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Lolium latent virus (LoLV) was recently detected in the USA for the first time in ryegrass hybrids ( Lolium perenne × Lolium multiflorum). The genome of one USA isolate, LoLV-US1, has now been fully sequenced. The positive strand genomic RNA is 7674 nucleotides (nt) long and is organized in five open reading frames (ORFs) encoding the replication-associated protein, the movement-associated triple gene block proteins and the coat protein (CP). The genome organization is similar to that of viruses in the genera Potexvirus and Foveavirus; however, analysis of the complete LoLV genomic sequence, phylogenetic analyses of the deduced amino acid (aa) sequences of the polymerase and the CP, presence of a putative ORF 6, and the in vivo detection of two CPs in equimolar amounts, highlight features peculiar to LoLV. These characteristics indicate that LoLV forms a monotypic group separate from existing genera and unassigned species within the family Flexiviridae, for which we propose the genus name Lolavirus. One-step RT-PCR was developed for quick and reliable LoLV detection. [ABSTRACT FROM AUTHOR]

The advantages of androgenesis from Lolium × Festuca hybrids as a means towards enhanced gene expression and capture of rare genetic variation are reviewed. New evidence is presented for the technique’s use in combination with introgression-mapping for targeting Festuca-derived genes for enhanced freezing-tolerance. As a starting point, a dihaploid genotype derived by androgenesis from a Lolium multiflorum × Festuca pratensis amphiploid (2 n = 4 x = 28) hybrid cultivar is used as female parent in a backcross breeding programme with L. multiflorum (2 n = 2 x = 14). A derivative of the backcross␣breeding programme was a genotype of L.␣multiflorum (2 n = 2 x = 14) incorporating a F.␣pratensis introgression on chromosome 4 that was more freezing-tolerant than Lolium. New evidence of the importance in Lolium and Festuca species of the adaptive capabilities of Photosystem II (PSII) in relation to subsequent freezing-tolerance, is presented. Non-photochemical quenching (NPQ) mechanisms for expulsion of excess light energy during cold acclimation are found in F. pratensis but not in L. multiflorum. Screens of a backcross population derived from an initial dihaploid genotype ( n + n = 14) produced by androgenesis from a L. multiflorum × F.␣pratensis amphiploid, indicate a direct relationship between cold acclimation induced increases in NPQ and freezing-tolerance. Preliminary evidence of a role for genes found on chromosome 4 of F. pratensis for increased NPQ expression, is presented. [ABSTRACT FROM AUTHOR]

Gray leaf spot (GLS) is a serious fungal disease caused by Magnaporthe grisea, recently reported on perennial ryegrass ( Lolium perenne L.), an important turfgrass and forage species. This fungus also causes rice blast and many other grass diseases. Rice blast is usually controlled by host resistance, but durability of resistance is a problem. Little GLS resistance has been reported in perennial ryegrass. However, greenhouse inoculations in our lab using one ryegrass isolate and one rice-infecting lab strain suggest presence of partial resistance. A high density linkage map of a three generation Italian × perennial ryegrass mapping population was used to identify quantitative trait loci (QTL) for GLS resistance. Potential QTL of varying effect were detected on four linkage groups, and resistance to the ryegrass isolate and the lab strain appeared to be controlled by different QTL. Of three potential QTL detected using the ryegrass isolate, the one with strongest effect for resistance was located on linkage group 3 of the MFB parent, explaining between 20% and 37% of the phenotypic variance depending on experiment. Another QTL was detected on linkage group 6 of the MFA parent, explaining between 5% and 10% of the phenotypic variance. The two QTL with strongest effect for resistance to the lab strain were located on linkage groups MFA 2 and MFB 4, each explaining about 10% of the phenotypic variance. Further, the QTL on linkage groups 3 and 4 appear syntenic to blast resistance loci in rice. This work will likely benefit users and growers of perennial ryegrass, by setting the stage for improvement of GLS resistance in perennial ryegrass through marker-assisted selection. [ABSTRACT FROM AUTHOR]

We describe an association mapping approach using natural populations of perennial ryegrass ( Lolium perenne L.) to identify molecular markers associated with heading date, an important trait affecting seasonal production, tillering, digestibility and grassland management regimes. Twenty-three natural populations originating from throughout Europe, with heading date phenotypes ranging from very early to very late, as well as three synthetic populations (varieties) were used for molecular marker genotyping using AFLP. In total, 589 polymorphic markers were identified. Hierarchical clustering, principal coordinate and other statistical analyses identified four outlying populations forming a clearly distinct sub-group. Removal of those four populations from the subsequent analysis reduced population sub-structure twofold. However, this made relatively little difference to the result of the association analysis. Linear regression identified three markers whose frequency of occurrence correlated with the heading date phenotype. Moreover, these markers were shown to be closely linked to each other within a major QTL on Chromosome 7, explaining 70% of the total variation in heading date. Pairwise linkage disequilibrium among them was also significant. These results suggest that association mapping approaches may be feasible in L. perenne, and that the use of natural populations could provide a useful source of genetic variation in traits of importance in crop improvement. [ABSTRACT FROM AUTHOR]

The two-locus gametophytic incompatibility system in perennial ryegrass (Lolium perenne L.) is not always fully effective: obligate selfing of plants sieves self-compatible pollen mutants, and self-fertility becomes fixed in subsequent generations. Self-compatibility (SC) was investigated in an F2 family. In vitro self-pollinations were analysed and recorded and plants were classified as being either partially or fully compatible. Distorted segregation ratios of markers on linkage group (LG) 5 were found, which indicate the possible presence of a gametophytic SC locus. Interval linkage analysis of pollen compatibility after selfing confirmed that this distortion was due to a locus (T) analogous to the S5 locus of rye. However, even though markers in this region were, on average, less than 1?cM apart, the minimum number of plants possessing the unfavoured allele was never less than 6%for any marker locus. We proved that this was because of the presence of another SC locus, exhibiting gametophytic selection, segregating in this population and identified by interval mapping analysis of compatibility classes of in vitro self-pollinations. This locus was located on LG1, and probably corresponds to the S locus. We show that the T locus, a relic of a multilocus system, functions through interaction with the S locus: F2 segregation of incompatibility phenotypes and linked markers demonstrated that the S/t pollen genotype combination, expected to be compatible on selfing, was sometimes incompatible. Further evidence is presented to show that this interaction must be dependent on yet another locus located on LG2. A prime candidate would be the Z incompatibility locus.Heredity (2005) 94, 356-363. doi:10.1038/sj.hdy.6800582 Published online 29 September 2004 [ABSTRACT FROM AUTHOR]

Extension of the vegetative growth phase through delay of flowering is an important goal in today’s breeding programs of both forage and turf grasses. In forage grasses, the stem and inflorescence production comprise a significant reduction in the digestibility, nutritional value and productivity of the crop, and in turf grasses the stems that start to emerge during the growth season suppress the formation of new shoots and affect the quality, density and persistence of the sward. We have tested the potential of the strong floral repressor LpTFL1 from perennial ryegrass (Lolium perenne L.) to manipulate the transition to flowering in red fescue (Festuca rubra L.), a cool-season turf grass. Expression of LpTFL1 from the constitutive maize ubiquitin promoter represses flowering in red fescue, and the flowering repression phenotype correlates well with the level of LpTFL expression. Transgenic lines showing low to intermediate expression of LpTFL1 flowered approximately two weeks later than the controls, and transgenic lines showing very high LpTFL1 expression levels still remained non-flowering after exposure to natural vernalization conditions (Danish winter) in two successive years. There were no other phenotypic effects associated with the LpTFL transgene expression during vegetative growth. However, there was a tendency towards an LpTFL1-mediated reduction in stem length among the flowering lines. Expression of a truncated LpTFL, caused by transgene rearrangements during the transformation, lead to increased flowering and stem production and a decrease in panicle size. This is to our knowledge the first report on full inhibition of floral development in a commercially important grass species. [ABSTRACT FROM AUTHOR]

For ryegrass and many forage crops, characterization of varieties is often difficult for two reasons: few of discriminant morphological traits and a great within-varieties variation. Futhermore, few molecular markers are publicly accessible. In this paper we describe two approaches for the development of 42 sequence-tagged-site (STS) markers. Firstly, 14 STS markers were developed from Lolium sequences found in data bases. Secondly, 28 STS markers were developed from sequences found in related species of Gramineae. Out of 42 STS markers developed, 85.8% yielded successfull amplification and 62% revealed a high level of polymorphism with an average of five alleles per locus. The analysis of amplicons reveals a high STS marker specificity, a high conservation in gene structure and a strong intron sequence homology between allelic forms. Moreover, the majority of the STS markers can be considered as "universal markers" because 81% of these STS markers amplified successfully across 20 related grass species. These results permit us to consider the use of these markers in synteny studies. [ABSTRACT FROM AUTHOR]

The level of similarity between the DNA of Lolium multiflorum and three Festuca species, F. arundinacea, F. pratensis and F. glaucescens, was analysed on Southern blots, using DNA–DNA hybridization, and also on chromosomes using genomic in situ hybridization (GISH). It was demonstrated that the close relationship between L. multiflorum and the allohexaploid F. arundinacea arises principally from the affinity of L. multiflorum to one of the ancestral progenitors of F. arundinacea, namely F. pratensis. Using probes made from total genomic DNA of L. multiflorum, and also of L. perenne, particular regions of high homology, described here as ‘GISH bands’ were identified on F. pratensis chromosomes. These GISH bands, in combination with a specific probe for rDNA, provide us with new markers for the identification of chromosomes within the F. pratensis complement. Restriction analysis within the rDNA repeat unit revealed additional information on the close phylogeny of L. multiflorum and F. pratensis. The rDNA restriction patterns confirm that F. arundinacea originated as a hybrid between F. pratensis and F. glaucescens. [ABSTRACT FROM AUTHOR]

Pollen microspores from a hybrid between Lolium multiflorum (2n=4x=28) and Festuca arundinacea (2n=6x=42) were cultured and over 200 androgenic green plants established. In the initial hybrid one chromosome from each of the five homoeologous groups was labelled by a distinct PGI/2 homoeoallele. Segregation of PGI/2 alleles in the male gametes and in the androgenic plants indicated chromosome pairing preferences in the hybrid at meiosis. The pentaploid Festulolium hybrid genome comprised two homologous sets of Lolium (Lm) chromosomes and the polyhaploid set of F. arundinacea, namely one genome of F. pratensis (Fp) and two of F. glaucescens (Fg and Fg1). There was a high degree of preferential chromosome pairing between the two homologous sets of L. multiflorum chromosomes, and each androgenic plant contained a Lolium PGI/2 allele. Results supported earlier work indicating that gamete viability depended on the presence of a complete Lolium genome. Genomic in situ hybridization (GISH) was carried out on mitotic chromosome preparations of two androgenic plants using the total genomic DNA of L. multiflorum as a probe. Recombinants were observed between chromosomes of all three genomes in the pentaploid hybrid, demonstrating the efficacy of the hybrid as a starting point in introgression breeding programmes. [ABSTRACT FROM AUTHOR]

Genomic in situ hybridization (GISH) on mitotic chromosome preparations of two diploid Lolium multiflorum-like drought-resistant plants derived from a L. multiflorum × Festuca arundinacea hybrid is described. With F. arundinacea DNA as probe, each introgression line was found to carry a single Festuca recombinant chromosome segment. In both plants, the introgressed Festuca chromosome segment was on the long arm of chromosome 2 of the L. multiflorum-like hybrid derivative. A DNA probe of E pratensis, which is one of the progenitor species of F. arundinacea, hybridized with the introgressed E arundinacea chromosome segment. It is likely that genes that confer drought resistance have been transferred from the F. pratensis subgenome of F. arundinacea to L. multiflorum. It follows that the F. pratensis chromosome in F. arundinacea which is homoeologous to chromosome 2 in L. multiflorum carries genes for drought resistance. [ABSTRACT FROM AUTHOR]

The inverse types of autoallotriploid of Lolium temulentum × L. multiflorum have different trivalent frequencies and the possible causes were investigated by analysis of synaptonemal complex (SC) formation at zygotene. There is less synchrony in axial element (AE) formation in the TMM type than TTM. There are two autonomous pairing sites (APS) in the terminal regions, one at the end of the AE (telomeric) and one in a subterminal position. In the TMM combination, the L. temulentum subterminal APS cannot compete effectively with the pair of L. multiflorum APSs at synapsis and SCs are initiated between L. multiflorum AEs, but in TTM the L. multiflorum site competes with the pair of L. temulentum sites and SCs are frequently formed between homoeologous AEs. The L. temulentum chromosomes seem to be later in reaching a ready-to-pair stage and the results are taken to support the idea that genome asynchrony can lead to preferential pairing of chromosomes in polyploids. [ABSTRACT FROM AUTHOR]

Perennial ryegrass (Lolium perenne) is a perennial crop used in temperate regions as forage. In L. perenne breeding programs, persistency is an important trait. Poor persistency results in sward degradation and associated yield and nutritive value losses. Breeders assess persistency of accessions using visual scoring in field plots during the 2nd or 3rd growing season. This evaluation system is easy and cheap but is not free from human bias. In this study, the correlation between the scoring done by different breeders was only 0.243. As an alternative we have developed a methodology to assess persistency of L. perenne breeding materials based on vegetation indices (VIs) derived from Unmanned Aerial Vehicle (UAV) imagery. The VIs Excess green (ExG2), Green Leaf Index and Normalized green intensity (GCC) were found to provide consistent results for flights carried out under different light conditions and were validated by ground reference information. The correlation between the VIs and the percentage of ground cover extracted from on-ground imagery was 0.885. To test the implementation of the method we compared the ExG2 value based approach to selection with a visual score based selection methodology as applied by two breeders. The breeding decisions of Breeder A agreed well with decisions based on ExG2 values (74.6%), but those of Breeder B displayed a lower agreement (54.0%). In contrast, agreement between decisions based on different flights was very high (91.6%). The methodology was validated for general applicability. In summary, the results demonstrate that basing persistency selection in L. perenne breeding programs on ExG2 values from UAV imagery is likely to be more objective in comparison to the currently-used visual scoring method. [ABSTRACT FROM AUTHOR]

Annual ( Lolium multiflorum Lam.) and perennial ( L. perenne L.) ryegrass are two common forage and turfgrass species grown throughout the world. Perennial ryegrass is most commonly used for turfgrass purposes, and contamination by annual ryegrass, through physical seed mixing or gene flow, can result in a significant reduction in turfgrass quality. Seed certifying agencies in the United States currently use a test called seedling root fluorescence (SRF) to detect contamination between these species. The SRF test, however, can be inaccurate and therefore, the development of additional markers for species separation is needed. Male and female molecular-marker linkage maps of an interspecific annual Ö perennial ryegrass mapping population were developed to determine the map location of the SRF character and to identify additional genomic regions useful for species separation. A total of 235 AFLP markers, 81 RAPD markers, 16 comparative grass RFLPs, 106 SSR markers, 2 isozyme loci and 2 morphological characteristics, 8-h flowering, and SRF were used to construct the maps. RFLP markers from oat and barley and SSR markers from tall fescue and other grasses allowed the linkage groups to be numbered, relative to the Triticeae and the International Lolium Genome Initative reference population P150/112. The three-generation population structure allowed both male and female maps to be constructed. The male and female maps each have seven linkage groups, but differ in map length with the male map being 537 cm long and the female map 712 cm long. Regions of skewed segregation were identified in both maps with linkage groups 1, 3, and 6 of the male map showing the highest percentage of skewed markers. The (SRF) character mapped to linkage group 1 in both the male and female maps, and the 8-h flowering character was also localized to this linkage group on the female map. In addition, the Sod-1 isozyme marker, which can separate annual and perennial ryegrasses, mapped to linkage group 7. These results indicate that Lolium linkage groups 1 and 7 may provide additional markers and candidate genes for use in ryegrass species separation. [ABSTRACT FROM AUTHOR]