2012 Annual Report 1a.Objectives (from AD-416): 1) Perform large-scale sequencing using 454 and Solexa sequencing technologies to generate a draft whole genome sequence of the fire ant Solenopsis invicta,. 2)To assemble and annotate draft genome,. 3)To perform comparative genomic analyses,. 4)To perform large-scale EST study to significantly increase the breadth and depth of transcriptome coverage of the fire ant Solenopsis invicta,. 5)To develop an oligo microarray for fire ants for gene expression studies using all available sequence data and. 6)To develop a custom assay for screening SNPs for linkage and population genomic studies of fire ants. 1b.Approach (from AD-416): We will generate >5 Gb of 454 sequence using DNA from a pooled sample of Gp-9B haploid males and a pooled sample of Gp-9b males from one Gp-9Bb queen. We will assemble the sequence data using 454 software (Newbler) in conjunction with in-house assembly/software pipelines available at the Swiss Institute for Bioinformatics. We will perform annotation, SNP identification, and comparative genomics analyses using our own analysis pipelines. We also will generate DNA sequence data for the transcriptome of S. invicta by sequencing normalized cDNA libraries using 454 pyrosequencing technology. All resulting sequences will be run through an automatic pipeline to assemble and preliminarily annotate the sequences. A 4 X 44K feature microarray will be developed and utilized for several 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). A SNP panel will be developed and utilized to construct a moderately dense linkage map. Finally, a web-based platform integrating these data in an easily accessible format will be developed. 3.Progress Report: This project is related to sub-objective 1.1 of the in-house project (Perform large-scale EST (expressed sequence tag) study to significantly increase the breadth and depth of transcriptome coverage using 454 pyrosequencing technology). The objectives of this cooperative research project are to generate a draft whole genome sequence of the fire ant, develop microarrays for gene expression studies, and develop a custom assay for screening single nucleotide polymorphisms (SNPs) for linkage and population genomics studies. During FY 2012 a complete draft genome of a second fire ant was generated, assembled and annotated, the results of which are summarized in a manuscript submitted to a peer-reviewed journal. Analyses of recombination in several families revealed a large non-recombining region that includes Gp-9 spanning almost 13 Megabases. The two variants (SB and Sb, which include Gp-9B and Gp-9b variants, respectively) are characterized by a large region of ca. 13 Mb (55% of the chromosome) where recombination is completely suppressed between SB and Sb. Recombination appears to occur normally between the SB chromosomes but is impossible between Sb chromosomes because Sb/Sb individuals are non-viable. Genomic comparisons revealed limited differentiation between SB and Sb, with all or at least the vast majority of the 616 genes identified in the non-recombining region present in the two variants. The reduced recombination between the two chromosomal regions can be explained at least partly by the increased number of repetitive elements and insertions in the non-recombining region of Sb compared with the homologous region of SB. Moreover, we identified an inversion in Sb, which may further suppress recombination.