Evolutionary and Functional Analysis of LEAFY COTYLEDON1 in Isoetes L. from China

LEAFY COTYLEDON1 (LEC1) is a HAP3 subunit of CCAAT-binding transcription factor. The species in the genus Isoetes L. from China include I. hypsophila , I. yunguiensis , I. sinensis , I. orientalis , and I. taiwenensis . In this study, we initially gathered substantial samples from wide geographic ranges in China. Then we explored the evolutionary characteristics and expression profiles of LEC1 in Isoetes from China. We amplified 57 unique LEC1 nucleotide sequences from 94 individuals. The value of nucleotide diversity was 0.01693 for all samples, and neutrality tests indicated that LEC1 is under purifying negative selection in the evolutionary process. AMOVA analysis revealed that the genetic variation occurred mainly among populations within species. Moreover, the non-synonymous to synonymous substitution rates for 41 unique LEC1 protein sequences further demonstrated the presence of negative selection acting on the species from China to fix amino acid residues. Phylogenetic analyses of nucleotide and protein sequences showed consistently that the trees consisted of two well-supported main clades, and that the haplotypes from H42 to H57 in I. hypsophila were exclusive. In addition, expression profile analysis revealed that LEC1 transcripts accumulated primarily in reproductive organs. Looking at drought tolerance, LEC1 transcripts increased markedly at 24 h after drought treatment and reached a peak at 48 h. RNA accumulation reoccurred at relatively low levels and was slightly elevated at 48 h after rehydration. Altogether, our analyses provide insight into the evolutionary process of LEC1, and our results might promote further studies of functional genes in Isoetes .