Transfer of clover cyst nematode resistance from Trifolium nigrescens Viv. to T. repens L. by interspecific hybridisation

 Resistance to clover cyst nematode (Heterodera trifolii) has been successfully transferred from Trifolium nigrescens to T. repens by interspecific hybridisation. A sterile triploid hybrid (H-6909-5, 2n=3x=24) was initially produced with the aid of embryo culture. The hybrid was chromosome-doubled from axillary meristems by an in vitro colchicine method. Three chromosome-doubled plants were obtained, and these showed a marked increase in pollen stainability from 10% in 3x H-6909-5 to an average of 89% (range 88–91%) in 6x H-6909-5. T. nigrescens was a source of clover cyst nematode resistance. A mean of 23 (range 0–150) cysts per plant was recorded for T. nigrescens in comparison to a mean of 150 (range 50–240) cysts per plant for T. repens. The 3x and 6x interspecific hybrids were shown to be as resistant as the most resistant. T. nigrescens genotype and were significantly lower in cyst number per gram of root dry weight than the susceptible T. nigrescens and T. repens genotypes. Received: 11 July 1997 / Accepted: 15 July 1997     

On the origin of the cyanogenic polymorphism in Trifolium repens L.

Trifolium repens L. and Trifolium nigrescens Viv. are two of the approximately six cyanogenic species known in the genus Trifolium. The two species are closely related: T. nigrescens is considered to be one of the diploid ancestors of the amphidiploid T. repens . We studied morphology, meiosis and the cyanogenic system in T. repens (amphidiploid), T. nigrescens (diploid) and their reciprocal hybrids. A comparison of the enzyme linamarase in the species and hybrids shows that there is a general resemblance between their linamarases. Immunological studies indicate that the linamarases must have a somewhat different three-dimensional structure. These facts are consistent with the view that T. nigrescens (or an ancestral form of this species) has donated the Li gene of T. repens . The other putative parent, T. occidentale has probably not donated an active Li gene. The hypothesis of the origin of the Li gene does not explain its polymorphism in European populations of T. repens , as T. nigrescens is monomorphic for cyanogenesis and amphidiploids do not segregate for genes which are homozygous dominant in one of the parents. Segregation for Li could be caused by a gene mutation or a small exchange between homeologous chromosomes. The latter event is more probable. A nigrescens-repens exchange would give rise to a chromosomal region with reduced homology to both parental chromosomes. The genes in the region of exchange will be tightly linked due to diminished cross-over frequency. It has been known for years that Li has effects on the vegetative and reproductive characters in T. repens and we have recently shown that these effects must be the result of genes linked to Li . As the associated characters influence the fitness of the cyanotypes, not only the origin but also the maintenance of the cyanogenic polymorphism is closely related to the evolutionary history of T. repens .     

Evidence of Three Subspecies in Trifolium nigrescens Viv.

Germplasm accessions of Trifolium nigrescens Viv. were foundto belong to three distinct taxa which differed in morphology,hybrid seed-set in crosses among themselves and with white clover(T. repens L.), chromosomal distribution of rDNA genes, andrDNA internally transcribed spacer (ITS) region sequences. Thesetaxonomic groups correspond with T. nigrescens ssp. nigrescens,T. nigrescens ssp. petrisavii var.petrisavii and var. meneghinianum.The existence of var. meneghinianum has been rejected or ignoredin recent taxonomic treatments. The present study indicatesthat it is very distinctive and warrants at least subspeciesstatus. Phylogenetic analysis based on ITS sequences and podmorphology placed ssp. nigrescens as progenitor to the others.ITS sequences placed white clover closer to ssp. petrisaviibut neither DNA sequences nor rDNA chromosome locations clearlyshowed whether var. petrisavii or var. meneghinianum was closerto white clover. In contrast, results of hybridization studiesshowed that ssp. nigrescens gave full seed-set when pollinatedwith T. repens whereas both ssp. petrisavii var. petrisaviiand ssp. petrisavii var. meneghinianum gave poor seed-set. Allthree taxa showed close relationships with white clover andnone can clearly be shown to be a direct ancestor of T. repens.Copyright 2001 Annals of Botany Company Fabaceae, Trifolium nigrescens, Trifolium repens, internal transcribed spacer region (ITS), nuclear ribosomal DNA, interspecific hybridization     

Selective grazing of acyanogenic white clover: Variation in behaviour among populations of the slug Deroceras reticulatum

Summary Collections of the slug Deroceras reticulatum were made from grassland sites containing contrasting frequencies of the cyanogenic morph of white clover, Trifolium repens. In choice chamber experiments, slugs obtained from sites with a low frequency of cyanogenic clover showed a significantly greater degree of selective eating of acyanogenic morphs than slugs taken from a site containing a high frequency of cyanogenic clover. Differences in selectivity between populations were caused both by differences in the rate of initiation of feeding on cyanogenic morphs, and by differences in the extent of damage once feeding had been initiated. The implications of these results for the cyanogenic polymorphism of T. repens are discussed.     

A microsatellite map of white clover

The white clover (Trifolium repens) nuclear genome (n=2x=16) is an important yet under-characterised genetic environment. We have developed simple sequence repeat (SSR) genetic markers for the white clover genome by mining an expressed sequence tag (EST) database and by isolation from enriched genomic libraries. A total of 2,086 EST-derived SSRs (EST-SSRs) were identified among 26,480 database accessions. Evaluation of 792 EST-SSR primer pairs resulted in 566 usable EST-SSRs. Of these, 335 polymorphic EST-SSRs, used in concert with 30 genomic SSRs, detected 493 loci in the white clover genome using 92 F₁ progeny from a pair cross between two highly heterozygous genotypes—364/7 and 6525/5. Map length, as estimated using the joinmap algorithm, was 1,144 cM and spanned all 16 homologues. The R (red leaf) locus was mapped to linkage group B1 and is tightly linked to the microsatellite locus prs318c. The eight homoeologous pairs of linkage groups within the white clover genome were identified using 96 homoeologous loci. Segregation distortion was detected in four areas (groups A1, D1, D2 and H2). Marker locus density varied among and within linkage groups. This is the first time EST-SSRs have been used to build a whole-genome functional map and to describe subgenome organisation in an allopolyploid species, and T. repens is the only Trifolieae species to date to be mapped exclusively with SSRs. This gene-based microsatellite map will enable the resolution of quantitative traits into Mendelian characters, the characterisation of syntenic relationships with other genomes and acceleration of white clover improvement programmes.     

An analysis of the costs and benefits of the cyanogenic system in Trifolium repens L.

Summary The effect of the cyanogenic glucosides linamarin and lotaustralin and their hydrolyzing enzyme linamarase was studied in a B₂ generation segregating for the genes Ac and Li. Plants containing the glucosides are protected against grazing by snails both in the seedling stage and as adult plants. In seedlings, however, there is a direct effect on survival, whereas in adult plants the leaf area of plants containing linamarin/lotaustralin is less reduced under intense grazing. Linamarase has no effect on grazing by snails, possibly as a result of the presence of -glucosidase activity in the gut of these animals. The genes Ac and Li, or genes tightly linked to them, have other effects as well: plants possessing one dominant Ac allele produce fewer flowers than homozygous ac plants. I compared this difference in flower production to the metabolic cost of producing the cyanogenic glucosides. The energy content of the difference in flower head production far exceeded the metabolic cost of cyanoglucoside production in Acac plants. It is possible that the cost of maintaining a certain level of cyanoglucosides is much more important for the plant than the initial cost of biosynthesis. The importance of the effects of Ac and Li in the maintenance of cyanogenic polymorphism in white clover is discussed.     

Influence of two selective factors on cyanogenesis polymorphism ofTrifolium repens L. in Darjeeling Himalaya

Cyanogenesis-the production of toxic hydrogen cyanide (HCN) by damaged tissue-inTrifolium repens L. (white clover), a type of most important pasture legume, has been studied at different elevations of Darjeeling Himalaya (latitude-27° 2′ 57″ N, longitude-88° 15′ 45″ E). Release of HCN takes place due to reaction between cyanogenic glucosides stored in vacuoles of the leaf cell and the corresponding enzyme β-glucosidase present in another compartment, often cell wall. Cyanogenesis, a defense system in plant, protects the clover from herbivore and inhibits grazing. Biochemical analysis showed the presence and absence of the cyanogenesis trait within the population in different proportions at different elevations. Acyanogenic individuals also showed variations with respect to presence or absence of either cyanogenic glucosides or β-glucosidase enzyme or both. The distribution of cyanogenic and acyanogenic plants was found in all places, but at lower altitudes (2084–2094 m) the dominating plants were cyanogenic whereas in higher altitude (2560 m) the dominating plants were acyanogenic. It was observed that blister beetle (Mylabris pustalata Thunb.) and the mollusc (Macrochlamys tusgurium Benson.) were the most common consumer of leaflets ofT. repens. Six categories of damage on white clover leaf by these animals were recorded. Our results suggest that the two selective factors or forces i.e. very cold temperature (harmful to cyanogenic plants) at higher altitude as well as indiscriminate but preferential predation (harmful to acyanogenic plants) interact to affect the system of cyanogenesis and also to cause the stable and protective polymorphism inT. repens rather than genotypic differences present among the plants.     

Genetic analysis of shikimate dehydrogenase allozymes in Trifolium repens L.

 A genetic analysis was carried out on progeny families from pair crosses among plants polymorphic for shikimate dehydrogenase (SDH) isozymes in white clover (Trifolium repens L.). SDH was controlled by two independently assorting disomic loci. This result is consistent with the presence of a single gene (Sdh) represented in this putative allotetraploid by one locus in each of the genomes. One of the Sdh loci is linked (6.0±2.0 cM) to the linamarase (Li) locus. There was no evidence for differentiation of the duplicate Sdh loci (both carry common alleles). White clover behaves genetically as a diploidised allotetraploid but the possibility of a low frequency of multivalent formation and homoeologous pairing has not been ruled out. The SDH locus is likely to be useful for the marker-assisted selection of agronomic traits. Received: 5 June 1997 / Accepted: 19 November 1997     

Restriction fragment length polymorphism segregation analysis of the Li locus in Trifolium repens L.

The Li locus in white clover controls the presence of cyanogenic -glucosidase (linamarase) activity in leaf tissue, such that plants homozygous for the null allele (li) have no linamarase activity in this tissue. The isolation of a cDNA clone from linamarase mRNA is described. The cDNA clone is used to further characterise alleles of the Li locus. Northern blot analysis shows that plants homozygous for the null allele (li li) produce very reduced levels of mRNA which hybridises to the cDNA. Heterozygous plants (Li li), which have intermediate levels of enzyme activity, produce intermediate levels of mRNA. Southern blot analysis of Hind III digested genomic DNA shows that the white clover genome contains three genes with homology to the linamarase cDNA and that at least two of these genes segregate independently. Analysis of the cosegregation of linamarase activity and the presence of genomic restriction fragments identifies the genomic sequence specifying linamarase structure and indicates either a structural or cis acting control function of the Li locus.     

A cyanogenic glucoside of Trifolium repens deters oviposition by the common grass yellow Eurema mandarina

The common grass yellow Eurema mandarina (Pieridae, Coliadinae) widely inhabits Japan, feeds on various fabaceous plants such as silktree (Albizia julibrissin) and uses d ‐pinitol, a cyclitol omnipresent in Fabaceae, as a primary oviposition stimulant. However, E. mandarina has a clear host preference within the Fabaceae; for example, white clover (Trifolium repens) is a nonhost despite containing d ‐pinitol. The present study aims to identify plant chemicals in white clover that inhibit oviposition of E. mandarina. Females lay very few eggs on T. repens foliage and plastic plant models treated with a methanolic extract of the foliage. The foliage extract is fractionated by successive extraction with chloroform, isobutanol and water. None of these fractions induce egg‐laying responses. The aqueous fraction is further separated into four subfractions (Tr‐3‐1 to Tr‐3‐4) by column chromatography. Among these subfractions, females show high egg‐laying responses to Tr‐3‐1, which is known to contain d ‐pinitol. Interestingly, Tr‐3‐2, when mixed with Tr‐3‐1, significantly decreases egg‐laying responses, indicating that it contains oviposition deterrents. Chemical analyses reveal that two cyanogenic glucosides, linamarin and lotaustralin, are the major constituents of Tr‐3‐2. Authentic linamarin does not elicit egg‐laying responses and significantly inhibits female oviposition when mixed with Tr‐3‐1 at the natural concentration. Although these cyanogenic glucosides are reported to synergistically induce oviposition of a coliadine species Colias erate on white clover, we conclude that linamarin acts as an oviposition deterrent for E. mandarina, restricts its host range and regulates their differential host acceptance.     

Genetic diversity in white clover and its progenitors as revealed by DNA fingerprinting

The genetic diversity and ancestral relationships of a number of Trifolium species was revealed by using the amplified fragment length polymorphism (AFLP) and the random amplified polymorphic DNA (RAPD) markers. Both markers produced few species-specific markers. Using distance and parsimony methods, in NTSYS-pc and PAUP software programs, we clearly differentiated the accessions of white clover from other closely related progenitors. The phylogenetic trees, produced by PAUP, also reinforced the close affinity of T. nigrescens and the allopolyploid white clover in support of former views that this diploid species could have been the donor of one of two genomes of the allotetraploid T. repens. In addition, the dendrograms, produced by NTSYS-pc, also indicated close affinity of T. nigrescens and T. occidentale to the accessions of T. repens. These data is congruent with karyological and phylogenetic affinities between the white clover and T. occidentale. The relationships between the examined accessions, in the T. repens gene pool, may be regarded to indicate the presence of shared alleles between T. repens, T. occidentale and T. uniflorum. Further, T. occidentale showed close phylogenetic relations to T. pallescens.     

Interaction between Sitona lepidus and red clover lines selected for formononetin content

Adult clover root weevil Sitona lepidus show a feeding preference for white clover Trifolium repens over red clover Trifolium pratense. The effects on S. lepidus of three red clover T. pratense lines, selected for high, medium, or low levels of the isoflavone formononetin in foliage, were compared in three experiments using white clover as a control. In a no‐choice slant board experiment, weevil larval weights were greater for larvae feeding on white clover roots than those feeding on roots of the red clovers. The effect of larval root herbivory on plant growth was similar for all four clovers. Following root herbivory, a large increase in root and shoot formononetin levels was observed in the high‐formononetin selection of red clover but little change in the low‐formononetin red clover. In a no‐choice experiment with sexually mature female adult weevils feeding on foliage of the four clovers, all the red clovers had increased weevil mortality. Female weevils eating the high‐formononetin red clover laid fewer eggs than weevils eating white clover. The red clover diet caused a large accumulation of abdominal fat and/or oil in the weevils, whereas weevils feeding on white clover did not accumulate fat/oil. When sexually immature adult weevils were given a choice of foliage from all four clovers, white clover was eaten preferentially, and the low‐formononetin red clover was preferred to the high‐formononetin red clover. The results suggest that formononetin and associated metabolites in red clover may act as chemical defences against adult S. lepidus and that distribution in forage legumes can be manipulated by plant breeding to improve root health.     

Genome mapping of white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) and comparative analysis within the Trifolieae using cross-species SSR markers

Allotetraploid white clover (Trifolium repens L.), a cool-season perennial legume used extensively as forage for livestock, is an important target for marker-assisted breeding. A genetic linkage map of white clover was constructed using simple sequence repeat (SSR) markers based on sequences from several Trifolieae species, including white clover, red clover (T. pratense L.), Medicago truncatula (Gaertn.) and soybean (Glycine max L.). An F₁ population consisting of 179 individuals, from a cross between two highly heterozygous genotypes, GA43 and Southern Regional Virus Resistant, was used for genetic mapping. A total of 1,571 SSR markers were screened for amplification and polymorphism using DNA from two parents and 14 F₁s of the mapping population. The map consists of 415 loci amplified from 343 SSR primer pairs, including 83 from white clover, 181 from red clover, 77 from M. truncatula, and two from soybean. Linkage groups for all eight homoeologous chromosome pairs of allotetraploid white clover were detected. Map length was estimated at 1,877 cM with 87% genome coverage. Map density was approximately 5 cM per locus. Segregation distortion was detected in six segments of the genome (homoeologous groups A1, A2, B1, B2, C1, and D1). A comparison of map locations of markers originating from white clover, red clover, and alfalfa (M. sativa L.) revealed putative macro-colinearity between the three Trifolieae species. This map can be used to link quantitative trait loci with SSR markers, and accelerate the improvement of white clover by marker-assisted selection and breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biochemical characterisation of the Li locus,which controls the activity of the cyanogenic β-glucosidase in Trifolium repens L.

The cyanogenic -glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300–103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.     

First RFLP linkage map of red clover (<Emphasis Type="Italic">Trifolium pratense</Emphasis> L.) based on cDNA probes and its transferability to other red clover germplasm

We constructed a genetic linkage map of red clover (Trifolium pratense L., 2n=2x=14) using RFLP markers from cDNA probes of a backcrossed mapping population, and investigated the transferability of the markers to other red clover germplasm. The map contains 157 RFLP markers and one morphological marker on seven linkage groups. The total map distance was 535.7 cM and the average distance between two markers was 3.4 cM. All of the cDNA probes of the map were hybridized to the fragments of genomic DNA from 12 plants derived from three varieties, and 87% of the cDNA probes detected polymorphic bands that corresponded to those of mapping parents. This result indicated that RFLP markers on the present map were transferable to the genome analysis of other red clover germplasm. This is the first report to construct a linkage map of Trifolium species; it should provide fundamental and useful genetic information relevant to the breeding of red clover and genus Trifolium.Communicated by H.C. Becker     

Population differences in<Emphasis Type="Italic"> Trifolium repens</Emphasis> L. response to ultraviolet-B radiation: foliar chemistry and consequences for two lepidopteran herbivores

White clover growing in New Zealand is experiencing increasing levels of ultraviolet-B (UV-B) radiation as a result of ozone depletion. We evaluated the effects of UV-B radiation on the foliar chemistry of two populations of white clover (Trifolium repens L.), ’Huia’ and ’Tienshan,’ and the consequences for the performance of armyworms (Spodoptera litura) and cutworms (Graphania mutans). Plants were grown in controlled environment rooms with and without supplemental UV-B radiation at a dose of 13.3 kJ m^–2 day^–1, corresponding to a 25% mid-summer ozone depletion above Palmerston North, New Zealand. In both white clover populations, UV-B radiation elicited changes in foliar chemistry, including slight increases in nitrogen concentrations and decreases in carbohydrate concentrations. In addition, the ’Huia’ population showed decreases in fiber concentrations and marked increases in cyanogenic activity. No change in UV-absorbing compounds was detected in either population. Long- and short-term feeding trials were conducted to assess dietary effects on insect growth, consumption, and food utilization. Changes in the performance of both insect species were generally small. The most pronounced effect was a 36% reduction in weight of S. litura after 2 weeks of feeding on Huia grown at high UV, but larval development times were only slightly prolonged and pupal weights were unaffected. S. litura short-term performance was affected by differences in white clover population. The long-term performance of G. mutans was not affected and its short-term performance (stadium duration and consumption rate) was only marginally affected by the high-UV treatment. We conclude that the effects of elevated UV-B radiation on white clover plant chemistry can be specific to certain plant populations. The differences in sensitivity of the two generalist insect species suggest that effects may also be specific to certain plant-herbivore associations. These results indicate that future UV-B herbivory studies should examine genotypic effects in both plants and animals. Received: 18 May 1999 / Accepted: 25 July 1999     

Genetic map-based location of the red clover (Trifolium pratense L.) gametophytic self-incompatibility locus

Red clover is a hermaphroditic allogamous diploid (2n = 2x = 14) with a homomorphic gametophytic self-incompatibility (GSI) system (Trifolium pratense L.). Red clover GSI has long been studied, and it is thought that the genetic control of GSI constitutes a single locus. Although GSI genes have been identified in other species, the genomic location of the red clover GSI-locus remains unknown. The objective of this study was to use a mapping-based approach to identify simple sequence repeats (SSR) that were closely linked to the GSI-locus. Previously published SSR markers were used in this effort (Sato et al. in DNA Res 12:301–364, 2005). A bi-parental cross was initiated in which the parents were known to have one self-incompatibility allele (S-allele) in common. S-allele genotypes of 100 progeny were determined through test crosses and pollen compatibility. Pseudo F₁ linkage analysis isolated the GSI-locus on red clover linkage-group one within 2.5 cM of markers RCS5615, RCS0810, and RCS3161. A second 256 progeny mapping testcross population of a heterozygous self-compatible mutant revealed that this specific self-compatible mutant mapped to the same location as the GSI-locus. Finally, 82 genotypes were identified whose parents putatively shared one S-allele in common from maternal halfsib families derived from two random mating populations in which paternal identity was determined using molecular markers. Unique S-allele identity in the two random mating populations was tentatively inferred based on haplotypes of two highly allelic linkage-group one SSR (RCS0810 and RCS4956), which were closely linked to each other and the GSI-locus. Paternally derived pollen haplotype linkage analysis of RCS0810 and RCS4956 SSR and the GSI-locus again revealed tight linkage at 2.5 and 4.7 cM between the GSI-locus and RCS0810 and RCS4956, respectively. The map-based location of the GSI-locus in red clover has many immediate applications to red clover plant breeding and could be useful in helping to sequence the GSI-locus.     

The arctic-alpine polyploids Cerastium alpinum and C. nigrescens (Caryophyllaceae) in a sympatric situation: breakdown of species integrity?

 The high polyploids Cerastium alpinum (8x) and C. nigrescens (12x) were investigated in a mixed population in central Norway to evaluate whether hybridisation has resulted in continuous variation in morphology and genetic markers, a hypothesis previously proposed to account for the extensive taxonomic confusion in this species group. Isozyme, fertility, and morphological (37 characters) variation were examined among 347, 265, and 237 plants, respectively. A PCO analysis based on 23 quantitative morphological characters identified two main groups, corresponding to C. alpinum and C. nigrescens. The groups were also clearly separated in isozyme markers, several qualitative morphological characters, and chromosome numbers. Only 20 plants (8.4%) were more or less intermediate in the PCO analysis. These plants had a parental or hybrid isozyme multilocus phenotype and typically few well-developed anthers, low pollen stainability, and no seed set. Several plants within the C. nigrescens group also had reduced pollen stainability. These results indicate that later-generation hybrids and/or backcrosses towards C. nigrescens are formed. Thus, interspecific, interploidal gene flow probably occurs, but at rates that are insufficient to break down species integrity. Received July 5, 2000 Accepted August 2, 2001     

Antiherbivore defenses alter natural selection on plant reproductive traits

While many studies demonstrate that herbivores alter selection on plant reproductive traits, little is known about whether antiherbivore defenses affect selection on these traits. We hypothesized that antiherbivore defenses could alter selection on reproductive traits by altering trait expression through allocation trade‐offs, or by altering interactions with mutualists and/or antagonists. To test our hypothesis, we used white clover, Trifolium repens, which has a Mendelian polymorphism for the production of hydrogen cyanide—a potent antiherbivore defense. We conducted a common garden experiment with 185 clonal families of T. repens that included cyanogenic and acyanogenic genotypes. We quantified resistance to herbivores, and selection on six floral traits and phenology via male and female fitness. Cyanogenesis reduced herbivory but did not alter the expression of reproductive traits through allocation trade‐offs. However, the presence of cyanogenic defenses altered natural selection on petal morphology and the number of flowers within inflorescences via female fitness. Herbivory influenced selection on flowers and phenology via female fitness independently of cyanogenesis. Our results demonstrate that both herbivory and antiherbivore defenses alter natural selection on plant reproductive traits. We discuss the significance of these results for understanding how antiherbivore defenses interact with herbivores and pollinators to shape floral evolution.