studies of FAW wherein samples must be imported from international sites but for other Lepidoptera and, perhaps, other Orders of insects.

Technical Abstract: Agricultural research in the 21st century has become a collaborative effort. Research on crop pests like Spodoptera frugiperda (J.E. Smith), commonly known as the fall armyworm (FAW), can involve international collaboration because it is a pest not only in the southern United States, but also in Latin and South America. Our interest to study the genetic variation of 24 subpopulations of FAW from the southern United States, Mexico, Puerto Rico, Brazil and Argentina required insect collection procedures that preserve the integrity of DNA for molecular genetic analysis. The samples were collected primarily from maize (Zea mays L.), but also included outliers collected from pigweed (Amaranthus sp.), Royal Paulownia (Paulownia tomentosa (Thunb.) Sieb. and Zucc. ex Steud.), lemon tree (Citrus limon (L.) Burm) and Bermuda grass (Cynodon dactylon (L.) Pers.). A common insect preservation technique is to place individual insects in 95% ethanol (ETOH). However, various regulations for shipping and the size of this insect size often prevent large sample sizes stored in ETOH from being imported. Genomic DNA from samples preserved in 95% ETOH, lyophilized and fresh insects was extracted and evaluated using DNA quantification and polymerase chain reaction–amplified fragment length polymorphism (PCR–AFLP). All three treatments yielded high-quality/high molecular weight (c. 70– 150 mg) DNA. No differences in quality of genomic DNA for AFLP analysis were observed. Lyophilization is a reliable tool to preserve FAW samples, which yields high-quality DNA for use in AFLP genetic analysis.

Last Modified: 9/21/2014
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