Floral Transcriptome Analyses of Four Paphiopedilum Orchids with Distinct Flowering Behaviors and Development of Simple Sequence Repeat Markers

Paphiopedilum orchids are evergreen perennials with distinct flowering behaviors; however, little genetic information hindered the understanding of these interesting flowering patterns. In this study, the floral transcriptomes of two Paphiopedilum species, Paphiopedilum concolor and Paphiopedilum hirsutissimum, and two popular cultivars were characterized. A total of 42.4–68.5 million clean reads were generated from these four Paphiopedilum orchid floral buds. De novo assembly yielded 73,201–79,086 unigenes with average lengths of 765–848 bp for these four floral transcriptomes, of which 42.5–43.1 % were annotated in public protein databases. With 8,019 unique protein-coding genes shared, similar function classifications were found among the four orchid floral transcriptomes, and 113 Paphiopedilum floral unigenes were found to be homologous to flowering genes in Arabidopsis. Of these flowering genes, many regulatory genes, namely, MIKC, NAC, MYB, WRKY, bZIP, bHLH transcription factors, and protein kinases and plant hormone signaling-related genes were identified. Notably, one gene, PIF4 may play vital roles for regulation flowering in warm seasons. A phylogeny clustering based on MIKC MADS-box gene family among Paphiopedilum, rice, and Arabidopsis classified the 61 Paphiopedilum members into 11 subfamilies, which provided some clues on floral organ specification and development in Paphiopedilum. Differentiation expression analysis among the four floral transcriptomes demonstrated that complicated molecular functions or biological processes, such as transmembrane transport, transcription regulation, signal transduction, defense response, etc., might be responsible for the differences among these orchid floral development processes, and partial of these expression genes were validated by qRT-PCR. Finally, 20,969 EST-SSRs were identified as potential molecular markers, and 15,137 SSRs were successful for primer designing. Of 62 candidate SSR primer pairs, 51 were successfully amplified with expected sizes and exhibited polymorphisms among 14 genotypes of Paphiopedilum orchids. Our data should not only provide valuable sequence resources for novel gene discovery and marker-assisted studies in Paphiopedilum but also give us some new insights into understanding of the molecular mechanisms of flowering and floral development in Paphiopedilum.