Transgenesis and Genomics in Molecular Breeding of Forage Plants

Forage plant breeding has been largely based on phenotypic selection following sexual recombination of natural genetic variation found between and within ecotypes. Advances in plant genetic manipulation over the last 15 years have provided convincing evidence that these powerful technologies can complement and enhance plant breeding programs. Significant progress in the establishment of the methodologies required for the molecular breeding of forage plants has been made. Examples of current products and approaches for the application of these methodologies to forage grass and legume improvement are outlined. Large-scale genomic analysis of many organisms is under way with human, arabidopsis and rice genome sequences almost completed. Forage plant breeding is just now entering the genome era. The plethora of new technologies and tools now available for high-throughput gene discovery and genome-wide gene expression analysis have opened up opportunities for innovative applications in the identification, functional characterisation and use of genes of value in forage production systems and beyond. Examples of these opportunities, such as ‘molecular phenotyping’, ‘symbio-genomics’ and ‘xeno-genomics’ are introduced

Ryegrass ASTRA: a Web-Based Resource for \u3cem\u3eLolium\u3c/em\u3e EST Analysis

Perennial ryegrass (Lolium perenne L.) is a major grass species of temperate pastoral agriculture

Clover ASTRA: a Web-Based Resource for Trifolium EST Analysis

White clover (Trifolium repens L.) is a major temperate forage legume

Gene Discovery and Molecular Dissection of Lignin Biosynthesis in Perennial Ryegrass (Lolium Perenne)

Lignification of plant cell walls has been identified as a major factor limiting forage digestibility. It limits the amount of digestible energy available to livestock, resulting in an incomplete utilisation of cellulose and hemicellulose by ruminant animals. Modification of the lignin profile of ryegrasses (Lolium spp.) and fescues (Festuca spp.) is undertaken through modulating the expression of genes encoding enzymes involved in the biosynthesis of monolignols

Gene Discovery and Molecular Dissection of Fructan Metabolism in Perennial Ryegrass (Lolium Perenne)

Fructans are the main soluble carbohydrate stored in up to a third of the vegetation of the earth, including the economically important temperate grasses. Fructans are polymers of fructose attached to a sucrose precursor. Perennial ryegrass (L. perenne L.) accumulates fructans of the inulin series, inulin neoseries and levan neoseries. Four enzymes are required to produce fructans of this profile: 1-SST (sucrose:sucrose 1- fructosyltransferase), 1-FFT (fructan:fructan 1-fructosyltransferase), 6G-FFT (6-glucose fructosyltransferase) and 6-FFT (fructan:fructan 6-fructosyltransferase) or 6-SFT (sucrose:fructan 6-fructosyltransferase) (Figure 1). Fructan biosynthetic enzymes have evolved from invertases and thus it is argued that fructan metabolism is an extension of sucrose metabolism. A high fructan content is a valuable resource in perennial ryegrass as it can be readily mobilised to sustain regrowth immediately after defoliation as well as adding to the nutritive value of the feed. However, the physiological role of fructans in grasses is not fully understood

An Intact Kidney Slice Model to Investigate Vasa Recta Properties and Function in situ

Background: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. Methods: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in ‘live’ kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. Results: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10–30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E2) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). Conclusions: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow

Plants Modify Biological Processes to Ensure Survival following Carbon Depletion: A Lolium perenne Model

BACKGROUND: Plants, due to their immobility, have evolved mechanisms allowing them to adapt to multiple environmental and management conditions. Short-term undesirable conditions (e.g. moisture deficit, cold temperatures) generally reduce photosynthetic carbon supply while increasing soluble carbohydrate accumulation. It is not known, however, what strategies plants may use in the long-term to adapt to situations resulting in net carbon depletion (i.e. reduced photosynthetic carbon supply and carbohydrate accumulation). In addition, many transcriptomic experiments have typically been undertaken under laboratory conditions; therefore, long-term acclimation strategies that plants use in natural environments are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Perennial ryegrass (Lolium perenne L.) was used as a model plant to define whether plants adapt to repetitive carbon depletion and to further elucidate their long-term acclimation mechanisms. Transcriptome changes in both lamina and stubble tissues of field-grown plants with depleted carbon reserves were characterised using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR data for select key genes indicated that plants reduced fructan degradation, and increased photosynthesis and fructan synthesis capacities following carbon depletion. This acclimatory response was not sufficient to prevent a reduction (P<0.001) in net biomass accumulation, but ensured that the plant survived. CONCLUSIONS: Adaptations of plants with depleted carbon reserves resulted in reduced post-defoliation carbon mobilization and earlier replenishment of carbon reserves, thereby ensuring survival and continued growth. These findings will help pave the way to improve plant biomass production, for either grazing livestock or biofuel purposes

Analysis of Expressed Sequence Tags of the Cyclically Parthenogenetic Rotifer Brachionus plicatilis

Background. Rotifers are among the most common non-arthropod animals and are the most experimentally tractable members of the basal assemblage of metazoan phyla known as Gnathifera. The monogonont rotifer Brachionus plicatilis is a developing model system for ecotoxicology, aquatic ecology, cryptic speciation, and the evolution of sex, and is an important food source for finfish aquaculture. However, basic knowledge of the genome and transcriptome of any rotifer species has been lacking. Methodology/Principal Findings. We generated and partially sequenced a cDNA library from B. plicatilis and constructed a database of over 2300 expressed sequence tags corresponding to more than 450 transcripts. About 20% of the transcripts had no significant similarity to database sequences by BLAST; most of these contained open reading frames of significant length but few had recognized Pfam motifs. Sixteen transcripts accounted for 25% of the ESTs; four of these had no significant similarity to BLAST or Pfam databases. Putative up- and downstream untranslated regions are relatively short and AT rich. In contrast to bdelloid rotifers, there was no evidence of a conserved trans-spliced leader sequence among the transcripts and most genes were single-copy. Conclusions/Significance. Despite the small size of this EST project it revealed several important features of the rotifer transcriptome and of individual monogonont genes. Because there is little genomic data for Gnathifera, the transcripts we found with no known function may represent genes that are species-, class-, phylum- or even superphylum-specific; the fact that some are among the most highly expressed indicates their importance. The absence of trans-spliced leader exons in this monogonont species contrasts with their abundance in bdelloid rotifers and indicates that the presence of this phenomenon can vary at the subphylum level. Our EST database provides a relatively large quantity of transcript-level data for B. plicatilis, and more generally of rotifers and other gnathiferan phyla, and can be browsed and searched at gmod.mbl.edu

Discovery of genes associated with fruit ripening in arica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded: chitinase, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species