2012 Annual Report
1a.Objectives (from AD-416):
Sequencing and profiling of functional transcripts constructed in expressed sequence tags (ESTs) of Coptotermes formosanus to fulfill the following objectives: a) Identifying and annotating of genes specifically associated with post-embryonic polyphenism (caste differentiation and development); b) Discovering of genes specifically responding to environmental cues and internal signals (pesticides, food sources, juvenile hormones, etc.); and c) Characterizing of genes uniquely involved in critical physiological pathways (digestion, molting, immunity, etc.). The fulfillment of these objectives would directly lead to achieving the following goals: a) providing biochemical/physiological/molecular bases for disruption of colony formation, development and survival; b) Discovering novel target site(s) that would be developed into new control strategies that could be incorporated into effective area-wide integrated management of Formosan subterranean termites.
The overall objectives of this cooperative research are to determine genes that are unique to termites - specifically reproductive and development associated genes e.g. nymph or soldier formation mechanisms with specific emphasis on Coptotermes formosanus while also examining cellulosic material hydrolyzing enzymes for agriculture and industry. We will also concentrate on private and public database and website development strategies.
1b.Approach (from AD-416):
A cDNA library representing expressed genes in each different developmental stage of Coptotermes formosanus has been constructed. To facilitate transcriptome analysis and rare gene discovery, repeated transcripts were proportionally removed from the cDNA library using the procedure of cDNA normalization. The cDNA library would contain approximately 400,000 independent clones, an estimate of at least 16X coverage of the entire expressed genes, provided that the protein-coding capacity of invertebrate genomes is in the range of 16,000 to 25,000 genes. The sequencing and gene data assembly of the cDNA library will be cooperatively conducted in JCVI. We will continue Sanger sequencing of EST clones and perform SoLID sequencing to determine differential gene expression. EST sequences will be compared against existing databases and annotated using the Basic Local Alignment Search Tool (BLAST). Batches of sequences will be sequentially released to GenBank for public access. Unique genes and singletons would be selected and gene expression analysis. Differentially-expressed genes or development-stage specific genes will be preferentially analyzed quantitatively (such as real-time PCR) or qualitatively (such as gene silencing by RNA interference).
Last year, J. Craig Venter Institute (JCVI) completed whole genome sequencing and assembly of the nuclear genome of the Formosan Subterranean termite, Coptotermes formosanus, and also finished sequencing of Ribonucleic acid (RNA) samples from two developmental stages. The genomic Deoxyribonucleic acid (DNA) was used to prepare several fragmentary, Paired End and Mate Pair libraries and sequenced using Illumina GA-II and HiSeq platforms. This resulted in sequence coverage of over 50x after the reads were filtered based on quality. The draft assembly of the genome, generated by the ALLPATHS-LG assembler, has 8,478 scaffolds containing 72,457 contigs. The total scaffold length is 891,902,903 bp. The contig N50 is 29.2 kbp while the scaffold N50 is 1.478 Mbp. This assembly is currently under annotation, while refinements to the assembly are ongoing. After the annotation is complete, we will compare it with that of the native subterranean termite Reticulitermes flavipes in order to further understand caste differentiation and development and provide information on cellulose digestion useful in cellulosic biofuel production.
The RNA samples sequenced were from the Female Alates and the Worker Termites and the yields were 83,102,604 reads and 13,281,961 reads respectively. While the RNA samples from Nymphs and Male Alates were sequenced in the previous year and produced 22,493,290 reads and 21,234,321 reads respectively, the RNA sample from the Pre-Soldier stage failed library preparation. The RNA-Seq data from the four stages will be analyzed and genes will be identified that may regulate processes associated with differentiation in order to determine possible biologically-based control measures for the termite.