Thiolytic screening method for exploring condensed tannin variation in a unique sainfoin germplasm bank

Abstract

This EU funded ‘HealthyHay’ project established a sainfoin (Onobrychis viciifolia) germplasm bank at NIAB, Cambridge, with 360 accessions from around the world. A screening method was developed to characterise tannins by thiolytic degradation [1] directly in green plants for the first time. The method was validated by separate analysis of unextractable, extractable and purified tannins using thiolysis, HPLC-GPC and MALDI-TOF MS. Most tannins (58 to 73% of the total) could be recovered after Toyopearl HW50 fractionation with water, aqueous methanol and acetone. The greatest losses during purification occurred amongst larger molecular weight tannins with mean degree of polymerisation (mDP) >18. The composition of water-, aqueous methanol- and acetone-soluble tannins differed considerably in their mDP and trans/cis ratios, but not in their prodelphinidin/procyanidin (PD/PC) ratios. Direct thiolysis revealed that the condensed tannin contents in this germplasm collection ranged from 0.6 to 2.8g/100 dry weight; mDP ranged from 12 to 84; PD/PC ratios from 53/47 to 95/5; and trans/cis ratios from 12/88 to 34/66. Detailed analysis of leaves and stems of the Perly variety, which was grown at INRA near Clermont-Ferrand (France) and harvested at three phenological stages between 2 June to 16 July, demonstrated a 2-fold higher tannin content and a 3-fold lower mDP in the less mature compared to mature plants. There was little change in PD/PC or trans/cis ratios. Leaf and stem tannins differed particularly in their mDP and PD/PC ratios. This screening method will underpin future breeding programmes to improve the nutritional and veterinary properties of sainfoin for animals and to reduce greenhouse gas emissions (NOx and CH4) from ruminants.