Michael D. Casler

Genetic modification of lignin concentration affects fitness of perennial herbaceous plants

Abstractu2002Populations of four perennial herbaceous species that were genetically modified for altered lignin content (or associated forage digestibility) by conventional plant breeding were evaluated for two agricultural fitness traits, plant survival and plant biomass, in three Northcentral USA environments for more than 4 years. Reduced lignin concentration or increased digestibility resulted in increased winter mortality in two of four species and reduced biomass in one species. Results from other experiment indicate that these apparent genetic correlations may be ephemeral, suggesting that selection for fitness can be successful within high-digestibility or low-lignin germplasm. Results indicate that perennial plants genetically engineered with altered lignin concentration or composition for use in livestock, pulp and paper, or bioenergy production should be evaluated for fitness in field environments prior to use in agriculture.

Relationship of lignin and esterified phenolics to fermentation of smooth bromegrass fibre

The importance of concentration and composition of lignin and esterified phenolics in forage cell walls was investigated relative to limitation of fibre fermentation. Smooth bromegrass (Bromus inermis Leyss.) leaf and stem tissues were analyzed for in vitro fermentability of detergent fibre fractions. The smooth bromegrass samples represented nine genotypes selected for high in vitro dry matter disappearance (IVDMD) and nine genotypes selected for low IVDMD. Lignin was an important predictor of fibre fermentation for stems but not for leaves. The relationships of stem neutral detergent fibre and hemicellulose, but not cellulose, fermentability to lignin concentration were different between the high and low IVDMD genotype groups. Nitrobenzene oxidation products were negatively correlated with leaf blade fibre fermentation. Stepwise multiple regression analysis, within IVDMD genotype groups, showed no relationship between fibre fermentation and lignin/phenolic measures for leaves, and primarily a lignin concentration effect in high IVDMD stems. Major differences exist in the importance of lignin concentration, lignin composition, and esterified phenolics concentrations to fibre fermentability of smooth bromegrass genotypes selected for divergent IVDMD.

The Wisconsin integrated cropping systems trial: Combining agroecology with production agronomy

Two large-scale (25 ha) trials were initiated in 1989 in Wisconsin to compare six alternative production systems regarding productivity, profitability, and environmental impact. The project was designed and is managed by a coalition of farmers, extension agents and research personnel. Deliberations between production-oriented and ecologically oriented team members resulted in a factorial design, with two enterprise types (cash grain and forage-livestock) and three levels of biological complexity. Statistical methods have been used to identify the most efficient plot size, plot shape, and block shape, and the optimal procedures for sampling soil characteristics. A uniformity year was allowed before initiation of the trial, and the start was staggered. We defined treatments as production strategies rather than a specific set of inputs, which led to a more flexible plot management program.

Genetic evidence suggests a widespread distribution of native North American populations of reed canarygrass

Reed canarygrass is an important agricultural crop thought to be native to Europe, Asia, and North America. However, it is one of the worst wetland invaders in North American wetlands. The native North American status has been supported by the circumstantial evidence of early botanical records and the dating and location of herbarium specimens. The lack of empirical evidence has left the North American native status of the species in doubt and prevented comparisons between native North American and Eurasian populations of the species. We utilized genetic markers to compare a wide range of European and Asian collections to DNA extracted from 38 early North American herbarium specimens. The genetic data confirm the presence of a distinct population present throughout North America in the early twentieth century, but not present in Europe or Asia, ranging from Alaska, USA to New Brunswick, Canada. These native North American populations of reed canarygrass are likely present throughout Alaska today, as one specimen was collected as recently as 1996, and may still be present in other regions of North America. Future research can utilize this dataset to determine the origin of present-day invasive populations in North American wetlands.

Forage quality of five cool-season grasses. II. Species effects

A 2-year field experiment was conducted on established stands of smooth bromegrass (Bromus inermis Leyss.), orchardgrass (Dactylis glomerata L.), timothy (Phleum pratense L.), tall fescue (Festuca arundinacea Schreb.) and reed canarygrass (Phalaris arundinacea L.) to evaluate maturity effects on quality. Herbage was sampled on seven dates during spring and early summer. Species did not differ in total N and all declined in N with shoot maturation. Timothy was highest in average in vitro dry matter disappearance (IVDMD) at 661 g kg−1 and orchardgrass was lowest at 615 g kg−1. Reed canarygrass and tall fescue declined more rapidly in IVDMD with maturation than timothy and smooth bromegrass. Orchardgrass had the highest average neutral detergent fiber (NDF) concentration at 624 g kg−1. Tall fescue had the lowest average NDF concentration, at 576 g kg−1, but reed canarygrass increased more rapidly in NDF with maturation than smooth bromegrass and timothy. Results of this experiment indicate that reed canarygrass and, to a certain extent, tall fescue changed more rapidly in IVDMD, NDF and acid detergent fibre (ADF) with maturation than the other species and thus would need to be utilized at an earlier stage of maturity to obtain forage of similar quality. Timothy and smooth bromegrass declined least rapidly in quality. Timothy had the highest average IVDMD, but tall fescue had a lower average NDF concentration indicating that species differences also exist in cell-wall digestibility.

Improving Selection in Forage, Turf, and Biomass Crops Using Molecular Markers

Selection of improved forage, turf, and bioenergy crops is optimized if measuring the phenotype of interest is rapid, inexpensive, and repeatable. Phenotyping remains the most difficult issue to resolve for many important traits, including biomass yield, abiotic stress tolerance, and long-term persistence. The identification of molecular markers may augment phenotypic selection if markers are identified that are closely linked to or at genes controlling the traits of interest. Simply inherited traits can be easily manipulated with marker assisted selection (MAS), but using markers in more complex situations requires additional thought. In this paper, we put the use of molecular markers into the context of typical perennial forage and turf breeding programs. Identifying markers based on bi-parental mapping populations is likely not the best way to implement a MAS program, although this approach is useful to introgress alleles from wild germplasm. Instead, a more practical approach may be the use of association mapping, measuring both phenotypes and markers directly on the plants in the breeding nursery. Complications of this method include the limited amount of information on linkage disequilibrium that is available for breeding populations, but the increasing availability of gene identification methods and the use of single nucleotide polymorphism (SNP) markers may enable the use of association mapping in many cases. Applying the information to breeding may be done to assist selection, to prescreen plants to determine those on which field-based phenotypic data will later be collected, and to make rapid off-season selections. The practical applications of markers to the breeding programs are discussed.

Development and application of a selection criterion for particle size breakdown of smooth bromegrass leaves

The rate of particle size breakdown in the reticulo-rumen is an important determinant of voluntary intake by ruminants. The objective of this study was to develop a selection criterion to measure particle size breakdown and to study its suitability for creating unique germplasm. Dried leaf tissue of smooth bromegrass (Bromus inermis Leyss.) was weighed and placed in a rubber drum, with 200 steel balls. The drum was rotated at constant speed, ball-milling the sample, after which the contents were sieved through a 1-mm screen. The particle size reduction index (PSRI) was determined as the weight of tissue passing through the 1-mm screen as a percentage of total sample weight. Two experiments focused on the optimal milling time, leading to the conclusion that a 30-s time was optimal. Five experiments focused on sample size and showed that sample sizes of ≥ 2.0 g adequately controlled precision. Repeatability of duplicate determinations was judged to be moderate in one experiment, suggesting that a single determination could characterize a sample of dried leaf tissue with a 24% reduction in precision compared to duplicate determinations. Another experiment indicated that genotype × harvest interaction was very important, but could be partly controlled by using two-stage selection. Finally, PSRI was not correlated with fiber digestibility, but was moderately and negatively correlated with fiber concentration. The results suggest that genetic variation exists for this selection criterion, it is repeatable, and it characterizes a physical property of grass leaves distinct from fiber digestibility or concentration.

Forage quality of five cool-season grasses. I. Cultivar effects

A field experiment was conducted over 2 years to evaluate five cultivars and four experimental synthetics of smooth bromegrass (Bromus inermis Leyss.), six cultivars of orchardgrass (Dactylis glomerata L.), four cultivars of timothy (Phleum pratense L.), three cultivars of tall fescue (Festuca arundinacea Schreb.) and two cultivars of reed canarygrass (Phalaris arundinacea L.). Herbage was sampled on seven dates during spring and early summer. Each treatment was replicated three times. Cultivars differed in nitrogen (N) concentration for orchardgrass, timothy and tall fescue. Timothy cultivars varied most in N, ranging from 24.7 g kg−7 for ‘Clair’ to 27.2 g kg−1 for ‘Verdant’. Within each species, the later maturing cultivars were generally highest in N. Cultivar differences in in vitro dry matter disappearance (IVDMD) were found for smooth bromegrass (range 41 g kg−1), orchardgrass (range 22 g kg−1) and timothy (range 27 g kg−1), but not for the other species. Cultivar effects for neutral detergent fiber were significant only for smooth bromegrass (range 27 g kg−1) which also differed in acid detergent fiber and hemicellulose. Cultivars of reed canarygrass, orchardgrass and timothy differed in cellulose, hemicellulose and acid-insoluble ash, respectively. Cultivar × date interactions were generally not significant except in 1982 when all species but orchardgrass had interaction effects for one or more constituents. These data indicate that grass cultivars differ in quality but that, within a species, cultivars generally respond similarly to advancing maturity.

Generation of transcript assemblies and identification of single nucleotide polymorphisms from seven lowland and upland cultivars of Switchgrass

Switchgrass is a North American perennial prairie species that has been used as a rangeland and forage crop and has recently been targeted as a potential biofuel feedstock species. Switchgrass, which occurs as tetraploid and octoploid forms, is classified into lowland or upland ecotypes that differ in growth phenotypes and adaptation to distinct habitats. Using RNA‐sequencing (RNA‐seq) reads derived from crown, young shoot, and leaf tissues, we generated sequence data from seven switchgrass cultivars, three lowland and four upland, to enable comparative analyses between switchgrass cultivars and to identify single nucleotide polymorphisms (SNPs) for use in breeding and genetic analysis. We also generated individual transcript assemblies for each of the cultivars. Transcript data indicate that subgenomes of octoploid switchgrass are not substantially different from subgenomes of tetraploids as expected for an autopolyploid origin of switchgrass octoploids. Using RNA‐seq reads aligned to the switchgrass Release 0 AP13 reference genome, we identified 1,305,976 high‐confidence SNPs. Of these SNPs, 438,464 were unique to lowland cultivars, but only 12,002 were found in all lowlands. Conversely, 723,678 SNPs were unique to upland cultivars, with only 34,665 observed in all uplands. Comparison of our high‐confidence transcriptome‐derived SNPs with SNPs previously identified in a genotyping‐by‐sequencing (GBS) study of an association panel revealed limited overlap between the two methods, highlighting the utility of transcriptome‐based SNP discovery in augmenting genome diversity polymorphism datasets. The transcript and SNP data described here provide a useful resource for switchgrass gene annotation and marker‐based analyses of the switchgrass genome.

Using variable importance measures to identify a small set of SNPs to predict heading date in perennial ryegrass

Prior knowledge on heading date enables the selection of parents of synthetic cultivars that are well matched with respect to time of heading, which is essential to ensure plants put together will cross pollinate. Heading date of individual plants can be determined via direct phenotyping, which has a time and labour cost. It can also be inferred from family means, although the spread in days to heading within families demands roguing in first generation synthetics. Another option is to predict heading date from molecular markers. In this study we used a large training population consisting of individual plants to develop equations to predict heading date from marker genotypes. Using permutation-based variable selection measures we reduced the marker set from 217,563 to 50 without impacting the predictive ability. Opportunities exist to develop a cheap assay to sequence a small number of regions in linkage disequilibrium with heading date QTL in thousands of samples. Simultaneous use of these markers in non-linkage based marker-assisted selection approaches, such as paternity testing, should enhance the utility of such an approach.