Aneuploids of perennial ryegrass (Lolium perenne)

Summary Aneuploid plants of perennial ryegrass (Lolium perenne L.) with 2n=15 to 30 chromosomes were obtained by crossing a near-triploid (2n=3x+1=22) with a diploid or on open-pollination with diploids and tetraploids. Aneuploids occurred with a frequency of 83% in near triploid × diploid progeny and 92% on open-pollination with diploid and tetraploid plants. Aneuploid plants with 15 to 18 chromosomes resembled diploids in morphology and those with 19 to 30 chromosomes were akin to tetraploids. Meiotic studies suggested that most aneuploid plants resulted from transmission of aneuploid egg cells (n=8 to 23). Aneuploid plants with 2n=27 to 30 chromosomes in the progeny of 22×14 cross originated from unreduced egg cells. Plants with 19 to 21 chromosomes were recovered only by immature seed culture. Aneuploid plants with 26 to 30 chromosomes and triploids (2n=21) had higher pollen fertility and bigger seeds than plants with 15 to 22 chromosomes.     

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.     

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.     

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     

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.     

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.     

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.     

Introduction of a self-compatible gene of Lolium temulentum L. to perennial ryegrass (Lolium perenne L.) for the purpose of the production of inbred lines of perennial ryegrass

Interspecific hybrids between self-compatible species, Lolium temulentum L. and self-incompatible species, L. perenne L. were obtained using embryo rescue. Two cycles of backcrossing of interspecific hybrids with L. perenne were carried out. A 1: 1 segregation ratio of self comatibility and incompatibility was observed in backcross generations. These segregation data confirmed that self-compatibility of L. temulentum was controlled by a single gene. It suggested that the self-compatible gene of L. temulentum could be introduced to self-incompatible plants of L. perenne through interspecific hybridization. It appears that utilization of a self-compatible gene of L. temulentum would be useful for production of inbred lines of L. perenne. The possibility of breeding procedures of perennial ryegrass using a self-compatible gene was discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

Components of resistance to ryegrass mosaic virus in a clone of Lolium perenne and their strain-specificity

Summary The extreme resistance to ryegrass mosaic virus (RMV) of a clone of Lolium perenne L. was due to a combination of two distinct types of resistance: resistance to infection and resistance to multiplication and movement of virus within the plant. Resistance to infection was quantitatively inherited and highly effective against three strains of RMV, while resistance to multiplication and movement was controlled by two complementary recessive genes and was effective against only two of the three RMV strains.     

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.     

Isozyme and quantitative traits polymorphisms in European provenances of perennial ryegrass (Lolium perenne L)

Twenty populations of Lolium perenne originating from a range of habitats in Europe were compared for isoenzyme polymorphisms and agronomically important quantitative traits in order to establish relationships of the levels of diversity with the origin of each population and to assess their suitability to be included in the European core collection of Lolium germplasm. Forty genotypes from each of the twenty populations and each genotype represented by three clonal propagules, were field planted in a fully randomized spaced plant design and fifteen quantitative characters including yield, persistency, reproductive and disease resistence characters were evaluated over a period of two years. Seven putative isozyme loci were assayed to compare the allozyme divergence of populations. The results of the isozyme survey indicate that 71–100% of the loci were polymorphic, 2.3–3.0 alleles/locus and the gene diversity was varying from 0.234–0.410. Of the total allelic diversity 94% remained within populations (H_s) whilst only 6% was distributed among populations (D_st). The differences between populations were determined on the basis of allele frequencies and multivariate analyses of quantitative characters. Populations significantly differed in their allele frequencies at all loci analysed. Random mating was predominant in all populations at most of the loci. The study revealed that the German accession BA 10998 was clearly distinct from the rest both in quantitative characters and allele frequencies. German accession BA 11015 with the lowest gene diversity showed the highest genetic variation for quantitative characters. However, no strict relationship was found between the genetic distance and the geograpical distribution of the populations. Among the quantitative characters, flowering time showed a strong relationship with the type of management practised at the collection site than their place of origin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of date of sampling and plant density upon size and digestibility of reproductive tillers of Lolium perenne L.

Summary Evaluation of forage germplasm digestibility is an important factor in screening programmes. The major problems involve the relevance of estimations made in spaced plants to plot performance, the effect of maturity, leaf: stem ratio and the choice of plant organ to be analysed. The use of stems of reproductive tiller proved successful in Lolium multiflorum. In the present experiment the effect of dates of sampling (date 1=28/6; date 2=5/7; date 3=12/7/83) and of plant densities (spaced plants and plots) on the digestibility of reproductive tillers of L. perenne cv. Marta 23 days after ear emergence was evaluated. After harvesting, the following characters were recorded on each tiller: tiller length and diameter, and spike and flag leaf length. Leaf blades and sheaths were then removed, the tillers dried and milled individually, and the in vitro digestibility assessed. The data were analysed by Analysis of Variance, Principal Components Analysis and Numerical Classification followed by a study of how the variables contributed to the derived group structure. Results: I-Plots: digestibility values were 63.4, 60.3 and 56.6% (P<0.01), respectively, for the sampling dates 1, 2 and 3. The Cramer Value (0.41) shows some correlation between tiller size and digestibility. Tillers from date 3 tend to be smaller than the others but the separation is not very clear. II-Spaced plants: digestibility values were 63.2, 60.0 and 60.8%, respectively, for dates 1, 2 and 3. Analysis of variance showed differences (P<0.01) between dates and also between genotypes. The Cramer Value (0.23) shows less correlation between tiller size and digestibility: however, tillers from dates 1 and 3 are clearly separated on the basis of size. Tillers from date 3 being smaller, date 2 tillers are located in between those from dates 1 and 3. III-Plots and single plants combined: Tillers from plots are larger than tillers from spaced plants in sampling dates 2 and 3. The Cramer Value for digestibility (0.14) is the lowest one. The results indicate differences in size and digestibility of reproductive tillers of L. perenne when contrasting situations are being compared, which suggest influence of environmental conditions upon the characters evaluated in the experiment. The differences are sufficiently high so that the use of this technique, when populations of L. perenne with wide variation in flowering dates are being evaluated, is not recommended.     

Useful variation in in vitro digestibility within perennial ryegrass

In vitro dry matter digestibility (DMD) and dry matter yield (DMY) of herbage from plots sown with three selected perennial ryegrass varieties with similar heading dates (Ba11778, Talbot and Gator) under a range of harvesting frequencies (2, 3, 4 and 5-weekly harvests) were compared over two harvest years (1994 and 1995). Varieties varied in mean percentage DMD over all harvests and harvesting frequencies by 3.4 and 3.5 units in 1994 and 1995 respectively. Varietal ranking in mean DMD over all harvests was the same under each harvesting frequency in both years: Ba11778 being the highest, Gator the lowest and Talbot intermediate. In 1995, Gator had a higher proportion of unsown species in the herbage than both Ba11778 and Talbot but this was only a minor factor contributing to the differences among the varieties in mean DMD over all harvests. Although varieties consistently ranked in the same order of mean DMD over all harvesting frequencies during 13 5-week periods, the range among varieties varied from less than 1 unit to 8.6 units. This range was greatest in mid-season of each year when mean DMD over all varieties was lowest. The minimum mean DMD over all harvesting frequencies during both years was 75.5%, 70.9% and 66.8% for Ba11778, Talbot and Gator respectively. The most digestible variety Ba11778 also had higher annual DMY than the other two varieties in both harvest years and under all four harvesting frequencies. These results indicate that there is sufficient useful genetic variation for DMD within perennial ryegrass to justify breeding for the trait and its routine assessment in official variety trials.     

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.     

Pattern of inheritance of a self-fertility gene in an autotetraploid perennial ryegrass (Lolium perenne) population

A mutation causing self-fertility (SF) in perennial ryegrass was studied at the tetraploid level. The aim of this work was to determine a) whether SF remains functional in a tetraploid population and b) whether the SF mutation expresses dominance in heterozygous pollen grains. A tetraploidized plant carrying SF alleles was self-pollinated to create a segregating F₂ population. In the F₂ individuals, pollen compatibility ranged between 38% and 84% showing that SF remained functional. The SF locus genotype was the main determinant of pollen compatibility explaining 78% of the variation. The observed segregation was significantly different from the expected under both SF being dominant or recessive models (P_(χ2)≤0.001) and tended to be intermediate between them, indicating partial dominance or additive gene action. The frequency of the different genotypes suggested that pollen grains homozygous for the mutation have a competitive advantage over heterozygous pollen and that pollen compatibility is affected by the interaction with additional loci. The implications of our results for breeding polyploid grasses are discussed.     

Hybrid production in Lolium perenne based on incompatibility

Summary SI-hybrids were produced by crossing 30 partially inbred lines in 3 sets of 5×5 factorial crosses. Annual dry matter yields from 3 successive years and 2 locations were on average 9.5 T/ha for the hybrids compared to 8.9 T/ha for their parental lines, with the best hybrid yielding 10.3 T/ha. Heterosis was up to 20%. Problems and prospects of SI-hybrid production in grasses are discussed.This paper is dedicated to Prof. Dr. F.W. Schnell on the occasion of his 80th birthday.     

Genetic variation in leaf epidermal cell size and shape in Lolium perenne

Summary Six diploid perennial ryegrass populations, two diploid F₁ hybrid families and the autotetraploid cv. Tove were grown in a glasshouse and the mean length and mean width of epidermal cells on the abaxial leaf surface of young fully expanded leaves compared. The diploids varied in cell length by 26%, in cell width by 9% and in length: width ratio by 39%. The cells of cv. Tove were 25% longer and 12% wider than those of the highest ranking diploid and the length: width ratio 9% higher. The DNA contents per telophase root-tip cell nucleus of a large-celled and a small-celled diploid population were similar, suggesting that the variation in epidermal cell size among diploids was due to the action of specific genes and not to differences in genome size. The mean epidermal cell dimensions of four of the above populations growing as plots in a field trial employing three levels of fertiliser nitrogen and four harvesting frequencies were measured on three occasions. Varietal ranking in cell dimensions was consistent over the different managements and dates, and agreed well with the glasshouse results. The results indicate that measurements of leaf epidermal cell length and width can be useful aids both in breeding for herbage yield and in distinguishing varieties with similar flowering dates.