The application of pattern analysis for the recognition of adaptation in a collection of Lolium multiflorum populations

Clustering procedures for the recognition of patterns of adaptation were applied to 43 introduced populations of Lolium multiflorum undergoing evaluation prior to use in breeding programmes. Regular analysis of variance of the productivity revealed considerable interaction between populations and the 15 cuts imposed. The clustering reduced this to a 12 group situation, which maintained 85% of the population variation and 81% of population × cut interaction. The twelve groups displayed differing patterns of adaptation which could be related to the ecological situations from where the populations derived.     

Mapping of EST-derived CAPS markers in Italian ryegrass (Lolium multiflorum Lam.)

New molecular markers derived from expressed sequence tag (EST) sequences were mapped on linkage maps of Italian ryegrass by a two-way pseudo-testcross strategy. cDNA sequences were obtained from various tissues of Italian ryegrass ( Lolium multiflorum ) and converted into cleaved amplified polymorphic sequence (CAPS) markers. Of 260 EST primer pairs that amplified a single band, 74 generated bands that showed clear polymorphisms among individuals of an F₁ mapping family. Of the 74 polymorphic marker loci, 69 were mapped on an Italian ryegrass linkage map previously constructed using amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), and simple sequence repeat (SSR) markers. The newly-developed EST-CAPS markers would be useful as an efficient tool to identify genetic markers and to identify candidate genes for quantitative trait loci (QTLs) associated with important traits in Italian ryegrass.     

A sampling technique for assessing digestibility of Italian ryegrass populations

Summary A sampling technique is proposed for rapid digestibility screening of herbage grass ecotypes. Its application in the characterisation of genetic resources is discussed. The technique aims to standardize maturity and circumvent the problems of variation in stem:leaf ratio and the use of single plants versus swards.A experiment comparing the method with a standard technique using eleven ecotypes plus two controls of Lolium multiflorum is described.     

The application of in vitro tiller induction in Lolium multiflorum

Summary Eight Lolium multiflorum genotypes were micropropagated for over one year. Using Murashige & Skoog's basal medium, various growth regulators (6-benzylaminopurine, indole acetic acid, adenine sulphate) were tested for their effect on micropropagation. Plantlet handling methods, culture conditions and culture procedures were also compared. Differences were observed in rates of micropropagation between plant genotypes. Under the best conditions for micropropagation, an average of approximately 50 tillers were produced in a 5–6 week period, compared with an estimated 8–15 tillers from soil-grown plants over the same period. Tillers must reach a certain developmental stage before they will grow independently, therefore the number of surviving propagules at subculture under micropropagation and in soil is about half the number of tillers produced. A proposed system for the micropropagation of Lolium multiflorum is described.     

Colchicine-induced heritable variation in cell size and chloroplast number in the leaf cells of inbred ryegrasses (Lolium perenne,L. multiflorum)

Summary A 3 hr period of treatment with a 0.2% aqueous solution of colchicine was given to one week old seedlings of 3 inbred lines of perennial ryegrass (Lolium perenne) and 4 lines of Italian ryegrass (L. multiflorum). The surviving plants developed as mixoploids and were self-pollinated. In the following generation (CT1) the seeds were grown without further treatment, and were screened cytologically at the seedling stage to separate out diploids from tetraploids. Only the diploids were kept, and in this CT1 generation comparisons were then made between controls (2x) and colchicine-treated diploids (C2x) for leaf mesophyll cell size and chloroplast number variation. Heritable differences were found such that the cell plan areas and/or the numbers of chloroplasts per cell were significantly greater in the C2x compared with the 2x treatment in 3 and 5 out of the 7 lines. In each case there was enhancement of chloroplast numbers independently of the variation in cell size.     

Breeding of Italian Ryegrass (Lolium multiflorum Lam.) for a low nitrate concentration by seedling test

In Japan, nitrate poisoning in ruminantscaused by the accumulation of nitrate inforage crops is a serious concern. In thisstudy, 3 cycles of phenotypic recurrentselection for nitrate concentration on adry matter basis were conducted for Italianryegrass (Lolium multiflorum Lam. cv.Nioudachi) to develop a breeding methodusing a seedling test. The seedling testinvolved the nitrate analysis of a shootfrom a plant grown for 6 or 8 weeks in thepresence of 50 mM KNO₃. The selectionintensity was approximately 0.07 for eachcycle. The realized heritability of thethird generation was calculated to be 0.21for seedlings and 0.57 for adult plants. Ina pot experiment, the nitrate concentrationof the adult plants of the third generationwas found to be 60% of the value for theoriginal population at an early headingstage. The reduction is related to theincrease in dry matter (DM) content as wellas the decrease in nitrate concentration ona fresh matter basis. The original andselected populations did not differ in theheading date. The concentrations of K, Nand P in the third generation decreasedwith the increase of DM content, whereas Caand Mg concentrations remained constant. Variation in the nitrate concentration wasfound among varieties on the market. Inconclusion, the ability to accumulatenitrate is a quantitative genetic trait ofItalian ryegrass and large-scale selectionfor the breeding of low nitrate populationsis possible using a seedling test.     

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.     

Identification of molecular markers linked with crown rust (Puccinia coronata f. sp. lolii) resistance in perennial ryegrass (Lolium perenne) using AFLP markers and a bulked segregant approach

Crown rust resistance is an important selection criterion in ryegrass breeding. The fungal disease caused by P. coronata causes yield loss and a reduced quality of the fodder crop. Molecular markers were used to unravel the genomic organization of crown rust resistance in a segregating L. perenne population. Two genomic regions involved in crown rust resistance were identified that together explained 35% of the phenotypic variance present. Bulked segregant analysis in combination with AFLP markers was a suitable method to identify DNA markers associated with genomic regions of major effect. One cluster of AFLP markers explained 6.1% of the variance and mapped to linkage group 2, a genomic region known to contain crown rust resistance genes. A second cluster of AFLP markers detected a novel genomic region of major effect that explained 27.7% of the phenotypic variance in crown rust resistance. This cluster was unlinked to the cluster on linkage group 2. Divergent selections performed within the segregating F₁ population on the basis of genotype and phenotype revealed that the markers associated with crown rust resistance identified in this study have potential for marker assisted selection. Selection of plants on the basis of markers was more straightforward than the selection on the basis of phenotype.     

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.     

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.     

Introgression of crown rust resistance from Festuca spp. into Lolium multiflorum

Crown rust, Puccinia coronata Corda, causes one of the most damaging foliar diseases in Italian ryegrass, Lolium multiflorum Lam. For introgression of crown rust resistance, highly resistant hybrids of the crosses Festuca arundinacea (2n = 6x = 42) ×L. multiflorum (2n = 4x = 28) and reciprocally, L. multiflorum (2n = 2x = 14) ×Festuca pratensis (2n = 4x = 28) and subsequently resistant recombinant individuals were used as female parents and susceptible cultivars of Italian ryegrass as male parents in three successive backcrosses. The BC₃ plants were selfed and crossed mutually. Uredospores of seven different crown rust isolates collected from plants of L. multiflorum, Lolium perenne, F. pratensis, F. arundinacea and L. multiflorum × F. pratensis hybrids were applied to identify the resistance or susceptibility of the parental species, backcrossed, selfed and intercrossed progenies. The various crown rust populations revealed a species-specific capability to infect plants of the Lolium-Festuca complex corresponding to the host species from which the spores originated. Selected BC⁹ plants, however, were found to be completely resistant to all crown rust populations tested. Successful introgression of the resistance was achieved from F. arundinacea as well as from F. pratensis. The resistance represents a dominant character, apparently based on a strong heterologous incompatibility between host and pathogen. In phenotype, bivalent formation during meiosis and in fertility, the novel germplasms are comparable with the L. multiflorum cultivars.     

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.     

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.     

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.     

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.     

Disturbed Mendelian segregations at isozyme marker loci in early backcrosses of Lolium multiflorum × Festuca pratensis hybrids to L. multiflorum

Summary The segregation of interspecific recombinant Festuca pratensis (Fp) alleles, introgressed into the germolasm of Lolium multiflorum (Lm), at four loci (PGI/2, AcP/2, GOT/3 and BAP) is described. Heterozygous (Lm/Fp) plants were backcrossed to L. multiflorum (2n=2×=14) and subsequent BC2 Lm/Fp sibling genotypes intercrossed.In crosses between BC1 heterozygous plants (Lm/Fp) used as males and L. multiflorum, there was a reduced transmission of the F. pratensis (Donor Parent) derived alleles in the populations with PGI/2 and AcP/2 marker loci compared to the reciprocal cross but the reduction was not significantly different in those with GOT/3 and BAP markers.Two PGI/2 marked BC2 half-sib families in particular exhibited a more extreme deficiency of Fp/Fp progeny plants than anticipated from the BC2 segregations indicating possible linkage to zygotic lethals. Deficiencies of F. pratensis alleles were, in most cases, less marked in BC2 half-sib families indicating that a further round of recombination had reduced the size of the introgressed chromosome segment or that deleterious linkages had been broken. A tendency towards heterozygote advantage was found in one BAP marked halfsib family.The significance for forage grass breeding of reduced transmission rates of Donor Parent alleles in early back-cross generations especially through the male gametes is discussed.     

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.     

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.     

The controlled introgression of Festuca arundinacea genes into Lolium multiflorum

Summary Using phosphoglucoisomerase (PGI/2) as a genetic marker, it has been shown to be possible to transfer genes from Festuca arundinacea into diploid Lolium multiflorum using the pentaploid hybrid L. multiflorum (4x) x F. arundinacea (6x). The pentaploid hybrid was sufficiently fertile to be used in reciprocal crosses with diploid. L. multiflorum. When used as the male parent, only two backcross generations were then required to reconstitute the diploid genotype. Intergeneric recombinants including a F. arundinacea PGI/2 allele were found among the diploid BC₂. Cytological data indicates that although the majority of chromosome associations involve only homologous Lolium chromosomes, associations involving Lolium and Festuca chromosomes also occur.Interpollinating the pentaploid hybrids prior to commencing a backcrossing programme increases the number of cycles of recombination and improves the chance of recovering intergeneric recombinants. The crossing programme described is proposed to be an effective method of introducing F. arundinacea genes into L. multiflorum.