Lotus tenuis x L. corniculatus interspecific hybridization as a means to breed bloat-safe pastures and gain insight into the genetic control of proanthocyanidin biosynthesis in legumes

Abstract

BackgroundProanthocyanidins (PAs) are secondary metabolites that strongly affect plant quality traits. The concentration and the structure of these metabolites influence the palatability and nutritional value of forage legumes. Hence, modulating PAs in the leaves of forage legumes is of paramount relevance for forage breeders worldwide. The lack of genetic variation in the leaf PA trait within the most important forage species and the difficulties in engineering this pathway via the ectopic expression of regulatory genes, prompted us to pursue alternative strategies to enhance this trait in forage legumes of agronomic interest. The Lotus genus includes forage species which accumulate PAs in edible organs and can thus be used as potential donor parents in breeding programs.ResultsWe recovered a wild, diploid and PA-rich population of L. corniculatus and crossed with L. tenuis. The former grows in an alkaline-salty area in Spain while the latter is a diploid species, grown extensively in South American pastures, which does not accumulate PAs in the herbage. The resulting interspecific hybrids displayed several traits of outstanding agronomic relevance such as rhizome production, PA levels in edible tissues sufficient to prevent ruminal bloating (around 5 mg of PAs/g DW), biomass production similar to the cultivated parent and potential for adaptability to marginal lands. We show that PA levels correlate with expression levels of the R2R3MYB transcription factor TT2 and, in turn, with those of the key structural genes of the epicatechin and catechin biosynthetic pathways leading to PA biosynthesis.ConclusionsThe L. tenuis x L. corniculatus hybrids, reported herein, represent the first example of the introgression of the PA trait in forage legumes to levels known to provide nutritional and health benefits to ruminants. Apart from PAs, the hybrids have additional traits which may prove useful to breed forage legumes with increased persistence and adaptability to marginal conditions. Finally, our study suggests the hybrids and their progeny are an invaluable tool to gain a leap forward in our understanding of the genetic control of PA biosynthesis and tolerance to stresses in legumes.

The R2R3-MYB TT2b and the bHLH TT8 genes are the major regulators of proanthocyanidin biosynthesis in the leaves of Lotus species

“…et Kit and Lotus corniculatus L., their interspecific hybrids (LH1; LH2; LH3 and LH4) and F 2 progeny (A27, A120, A186, B3, B5 and B186) were previously employed to clone and study the expression of both early and late genes of the flavonoid pathways as well as the relationship between their expression and the overall accumulation of soluble and insoluble PAs (Escaray et al 2014). Here, we used these tools to clone and assess the expression levels of master genes related to PA biosynthesis and transport.…”

“…The parental plants (L. corniculatus and L. tenuis), the selected F 1 hybrid plants (LH1, LH2, LH3 and LH4) and the selected F2 were propagated by cuttings and grown either under growth chamber condition, for the parental plants and F 1 hybrids, or in pots under field condition for the parental plants and F 2 hybrids, as previously reported (Escaray et al 2014). …”

“…Here, we used these tools to clone and assess the expression levels of master genes related to PA biosynthesis and transport. In essence, plant material, plant growing conditions and cDNA synthesis were as previously described (Escaray et al 2014). Briefly, the L. tenuis parent plants are from a commercial variety (cv.…”

“…(Escaray et al 2014). To reach this goal, we cloned the R2R3-MYBs TT2a, TT2b, TT2c, MYB14 and PAP2, the bHLH TT8, the WDR TTG1 and the multidrug and toxic compound extrusion (MATE1) genes from both parental plants and assessed whether their expression correlated with those of the structural genes and the levels of PAs in the leaves from parents down to F 2 plants with contrasting levels of these metabolites.…”

Production and transcriptional regulation of proanthocyanidin biosynthesis in forage legumes

“…For example, the L. tenuis×L. corniculatus hybrids displayed the PA levels in edible tissues sufficient to prevent ruminal bloating (Escaray et al 2014). 6.…”