Sequencing-Based Approaches Reveal Low Ambient Temperature-Responsive and Tissue-Specific MicroRNAs in Phalaenopsis Orchid

Plant small RNAs (smRNAs) are short, non-coding RNA molecules that mediate RNA silencing and regulate a group of genes involved in plant development and responses to environmental stimuli. Low temperature is necessary to initiate stalk development in the orchid Phalaenopsis aphrodite subsp. formosana. To identify smRNAs in Phalaenopsis responding to low temperatures, a smRNA profiling analysis using high-throughput sequencing technology was performed. Subsequent bioinformatic analysis was applied to categorize the miRNAs identified. A total of 37,533,509 smRNA reads yielded 11,129 independent orchid miRNA sequences, representing 329 known miRNA families identified in other plant species. Because the genomic resources available for Phalaenopsis are limited, a transcriptomic database was established using deep sequencing data sets to identify miRNAs precursors and their target transcripts. Comparing small RNAs and the transcriptomic database, 14 putative miRNA precursors of 10 miRNA families were identified, as were hundreds of putative targets. Comparing sequencing data and smRNA northern hybridization results identified miR156, miR162, miR528 and miR535 as low temperature-induced miRNAs. In addition, tissue-specific expression of these miRNAs was investigated. It was concluded that miR156 and miR172 may be components of a regulatory pathway mediating transition from the vegetative to the reproductive phase in Phalaenopsis. The smRNA and transcriptomic databases could be the foundations for further research aimed at elucidating the control of the flowering time in orchids

Complete chloroplast genome of Oncidium Gower Ramsey and evaluation of molecular markers for identification and breeding in Oncidiinae

<p>Abstract</p> <p>Background</p> <p><it>Oncidium </it>spp. produce commercially important orchid cut flowers. However, they are amenable to intergeneric and inter-specific crossing making phylogenetic identification very difficult. Molecular markers derived from the chloroplast genome can provide useful tools for phylogenetic resolution.</p> <p>Results</p> <p>The complete chloroplast genome of the economically important <it>Oncidium </it>variety <it>Onc</it>. Gower Ramsey (Accession no. GQ324949) was determined using a polymerase chain reaction (PCR) and Sanger based ABI sequencing. The length of the <it>Oncidium </it>chloroplast genome is 146,484 bp. Genome structure, gene order and orientation are similar to <it>Phalaenopsis</it>, but differ from typical Poaceae, other monocots for which there are several published chloroplast (cp) genome. The <it>Onc</it>. Gower Ramsey chloroplast-encoded <it>NADH dehydrogenase </it>(<it>ndh</it>) genes, except <it>ndhE</it>, lack apparent functions. Deletion and other types of mutations were also found in the <it>ndh </it>genes of 15 other economically important Oncidiinae varieties, except <it>ndhE </it>in some species. The positions of some species in the evolution and taxonomy of Oncidiinae are difficult to identify. To identify the relationships between the 15 Oncidiinae hybrids, eight regions of the <it>Onc</it>. Gower Ramsey chloroplast genome were amplified by PCR for phylogenetic analysis. A total of 7042 bp derived from the eight regions could identify the relationships at the species level, which were supported by high bootstrap values. One particular 1846 bp region, derived from two PCR products (<it>trnH</it>^GUG-<it>psbA </it>and <it>trnF</it>^GAA-<it>ndhJ</it>) was adequate for correct phylogenetic placement of 13 of the 15 varieties (with the exception of <it>Degarmoara </it>Flying High and <it>Odontoglossum </it>Violetta von Holm). Thus the chloroplast genome provides a useful molecular marker for species identifications.</p> <p>Conclusion</p> <p>In this report, we used <it>Phalaenopsis. aphrodite </it>as a prototype for primer design to complete the <it>Onc</it>. Gower Ramsey genome sequence. Gene annotation showed that most of the <it>ndh </it>genes inOncidiinae, with the exception of <it>ndhE</it>, are non-functional. This phenomenon was observed in all of the Oncidiinae species tested. The genes and chloroplast DNA regions that would be the most useful for phylogenetic analysis were determined to be the <it>trnH</it>^GUG-<it>psbA </it>and the <it>trnF</it>^GAA-<it>ndhJ </it>regions. We conclude that complete chloroplast genome information is useful for plant phylogenetic and evolutionary studies in <it>Oncidium </it>with applications for breeding and variety identification.</p

Altered Gene Regulatory Networks Are Associated With the Transition From C3 to Crassulacean Acid Metabolism in Erycina (Oncidiinae: Orchidaceae)

Crassulacean acid metabolism (CAM) photosynthesis is a modification of the core C3 photosynthetic pathway that improves the ability of plants to assimilate carbon in water-limited environments. CAM plants fix CO2 mostly at night, when transpiration rates are low. All of the CAM pathway genes exist in ancestral C3 species, but the timing and magnitude of expression are greatly altered between C3 and CAM species. Understanding these regulatory changes is key to elucidating the mechanism by which CAM evolved from C3. Here, we use two closely related species in the Orchidaceae, Erycina pusilla (CAM) and Erycina crista-galli (C3), to conduct comparative transcriptomic analyses across multiple time points. Clustering of genes with expression variation across the diel cycle revealed some canonical CAM pathway genes similarly expressed in both species, regardless of photosynthetic pathway. However, gene network construction indicated that 149 gene families had significant differences in network connectivity and were further explored for these functional enrichments. Genes involved in light sensing and ABA signaling were some of the most differently connected genes between the C3 and CAM Erycina species, in agreement with the contrasting diel patterns of stomatal conductance in C3 and CAM plants. Our results suggest changes to transcriptional cascades are important for the transition from C3 to CAM photosynthesis in Erycina

NDH Expression Marks Major Transitions in Plant Evolution and Reveals Coordinate Intracellular Gene Loss

Key innovations have facilitated novel niche utilization, such as the movement of the algal predecessors of land plants into terrestrial habitats where drastic fluctuations in light intensity, ultraviolet radiation and water limitation required a number of adaptations. The NDH (NADH dehydrogenase-like) complex of Viridiplantae plastids participates in adapting the photosynthetic response to environmental stress, suggesting its involvement in the transition to terrestrial habitats. Although relatively rare, the loss or pseudogenization of plastid NDH genes is widely distributed across diverse lineages of photoautotrophic seed plants and mutants/transgenics lacking NDH function demonstrate little difference from wild type under non-stressed conditions. This study analyzes large transcriptomic and genomic datasets to evaluate the persistence and loss of NDH expression across plants. Results: Nuclear expression profiles showed accretion of the NDH gene complement at key transitions in land plant evolution, such as the transition to land and at the base of the angiosperm lineage. While detection of transcripts for a selection of non-NDH, photosynthesis related proteins was independent of the state of NDH, coordinate, lineage-specific loss of plastid NDH genes and expression of nuclear-encoded NDH subunits was documented in Pinaceae, gnetophytes, Orchidaceae and Geraniales confirming the independent and complete loss of NDH in these diverse seed plant taxa. Conclusion: The broad phylogenetic distribution of NDH loss and the subtle phenotypes of mutants suggest that the NDH complex is of limited biological significance in contemporary plants. While NDH activity appears dispensable under favorable conditions, there were likely sufficiently frequent episodes of abiotic stress affecting terrestrial habitats to allow the retention of NDH activity. These findings reveal genetic factors influencing plant/environment interactions in a changing climate through 450 million years of land plant evolution.National Science Foundation IOS-1027259Innovative Translational Agricultural Research Administrative OfficeIntegrative Biolog

Complete Chloroplast Genome Sequence of an Orchid Model Plant Candidate: Erycina pusilla Apply in Tropical Oncidium Breeding

Oncidium is an important ornamental plant but the study of its functional genomics is difficult. Erycina pusilla is a fast-growing Oncidiinae species. Several characteristics including low chromosome number, small genome size, short growth period, and its ability to complete its life cycle in vitro make E. pusilla a good model candidate and parent for hybridization for orchids. Although genetic information remains limited, systematic molecular analysis of its chloroplast genome might provide useful genetic information. By combining bacterial artificial chromosome (BAC) clones and next-generation sequencing (NGS), the chloroplast (cp) genome of E. pusilla was sequenced accurately, efficiently and economically. The cp genome of E. pusilla shares 89 and 84% similarity with Oncidium Gower Ramsey and Phalanopsis aphrodite, respectively. Comparing these 3 cp genomes, 5 regions have been identified as showing diversity. Using PCR analysis of 19 species belonging to the Epidendroideae subfamily, a conserved deletion was found in the rps15-trnN region of the Cymbidieae tribe. Because commercial Oncidium varieties in Taiwan are limited, identification of potential parents using molecular breeding method has become very important. To demonstrate the relationship between taxonomic position and hybrid compatibility of E. pusilla, 4 DNA regions of 36 tropically adapted Oncidiinae varieties have been analyzed. The results indicated that trnF-ndhJ and trnH-psbA were suitable for phylogenetic analysis. E. pusilla proved to be phylogenetically closer to Rodriguezia and Tolumnia than Oncidium, despite its similar floral appearance to Oncidium. These results indicate the hybrid compatibility of E. pusilla, its cp genome providing important information for Oncidium breeding

Evaluation of abiotic stress tolerance and physiological characteristics of potato (Solanum tuberosum L. cv. Kennebec) that heterologously expresses the rice Osmyb4 gene

MYB transcription factors are involved in diverse biochemical and physiological processes, including hormone signaling, defense, and stress responses. In the present study, we developed a transgenic potato (Solanum tuberosum L. cv. Kennebec) expressing the rice Osmyb4 gene, which encodes the transcription factor MYB4. The transgene was under the control of either the constitutive CaMV35S promoter or the stress-induced Arabidopsis COR15a promoter. The potential involvement of MYB4 in certain physiological processes and the abiotic stress response in the potato was evaluated. The transgenic plants did not exhibit growth retardation, and they showed no significant difference (P < 0.05) in tuber yield from that of non-transgenic wild-type plants. Although the chlorophyll a and b as well as the anthocyanin contents of the six transgenic lines were similar to those of the wild type, the transgenic line S2 presented a significantly higher carotenoid content. The total sugar contents of the lines S2 and M48 were significantly higher than that of the wild-type plants. S2 and M48 were significantly more tolerant of salinity than the wild type, according to measured growth parameters. Transgenic plants grown under a high concentration of boric acid (3 mM) exhibited greater survival rates than non-transgenic control plants. On the other hand, the transgenic plants did not show an improvement in freezing tolerance. Overall, our results indicated that MYB4 may affect diverse processes such as carotenoid biosynthesis, sugar metabolism, and salinity tolerance in potato, and that it may be an upstream regulatory element of these processes

The Application Of The Chloroplast Genome Of Oncidium In Plant Identification And Breeding In Oncidiinae

Oncidium spp. is an important orchid species among cut flowers. However, determining their phylogenetic relationships and identifying varieties are very difficult. Molecular markers can provide useful tools for phylogenetic resolution, the chloroplast being one resource for those markers. The complete chloroplast genome of Onc. Gower Ramsey (Accession no. GQ324949) was determined. The size of the Oncidium chloroplast genome is 146,484 bp. Interesting, Onc. Gower Ramsey chloroplast encoded NADH dehydrogenase (ndh) genes, except ndhE, have no functions. Based on this information, eight regions of the Onc. Gower Ramsey chloroplast genome were amplified by polymerase chain reaction for phylogenetic analysis to identify the relationships between the 15 Oncidiinae hybrids. The results confirm that the chloroplast genome provides a useful molecular marker for species identification

Biotechnology of ornamental plants: when beauty joins science—preface from the editors

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