Analysis of expressed sequence tags and alternative splicing genes in sacred lotus (Nelumbo nucifera Gaertn.)

Sacred lotus, Nelumbo nucifera (Gaertn.), a basal eudicot of agricultural, medicinal, cultural, and religious importance, is an understudied species at the molecular level. Expressed sequence tags (ESTs) are particularly useful for gene identification, genome annotation and alternative splicing (AS) study. We generated 626,137 reads by 454 pyrosequencing of a cDNA library, which was constructed from mRNA collected from 7 different tissues of sacred lotus. The reads were assembled into 16,349 non-redundant EST contigs with an average length of 715 bp. Aligning ESTs to sacred lotus genome revealed the internal exons and introns had an average size of 140 and 2,005 bp, respectively. The average intron length in the sacred lotus is much larger than most sequenced plant genomes. We also identified 174 AS events involving 161 genes. Classification of the AS events showed that 62.6% of the AS events were retained introns, 8.0% were alternative acceptor sites, 7.5% were alternative donor sites, 4.0% were skipped exons, and 17.8% had "complex events" which had more than one basic event. Comparison of AS genes in sacred lotus, rice and Arabidopsis identified 7 commonly conserved genes between lotus and rice and 9 between sacred lotus and Arabidopsis. A gene encoding a 40S ribosomal protein was commonly conserved in all three species. These AS events and annotated ESTs are available through the Plant Alternative Splicing Database website (http://proteomics.ysu.edu/altsplice/plant/).