Fingerprint identification of white clover cultivars based on SSR molecular markers.

White clover (Trifolium repens L.) is an important perennial legume forage with high productivity and quality. To strengthen the basic research on the genetic characteristics, fingerprint identification and adaptability of white clover germplasm resources, Simple sequence repeat (SSR) molecular markers were applied to 10 white clover cultivars to assess the genetic diversity and related lines of white clover at the molecular level in order to lay a theoretical foundation for the selection of high-quality seeds and cultivars of white clover. A total of 120 different bands were amplified by 29 pairs of SSR primers with good polymorphism, of which 103 (89.5%) were polymorphic. Meanwhile, the PIC of each primer was 0.181-0.588, with an average of 0.329. Analysis of molecular variance revealed that 57% of the genetic variation occurred within cultivars and 43% occurred among cultivars. The results of cluster analysis and the principal coordinate analysis revealed that the parental relationships of the 10 cultivars, with the 'Purple' cultivar very distantly related to the other 9 cultivars and the closest parental relationship between 'Ladino' and 'Sulky'. The fingerprints constructed by three representative primers (gtrs679, gtrs319, and gtrs678) have a strong identification ability. In summary, the SSR markers had good polymorphism and could be used for DNA fingerprint analysis of white clover cultivars.

Pedigree analysis of pre-breeding efforts in Trifolium spp. germplasm in New Zealand.

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.

Agronomic and qualitative characterization of multi-cut berseem clover (Trifolium alexandrinum L.) cultivars.

BACKGROUND: Berseem clover is the main forage crop grown in Mediterranean regions. There are plenty of cultivars that possess variability in their productivity and quality among the different cuts. Therefore, accurate agronomic and qualitative characterization is crucial for selecting the most promising cultivars for breeding and feeding purposes. In the present study, the agronomic characteristics, ruminal degradability and fermentation measures of five cuts of the five most prominent Egyptian beseem clover cultivars (Helaly, Serw, Giza6, Gemmeza1 and Sakha4) were evaluated. RESULTS: The Giza6 cultivar produced a significantly higher fresh yield and high whole plant dry matter content on the 3rd cut. Giza6 was among the superior cultivars in crude protein content and had the highest values of gas production at the 3rd cut, and ruminal degraded acid detergent fiber and propionate concentrations at the 2nd cut, at the same time as presenting the lowest ammonia concentrations at the 3rd and 5th cuts. Overall, the 3rd cut produced significantly higher fresh yield across all cultivars, except for Sakha4. The 3rd cut also produced a higher leaf to stem ratio compared to the 4th and 5th cuts. The 1st three cuts had higher degraded neutral detergent fiber than the 4th and 5th cuts. The 1st cut was characterized by low propionate concentration in the Helaly and Gemmeza1 cultivars compared to the other cuts. CONCLUSION: Expanded production of the high-yielding, high-quality cultivar Giza6 is recommended. When harvesting, three cuts is optimal for berseem clover cultivation for breeding and feeding programs. © 2020 Society of Chemical Industry.

Comparative Mitogenome Analysis of the Genus Trifolium Reveals Independent Gene Fission of ccmFn and Intracellular Gene Transfers in Fabaceae.

The genus Trifolium is the largest of the tribe Trifolieae in the subfamily Papilionoideae (Fabaceae). The paucity of mitochondrial genome (mitogenome) sequences has hindered comparative analyses among the three genomic compartments of the plant cell (nucleus, mitochondrion and plastid). We assembled four mitogenomes from the two subgenera (Chronosemium and Trifolium) of the genus. The four Trifolium mitogenomes were compact (294,911-348,724 bp in length) and contained limited repetitive (6.6-8.6%) DNA. Comparison of organelle repeat content highlighted the distinct evolutionary trajectory of plastid genomes in a subset of Trifolium species. Intracellular gene transfer (IGT) was analyzed among the three genomic compartments revealing functional transfer of mitochondrial rps1 to nuclear genome along with other IGT events. Phylogenetic analysis based on mitochondrial and nuclear rps1 sequences revealed that the functional transfer in Trifolieae was independent from the event that occurred in robinioid clade that includes genus Lotus. A novel, independent fission event of ccmFn in Trifolium was identified, caused by a 59 bp deletion. Fissions of this gene reported previously in land plants were reassessed and compared with Trifolium.

Five species, many genotypes, broad phenotypic diversity: When agronomy meets functional ecology.

PREMISE OF THE STUDY: Current ecological theory can provide insight into the causes and impacts of plant domestication. However, just how domestication has impacted intraspecific genetic variability (ITV) is unknown. We used 50 ecotypes and 35 cultivars from five grassland species to explore how selection drives functional trait coordination and genetic differentiation. METHODS: We quantified the extent of genetic diversity among different sets of functional traits and determined how much genetic diversity has been generated within populations of natural ecotypes and selected cultivars. KEY RESULTS: In general, the cultivars were larger (e.g., greater height, faster growth rates) and had larger and thinner leaves (greater SLA). We found large (average 63%) and trait-dependent (ranging from 14% for LNC to 95.8% for growth rate) genetic variability. The relative extent of genetic variability was greater for whole-plant than for organ-level traits. This pattern was consistent within ecotypes and within cultivars. However, ecotypes presented greater ITV variability. CONCLUSIONS: The results indicated that genetic diversity is large in domesticated species with contrasting levels of heritability among functional traits and that selection for high yield has led to indirect selection of some associated leaf traits. These findings open the way to define which target traits should be the focus in selection programs, especially in the context of community-level selection.

Characterization of new transposable element sub-families from white clover (Trifolium repens) using PCR amplification.

Transposable elements (TEs) dominate the landscapes of most plant and animal genomes. Once considered junk DNA and genetic parasites, these interspersed, repetitive DNA elements are now known to play major roles in both genetic and epigenetic processes that sponsor genome variation and regulate gene expression. Knowledge of TE consensus sequences from elements in species whose genomes have not been sequenced is limited, and the individual TEs that are encountered in clones or short-reads rarely represent potentially canonical, let alone, functional representatives. In this study, we queried the Repbase database with eight BAC clones from white clover (Trifolium repens), identified a large number of candidate TEs, and used polymerase chain reaction and Sanger sequencing to create consensus sequences for three new TE families. The results show that TE family consensus sequences can be obtained experimentally in species for which just a single, full-length member of a TE family has been sequenced.

Plant-mediated green synthesis of silver nanoparticles using Trifolium resupinatum seed exudate and their antifungal efficacy on Neofusicoccum parvum and Rhizoctonia solani.

In recent years, biosynthesis and the utilisation of silver nanoparticles (AgNPs) has become an interesting subject. In this study, the authors investigated the biosynthesis of AgNPs using Trifolium resupinatum (Persian clover) seed exudates. The characterisation of AgNPs were analysed using ultraviolet-visible spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM) and Fourier transform infra-red spectroscopy. Also, antifungal efficacy of biogenic AgNPs against two important plant-pathogenic fungi (Rhizoctonia solani and Neofusicoccum Parvum) in vitro condition was evaluated. The XRD analysis showed that the AgNPs are crystalline in nature and have face-centred cubic geometry. TEM images revealed the spherical shape of the AgNPs with an average size of 17 nm. The synthesised AgNPs were formed at room temperature and kept stable for 4 months. The maximum distributions of the synthesised AgNPs were seen to range in size from 5 to 10 nm. The highest inhibition effect was observed against R. solani at 40 ppm concentration of AgNPs (94.1%) followed by N. parvum (84%). The results showed that the antifungal activity of AgNPs was dependent on the amounts of AgNPs. In conclusion, the AgNPs obtained from T. resupinatum seed exudate exhibit good antifungal activity against the pathogenic fungi R. solani and N. Parvum.

The single evolutionary origin of chlorinated auxin provides a phylogenetically informative trait in the Fabaceae.

Chlorinated auxin (4-chloroindole-3-acetic acid, 4-Cl-IAA), a highly potent plant hormone, was once thought to be restricted to species of the tribe Fabeae within the Fabaceae, until we recently detected this hormone in the seeds of Medicago, Melilotus and Trifolium species. The absence of 4-Cl-IAA in the seeds of the cultivated species Cicer aeritinum from the Cicerae tribe, immediately basal to the Fabeae and Trifolieae tribes, suggested a single evolutionary origin of 4-Cl-IAA. Here, we provide a more robust phylogenetic placement of the ability to produce chlorinated auxin by screening key species spanning this evolutionary transition. We report no detectable level of 4-Cl-IAA in Cicer echinospermum (a wild relative of C. aeritinum) and 4 species (Galega officinalis, Parochetus communis, Astragalus propinquus and A. sinicus) from tribes or clades more basal or sister to the Cicerae tribe. We did detect 4-Cl-IAA in the dry seeds of 4 species from the genus Ononis that are either basal to the genera Medicago, Melilotus and Trigonella or basal to, but still within, the Fabeae and Trifolieae (ex. Parochetus) clades. We conclude that the single evolutionary origin of this hormone in seeds can be used as a phylogenetically informative trait within the Fabaceae.

Antioxidant action of six Trifolium species in blood platelet experimental system in vitro.

This study includes a comparative evaluation of antioxidant effects of plant extracts (1.5-50.0 µg/ml), derived from six clover (Trifolium) species: T. alexandrinum L., T. fragiferum L., T. hybridum L., T. incarnatum L., T. resupinatum var. majus Boiss., and T. resupinatum var. resupinatum L. Chemical profiles of the extracts contained three or four groups of (poly)phenolic compounds such as phenolic acids, clovamides, isoflavones, and other flavonoids. Antioxidant properties of Trifolium extracts were assessed as the efficacy to reduce oxidative and nitrative damage to blood platelets, exposed to 100 µM peroxynitrite-induced oxidative stress in vitro. Antioxidant actions of the examined extracts were determined by the following biomarkers of oxidative stress: thiol groups, 3-nitrotyrosine, lipid hydroperoxides, and thiobarbituric acid-reactive substances (TBARS). Despite the significant differences in the chemical composition (the total phenolic concentrations varied between 11.30 and 52.55 mg/g of dry mass) of Trifolium extracts, we observed noticeable protective effects of almost all tested plant preparations. The T. alexandrinum extract, containing the highest concentration of phenols, was the most effective antioxidant among the tested extracts. On the other hand, the T. incarnatum extract, which contained a comparable total phenolic content (49.77 mg/g), was less efficient in prevention of tyrosine nitration and generation of TBARS. These findings indicate on the important role of individual phenolic components of the examined clover extracts for the final antioxidative effects. Antioxidative properties of the remaining extracts were noticeably weaker.

Evolutionary origin of highly repetitive plastid genomes within the clover genus (Trifolium).

BACKGROUND: Some clover species, particularly Trifolium subterraneum, have previously been reported to have highly unusual plastomes, relative to closely related legumes, enlarged with many duplications, gene losses and the presence of DNA unique to Trifolium, which may represent horizontal transfer. In order to pinpoint the evolutionary origin of this phenomenon within the genus Trifolium, we sequenced and assembled the plastomes of eight additional Trifolium species widely sampled from across the genus. RESULTS: The Trifolium plastomes fell into two groups: those of Trifolium boissieri, T. strictum and T. glanduliferum (representing subgenus Chronosemium and subg. Trifolium section Paramesus) were tractable, assembled readily and were not unusual in the general context of Fabeae plastomes. The other Trifolium species ("core Trifolium") proved refractory to assembly mainly because of numerous short duplications. These species form a single clade, which we call the "refractory clade" (comprising subg, Trifolium sections Lupinaster, Trifolium, Trichocephalum, Vesicastrum and Trifoliastrum). The characteristics of the refractory clade are the presence of numerous short duplications and 7-15% longer genomes than the tractable species. Molecular dating estimates that the origin of the most recent common ancestor (MRCA) of the refractory clade is approximately 13.1 million years ago (MYA). This is considerably younger than the estimated MRCA ages of Trifolium (c. 18.6 MYA) and Trifolium subg. Trifolium (16.1 MYA). CONCLUSIONS: We conclude that the unusual repetitive plastome type previously characterized in Trifolium subterraneum had a single origin within Trifolium and is characteristic of most (but not all) species of subgenus Trifolium. It appears that an ancestral plastome within Trifolium underwent an evolutionary change resulting in plastomes that either actively promoted, were permissive to, or were unable to control, duplications within the genome. The precise mechanism of this important change in the mode and tempo of plastome evolution deserves further investigation.

Evaluation of a diverse red clover collection for clover rot resistance (Sclerotinia trifoliorum).

Sclerotinia trifoliorum Erikks. causes clover rot (clover cancer, Sclerotinia crown and root rot), an important disease in European red clover crops (Trifolium pratense L). The fungus infects plants in autumn through ascospores and entire fields can be destroyed by early spring. Although previous studies have evaluated various red clover populations for clover rot resistance, screening was often performed with one local isolate on just a few local varieties, often cultivars. Until today, no large collections of diverse red clover accessions have been screened. In this study, we studied the variation in clover rot susceptibility among 122 red clover accessions, including 85 accessions from the NPGS-USDA core collection. Cultivars (both diploid and tetraploid), landraces and wild accessions were included and different S. trifoliorum isolates were used. In a field experiment, plant yield, branching and susceptibility to mildew, rust and virus disease were scored for 122 red clover accessions. A similar collection of germplasm was screened for clover rot resistance by a bio-test on young plants using a mixture of five aggressive S. trifoliorum isolates. The effects of the variety type, ploidy level, growth habit, resistance to other diseases and levels of isoflavones (available for the NPGS-USDA collection) on clover rot susceptibility were determined. Possible sources of resistance were identified. Our red clover accessions differed significantly in susceptibility but no accession was completely resistant Three accessions (Maro, Tedi and No. 292) were significantly less susceptible than the other accessions. Intensive branching or a prostrate growth habit did not render plants more resistant. Accessions resistant to mildew or viruses were not more resistant to clover rot and accessions with high levels of isoflavones were not better protected against clover rot. On the other hand, tetraploid cultivars were on average 10% less susceptible than diploid cultivars. Cultivars were generally less susceptible than landraces and wild accessions. Allocating sources of resistance for breeding purposes is difficult. The best way to improve clover rot resistance may be to select and intercross resistant plants from cultivars with low susceptibility.

Eleven microsatellite markers for the mountain clover Trifolium montanum (Fabaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed in Trifolium montanum to investigate pollen dispersal patterns at landscape scale with a pollen-pool analysis (indirect parentage analysis) as well as in an experimental set-up with a direct paternity analysis. METHODS AND RESULTS: Screening 46 microsatellites developed for T. repens yielded four markers usable in T. montanum. Seven additional ones have been developed specifically for the target species using a 454-sequencing approach. All markers were polymorphic, with an allele number ranging from two to 45 based upon 254 individuals sampled from four populations, and an exclusion probability of 0.999. CONCLUSIONS: These markers proved a useful and reliable molecular tool for use in population genetics and parentage studies of this common grassland herb.

Experimental evidence for the ancestry of allotetraploid Trifolium repens and creation of synthetic forms with value for plant breeding.

BACKGROUND: White clover (Trifolium repens) is a ubiquitous weed of the temperate world that through use of improved cultivars has also become the most important legume of grazed pastures world-wide. It has long been suspected to be allotetraploid, but the diploid ancestral species have remained elusive. Putative diploid ancestors were indicated by DNA sequence phylogeny to be T. pallescens and T. occidentale. Here, we use further DNA evidence as well as a combination of molecular cytogenetics (FISH and GISH) and experimental hybridization to test the hypothesis that white clover originated as a hybrid between T. pallescens and T. occidentale. RESULTS: T. pallescens plants were identified with chloroplast trnL intron DNA sequences identical to those of white clover. Similarly, T. occidentale plants with nuclear ITS sequences identical to white clover were also identified. Reciprocal GISH experiments, alternately using labeled genomic DNA probes from each of the putative ancestral species on the same white clover cells, showed that half of the chromosomes hybridized with each probe. F1 hybrids were generated by embryo rescue and these showed strong interspecific chromosome pairing and produced a significant frequency of unreduced gametes, indicating the likely mode of polyploidization. The F1 hybrids are inter-fertile with white clover and function as synthetic white clovers, a valuable new resource for the re-incorporation of ancestral genomes into modern white clover for future plant breeding. CONCLUSIONS: Evidence from DNA sequence analyses, molecular cytogenetics, interspecific hybridization and breeding experiments supports the hypothesis that a diploid alpine species (T. pallescens) hybridized with a diploid coastal species (T. occidentale) to generate tetraploid T. repens. The coming together of these two narrowly adapted species (one alpine and the other maritime), along with allotetraploidy, has led to a transgressive hybrid with a broad adaptive range.

Comparison of genome structure between white clover and Medicago truncatula supports homoeologous group nomenclature based on conserved synteny.

Computational analysis has been used to align the genetic map of white clover (Trifolium repens L.) with the draft genome sequence of the model legume species Medicago truncatula Gaertn. In silico comparison based on white clover expressed sequence tags that contain simple sequence repeat loci revealed substantial macrosynteny between the genomes of these two species, which are closely related within the Trifolieae tribe of the Fabaceae family. Six of the eight homoeologous chromosome groups (HGs) of allotetraploid white clover show predominant relationships with single M. truncatula (Mt) chromosomes, while the two remaining groups may have participated in an evolutionary reciprocal translocation event. On this basis, a new chromosome nomenclature system for allotetraploid white clover is proposed such that HG A = 3, HG B = 8, HG C = 7, HG D = 4, HG E = 1, HG F = 2, HG G = 5, and HG H = 6. A rationalized linkage map ordering system has also been demonstrated. Improved knowledge of the relationships between agricultural and model forage legume genomes will facilitate prediction of gene location for key agronomic traits for pasture production.

Molecular phylogenetics of the clover genus (Trifolium--Leguminosae).

Trifolium, the clover genus, is one of the largest genera of the legume family. We conducted parsimony and Bayesian phylogenetic analyses based on nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL intron sequences obtained from 218 of the ca. 255 species of Trifolium, representatives from 11 genera of the vicioid clade, and an outgroup Lotus. We confirm the monophyly of Trifolium, and propose a new infrageneric classification of the genus based on the phylogenetic results. Incongruence between the nrDNA and cpDNA results suggests five to six cases of apparent hybrid speciation, and identifies the putative progenitors of the allopolyploids T. dubium, a widespread weed, and T. repens, the most commonly cultivated clover species. Character state reconstructions confirm 2n=16 as the ancestral chromosome number in Trifolium, and infer a minimum of 19 instances of aneuploidy and 22 of polyploidy in the genus. The ancestral life history is hypothesized to be annual in subgenus Chronosemium and equivocal in subgenus Trifolium. Transitions between the annual and perennial habit are common. Our results are consistent with a Mediterranean origin of the genus, probably in the Early Miocene. A single origin of all North and South American species is hypothesized, while the species of sub-Saharan Africa may originate from three separate dispersal events.

A lineage-specific centromeric satellite sequence in the genus Trifolium.

We report the molecular structure, genomic organization, chromosomal distribution and evolutionary dynamics of TrR350, a satellite DNA isolated from the forage legume white clover (Trifolium repens L.; 2n = 4 x = 32). The basic repeating unit is an A+T rich 350 bp Hin dIII fragment with a complex dimeric structure consisting of an internal direct repeat of 156 bp packed between unrelated flanking sequences. Each 156 bp repeat has a conserved 24 bp motif repeating at two places. Most of the 24 bp short repeating units enclose a pentanucleotide CAAAA motif, presumed to be involved in breakage-reunion mechanism of tandemly repeating arrays. The dimers share high sequence homology among themselves while monomers within dimers show significant sequence divergence. Genomic Southern hybridization and/or fluorescence in situ hybridization (FISH) on 17 Trifolium species/subspecies revealed that it is a lineage-specific repeat confined to several species within the section Lotoidea originating in the Mediterranean region. The uniform length of the basic repeating unit and the centromeric localization in most of the species harbouring it reflects its extensive conservation in the lineage. However, the Hin dIII restriction profile in seven species also indicated independent evolution of this repeat.