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 EST–RFLP loci in the F1(NA6 × AU6) population. A comprehensive set of EST–SSR 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 EST–RFLP and 71 EST–SSR loci with a total map length of 963 cM, while the AU6 genetic map contains 67 EST–RFLP and 58 EST–SSR 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.

Response of perennial ryegrass (Lolium perenne) to renovation in Australian dairy pastures

This study reports on the effect of oversowing perennial ryegrass (Lolium perenne L.) into a degraded perennial ryegrass and white clover (Trifolium repens L.) pasture to extend its productive life using various intensities of seedbed preparation. Sites in New South Wales (NSW), Western Australia (WA), South Australia (SA) and Tasmania (Tas.) were chosen by a local group of farmers as being degraded and in need of renovation. Control (nil renovation) and medium (mulch and graze, spray with glyphosphate and sow) renovation treatments were common to all sites whereas minimum (mulch and graze, and sow) and full seedbed (graze and spray with glyphosphate and then full seedbed preparation) renovation were imposed only at some sites. Plots varied in area from 0.14 to 0.50 ha, and were renovated then sown in March or April 2000 and subsequently grazed by dairy cows. Pasture utilisation was estimated from pre- and post-grazing pasture mass assessed by a rising plate pasture meter. Utilised herbage mass of the renovated treatments was significantly higher than control plots in period 1 (planting to August) and 2 (first spring) at the NSW site only. There was no difference among treatments in period 3 (first summer) at any site, and only at the WA and NSW sites in period 4 (March to July 2001) was there a response to renovation. As a result, renovation at the NSW site only significantly increased ryegrass utilisation over the whole experimental period. Ryegrass plant density was higher at the NSW, WA (excluding minimum renovation) and Tas. (excluding full renovation) sites 6 months after renovation but this was only sustained for 12 months for the minimum and medium treatments at the NSW and Tas. sites, respectively, presumably due to reduced competition from naturalised C4 summer grasses [kikuyu (Pennisetum clandestinum) and paspalum (Paspalum dilatatum)] in NSW At the NSW, WA and SA sites, the original ryegrass plant density was low (<35 plants/m2) compared with the Tas. site where density was around 185/m2. The response to renovating a degraded perennial ryegrass pasture varied between sites in Australia. Positive responses were generally small and were most consistent where renovation removed competing C4 summer grasses.

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

Sk?t, L., Humphreys, J., Humphreys, M. O., Thorogood, D., Gallagher, J. A., Sanderson, R., Armstead, I. P., Thomas, I. D. (2007). Association of candidate genes with flowering time and water-soluble carbohydrate content in Lolium perenne (L.). Genetics, 177 (1), 535-547. Sponsorship: BBSRC RAE2008

Effects of a stay-green mutation on plant nitrogen relations in Lolium perenne during N starvation and after defoliation

Macduff, J. H., Humphreys, M. O., Thomas, Howard (2002). Effects of a stay-green mutation on plant nitrogen relations in Lolium perenne during N starvation and after defoliation. Annals of Botany, 89 (1), 11-21. Sponsorship: BBSRC RAE2008

An association mapping approach to identify flowering time genes in natural populations of Lolium perenne (L.)

Sk?t, L., Humphreys, M. O., Armstead, I. P., Heywood, S., Sk?t, K. P., Sanderson, R., Thomas, I. D., Chorlton, K. H., & Sackville Hamilton, N. R. (2005). An association mapping approach to identify flowering time genes in natural populations of Lolium perenne (L.). Molecular Breeding, 15(3), 233-245. Sponsorship: BBSRC RAE2008

Differences in growth and nitrogen productivity between a stay-green genotype and a wild-type of Lolium perenne under limiting relative addition rates of nitrate supply

James Macduff, Neil Raistrick and Mervyn Humphreys (2002). Differences in growth and nitrogen productivity between a stay-green genotype and a wild-type of Lolium perenne under limiting relative addition rates of nitrate supply. Physiologia Plantarum, 116 (1), 52-61. Sponsorship: BBSRC RAE2008

Análisis comparativo del crecimiento y la partición de asimilatos de plantulas de Senecio madagascariensis, Crepis capillaris, Trifolium repens y Lolium perenne

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Thermogravimetric evaluation of perennial ryegrass (Lolium perenne) for the prediction of in vitro dry matter digestibility

Thermogravimetry (TG) can be used for assessing the compositional differences in grasses that relate to dry matter digestibility (DMD) determined by pepsin-cellulase assay. This investigation developed regression models for predicting DMD of herbage grass during one growing season using TG results. The calibration samples were obtained from a field trial of eight cultivars and two breeding lines. The harvested materials from five cuts were analysed by TG to identify differences in the combustion patterns within the range of 30-600 degrees C. The discrete results including weight loss, peak height, area, temperature, widths and residue of three decomposition peaks were regressed against the measured DMD values of the calibration samples. Similarly, continuous weight loss results of the same samples were also utilised to generate DMD models. The r(2) for validation of the discrete and the best continuous models were 0.90 and 0.95, respectively, and the two calibrations were validated using independent samples from 24 plots from a trial carried out in 2004. The standard error for prediction of the 24 samples by the discrete model (4.14%) was higher than that by the continuous model (2.98%). This study has shown that DMD of grass could be predicted from the TG results. The benefit of thermal analysis is the ability to detect and show changes in composition of cell wall fractions of grasses during different cuts in a year.

The effect of soil pH on rhizosphere carbon flow of Lolium perenne

Perennial rye-grass plants were grown at 15°C in microcosms containing soil sampled from field plots that had been maintained at constant pH for the last 30 years. Six soil pH values were tested in the experiment, with pH ranging from 4.3-6.5. After 3 weeks growth in the microcosms, plant shoots were exposed to a pulse of ¹⁴C-CO₂. The fate of this label was determined by monitoring ¹⁴C-CO₂ respired by the plant roots/soil and by the shoots. The ¹⁴C remaining in plant roots and shoots was determined when the plants were harvested 7 days after receiving the pulse label. The amount of ¹⁴C (expressed as a percentage of the total ¹⁴C fixed by the plant) lost from the plant roots increased from 12.3 to 30.6% with increasing soil pH from 4.3 to 6. Although a greater percentage of the fixed ¹⁴C was respired by the root/soil as soil pH increased, plant biomass was greater with increasing soil pH. Possible reasons for observed changes in the pattern of ¹⁴C distribution are discussed and, it is suggested that changes in the soil microbial biomass and in plant nitrogen nutrition may, in particular be key factors which led to increased loss of carbon from plant roots with increasing soil pH. © 1990 Kluwer Academic Publishers.

Carbon distribution within the plant and rhizosphere in laboratory and field-grown Lolium perenne at different stages of development

Carbon distribution within perennial ryegrass was determined at different stages of plant development, by pulse-labelling laboratory and field-grown plants with ¹⁴C-CO₂. During the early stages of growth (23-51 days), C distribution of laboratory grown plants was not markedly affected by plant age, with 12.4-24% of net assimilated label lost into the soil as root-soil respiration. The percentage of net assimilate translocated below ground was 20-28% during this stage of growth. At 65 days, the percentage of the label translocated below ground decreased to 8.1% of the net assimilate, with a subsequent decrease in root-soil respiration to 3.9%. The ability of the plant to fix the label (expressed in MBq g^-1 oven dry total plant weight) decreased steadily as the plants aged. When the 30 day old plants were subjected to water stress (soil water potential -1.5 MPa) for 2 days before pulse-labelling, root-soil respiration of the pulse-label decreased compared with plants grown at field capacity. The distribution of a ¹⁴C pulse-label within perennial ryegrass grown under field conditions was found to be dependent on the age of the plants. For 4 week old plants, 67% of net assimilated label was translocated below ground, with 64.8% of this respired by the roots and soil. Less label was translocated below ground at subsequent pulse-labels from weeks 8 to 24. The proportion of label translocated below ground respired by the roots and soil also decreased. The investment of label in the plant shoots was found to be greater in field grown plants as compared to plants of the same age grown in a controlled, laboratory environment. © 1990.

Carbon distribution within the plant and rhizosphere for Lolium perenne subjected to anaerobic soil conditions

Perennial ryegrass was subjected to a range of anaerobic treatments. The distribution of C within the plant was determined by pulse labelling the shoots with ¹⁴C-CO₂. A 5 h anaerobic period before pulse labelling reduced by 2.5-10 times the ¹⁴C remaining in the plants and released into the soil. The distribution of the ¹⁴C within the plant was also affected by anaerobiosis. Short periods of anaerobiosis (5 or 10 h) led to increased root-soil ¹⁴C respiration (monitored for 7 days). A longer period of anaerobiosis (48 h) initially inhibited root-soil ¹⁴C respiration, but when aerobiosis was restored. 57% of the total ¹⁴C fixed by the plant was respired by the roots-soil during the following 7 days compared to 19% for the aerobic control. There was a two-thirds reduction in the percentage C retained by the plants stressed for the 48 h compared to the aerobic control. At harvest, all anaerobic treatments were associated with more ¹⁴C remaining in the soil as a proportion of the total ¹⁴C fixed by the plant compared to the aerobic control. © 1990.

Distribution of assimilated carbon within the plant and rhizosphere of Lolium perenne:Influence of temperature

Perennial rye-grass plants were pulse labelled with [¹⁴C]-CO₂ over a range of temperatures (5-25°C). The fate of the label was determined within the plant and soil. The temperature at which plants were pulse labelled had a marked effect on the distribution of the label within the plant and soil system. Root-soil respiration increased from 5.7 to 24.15% when expressed as a percentage of net assimilated label. The percentage of label remaining in the plant root and in the soil was greater at 5 and 25°C, with a minimum for both these components at 15°C. At 15°C the percentage of net assimilated label that remained in the shoots was greater than at other temperatures, with this percentage decreasing at the lower and higher temperatures. © 1989.

Charakterisierung der mikrobiellen Population und Untersuchungen zum Quorum Sensing in der Rhizosphäre vom Weidelgras (Lolium perenne) auf einer Rückstandshalde der Kaliindustrie

Im Rahmen dieser Dissertation wurden die Dynamik und die Kommunikation innerhalb der mikrobiellen Population der Rhizosphäre von Deutschem Weidelgras (Lolium perenne) untersucht, welches auf einer teilweise rekultivierten Rückstandshalde der Kaliindustrie wuchs. Um die niederschlagsbedingte Auswaschung von Salzen zu reduzieren, wird die Rückstandshalde des Kaliwerks Sigmundshall (in Bokeloh bei Hannover) schrittweise mit dem technogenen Abdecksubstrat REKAL/SAV ummantelt. Dieses weist eine hohe Standfestigkeit und Wasserspeicherkapazität auf und kann zudem begrünt werden, wofür als Pionierpflanze Lolium perenne dient. Durch diese Rekultivierung wird Niederschlag besser gespeichert und über Evapotranspiration wieder in die Luft abgegeben, was letztendlich die Bildung von Salzwasser vermindert. Da das Abdecksubstrat neben alkalischem pH-Wert auch teilweise hohe Schwermetallkonzentrationen aufweist, sollte in der vorliegenden Arbeit erstmals die mikrobielle Rhizosphären-Gemeinschaft in diesem extremen Habitat mittels einer kulturunabhängigen Methode erforscht werden. Zudem wurden erste Untersuchungen angestellt, ob im Substrat die zelldichte-abhängige bakterielle Kommunikation (Quorum Sensing) nachgewiesen werden kann. Mittels extrahierter Gesamt-DNA wurde anhand der 16S rDNA die Analyse des „Terminalen Restriktonsfragmentlängenpolymorphismus“ (TRFLP) verwendet, um die komplexe bakterielle Rhizosphären-Gemeinschaft unter zeitlichen und lokalen Aspekten zu vergleichen. Auftretende Veränderungen bei den bakteriellen Populationen der jeweiligen Proben wurden durch eine Zu- oder Abnahme der auch als Ribotypen bezeichneten terminalen Restriktionsfragmente (TRF) erfasst. Hierbei zeigten sich am Südhang der Halde während der Sommermonate der Jahre 2008 und 2009 zwar Schwankungen in den bakteriellen Gemeinschaftsprofilen, es lagen jedoch keine eindeutigen Dynamiken vor. Im Vergleich zum Südhang der Halde wies der Nordhang eine höhere Ribotyp-Diversität auf, was mit der fortgeschritteneren Rekultivierung dieses Haldenabschnitts zusammenhängen könnte. Zusätzlich wurden Bakterien aus der Rhizosphäre von Lolium perenne isoliert und mithilfe der Biosensoren Agrobacterium tumefaciens A136 pCF218 pCF372 und Chromobacterium violaceum CV026 auf die Produktion von N-Acylhomoserinlactonen (AHLs) überprüft. Diese AHLs werden von Gram-negativen Mikroorganismen als Signalmoleküle verwendet, um ihre Genexpression zelldichteabhängig zu kontrollieren. Von den 47 getesteten Gram-negativen Rhizosphärenisolaten konnten nur bei einem reproduzierbar AHL-Moleküle mithilfe des Reporterstamms A. tumefaciens nachgewiesen werden. Der AHL-Produzent wurde als Pseudomonas fluorescens identifiziert. Mittels dünnschichtchromatographischer Analysen konnten die extrahierten bakteriellen AHL-Moleküle den N-Octanoyl-L-homoserinlactonen zugeordnet werden.

Determination of the impact of continuous defoliation of Lolium perenne and Trifolium repens on bacterial and fungal community structure in rhizosphere soil

To determine the effects of defoliation on microbial community structure, rhizosphere soil samples were taken pre-, and post-defoliation from the root tip and mature root regions of Trifolium repens L. and Lolium perenne L. Microbial DNA isolated from samples was used to generate polymerase chain reaction-denaturing gradient gel electrophoresis molecular profiles of bacterial and fungal communities. Bacterial plate counts were also obtained. Neither plant species nor defoliation affected the bacterial and fungal community structures in both the root tip and mature root regions, but there were significant differences in the bacterial and fungal community profiles between the two root regions for each plant. Prior to defoliation, there was no difference between plants for bacterial plate counts of soils from the root tip regions; however, counts were greater in the mature root region of L. perenne than T. repens. Bacterial plate counts for T. repens were higher in the root tip than the mature root region. After defoliation, there was no effect of plant type, position along the root or defoliation status on bacterial plate counts, although there were significant increases in bacterial plate counts with time. The results indicate that a general effect existed during maturation in the root regions of each plant, which had a greater impact on microbial community structure than either plant type or the effect of defoliation. In addition there were no generic consequences with regard to microbial populations in the rhizosphere as a response to plant defoliation.