K. F. Smith

Functional Analyses of Caffeic Acid O-Methyltransferase and Cinnamoyl-CoA-Reductase Genes from Perennial Ryegrass (Lolium perenne)

The authors show enhanced digestibility of cinnamoyl CoA-reductase and caffeic acid O-methyltransferase-deficient perennial ryegrass plants grown under glasshouse and field conditions. This indicates that both of these lignin biosynthetic genes are promising targets for transgenic approaches aiming to enhance forage quality and improve feedstock plants for biofuel production. Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference–mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.

AFLP analysis of genetic diversity within and between populations of perennial ryegrass (Lolium perenne L.)

Amplified fragment length polymorphism (AFLP) analysis has been used to measure genetic diversity in perennial ryegrass (Lolium perenne L.) and to relate intra- and interpopulation variation to breeding history. Cluster analysis of AFLP data from contrasting populations showed features consistent with the origins of these varieties. Significant differences in intrapopulation diversity were detected and partial separation of different cultivars was observed. Restricted base cultivars, derived from small numbers of foundation clones, were suitable for this type of study, allowing near complete discrimination of closely related cultivars. Analysis of bulked samples was based on the pooling of genomic DNA from 20 individuals from 6 selected populations. Cluster analysis of AFLP data from bulked samples produced a phenogram showing relationships consistent with the results of individual analysis. AFLP profiling provides an important tool for the detection and quantification of genetic variation in perennial ryegrass.

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.

Fine-scale comparative genetic and physical mapping supports map-based cloning strategies for the self-incompatibility loci of perennial ryegrass (lolium perenne L.)

Perennial ryegrass is an obligate outbreeding pasture grass of the Poaceae family, with a two-locus (S and Z) gametophytic self-incompatibility (SI) mechanism. This system has provided a major obstacle to targeted varietal development, and enhanced knowledge is expected to support more efficient breeding strategies. Comparative genetics and physical mapping approaches have been developed to permit molecular cloning of the SI genes. SI gene-linked genetic markers based on heterologous cDNA restriction fragment length polymorphisms (RFLPs) and homologous genomic DNA-derived simple sequence repeats (SSRs) were converted to single nucleotide polymorphism (SNP) format for efficient genotyping. Genetic mapping identified the location of SI loci and demonstrated macrosynteny between related grass species. S- and Z-linked bacterial artificial chromosome (BAC) clones were sequenced using massively parallel pyrosequencing technology to provide the first physical mapping data for Poaceae SI loci. The sequence assembly process suggested a lower prevalence of middle repetitive sequences in the Z locus region and hence precedence for positional cloning strategy. In silico mapping using data from rice, Brachypodium distachyon and Sorghum revealed high sequence conservation in the vicinity of the Z locus region between SI and self-compatible (SC) grass species. Physical mapping identified a total of nine genes encoded in the Z locus region. Expression profiling and nucleotide diversity assessment identified two Z-linked genes, LpTC116908 and LpDUF247, as plausible candidates for the male and female determinants of the S-Z SI system.

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.

Spatial analysis of forage grass trials across locations, years, and harvests.

field trials. The use of row-column analysis or neighbor analysis has been shown to increase the precision of a Spatial analyses of yield trials are a powerful method of adjusting large number of grain yield trials (Cullis and Gleeson, treatment means for spatial variation and improving statistical precision of mean estimation. Because yield trials are typically repeated 1989; Cullis and Gleeson, 1991; Kempton et al., 1994). across multiple locations and years, spatial analysis methods must Recent analyses of forage grass cultivar trials of a be adapted for combined analyses across locations and years. The range of cool season forage species have shown that it objective of this study was to evaluate the relative efficiency of nearest is possible to improve the precision of cultivar yield neighbor analysis (NNA) across locations and years for several perenestimates within a location through both optimizing the nial forage grass trials. Three spatial adjustment methods were develnumber of replicates sown, based on the likely differoped: preadjustment based on total forage yield, postadjustment ences between the cultivars under test, and utilizing based on total forage yield, and preadjustment based on forage yield statistical analyses that account for spatial variability in of individual harvests. For cool-season grasses on a multiple-harvest plot yield (Casler, 1999a,b; Smith and Kearney, 2002). management, NNA had relative efficiencies of 105 to 135% across When RCB designs were compared with lattice designs locations, years, and trials. Within trials, there was some consistency across harvests, resulting in greater improvements in precision for and NNA in a comparison of 27 perennial cool-season adjustment based on total yield. Across locations and years, the three grass trials, NNA was shown to provide more precise spatial adjustment methods always ranked the same in relative effiestimates of mean forage yield than either the lattice ciency: preadjustment by harvest preadjustment of total yield or RCB designs (Casler, 1999b). The improvements in postadjustment of total yield. The advantage of the preadjustment precision of entry means were shown to be incremental methods was likely due to fitting heterogeneous slopes (adjustment with an average improvement in precision of 15% due factors) across locations, years, and/or harvests. In contrast, trials to the use of RCB designs, an additional 17% due to with a single-harvest management for biomass production always had the lattice analysis, and a further 22 to 30% due to trend relatively low relative efficiency of NNA. Trial operators should assess analysis or NNA (Casler, 1999b). the relative efficiency of NNA on early harvests from all locations These improvements in the precision of the estimawithin a trial and if the relative efficiencies are large, they should consider the use of NNA across locations and years to adjust entry tion of cultivar herbage yield are of great importance means. given the rapid increase in the number of forage grass cultivars on the market, the relatively low rates of genetic gain for forage yield (0.1–0.5% yr 1; Van Wijk and Reheul, 1991; Casler, 1998; Casler et al., 2000), and P forage grass species are routinely tested the reduction in funds available for cultivar testing in a for improvements in forage yield through the use of number of countries. Nearest neighbor adjustment of replicated plot trials in a number of years and locations. cultivar means for individual trials provides improved These cultivar evaluation programs are essential for precision for cultivar means, but does not provide a making choices between forage grass cultivars and also direct assessment of cultivar environment interactions, for assessing whether new cultivars are broadly suited which require a combined analysis across locations or across a range of environments or possess more specific years. Supplemental analysis of adjusted cultivar means adaptation to certain environmental niches. could provide this information (Cullis et al., 1998), but The majority of forage cultivar evaluation trials are would not provide a test of each cultivar environment sown in a randomized complete block (RCB) design component (location, year, and location year). Thus, (APPEC, 1996). While the RCB design may be an effecthe need still exists to develop techniques to allow for tive way of controlling spatial variation in field trials in analysis of spatially adjusted means across environments one direction, it is ineffective when the spatial variability and years as forage cultivar trials are usually conducted is continuous in two directions, leading to considerable across 2 to 3 yr in a number of locations (Casler, within-block variability (Lin et al., 1993). There has a 1999a,b). been a marked change in the way that multienvironment The objective of this study was to evaluate several trial data from annual grain crop variety testing trials methods to use NNA to account for spatial variability are analyzed with a move toward spatial analysis (Gleein the yields of forage plots from nine separate cultivar son and Cullis, 1987; Cullis and Gleeson, 1989) to better evaluation trials conducted across locations and years. accommodate the plot-to-plot variation observed in The trials cover two distinct classes of forage cultivar evaluations: multiple-harvest hay trials of cool-season K.F. Smith, Agriculture Victoria, CRC for Molecular Plant Breeding, Pastoral and Veterinary Inst., Private Bag 105, Hamilton, VIC 3300, grasses, for which season-total forage yield is the trait Australia; M.D. Casler, USDA-ARS, U.S. Dairy Forage Research of interest, and single-harvest biomass trials of a warmCenter, Madison, WI 53706-1108. Received 19 Feb. 2003. *Correseason grass. sponding author (mdcasler@wisc.edu). Abbreviations: LSR, least significant range; NNA, nearest neighbor Published in Crop Sci. 44:56–62 (2004).  Crop Science Society of America analysis; Pre-IH, preadjustment by individual harvests; RCB, randomized complete block. 677 S. Segoe Rd., Madison, WI 53711 USA

Growth and carbon partitioning in perennial ryegrass ( Lolium perenne ) cultivars selected for high water-soluble carbohydrate concentrations

Perennial ryegrass (Lolium perenne L.) cultivars with increased water-soluble carbohydrate (WSC) concentrations were evaluated under controlled environment conditions. The growth and carbon partitioning of these cultivars was compared with standard cultivars during vegetative growth. The high WSC cultivars had shoot growth rates that were not significantly different from the standard cultivars, confirming that the extra WSC in these cultivars was not made available through reductions in yield potential. The extra WSC stored in these cultivars coincided with lower concentrations of neutral detergent fibre in the dry matter. When the cultivars were grown in hydroponic solution the high WSC cultivars Aurora and Ba10727 were found to also have less root mass and a lower root: shoot ratio than the standard cultivars. However, this trait was not consistent across all high WSC cultivars with Cariad having the same root:shoot ratio as the standard cultivars at the end of the experiment. The reduction in the root mass of the cultivars Aurora and Ba10727 was far greater than necessary to provide the extra carbon stored as WSC in these cultivars. The implications of these results for the breeding of cultivars of perennial ryegrass with increased WSC concentrations are discussed.

Molecular characterisation and genetic mapping of candidate genes for qualitative disease resistance in perennial ryegrass (Lolium perenne L.)

BackgroundQualitative pathogen resistance in both dicotyledenous and monocotyledonous plants has been attributed to the action of resistance (R) genes, including those encoding nucleotide binding site – leucine rich repeat (NBS-LRR) proteins and receptor-like kinase enzymes. This study describes the large-scale isolation and characterisation of candidate R genes from perennial ryegrass. The analysis was based on the availability of an expressed sequence tag (EST) resource and a functionally-integrated bioinformatics database.ResultsAmplification of R gene sequences was performed using template EST data and information from orthologous candidate using a degenerate consensus PCR approach. A total of 102 unique partial R genes were cloned, sequenced and functionally annotated. Analysis of motif structure and R gene phylogeny demonstrated that Lolium R genes cluster with putative ortholoci, and evolved from common ancestral origins. Single nucleotide polymorphisms (SNPs) predicted through resequencing of amplicons from the parental genotypes of a genetic mapping family were validated, and 26 distinct R gene loci were assigned to multiple genetic maps. Clusters of largely non-related NBS-LRR genes were located at multiple distinct genomic locations and were commonly found in close proximity to previously mapped defence response (DR) genes. A comparative genomics analysis revealed the co-location of several candidate R genes with disease resistance quantitative trait loci (QTLs).ConclusionThis study is the most comprehensive analysis to date of qualitative disease resistance candidate genes in perennial ryegrass. SNPs identified within candidate genes provide a valuable resource for mapping in various ryegrass pair cross-derived populations and further germplasm analysis using association genetics. In parallel with the use of specific pathogen virulence races, such resources provide the means to identify gene-for-gene mechanisms for multiple host pathogen-interactions and ultimately to obtain durable field-based resistance.

Identification of homologous, homoeologous and paralogous sequence variants in an outbreeding allopolyploid species based on comparison with progenitor taxa.

The combination of homologous, homoeologous and paralogous classes of sequence variation presents major challenges for SNP discovery in outbreeding allopolyploid species. Previous in vitro gene-associated SNP discovery studies in the allotetraploid forage legume white clover (Trifolium repens L.) were vulnerable to such effects, leading to prohibitive levels of attrition during SNP validation. Identification of T. occidentale and T. pallescens as the putative diploid progenitors of white clover has permitted discrimination of the different sequence variant categories. Amplicons from selected abiotic stress tolerance-related genes were obtained using mapping family parents and individuals from each diploid species. Following cloning, progenitor comparison allowed tentative assignment of individual haplotypes to one or other sub-genome, as well as to gene copies within sub-genomes. A high degree of coincidence and identity between SNPs and HSVs was observed. Close similarity was observed between the genome of T. occidentale and one white clover sub-genome, but the affinity between T. pallescens and the other sub-genome was weaker, suggesting that a currently uncharacterised taxon may be the true second progenitor. Selected validated SNPs were attributed to individual sub-genomes by assignment to and naming of homoeologous linkage groups, providing the basis for improved genetic trait-dissection studies. The approach described in this study is broadly applicable to a range of allopolyploid taxa of equivocal ancestry.

The effects of waterlogging on growth, photosynthesis and biomass allocation in perennial ryegrass ( Lolium perenne L.) genotypes with contrasting root development

Perennial ryegrass ( Lolium perenne L.) is often subject to transient waterlogging during winter under dryland conditions and summer when flood-irrigated. Despite this, little is known about the physiological responses of perennial ryegrass genotypes to waterlogging. In a pot experiment, four perennial ryegrass genotypes with contrasting root growth characteristics were subjected to waterlogging for 0, 3, 7, 14, 21 or 28 days. The masses of roots and shoots of the genotype Aurora6 were not significantly ( P >0·05) reduced by waterlogging, throughout the experiment. All other genotypes exhibited reductions in root and shoot biomass between 14 and 21 days after waterlogging was imposed. The masses of laminae and roots of susceptible genotypes were reduced by up to 70% after 28 days of waterlogging. Aurora6 was also able to maintain photosynthesis for longer into the waterlogging period. However, after 28 days of waterlogging, photosynthesis of all genotypes was reduced by 30–50%. The waterlogging tolerance of Aurora6 was not due to its relatively poor root growth, as its progeny (2178), which also had poor root growth under control conditions, was susceptible to waterlogging. These findings show that there is variation in physiological processes and herbage yield of perennial ryegrass under waterlogged conditions. The implications of these findings for the genetic improvement of waterlogging tolerance of perennial ryegrass are discussed.