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Condensed tannins (CT) are highly desirable in forage as they sequester dietary protein and reduce bloat and methane emissions in ruminants. However, the widely used forage legume white clover ( Trifolium repens ) only produces CTs in flowers and trichomes and at levels too low to achieve therapeutic effects. Genetic transformation with transcription factor Ta-MYB14-1 from Trifolium arvense was effective in inducing CTs to 0.6% of leaf dry matter. CT synthesis has been elevated further by crossing the primary white clover transgenic line with wild type genotypes producing the related phenylpropanoids, anthocyanins. CT levels in leaves were highest under the anthocyanin leaf marks associated with the "red midrib" trait; however, there was no evidence for CT accumulation in leaf sections with the "red V" anthocyanin marking. Ta-MYB14-1 was stably inherited in two generations of crosses, and T2 progeny produced up to 3.6-fold higher CTs than the T0 parent. The profile of small CT oligomers such as dimers and trimers was consistent in T0, T1, T2, and BC2 progeny and consisted predominantly of prodelphinidins (PD), with lesser amounts of procyanidins (PC) and mixed PC:PD oligomers.

Perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) are forage species of the grass family (Poaceae) that are key components of temperate pasture-based agricultural systems. Proanthocyanidins (PAs) are oligomeric flavonoids that, when provided as part of a farm animal's diet, have been reported to improve animal production and health. Up to now, forage grasses have been deemed not to produce PAs. This paper reports for the first time the detection of polymerized PAs in aqueous methanolic extracts of seed tissue of both perennial ryegrass and tall fescue, using LC-MS/MS. We have determined the structure of the PAs to be trans-flavan-3-ol-based, consisting predominately of afzelechin and catechin and linked primarily by B-type bonds. Investigations into the leaf tissue of both species failed to detect any PAs. This discovery opens the possibility of using genetic engineering tools to achieve tannin accumulation in leaf tissue of perennial ryegrass and tall fescue.

Background: White 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., Results: This 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., Conclusions: This 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.

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.