Study of the genetic potential of autochthonous populations of perennial ryegrass (Lolium perenne L.) for use in breeding purposes

BackgroundPyrrolizidine alkaloids (PAs) are a class of secondary metabolites that function as feeding deterrents in a range of different plant species. In perennial ryegrass (Lolium perenne L.) the only PAs that have been identified are the thesinine-rhamnoside group, which displays significant genetic variation. Homospermidine synthase (HSS) has evolved from deoxyhypusine synthase (DHS) and catalyses the first step in the PA pathway, making it a key candidate for the investigation of genes influencing observed PA trait variation.ResultsDuring PCR amplification and sequence analysis of DHS we identified two putative HSS genes in perennial ryegrass. One of the genes (LpHSS1) was absent in some perennial ryegrass plants. Thesinine-rhamnoside levels were measured using liquid chromatography coupled with mass spectrometry in a diverse association mapping population, consisting of 693 plants free of fungal endophytic symbionts. Association tests that accounted for population structure identified a significant association of absence of the LpHSS1 gene with lower levels of thesinine-rhamnoside PAs. HSS-like gene sequences were identified for other grass species of the Poaceae, including tall fescue, wheat, maize and sorghum.ConclusionHSS is situated at the crucial first step in the PA pathway making it an important candidate gene for investigation of involvement in PA phenotypic variation. In this study, PA level in perennial ryegrass was strongly associated with the presence or absence of the LpHSS1 gene. A genetic marker, developed for the presence/absence of LpHSS1, may be used for marker-assisted breeding to either lower or increase PAs in breeding populations of perennial or Italian ryegrass to investigate a potential role in the deterrence of herbivore pests. The presence of HSS-like genes in several other Poaceae species suggests that PA biosynthesis may occur in plant family members beyond perennial ryegrass and tall fescue and identifies a potential route for manipulating PA levels.

Perennial ryegrass (Lolium perenne) has developed resistance to glyphosate within New Zealand vineyards following many years of herbicide application. The objectives of this work were to confirm resistance within two populations obtained from affected vineyards, and to determine the mechanism of resistance to glyphosate. Population O was confirmed to have a 25-fold resistance to glyphosate, whereas population J had a sevenfold resistance. Results of genotyping assays demonstrated a single nucleotide substitution at codon 106 of 5-enolpyruvylshikimate-3-phosphate synthase in population O but not population J. Glyphosate-resistant and glyphosate-susceptible populations did not differ in glyphosate absorption. However, in both resistant populations, much more of the absorbed (14) C-glyphosate remained in the treated leaf than occurred in the susceptible population. Significantly more glyphosate was found in the pseudostem region of susceptible plants compared with resistant plants. Both target-site and non-target-site mechanisms of glyphosate resistance were found in the perennial ryegrass population with 25-fold resistance, whereas only the non-target-site mechanism of resistance was found in the population with sevenfold resistance. This is the first study of the mechanism of glyphosate resistance in perennial ryegrass.

Self-incompatibility (SI) is a mechanism that many flowering plants employ to prevent fertilisation by self- and self-like pollen ensuring heterozygosity and hybrid vigour. Although a number of single locus mechanisms have been characterised in detail, no multi-locus systems have been fully elucidated. Historically, examples of the genetic analysis of multi-locus SI, to make analysis tractable, are either made on the progeny of bi-parental crosses, where the number of alleles at each locus is restricted, or on crosses prepared in such a way that only one of the SI loci segregates. Perennial ryegrass (Lolium perenne L.) possesses a well-documented two locus (S and Z) gametophytic incompatibility system. A more universal, realistic proof of principle study was conducted in a perennial ryegrass population in which allelic and non-allelic diversity was not artificially restricted. A complex pattern of pollinations from a diallel cross was revealed which could not possibly be interpreted easily per se, even with an already established genetic model. Instead, pollination scores were distilled into principal component scores described as Compatibility Components (CC1-CC3). These were then subjected to a conventional genome-wide association analysis. CC1 associated with markers on linkage groups (LGs) 1, 2, 3, and 6, CC2 exclusively with markers in a genomic region on LG 2, and CC3 with markers on LG 1. BLAST alignment with the Brachypodium physical map revealed highly significantly associated markers with peak associations with genes adjacent and four genes away from the chromosomal locations of candidate SI genes, S- and Z-DUF247, respectively. Further significant associations were found in a Brachypodium distachyon chromosome 3 region, having shared synteny with Lolium LG 1, suggesting further SI loci linked to S or extensive micro-re-arrangement of the genome between B. distachyon and L. perenne. Significant associations with gene sequences aligning with marker sequences on Lolium LGs 3 and 6 were also identified. We therefore demonstrate the power of a novel association genetics approach to identify the genes controlling multi-locus gametophytic SI systems and to identify novel loci potentially involved in already established SI systems.

Field surveys were conducted across the Blacklands region of Texas during 2016 and 2017 to document the distribution of herbicide-resistant Lolium spp. infesting winter wheat production fields in the region. A total of 68 populations (64 Italian ryegrass, four perennial ryegrass) were evaluated in a greenhouse for sensitivity to herbicides of three different modes of action: an acetolactate synthase (ALS) inhibitor (mesosulfuron-methyl), two acetyl-coenzyme-A carboxylase (ACCase) inhibitors (diclofop-methyl and pinoxaden), and a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor (glyphosate). Herbicides were applied at twice the label-recommended rates for mesosulfuron-methyl (29 g ai ha−1), diclofop-methyl (750 g ai ha−1), and pinoxaden (118 g ai ha−1); and at the recommended rate for glyphosate (868 g ae ha−1). The herbicide screenings were followed by dose-response assays of the most-resistant ryegrass population for each herbicide at eight rates (0.5, 1, 2, 4, 8, 16, 32, and 64×), compared with a susceptible population at six rates (0.0625, 0.125, 0.25, 0.5, 1, and 2×). The initial screening and dose-response experiments were conducted in a completely randomized design with three replications and two experimental runs. Survivors (<80% injury) were characterized as highly resistant (0% to 20% injury) or moderately resistant (21% to 79%). Results showed that 97%, 92%, 39%, and 3% of the Italian ryegrass populations had survivors to diclofop-methyl, mesosulfuron-methyl, pinoxaden, and glyphosate treatments, respectively. Of the four perennial ryegrass populations, three were resistant to diclofop-methyl and mesosulfuron-methyl, and one was resistant to pinoxaden as well. Perennial ryegrass populations did not exhibit any resistance to glyphosate. Dose-response assays revealed 37-, 196-, and 23-fold resistance in Italian ryegrass to mesosulfuron-methyl, diclofop-methyl, and pinoxaden, respectively, compared with a susceptible standard. One Italian ryegrass population exhibited three-way multiple resistance to ACCase-, ALS-, and EPSPS-inhibitors. The proliferation of multiple herbicide–resistant ryegrass is a challenge to sustainable wheat production in Texas Blacklands and warrants diversified management strategies.

The mechanisms of a plant to resist water stress can be divided into 2 components: avoidance and tolerance. However, reliable methods for measuring these components have not been established. We qualitatively evaluated the avoidance and tolerance abilities of shoots in diploid and tetraploid populations of perennial ryegrass (Lolium perenne L.) by continuously measuring their water contents and mortality rates under severe dehydration stress. Plant age greatly influenced dehydration resistance. Younger (30 days old) plants showed lower resistance to dehydration stress than did older plants (60 days old) because of both their lower avoidance and tolerance abilities. The two populations showed contrasting patterns of response to dehydration stress. The tetraploid population (Condesa) had higher avoidance and lower tolerance than did the diploid population (Manhattan II), and different combinations of the 2 components resulted in similar degrees of resistance in the 2 populations. The results of experiments demonstrated the importance of distinction between avoidance and tolerance abilities for studies on plant water stress.

ABSTRACTAssignment or exclusion of an individual to specific populations or cultivars based on molecular genetic markers provides an attractive approach for varietal identification at the individual level in cross‐pollinated plant species. The objectives of this study were (i) to explore the molecular diversity and relationships between Australasian perennial ryegrass (Lolium perenne L.) populations; (ii) to investigate accuracy of assignment of individuals to different types of populations including ecotypic, nonrestricted‐ and restricted‐based cultivars; and (iii) to determine the effect of variable number of SSR loci and different statistical analysis methods on assignment accuracy. Eight forage perennial ryegrass populations comprising 48 individual plants per population were genotyped with 29 simple sequence repeat (SSR) marker loci. The number of alleles per locus ranged from 3.72 to 6.76. The mean observed heterozygosity varied from 0.419 to 0.538. Various genetic distance estimates and clustering methods obtained results consistent with breeding history. Genetic variation among (8.7%) and within populations (91.3%) was significant. Accuracy of individual assignment differed by population type, and it was higher (>90%) for restricted‐based cultivars than for ecotypic and nonrestricted‐based populations. These results indicate that SSR marker profiles can be effectively used to assign individuals for outbreeding populations such as perennial ryegrass. The approach used in this study may be useful for ryegrass germplasm management issues such as cultivar identification at the individual level.

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 x 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.

For the development of mechanistic models of herbage digestibility, quantitative insight into the effects of age, temperature and cultivar on digestibility characteristics of individual plant parts is needed. Towards that goal, glasshouse experiments were conducted at day/night temperatures of 13/8, 18/13 and 23/18 °C with vegetative and reproductive crops of two populations of perennial ryegrass (Lolium perenne L) selected for differences in leaf blade digestibility. Cell wall content (CWC) and true cell wall and organic matter digestibility (CWD and OMD) of vegetative and reproductive tillers were related to dimensions, mass, CWC and digestibility of separate plant parts. Compared with the vegetative tillers, the reproductive tillers had higher rates of leaf appearance, organic matter growth and CWD decline. Strikingly, for both tiller types, no direct effect of temperature on whole tiller CWD was observed, since temperature effects could be eliminated completely by relating CWD to development stage (DVS) expressed as number of leaves appearing on the main tiller. Temperature effects on CWD were restricted to its influence on tiller development rate only. The decline of CWD of individual plant parts with DVS in the reproductive tillers could be described with a negative exponential curve, which reached an asymptote that was higher for leaf blades (755 g kg−1) than for leaf sheaths (491 g kg−1) and stem internodes (230 g kg−1). However, all plant parts in both tiller types had the same fractional CWD decline rate of 0.395 per leaf appearance interval, independent of plant part insertion level, population or temperature. Differences between temperature treatments in OMD were caused by the higher CWC of plant parts at higher temperature, due to a stronger decline of the specific organic matter mass than of the specific cell wall mass of plant parts at increasing temperature. Differences in whole tiller OMD between populations were observed only for vegetative tillers and were also caused by differences in CWC. It is concluded that temperature increase accelerated both the tiller development rate and the rate of decline of CWD during aging to the same extent, whereas plant parts responded similarly in the fractional CWD decline pattern as a function of DVS. These trends offer unique possibilities for modelling grass digestibility under contrasting temperature regimes.© 2003 Society of Chemical Industry

A genetic approach to the multivariate differentiation of perennial ryegrass ( Lolium perenne L.) populations

Differences have been shown between ryegrass and fescue within the Festulolium subline introgression family for fructan synthesis, metabolism, and polymer-size traits. It is well-established that there is considerable variation for water-soluble carbohydrate and fructan content within perennial ryegrass. However there is much still to be discovered about the fructan polymer pool in this species, especially in regard to its composition and regulation. It is postulated that similar considerable variation for polymer traits may exist, providing useful polymers for biorefining applications. Seasonal effects on fructan content together with fructan synthesis and polymer-size traits have been examined in diverse perennial ryegrass material comprising contrasting plants from a perennial ryegrass F2 mapping family and from populations produced by three rounds of phenotypic selection. Relationships with copy number variation in candidate genes have been investigated. There was little evidence of any variation in fructan metabolism across this diverse germplasm under these conditions that resulted in substantial differences in the complement of fructan polymers present in leaf tissue at high water-soluble carbohydrate concentrations. The importance of fructan synthesis during fructan accumulation was unclear as fructan content and polymer characteristics in intact plants during the growing season did not reflect the capacity for de novo synthesis. However, the retention of fructan in environmental conditions favoring high sink/low source demand may be an important component of the high sugar trait and the roles of breakdown and turnover are discussed.

Three quick tests were evaluated for detecting glyphosate-resistant biotypes of Italian ryegrass and perennial ryegrass. Biotypes of these two species were used that were 13.4 and 7.3 times more resistant to glyphosate, respectively, than susceptible biotypes when assessed using a sprayed pot study. One assay exposed germinating seeds of resistant and susceptible populations for 8 days to different concentrations of glyphosate. Measurements of reduction in seedling root growth gave an estimate of resistance magnitude of 10.8 and 8.9 for Italian ryegrass and perennial ryegrass, respectively, similar to that from the sprayed pot study. Two other assays were tested and were able to differentiate resistant and susceptible biotypes. One measured the relative levels of shikimic acid in leaves following exposure to glyphosate; four to seven times more shikimic acid accumulated in glyphosate-susceptible populations of Italian and perennial ryegrass than in resistant populations. The other assay involved growing tillers of the plants in glyphosate solutions and the results showed that at glyphosate concentrations of 10 and 40 mg/L, the plantlets of resistant plants showed negligible visual injury and growth reduction compared with susceptible ones. Results obtained from these assays were not affected by whether the glyphosate formulation used was an isopropylamine salt or potassium salt.

Analysis of the competitive interactions among a set of white clover and perennial ryegrass populations indicated that the intra-specific pressures exerted by the white clover plants upon themselves were significantly greater than the inter-specific pressures they exerted upon the perennial ryegrass. This partitioning of competitive effects could not be carried out on the ryegrass populations, however, because the data required the fitting of separate models to monocultures and duocultures. Although not significant at this stage of the experiment, trends were detected among some of the clover-ryegrass duocultures that could be related to their previous coexistence. The results are briefly discussed in relation to the problems surrounding the measurement of competitive effects among species that ideally require very different managements.

The number of cultivars of perennial ryegrass has steadily increased since the early 1960s. Perennial ryegrass has a self‐incompatible, cross‐pollinated breeding system and thus, each cultivar is a heterogeneous population of individual genotypes. The ability to accurately distinguish large numbers of genetically heterogeneous populations from one another, based solely on morphological data and agronomic performance, is limited. Random amplified polymorphic DNA (RAPD) markers offer a nearly unlimited supply of molecular traits on which to base distinctive characterizations of plant populations. Analysis of molecular variance (AMOVA) provides a statistical tool which partitions molecular marker variation within and among populations and performs significance testing of differences. The objective of this research was to survey RAPD marker variation within and among populations of perennial ryegrass to characterize breeding germplasm and commercial cultivars using AMOVA. A range of genetic diversity was represented by choosing 18 grass populations that included related crosses, derived selections, and unrelated ecotypes. For each population, 10 individuals were examined for 33 RAPD markers produced with two primers. Markers present in all individuals of a particular population but not present in any other individual of another population (fixed marker difference) were not observed among the 18 grass populations, so characterizations were based on marker frequency differences among populations. RAPD markers analyzed with AMOVA closely reflected the breeding histories of the perennial ryegrass germplasm or commercial cultivars. This was particularly true for relationships structured on a population basis. Results indicated that high selection intensity substantially altered the distribution of RAPD markers between parent and progeny populations thereby obscuring measurements of genetic relatedness. Furthermore, this study observed a limited ability to separate closely related populations. Evidence for the narrow germplasm base of turfgrass cuitivars was observed; although, many perennial ryegrass populations appeared to retain levels of within population variability comparable with that of land race ecotypes. The methods of analysis presented should provide a supplement to traditional morphological and agronomic data for plant variety protection and for measuring genetic diversity within breeding programs.

Severe outbreaks of toxicosis caused by the natural endophyte Neotyphodium lolii in perennial ryegrass (Lolium perenne L.) have occasionally resulted in heavy loss of livestock in Victoria and Tasmania. Meteorological records were examined from locations where acute toxicosis was recorded. High rainfall (>350 mm) in spring–summer was an obvious common feature; typically, it prolonged the period of high growth rate of pasture, increasing predicted spring–summer (September–March) growth (by ~36%) and increasing the predicted digestibility of organic matter of summer pasture (1 February) by 2.7% units (up to 55.6%). In years when severe toxicosis occurred, such rainfall was accompanied by dry, warm conditions in March and April, viz. mean maximum daily temperatures on the mainland of ≥21°C (March) and ≥18°C (April). During summer–autumn 2002, pastures from 10 Victorian farms on which acute perennial ryegrass toxicosis resulted in the loss of >7000 sheep/deer, were found to be dominated by perennial ryegrass. The ryegrass endophyte-produced toxins, ergovaline and lolitrem B, were detected in 74% and 100%, respectively, of composite faecal samples collected from acutely affected animals. The populations of perennial ryegrass all tested positive for Neotyphodium lolii endophyte, with a mean infection frequency of 90.5% (s.e.m. 1.81). The mean mycelial mass in basal tillers of perennial ryegrass was estimated at 66.00 (s.e.m. 3.40) from a sample of 20 plants, each of which was assessed on a scale of 1–5. Mycelial mass accounted for 19–20% of the variation in the concentration of ergovaline and lolitrem B in perennial ryegrass. Toxin concentrations in the plants where acute toxicosis was observed exceeded the tolerance levels for sheep (ergovaline 0.8 mg/kg; lolitrem B 1.8 mg/kg) in the minor (green) fraction of the plant in all but one affected pasture for ergovaline and all but two for lolitrem B. Maximum concentrations recorded were 4.3 and 4.6 mg/kg for ergovaline and lolitrem B respectively. Ergovaline increased in an asymptotic relationship with lolitrem B, which accounted for 45% of the variation in ergovaline. The concentrations of ergovaline and lolitrem B in the whole plant (green and dead fractions combined) were not greater than those recorded from randomly sampled pastures in earlier seasons, when only occasional mild cases of toxicosis were reported. Further examination of stored grass samples collected during the 2002 outbreak recently revealed unidentified peaks on the chromatograms for both ergovaline and lolitrem B; peaks that are not seen on chromatograms for toxic perennial ryegrass from New Zealand, where the expression of perennial ryegrass toxicosis is usually milder and rarely fatal.

Two populations of perennial ryegrass (Lolium perenne L.) S23, selected for contrasting rates of yield and mature leal dark respiration, were used in this study. Since previous work showed that yield decreased severely in population GL66 due to mechanical perturbation, possible effects on respiration rates were examined. Apart from a transient increase in population GL72, there was no effect on the respiration rate but mechanical perturbation did affect other processes. Handling caused transpiration ratesinthe light to increas for GL72, and in the dark to decrease for GL66. In the logger term, the nitrogen content decreased in handled plants grown at high density. These results emphasize that handling plants should be reduced to a minimum since it might have major effect on several processes. It is concluded that the original selection for the Lolium population was indeed on contrasting rates in yield and respiration. Furthermore it is argued that the existence of low‐ and high‐yielding genotypes can be attributed to the dissimilar responses of different genotypes to mechanical influence.