Expression profiles of miRNAs in the genesis of Populus euphratica Oliv. heteromorphic leaves

The poplar species Populus euphratica Oliv. exhibits a typical heterophylly; lanceolate and broad-oval leaves grow on young and adult trees, respectively. It is therefore an ideal model species for studying leaf shape development. MicroRNAs (miRNAs) have important regulatory functions in plant growth and development, such as the developmental regulation of adaxial-abaxial polarity. To investigate the miRNAs involved in the development of the apical-basal and medial-lateral axes of P. euphratica leaves, an unfolded piece method and microscopy measuring techniques were employed to study the leaf shape variations between lanceolate and broad-oval leaves within buds. We found that the leaf shape of P. euphratica is determined between the first and the third or fifth macroscopic leaf during the germination period. The first three tender leaves of lanceolate and broad-oval leaf buds were obtained to construct six cDNA libraries from small RNAs. MicroRNAs were identified using high-throughput sequencing, and 165 novel miRNAs were found. By comparing the expression levels of the miRNAs between these two kinds of samples (lanceolate and broad-oval leaves), we found that nine miRNAs (six novel miRNAs and three conserved miRNAs) and their 113 target genes are involved in the development of the apical-basal and medial-lateral axes. The expression profiles of these miRNAs and their targets were further validated by quantitative reverse transcription polymerase chain reaction. Moreover, functional information on the targets was annotated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases.