AFLP analysis of genetic diversity within and between populations of perennial ryegrass (Lolium perenne L.)

Amplified fragment length polymorphism (AFLP) analysis has been used to measure genetic diversity in perennial ryegrass (Lolium perenne L.) and to relate intra- and interpopulation variation to breeding history. Cluster analysis of AFLP data from contrasting populations showed features consistent with the origins of these varieties. Significant differences in intrapopulation diversity were detected and partial separation of different cultivars was observed. Restricted base cultivars, derived from small numbers of foundation clones, were suitable for this type of study, allowing near complete discrimination of closely related cultivars. Analysis of bulked samples was based on the pooling of genomic DNA from 20 individuals from 6 selected populations. Cluster analysis of AFLP data from bulked samples produced a phenogram showing relationships consistent with the results of individual analysis. AFLP profiling provides an important tool for the detection and quantification of genetic variation in perennial ryegrass. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ecotypes of Italian ryegrass from Swiss permanent grassland outperform current recommended cultivars

Italian ryegrass (Lolium multiflorum Lam. ssp. italicum Volkart ex Schinz et Keller) thrives well in permanent grassland in mild and moist regions of Switzerland. Ecotypes of Italian ryegrass from Swiss permanent meadows have often been used successfully as the base material of breeding programmes. However, their agronomic performance per se has never been studied in detail. Therefore, field trials were conducted to assess the potential of this semi-natural genetic resource for use in breeding. Twenty ecotype populations were collected in different geographical regions in 2003 and seed was increased. Plot trials with 20 ecotype populations were conducted 2005–2007 at three locations, supplemented by row trials at two additional locations, and including four currently recommended cultivars as controls. Average performance in terms of yield, vigour and resistance to snow mould of ecotypes was superior to the cultivars. Depending on the characteristic, between three and 16 of the 20 ecotypes performed significantly better than the cultivars mean. However, resistance against crown rust of all ecotypes was significantly poorer than that of the cultivars. Correlation and regression analysis showed that more eastern and more northern collecting sites harboured better performing ecotypes. However, the most consistent site related factor favouring performance of the ecotypes in terms of yield, vigour, snow mould and Xanthomonas resistance was a low abundance of species which are typical of Arrhenatheretum type meadows. Data from the plot trials were related to results obtained with spaced plants at a single site, either in a seedling nursery or in the greenhouse. Resistance against crown rust was extremely well predicted by spaced plant observations. Xanthomonas resistance observed after artificial inoculation in the greenhouse was also significantly, but less closely correlated with field observations. An early date of ear emergence as observed on spaced plants was associated with high annual yield in the plot trials. It is concluded that in intensively managed habitats, natural selection has resulted in ecotype populations of Italian ryegrass which are highly promising for use in breeding. Individual plant selection for resistance can be expected to efficiently reduce their high susceptibility to crown rust.     

Spaced-plant traits related to seed yield in plots of perennial ryegrass (Lolium perenne L.)

Summary Seed yield in perennial ryegrass is low and selection criteria for high seed production are lacking. Indirect selection in spaced plants would be efficient, but spaced-plant traits need to be identified that correlate with seed yield in drilled plots. Spaced plants were studied of nine perennial ryegrass cultivars with a different seed production when grown in plots. Genotypic variation within cultivars for seed yield components and other traits was assessed in four environments in replicated clonal experiments. Extensive genotypic variation for most traits was present within each cultivar. Based on cultivar means of 25 or 50 genotypes, significant differences among cultivars were found for most traits. Cultivar-year interactions were found for most traits, but no significant cultivar-location interactions were generally found. Spaced-plant traits in general showed poor correlation to corresponding traits in drilled plots. Cultivars with contrasting seed yield in plots could not be distinguished on the basis of their spaced-plant traits and therefore no criteria for indirect selection for seed production in drilled plots could be identified in spaced plants.     

Breeding two base populations of perennial ryegrass

Summary A program designed to develop two base populations of perennial ryegrass was initiated in 1985 in Galicia. Multitrait mass selection by a base index was practised in two genetic resources groups of wild Galician populations. Plants selected were intercrossed and the two resulting base populations (F and G) were evaluated in two locations at the spaced plant level and in plot trials with the aim of estimating the genetic parameters and the possibilities for future breeding. The two populations did not show significant differences from commercial cultivars. Production characters had low heritabilities (0.01–0.32) in both locations. Seasonal yields had medium to high family heritabilities (0.36–0.73). For the economic traits chosen, the expected genetic advances were important in both populations for summer and autumn growth in Mabegondo and spring and summer-autumn dry matter yield (1^st and 3^rd year) in Puebla de Brollón. Expected genetic advance for early spring growth was 0.62 in population F. In the autumn of 1991 equal numbers of plants, selected at each location by combined family and mass selection from each of these populations, were planted in two polycrosses with the aim of improving the genetic basis and adaptation of these populations.     

Genetic variation in a Norwegian germplasm collection of white clover (Trifolium repens L.)

Genetic variation and genotype × environment interactions for important agronomic characters were estimated for eleven Norwegian populations of white clover (Trifolium repens L.), originating from between 58^°52′N and 69^°30′N latitude, and from altitudes of 10 to 450 m a.s.l. The populations were evaluated in monoculture clonal field plots for two years at two locations in Norway. The medium-leaved Danish cultivar Milkanova was also included in the experiment as a standard market cultivar. Considerable variation between populations was observed for winter survival, spring growth, morphological characteristics, dry matter yield, general performance, earliness and seed yield. Winter survival, measured as plant cover after two years, was significantly different at the two locations. All the populations had sufficient winter survival at the southern, continental highland site (61^°07′N).At the northern coastal location (69^°30′N),where the winter climate is unstable with physical stresses such as frost, ice-encasement and water-logging frequently occurring, only the northernmost, coastal populations showed sufficient winter survival. Highly significant interactions were found between populations and locations, and between populations and years for all characters measured at both locations and in both years, respectively. Interaction effects between populations, locations and years were significant only for leaflet length. The largest leaf lamina's were observed in the most southern populations. However, genotypes possessing relatively long leaflets were also found in the northernmost populations, although at low frequencies. In general, the populations originating from the northernmost locations had lower biomass production, and were smaller-leaved, more prostrate and exhibited shorter internodes and stolons compared to the populations from South and Middle Norway. These populations also initiated inflorescences earliest, and were among the lowest seed yielding populations. Otherwise, no clear clinal variation in growth characteristics was observed which could be related to the latitude or altitude of origin of the populations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variation in forage grasses as hosts of the root-knot nematode Meloidogyne naasi (Franklin) and selection for resistance in ryegrass

Summary Reactions of 13 grasses to Meloidogyne naasi varied with species; ryegrasses, fescues and their hybrids were generally susceptible and cocksfoot and timothy resistant. Marked variation in host resistance levels occurred between genotypes within cultivars.Selection of single plants, followed by tests on replicate tillers, identified resistant and susceptible genotypes in both Italian and perennial ryegrass cultivars. Resistant plants had few nematode-induced galls and fewer females and eggs than susceptibles. There was more or less continuous variation, with many genotypes intermediate between extremes of resistance and susceptibility. Selected resistant and susceptible genotypes are of use in assessing variation in nematode populations and as controls for breeding and selection programmes.     

Value of permanent grassland habitats as reservoirs of Festuca pratensis Huds. and Lolium multiflorum Lam. populations for breeding and conservation

European natural and semi-natural grassland form reservoirs of genetic resources containing highly adapted and variable ecotype populations of forage plants. Variation within these reservoirs is stimulated by variation in natural and anthropogenic site-related factors. Changes in agricultural practices lead to the loss of many characteristic habitats. In order to preserve resources for breeding, targeted conservation strategies for germplasm in gene banks (ex-situ) or on site (in-situ) are needed. In order to define site-related criteria for the potential of habitats to preserve valuable resources for breeding and conservation, 38 different habitats across Switzerland were selected to collect Festuca pratensis Huds. and Lolium multiflorum Lam. ecotype populations. Phenotypic variation and population differentiation of 60 single plants were evaluated in a field experiment using 16 morpho-physiological traits. For F. pratensis, ecotype populations and cultivars were clearly separated and there was a significant correlation between diversity of morphological traits and geographic location of sampling sites. For L. multiflorum no clear separation of ecotype populations and cultivars was observed suggesting gene flow from adjacent temporary leys into permanent grassland. Several ecotype populations were superior to cultivars in important traits such as early heading or resistance to winter damage, indicating the importance of natural habitats as a reservoir of genetic resources for breeding. In conclusion, maintenance of permanent grassland in contrasting environments appears to be a promising strategy for preserving valuable genetic variation of forage grasses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phenotypic variation in local populations and cultivars of meadow fescue – potential for improving cultivars by utilizing wild germplasm

The utilization of gene bank accessions for breeding purposes is generally hampered by a lack of sufficient genetic and phenotypic characterization. Results are given here of a phenotypic analysis of 30 Norwegian local populations and 10 Nordic cultivars of meadow fescue, and a combined analysis of phenotypic data with previously published molecular data based on 95 amplified fragment length polymorphic markers. Seventeen phenological and morphological, and two derived characters, were assessed on 2264 individual plants. Previous molecular diversity analyses have shown that the Norwegian local populations can be divided into three groups based on a likely history of the introduction of the species into Norway. A similar pattern was evident from relationships between the mean phenotypic values of populations and cultivars. Such a combined phenotypic and genetic characterization of germplasm can facilitate the use of gene bank material in breeding, as breeders can use these data to select populations with extreme values of a field trait, along with markers which represent potential predictors of phenotypic value.     

Molecular characterization of genetic diversity in European germplasm of perennial ryegrass

Summary Perennial ryegrass (Lolium perenne L.) is the most important grass species for temperate grassland agriculture. The level and distribution of genetic variation in gene bank ecotype collections is still largely unknown but of great interest for the planning of breeding programs. The objectives of this study were to (i) assess the molecular diversity of Polish ecotypes of perennial ryegrass, and (ii) compare the relationship between this group and German ecotypes and European cultivars investigated previously. A total number of 166 polymorphic marker bands were detected among the 171 individual plants of the 9 Polish ecotypes. In a joint analysis with 9 Polish and 22 German ecotypes, and 22 European cultivars 172 polymorphic RAPD markers could be found. Genetic distance among the Polish ecotypes ranged from 0.31 to 0.51, while for all 53 populations a broader range was detected (0.25–0.67). An analysis of molecular variance (AMOVA) revealed a much larger variation within populations (71%) than among them (29%). The Polish ecotypes contained the highest within population variation (74%). The largest among group difference (15%) was found between the Polish ecotypes versus all other accessions. We conclude that the Polish ecotypes represent a valuable genetic resource for enlarging the genetic variation in the West European germplasm pool of perennial ryegrass.     

Association of agronomic traits with isozyme loci in perennial ryegrass (Lolium perenne L.)

Summary Inbred lines possessing different genotypes at three isozyme loci, PGI-2, GOT-3 and ACP-1, were produced by two generations of selfing from two contrasting cultivars of perennial ryegrass. Four separate F₂ populations were obtained by selfing or crossing F₁ plants from crosses between cultivar inbred lines. F₂ plants were scored for genotype at the three isozyme loci and for eight agronomic traits. The three isozyme loci showed independent segregation confirming that they belonged to separate linkage groups. A consistent association was observed between water soluble carbohydrate content and genotype at the PGI-2 locus. Some association was also detected between genotype at the ACP-1 locus and heading date. The relevance of these observations to ryegrass breeding programmes is discussed.     

Mitochondrial DNA analysis reveals cytoplasmic variation within a single cultivar of perennial ryegrass (Lolium perenne L.)

Summary The extent of mitochondrial DNA restriction fragment length polymorphisms among individual plant samples of perennial ryegrass was determined. A total of 72 plants from three cultivars Yorktown II, S23 and Riikka were surveyed using three restriction enzymes (BamHI,EcoRI andHindIII) and three mitochondrial gene probes (coxI, coxIII andnad9). Polymorphisms were noted within each of the two cultivars Yorktown II and S23, whereas in Riikka no variation was detected. It seems most likely that the mitochondrial genome diversity within the same cultivar has resulted from non-homogeneous ancestor cytoplasms. The hybridization-based assay employed is simple to perform, gives unambiguous results, and may thus be used in mass screening of perennial ryegrass populations for breeding purpose.     

Temperature Before Cold Acclimation Affects Cold Tolerance and Photoacclimation in Timothy (Phleum pratense L.), Perennial Ryegrass (Lolium perenne L.) and Red Clover (Trifolium pratense L.)

The expected temperature rise in late summer/early autumn can change the conditions for acclimation and affect the winter survival of perennial crops. This study examined the effect of the temperature just before the onset of cold acclimation (pre‐acclimation) on freezing tolerance of timothy (Phleum pratense L.), perennial ryegrass (Lolium perenne L.) and red clover (Trifolium pratense L.) populations (both cultivars and breeding populations) adapted to either northern or southern parts of Norway. Using phytotron experiments, we studied whether increasing pre‐acclimation temperature delays growth cessation, affects photoacclimation and reduces freezing tolerance. Furthermore, we assessed whether these effects were related to the latitudinal adaptation of the plant material. The results showed that a rise in pre‐acclimation temperature decreased both cold acclimation capacity and photoacclimation in these species. This affected the freezing tolerance, which was reduced significantly more in northern‐adapted population of timothy and perennial ryegrass compared with southern‐adapted populations. Red clover was less affected by temperature changes than the grasses.     

Causal relationships between inbreeding, seed characteristics and plant performance in leek (Allium porrum L.)

Leek (Allium porrum L.) is an important outdoor vegetable in West Europe, where it is cultivated on about 30,000 ha. Most commercial cultivars of leek are open pollinated. One of the major problems with the crop is poor uniformity. Leek is an outbreeding species with up to 20% self-fertilisation. Much of the variation of open pollinated cultivars is explained by the strong sensitivity to in breeding depression after selfing. In this study we attempt to analyse in a systematic way the relationship between degree of selfing and agricultural performance in leek. The results obtained confirm a negative correlation between selfing and somea gricultural aspects important for vigour such as seedling emergence, plant growth and plant fresh yield. Also negative correlations between degree of selfing and seed individual weight and between selfing and seed individual size were found. The negative relationship between inbreeding and plant weight at harvest is further confirmed in an experiment in which the origin (cross-pollination orself-pollination) of each individual offspring plant analysed was determined using AFLP-markers. Finally, the correlation between chlorophyll deficiency genes and the loss of vigour that follows selfing was investigated. The results obtained demonstrate a significant decrease of pigment content in two generations of selfing. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spaced plant evaluation of Mediterranean germplasm collections of perennial ryegrass

Summary A sample of 50 wild populations of perennial ryegrass (12 from northern Italy and 38 from Corsica) was tested with controls as spaced plant nurseries in two locations in France. Multivariate analyses, such as principal component analysis, hierarchical clustering and factorial discriminant analysis were used to describe the genetic variability of such Mediterranean material.On the whole, Mediterranean populations do not appear to be more promosing than the north European varieties. However, some populations from Corsica could be used to develop genetic resources for the Mediterranean area.The interest of each cluster is discussed and geographical repartition of clusters is shown on country maps.The factorial discriminant analysis gives a linear discriminant Fisher function which clearely discriminates two groups of populations according to their country of origin.     

Genetic diversity in European perennial ryegrass cultivars investigated with RAPD markers

Perennial ryegrass (Lolium perenne L.) is the most important grass species for temperate grassland agriculture. The genetic relationship and distance among cultivars is largely unknown but of great interest for breeding programmes. The objectives of this study were to (i) investigate the molecular variation and structure of population cultivars, (ii) describe the relationship among cultivars in terms of the modified Rogers’ distance, and (iii) determine the minimum sample size required for characterization of cultivars of L. perenne using random amplified polymorphic DNA (RAPD) markers. A total of 22 ryegrass cultivars, mainly of European origin, were investigated with RAPD markers. The minimum sample size required for the characterization of cultivars was about 20 individuals per population. An analysis of molecular variance (AMOVA) revealed a much larger genetic variation within cultivars (66%) than between them (34%).     

Inheritance of anther-culture response in perennial ryegrass (Lolium perenne L.)

Genetic control of the capacity to respond to anther culture in perennial ryegrass was studied in F₁ offspring from crosses between 11 clones selected for anther-culture response (‘inducers’) and 10 clones selected from breeding material on the basis of good agronomic performance. Large differences in anther-culture response were observed between the two types of parent, with inducer clones producing an average of 10.7 green plants per 100 anthers, compared with only 0.3 for breeding clones. Hybrid populations produced an average of 2.2 green plants per 100 plated anthers, which is 7.3 times the response of the breeding material. This improvement was mainly due to a 9.4-fold increase in the percentage of green plants regenerated from hybrid populations to 4.7% compared to an average of 0.5% from breeding clones. Most of the GCA (general combining ability) in the experiment was contributed by the breeding material, constituting 55.8, 100.0 and 36.7% of genetic variation among hybrids for embryo formation, regeneration and green plant percentage, respectively. Any remaining genetic variation among hybrids was predominantly due to SCA (specific combining ability) effects, with percentages of 44.2 and 63.3%, respectively, for embryo and green plant formation. These results are discussed with respect to the possible exploitation of genes controlling anther-culture response for haploid production in breeding programmes of perennial ryegrass.     

Genetic structure of landrace populations of the New World grain amaranths

Summary Grain amaranth populations from their centers of origin in the New World had shown a pattern of allozyme variation that suggested most landraces to be highly homozygous mixtures of genotypes. To compare this pattern of variation with the variation for morphological traits, 15 selfed families from each of six populations were grown in a replicated field experiment. Four pigmentation traits known to be smiply inherited were scored along 18 other morphological traits. Populations varied for the amount of polymorphism for marker loci, and exhibited little heterozygosity. Analysis of variance for the quantitative traits showed significant interpopulation differences for each of the observed characters. Populations differed for the number of metric traits showing significant between-family differences for just one of the metric traits whereas another had between-family differences for all ten. These results suggested high levels of homozygosity within these landraces; thus, variation for quantitative traits conformed well with the allozyme variation patterns.Stepwise multiple regression of all characters on yield as the dependent variable was used to compare the relative contributions of specific characters to yield within individual populations. Plant height, days to flowering, and leaf length were included in the regression equations for 6, 5, and 5, of the populations, respectively. Days to floweing was negatively correlated with yield, while plant height and leaf length were positively correlated. These correlations suggest the potential for developing early flowering, high yielding cultivars having short stature, selected by breaking its correlation with yield. Several breeding strategies based on these findings on genetic resources are discussed briefly.     

Breeding winter hardy grasses

Summary Perennial grasses are vital for Norwegian agricultural production. The nature and extent of winter damage on grasslands is highly dependent on climatic conditions, and determines both persistency and yield. Physical stresses such as frost and ice encasement predominate in coastal regions with an unstable winter climate, while biotic stresses such as low temperature fungi are more common in the inland regions. Development of hardening depends on plant adaptation and climatic conditions during autumn and winter. New winter-hardy cultivars should be bred for wide adaptation to winter stresses. The genetic background for the most important character, freezing tolerance, seems to be of polygenic nature with mainly additive gene action. Selection for increased freezing tolerance has been effective over generations in grasses, and in most grass species ample variation still exists to be exploited by breeding. However, in some species like perennial ryegrass, modern biotechnological methods should be used to improve freezing tolerance and winter hardiness.     

Breeding value of base populations derived from 'contiguous' clusters in perennial ryegrass

Five broad-based breeding populations were produced by polycrossing natural populations sampled in five clusters developed by a hierarchical method with a geographic-contiguity constraint. As a result, each of these breeding populations originates from a single region and the populations within a cluster are more likely to share the same alleles with similar frequencies. A single generation of intercrossing would therefore be sufficient to establish a panmixia equilibrium. These populations were tested for dry-matter yield in plots and compared to control cultivars in two locations. There were some trends of specific adaptation related to the geographic origin of populations, which, on average, compared well to controls. Their half-sib families were evaluated as spaced plants in one location. This allowed genetic parameters and expected response to selection to be computed. Three out of five breeding populations appeared to be promising for the development of new, improved synthetic varieties.     

Inbreeding depression and single cross hybrids in leeks (Allium ampeloprasum ssp. porrum)

Summary All current commercial cultivars of leeks are open-pollinated and one of the major problems with the crop is poor uniformity with much of the variation being genetic in origin. Inbred lines and single cross hybrids were produced to try to reduce the genetic variation. Inbreds were generated by single seed descent from 5400 plants taken from six commercial cultivars and performance data for uniformity, yield and quality are presented for the S₁-S₃ generations. A few relatively vigorous inbred lines were obtained but overall, inbreeding depression was very severe with no compensating increase in uniformity as measured by coefficients of variation. In contrast, the experimental hybrids gave significant uniformity, yield and quality benefits compared to open-pollinated commercial cultivars and can be used as the basis for developing a range of commercial hybrid cultivars.Abbreviations SSD Single Seed Descent - HRI Horticulture Research International