Catalog of Erycina pusilla miRNA and categorization of reproductive phase-related miRNAs and their target gene families

Abstract

The orchid Erycina pusilla has a short life cycle and relatively low chromosome number, making it a potential model plant for orchid functional genomics. To that end, small RNAs (sRNAs) from different developmental stages of different organs were sequenced. In this miRNA mix, 33 annotated miRNA families and 110 putative miRNA-targeted transcripts were identified in E. pusilla. Fifteen E. pusilla miRNA target genes were found to be similar to those in other species. There were putative novel miRNAs identified by 3 different strategies. The genomic sequences of the four miRNAs that were identified using rice genome as the reference can form the stem loop structure. The t0000354 miRNA, identified using rice genome sequences and a Phalaenopsis study, had a high read count. The target gene of this miRNA is MADS (unigene30603), which belongs to the AP3-PI subfamily. The most abundant miRNA was E. pusilla miR156 (epu-miR156), orthologs of which work to maintain the vegetative phase by repressing the expression of the SQUAMOSA promoter-binding-like (SPL) transcription factors. Fifteen genes in the E. pusilla SPL (EpSPL) family were identified, nine of which contained the putative epu-miR156 target site. Target genes of epu-miR172, also a key regulator of developmental changes in the APETALA2 (EpAP2) family, were identified. Experiments using 5'RLM-RACE demonstrated that the genes EpSPL1, 2, 3, 4, 7, 9, 10, 14 and EpAP2-9, -10, -11 were regulated by epu-miR156 and epu-miR172, respectively.

The Analysis of the Inflorescence miRNome of the Orchid Orchis italica Reveals a DEF-Like MADS-Box Gene as a New miRNA Target

“…Both these miRNAs are homologs of miR5179 of Oryza sativa and Brachypodium distachyon whose function is unknown. Recently, an orchid-specific miRNA that putatively cleaves the transcripts of class B MADS-box genes was identified in E. pusilla and P. aphrodite , without any further characterization [11]. Using a modified 5′ RACE protocol, we successfully amplified a fragment of the expected size (129 bp) and sequence representing the cleavage product of miR5179 on the Oita DEF2 transcript of O. italica (Figure 5), thus confirming that this class B MADS-box gene is a target of the homolog of miR5179 in O. italica .…”

“…Recently, the high-throughput sequencing approach has been applied to the study of miRNAs in two orchid species, Phalaenopsis aphrodite and Erycina pusilla , both belonging to the sub-family Epidendroideae (Orchidaceae) [10], [11]. Conserved and novel miRNAs have been identified in both species, and some differences between the two species have been detected.…”

A putative miR172-targeted CeAPETALA2-like gene is involved in floral patterning regulation of the orchid Cymbidium ensifolium

“…Hundreds of miRNA families have been identified as playing crucial roles in many developmental and physiological processes in different plant species (Jones-Rhoades and Bartel, 2004;Zhang et al, 2006). However, despite reports on small RNA sequencing in orchid, no previous studies have investigated the function of miRNAs in orchid species Lin et al, 2013).…”

Integrated mRNA and microRNA transcriptome variations in the multi-tepal mutant provide insights into the floral patterning of the orchid Cymbidium goeringii

“…Recently, a few studies have also examined miRNAs in orchids, which are characterized by highly diversified floral structures and pollination strategies. Examples such as Phalaenopsis aphrodite , Erycina pusilla and Dendrobium officinale belonging to the sub-family Epidendroideae have been sequenced [24–27]. Both conserved and novel miRNAs have been identified in these species.…”