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Seed size is an important yield and quality-determining trait in higher plants and is also crucial to their evolutionary fitness. In African yam bean (AYB), seed size varies widely among different accessions. However, the genetic basis of such variation has not been adequately documented. A genome-wide marker-trait association study was conducted to identify genomic regions associated with four seed size traits (seed length, seed width, seed thickness, and 100-seed weight) in a panel of 195 AYB accessions. A total of 5416 SNP markers were generated from the diversity array technology sequence (DArTseq) genotype-by-sequencing (GBS)- approach, in which 2491 SNPs were retained after SNP quality control and used for marker-trait association analysis. Significant phenotypic variation was observed for the traits. Broad-sense heritability ranged from 50.0% (seed width) to 66.4% (seed length). The relationships among the traits were positive and significant. Genome-wide association study (GWAS) using the general linear model (GLM) and the mixed linear model (MLM) approaches identified 12 SNP markers significantly associated with seed size traits across the six test environments. The 12 makers explained 6.5–10.8% of the phenotypic variation. Two markers (29420334|F|0-52:C>G-52:C>G and 29420736|F|0-57:G>T-57:G>T) with pleiotropic effects associated with seed width and seed thickness were found. A candidate gene search identified five significant markers (100026424|F|0-37:C>T-37:C>T, 100041049|F|0-42:G>C-42:G>C, 100034480|F|0-31:C>A-31:C>A, 29420365|F|0-55:C>G-55:C>G, and 29420736|F|0-57:G>T-57:G>T) located close to 43 putative genes whose encoding protein products are known to regulate seed size traits. This study revealed significant makers not previously reported for seed size in AYB and could provide useful information for genomic-assisted breeding in AYB.

Background: Bambara groundnut accessions over the years had been unstable in its yield potentials with contrasting yield inferences, factors not limited to variety, soil types and nutrients, climate change, time had been bottlenecks for scientist to breakthrough in stability research. Field experiments were conducted in three years at two locations to assess and identify accessions with stable yield and determine the relationship between yield and yield components. Methods: Seeds of 100 accessions of Bambara groundnut of Nigerian origin were obtained from the genebank of the Genetic Resources Center, IITA in 2017. Randomized complete block design (RCBD) was used in the evaluation for three years in two locations. PROC ANOVA procedure on the statistical analytical system (SAS, 9.4) was used to determine significant differences and stability equation was used to identify yield stable index. Result: Correlation showed that seven yield contributing traits had positive and significant correlation with yield per hectare. Yield stability analysis indicated nine of the selected accessions were stable in the expression of its yield potentials in the environments and years. This study showed that selection for high yielding and stable accessions could be useful for further utilization of genetic resources in achieving food security.

Taro and new cocoyam are root and leaf crops commonly grown in tropical to warm temperate regions. In Togo, they are neglected and underutilised. Here we report the genetic diversity of 26 accessions of taro and 101 accessions of new cocoyam. Analysis of simple sequence repeats revealed low polymorphic information content of 0.43 and 0.25 in taro and new cocoyam, respectively. PCA scatterplots and Neighbour Joining dendrograms based on the SSR data clustered accessions into groups that more-or-less correspond to morphological diversity in both species. AMOVA within and between morphological groups revealed greater variances within groups than between. This indicates weak genetic differentiation between morphological groups, particularly for taro. Genetic diversity was greater among taro cultivars. Taro has a longer history of introduction and dispersal in Africa, and has had more opportunity for multiple introduction and local cultivar development. Different strategies are suggested for future development of these crops in Togo and Africa. For taro, further studies of existing diversity and recent experimental introduction, has spread widely in Africa with little genetic diversity. For this crop, international collaboration is needed to clarify taxonomy, and to introduced further cultivars for evaluation under local conditions in Africa.

Bambara groundnut is one of the crops with inadequate molecular research to show its full potentials. Previous studies showed morphological diversity with inadequate information to confirm genetic variations. In the quest to reveal the genetic potentials, deoxyribonucleic acid (DNA) of the selected accessions were extracted through leaf samples at 3 weeks old, using Dellaporta Miniprep for Plant DNA Isolation procedure. The high quality DNA was sequenced using Diversity Arrays Technology (DArT) markers to unlock diversity among Bambara groundnut of Nigerian origin. Cluster analysis (neighbor-joining clustering) of the single nucleotide polymorphisms (SNP’s) were used to generate sub-population to show relatedness and differences. Seven sub-populations were generated with 5,927 (50.13 %) high quality DArT markers out of the 11, 821 SNPs generated. This revealed high genetic diversity existed among the selected Bambara groundnut accessions in Nigeria. This also revealed that DArT markers were highly efficient in classifying the accessions based on molecular expressions. This study also identified markers responsible for genetic variation that could facilitate the characterization of larger collections for further utilization of genetic resources and most importantly Bambara groundnut for the purpose of crop improvement.

In plant breeding and agricultural research, biplot analysis has become an important statistical technique. The goal of this study was to find the winning genotype(s) for the test settings in a part of the Southwest region of Nigeria, as well as to investigate the nature and extent of genotype X environment interaction (GEI) effects on Bambara groundnut (BGN) production. The experiment was carried out in four environments (two separate sites, Ibadan and Ikenne, for two consecutive years, 2018 and 2019) with ninety-five BGN accessions. According to the combined analysis of variance over environments, genotypes and GEI both had a substantial (p &lt, 0.001) impact on BGN yield. The results revealed that BGN accessions performed differently in different test conditions, indicating that the interaction was crossover in nature. The results revealed that BGN accessions performed differently in different test conditions, indicating that the interaction was crossover in nature. To examine and show the pattern of the interaction components, biplots with the genotype main effect and genotype X environment interaction (GEI) were used. The first two PCs explained 80% of the total variation of the GGE model (i.e., G + GE) (PC1 = 48.59%, PC2 = 31.41%). The accessions that performed best in each environment based on the “which-won-where” polygon were TVSu-2031, TVSu-1724, TVSu-1742, TVSu-2022, TVSu-1943, TVSu-1892, TVSu-1557, TVSu-2060, and TVSu-2017. Among these accessions, TVSu-2017, TVSu-1557, TVSu-2060, TVSu-1892, and TVSu-1943 were among the highest-yielding accessions on the field. The adaptable accessions were TVSu-1763, TVSu-1899, TVSu-2019, TVSu-1898, TVSu-1957, TVSu-2021, and TVSu-1850, and the stable accessions were TVSu-1589, TVSu-1905, and TVSu-2048. In terms of discriminating and representativeness for the environments, Ibadan 2019 is deemed to be a superior environment. The selected accessions are recommended as parental lines in breeding programs for grain yield improvement in Ibadan or Ikenne or similar agro-ecological zones.

Adegboyega TT, Abberton MT, Abdelgadir AH, Mahamadi D, Olaniyi OA, Ofodile S, Babalola OO. 2021. Variation in winged bean (Psophocarpus tetragonolobus) growth parameters, seed yield, nodulation, and nitrogen fixation. Asian J Agric 5: 61-71. Underutilized legumes are widely distributed in tropical agriculture and associated with low yield. Thus they have not really been fully explored due to lack of research investment, breeding programs targeting crop improvement, marketing, and low awareness of nutritional benefits. This study was conducted to determine the variation in growth parameters, seed yield, nodulation, and nitrogen fixation in winged bean (Psophocarpus tetragonolobus) germplasms. High genotypic and phenotypic coefficients of variances were observed in traits evaluated. The combined analysis of variance for five variables of 25-winged bean accessions showed that replication by year interaction was statistically significant (p ? 0.0001) for nodule parameters and dry shoot weight while it was not significant (p = > 0.05) for dry root weight, and total biomass. Significant variations (p = ? 0.05) were observed among the accessions on some growth parameters. The genetic variability of winged beans could be carefully exploited to provide higher grain yield, as well as other economic and important traits to boost food security and conservation of the plant genetic materials.

Yam (Dioscorea spp.) is a monocotyledonous herbaceous vine plant grown in the tropics and subtropics. It is a multi-species plant with varied intra- and interspecific ploidy levels. Of the 600 species, 11 are cultivated staple supporting the livelihood of over 300 million people. The paucity of information on ploidy and the genomic constitution is a significant challenge to the crop’s genetic improvement through crossbreeding. The objective of this study was to investigate the ploidy levels of 236 accessions across six cultivated and two wild species using chromosome counting, flow cytometry and genotyping-based ploidy determination methods. Results obtained from chromosome counting and genotyping-based ploidy determination were in agreement. In majority of the accessions, chromosome counting and flow cytometry were congruent, allowing future rapid screening of ploidy levels using flow cytometry. Among cultivated accessions, 168 (71%) were diploid, 50 (21%) were triploid, and 12 (5%) were tetraploid. Two wild species included in the study were diploids. Resolution of ploidy level in yams offers opportunities for implementing successful breeding programmes through intra- and inter-specific hybridization.

White yam (Dioscorearotundata Poir.) is one of the most important tuber crops in West Africa, where it is indigenous and represents the largest repository of biodiversity through several years of domestication, production, consumption, and trade. In this study, the genotyping-by-sequencing (GBS) approach was used to sequence 814 genotypes consisting of genebank landraces, breeding lines, and market varieties to understand the level of genetic diversity and pattern of the population structure among them. The genetic diversity among different genotypes was assessed using three complementary clustering methods, the model-based admixture, discriminant analysis of principal components (DAPC), and phylogenetic tree. ADMIXTURE analysis revealed an optimum number of four groups that matched with the number of clusters obtained through phylogenetic tree. Clustering results obtained from ADMIXTURE analysis were further validated using DAPC-based clustering. Analysis of molecular variance (AMOVA) revealed high genetic diversity (96%) within each genetic group. A network analysis was further carried out to depict the genetic relationships among the three genetic groups (breeding lines, genebank landraces, and market varieties) used in the study. This study showed that the use of advanced sequencing techniques such as GBS coupled with statistical analysis is a robust method for assessing genetic diversity and population structure in a complex crop such as white yam.

Yam (Dioscorea spp.) is a monocotyledonous herbaceous vine plant grown in the tropics and subtropics. It is a multi-species plant with varied intra- and interspecific ploidy levels. Of the 600 species, 11 are cultivated supporting the livelihood of over 300 million people. The paucity of information on ploidy and the genomic constitution is a significant challenge to the crop’s genetic improvement through crossbreeding. The objective of this study was to investigate the ploidy levels of 236 accessions across six cultivated and two wild species using chromosome counting, flow cytometry and genotyping-based ploidy determination methods. Results obtained from chromosome counting and genotyping-based ploidy determination were in agreement. In majority of the accessions, chromosome counting and flow cytometry were congruent, allowing future rapid screening of ploidy levels using flow cytometry. Among cultivated accessions, 168 (71%) were diploid, 50 (21%) were triploid, and 12 (5%) were tetraploid. Two wild species included in the study were diploids. Resolution of ploidy level in yams offers opportunities for implementing successful breeding programmes through intra- and interspecific hybridization.

Pollock, C. J., Humphreys, M. O., Morris, P., Abberton, M. T., Humphreys, M. W. (2005). Biotechnological approaches to temperate forage improvement. Grassland Future Perspectives, Reynolds, S. Food and Agriculture Organization, Rome, 281-302

White clover is a short-lived perennial whose persistence is greatly affected by abiotic stresses, particularly drought. The aim of this work was to characterize its molecular response to water deficit and recovery following re-hydration to identify targets for the breeding of tolerant varieties. We created a white clover reference transcriptome of 16,193 contigs by deep sequencing (mean base coverage 387x) four Suppression Subtractive Hybridization (SSH) libraries (a forward and a reverse library for each treatment) constructed from young leaf tissue of white clover at the onset of the response to drought and recovery. Reads from individual libraries were then mapped to the reference transcriptome and processed comparing expression level data. The pipeline generated four robust sets of transcripts induced and repressed in the leaves of plants subjected to water deficit stress (6,937 and 3,142, respectively) and following re-hydration (6,695 and 4,897, respectively). Semi-quantitative polymerase chain reaction was used to verify the expression pattern of 16 genes. The differentially expressed transcripts were functionally annotated and mapped to biological processes and pathways. In agreement with similar studies in other crops, the majority of transcripts up-regulated in response to drought belonged to metabolic processes, such as amino acid, carbohydrate, and lipid metabolism, while transcripts involved in photosynthesis, such as components of the photosystem and the biosynthesis of photosynthetic pigments, were up-regulated during recovery. The data also highlighted the role of raffinose family oligosaccharides (RFOs) and the possible delayed response of the flavonoid pathways in the initial response of white clover to water withdrawal. The work presented in this paper is to our knowledge the first large scale molecular analysis of the white clover response to drought stress and re-hydration. The data generated provide a valuable genomic resource for marker discovery and ultimately for the improvement of white clover.

Clovers (Trifoliumspecies) are a large and widespread genus of legumes. Recent work supports the Mediterranean origin of the genus in the Early Miocene period, and centres of diversity for clovers occur in the Eastern Mediterranean, East Africa and South America. A number of clovers are of agricultural importance as forage species in grassland agriculture around the world, particularly in temperate areas. White clover (Trifolium repensL.) is the most important legume of grazed pastures, and red clover (T. pratense) is widely cut and conserved as a winter feed. We consider the current state of collected resources in clovers, which have been collated in the most important databases. These are European Internet Search Catalogue (EURISCO), germplasm resources information network and system-wide information network for genetic resources together with the collection held at AgResearch, New Zealand. In total, collections have been made of 204 species with 48 species having more than 100 accessions in these databases. As expected, the majority of accessions are of the agriculturally important species. The geographical origin of collected material is outlined and, for the major species, accessions are broken down according to their status, e.g. wild population, breeders' lines. We then describe some of the ways in which genetic resources of white and red clovers and their relatives have been used in the breeding of these species. These include introgression of stress tolerance traits, targetting improvements in resource use efficiency and increasing seed yield.

Ruminant farming is an important component of the human food chain. Ruminants can use offtake from land unsuitable for cereal crop cultivation via interaction with the diverse microbial population in their rumens. The rumen is a continuous flow fermenter for the digestion of ligno-cellulose, with microbial protein and fermentation end-products incorporated by the animal directly or during post-ruminal digestion. However, ruminal fermentation is inefficient in capturing the nutrient resource presented, resulting in environmental pollution and generation of greenhouse gases. Methane is generated as a consequence of ruminal fermentation and poor retention of ingested forage nitrogen causes nitrogenous pollution of water and land and contributes to the generation of nitrous oxide. One possible cause is the imbalanced provision of dietary substrates to the rumen micro-organisms. Deamination of amino acids by ammonia-producing bacteria liberates ammonia which can be assimilated by the rumen bacteria and used for microbial protein synthesis. However, when carbohydrate is limiting, microbial growth is slow, meaning low demand for ammonia for microbial protein synthesis and excretion of the excess. Protein utilisation can therefore be improved by increasing the availability of readily fermentable sugars in forage or by making protein unavailable for proteolysis through complexing with plant secondary products. Alternatively, realisation that grazing cattle ingest living cells has led to the discovery that plant cells undergo endogenous, stress-mediated protein degradation due to the exposure to rumen conditions. This presents the opportunity to decrease the environmental impact of livestock farming by using decreased proteolysis as a selection tool for the development of improved pasture grass varieties.

White clover (Trifolium repens L.) is a forage legume of considerable economic importance in temperate agricultural systems. It has a strong self-incompatibility system. The molecular basis of self-incompatibility in T. repens is unknown, but it is under the control of a single locus, which is expressed gametophytically. To locate the self-incompatibility locus (S locus) in T. repens, we carried out cross-pollination experiments in an F(1) mapping population and constructed a genetic linkage map using amplified fragment length polymorphism and simple sequence repeat markers. As the first step in a map-based cloning strategy, we locate for the first time the S locus in T. repens on a genetic linkage map, on the homoeologous linkage group pair 1 (E), which is broadly syntenic to Medicago truncatula L. chromosome 1. On the basis of this syntenic relationship, the possibility that the S locus may or may not possess an S-RNase gene is discussed.

Island populations are often thought to be more susceptible to the loss of genetic diversity as a consequence of limited population size and genetic drift, greater susceptibility to detrimental stochastic events and low levels of immigration. However the geographic isolation of islands may create refuges for native crop species whose genetic diversity is threatened from the genetic erosion occurring in mainland areas as a result of crop-wild gene flow and genetic swamping. Many UK islands remain uncharacterised in terms of plant genetic diversity. In this study we compared the genetic diversity of mainland populations and landraces of Trifolium repens with wild populations collected from the islands surrounding the UK, including the island of Hirta in the St Kildan archipelago. Individuals from St Kilda represent a unique conservation resource, with populations both highly differentiated from UK mainland populations and genetically distinct from cultivated varieties, whilst able to retain diversity through limited human influence on the islands. In contrast, there is relative genetic similarity of wild UK populations to cultivated forms highlighted in mainland populations, but with geographic barriers preventing complete homogenisation of the mainland UK genepool. We underline the need for conservation priorities to include common species that are threatened by gene flow from cultivation, and draw attention to the potential of islands to preserve natural levels of genetic diversity.

Moorby, J. M., Kingston-Smith, A. H., Abberton, M. T., Humphreys, M. O., Theodorou, M. K. (2009). Improvement of forages to increase the efficiency of nitrogen and energy use by ruminants. Pages 39-65 in: Garnsworthy, P. C., Wiseman, J. (Eds). Recent Advances in Animal Nutrition 2008. EAN: 9781904761044 University of Nottingham, 42nd Feed Conference, September 2-4 2008.

Interspecific hybrids between white clover and the annual, profuse flowering ball clover have been developed as a strategy to improve the potential seed yield of white clover. The seed yield and seed yield components of advanced white clover x ball clover hybrids were compared with white clover in replicated field experiments over two harvest years. The backcross 3 hybrid had more reproductive nodes per stolon than the control variety 'AberHerald' which was reflected in more inflorescences per unit area but inflorescences with fewer florets and seeds. The hybrid had a greater potential seed yield than the control variety in 2002 but not 2001. Backcross 3 hybrids have also been produced across the range of leaf size categories. Medium and large leaved hybrids produced significantly more inflorescences and a higher potential seed yield than control varieties of comparable leaf size in both harvest years. The small leaved hybrid was not significantly greater than the control variety 'AberDale' in either inflorescence production or seed yield.

Two field experiments were carried out to analyze the performance of white clover varieties differing in their resistance to stem nematode. Varieties were compared under grazing and cutting regimes in mixed swards with or without the addition of nematode and dry matter yields of white clover and perennial ryegrass recorded over a 3-year period. The resistant variety did not show a yield advantage in the absence of nematode but did so in the presence of nematode in the first year and for several of the sampling dates in the second year. White clover yields under grazing were significantly less than under cutting in both experiments. The hypothesis that the additional stress of grazing would increase the benefits of resistance relative to a cutting management was not supported.

The genus Trifolium (Leguminosae) or clovers includes a number of agriculturally important forage species. In particular Trifolium repens (white clover) and Trifolium pratense (red clover) are widely used in temperate livestock agriculture. Interspecific crossing programmes within this genus have been carried out for more than 50 years, in many cases involving embryo rescue or ovule culture techniques. In general, the aims of these have been twofold: to aid understanding of evolutionary relationships within the genus and to introgress useful traits, predominantly into white or red clover. In the case of white clover, the greatest efforts have been made with respect to crossing with Trifolium nigrescens and Trifolium ambiguum. Novel germplasm has been developed and useful traits transferred, although no material has yet been commercialized. The main emphasis with respect to red clover has been increasing longevity through crosses with more persistent species, particularly Trifolium medium. Again, this work has not yet resulted in new varieties. Wider crossing programmes, allied in recent years to comparisons at the DNA level, have shed considerable light on the systematics and evolution of the genus. The future of interspecific hybridization for improvement of the agriculturally important clovers and the role of molecular approaches in this are discussed.

White clover ( Trifolium repens L.) is a forage legume widely used in combination with grass in pastures because of its ability to fix nitrogen. We have constructed a bacterial artificial chromosome (BAC) library of an advanced breeding line of white clover. The library contains 37 248 clones with an average insert size of approximately 85 kb, representing an approximate 3-fold coverage of the white clover genome based on an estimated genome size of 960 Mb. The BAC library was pooled and screened by polymerase chain reaction (PCR) amplification using both white clover microsatellites and PCR-based markers derived from Medicago truncatula , resulting in an average of 6 hits per marker; this supports the estimated 3-fold genome coverage in this allotetraploid species. PCR-based screening of 766 clones with a multiplex set of chloroplast primers showed that only 0.5% of BAC clones contained chloroplast-derived inserts. The library was further evaluated by sequencing both ends of 724 of the clover BACs. These were analysed with respect to their sequence content and their homology to the contents of a range of plant gene, expressed sequence tag, and repeat element databases. Forty-three microsatellites were discovered in the BAC-end sequences (BESs) and investigated as potential genetic markers in white clover. The BESs were also compared with the partially sequenced genome of the model legume M. truncatula with the specific intention of identifying putative comparative-tile BACs, which represent potential regions of microsynteny between the 2 species; 14 such BACs were discovered. The results suggest that a large-scale BAC-end sequencing strategy has the potential to anchor a significant proportion of the genome of white clover onto the gene-space sequence of M. truncatula.

Red clover (Trifolium pratense L.) is a globally significant forage legume in pastoral livestock farming systems. It is an attractive component of grassland farming, because of its high yield and protein content, nutritional value and ability to fix atmospheric nitrogen. Enhancing its role further in sustainable agriculture requires genetic improvement of persistency, disease resistance, and tolerance to grazing. To help address these challenges, we have assembled a chromosome-scale reference genome for red clover. We observed large blocks of conserved synteny with Medicago truncatula and estimated that the two species diverged ~23 million years ago. Among the 40,868 annotated genes, we identified gene clusters involved in biochemical pathways of importance for forage quality and livestock nutrition. Genotyping by sequencing of a synthetic population of 86 genotypes show that the number of markers required for genomics-based breeding approaches is tractable, making red clover a suitable candidate for association studies and genomic selection., Scientific Reports, 5, ISSN:2045-2322

Fothergill, M., Abberton, M. T., Collins, R. P., Michaelson-Yeates, T. P. T., Williams, T. A., Marshall, A. H., Rees, M. E. (2006). Breeding forage legumes for the less favoured areas. International Journal of Biodiversity Science and Management, 2, (3), 209-212. 'Sustainable Uplands: Future Scenarios for People, Environment and Landscape' - a Future Vision Conference and Workshop for the Uplands, ICU-C Cumbria, June 2006, Soane, I., Shaw, H.(Eds).

White clover (Trifolium repens L.) and red clover (T. pratense L.) are the most important legumes of temperate pastures. The former is used largely in systems based around sheep or cattle grazing and is grown together with a companion grass. Breeding aims to optimize the white clover contribution to the sward. This means that yield per se is not the aim but rather to take full advantage of the benefits of white clover; in particular, nitrogen fixation, high protein content, digestibility, mineral content and high intake. The objective is an agronomically and, as far as possible, nutritionally balanced sward, thus persistence of white clover and yield stability over a number of years are key goals. A considerable focus of germplasm improvement has therefore been overcoming biotic and abiotic stresses to clover performance. The former include not only pests and diseases but also the impact of the ruminant animal and the competitive interaction with the companion grass, while abiotic stress could be loosely defined as ‘winter hardiness’ and ‘summer survival’ depending on the site. In recent years the focus of many breeding efforts has shifted to give more consideration to the effects of variation within white clover germplasm on animal performance and the environment. Beneficial effects on productivity have been known for many years, but recent studies of the impact of forage diets on meat and milk quality have opened up new opportunities for improvement. Diffuse pollution of nitrogen and phosphorus from agricultural sources is high on the environmental protection agenda of many governments. Breeding efforts are now being made to reduce the contribution of clovers to both direct (leaching) and indirect (through animal returns) pollution. In particular, recent insights into mechanisms affecting protein breakdown in the rumen and silo offer new prospects for breeding interventions to reduce environmental impacts.Molecular marker methods are being developed in white clover and the transfer and use of resources and information accumulating in the model legumes Medicago truncatula and Lotus japonicus is likely to be a major route by which the power of genomic approaches is translated into forage legume improvement. Hybrids of white clover and related species have been developed to introgress key traits; namely, drought tolerance, grazing tolerance of large leaf types and enhanced seed yield, for which only limited genetic variation is present within the white clover gene pool.Red clover is less persistent than white clover, is typically cut three or more times in a season and is used to make silage for winter feed. Although it is often grown with a companion grass, monocultures are common and yield per se as well as persistency and pest and disease resistance are major breeding aims. Fewer agronomic studies and less germplasm improvement have been carried out in this species and molecular studies are not as well advanced although, as with white clover, future developments are likely to benefit greatly from a close relationship to model legumes. Red clover brings considerable benefits in terms of animal production and meat and milk quality. These aspects, alongside approaches to reduce nitrogenous pollution from the silo, represent considerable opportunities for variety development.

Marshall, A. H., Williams, T. A., Olyott, P., Abberton, M. T., Michaelson-Yeates, T. P. T. (2005). Forage yield and persistency of Trifolium repens x Trifolium nigrescens hybrids under rotational sheep grazing. Grass and Forage Science, 60, (1), 68-73 KEYWORDS: Trifolium repens ? Trifolium nigrescens ? dry matter production ? rotational sheep grazing ? persistency

Marshall, A. H., Williams, T. A., Abberton, M. T., Michaelson-Yeates, T. P. T., Olyott, P., Powell, H. G. (2004). Forage quality of white clover (Trifolium repens L.), Caucasian clover (T. ambiguum M. Bieb.) hybrids and their grass companion when grown over three harvest years. Grass and Forage Science, 59, (1), 91-99

A framework genetic map of white clover (Trifolium repens L.) has been constructed using an F2 progeny set derived from the intercross of fourth and fifth generation inbred genotypes carrying a self-fertile mutation (Sf). White clover SSR (TRSSR) and AFLP markers were used to derive a map with 135 markers (78 TRSSR loci and 57 AFLP loci) assigned to 18 linkage groups (LGs) with a total map length of 825 cm. Sixteen of these LGs are presumed to correspond to the 16 chromosomes of the white clover karyotype. A limited number of multiple loci were detected. Substantial segregation distortion was observed for both mapped and unmapped loci, with a bias towards heterozygous types and a preponderance of distorted markers on certain LGs. This observation, along with a high degree of residual heterozygosity within the inbred parental genotypes, suggests that reduced individual fitness due to loss of heterosis is a major effect for white clover and will limit the applicability of F2 or backcross mapping strategies for this species. A core set of map-assigned co-dominant, single locus SSR markers has been defined for whole genome scans of genetic variability in white clover.

Marshall, A. H., Abberton, M. T., Williams, T. A., Michaelson-Yeates, T. P. T., Powell, H. G. (2003). Forage quality of Trifolium repens L. x T. nigrescens Viv. hybrids. Grass and Forage Science, 58, (3), 295-301 Sponsorship: Department for the Environment, Food and Rural Affairs (DEFRA)

Marshall, A. H., Williams, T. A., Abberton, M. T., Michaelson-Yeates, T. P. T., Powell, H. G. (2003). Dry matter production of white clover (Trifolium repens L.), Caucasian clover (T. ambiguum M. Bieb.) and their associated hybrids when grown with a grass companion over 3 harvest years. Grass and Forage Science, 58, (1), 63-69.

Williams, T. A., Abberton, M. T., Rhodes, I. (2003). Performance of white clover varieties combined in blends and alone when grown with ryegrass under sheep and cattle grazing. Grass and Forage Science, 58, (1), 90-93. Sponsorship: Germinal Holdings Ltd

Three experiments were carried out to investigate the dry matter production of nine white clover (Trifolium repens L.) varieties grown with the same variety of perennial ryegrass (Lolium perenne L.) and subjected to rotational grazing by sheep over a period of 10 years. The experiments differed only in the nitrogen fertilizer added: no nitrogen, 100 kg/ha per year and 200 kg/ha per year. Dry matter (DM) yields of white clover and perennial ryegrass components of the sward were recorded at approximately 6-week intervals during the growing season.Without applied nitrogen, 10-year mean white clover annual DM yields ranged from 2·89–4·54 t/ha. Mean clover content for all varieties ranged from 31–50% for the first 9 years, declining to 20% in the 10th year. The most consistently high yielding variety, AberVantage, gave yields greater than 3·5 t/ha in every year. Similar results were observed for the variety Aran. The 10-year mean annual DM yield of the perennial ryegrass companion (variety Talbot) ranged from 4·41–5·57 t/ha depending on the white clover variety in the mixture. Variation in perennial ryegrass DM yield was of a similar range and pattern as for white clover.In the experiment in which 100 kg N/ha per year was added, 10-year mean white clover annual DM yields ranged from 2·27–3·87 t/ha. The varieties AberVantage and Aran showed yields that fell below 3 t/ha per year in only 1 or 2 years, respectively. Mean clover content for all varieties ranged from 26–38% for the first 9 years, declining to 17·5% in the 10th year. There was no consecutive annual decline in white clover DM yields. The 10-year mean annual DM yields of the perennial ryegrass companion ranged from 6·03–7·57 t/ha.With applied nitrogen fertilizer of 200 kg N/ha per year (Experiment 3), 10-year mean white clover annual DM yields ranged from 1·21–2·67 t/ha. Mean clover content ranged from 12–27% for the 10 years. AberVantage and Aran showed yields consistently above 2 t/ha. No general decline in white clover DM yields was observed although some varieties showed consecutive annual reductions in years 8, 9 and 10. The 10-year mean annual DM perennial ryegrass yields ranged from 7·27–8·68 t/ha.The results show that, under rotational grazing by sheep, white clover can give reliably high yields over a 10-year period. With moderate to high applications of nitrogen fertilizer white clover yields were lower than without nitrogen but they were consistent and sufficient to make a considerable impact on forage quality. There was no evidence for a general decline in clover content until the 10th year of the experiment and no cyclical pattern of annual variation in white clover yield was observed. The implications of these results for the role of white clover in UK agriculture are discussed.

Bulked segregant analysis (BSA) using the technique of amplified fragment length polymorphism (AFLPTM) was carried out on backcross hybrids between the important pasture species white clover and its relative Trifolium ambiguum. The introgression of the rhizomatous trait fromTrifolium ambiguum, previously shown to be associated with enhanced drought tolerance but which takes eighteen months to manifest itself, was analysed in a series of third generation backcross families with white clover as the recurrent parent. Eleven families differed in the proportions of plants classified according to rhizome content. Three of these families were selected for BSA-AFLP based on the creation of separate DNA pools from plants with large amounts of rhizomes or no rhizomes. Following an initial screen with119 primer combinations, 53 combinations were selected on the basis of the number of clear, reproducible polymorphisms they produced. Polymorphic bands were found that distinguished between rhizomatous and non-rhizomatous bulks in the three families. However, only one polymorphic band was found that unambiguously reflected the presence of the rhizomatous trait in individual plants in these families. This polymorphic band was also shown to be present in the BC2 parent, the BC1 and the original T. ambiguum parent. Given the aneuploid nature of the BC2 plants this band would appear to be a chromosome-specific marker tightly linked to the locus controlling rhizome development. The identification of a consistent marker for the rhizomatous trait opens up the possibility of applying marker assisted introgression and thereby facilitating the rapid incorporation of this trait into diverse germplasm.

Interspecific hybridisation with Trifolium nigrescens Viv. is a possible strategy to improve the reproductive potential of white clover (Trifolium repens L.). Following the development of a fertile F1 hybrid, three generations of backcrossing have been carried out usingT. repens as the recurrent parent. Vegetative characteristics, stolon growth and seed yield components of the backcross (BC) 2 and 3 generations, as well as the parental species were measured on spaced plants grown in the field. Leaf size and plant spread of the BC2 and BC3 generations were less than T. repens but there was no difference in plant fresh weight. Numbers of inflorescences per plant and florets per inflorescence of the backcrosses were greater than T. repens however this was not reflected indifferences in seed yield per plant asT. repens had more seeds per floret and per plant than the backcrosses. Differences in stolon length, the proportion of flowering nodes and the pattern of axillary bud development were observed between T. repens and the backcrosses. Significant variation among the BC 3 generation for vegetative and reproductive traits was observed. Individual plants among the BC 3 generation were identified that combine high forage yield, substantial inflorescence production and good fertility, and these will form the basis of further selection.

Two generations of novel backcross hybrids between white clover (Trifolium repens L.) and the related species Trifolium ambiguum,with white clover as the recurrent parent, were analysed for forage quality characteristics. Water soluble carbohydrate (WSC) levels, nitrogen concentrations (%N), and dry matter digestibility (DMD) were recorded for the hybrids and parental species grown in deep soil bins with and without a companion grass. Half the experimental material was subjected to moisture stress by being left unwatered through the four-week period when measurements were made. Four such four -week cycles of drought were imposed. The first and second generation backcrosses had the highest values for WSC, but were lowest for N%. There was no difference between the different legume lines (parents and backcrosses) for DMD. No effects of either moisture stress or presence of a grass companion on quality traits were observed. The WSC and %N of the grass component differed significantly depending on which legume line it was grown with. The importance of these results for the potential use of these hybrids in agriculture is discussed.

Macduff, J. H., Abberton, M. T., Raistrick, N., Michaelson-Yeates, T. P. T. (2002). Low nitrate uptake white clover genotype - Nitrogen relations of a low nitrate uptake inbred line of white clover (Trifolium repens L.). Plant and Soil, 238, (2), 191-204 Sponsorship: MAFF; BBSRC

Background Red clover (Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress. Results Plants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A de novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing ‘transcript tags’. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress. Conclusions This de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-453) contains supplementary material, which is available to authorized users.

Williams, T. A., Abberton, M. T., Thornley, W. J., Rhodes, I. (2001). No relationship between leaf size and yield in medium leaf size white clover varieties under rotational sheep grazing and cutting. Grass and Forage Science, 56, (4), 412-417. Sponsorship: UK Ministry of Agriculture, Fisheries and Food

Williams, T. A., Abberton, M. T., Thornley, W. J., Rhodes, I. (2001). Relationships between the yield of perennial ryegrass and of small-leaved white clover under cutting or continuous grazing by sheep. Grass and Forage Science, 56, (3), 231-237. Sponsorship: MAFF