De Novo Creation of Two Novel Spliceosomal Introns of RECG1 by Intronization of Formerly Exonic Sequences in Orchidaceae.

Spliceosomal introns are a key characteristic of eukaryotic genes. However, the origins and mechanisms of new spliceosomal introns remain elusive, and definitive case studies documenting intron creation are still limited. This study examined the RECG1 genes of 49 land plants, including 21 orchids and 28 non-orchid species. Sequence comparison revealed that the fourth intron of Gastrodia and Platanthera (Orchidaceae) is a newly gained spliceosomal intron, originating from the intronization of former exonic sequences. This intronization event was accompanied by the creation of novel recognizable GT/AG splice sites. In contrast, other orchid species lack the corresponding splice sites in the counterpart regions. Moreover, the secondary and tertiary protein structures implied that the intronization events do not affect the protein function. Given the diverse trophic modes of the two genera, we infer that relaxed selection may have contributed to the fluidity of gene structures. This study provides a typical example of de novo lineage-specific intron creation via intronization in orchids supported by multiple lines of evidence, and the two intronization events occurred independently in the same gene. This research enhances our understanding of gene evolution in orchids and provides valuable insights that may assist the annotation of structurally complex genes.