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1.  Somatic embryogenesis, tetraploidy, and variant leaf morphology in transgenic diploid strawberry (Fragaria vesca subspecies vesca ‘Hawaii 4’) 
BMC Plant Biology  2014;14:23.
The diploid (2n = 2x = 14) strawberry model plant Fragaria vesca ssp. vesca ‘Hawaii 4’ was employed for functional analysis of expressed DNA sequences initially identified as being unique to Fragaria and of unknown or poorly understood function. ‘Hawaii 4’ is prominent in strawberry research due to its ease of Agrobacterium-mediated transformation and regenerability, and its status as the source of the first complete strawberry genomic sequence. Our studies of a set of transformants have documented intriguing, construct-associated effects on leaf morphology, and provide important and unexpected insights into the performance of the ‘Hawaii 4’ transformation and regeneration system.
Following Agrobacterium-mediated transformation of leaf explants with gene constructs carried by Gateway® vectors, plants were regenerated using a modified version of an established ‘Hawaii 4’ protocol. Expanding upon the findings of prior studies, we documented that plantlet regeneration was occurring via a somatic embryogenic rather than an organogenic developmental pathway. Among transformants, several variations in leaf morphology were observed. Unexpectedly, a particular leaf variant type, occurring in ~17% of all regenerants independent of construct type, was found to be attributable to tetraploidy. The tetraploidy-associated alteration in leaf morphology could be differentiated from the leaf morphology of diploid regenerants on the basis of a quantitative ratio of leaf dimensions: B/A, where B is the width of the central leaflet and A is the overall width of the trifoliate leaf. Variant effects on leaf morphology of four different transgenic constructs were also documented, and were in all cases distinguishable from the effects of tetraploidy.
These results define opportunities to optimize the existing ‘Hawaii 4’ protocol by focusing on treatments that specifically promote somatic embryogenesis. The reported morphological metric and descriptions will guide future transgenic studies using the ‘Hawaii 4’ model system by alerting researchers to the potential occurrence of polyploid regenerants, and to differentiating the effects on leaf morphology due to polyploidy versus transgenic manipulations. Finally, an intriguing spectrum of leaf morphology alterations resulting from manipulation of expressed sequences of uncertain function is documented, providing a foundation for detailed studies of the respective genes and their functional roles.
PMCID: PMC3898059  PMID: 24418064
Fragaria; Strawberry; Transgenes; Leaf morphology; Tetraploidy; Somatic embryogenesis
2.  Whole genome wide expression profiles of Vitis amurensis grape responding to downy mildew by using Solexa sequencing technology 
BMC Plant Biology  2010;10:234.
Downy mildew (DM), caused by pathogen Plasmopara viticola (PV) is the single most damaging disease of grapes (Vitis L.) worldwide. However, the mechanisms of the disease development in grapes are poorly understood. A method for estimating gene expression levels using Solexa sequencing of Type I restriction-endonuclease-generated cDNA fragments was used for deep sequencing the transcriptomes resulting from PV infected leaves of Vitis amurensis Rupr. cv. Zuoshan-1. Our goal is to identify genes that are involved in resistance to grape DM disease.
Approximately 8.5 million (M) 21-nt cDNA tags were sequenced in the cDNA library derived from PV pathogen-infected leaves, and about 7.5 M were sequenced from the cDNA library constructed from the control leaves. When annotated, a total of 15,249 putative genes were identified from the Solexa sequencing tags for the infection (INF) library and 14,549 for the control (CON) library. Comparative analysis between these two cDNA libraries showed about 0.9% of the unique tags increased by at least five-fold, and about 0.6% of the unique tags decreased more than five-fold in infected leaves, while 98.5% of the unique tags showed less than five-fold difference between the two samples. The expression levels of 12 differentially expressed genes were confirmed by Real-time RT-PCR and the trends observed agreed well with the Solexa expression profiles, although the degree of change was lower in amplitude. After pathway enrichment analysis, a set of significantly enriched pathways were identified for the differentially expressed genes (DEGs), which associated with ribosome structure, photosynthesis, amino acid and sugar metabolism.
This study presented a series of candidate genes and pathways that may contribute to DM resistance in grapes, and illustrated that the Solexa-based tag-sequencing approach was a powerful tool for gene expression comparison between control and treated samples.
PMCID: PMC3017854  PMID: 21029438
3.  An examination of targeted gene neighborhoods in strawberry 
BMC Plant Biology  2010;10:81.
Strawberry (Fragaria spp.) is the familiar name of a group of economically important crop plants and wild relatives that also represent an emerging system for the study of gene and genome evolution. Its small stature, rapid seed-to-seed cycle, transformability and miniscule basic genome make strawberry an attractive system to study processes related to plant physiology, development and crop production; yet it lacks substantial genomics-level resources. This report addresses this deficiency by characterizing 0.71 Mbp of gene space from a diploid species (F. vesca). The twenty large genomic tracks (30-52 kb) captured as fosmid inserts comprise gene regions with roles in flowering, disease resistance, and metabolism.
A detailed description of the studied regions reveals 131 Blastx-supported gene sites and eight additional EST-supported gene sites. Only 15 genes have complete EST coverage, enabling gene modelling, while 76 lack EST support. Instances of microcolinearity with Arabidopsis thaliana were identified in twelve inserts. A relatively high portion (25%) of targeted genes were found in unanticipated tandem duplications. The effectiveness of six FGENESH training models was assessed via comparisons among ab initio predictions and homology-based gene and start/stop codon identifications. Fourteen transposable-element-related sequences and 158 simple sequence repeat loci were delineated.
This report details the structure and content of targeted regions of the strawberry genome. The data indicate that the strawberry genome is gene-dense, with an average of one protein-encoding gene or pseudogene per 5.9 kb. Current overall EST coverage is sparse. The unexpected gene duplications and their differential patterns of EST support suggest possible subfunctionalization or pseudogenization of these sequences. This report provides a high-resolution depiction of targeted gene neighborhoods that will aid whole-genome sequence assembly, provide valuable tools for plant breeders and advance the understanding of strawberry genome evolution.
PMCID: PMC2890015  PMID: 20441596
4.  Rapid and accurate pyrosequencing of angiosperm plastid genomes 
BMC Plant Biology  2006;6:17.
Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20) System (454 Life Sciences Corporation), to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae) and Platanus occidentalis (Platanaceae).
More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions.
Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy observed in the GS 20 plastid genome sequence was generated for a significant reduction in time and cost over traditional shotgun-based genome sequencing techniques, although with approximately half the coverage of previously reported GS 20 de novo genome sequence. The GS 20 should be broadly applicable to angiosperm plastid genome sequencing, and therefore promises to expand the scale of plant genetic and phylogenetic research dramatically.
PMCID: PMC1564139  PMID: 16934154
5.  Design and fabrication of adjustable red-green-blue LED light arrays for plant research 
BMC Plant Biology  2005;5:17.
Although specific light attributes, such as color and fluence rate, influence plant growth and development, researchers generally cannot control the fine spectral conditions of artificial plant-growth environments. Plant growth chambers are typically outfitted with fluorescent and/or incandescent fixtures that provide a general spectrum that is accommodating to the human eye and not necessarily supportive to plant development. Many studies over the last several decades, primarily in Arabidopsis thaliana, have clearly shown that variation in light quantity, quality and photoperiod can be manipulated to affect growth and control developmental transitions. Light emitting diodes (LEDs) has been used for decades to test plant responses to narrow-bandwidth light. LEDs are particularly well suited for plant growth chambers, as they have an extraordinary life (about 100,000 hours), require little maintenance, and use negligible energy. These factors render LED-based light strategies particularly appropriate for space-biology as well as terrestrial applications. However, there is a need for a versatile and inexpensive LED array platform where individual wavebands can be specifically tuned to produce a series of light combinations consisting of various quantities and qualities of individual wavelengths. Two plans are presented in this report.
In this technical report we describe the practical construction of tunable red-green-blue LED arrays to support research in plant growth and development. Two light fixture designs and corresponding circuitry are presented. The first is well suited for a laboratory environment for use in a finite area with small plants, such as Arabidopsis. The second is expandable and appropriate for growth chambers. The application of these arrays to early plant developmental studies has been validated with assays of hypocotyl growth inhibition/promotion and phototropic curvature in Arabidopsis seedlings.
The presentation of these proven plans for LED array construction allows the teacher, researcher or electronics aficionado a means to inexpensively build efficient, adjustable lighting modules for plant research. These simple and effective designs permit the construction of useful tools by programs short on electronics expertise. These arrays represent a means to modulate precise quality and quantity in experimental settings to test the effect of specific light combinations in regulating plant growth, development and plant-product yield.
PMCID: PMC1198233  PMID: 16117835
6.  Expressed sequence tags (ESTs) and simple sequence repeat (SSR) markers from octoploid strawberry (Fragaria × ananassa) 
BMC Plant Biology  2005;5:12.
Cultivated strawberry (Fragaria × ananassa) represents one of the most valued fruit crops in the United States. Despite its economic importance, the octoploid genome presents a formidable barrier to efficient study of genome structure and molecular mechanisms that underlie agriculturally-relevant traits. Many potentially fruitful research avenues, especially large-scale gene expression surveys and development of molecular genetic markers have been limited by a lack of sequence information in public databases. As a first step to remedy this discrepancy a cDNA library has been developed from salicylate-treated, whole-plant tissues and over 1800 expressed sequence tags (EST's) have been sequenced and analyzed.
A putative unigene set of 1304 sequences – 133 contigs and 1171 singlets – has been developed, and the transcripts have been functionally annotated. Homology searches indicate that 89.5% of sequences share significant similarity to known/putative proteins or Rosaceae ESTs. The ESTs have been functionally characterized and genes relevant to specific physiological processes of economic importance have been identified. A set of tools useful for SSR development and mapping is presented.
Sequences derived from this effort may be used to speed gene discovery efforts in Fragaria and the Rosaceae in general and also open avenues of comparative mapping. This report represents a first step in expanding molecular-genetic analyses in strawberry and demonstrates how computational tools can be used to optimally mine a large body of useful information from a relatively small data set.
PMCID: PMC1182381  PMID: 15985176

Results 1-6 (6)