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Allotetraploid white clover (Trifolium repens) formed during the last glaciation through hybridisation of two European diploid progenitors from restricted niches: one coastal, the other alpine. Here, we examine which hybridisation‐derived molecular events may have underpinned white clover's postglacial niche expansion. We compared the transcriptomic frost responses of white clovers (an inbred line and an alpine‐adapted ecotype), extant descendants of its progenitor species and a resynthesised white clover neopolyploid to identify genes that were exclusively frost‐induced in the alpine progenitor and its derived subgenomes. From these analyses we identified galactinol synthase, the rate‐limiting enzyme in biosynthesis of the cryoprotectant raffinose, and found that the extant descendants of the alpine progenitor as well as the neopolyploid white clover rapidly accumulated significantly more galactinol and raffinose than the coastal progenitor under cold stress. The frost‐induced galactinol synthase expression and rapid raffinose accumulation derived from the alpine progenitor likely provided an advantage during early postglacial colonisation for white clover compared to its coastal progenitor. Summary statement: Cold induction of galactinol synthase expression and raffinose accumulation are likely alpine progenitor‐derived traits contributing to postglacial niche expansion in allotetraploid white clover. [ABSTRACT FROM AUTHOR]

Red clover (Trifolium pratense L.), a key perennial pastoral species used globally, can strengthen pastural mixes to withstand increasingly disruptive weather patterns from climate change. Breeding selections can be refined for this purpose by obtaining an in-depth understanding of key functional traits. A replicated randomized complete block glasshouse pot trial was used to observe trait responses critical to plant performance under control (15% VMC), water deficit (5% VMC) and waterlogged conditions (50% VMC) in seven red clover populations and compared against white clover. Twelve morphological and physiological traits were identified as key contributors to the different plant coping mechanisms displayed. Under water deficit, the levels of all aboveground morphological traits decreased, highlighted by a 41% decrease in total dry matter and 50% decreases in both leaf number and leaf thickness compared to the control treatment. An increase in root to shoot ratio indicated a shift to prioritizing root maintenance by sacrificing shoot growth, a trait attributed to plantwater deficit tolerance. Under waterlogging, a reduction in photosynthetic activity among red clover populations reduced several morphological traits including a 30% decrease in root dry mass and total dry matter, and a 34% decrease in leaf number. The importance of root morphology for waterlogging was highlightedwith low performance of red clover: there was an 83% decrease in root dry mass compared to white clover which was able to maintain root dry mass and therefore plant performance. This study highlights the importance of germplasm evaluation across water stress extremes to identify traits for future breeding programs. [ABSTRACT FROM AUTHOR]

White clover (Trifolium repens) is integral to mixed pastures in New Zealand and temperate agriculture globally. It provides quality feed and a sustainable source of plant-available nitrogen (N) via N-fixation through symbiosis with soil-dwelling Rhizobium bacteria. Improvement of N-fixation in white clover is a route to enhancing sustainability of temperate pasture production. Focussing on seedling growth critical for crop establishment and performance, a population of 120 half-sibling white clover families was assessed with either N-supplementation or N-fixation via inoculation with a commercial Rhizobium strain (TA1). Quantitative genetic analysis identified significant (p < 0.05) family additive genetic variance for Shoot and Root Dry Matter (DM) and Symbiotic Potential (SP), and Root to Shoot ratio. Estimated narrow-sense heritabilities for above-ground symbiotic traits were moderate (0.24-0.33), and the strong (r = 0.97) genetic correlation between Shoot and Root DM indicated strong pleiotropy or close linkage. The moderate (r = 0.47) phenotypic correlation between Shoot DM under symbiosis vs. under N-supplementation suggested plant growth with mineral-N was not a strong predictor of symbiotic performance. At 5% among-family selection pressure, predicted genetic gains per selection cycle of 19 and 17% for symbiotic traits Shoot DM and Shoot SP, respectively, highlighted opportunities for improved early seedling establishment and growth under symbiosis. Single and multi-trait selection methods, including a Smith-Hazel index focussing on an ideotype of high Shoot DM and Shoot SP, showed commonality of top-ranked families among traits. This study provides a platform for proof-of-concept crosses to breed for enhanced seedling growth under Rhizobium symbiosis and is informative for other legume crops. [ABSTRACT FROM AUTHOR]

Pedigrees provide suitable input for tracking breeding crosses and monitor plants and animal populations. Pedigree analysis is an essential tool to visualize and describe population structure and genetic diversity. Pasture plant breeding relies on a range of strategies from closed system recurrent selection using elite cultivars to diversity‐focused programs using ecotypes and wild germplasm as parents. There is currently no published pedigree analysis of white clover (Trifolium repens L.), despite over a century of breeding efforts in New Zealand. A pedigree map was constructed for white clover germplasm stored in the Margot Forde Germplasm Centre located in Palmerston North, New Zealand. Inbreeding and kinship coefficients and the effective number of founders through time were estimated to assess genetic relatedness in the reference population. A total of 15,265 accessions were included in the map. The maximum number of traced generations was 15, and the mean completeness of parentage for the reference population was 72%. The mean number of offspring was 5.14. The origin of founding and introduced accessions was assessed. The effective number of founders was 175.68 or 17.5% of the total number of reference founders. Founding accessions and influencing ancestors were identified. One of the earliest parental accessions identified was 'North Canterbury Type I' from 1941. Relatedness was determined using the k coefficient, with k = 1 as complete relatedness. The overall mean relatedness was k = 0.002. Overall mean inbreeding level was 0.39%. Relatedness and inbreeding coefficients revealed distinct germplasm pools formed across time that are of interest to pre‐breeding efforts. [ABSTRACT FROM AUTHOR]

* Background and aims Traits related to root depth distribution were examined in Trifolium repens x T. uniflorum backcross 1 (BC1) hybrids to determine whether root characteristics of white clover could be improved by interspecific hybridization. * Methods Two white clover cultivars, two T. uniflorum accessions and two BC1 populations were grown in 1 -m deep tubes of sand culture. Maximum rooting depth and root mass distribution were measured at four harvests over time, and root distribution data were fitted with a regression model to provide measures of root system shape. Morphological traits were measured at two depths at harvest 3. * Key Results Root system shape of the hybrids was more similar to T. uniflorum than to white clover. The hybrids and T. uniflorum had a higher rate of decrease in root mass with depth than white clover, which would result in higher proportions of root mass in the upper profile. Percentage total root mass at 100-200 mm depth was higher for T. uniflorum than white clover, and for Crusader BC1 than 'Crusader'. Roots of the hybrids and T. uniflorum also penetrated deeper than those of white clover. T. uniflorum had thicker roots at 50-100 mm deep than the other entries, and more of its fine root mass at 400-500 mm. The hybrids and white clover had more of their fine root mass higher in the profile. Consequently, T. uniflorum had a higher root length density at 400-500 mm than most entries, and a smaller decrease in root length density with depth. * Conclusions These results demonstrate that rooting characteristics of white clover can be altered by hybridization with T. uniflorum, potentially improving water and nutrient acquisition and drought resistance. Root traits of T. uniflorum are likely to be adaptations to soil moisture and fertility in its natural environment. [ABSTRACT FROM AUTHOR]

The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor ( KPI) genes isolated from white clover ( Trifolium repens; designated Tr- KPI1, Tr- KPI2, Tr- KPI4 and Tr- KPI5) has been investigated to determine their wider functional role., The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr- KPI1, Tr- KPI2, Tr- KPI4 and Tr- KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr- KPI1 and Tr- KPI4 over-expressing lines., RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr- KPI1, Tr- KPI2 and Tr- KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes., These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr- KPI2, may occupy a central role in the maintenance of a cellular homeostasis. [ABSTRACT FROM AUTHOR]

This study investigated drought responses in first (BC 1 ) and second (BC 2 ) backcross generation hybrids of Trifolium repens L. × T. uniflorum L., and compared these to high-yielding T. repens L. (white clover) cultivars. Plant attributes included accumulation of leaf phenolics as well as other biochemical and physiological water stress responses. Measurements were made across broad clover types as well as on a subset of related material. Under drought, net photosynthesis decreased by 44–48% and transpiration rates decreased by 60% in the BC 2 family and in the white clover parent, but were unaltered in the BC 1 family. Drought-induced decreases in leaf water potential were more pronounced in the BC 1 family (−47%) than the BC 2 (−31%) and parental (−28%) material. Quercetin glycoside accumulation generally increased 2–3 fold under drought. Compared with the BC 2 family and with parent plants, the BC 1 family had 2.5–4x higher constitutive kaempferol glycoside levels, and 1.5–2.5x higher kaempferol glycoside accumulation under drought. Constitutive kaempferol glycoside accumulation was related to reduced senescence and to less pronounced decreases in shoot DW under drought stress. Hydroxycinnamic acid accumulation increased by 56–73% under water stress, particularly for the BC 1 generation. These results identify physiological and biochemical traits that help explain drought resistance of T. repens × T. uniflorum hybrids. The findings suggest merit for the selection of BC 1 families with increased accumulation of protective phenolic compounds in breeding programmes for improved drought resistance. [ABSTRACT FROM AUTHOR]

Highlights: [•] The experiment showed repeated highly significant variation for the measured traits. [•] There was only a weak negative correlation between biomass and flavonols. [•] Both increased stress protective metabolites and selection for high herbage yield. [•] The flavonoid biochemical pathway occurs in all crop plants. [•] This finding could improve other economically important crops. [Copyright &y& Elsevier]

Abstract: Pasture plants such as Trifolium repens L. (white clover) are exposed to high levels of ultraviolet-B (UV-B) radiation in summer, as well as to frequent defoliation events from grazing animals and pests. This study examined responses in two T. repens populations exposed to 16 weeks supplementation of 0 or 13.3kJm−2 d−1 UV-B radiation under controlled environmental conditions. During that period, plants were exposed to two large defoliation events that lasted two and three weeks, respectively. We investigated a number of leaf morphological characteristics, photochemical attributes, as well as aspects of cellular leaf structure. In particular, we sought to explore whether possible differences in these attributes between the two T. repens populations could be related to their UV-B responsiveness. Leaf dry mass decreased by 16% in the UV-B-sensitive cultivar ‘Huia’ under UV-B, whereas the tolerant ecotype ‘Tienshan’ was unaffected. This differential UV-B response was related to constitutive differences in leaf mass and in leaf area between the two populations. UV-B did not affect specific leaf mass, whereas leaf dry matter content was reduced by 8% in response to UV-B. Measurements of chlorophyll fluorescence revealed no significant effects of UV-B on photochemistry. Results from light microscopy showed that the cellular leaf structure of the T. repens populations was not damaged by UV-B. Population-specific structural features included more dome-shaped epidermal cells for ‘Tienshan’. We conclude that differential UV-B-responses in T. repens populations can occur after defoliation pressure and can be related to differences in leaf characteristics. [Copyright &y& Elsevier]

White clover (Trifolium repens L.) is an important pasture legume in temperate regions, but growth is often strongly reduced under summer drought. Cloned individuals from a full-sib progeny of a pair cross between two phenotypically distinct white clover populations were exposed to water deficit in pots under outdoor conditions for 9 weeks, while control pots were maintained at field capacity. Water deficit decreased leaf water potential by more than 50% overall, but increased the levels of the flavonol glycosides of quercetin (Q) and the ratio of quercetin and kaempferol glycosides (QKR) by 111% and by 90%, respectively. Water deficit reduced dry matter (DM) by 21%, with the most productive genotypes in the controls showing the greatest proportional reduction. The full-sib progeny displayed a significant increase in the root : shoot ratio by 53% under water deficit. Drought-induced changes in plant morphology were associated with changes in Q, but not kaempferol (K) glycosides. The genotypes with high QKR levels reduced their DM production least under water deficit and increased their Q glycoside levels and QKR most. These data show, at the individual genotype level, that increased Q glycoside accumulation in response to water deficit stress can be positively associated with retaining higher levels of DM production. [ABSTRACT FROM AUTHOR]

This study used nine populations of Trifolium repens L. (white clover) to investigate possible relationships between plant morphological attributes and responses to ultraviolet-B (UV-B) radiation. Plants were exposed to 0 or 13.3 kJ·m−2·day−1 UV-B for 12 weeks. Drought was applied in parallel to these treatments during the last 4 weeks of the experiment to test whether limited moisture availability would alter morphological UV-B responses. UV-B affected plant morphology under well-watered conditions, reducing leaf size by 15%, leaf number by 5% and stolon elongation by 19%. The number of leaf primordia in the apical bud was decreased by 4% under UV-B, and by 12% under drought. In drought-exposed plants, leaf size was reduced by 50%, leaf number by 30% and stolon elongation by 60%. In addition, drought reduced specific leaf area (SLA) by 33% and increased leaf percentage dry mass (PDM) by 40%. UV-B-induced reduction in plant biomass in the T. repens populations was associated with higher plant productivity and this was further linked to larger leaf size as well as to lower PDM. In conclusion, the findings suggest that morphological attributes conferring fast potential growth under productive conditions carry a cost in the form of lower biomass accumulation under UV-B. [ABSTRACT FROM AUTHOR]

An outdoor study was conducted to examine relationships between plant productivity and stress-protective phenolic plant metabolites. Twenty-two populations of the pasture legume white clover were grown for 4½ months during spring and summer in Palmerston North, New Zealand. The major phenolic compounds identified and quantified by HPLC analysis were glycosides of the flavonoids quercetin and kaempferol. Multivariate analysis revealed a trade-off between flavonoid accumulation and plant productivity attributes. White clover populations with high biomass production, large leaves and thick tap roots showed low levels of quercetin glycoside accumulation and low quercetin:kaempferol ratios, while the opposite was true for less productive populations. The latter included stress-resistant ecotypes from Turkey and China, and the analysis also identified highly significant positive relationships of quercetin glycoside accumulation with plant morphology (root:shoot ratio). Importantly, a high degree of genetic variation was detected for most of the measured traits. These findings suggest merit for considering flavonoids such as quercetin as potential selection criteria in the genetic improvement of white clover and other crops. [ABSTRACT FROM AUTHOR]

ABSTRACT This study used comparisons across nine populations of Trifolium repens (white clover) in conjunction with drought to examine physiological responses to ultraviolet-B radiation (UV-B). Plants were exposed for 12 weeks to supplementation with 13.3 kJ m-2 d-1 UV-B, accompanied by 4 weeks of drought under controlled environmental conditions. UV-B increased the levels of UV-B-absorbing compounds and of flavonol glycosides and this effect was synergistically enhanced by water stress. These changes were more pronounced for the ortho -dihydroxylated quercetin, rather than the monohydroxylated kaempferol glycosides. UV-B increased leaf water potential (ψ L ) by 16% under drought and proline levels by 23% under well-watered conditions. The intraspecific comparisons showed that higher UV-B-induced levels of UV-B-absorbing compounds, of quercetin glycosides and of ψ L were linked to lower plant productivity and to higher UV-B tolerance under well-watered conditions. These findings suggest that: (1) slow-growing T. repens ecotypes adapted to other stresses have higher capacity for physiological acclimation to UV-B; and (2) that these attributes also contribute to decreased UV-B sensitivity under drought. [ABSTRACT FROM AUTHOR]

Abstract Nine populations of white clover (Trifolium repens L.) were grown for 12 weeks with supplemental application of 13.3 kJ m-2 d-1 ultraviolet-B (UV-B) radiation under controlled environmental conditions. Drought was applied during the last four weeks of the experiment. Under well-watered conditions, UV-B decreased white clover growth on average by 20%. Cultivars bred for agricultural performance were sensitive to UV-B, while slow-growing ecotypes were UV-B-tolerant. After four weeks of water stress, there were no significant population differences in UV-B responsiveness. UV-B sensitivity decreased with increasing exposure to drought and with longer duration of UV-B irradiation, suggesting that the direction and extent of the UV-B 3× drought interaction depends on the duration of stress. The population comparisons indicate that low constitutive growth rate and adaptation to other forms of stress may be related to UV-B tolerance under well-watered conditions, but not during extended periods of drought. [ABSTRACT FROM AUTHOR]

ABSTRACT White clover (Trifolium repens L.) is experiencing increased levels of ultraviolet-B (UV-B) radiation in temperate pastures due to the depletion of the stratospheric ozone layer. Based on 17 morphological, morphogenetic and physiological attributes, this study analysed the consequences of enhanced UV-B on 26 white clover populations using principal components analysis (PCA). After 18 d of exposure to 13·3 kJ m-2 d-1 UV-B in controlled environments, UV-B significantly decreased above-ground and below-ground plant growth attributes, epidermal cell surface area and maximum quantum efficiency of photosystem II photochemistry (Fv/Fm). Aspects of cell division and cell expansion both were negatively affected by UV-B. Stomatal density, specific leaf mass, root-to-shoot ratio and levels of UV-B-absorbing compounds increased in response to UV-B. In the multivariate analysis, the main dimension of UV-B sensitivity was characterized by changes in plant growth attributes. Alterations in partitioning within and between plant organs constituted a secondary tier of UV-B responsiveness. Plant characteristics related to UV-B tolerance included lower growth rate, smaller epidermal cell surface area and higher UV-B-induced levels of UV-B-absorbing compounds. The results suggest overall UV-B tolerance for slower-growing populations from less productive habitats with higher natural UV-B irradiance. [ABSTRACT FROM AUTHOR]

White clover growing in New Zealand is experiencing increasing levels of ultraviolet-B (UV-B) radiation as a result of ozone depletion. We evaluated the effects of UV-B radiation on the foliar chemistry of two populations of white clover (Trifolium repens L.), ’Huia’ and ’Tienshan,’ and the consequences for the performance of armyworms (Spodoptera litura) and cutworms (Graphania mutans). Plants were grown in controlled environment rooms with and without supplemental UV-B radiation at a dose of 13.3 kJ m–2 day–1, corresponding to a 25% mid-summer ozone depletion above Palmerston North, New Zealand. In both white clover populations, UV-B radiation elicited changes in foliar chemistry, including slight increases in nitrogen concentrations and decreases in carbohydrate concentrations. In addition, the ’Huia’ population showed decreases in fiber concentrations and marked increases in cyanogenic activity. No change in UV-absorbing compounds was detected in either population. Long- and short-term feeding trials were conducted to assess dietary effects on insect growth, consumption, and food utilization. Changes in the performance of both insect species were generally small. The most pronounced effect was a 36% reduction in weight of S. litura after 2 weeks of feeding on Huia grown at high UV, but larval development times were only slightly prolonged and pupal weights were unaffected. S. litura short-term performance was affected by differences in white clover population. The long-term performance of G. mutans was not affected and its short-term performance (stadium duration and consumption rate) was only marginally affected by the high-UV treatment. We conclude that the effects of elevated UV-B radiation on white clover plant chemistry can be specific to certain plant populations. The differences in sensitivity of the two generalist insect species suggest that effects may also be specific to certain plant-herbivore associations. These results indicate that future UV-B herbivory studies should examine genotypic effects in both plants and animals. [ABSTRACT FROM AUTHOR]

Background: Prebreeding in plants is the activity designed to identify useful characteristics from wild germplasm and its integration in breeding programs. Prebreeding aims to introduce new variation into the populations of a species of interest. Pedigree analysis is a valuable tool for evaluation of variation in genebanks where pedigree maps are used to visualize and describe population structure and variation within these populations. Margot Forde Germplasm Centre (MFGC) is New Zealand's national forage genebank and holds a collection of ~ 75 species of the genus Trifolium, of which only a dozen have been taken through prebreeding programs. The main objective of this study was to construct pedigree maps and analyse patterns of relatedness for seven minor Trifolium species accessions contained at the MFGC. These species are Trifolium ambiguum, Trifolium arvense, Trifolium dubium, Trifolium hybridum, Trifolium medium, Trifolium subterraneum and the Trifolium repens x Trifolium occidentale interspecific hybrids. We present a history of Trifolium spp. prebreeding in New Zealand and inform breeders of possible alternative forage species to use. Results: Pedigree data from accessions introduced between 1950 and 2016 were used and filtered based on breeding activity. Kinship levels among Trifolium spp. remained below 8% and no inbreeding was found. Influential ancestors that contributed largely to populations structure were identified. The Australian cultivar 'Monaro' had a strong influence over the whole population of accessions in T. ambiguum. T. subterraneum and T. repens x T. occidentale had the largest number of generations (3). T. ambiguum and T. medium had the highest cumulative kinship across the decades. Conclusions: We conclude that there are high levels of diversity in the seven Trifolium spp. studied. However, collection and prebreeding efforts must be strengthened to maximize utilization and bring useful genetic variation. [ABSTRACT FROM AUTHOR]