Fu-Hui Wu

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

BackgroundOncidium 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.ResultsThe complete chloroplast genome of the economically important Oncidium variety Onc. Gower Ramsey (Accession no. GQ324949) was determined using a polymerase chain reaction (PCR) and Sanger based ABI sequencing. The length of the Oncidium chloroplast genome is 146,484 bp. Genome structure, gene order and orientation are similar to Phalaenopsis, but differ from typical Poaceae, other monocots for which there are several published chloroplast (cp) genome. The Onc. Gower Ramsey chloroplast-encoded NADH dehydrogenase (ndh) genes, except ndhE, lack apparent functions. Deletion and other types of mutations were also found in the ndh genes of 15 other economically important Oncidiinae varieties, except ndhE 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 Onc. 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 (trnHGUG -psbA and trnFGAA-ndhJ) was adequate for correct phylogenetic placement of 13 of the 15 varieties (with the exception of Degarmoara Flying High and Odontoglossum Violetta von Holm). Thus the chloroplast genome provides a useful molecular marker for species identifications.ConclusionIn this report, we used Phalaenopsis. aphrodite as a prototype for primer design to complete the Onc. Gower Ramsey genome sequence. Gene annotation showed that most of the ndh genes inOncidiinae, with the exception of ndhE, 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 trnHGUG-psbA and the trnFGAA-ndhJ regions. We conclude that complete chloroplast genome information is useful for plant phylogenetic and evolutionary studies in Oncidium with applications for breeding and variety identification.

Complete nucleotide sequence of Dendrocalamus latiflorus and Bambusa oldhamii chloroplast genomes.

Although bamboo is one of the most important woody crops in Asia, information on its genome is still very limited. To investigate the relationship among Poaceae members and to understand the mechanism of albino mutant generation in vitro, the complete chloroplast genome of two economically important bamboo species, Dendrocalamus latiflorus Munro and Bambusa oldhamii Munro, was determined employing a strategy that involved polymerase chain reaction (PCR) amplification using 443 novel primers designed to amplify the chloroplast genome of these two species. The lengths of the B. oldhamii and D. latiflorus chloroplast genomes are 139,350 and 139,365 bp, respectively. The organization structure and the gene order of these two bamboos are identical to other members of Poaceae. Highly conserved chloroplast genomes of Poaceae facilitated sequencing by the PCR method. Phylogenetic analysis using both chloroplast genomes confirmed the results obtained from studies on chromosome number and reproductive organ morphology. There are 23 gaps, insertions/deletions > 100 bp, in the chloroplast genomes of 10 genera of Poaceae compared in this study. The phylogenetic distribution of these gaps corresponds to their taxonomic placement. The sequences of these two chloroplast genomes provide useful information for studying bamboo evolution, ecology and biotechnology.

Screening a cDNA Library for Protein–Protein Interactions Directly in Planta

This article presents a method to screen a plant cDNA library for genes encoding proteins that interact with a bait protein directly in plants using bimolecular fluorescence complementation technology. Proof-of-concept experiments identified both known and novel Arabidopsis thaliana proteins important for Agrobacterium-mediated plant transformation. Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein– or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ∼2 × 105 cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.

Analysis of the expression of BohLOL1, which encodes an LSD1-like zinc finger protein in Bambusa oldhamii.

A cDNA, BohLOL1, encoding a protein containing three zf-LSD1 (zinc finger-Lesions Simulating Disease resistance 1) domains was cloned from growing bamboo (Bambusa oldhamii) shoots. A phylogenetic analysis revealed that BohLOL1 is a homolog of Arabidopsis LSD1 and LOL1 (LSD-one-like 1), which have been reported to act antagonistically in controlling cell death via the maintenance of reactive oxygen species homeostasis. The BohLOL1 gene was differentially expressed in various bamboo shoot tissues and was upregulated in shoots with higher rates of culm elongation. The expression level of this gene in multiple shoots of bamboo, which were cultured in vitro, was also upregulated by auxins, cytokinins, pathogen infection, 2,6-dichloroisonicotinic acid (a functional analog of salicylic acid), and hydrogen peroxide. The results suggest that BohLOL1 participates in bamboo growth and in the response to biotic stress. The DNA-binding assays and subcellular localization studies demonstrated that BohLOL1 is a nuclear DNA-binding protein. BohLOL1 might function through protein-DNA interactions and thus affect the expression of its target genes. The results of this study extend the role of plant LSD1 and LOL1 proteins from the regulation of cell death to cell growth. The growth-dependent up-regulation of BohLOL1 expression, which uniquely occurs in growing bamboo, might be one of the critical factors that contribute to the rapid growth of this remarkable plant.

Establishment of a cDNA library from Bambusa edulis Murno in vitro-grown shoots

An expressed sequence tag (EST) analysis approach was undertaken to identify the genes involved in photosynthesis and metabolism of Bambusa edulis Murno, an important bamboo crop species in Asia. The cDNA library was constructed from in vitro-grown multiple shoots. Sequencing of the cDNA clones generated 987 5′-end high-quality expressed sequence tags (ESTs, Accession number: FG551848–FG552834), of which 792 were revealed by sequence comparison to be unique. A BLASTX analysis showed that 645 of these genes were similar to genes present in the NCBI database. A total of 389 genes were most similar to rice genes. Nine genes had an E-value of zero. Functional classification of these genes by the Munich Information Center for Protein Sequences (MIPS) showed that most of the genes were related to metabolism and subcellular localization processes. PSI assembly protein (Beycf3) expression was absent in the albino mutant, while PSII 43-kDa protein (BepsbC), chlorophyll synthase (BeCAO) and PSII 10-kDa protein (BePsbR) expression were reduced. These data suggest that in this chloroplast genome-aberrated mutant, both chloroplast-encoded and nucleus-encoded genes were repressed and that this library will be useful for further investigation of bamboo.

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration

Summary Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.