Novel markers for drought resistance in white clover

Introduction. White clover (Trifolium repens L.) is an out-crossing allotetraploid (2n=4x=32) that acts like a diploid, also called amphidiploid. White clover is a forage crop with a high level of genetic heterogeneity, which allows it to adapt to a wide range of environments. However, growth of white clover is often impaired by summer drought. New cultivars need to adapt to higher levels of abiotic stress to survive and persist. Recent research in white clover populations demonstrated that levels of a secondary metabolite, glycosides of the phenolic compound quercetin (Q), were positively associated with abiotic stress resistance, and inversely related to dry matter (DM) production. Molecular plant breeding methods using DNA markers and quantitative trait loci (QTL) analysis can increase breeding efficiency using information on genotype to complement phenotype, and identify genes controlling complex traits. Aims. The objectives of this study were (i) to investigate genetic control of the complex traits Q glycosides and DM production in contrasting environments, (ii) to test the inverse correlation between Q glycosides and DM production, and (iii) to identify DNA markers associated with quantitative trait loci (QTL) for use in marker-assisted selection (MAS). Materials and methods. An F1 mapping population (n = 190) created from a paired cross between two highly heterozygous white clover genotypes sampled from New Zealand cultivar “Kopu II” and Chinese ecotype “Tienshan” was used for phenotyping and genotyping. Phenotyping consisted of three parts: 1) in pots under outside conditions in separate pilot and phenotyping experiments, 2) in pots outside under imposed water deficit, and 3) in the field in two environments contrasting in soil moisture. Important traits were measured, including morphological, e.g. root DM, shoot DM, total DM, root-to-shoot DM ratio, stolon density, leaf size; biochemical, glycoside levels of the flavonols quercetin and kaempferol (K), Q:K glycoside ratio (QKR); as well as physiological, e.g. water potential, stomatal conductance and carbon isotope discrimination. Genotyping was investigated by means of single sequence repeat (SSR) markers and diversity array technology (DArT) markers from the genomes of Trifolium repens, Trifolium occidentale and Medicago truncatula. Results. Phenotyping in pot studies revealed sufficient genetic variation in the population, and only a weak albeit significant (P< 0.001) correlation between dry matter yield and Q, with levels of one explaining less than 10% of variation in the other. Imposed water deficit decreased leaf water potential by more than half overall, Q glycosides increased more than twofold and the ratio of quercetin to kaempferol glycosides increased preferentially towards Q. Furthermore, the most productive genotypes in the controls showed the greatest proportional reduction, the root:shoot ratio increased by half and individuals with high QKR levels reduced their biomass least under water deficit, and in turn increased their Q glycosides and QKR most. In the field studies, water deficit significantly reduced carbon isotope discrimination, Q glycosides were preferentially synthesised over K glycosides, accumulation of Q glycosides was related to retaining biomass, and stolon density was inversely related to stolon numbers. Genotyping resulted in a large number of DNA markers associated with genome regions influencing traits in the population. QTL effects were consistent over pot- and field experiments in some cases. Suitable markers were used to build a partial linkage map to better test for chromosomal locations containing QTLs associated with the traits of interest. Interaction between Q glycosides and DM QTLs was analysed. Discussion and conclusions. The data show, for the first time, that at the individual genotype level increased Q glycoside accumulation in response to water deficit is associated with retaining higher levels of biomass production. These findings are the first indication that forage populations that are both high yielding and show high Q glycoside levels are possible. The single locus discoveries of marker-trait associations provide a basis for enhanced plant breeding efficiency for these traits, and specific sources of new variation for economically significant traits. Overall, the findings can be used to better understand the physiological underpinnings of water stress relations in forage plants, and improve gain from selection in white clover by increasing the precision with which improved plants and populations are identified.