1a. Objectives (from AD-416)
1. Perform large-scale EST study to significantly increase the breadth and depth of transcriptome coverage of the fire ant Solenopsis invicta using 454 pyrosequencing technology. 2. Develop an oligo microarray for fire ants for gene expression studies using all available EST sequence data (including new data from objective 1). 3. Develop a custom assay for screening SNPs for linkage and population genomic studies of fire ants.
1b. Approach (from AD-416)
DNA sequence data will be generated for the transcriptome of S. invicta by sequencing three normalized cDNA libraries using 454 pyrosequencing technology. All resulting sequences will be run through an automatic pipeline to assemble and preliminarily annotate the sequences. Microarray development will occur in two phases. First, a pilot array will be constructed to identify the best probes for the unigenes identified from the sequencing efforts (244K feature microarray). The best performing probes (and correct orientation) for each sequence then will be synthesized along with positive and negative controls on a 4 X 44K feature microarray, which will be the platform used for subsequent studies. This array will be utilized for four experiments focusing on innate immunity, reproductive physiology, and social organization (including studies of Gp-9, a single gene associated with social organization in this ant). Array probes, layout, and purchase information will be made publicly available on an updated version of a fire ant database. Intraspecific SNPs will be identified using pipeline filtering methods developed for this purpose. A SNP panel consisting of 1536 SNPs will be developed and utilized to construct a moderately dense linkage map, to identify genes linked to Gp-9, to determine whether gametic disequilibrium between Gp-9, SNPs and co-expressed genes exists, and to determine whether these candidate genes are consistently associated with social form as has been shown for Gp-9. Finally, a web-based platform integrating these data in an easily accessible format will be developed.
3. Progress Report
This project is related to Objective 1 of this in-house project: Develop functional genomic resources and employ these resources to examine the genetic basis of biological traits that can potentially be used for biologically based control, including implications for the geographic origins of infestations. The goal of this project is to develop new functional genomic resources for the fire ant S. invicta. A pilot oligo microarray was constructed and used to identify the most appropriate oligo sequences for development of a microarray that includes every potentially transcribed gene region in fire ants. This newly developed array will be used for a number of gene expression studies outlined in Agriculture and Food Research Initiative Grant proposal. Necessary samples for two comparative gene expression studies were collected and archived. Progress on this project was monitored by means of frequent email communications, telephone communications, and visits to the ARS scientist’s laboratory by the collaborator.