Chris S. Jones

Expression of the R2R3-MYB Transcription Factor TaMYB14 from Trifolium arvense Activates Proanthocyanidin Biosynthesis in the Legumes Trifolium repens and Medicago sativa

Proanthocyanidins (PAs) are oligomeric flavonoids and one group of end products of the phenylpropanoid pathway. PAs have been reported to be beneficial for human and animal health and are particularly important in pastoral agricultural systems for improved animal production and reduced greenhouse gas emissions. However, the main forage legumes grown in these systems, such as Trifolium repens and Medicago sativa, do not contain any substantial amounts of PAs in leaves. We have identified from the foliar PA-accumulating legume Trifolium arvense an R2R3-MYB transcription factor, TaMYB14, and provide evidence that this transcription factor is involved in the regulation of PA biosynthesis in legumes. TaMYB14 expression is necessary and sufficient to up-regulate late steps of the phenylpropanoid pathway and to induce PA biosynthesis. RNA interference silencing of TaMYB14 resulted in almost complete cessation of PA biosynthesis in T. arvense, whereas Nicotiana tabacum, M. sativa, and T. repens plants constitutively expressing TaMYB14 synthesized and accumulated PAs in leaves up to 1.8% dry matter. Targeted liquid chromatography-multistage tandem mass spectrometry analysis identified foliar PAs up to degree of polymerization 6 in leaf extracts. Hence, genetically modified M. sativa and T. repens plants expressing TaMYB14 provide a viable option for improving animal health and mitigating the negative environmental impacts of pastoral animal production systems.

Future options and targets for pasture plant breeding in New Zealand

Abstract Industries based on pastoral farming have increased their contribution to GDP from 13.5 to 17% since 1990 as the result of markedly intensified farming practices. In the future, we predict that this intensification will continue but, at the same time, there will be an emergence of an efficient, lower‐input farming sector with almost no environmental footprint. Both sectors will require continuing input by pasture plant breeders. Over the past 20 years, development of pasture cultivars has become totally industry funded, with support from Crown funding for basic research. There have been several key advances in pasture plant breeding including new methods for using exotic germplasm and secondary gene‐pools, modification of grass‐en‐dophyte associations, breeding for specific environments and the successful adoption of international breeding programmes. The emergence of genom‐ics, marker‐assisted selection (MAS) and genetic modification (GM) offer considerable promise for future development of pasture cultivars. Future grass breeding, aided by MAS and GM of both plants and endophytes, will place strong emphasis on feeding value for optimal animal performances, especially in intensive systems. There will also be development of grass types adapted to efficient, lower‐input farming systems that will have minimal environmental impacts. White clover breeding has had a period of unprecedented input built on a strong foundation of knowledge and germplasm. For intensive agriculture, future breeding will emphasise disease and pest resistance, improved feed quality, seed production and the development of hybrid cultivars. For lower‐input sustainable systems, breeding will aim to broaden the adaptation of clover to semi‐arid and other marginal environments. This will involve increasing use of related clover species in interspecific hybrids, and selection for improved phosphate efficiency, N‐fixation and drought tolerance. DNA technologies will provide an increasingly valuable contribution to these products but the predominant effort will involve plant breeding methods that re‐combine and select whole genomes. New Zealand farming is based on a small number of pasture plant species and, despite the expanded use of the herbs chicory and plantain, this number has reduced with intensification. It is predicted that some expansion of the species base will be needed in future to cope with climate warming and the development of a sustainable low‐input farming sector. In particular, subtropical grasses should be investigated more thoroughly along with ways of enabling (perhaps new) legumes to be used with them. Despite a relatively enlightened germplasm introduction programme, the germplasm base of most pasture species is inadequate and requires continued effort to locate and import new materials from diverse international sources. International research networks have been developed in recent years and these will increasingly contribute to future pasture plant breeding progress. The current biosecurity and Hazardous Substances and New Organisms regulatory environment is not conducive to timely research and innovation on new species for agriculture and needs reconsideration by law makers.

The isolation of RNA from raspberry (Rubus idaeus) fruit

Previous attempts to extract high-quality, total RNA from raspberry (Rubus idaeus) fruits using published protocols have proven to be unsuccessful. Even the use of protocols developed for the extraction of RNA from other fruit tissue has resulted in low yields (1) or the isolation of degraded RNA (2). Here, we report on the development of a quick and simple method of extracting total RNA from raspberry fruit. Using this method, high yields of good quality, undegraded RNA were obtained from fruit at all stages of ripening. The RNA is of sufficient quality for northern analysis and cDNA library construction.

Profiling of changes in gene expression during raspberry (Rubus idaeus) fruit ripening by application of RNA fingerprinting techniques

Abstract. Processes that contribute to the overall phenomenon of fruit ripening are not well understood for many soft fruit species, including raspberry (Rubus idaeus L.) Recent biochemical data implicate ethylene and a range of cell wall hydrolases in ripening processes. However, the genes encoding these activities, and others related to ripening, remain to be characterised. With the advent of high-throughput RNA-fingerprinting techniques it is possible to characterise rapidly the changes in gene expression during developmental processes. This paper describes the application of two RNA-fingerprinting techniques (cDNA amplified fragment length polymorphism and differential display reverse transcription-polymerase chain reaction) to the ripening fruit of Rubusi daeus. Copy-DNA tags were isolated representing 34 genes, up-regulated during fruit ripening. The expression profiles of these genes were determined by RNA-blot analysis and their sequences were compared with those in public databases. Potential roles for some of these gene products are considered, providing valuable insights into the processes that underpin fruit ripening. Many of the cDNAs isolated in this study provide tools for the biotechnological improvement of fruit quality.

An integrated genetic linkage map for white clover (Trifolium repens L.) with alignment to Medicago

BackgroundWhite clover (Trifolium repens L.) is a temperate forage legume with an allotetraploid genome (2n=4×=32) estimated at 1093 Mb. Several linkage maps of various sizes, marker sources and completeness are available, however, no integrated map and marker set has explored consistency of linkage analysis among unrelated mapping populations. Such integrative analysis requires tools for homoeologue matching among populations. Development of these tools provides for a consistent framework map of the white clover genome, and facilitates in silico alignment with the model forage legume, Medicago truncatula.ResultsThis is the first report of integration of independent linkage maps in white clover, and adds to the literature on methyl filtered GeneThresher®-derived microsatellite (simple sequence repeat; SSR) markers for linkage mapping. Gene-targeted SSR markers were discovered in a GeneThresher® (TrGT) methyl-filtered database of 364,539 sequences, which yielded 15,647 SSR arrays. Primers were designed for 4,038 arrays and of these, 465 TrGT-SSR markers were used for parental consensus genetic linkage analysis in an F1 mapping population (MP2). This was merged with an EST-SSR consensus genetic map of an independent population (MP1), using markers to match homoeologues and develop a multi-population integrated map of the white clover genome. This integrated map (IM) includes 1109 loci based on 804 SSRs over 1274 cM, covering 97% of the genome at a moderate density of one locus per 1.2 cM. Eighteen candidate genes and one morphological marker were also placed on the IM. Despite being derived from disparate populations and marker sources, the component maps and the derived IM had consistent representations of the white clover genome for marker order and genetic length. In silico analysis at an E-value threshold of 1e-20 revealed substantial co-linearity with the Medicago truncatula genome, and indicates a translocation between T. repens groups 2 and 6 relative to M. truncatula.ConclusionsThis integrated genetic linkage analysis provides a consistent and comprehensive linkage analysis of the white clover genome, with alignment to a model forage legume. Associated marker locus information, particularly the homoeologue-specific markers, offers a new resource for forage legume research to enable genetic analysis and improvement of this forage and grassland species.

Association of climatic stress with blight on Chinese chestnut in the eastern United States.

JONES, C., G. J. GRIFFIN, and J. R. ELKINS. 1980. Association of climatic stress with blight on Chinese chestnut in the eastern United States. Plant Disease 64:1001-1004. In commercial and home plantings of Chinese chestnut in Georgia, North Carolina, Tennessee, Virginia, West Virginia, and New York, 23% of the trees had main stem cankers incited by the chestnut blight fungus, Endothiaparasitica. The average main stem canker size was 28 X 55 cm. Fifteen percent of the trees had infection over 50% or more of the limb circumferences. Only two blighted trees were killed. In general, main stem canker incidence (13-93%) was higher in plantings of the Appalachian Mountain region than in plantings of the Piedmont region (2-13% incidence). Trees that were damaged most by E. parasitica cankers were located in high-wind and cold-winter areas of the Appalachian Mountains.

Functional characterisation and transcript analysis of an alkaline phosphatase from the arbuscular mycorrhizal fungus Funneliformis mosseae.

Alkaline phosphatases (ALP) in arbuscular mycorrhizal (AM) fungi have been suggested to be involved in transfer of phosphate from the mycorrhizal fungus to the host plant, but exact mechanisms are still unknown, partially due to the lack of molecular information. We isolated a full-length cDNA (FmALP) from the AM fungus Funneliformis mosseae (syn. Glomus mosseae) showing similarity with putative ALP genes from Rhizophagus intraradices (syn. Glomus intraradices) and Gigaspora margarita. For functional characterisation FmALP was expressed heterologously in the yeast Pichia pastoris. The recombinant FmALP protein had a pH optimum of 9.5, and catalysed the hydrolysis of glycerolphosphate and, to a lesser extent of glucose-1- and 6-phosphate, confirming it to be an alkaline phosphatase belonging to the family of alkaline phosphomonoesterases (EC 3.1.3.1). FmALP did not catalyse the hydrolysis of ATP or polyP. Relative FmALP transcript levels were analysed in intra- and extraradical hyphae isolated from F. mosseae infected ryegrass (Lolium perenne) using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). FmALP was highly expressed in intraradical hyphae at low P(i) supply, and its expression was repressed by high P(i) supply. Taken together this study provides evidence for mycorrhizal alkaline phosphatases playing a role in P mobilisation from organic substrates under P starvation conditions.

Development of Trifolium occidentale as a Plant Model System for Perennial Clonal Species

Trifolium occidentale D.E. Coombe is a diploid, clonal perennial clover that is very closely related to white clover (T. repens L.). It has been previously reported to be self-pollinating and lacking in genetic diversity. However, new collections, especially in Spain and Portugal, have revealed that cross-pollinating populations with substantial genetic diversity do exist. This has led to T. occidentale being investigated as a potential genetic model species to facilitate the application of genomic methods for the improvement of white clover. Investigations have shown that T. occidentale has many attributes that make it suitable as a genetic model for white clover. It forms hybrids with white clover and the chromosomes of the two species pair and recombine at meiosis. Phylogenetic research shows that it is a very close relative, and probably an ancestor, of white clover. A framework linkage map based on SSR markers has shown it to be highly syntenic with white clover. A protocol for efficient transformation has been developed. An effective EMS mutagenesis method has been demonstrated by the induction of a high frequency of condensed tannin negative mutants. The clonal nature of T. occidentale is not shared by other dicotyledonous model species. It may, therefore, be useful for the genomic characterisation of traits associated with clonal growth and perenniality in this wider class of plants.

Cloning of three genes up-regulated in ripening raspberry fruit (Rubus idaeus cv. Glen Clova)

Summary cDNA clones of three genes, up-regulated as raspberry ( R. idaeus cv. Glen Clova) fruit ripen, were isolated by differential screening of a cDNA library constructed from RNA extracted from ripe fruit. The expression patterns of these genes were determined in ripening fruit and in other tissues of the raspberry plant. When compared with sequences in databases, the genes were identified on the basis of sequence similarity. The predicted polypeptides coded for by the three clones show similarity to major latex proteins, a class of fruit-specific metallothionein-like proteins, and fruit-ripening associated endo-polygalactu-ronases, respectively. The potential roles of these gene products in fruit-ripening is discussed.

Plant vigour at establishment and following defoliation are both associated with responses to drought in perennial ryegrass ( Lolium perenne L.)

Highlight text We have developed a new methodology to assess individual perennial ryegrass plant performance under moisture stress and identified QTLs associated with improved performance during drought in this important forage species.