G. A. Kearney

Individual and multi-environment combined analyses identify QTLs for morphogenetic and reproductive development traits in white clover (Trifolium repens L.)

White clover (Trifolium repens L.) is a key component legume of temperate pasture agriculture and an important target for molecular marker-assisted plant breeding. A genetic map of white clover has been used to assess genetic control of agronomically important traits that vary in the F2(I.4R×I.5J) mapping family. Phenotypic analysis was performed for a range of vegetative morphogenesis traits (such as leaf area, internode length, plant height and plant spread) and reproductive morphogenesis and development traits (such as flowering date, floral intensity and seed yield), with both spatial and temporal replication. A multi-environment combined analysis (combined analysis) has been performed for traits assessed across multiple experimental datasets in order to identify consistent genetic effects. Quantitative trait locus (QTLs) were detected for the majority of traits, and the locations and magnitudes of QTL effects were compared between individual and combined analyses. This molecular genetic dissection of agronomic traits in white clover provides the basis for equivalent studies in more complex populations, design of marker-assisted selection strategies and comparative genetics with model legume species. Selection for QTLs derived from the combined analysis will permit robust improvement of phenotypic traits over different environments.

The effects of ploidy and seed mass on the emergence and early vigour of perennial ryegrass (Lolium perenne L.) cultivars

Genetic variation for seed mass and components of early vigour were measured on 120 seedlings of each of 18 diploid and 27 tetraploid perennial ryegrass (Lolium perenne L.) cultivars. Seeds of tetraploid cultivars were on average heavier (3.8 mg) than seed of diploid cultivars (2.4 mg). However, there was variation for mean seed mass both within and between ploidy classes and within cultivars. The components of early vigour measured on each of the seedlings were: date of emergence, leaf appearance rate, seedling height (10 days after germination), leaf length and width and dry matter at 33 days after emergence. Mean seed mass of a cultivar was significantly (P<0.05) positively correlated with seedling height, leaf length, leaf width, shoot length and shoot mass. Individual seed mass within a cultivar was significantly associated with all components of early vigour, except the date of emergence of the second true leaf. There was no difference in the rate of emergence of tetraploid and diploid cultivars, although tetraploid cultivars tended to have longer and wider leaves, greater seedling lengths and fewer leaves and tillers than diploid cultivars. There was also evidence of genetic variation for seedling vigour components in perennial ryegrass after seed mass and ploidy effects had been removed during analysis. These data suggest that seed mass has a large positive effect on early vigour of both diploid and tetraploid cultivars of perennial ryegrass and that, in the absence of any specific knowledge of the early vigour characteristics of a given cultivar, seed mass could be used as a surrogate when choosing between cultivars with similar adaptation and performance.

Assessment of gene flow using tetraploid genotypes of perennial ryegrass (Lolium perenne L.)

Ryegrass species are among the most important species in sown pastures, turf settings, and weed populations worldwide. Perennial ryegrass (Lolium perenne L.) is an outcrossing, wind-pollinated grass. Recent research has demonstrated the feasibility of developing transgenic perennial ryegrass varieties. In order to model the consequences of gene flow from transgenic grass genotypes in a field situation, the model non-transgenic trait of fertility among autotetraploid genotypes was chosen. Gene flow over distance and direction from a donor plot to surrounding sexually compatible recipient plants was studied. Reproductive isolation was achieved through the fertility barrier that arises between tetraploid and diploid ryegrass genotypes, despite the presence of diploid plants in a meadow situation. Fertility was used as an indication of effective gene flow over distance and direction. Measures of the fertility of recipient plants included total seed production (TSP), floret site utilisation (FSU), and relative fertility of recipient plants as a percentage of those within the donor plot (RF%). A leptokurtic distribution for gene flow was identified, with differences in the rate of decline over distance depending on direction. Simple sequence repeat (SSR) polymorphism was used to identify the paternity of progeny plants. The proportional representation of parents among the progeny was not significantly different from that expected due to the numerical representation of the different donor parent genotypes. The results of this research will have important implications for risk analysis prior to the field release of transgenic ryegrasses, fescues, and other pasture grass species, and for seed production in terms of cultivar purity and optimum isolation distance.

Evaluation of herbage yield in a forage grass breeding program: comparison of visual rating versus measurement in single-row plots or swards

To refine selection methods for a perennial ryegrass (Lolium perenne L.) breeding program, half-sib families and commercial cultivars were evaluated for 3 years with treatments sown as both single-drill rows or swards. Dry matter yield of the perennial ryegrass treatments was evaluated several times in each year as a visual score which was subsequently calibrated against a regression determined by cutting a subset of plots or by cutting all plots. Thus, the experiment evaluated 2 aspects of herbage-yield determination in a perennial ryegrass breeding program: (i) the use of visual estimates of herbage yield to reduce the time spent cutting plots, and (ii) the use of single-row plots compared with swards. The correlation (either as Pearsons correlation coefficient, or Spearmans rank correlation coefficient) between visual estimates of herbage yield was always significant (P<0.01), with the exception of the rank correlation for sward plots in the summer 1995 (r = 0.4; P<0.05). However, the extent of the correlation varied (r = 0.4–0.9), and at some harvests calibrated visual ratings only explained a small proportion of the variance observed in harvested dry matter yields. These data suggest that visual ratings of herbage yield would be accurate enough to be used to detect large differences between families, breeding lines, cultivars or accessions of perennial ryegrass. However, when differences between lines are likely to be small, then harvesting all plots would give a more accurate estimate of the yield of perennial ryegrass lines. Likewise, the herbage yield of perennial ryegrass in single-row plots was significantly correlated with the herbage yield of perennial ryegrass sown as swards (P<0.01 or P<0.05). However, the correlation was again variable leading to the conclusion that evaluation of perennial ryegrass as single-row plots was not always an accurate indicator of sward yield. For those 4 (of 13) harvests over 3 years where the interaction between sward yield and row yield of the perennial ryegrass lines was significant (P<0.05), this interaction was shown not to be due to significant rank changes but rather to an increase in the differences of yield in swards or yield in single-row plots. We conclude that the harvesting of swards was the most reliable method of estimating the dry matter yield of perennial ryegrass cultivars. However, significant correlations between visual rating of treatments, or yield in single-row plots and measured yield as swards illustrated that these methods (visual ratings and single-plot yields) could be used to reduce the cost of evaluating differences in the herbage yield potential of perennial ryegrass, especially when these differences were likely to be large or when seed is limited, such as during the evaluation of accessions.

The distribution of errors associated with genotype and environment during the prediction of the water-soluble carbohydrate concentration of perennial ryegrass cultivars using near infrared reflectance spectroscopy

Significant deviations associated with site or cultivars within sites were detected in 4 of 6 independent near infrared reflectance spectroscopy (NIRS) calibrations developed to predict water-soluble carbohydrate (WSC) concentrations in perennial ryegrass herbage harvested from 2 sites. These effects were observed both when calibration subsets were selected on the basis of spectral characteristics, and when calibration sets were balanced with respect to a priori knowledge of the structure of the data set. However, there were also instances when non-random deviations were not significant, demonstrating that it was possible to develop broadly based NIRS calibrations to predict WSC in perennial ryegrass. Deviations between NIRS predictions and reference values should be monitored, with reference to the structure of the experiment from which the samples were derived, before NIRS estimates of WSC concentration are used in agronomy or plant breeding.

Improving the power of pasture cultivar trials to discriminate cultivars on the basis of differences in herbage yield

A review of 7 recently published perennial ryegrass cultivar trials (from 6 contrasting environments) with data expressed as an aggregate of seasonal harvests (autumn, winter, spring, and summer) revealed that the l.s.d. (P = 0.05) varied between 4 and 255% of the mean herbage yield of the trial in a given season, with 56 of 72 data points having an l.s.d. (P = 0.05) >10% of the trial mean. Power analysis of a perennial ryegrass trial that was conducted at Heywood (Vic.) from 1997 to 1998, with a 16% apparent difference in the total yield of a new synthetic and commercial cultivars, demonstrated that this difference would have only been detected 45% of the time. However, if the number of replications in the trial was increased from 4 to 8, then it was predicted that this difference would have been detected 70% of the time. In response to the data from this experiment, a trial was sown in 1999 that compared 4, 6, and 8 replicates to detect differences in the herbage yield of perennial ryegrass cultivars. In this trial, differences that were detected (P < 0.05) with 8 replicates would have routinely gone undetected when 4 or 6 replicate combinations were used. The use of a row–column design on the 8 replicates of the trial reduced the error variance of the trial by 5–12%, depending on the harvest. It was concluded that current pasture cultivar trials are routinely failing to detect differences between cultivars at an adequate level, given the rates of genetic gain in pasture species. In order to increase this precision, the number of replicates in a trial should be optimised on the basis of past data and the likely difference between control and test cultivars. Once the number of replications in a trial has been optimised then the use of row–column, or nearest neighbour designs, and analysis, will further increase precision for little extra cost.

Evaluation of endophyte toxin production and its interaction with transgenic perennial ryegrass (Lolium perenne L.) with altered expression of fructosyltransferases

Alkaloid concentration of perennial ryegrass herbage is affected by endophyte strain and host plant genotype. However, previous studies suggest that associations between host and endophyte also depends on environmental conditions, especially those affecting nutrient reserves and that water-soluble carbohydrate (WSC) concentration of perennial ryegrass plants may influence grass-endophyte associations. In this study a single transgenic event, with altered expression of fructosyltransferase genes to produce high WSC and biomass, has been crossed into a range of cultivar backgrounds with varying Epichloë endophyte strains. The effect of the association between the transgenic trait and alkaloid production was assessed and compared with transgene free control populations. In the vast-majority of comparisons there was no significant difference between alkaloid concentrations of transgenic and non-transgenic plants within the same cultivar and endophyte backgrounds. There was no significant difference between GOI+ (gene of interest positive) and GOI− (gene of interest negative) populations in Janthritrem response. Peramine concentration was not different between GOI+ and GOI− for 10 of the 12 endophytes-cultivar combinations. Cultivar Trojan infected with NEA6 and Alto with SE (standard endophyte) exhibited higher peramine and lolitrem B (only for Alto SE) concentration, in the control GOI− compared with GOI+. Similarly, cultivar Trojan infected with NEA6 and Alto with NEA3 presented higher ergovaline concentration in GOI−. Differences in alkaloid concentration may be attributable to an indirect effect in the modulation of fungal biomass. These results conclude that the presence of this transgenic insertion, does not alter the risk (toxicity) of the endophyte–grass associations. Endophyte–host interactions are complex and further research into associations with high WSC plant should be performed in a case by case basis.