Andrew A. Hopkins

Molecular Breeding of Forage and Turf

Quality traits are major targets for the biotechnological improvement of forage crops. Many years of research have identified lignin as an impediment to digestibility, lack of condensed tannins as promoting pasture bloat and limiting nitrogen nutrition, and triterpene saponins as anti-palatability factors. Recent progress on understanding and manipulating the biosynthetic pathways leading to lignin, condensed tannins and saponins will facilitate engineering of alfalfa and other forage crops for reduced bloating potential and improved digestibility and palatability. Developments in genomics technology, centered on selected model species, have accelerated the pace of gene discovery in secondary metabolism and other complex pathways in plants. The rapidly emerging genomics resources for model species such as the legume Medicago truncatula will impact many aspects of forage improvement, with, in the case of M. truncatula, direct and immediate relevance for alfalfa. The first biotech products to reach the farm have been modified for input traits such as insect or herbicide resistance. Millions of acres in the US are now planted with genetically modified corn, soybean and cotton, but such products have met with resistance from environmentalists and the public in Europe and elsewhere. Forage crops with genetically improved quality (output) traits will benefit both the health of the animals that consume them and the environment through reductions in waste excretion and greenhouse gas emission. Policymakers should be made aware of these attributes. Genetically improved forage crops represent a unique opportunity for demonstrating the global benefits of biotechnology.

Field performance of transgenic tall fescue (Festuca arundinacea Schreb.) plants and their progenies

Abstract. Tall fescue (Festuca arundinacea Schreb.) is a hexaploid, outcrossing grass species widely used for forage and turf purposes. Transgenic tall fescue plants were generated by biolistic transformation of embryogenic cell suspension cultures that were derived from single genotypes of widely used cultivar Kentucky-31. Primary transgenics from two genotypes, their corresponding regenerants from the same genotypes and control seed-derived plants were transferred to the field and evaluated for 2 years. Progenies of these three classes of plants were obtained and evaluated together with seed-derived plants in a second field experiment. The agronomic characteristics evaluated were: heading date, anthesis date, height, growth habit, number of reproductive tillers, seed yield and biomass. The agronomic performance of the primary transgenics and regenerants was generally inferior to that of the seed-derived plants, with primary transgenics having fewer tillers and a lower seed yield. However, no major differences between the progenies of transgenics and the progenies of seed-derived plants were found for the agronomic traits evaluated. Primary transgenics and regenerants from the same genotype were more uniform than plants from seeds. Progenies of transgenics performed similarly to progenies of the regenerants. The addition of a selectable marker gene in the plant genome seems to have had little effect on the agronomic performance of the regenerated plants. No indication of weediness of the transgenic tall fescue plants was observed. Our results indicate that outcrossing grass plants generated through transgenic approaches can be incorporated into forage breeding programs.

Characterization of Epichloë coenophiala within the US: are all tall fescue endophytes created equal?

Tall fescue (Lolium arundinaceum) is a valuable and broadly adapted forage grass that occupies approximately 14 million hectares across the United States. A native to Europe, tall fescue was likely introduced into the US around the late 1800s. Much of the success of tall fescue can be attributed to Epichloë coenophiala (formerly Neotyphodium coenophialum) a seed borne symbiont that aids in host persistence. Epichloë species are capable of producing a range of alkaloids (ergot alkaloids, indole-diterpenes, lolines, and peramine) that provide protection to the plant host from herbivory. Unfortunately, most tall fescue within the US, commonly referred to as “Kentucky-31” (KY31), harbors the endophyte E. coenophiala that causes toxicity to grazing livestock due to the production of ergot alkaloids. Molecular analyses of tall fescue endophytes have identified four independent associations, representing tall fescue with E. coenophiala, Epichloë sp. FaTG-2, Epichloë sp. FaTG-3, or Epichloë sp. FaTG-4. Each of these Epichloë species can be further distinguished based on genetic variation that equates to differences in the alkaloid gene loci. Tall fescue samples were evaluated using markers to simple sequence repeats (SSRs) and alkaloid biosynthesis genes to determine endophyte strain variation present within continental US. Samples represented seed and tillers from the Suiter farm (Menifee County, KY), which is considered the originating site of KY31, as well as plant samples collected from 14 states, breeders seed and plant introduction lines (National Plant Germplasm System, NPGS). This study revealed two prominent E. coenophiala genotypes based on presence of alkaloid biosynthesis genes and SSR markers and provides insight into endophyte variation within continental US across historical and current tall fescue samples.

Response of Tall Fescue Genotypes to a New Strain of Brome mosaic virus

Brome mosaic virus (BMV) infects many different species within the Poaceae family. A new strain of BMV, named F-BMV, was identified in a tall fescue (Festuca arundinacea Schreb.) plant. Here, we report the identification and characterization of tall fescue plants resistant to F-BMV, and the effects of F-BMV infection on their growth and development. Susceptible plants infected with F-BMV produced 40% fewer tillers and 42% less dry matter compared with virus-resistant plants in a greenhouse study. In the field, susceptible plants infected with F-BMV produced 25% fewer tillers, 36% less dry matter, 10% less plant height, and 40% lower seed yield compared with virus-resistant plants. In a field evaluation of a tall fescue mapping population, the virus symptom scores were negatively correlated with production of dry matter (r = -0.55), plant height (r = -0.55), and seed yield (r = -0.33). Thus, F-BMV has the potential to cause significant economic damage to susceptible tall fescue plants. These results indicate that the virus can present a serious challenge for long-term maintenance of valuable plant materials. A survey of tall fescue plants from Arkansas, Oklahoma, and Oregon indicated that the prevalence F-BMV in the field was very low.

Field Evaluation and Risk Assessment of Transgenic Tall Fescue (Festuca arundinacea) Plants

Tall fescue (Festuca arundinacea Schreb.) is an outcrossing hexaploid grass species widely grown for forage and turf purposes. Transgenic tall fescue plants were generated by biolistic transformation of embryogenic cell suspension cultures of the commonly used cultivar Kentucky-31. T1 and T2 progenies were obtained after reciprocal crosses between transgenic and untransformed control plants. Molecular analysis of the progenies revealed stable meiotic transmission of transgenes following Mendelian rules in transgenic tall fescue. Agronomic performance of the primary transgenics and primary regenerants under field conditions were generally inferior to seed-derived plants, with primary transgenics having fewer tillers and lower seed yield. However, no major differences between the progenies of transgenics and the progenies of seed-derived plants were found for the agronomic traits evaluated. The addition of a selectable marker gene in the plant genome seems to have little effect on the agronomic performance of the regenerated plants. No indication of weediness was observed for the transgenic tall fescue plants. An experiment on pollen dispersal has also been carried out using transgenic tall fescue in a central plot, surrounded by untransformed recipient plants in a wagon wheel design. The highest transgene frequencies, 0.88% at 50 m and 0.59% at 100 m, were observed north of the central plot, the prevailing wind direction. Issues regarding experimental design for gene flow studies and future directions on risk assessment of forage and turf grasses are discussed.

Linkage Maps of a Mediterranean Continental Tall Fescue Population and their Comparative Analysis with Other Poaceae Species

Temperate grasses belonging to the Festuca–Lolium complex are important throughout the world in pasture and grassland agriculture. Tall fescue (Festuca arundinacea Schreb.) is the predominant species in the United States, covering approximately 15 million ha. Tall fescue has distinctive morphotypes, two of which are Continental (summer active) and Mediterranean (summer semidormant). This is the first report of a linkage map created for Mediterranean tall fescue, while updating the Continental map with additional simple sequence repeat and sequence‐tagged site markers. Additionally, this is the first time that diversity arrays technology (DArT) markers were used in the construction of a tall fescue map. The male parent (Continental), R43‐64, map consisted of 594 markers arranged in 22 linkage groups (LGs) and covered a total of 1577 cM. The female parent (Mediterranean), 103‐2, map was shorter (1258 cM) and consisted of only 208 markers arranged in 29 LGs. Marker densities for R43‐64 and 103‐2 were 2.65 and 6.08 cM per marker, respectively. When compared with the other Poaceae species, meadow fescue (F. pratensis Huds.), annual ryegrass (L. multiflorum Lam.), perennial ryegrass (L. perenne L.), Brachypodium distachyon (L.) Beauv., and barley (Hordeum vulgare L.), a total of 171 and 98 orthologous or homologous sequences, identified by DArT analysis, were identified in R43‐64 and 103‐2, respectively. By using genomic in situ hybridization, we aimed to identify potential progenitors of both morphotypes. However, no clear conclusion on genomic constitution was reached. These maps will aid in the search for quantitative trait loci of various traits as well as help define and distinguish genetic differences between the two morphotypes.

Biotechnological Improvement of Forage Crops

Forages play a unique role in agriculture and the environment; they contribute through animals to our food supply. Genetic improvement is one of the most effective ways to improve forage productivity. The potential of biotechnology in the development of improved forage grass cultivars has been recognized in recent years. Transgenic approaches are expected to complement or accelerate conventional breeding, since they offer the opportunity to generate unique genetic variation that would otherwise be absent or low in heritability.