INTERSPECIFIC HYBRIDIZATION IN THE LOTUS GENUS TO BUILD FORAGE VARIETIES WITH ENHANCED NUTRITIONAL VALUE: MOLECULAR AND FUNCTIONAL EVIDENCES

Proanthocyanidins (PA) are oligomeric and polymeric end products of the flavonoid pathway based on epicatechin and/or catechin units and leucoanthocyanidins. PA strongly affect palatability and nutritive value of forage legumes. Whereas high PA concentrations in the forage are detrimental for the animals, moderate levels of these metabolites prevent proteolysis during ensiling and rumen fermentation, thereby protecting ruminants against pasture bloat and decreasing methane emission by livestock. Among forage legumes only a few species are capable of synthesizing PAs into the foliage. Unfortunately, largely grown legumes such as alfalfa, Lotus tenuis or clovers are not in this list. Hence, interventions promoting the accumulation of moderate amount of PA in these forage species is of the outmost importance, both from an environmental and agronomical point of view. Although both regulatory and structural genes of the PA pathway have been cloned in model and crop legumes, any GMO-based approaches have so far basically failed in inducing ex novo PA accumulation in legume cells naturally uncommitted to accumulate these metabolites. Hence, aiming to produce bloat-safe legume varieties and dissect the genetic control and molecular determinants of PA biosynthesis, we crossed plants belonging to a L. tenuis variety largely grown in South America with plants from a wild diploid population of L. corniculatus that accumulates high levels of foliage PA. Notably, hybrid F1 plants showed an intermediate level of leaf PA with respect to the two parents that displayed a contrasting phenotype for this trait. F1 plants were fertile and a F2 population, consisting of more than 200 individuals, was generated by free F1 intercrossing. Spectrophotometric quantification of PAs in these plants and in vitro digestibility tests of forage from F1 and F2 plants are ongoing. qRT-PCR based analyses of structural and regulatory genes showed that PA accumulation in leaves and stems of F1 and F2 plants nicely correlated with expression levels of genes deputed to the synthesis of catechins (LAR), epicatechins (ANR) and leucoanthocyanidins (DFR, ANS) as well as with some members of the TT2 (R2R3MYB) and bHLH gene families. This study highlights the importance of recovering wild populations to enrich Lotus varieties with useful traits and it is the first example of transferring the PA trait between forage legumes.