Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.)

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic ESTRFLP loci in the F₁(NA₆ í AU₆) population. A comprehensive set of ESTSSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA₆ genetic map contains 88 ESTRFLP and 71 ESTSSR loci with a total map length of 963 cM, while the AU₆ genetic map contains 67 ESTRFLP and 58 ESTSSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.An erratum to this article can be found at M.J. Faville and A.C. Vecchies contributed equally to this work.     

QTL analysis and comparative genomics of herbage quality traits in perennial ryegrass (Lolium perenne L.)

Genetic control of herbage quality variation was assessed through the use of the molecular marker-based reference genetic map of perennial ryegrass (Lolium perenne L.). The restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) and genomic DNA-derived simple sequence repeat-based (SSR) framework marker set was enhanced, with RFLP loci corresponding to genes for key enzymes involved in lignin biosynthesis and fructan metabolism. Quality traits such as crude protein (CP) content, estimated in vivo dry matter digestibility (IVVDMD), neutral detergent fibre content (NDF), estimated metabolisable energy (EstME) and water soluble carbohydrate (WSC) content were measured by near infrared reflectance spectroscopy (NIRS) analysis of herbage harvests. Quantitative trait locus (QTL) analysis was performed using single-marker regression, simple interval mapping and composite interval mapping approaches, detecting a total of 42 QTLs from six different sampling experiments varying by developmental stage (anthesis or vegetative growth), location or year. Coincident QTLs were detected on linkage groups (LGs) 3, 5 and 7. The region on LG3 was associated with variation for all measured traits across various experimental datasets. The region on LG7 was associated with variation for all traits except CP, and is located in the vicinity of the lignin biosynthesis gene loci xlpomt1 (caffeic acid-O-methyltransferase), xlpccr1 (cinnamoyl CoA-reductase) and xlpssrcad 2.1 (cinnamyl alcohol dehydrogenase). Comparative genomics analysis of these gene classes with wheat (Triticum aestivum L.) provides evidence for conservation of gene order over evolutionary time and the basis for cross-specific genetic information transfer. The identification of co-location between QTLs and functionally associated genetic markers is critical for the implementation of marker-assisted selection programs and for linkage disequilibrium studies, which will enable future improvement strategies for perennial ryegrass.     

Gene-associated single nucleotide polymorphism discovery in perennial ryegrass (Lolium perenne L.)

Molecular genetic marker development in perennial ryegrass has largely been dependent on anonymous sequence variation. The availability of a large-scale EST resource permits the development of functionally-associated genetic markers based on SNP variation in candidate genes. Genic SNP loci and associated haplotypes are suitable for implementation in molecular breeding of outbreeding forage species. Strategies for in vitro SNP discovery through amplicon cloning and sequencing have been designed and implemented. Putative SNPs were identified within and between the parents of the F₁(NA₆ × AU₆) genetic mapping family and were validated among progeny individuals. Proof-of-concept for the process was obtained using the drought tolerance-associated LpASRa2 gene. SNP haplotype structures were determined and correlated with predicted amino acid changes. Gene-length LD was evaluated across diverse germplasm collections. A survey of SNP variation across 100 candidate genes revealed a high frequency of SNP incidence (c. 1 per 54 bp), with similar proportions in exons and introns. A proportion (c. 50%) of the validated genic SNPs were assigned to the F₁(NA₆ × AU₆) genetic map, showing high levels of coincidence with previously mapped RFLP loci. The perennial ryegrass SNP resource will enable genetic map integration, detailed LD studies and selection of superior allele content during varietal development.     

Isolation of viable egg cells of perennial ryegrass (Lolium perenne L.)

Summary Isolation of viable egg cells of perennial ryegrass (Lolium perenne L.) has been accomplished. After an enzyme incubation, ovules disintegrated into loose cells upon mechanical manipulation. The egg cells could be identified between the bulk of sporophytic cells derived from the macerated ovules. The morphology of the isolated egg cell corresponds to the morphology of the egg cell in situ and is comparable to the morphology of egg cells of other monocotyledons and angiosperms. Two hours after isolation the egg cells were still viable. The protocol proved reproducible and the yield was determined at 10%.     

Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Infection rates of perennial ryegrass seeds (Lolium perenne L.) with different fungi

Authors made investigations to map occurrence of different fungal pathogens on seeds of varieties from different grass species. This work presents data on three cultivars of perennial ryegrass (Lolium perenne L.). When seeds were germinated, eight genuses of fungi were identified belonging to the Deuteromycota. Alternaria was the most frequently occurred genus. Drechslera, Bipolaris, Cladosporium, Stemphylium, and Epicoccum spp. has also been found in the seed samples. Two genus of fungal pathogens (Embellisia and Septonema) were identified which were not mentioned in Hungary so far.     

Cytological characterization of isolated sperm cells of perennial ryegrass (Lolium perenne L.)

Summary The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.     

Effects of sheath tube length on leaf development in perennial ryegrass (Lolium perenne L.)

Position in and contribution of leaf laminae to the canopy of forage grasses are important both in determining herbage growth rates and intake rate by grazing animals. These canopy characteristics are controlled by the way dry matter is apportioned between sheath and lamina in growing leaves. The objective of this work was to determine how the development of individual leaves is affected by altering the effective length of the psuedostem tube, on the assumption that the light environment within the tube varied. The development of a leaf from initiation at the apex to maturity was followed by successive destructive dissections of tillers. Vertical incisions were made in the pseudostem of each tiller to three different depths. The three treatments imposed were — no incision (control), moderate and severe incision of the sheath length. Destructive harvests of tillers followed 3, 6, 12 and 24 days after imposition of treatments. Incision resulted in the length of the monitored leaf being reduced significantly at all harvests, and differentiation of the sheath beginning earlier. The length reduction reflected a reduction in both cell size and cell number and the effects were evident at the earliest harvest. The data support the theory that leaf size and timing of onset of sheath development are influenced by the environment of the developing leaf. The present results indicate that sheath tube length affects leaf development and suggests that the effects are substantially explained by a direct light effect on the location and depth of the elongation zone.     

QTL mapping of vernalization response in perennial ryegrass (Lolium perenne L.) reveals co-location with an orthologue of wheat VRN1

The objective of this study was to map quantitative trait loci (QTL) for the vernalization response in perennial ryegrass (Lolium perenne L.). The mapping population consisted of 184 F₂ genotypes produced from a cross between one genotype of a synthetic perennial ryegrass variety Veyo and one genotype from the perennial ryegrass ecotype Falster. Veyo and Falster were chosen among four different populations because of their contrasting vernalization requirements. In total, five QTL for the vernalization response, measured as days to heading, were identified and mapped to linkage groups (LG) LG2, LG4, LG6 and LG7. Individually, these QTL explained between 5.4 and 28.0% of the total phenotypic variation. The overall contribution of these five QTL was 80% of the total phenotypic variation. A putative orthologue of Triticum monococcum VRN1 was amplified from genomic DNA from perennial ryegrass. PCR fragments covering the proximal part of the promoter and the 5 end of the orthologue were subsequently PCR-amplified from both parents of the mapping population and shown to possess 95% DNA sequence identity to VRN1. Several polymorphisms were identified between Veyo and Falster in this fragment of the putative VRN1 orthologue. A CAPS marker, vrn-1, was developed and found to co-segregate with a major QTL on LG4 for the vernalization response. This indicates that the CAPS marker vrn-1 could be located in an orthologous gene of the wheat VRN1.     

Effects of genotype and temperature on pollen tube growth in perennial ryegrass (Lolium perenne L.)

Summary Low yield in seed crops of perennial ryegrass is related to low fertilization efficiency and low temperature during anthesis. To study the effect of genotype and temperature on pollen performance, we conducted greenhouse experiments at controlled temperatures. Individual florets of four genotypes that are known to differ in seed production were hand pollinated at four temperatures (14°, 18°, 22°, 26° C) both in vivo and via a semiin-vitro method involving excised florets on agar. Pollen germination and tube growth were determined with UV-fluorescence microscopy and scored in six classes at 2 h after pollination in vitro and after 0.5, 2 and 5 h in vivo. In vitro, both genotype and temperature had a significant effect on the performance of self-pollen. Pollen tube growth increased with temperature. In cross-pollinations, the pistil parent had a significant effect on pollen tube growth, and there was also a significant pistil-by-temperature interaction. In vivo, genotype and temperature significantly affected pollen performance. The genotype-by-temperature interaction was only significant 5 h after pollination. One genotype with low seed yield was pseudoself-compatible and was a relatively poor mother after cross-pollination. The effects of genotype and temperature on the growth of self-pollen might be exploited in a breeding programme.A.G. Stephenson was on a sabbattical leave at SVP in 1987     

Identification of QTLs for morphological traits influencing waterlogging tolerance in perennial ryegrass (Lolium perenne L.)

Perennial ryegrass is a globally cultivated obligate outbreeding diploid species (2n = 2x = 14) which is subjected to periods of waterlogging stress due to flood irrigation during winter and the lead-up to summer. Reduction of oxygen supply to root systems due to waterlogging produces consequent deleterious effects on plant performance. Framework genetic maps for a large-scale genetic mapping family [F?(NA(x) × AU?)] were constructed containing 91 simple sequence repeat and 24 single nucleotide polymorphism genetic markers. Genetic trait dissection using both control and waterlogging treatments was performed in the glasshouse, a total of 143 maximally recombinant genotypes being selected from the overall sib-ship and replicated threefold in the trial. Analysis was performed for nine quantitative morphological traits measured 8 weeks after stress treatments were applied. A total of 37 quantitative trait loci (QTLs) were identified; 19 on the NA(x) parental genetic map, and 18 on the AU? parental genetic map. Regions of particular interest were identified on linkage groups (LGs) 4 and 3 of the respective maps, which have been targeted for further analysis by selection of critical recombinants. This first study of genetic control of waterlogging tolerance in ryegrasses has important implications for breeding improvement of abiotic stress adaptation.     

Association of candidate genes with flowering time and water-soluble carbohydrate content in Lolium perenne (L.)

We describe a candidate gene approach for associating SNPs with variation in flowering time and water-soluble carbohydrate (WSC) content and other quality traits in the temperate forage grass species Lolium perenne. Three analysis methods were used, which took the significant population structure into account. First, a linear mixed model was used enabling a structured association analysis to be incorporated with the nine populations identified in the structure analysis as random variables. Second, a within-population analysis of variance was performed. Third, a tree-scanning method was used, in which haplotype trees were associated with phenotypes on the basis of inferred haplotypes. Analysis of variance within populations identified several associations between WSC, nitrogen (N), and dry matter digestibility with allelic variants within an alkaline invertase candidate gene LpcAI. These associations were only detected in material harvested in one of the two years. By contrast, consistent associations between the L. perenne homolog (LpHD1) of the rice photoperiod control gene HD1 and flowering time were identified. One SNP, in the immediate upstream region of the LpHD1 coding sequence (C-4443-A), was significant in the linear mixed model. Within-population analysis of variance and tree-scanning analysis confirmed and extended this result to the 2118 polymorphisms in some of the populations. The merits of the tree-scanning method are compared to the single SNP analysis. The potential usefulness of the 4443 SNP in marker-assisted selection is currently being evaluated in test crosses of genotypes from this work with turf-grass varieties.     

Development of phosphogluco-isomerase isozymes in perennial ryegrass (Lolium perenne)

The development of isozymes of phosphogluco-isomerase (PGI; D-glucose-6-phosphate ketol isomerase EC 5.3.1.9.) in perennial ryegrasses was followed from dry seed through to leaf senescence using starch gel electrophoretic separations. Root isozymes were also examined. Two forms of the enzyme were found, one (PGI/2) being present in all tissues and at all stages of the life cycle. The other (PGI/1) had two zones of activity, one of which was detected only in light-exposed tissue. Normal development of this form could be inhibited by growing seedlings on distilled water. Some alleles of PGI/2 not previously reported for ryegrasses are also described.     

Acclimation to freezing temperatures in perennial ryegrass (Lolium perenne)

The increasing demands being placed on natural grasslands in the era following the appearance of Bovine Spongiform Encephalitis require that forage crops provide a reliable extended season of growth, combined with good winter survival to ensure sward longevity. The ability to tolerate sub-zero temperatures is integral to the survival of perennial forages. Since the development of freezing tolerance is crucial to the survival and productivity of over-wintering crops, forage breeding programmes require an improved understanding of the individual characteristics that contribute to tolerance to sub-zero temperatures. Photosynthesis, carbohydrate content and changes in protein composition were investigated in two varieties of Lolium perenne which differ in their response to growth at low temperature.