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ABSTRACT: These studies were designed to test if a binary vector containing the gfp, bar and oxalate oxidase genes could transform American chestnut somatic embryos; to see if a desiccation treatment during co-cultivation would affect the transformation frequency of different American chestnut somatic embryo clones; to explore the effects of more rapid desiccation; and to see if the antibiotics used to kill the Agrobacterium were interfering with the regeneration of the somatic embryos. Two days of gradual desiccation was found to significantly enhance transient GFP expression frequency. When this treatment was tested on six American chestnut clones, five were transformed and four of these remained embryogenic. Transformation was confirmed by Southern hybridization. Phenotypically normal transgenic shoots were regenerated and rooted. Vascular tissue specific expression of the oxalate oxidase gene was detected in one transgenic line. Carbenicillin, cefotaxime, and tricarcillin were found to not interfere with the regeneration of transformed embryos.
Plant Cell Tissue and Organ Culture 04/2012; 84(1):69-79. · 3.09 Impact Factor
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ABSTRACT: FLORICAULA/LEAFY (FLO/LFY) plays an important role in the reproductive transition and controls flower spatial patterning by inducing the expression
of the ABC floral organ identity genes. In this study, we sequenced two bacterial artificial chromosomes harboring a FLO/LFY and three other genes from yellow-poplar (Liriodendron tulipifera L.) and compared the gene order in this locus between several species. Besides the conserved terminal domains, key residues
involved in interactions with DNA bases, backbone, and in dimerization were also conserved in the yellow-poplar FLO/LFY. Phylogenetic
analysis of the FLO/LFY amino acid sequences placed yellow-poplar closer to eudicots than to monocotyledonous species. We
found that gene content and order in this region of the yellow-poplar genome was more similar to corresponding regions in
Vitis vinifera L., Carica papaya L., Populus trichocarpa Torr. & Gray, and Ricinus communis L., regardless of the evolutionary relationship. In addition, evidence for transposition, large insertions, and duplications
were found, suggesting multiple and complex mechanisms of basal angiosperm genome evolution.
KeywordsMicrosynteny–Gene content–Gene organization–Genome evolution–
Liriodendron
–Yellow-poplar
Tree Genetics & Genomes 04/2012; 7(2):373-384. · 2.34 Impact Factor
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Haiying Liang,
Saravanaraj Ayyampalayam,
Norman Wickett,
Abdelali Barakat,
Yi Xu,
Lena Landherr,
Paula E. Ralph,
Yuannian Jiao,
Tao Xu,
Scott E. Schlarbaum,
Hong Ma,
James H. Leebens-Mack,
Claude W. dePamphilis
[show abstract]
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ABSTRACT: Liriodendron tulipifera L., a member of Magnoliaceae in the order Magnoliales, has been used extensively as a reference species in studies on plant
evolution. However, genomic resources for this tree species are limited. We constructed cDNA libraries from ten different
types of tissues: premeiotic flower buds, postmeiotic flower buds, open flowers, developing fruit, terminal buds, leaves,
cambium, xylem, roots, and seedlings. EST sequences were generated either by 454 GS FLX or Sanger methods. Assembly of almost
2.4 million sequencing reads from all libraries resulted in 137,923 unigenes (132,905 contigs and 4,599 singletons). About
50% of the unigenes had significant matches to publically available plant protein sequences, representing a wide variety of
putative functions. Approximately 30,000 simple sequence repeats were identified. More than 97% of the cell wall formation
genes in the Cell Wall Navigator and the MAIZEWALL databases are represented. The cinnamyl alcohol dehydrogenase (CAD) homologs identified in the L. tulipifera EST dataset showed different expression levels in the ten tissue types included in this study. In particular, the LtuCAD1 was found to partially recover the stiffness of the floral stems in the Arabidopsis thaliana CAD4 and CAD5 double mutant plants, of the LtuCAD1 in lignin biosynthesis. L. tulipifera genes have greater sequence similarity to homologs from other woody angiosperm species than to non-woody model plants. This
large-scale genomic resour"HistryDatesce will be instrumental for gene discovery, cDNA microarray production, and marker-assisted
breeding in L. tulipifera, and strengthen this species' role in comparative studies.
KeywordsEST database–Xylogenesis–
Liriodendron
–Yellow-poplar–Magnoliaceae
Tree Genetics & Genomes 04/2012; 7(5):941-954. · 2.34 Impact Factor
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Haiying Liang,
Eric G. Fang,
Jeffrey P. Tomkins,
Meizhong Luo,
David Kudrna,
Hye Ran Kim,
K. Arumuganathan,
Shaying Zhao,
James Leebens-Mack,
Scott E. Schlarbaum,
Jo Ann Banks,
Claude W. dePamphilis,
Dina F. Mandoli,
Rod A. Wing,
John E. Carlson
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ABSTRACT: Liriodendron tulipifera L., a member of the Magnoliaceae, occupies an important phylogenetic position as a basal angiosperm that has retained numerous
putatively ancestral morphological characters, and thus has often been used in studies of the evolution of flowering plants
and of specific gene families. However, genomic resources for these early branching angiosperm lineages are very limited.
In this study, we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from L. tulipifera. Flow cytometry estimates that this nuclear genome is approximately 1,802Mbp per haploid genome (±16 SD). The BAC library
contains 73,728 clones, a 4.8-fold genome coverage, with an average insert size of 117kb, a chloroplast DNA content of 0.2%,
and little to no bacterial sequences nor empty vector content clones. As a test of the utility of this BAC library, we screened
the library with six single/low-copy genic probes. We obtained at least two positive clones for each gene and confirmed the
clones by DNA sequencing. A total of 182 paired end sequences were obtained from 96 of the BAC clones. Using BLAST searches,
we found that 25% of the BAC end sequences were similar to DNA sequences in GenBank. Of these, 68% shared sequence with transposable
elements and 25% with genes from other taxa. This result closely reflected the content of random sequences obtained from a
small insert genomic library for L. tulipifera, indicating that the BAC library construction process was not biased. The first genomic DNA sequences for Liriodendron genes are also reported. All the Liriodendron genomic sequences described in this paper have been deposited in the GenBank data library. The end sequences from shotgun
genomic clones and BAC clones are under accession DU169330–DU169684. Partial sequences of Gigantea, Frigida, LEAFY, cinnamyl alcohol dehydrogenase, 4-coumarate:CoA ligase, and phenylalanine ammonia-lyase genes are under accession DQ223429–DQ223434.
Tree Genetics & Genomes 04/2012; 3(3):215-225. · 2.34 Impact Factor
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Haiying Liang,
John E. Carlson,
James H. Leebens-Mack,
P. Kerr Wall,
Lukas A. Mueller,
Matyas Buzgo,
Lena L. Landherr,
Yi Hu,
D. Scott DiLoreto,
Daniel C. Ilut,
Dawn Field,
Steven D. Tanksley,
Hong Ma,
Claude W. dePamphilis
[show abstract]
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ABSTRACT: Liriodendron tulipifera L. was selected by the Floral Genome Project for identification of new genes related to floral diversity in basal angiosperms.
A large, non-normalized cDNA library was constructed from premeiotic and meiotic floral buds and sequenced to generate a database
of 9,531 high-quality expressed sequence tags. These sequences clustered into 6,520 unigenes, of which 5,251 were singletons,
and 1,269 were in contigs. Homologs of genes regulating many aspects of flower development were identified, including those
for organ identity and development, cell and tissue differentiation, and cell-cycle control. Almost 5% of the transcriptome
consisted of homologs to known floral gene families. Homologs of most of the genes involved in cell-wall construction were
also recovered. This provides a new opportunity for comparative studies in lignin biosynthesis, a trait of key importance
in the evolution of land plants and in the utilization of fiber from economically important tree species, such as Liriodendron. Also of note is that 1,089 unigenes did not match any sequence in the public databases, including the complete genomes of
Arabidopsis, rice, and Populus. Some of these novel genes might be unique in basal angiosperm species and, when better characterized, may be informative
for understanding the origins of diverged gene families. Thus, the Liriodendron expressed sequence tag database and library will help bridge our understanding of the mechanisms of flower initiation and
development that are shared among basal angiosperms, eudicots, and monocots, and provide new opportunities for comparative
analysis of gene families across angiosperm species.
Tree Genetics & Genomes 04/2012; 4(3):419-433. · 2.34 Impact Factor
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Yuannian Jiao,
Norman J Wickett,
Saravanaraj Ayyampalayam,
André S Chanderbali,
Lena Landherr,
Paula E Ralph,
Lynn P Tomsho,
Yi Hu, Haiying Liang,
Pamela S Soltis,
Douglas E Soltis,
Sandra W Clifton,
Scott E Schlarbaum,
Stephan C Schuster,
Hong Ma,
Jim Leebens-Mack,
Claude W dePamphilis
[show abstract]
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ABSTRACT: Whole-genome duplication (WGD), or polyploidy, followed by gene loss and diploidization has long been recognized as an important evolutionary force in animals, fungi and other organisms, especially plants. The success of angiosperms has been attributed, in part, to innovations associated with gene or whole-genome duplications, but evidence for proposed ancient genome duplications pre-dating the divergence of monocots and eudicots remains equivocal in analyses of conserved gene order. Here we use comprehensive phylogenomic analyses of sequenced plant genomes and more than 12.6 million new expressed-sequence-tag sequences from phylogenetically pivotal lineages to elucidate two groups of ancient gene duplications-one in the common ancestor of extant seed plants and the other in the common ancestor of extant angiosperms. Gene duplication events were intensely concentrated around 319 and 192 million years ago, implicating two WGDs in ancestral lineages shortly before the diversification of extant seed plants and extant angiosperms, respectively. Significantly, these ancestral WGDs resulted in the diversification of regulatory genes important to seed and flower development, suggesting that they were involved in major innovations that ultimately contributed to the rise and eventual dominance of seed plants and angiosperms.
Nature 05/2011; 473(7345):97-100. · 36.28 Impact Factor
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ABSTRACT: GIGANTEA plays an important role in the control of circadian rhythms and photoperiodic flowering. The GIGANTEA gene has been studied in various species, but not in basal angiosperms. Moreover, to the best of our knowledge, no study of the genome organization of a basal angiosperm has yet been published. In this study, we sequenced a bacterial artificial chromosome (BAC) harboring GIGANTEA from yellow-poplar (Liriodendron tulipifera L.) and compared the genomic organization of this gene in yellow-poplar with that in other species from various angiosperm clades. This is the first report on the gene structure and organization of a large contig in any basal angiosperm species. The BAC clone, covering a region of approximately 122 kb from the yellow-poplar genome, was sequenced and assembled by coupling the 454 pyrosequencing technology with ABI capillary sequencing. In addition to GIGANTEA, the gene RPS18.A (encoding ribosomal protein S18.A) was found in this segment of the genome. We found that gene content and order in this region of the yellow-poplar genome were similar to those in the corresponding region in eudicots but not in Oryza sativa and Sorghum bicolor, implying that clustering of the GIGANTEA and RPS18.A genes is ancestral and separation of the genes occurred after the phylogenetic split of monocots from dicots. Phylogenetic analysis of GIGANTEA amino acid sequences placed yellow-poplar closer to eudicots than to monocots. In addition, evidence for transposition and large insertions and duplications was found, suggesting multiple and complex mechanisms of basal angiosperm genome evolution.
Genome 07/2010; 53(7):533-44. · 1.65 Impact Factor
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P Kerr Wall,
Jim Leebens-Mack,
André S Chanderbali,
Abdelali Barakat,
Erik Wolcott, Haiying Liang,
Lena Landherr,
Lynn P Tomsho,
Yi Hu,
John E Carlson,
Hong Ma,
Stephan C Schuster,
Douglas E Soltis,
Pamela S Soltis,
Naomi Altman,
Claude W dePamphilis
[show abstract]
[hide abstract]
ABSTRACT: We have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG) ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19). We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica) and the magnoliid avocado (Persea americana) using a variety of methods for cDNA synthesis.
The Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB), 119,518 (88.7%) mapped exactly to known exons, while 1,117 (0.8%) mapped to introns, 11,524 (8.6%) spanned annotated intron/exon boundaries, and 3,066 (2.3%) extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics.
NG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents significant challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the absence of physical clones. These problems may be overcome by hybrid sequencing strategies using a mixture of sequencing methodologies, by new assemblers, and by sequencing more deeply. Sequencing and microarray outcomes from multiple experiments suggest that our simulator will be useful for guiding NG transcriptome sequencing projects in a wide range of organisms.
BMC Genomics 09/2009; 10:347. · 4.07 Impact Factor
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ABSTRACT: The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm.
Plant Cell Reports 08/2007; 26(7):977-87. · 2.27 Impact Factor
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ABSTRACT: A gene cassette, p35S-CNO, was designed to express three gene products driven by a single constitutive CaMV 35S promoter. The individual coding regions were linked in frame to produce a single polyprotein, using spacer sequences encoding a specific heptapeptide cleavage recognition site (ENLYFQS) for the nuclear-inclusion-a (NIa) proteinase of tobacco etch virus (TEV). The protein coding sequences used were: a Trichoderma harzinum endochitinase, a truncated NIa proteinase of TEV, and a wheat oxalate oxidase. When p35S-CNO construct was tested in Arabidopsis thaliana, the polyprotein was properly cleaved after translation and the products exhibited functional enzymatic activity in vivo.
Biotechnology Letters 04/2005; 27(6):435-42. · 1.68 Impact Factor
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ABSTRACT: Plasmids, pCA1 and pCWEA1, carrying antimicrobial peptide gene(s), Ac-AMP1.2 and ESF12, were used to transform hybrid poplar clones Ogy and NM6. Peptide Ac-AMP1.2 is an analog of Ac-AMP1 which is one of the smallest chitin-binding proteins. Synthetic peptide ESF12 mimics the amphipathic -helix found in magainins. Transgene mRNA was detected in the transformed plants. When evaluated for resistance to hybrid poplar pathogen Septoria musiva with an invitro leaf disk assay, the transformed Ogy plants showed significantly increased pathogen resistance as compared to the untransformed Ogy.
Biotechnology Letters 02/2002; 24(5):383-389. · 1.68 Impact Factor
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ABSTRACT: A cDNA clone of a wheat germin-like oxalate oxidase (OxO) gene regulated by the constitutive CaMV35S promoter was expressed in a hybrid poplar clone, Populus euramericana (`Ogy'). Previous studies showed that OxO is likely to play an important role in several aspects of plant development, stress response, and defense against pathogens. In order to study this wheat oxalate oxidase gene in woody plants, the expression of this gene and the functions of the encoded enzyme were examined in vitro and invivo in transgenic `Ogy'. The enzyme activity in the transformed `Ogy' was visualized by histochemical assays and in SDS-polyacrylamide gels. It was found that the wheat OxO gene is expressed in leaves, stems, and roots of the transgenic `Ogy' plants and the encoded enzyme is able to break down oxalic acid. Transgenic `Ogy' leaves were more tolerant to oxalic acid as well as more effective in increasing the pH in an oxalic acid solution when compared to untransformed controls. In addition, when leaf disks from `Ogy' plants were inoculated with conidia of the poplar pathogenic fungus Septoria musiva, which produces oxalic acid, the OxO-transformed plants were more resistant than the untransformed controls.
Plant Molecular Biology 03/2001; 45(6):619-629. · 4.15 Impact Factor
