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.     

Tracing of 2n egg occurrence in perennial ryegrass (Lolium perenne L.) using interploidy crosses

Several genotypes of Lolium perenne (2n = 2x = 14) were screenedfor the production of 2n eggs. In each cross a diploid genotype, used asfemale parent, was pollinated with a tetraploid genotype. All interploidycrosses were made in isolation cages. Flow cytometrical analysis of theprogeny of 154 diploid plants revealed 28 tetraploid descendants. Theseoffspring plants were harvested on 15 different diploid mother plants.Amplified Fragment Length Polymorphism (AFLP) markers were used tocheck the parenthood of these tetraploids. Two seedlings died, theremaining plants could, after AFLP fingerprinting, be identified as meiotictetraploids. The results obtained allowed to conclude that the tetraploidprogeny plants harvested on 15 diploid mother plants were the result ofthe fertilisation of an unreduced egg by a reduced pollen grain of thetetraploid plant.     

Morphological variation in Dutch perennial ryegrass (Lolium perenne L.) populations,in relation to environmental factors

Summary Twenty-one Dutch Lolium perenne populations, fifteen European populations and six L. perenne cultivars were compared for morphological variation. Dutch populations clearly differed from the European populations and the cultivars. Dutch populations generally had reduced plant length and smaller leaves. For other characters, e.g. date of ear emergence, the Dutch populations showed as much variation as the European populations and cultivars they were compared with in this trial. Correlations between morphology and environmental factors at the site of origin were significant in several cases but were generally weak, and dependent on the set of populations studied. In order to maximize the phenotypic differences between the collected populations, soil type and management type appeared to be the most important factors for the choice of collection sites in the Netherlands. To determine suitable collection sites all over Europe, other factors like precipitation, latitude, altitude and temperature factors were also important. For subsampling of the locations, variation in management type within the location determined whether or not phenotypic different samples could be collected. The extensive use of cultivars in the Netherlands does not seem to have prevented the formation of distinct populations. Therefore in situ conservation of grassland seems a good alternative for genetic conservation of L. perenne in the Netherlands.Also associated with the WAU Department of Plant Taxonomy, P.O. Box 8010, 6700 ED, Wageningen, The Netherlands     

Testing perennial ryegrass (Lolium perenne L.) as spaced plants in swards

Summary A method of testing spaced plants in swards of another grass crop is described. The procedure provides information on sward performance at each stage of the breeding scheme. A large number of entries may be included, together with whatever cutting frequency.When used for perennial ryegrass, the method favours the selection of productive and persistent plants.     

A relationship between heading date and in vitro tillering propensity in perennial ryegrass (Lolium perenne L.)

Summary In vitro tillers of over 1000 genotypes from nine contrasting Lolium perenne cultivars were cultured on various media containing the synthetic cytokinin, 6-benzylaminopurine (BAP), in order to determine the effect on tillering. The cultures were assessed after seven weeks for survival, tiller production, size and vigour. Added auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and -napthaleneacetic acid (NAA) had no consistent beneficial effects.A significant relationship was found between in vitro tiller production and cultivar heading date. Genotypes of early-heading, less persistent cultivars produced more tillers than genotypes from late heading more persistent cultivars. This was consistent with the generally higher in vitro tiller production in the biennial species Lolium multiflorum cultured on similar media.     

Bulked AFLP analysis for the assessment of genetic diversity in white clover (Trifolium repens L.)

The use of bulked leaf samples from individual plants for amplified fragment length polymorphism (AFLP) analysis was evaluated as a tool for assessment of genetic diversity in white clover (Trifolium repens L.). Bulking of leaf samples produced slightly simpler AFLP profiles compared to the combined profiles of individual plants from the same cultivar. Approximately 90% of bands which were present in individual plants were present in bulked samples of the same cultivar. The majority of those absent were rare bands, shared by less than 25% of individual plants. Replicate bulk samples gave almost identical banding patterns, demonstrating the robustness of the bulked AFLP technique. Cluster analysis of AFLP data derived from individual plants resulted in a phenogram similar to that produced from data derived from bulked samples of the same plants. AFLP analysis of bulked samples detected significant amounts of genetic variability among 52 cultivars and accessions with genetic similarity values ranging from 0.42 to 0.92. However, cluster analysis of AFLP data only partially reflected the geographic origin of cultivars and accessions and was not congruent with cluster analysis based on variation for morphophysiological characters. Bulked AFLP analysis provides a powerful tool for rapid assessment of genetic variability in white clover and may also be used for cultivar identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

Heritability estimates of spaced-plant traits in three perennial ryegrass (Lolium perenne L.) cultivars

Summary Seed yield in perennial ryegrass is low and unpredictable. Spaced-plant traits suitable for indirect selection for total seed yield in drilled plots would be very useful. The objectives of this investigation were to evaluate genetic variation for seed yield components and other traits among clones from three perennial ryegrass cultivars differing in seed yield and their open-pollinated progenies. Per cultivar, a random set of 50 genotypes was cloned and on each genotype seed was generated by open pollination. Clonal ramets of the parents were observed for 17 traits in 1986 at two locations. In 1987 and 1988, parents and progenies were observed as single plants in a randomized complete block design with two replications. There was little cultivar-environment interaction for most traits. The parents differed significantly for almost all traits. Half-sib (HS) families differed for only three to five traits. Broad-sense heritabilities (h² _b), based on variance components of the parents, were moderate to high; earliness had the highest h_b ². Narrow-sense heritabilities (h² _n), based on variance components among HS-families, were low to moderate and mostly not significant; for most traits h² _n estimates varied between years and cultivars. Flag leaf width and date of first anthesis showed the highest h² _n. Narrow-sense heritability estimates from parent-offspring regressions (h² _nPO) ranged from non-significant to high, depending on year and cultivar; they were generally higher than the corresponding h² _n estimates. Generally, h² _nPO was highest for earliness, flag leaf width, ear length and the number of spikelets per ear. Breeding methods that capitalize on additive genetic variance, such as mass selection, should result in improvement for these traits.     

Assessment of perennial ryegrass (Lolium perenne L.) for breeding. I Freezing tolerance

Summary Assessments of freezing tolerance, estimated in terms of temperature at which 50% mortality occurs (=LT₅₀), were obtained for 86 Lolium perenne accessions using a glycol bath test on naturally hardened field plants. Significant correlations were obtained between LT₅₀ and both heading dates of accessions and minimum temperature of the coldest month near their sites of origin. The effects of temperature and reproductive development on the hardening/dehardening of plants and their growth after freezing are discussed.     

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.     

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.     

Variation in digestibility in nine populations of perennial ryegrass (Lolium perenne) and its genetic basis

Summary Thirty five individuals from each of nine perennial ryegrass populations were sampled from set-stocked and zero-grazed swards and from an old permanent pasture. The swards were 6, 18, 30 months and 11 years old when sampled. The permanent pasture was at least 40 years old.Dry matter digestibility (DMD) and organic matter digestibility (OMD) were determined for green leaf material harvested 4 days after ear emergence. Significant differences were found between the populations, but genotypic differences were found only for OMD. The largest proportion of the total variation was due to differences between blocks. In the zero-grazed populations, DMD and OMD declined significantly with increasing sward age. This was not found for the set-stocked swards. Two sets of pair crosses, from which mid-parent, progeny regressions could be determined, were carried out.The regressions of offspring on mid-parent for set-stocked and zero-grazed swards were used to assess the presence of additive genetic variation and to estimate narrow sense heritability of DMD. The coefficients of correlation, 0.122 and 0.226 respectively, were non-significant, indicating an absence of additive genetic variation.     

Assessment of perennial ryegrass (Lolium perenne L.) for breeding. II. Components of winter hardiness

Summary When breeding for good winter hardiness a broad range of adaptations must be considered. Such a multitrait approach requires some understanding of physiological and genetical relationships between relevant traits.Fourteen winter related characters were measured on field grown plants from 86 perennial ryegrass accessions originating from a wide range of climates. Genetic components of variance and covariance were extracted from the data and a principal components analysis carried out. The first three independent components accounted for 80% of the variance represented by the genetic correlations and were broadly identified as winter growth, winter hardiness and early spring activity factors. None of the accessions exhibited a combination of extreme winter growth and winter hardiness although several with excellent winter hardiness showed better than average winter growth. Amounts of water soluble carbohydrate maintained through the winter appeared important for good survival.It was concluded that a selection index approach may prove useful in future breeding programmes as further information concerning physiological relationships becomes available.     

Stability of esterases and total proteins during seed storage of perennial ryegrass (Lolium perenne L.) and their use for cultivar discrimination

Polymorphisms in electrophoretic patterns obtained by isoelectric focusing (IEF) were examined to evaluate their suitability for cultivar identification in perennial ryegrass (Lolium perenne L.). It was possible to discriminate 64 (94%) of 68 cultivars by combining results from esterase and total protein analysis. Discrimination was based on quantitative differences (relative band intensity) rather than on qualitative differences (presence or absence of bands). Esterase patterns from different recent (fresh to seven years old) seed lots of the same perennial ryegrass cultivars were very stable. Occasionally, minor differences in band intensity were observed between recent and old (up to 30 years old) seed lots of a cultivar. Storage of meal samples up to two years at −20 ^°C had no effect on the total protein patterns. No correlation was found between esterase patterns and ploidy level, cultivar type (pasture or turf), heading date or breeding company. Esterase patterns appeared to be unsuitable markers for the selection of reference cultivars for distinctiveness, uniformity, and stability (DUS) testing, because no correlation was found between cultivars on the basis of esterase banding patterns and morphological characteristics. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Seed yield related to crop development and to yield components in nine cultivars of perennial ryegrass (Lolium perenne L.)

Summary We had previously found differences for seed yield among nine perennial ryegrass cultivars which were not associated with variation for seed weight. To detect the physiological basis of these genetic differences for seed yield, growth analyses were carried out. We related crop development and components of seed yield to seed yield during three years on clay and sandy soil. No significant differences occurred among cultivars for accumulation and partitioning of dry matter or the pattern of tiller production. Seed yield of the cultivars was not associated with ear number or total dry matter yield of the seed crop. Seed yield was more correlated with the number of seeds per unit area than with seed weight. The number of seeds as calculated after harvest from seed yield and seed weight was much lower than the number of seeds as estimated prior to harvest from seed yield components. The number of spikelets differed significantly among the cultivars, but the ranking was different from that for seed yield. The physiological basis of the genetic differences for seed yield is not clear. Implications for breeding perennial ryegrass are discussed.     

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.     

Molecular genetic diversity within and among German ecotypes in comparison to European perennial ryegrass cultivars

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 programmes. The objectives of this study were to (i) assess the molecular variance and population structure of German ecotypes at the regional and population level, (ii) assign ecotypes to germplasm pools and (iii) compare the relationship between German ecotypes and previously‐investigated European cultivars of perennial ryegrass, A total of 22 ecotypes originating from three geographic areas in Germany. each with a sample size of 20 individual plants, were investigated with 156 polymorphic RAPD markers. Genetic distance among ecotypes ranged from 0,27 to 0.48, An analysis of molecular variance (AMOVA) revealed a much larger variation within populations (71%) than among them (29%). Ecotypes from North Germany were significantly different from those of South and Middle Germany. Thus, two distinct germplasm pools could be identified. The 22 ecotypes and 22 previously investigated cultivars shared 98% of the molecular variance.     

Co-adaptation between neighbours? A case study with Lolium perenne genotypes

Summary Co-adaptation between neighbouring genotypes of Lolium perenne was examined in a pot experiment over an 18 month period. Competitive interactions in pure and mixed stands were examined using 9 genotype pairs (established as neighbours from electrophoresis data), 7 from a 10 year-old, and 2 from a 40+ year-old pasture. Combinations of each pair of neighbours grown together, and each grown with 5 randomly chosen alien (non-neighbour) genotypes sampled from the same pastures were also assessed. Seven harvests of green matter produced above 20 mm were made, and 50–135 kg N ha^-1 was administered after each harvest. Data for dry weight at each harvest for pure and mixed stands, and Relative Yield Totals (RYT) were obtained for neighbours and aliens in each treatment.In 6 of the 9 pairs of neighbours grown as pure stands, the yields of neighbours did not differ significantly at the final harvest. In 8 of the 9 pairs grown as mixtures of pairs of neighbours, the yield of one of the neighbours was significantly reduced and one neighbour had been eliminated. Over the last 5 harvests, yields from (alien + neighbour) mixtures had the greatest dry matter yields. No suppression of one component by the other was recorded for this (alien + neighbour) combination.Based upon RYT estimates, neighbours did not yield more when grown together than when grown separately with aliens. There was no relationship between original genotype size in their fields of origin and dry matter yield in this experiment.This experiment provides no evidence for he evolution of co-adaptation between surviving neighbours in the two pastures from which these individuals were sampled. The genotypic architecture of pasture would seem to be the product of random and/or competitive exclusion of individual genotypes, rather than the evolution of co-adapted groups of individuals through time. The consequences of extreme depletion of genetic variation in the first two years after establishment, the very low rates of seedling establishment in established pastures, and the impact of the grazing animal would suggest that there is little likelihood of or potential for, co-adaptation evolving between neighbours.     

Allozyme differentiation of European populations and cultivars of Lolium perenne L., and the relation to ecogeographical factors

Summary Sixty Lolium perenne populations were screened for allozyme diversity at five loci. Objective was to determine whether allozyme diversity could be used as selection criterion for genebank accessions of L. perenne. Subsampling of locations was tested with allozyme analysis to determine whether genetically different populations could be collected at one location. Correlations between allelic frequencies and environmental factors and morphological data were established, to find ecogeographical patterns in the observed variation. Results indicated that with few examples each allelic variant could be observed in each population screened; very few unique alleles were found. Differences between populations were largely due to differences in allelic frequencies. Few correlations were found with environmental factors and morphological data. For some allele frequencies a north-south cline was observed. Generally, allozymic data of the five screened loci did not appear to be useful for the selection of accessions for genebank storage. Significant genotypic differences between populations collected at one location could be established. In general these results agreed with earlier results concerning phenotypic variation in the same populations.     

Intraspecific phenotypic variation associated with nuclear DNA content in Lolium perenne L.

The variation in nuclear DNA content and its association with phenotypic traits were examined in 15 cultivated populations of perennial ryegrass (Lolium perenne L.), including herbage and turf populations. Nuclear DNA contents of 15 populations were measured by a flow cytometric method using DAPI (4′-6-diamidino-2-phenylindole) as a fluorochrome. DNA contents were also measured using PI (propidium iodide) for six populations that showed large differences in DAPI values to confirm the difference. There were significant differences in nuclear DNA contents among the populations for both dyes. Of the total variation in nuclear DNA content measured with DAPI, 29% was ascribed to the inter-population variation, 46% was ascribed to the intra-population variation, 24% was ascribed to the random error variation. Herbage populations tended to have larger DNA content than did turf populations. 2C DNA content was positively correlated with cell size, seed size and single leaf size, although significant correlations were mainly due to a small number of populations with large and small DNA contents. These results suggest that intraspecific variation in nuclear DNA content plays important roles in determining phenotypic differences between cultivated populations of L. perenne. This revised version was published online in August 2006 with corrections to the Cover Date.