This thesis describes the development and biological applications of GeneSeqer, which is a homology-based gene prediction program by means of spliced alignment. Additionally, a program named MyGV was written in JAVA as a browser to visualize the output of GeneSeqer. In order to test and demonstrate the performance, GeneSeqer was utilized to map 176,915 Arabidopsis EST sequences on the whole genome of Arabidopsis thaliana, which consists of five chromosomes, with about 117 million base pairs in total. All results were parsed and imported into a MySQL database. Information that was inferred from the Arabidopsis spliced alignment results may serve as valuable resource for a number of projects of special scientific interest, such as alternative splicing, non-canonical splice sites, mini-exons, etc. We also built AtGDB (Arabidopsis thaliana Genome DataBase, http://www.plantgdb.org/AtGDB/) to interactively browse EST spliced alignments and GenBank annotations for the Arabidopsis genome. Moreover, as one application of the Arabidopsis EST mapping data, U12-type introns were identified from the transcript-confirmed introns in the Arabidopsis genome, and the characteristics of these minor class introns were further explored.