Quantitative genetic analysis reveals potential to breed for improved white clover growth in symbiosis with nitrogen-fixing Rhizobium bacteria

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

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

Epistatic Association Mapping in Homozygous Crop Cultivars

The genetic dissection of complex traits plays a crucial role in crop breeding. However, genetic analysis and crop breeding have heretofore been performed separately. In this study, we designed a new approach that integrates epistatic association analysis in crop cultivars with breeding by design. First, we proposed an epistatic association mapping (EAM) approach in homozygous crop cultivars. The phenotypic values of complex traits, along with molecular marker information, were used to perform EAM. In our EAM, all the main-effect quantitative trait loci (QTLs), environmental effects, QTL-by-environment interactions and QTL-by-QTL interactions were included in a full model and estimated by empirical Bayes approach. A series of Monte Carlo simulations was performed to confirm the reliability of the new method. Next, the information from all detected QTLs was used to mine novel alleles for each locus and to design elite cross combination. Finally, the new approach was adopted to dissect the genetic basis of seed length in 215 soybean cultivars obtained, by stratified random sampling, from 6 geographic ecotypes in China. As a result, 19 main-effect QTLs and 3 epistatic QTLs were identified, more than 10 novel alleles were mined and 3 elite parental combinations, such as Daqingdou and Zhengzhou790034, were predicted

Variation for constitutive flavonols and morphological traits in a new white clover population

Breeding forage legumes combining high levels of stress-protective secondary metabolites and high herbage yield are possible. Previous findings suggested a trade-off between flavonol glycosides and biomass production in white clover (Trifolium repens L.), with population specific evidence indicating that association. The present study used a novel white clover first filial (F₁) reciprocal cross (n=130, 3 replications in pots) between the productive cultivar "Kopu II" and the cold- and drought stress-resistant population "Tienshan". The conditions were non-limiting as the plants were watered at regular intervals as needed, to establish a baseline for both constitutive flavonol glycosides and above-ground biomass production. This study showed that the phenotypic correlation between the traits quercetin glycosides (Q) and shoot dry matter (SDM) although significant (P<0.001) was weak with r²=0.0903 (9%). This suggests the possibility of improving white clover performance by increasing the levels of stress protective metabolites in tandem with selection for high yield. At a genotype level, constitutive quercetin (Q) glycoside accumulation in this white clover line is not a major constraint on DM production in the absence of moisture stress. This indicates that combining high DM yield and high constitutive levels of Q glycosides for abiotic stress protection is possible. This finding is significant to overcoming a key challenge in plant breeding: combining stress tolerance with increased herbage production. © 2014 Elsevier B.V

Genotype × environment analysis of flavonoid accumulation and morphology in white clover under contrasting field conditions

White clover (Trifolium repens) is a beneficial legume in temperate pastures as a provider of high quality feed to stock and nitrogen to grassland. However, its growth is often strongly impaired by summer drought. The aim of this study was to investigate the association of flavonoids and biomass production in environments contrasting in moisture availability, and to identify trait ideotypes to be used in breeding plants for stress environments. In two separate field experiments, cloned individuals from a mapping population consisting of full sib progeny of a bi-parental cross were exposed to two contrasting field environments: water-limited (WL) and water sufficient (WS). Most measured morphological, physiological traits and flavonol glycoside accumulation were affected by the contrasting WL and WS environments. WL decreased carbon isotope discrimination by 5.5%. Under WL the levels of quercetin glycosides (Q) and of the quercetin:kaempferol glycoside ratio (QKR) increased by 62% and by 36%, respectively, relative to the WS environment. The WL environment reduced shoot dry matter (SDM) by 68%, with the most productive genotypes showing the greatest proportional reduction. Stolon numbers per plant decreased by 48% under WL, but stolon density (stolon number m ⁻² canopy area) increased by 48%. In principal component analysis, drought-induced changes in plant morphology were closely aligned to changes in Q, but not kaempferol glycosides. The increased induction of Q accumulation in response to water deficit stress was related to retaining higher levels of SDM production. This study also enabled the identification of individual plant trait combinations useful for white clover breeding programs focussed on developing improved cultivars for dryland environments. © 2012 Elsevier B.V

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

Application of molecular marker assayed paternity in a white clover breeding program

The use of molecular tools, coupled with improvements in paternity assignment computation, offer breeders a cost-effective and readily implementable breeding tool to improve the rate of genetic gain in forages. Molecular marker paternity assays may improve genetic gain in white clover (Trifolium repens L.) half-sib family breeding programs by utilizing a larger coefficient of additive genetic variation. To evaluate the potential to improve breeding outcomes by use of a molecular marker paternity assay in allotetraploid white clover (2n = 4x = 32), seven single-locus, homoeologue-specific simple sequence repeat markers were genotyped and analyzed on 1277 half-sib progeny and their 20 parents from a polycross pollinated by bumble bees (Bombus sp.). Paternity was assigned to 88.9% of progeny using a likelihood algorithm implemented in Cervus. Estimates of additive genetic variances from multisite, space-planted field experiments established in perennial ryegrass (Lolium perenne L.) were used to predict selection responses. Joint maternal and molecular-marker-assayed paternal half-sib family selection offered improved theoretical selection gains relative to conventional maternal half-sib family selection for herbage yield and trifoliate center leaflet width. The estimated selection responses based on empirical parameters demonstrated the theoretical wisdom of incorporating molecular-marker-assayed paternity in white clover breeding programs as an effective tool to improve selection responses per cycle of selection

Effect of traditional soybean breeding on water use strategy in arid and semi-arid areas

Water is the main factor determining yield performance under drought conditions. Traditional soybean breeding has significantly increased grain yield under drought, but its effect on water use strategies and associated traits are not well understood. Field and pot experiments with new cultivars and landrace soybean genotypes were undertaken to identify the effect of soybean breeding on water use strategies and related leaf and root functional traits under different water regimes. The new cultivars, on average, had 33.9 %, 45.1 % and 169 % higher grain yields and 47.9 %, 98.4 % and 244 % higher water use efficiencies for grain yield (WUEG) but 8.8 %, 38.1 % and 19.6 % lower water use than the landraces in the field and pot experiments 1 and 2, respectively. Stomatal conductance decreased in the new cultivars at higher soil water contents than the landraces during soil drying. The new cultivars, on average, had a 22.9 % lower root fraction, 21.5 % lower leaf fraction, 20.6 % lower stem fraction and 69.8 % higher pod fraction than landraces, which had 21.7 % and 26.8 % lower root hydraulic conductance at the R2 and R6 stages, respectively, than the landraces. Our results show that (1) traditional soybean breeding has changed the water use strategy from profligate to conservative and improved yield performance and WUEG, (2) the high sensitivity of stomatal conductance to water stress is coordinated with lower root hydraulic conductance to limit water use, and (3) selecting new cultivars with high grain yield but low water use may a new way to improve yield performance under water-limited conditions

Characterisation of the morphological variation for seed traits among 537 germplasm accessions of common vetch (<i>Vicia sativa</i> L.) using digital image analysis

<p>Common vetch (<i>Vicia sativa</i> L.) is an important forage legume crop. Our study was focused on estimation of genotypic variation for seed traits among germplasm accessions within the <i>V. sativa</i> and the two subspecies of common vetch; <i>V. sativa</i> (402 accessions), <i>V. sativa</i> subsp. <i>sativa</i> (105 accessions) and <i>V. sativa</i> subsp. <i>nigra</i> (30 accessions). The seed traits measured were straight length, straight width, width to length ratio, curved length, curved width, perimeter, hilum length, 100-seed weight and seed shape. The seed trait data were analysed using REML in GenStat and the resulting accession-by-trait BLUP mean matrices were summarised using a combination of cluster and principal component analysis, presented as biplots. There was significant (<i>P</i> < 0.05) genotypic variation among germplasm accessions, within each subspecies for all the traits measured. The calculated seed trait repeatability (<i>R</i>) provided a rough estimate of the upper limit of genotypic variation among the accessions within the <i>V. sativa</i> and the two subspecies. The magnitude and type of association among the seed traits shown in the biplots were supported by the estimated phenotypic correlation coefficients. The germplasm accessions within the groups identified in <i>V. sativa</i>, <i>V. sativa</i> subsp<i>. sativa</i> and <i>V. sativa</i> subsp<i>. nigra</i> will provide valuable genetic diversity for taxonomic studies and breeding associated with the seed morphological traits reported in our investigation.</p

Improving white clover for Australasia

Improving the genetic merit of temperate forage legumes helps ensure profitability and sustainability of our Australasian pastoral industries. Today’s plant breeders are supported by a range of underpinning research activities including genetic resources exploration and enhancement, plant physiology, plant health, feed quality, agronomy, quantitative genetics and plant biotechnology; and have collaborative interfaces with animal and farm systems science. Lifting the rate of gain by integration of molecular tools, innovative breeding strategies, and new genetic resources is the major objective of our white clover breeding network. This paper, presented at the Australasian Grassland Association’s recent Legume Symposium, focuses on the key research and development achievements in white clover breeding for Australasia, and on the success and future of an Australasian collaboration to breed improved cultivars for the region’s temperate environments. The paper reports on successful developments in the areas of improving white clover root systems for phosphate uptake, pest tolerance, development of novel inter-specific hybrids and marker-aided breeding. The successful trans-Tasman collaboration in white clover breeding and future work is also discussed