White Clover Populations Research Articles

Breeding strategies for forage and grass improvement

Breeding strategies for forage and grass improvement

Phosphorus efficiency of naturalized Chilean white clover in a grazed field trial

AbstractNine naturalized white clover populations and two cultivars (Huia and Will) were evaluated at two soil phosphorus ( P ) levels (6 and 20 P mg kg−1) to characterize them for DM production and P efficiency, and to facilitate the selection of suitable genotypes to produce cultivars for P‐deficient marginal soils. The study was carried out in Chile during 2007–2010 under field grazing conditions using a randomized complete factorial block design with three replicates. The clover was grown with perennial ryegrass. Botanical composition and herbage DM production were recorded, phosphorus concentrations in soil and in clover and ryegrass herbage were determined, and P absorption (PAE) and P utilization (PUE) efficiency were calculated. Population 8‐1‐X yielded more DM under the low than under the high soil‐ P level and was the highest yielding clover in the low soil‐ P treatment. It also had the highest PAE (0·6 kg P ha−1 per mg kg−1 Olsen P) under low P availability. Dry‐matter yield of 9‐2‐X was statistically similar to 8‐1‐X and Huia at low P level; however, because of its lower P absorption, it had the highest PUE (455 kg DM ha−1 per kg ha−1 absorbed P). Furthermore, the highest yield of ryegrass occurred when grown with 8‐1‐X, and this combination gave the maximum total yield. These two populations have potential for inclusion in breeding programmes.

Multivariate associations of flavonoid and biomass accumulation in white clover (Trifolium repens) under drought.

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.

Tradeoff between Biomass and Flavonoid Accumulation in White Clover Reflects Contrasting Plant Strategies

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.

Plant responses to defoliation and relationships with pasture persistence

The effects of defoliation on plant morphology and the structure of perennial ryegrass and white clover populations are reviewed with reference to the persistence of yield in sown pastures. Maintenance of high densities of perennial ryegrass tillers and white clover stolons is fundamental to persistence. Tiller and stolon population densities are subject to within- and between-year variation, and are strongly influenced by defoliation management through processes such as competition for light, and size-density trade-offs mediated by self-thinning processes. Spring is a time when high tiller/stolon appearance and death rates occur simultaneously. Spring is also the time when clover populations undergo rapid structural change as large clover plants fragment to release small, unbranched plants with limited ability to compete for light with grasses. The central role of reproductive stem development in perennial ryegrass for summer survival of tillers and autumn re-establishment of tiller populations also highlights the importance of defoliation management during the spring for population persistence. Examples of the application of tactical defoliation management in spring, and at other times of the year, to manipulate stolon and tiller densities are presented. In some instances, such tactics may improve animal production potential, as well as the persistence of new cultivars. It is notable that the New Zealand literature on this topic is mostly more than 15 years old. Recent changes in farming systems and plant genotypes (e.g. tetraploidy in perennial ryegrass, late flowering perennial ryegrass) may require a reanalysis of established principles and practices related to defoliation x plant interactions in grazed pastures to ensure best possible pasture yield persistence and profitability of grazing systems. Keywords: grazing management, perennial ryegrass, persistence, population dynamics, white clover

Water use efficiency and associated physiological traits of nine naturalized white clover populations in Chile

With 1 figure and 4 tables AbstractWater use efficiency (WUE) of nine naturalized white clover (Trifolium repens L.) populations was evaluated to select parental genotypes for future breeding programmes and to identify associated physiological traits that could be used as selection criteria. A greenhouse experiment was carried out during the spring of 2007 in Chillán, Chile (36º03′S, 72º07′W). The nine naturalized populations plus two controls were planted in pots containing soil as substrate. The plants were grown under two soil water treatments: with water stress (−1 MPa) and without water stress (−0.01 MPa). The experiment was arranged in a randomized complete block design with three replicates. Transpirated water (T) was recorded daily by weighing the pots, shoot DM (leaves + petioles + stolons) production was measured and WUE was calculated (shoot DM/T). Stomatal conductance (gs), chlorophyll content, relative water content, leaf 13C isotopic discrimination, canopy temperature and stolon water potential were also evaluated. The WUE varied significantly (P = 0.05) among populations and increased under drought conditions because of gs reduction. A high WUE was associated with populations that had a low leaf area DM investment and high stolon DM investment.

Genetic variation of white clover (Trifolium repens L.) collections from China detected by morphological traits, RAPD and SSR.

White clover is an important legume naturalized in China. But genetic variation of Chinese local white clover germplasm has not been reported. There is no information for parents’ selection and genetic resource conservation. The objective of this study was to investigate and characterize genetic variation of ten local white clover germplasms collected from different geographical regions of China. Morphological data analysis of white clover populations was coupled with molecular markers of random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) to investigate the genetic relationships among white clover germplasms of China, and four commercial cultivars were included for a comparison. The results revealed that the populations showed diverse morphological traits, RAPD and SSR patterns. Despite the certain similarities among the dendrograms of particular populations, the overall correlations among the distance matrices appeared to be rather low. In the present study, cluster analysis only slightly reflected the geographical origins of populations, and the dendrogram employing SSR data showed the closest agreement with collections’ geographical origins. The analysis of molecular variance (AMOVA) analysis based on both RAPD and SSR markers revealed higher levels of intra-population variation than inter-population variation. Neither RAPD nor SSR based grouping showed obvious agreement with variation of leaf sizes and other morphological traits. Our results suggest that the combination of morphological traits and SSR analysis could be a useful method for assessing genetic variation in white clover, and could be helpful for breeders to plan crosses for positive traits.

Development of white clover populations with higher concentrations of water soluble carbohydrate

Water soluble carbohydrate (WSC) provides readily available energy in the rumen that improves the efficiency of crude protein (CP) utilisation, partitioning of dietary N towards animal growth, and reduces the loss of N as urea.

Attributes related to seasonal herbage growth in white clover

When breeding temperate forage species is investigated, some attributes such as herbage accumulation and seasonal growth patterns have to be considered. To modify some of these traits, knowledge of the detailed process might be useful. In order to evaluate seasonal growth of contrasting white clover populations an experiment was carried out. Treatments were five cultivars and three local populations collected in Argentina. Weekly measures were taken during each season to calculate leaf appearance and flower appearance rates, stolon growing rate and dry matter (DM) net accumulation. Different germplasm of this species have different mechanisms for DM accumulation. Leaf size, more than leaf appearance rate, was the variable with most differences among germplasm and mostly related to dry matter accumulation. During spring and summer, inflorescences production had important effects on growing stolon rate, and differences among germplasm were evident. Some local populations showed favourable attributes that could be useful for breeding.

Variation in flooding-induced morphological traits in natural populations of white clover (Trifolium repens) and their effects on plant performance during soil flooding.

Soil flooding leads to low soil oxygen concentrations and thereby negatively affects plant growth. Differences in flooding tolerance have been explained by the variation among species in the extent to which traits related to acclimation were expressed. However, our knowledge of variation within natural species (i.e. among individual genotypes) in traits related to flooding tolerance is very limited. Such data could tell us on which traits selection might have taken place, and will take place in future. The aim of the present study was to show that variation in flooding-tolerance-related traits is present among genotypes of the same species, and that both the constitutive variation and the plastic variation in flooding-induced changes in trait expression affect the performance of genotypes during soil flooding. Clones of Trifolium repens originating from a river foreland were subjected to either drained, control conditions or to soil flooding. Constitutive expression of morphological traits was recorded on control plants, and flooding-induced changes in expression were compared with these constitutive expression levels. Moreover, the effect of both constitutive and flooding-induced trait expression on plant performance was determined. Constitutive and plastic variation of several morphological traits significantly affected plant performance. Even relatively small increases in root porosity and petiole length contributed to better performance during soil flooding. High specific leaf area, by contrast, was negatively correlated with performance during flooding. The data show that different genotypes responded differently to soil flooding, which could be linked to variation in morphological trait expression. As flooded and drained conditions exerted different selection pressures on trait expression, the optimal value for constitutive and plastic traits will depend on the frequency and duration of flooding. These data will help us understanding the mechanisms affecting short- and long-term dynamics in flooding-prone ecosystems.

Population Dynamics of Broadleaf Weeds in Turfgrass as Influenced by Chemical and Biological Control Methods

A 3-yr field study was conducted to determine the effect of a biological control agent,Sclerotinia minorJagger, and a common herbicide, Killex, on the population dynamics of dandelion and other broadleaf species and on the dandelion seed bank. Treatments were applied as one spring, one early autumn, or a spring plus an early autumn treatment per year. The response of the dandelion population to a spring herbicide treatment was similar to two applications per year (spring and early autumn). Significantly less dandelion control occurred after the first early autumn application of the herbicide. Two weeks after application, spring or early autumn treatments withS. minorwere equally effective in suppressing dandelions. In the second year of the two applications per year ofS. minortreatment, weed control was equivalent to the herbicide. By the third year of the one spring application ofS. minor, weed control was equivalent to the herbicide. Generally over the study period, the early autumn application ofS. minorwas less effective than the spring or the spring and early autumn applications. TheS. minortreatments significantly reduced the dandelion seed bank, and this effect was not significantly different from the Killex herbicide treatment. The rate, frequency, and seasonal timing of application had no effect on the dandelion seed-bank size, but terminating the application would gradually replenish the seed bank. Populations of white clover, broadleaf plantain, birdsfoot trefoil, and common ragweed were similarly suppressed by either theS. minoror the herbicide treatments. Yellow woodsorrel significantly increased after 1 yr of herbicide treatment compared with theS. minorand untreated control treatments, indicating a possible weed species shift. Turf quality was improved because of the herbicide andS. minortreatments, but grass injury and smooth crabgrass invasion were recorded in 17% of herbicide-treated plots.

Forest understory clover populations in enriched CO 2 and O 3 atmospheres: Interspecific, intraspecific, and indirect effects

The effects of CO 2 and tropospheric O 3 on forest trees are increasingly the subject of experimental evaluation. Little is known, however, about the effects of these gases on understory plant taxa. At the Aspen free-air CO 2 and O 3 enrichment (Aspen FACE) site we assessed colonization and establishment of two common forest understory species, red ( Trifolium pratense) and white ( Trifolium repens) clover. To better understand these natural patterns in red clover, the more responsive of the two clover species, we also assessed intraspecific variation in growth performance to altered atmospheric conditions. Natural red clover populations were larger in enriched CO 2 atmospheres, whereas white clover populations showed no response to CO 2. Neither species showed beneficial or detrimental responses to enriched O 3 atmospheres. Nine red clover genotypes exhibited similar, but counterintuitive, decreases in shoot and root biomass, and increases in foliar nitrogen concentrations and root nodule numbers, under elevated CO 2. We suggest that in enriched CO 2 habitats, increased shading from rapidly growing trees offsets the positive benefits of CO 2 for understory plant performance. Enriched CO 2 and O 3 atmospheres may have large direct and indirect effects on colonization, establishment, and performance of common understory plants. Such changes may in turn alter forest community and ecosystem dynamics.

Forage Improvement via Marker-Assisted Selection

The use of DNA markers to accelerate genetic improvement of forages presents a unique set of opportunities, challenges, and benefits. Our experiments in full-sib mapping populations of white clover and perennial ryegrass have detected >75 quantitative trait loci (QTLs), each with multiple marker:trait associations at specific locations in either the perennial ryegrass or white clover genome. A subset of these QTL are robust (detected in multiple years / sites / populations) and exert a substantial influence on performance, warranting exploration of development for application in Marker-assisted Selection (MAS) breeding programmes. Ryegrass QTLs associated with herbage yield, seed yield, plant size and habit, cold tolerance, seasonal regrowth, and disease have been identified, whereas QTL discovery in white clover has been focused on reproductive traits. Markers from two white clover QTLs were used to develop marker assays suitable for selection of parental plants with superior breeding value for seed yield potential. Tandem testing of the two assays over two field seasons and eight populations indicates that substantial change in seed yield may be achieved (up to 90% increase), and that the marker / allele / phase relationships to plant performance are population specific. These data point to an opportunity to develop selection tools on a population specific basis, and to a challenge to implement MAS approaches tailored for open-pollinated population breeding systems.

Genetic differentiation in white clover (Trifolium repens) populations during 8 years of contrasting phosphorus supply in New Zealand hill country

Genetic differentiation under the selection force of variable soil P availability was measured in populations of two white clover cultivars introduced to a grazed, low fertility grassland site. The resulting mixed grass‐clover pastures subsequently received contrasting levels of phosphorus (P) fertiliser for 8 years. Afield‐based reciprocal transplant experiment and two controlled‐environment experiments were conducted using plants removed from the 8‐year field experiment and appropriate controls to test the hypotheses that: (1) genetic differentiation occurred within the white clover populations in response to variation in P supply; and (2) surviving plants which demonstrated genetic differentiation under high‐P conditions would continue to do this when grown in mixtures with plant material removed from unsown pasture. This study demonstrated that genetic differentiation did occur. In the reciprocal transplant experiment, when returned to a low‐P environment the mean of the populations removed from a low‐P site yielded more than the mean population removed from a high‐P site. Genetic differentiation was demonstrated most strongly with ‘Grasslands Huia’. ‘Huia’ survivors growing in the high‐P source environment retained the potential of the original seed line to respond to P additions, but this potential was lost in ‘Huia’ populations grown under low‐P fertility. In contrast, ‘Grasslands Tahora’ did not retain the potential of the original seed line to respond to additional P and behaved similarly to the resident material. High‐P inputs led to the retention of ‘Huia’ genotypes with the capacity for high growth rates and superior competitive ability. This competitive ability was demonstrated when resident plants grown in a mixture with ‘Huia’ plants removed from the high‐P source treatment suffered reductions in yield compared with their yield in pure pots. The drift by both ‘Tahora’ and ‘Huia’ away from the potential of the original seed attributes towards the resident type demonstrates the adaptive nature of the traits found in the resident population that favour a conservative growth strategy. P availability does not appear to be a dominant selection factor where new germplasm has been introduced to pastures with the objective of stimulating production; maintenance of fertility levels may assist in retaining a full complement of P‐responsive genotypes.

Genetic diversity in perennial ryegrass and white clover among old Dutch grasslands as compared to cultivars and nature reserves

To support conservation policies for old Dutch grasslands that are still in agricultural use, morphological variation and AFLP-based (amplified fragment length polymorphism-based) genetic diversity was studied in perennial ryegrass and white clover populations and compared with the diversity in reference varieties. In addition, AFLP variation was also studied in grasslands located in nature reserves. From principal component analysis (PCA), it appeared that date of ear emergence in perennial ryegrass and characters related to plant vigour in white clover were the main morphological characters separating the reference varieties from the old Dutch grassland populations, and some of the grassland populations from each other. In both species, intrapopulation variation was lower for the reference varieties. Lower heterogeneity within the reference varieties was also found in the AFLP analysis. All common AFLP's observed in old Dutch grasslands could also be found in the reference varieties and nature reserves. Only a small number of low-frequency alleles found in old Dutch grasslands were absent from the other two groups. However, band frequencies of markers could vary considerably between populations, which may have been caused by selection. Analysis of the AFLP data by PCA distinguished the majority of reference varieties from the old Dutch grasslands, and showed genetic differentiation only between some grasslands. Comparison of old Dutch grasslands with grasslands in nature reserves indicated that basically the same range of genetic variation is covered by the two groups. Our study indicates that the Netherlands harbour a more or less continuous population for major parts of the diversity of perennial ryegrass and white clover. It was concluded that no specific conservation measures are presently needed to maintain genetic diversity of perennial ryegrass and white clover occurring in old Dutch grasslands.

Inheritance of the Isozyme Isocitrate Dehydrogenase (IDH) in a Natural Population of Polyploid White Clover (Trifolium Repens L.)

An electrophoretic study of segregation of isocitrate dehydrogenase (IDH) was undertaken in polyploid white clover (Trifolium repens L.). The study aimed to test the conflicting theories on enzyme inheritance patterns in this species. A number of controlled sellings and reciprocal crosses were performed as well as a study of distribution of alleles in a natural Danish population of white clover. The result of the selfmg experiment was that the chosen material is strongly self-incompatible. The offspring from the crosses showed that two isoloci were present and that the alleles in these segregate in a Mendelian fashion. Five different IDH-phenotypes, composed of two alleles, where found in the natural population. This contrasts with previous findings, for this and other allotetraploid species, of one variant and one invariant isolocus. Maximum likelihood analysis of the distribution of these phenotypes showed that the two alleles were equally common on an overall population basis. Each of these alleles were, however, most frequent in one of the isoloci. The findings reported here strongly support the theory that white clover is an allotetraploid, showing disomic inheritance.

Identification of white clover (Trifolium repens L.) cultivars using molecular markers

The different molecular analysis for specification of white clover (Trifolium repens L.) populations was studied between 2002 and 2003. RAPD, SSR (microsatellites), rDNA and PCR-RFLP markers were used for this study. The high genetic variation was detected among the cultivars but also within the cultivars by RAPD markers. For this reason RAPD markers were not found as a suitable marker system for determination of white clover cultivars. The distribution of low genetic variation of rDNA and PCR-RFLP markers was not able to differentiate cultivars. SSR and rDNA markers did not show variability of patterns within one cultivar. The different sizes of PCR fragments were obtained after amplification with microsatellite primers. SSR markers are therefore suggested as the suitable markers for the identification of different T. repens cultivars.

White clover (Trifolium repens) and associated viruses in the subalpine region of south-eastern Australia: implications for GMO risk assessment

Over the past several years, increased emphasis has been placed on conducting comprehensive ecological-risk assessments of virus-resistant genetically modified organisms (GMOs) prior to their release into the environment. In this paper we report on the first stage in our assessment of the level of risk posed by virus-resistant transgenic Trifolium repens L. (white clover) to native plant communities in south-eastern Australia. We investigated the distribution, abundance and phytosociological characteristics of naturalised T. repens populations in two areas in the subalpine region of New South Wales (NSW) and the Australian Capital Territory (ACT), and determined the distribution and abundance of Alfalfa mosaic virus, Clover yellow vein virus and White clover mosaic virus in 31 populations of white clover in this region. We found that T. repens is a significant component of Poa grasslands and Eucalyptus–Poa woodlands in the subalpine region, but is absent or rare in Eucalyptus species forests and Carex–Poa species bogs. Clover yellow vein virus was by far the most common virus in the study area, being present in 18% of T. repens plants across a wide range of plant communities. Alfalfa mosaic virus and White clover mosaic virus were each recorded in only one white-clover population growing in a native plant community. We conclude that white clover is a significant constituent of subalpine grasslands and woodlands in the region studied, and that of the viruses investigated, Clover yellow vein virus is the most abundant and widespread.

Morphological Variations in the White Clover Population Collected from the Northern-Tohoku Region : 2. Relationships among plant characteristics and between the variation pattern in characteristics and geographical factors

Morphological Variations in the White Clover Population Collected from the Northern-Tohoku Region : 2. Relationships among plant characteristics and between the variation pattern in characteristics and geographical factors

Effects of white clover cultivar and companion grass on winter survival of seedlings in autumn‐sown swards

AbstractThe aim was to study the effects of white clover cultivar and combinations with perennial ryegrass cultivars on seedling establishment in autumn‐sown swards and on winter survival of seedlings. Large‐leaved white clover cv. Alice and small‐leaved white clover cv. Gwenda, and an erect and a prostrate perennial ryegrass cultivar were sown in autumn in pure stands and as four binary grass‐clover mixtures. Mixtures of white clover cv. Huia and Aberherald with perennial ryegrass were also sown. Companion grasses had no significant impact on the establishment of white clover. The number of seedlings of white clover cv. Alice in mixtures (335 m−2) was higher than cv. Gwenda (183 m−2) and pure swards had similar white clover population densities as mixed swards. White clover cv. Huia tended to have more seedlings than Aberherald (355 and 205 m−2 respectively). No stolons were produced prior to a severe winter, because of the late sowing date. Winter survival of clover seedlings was 0·56 in mixtures and 0·69 in pure stands, irrespective of white clover or companion grass cultivar. Stolon development of white clover in autumn is often considered essential for overwintering survival and spring growth. In this study, there was considerable survival of the non‐stoloniferous tap‐rooted seedlings of all four clover cultivars despite a severe winter.