Brachypodium distachyon. A new model system for functional genomics in grasses

Abstract

A new model for grass functional genomics is described based on Brachypodium distachyon, which in the evolution of the Pooideae diverged just prior to the clade of "core pooid" genera that contain the majority of important temperate cereals and forage grasses. Diploid ecotypes of B. distachyon (2n = 10) have five easily distinguishable chromosomes that display high levels of chiasma formation at meiosis. The B. distachyon nuclear genome was indistinguishable in size from that of Arabidopsis, making it the simplest genome described in grasses to date. B. distachyon is a self-fertile, inbreeding annual with a life cycle of less than 4 months. These features, coupled with its small size (approximately 20 cm at maturity), lack of seed-head shatter, and undemanding growth requirements should make it amenable to high-throughput genetics and mutant screens. Immature embryos exhibited a high capacity for plant regeneration via somatic embryogenesis. Regenerated plants display very low levels of albinism and have normal fertility. A simple transformation system has been developed based on microprojectile bombardment of embryogenic callus and hygromycin selection. Selected B. distachyon ecotypes were resistant to all tested cereal-adapted Blumeria graminis species and cereal brown rusts (Puccinia reconditia). In contrast, different ecotypes displayed resistance or disease symptoms following challenge with the rice blast pathogen (Magnaporthe grisea) and wheat/barley yellow stripe rusts (Puccinia striformis). Despite its small stature, B. distachyon has large seeds that should prove useful for studies on grain filling. Such biological characteristics represent important traits for study in temperate cereals.

Figure 1

A, Schematic phylogenetic relationship of B. distachyon to other Poaceae adapted from the data presented by Catalan et al. (1995, 1997; Catalan and Olmstead, 2000). B, Nuclear genome sizes (Mbp/1C) in different grass genera. Data derived from The Kew Gardens Angiosperm C- value database (http://www.rbgkew.org/cval/homepage.html).

Figure 2

Somatic and meiotic chromosomes of B. distachyon ABR1 showing light micrograph of the somatic chromosome complement (A) in which the five pairs of chromosomes are readily identified and fluorescence in situ hybridization (FISH; B) of 5S rDNA (red) and 45S rDNA (green) to the five pairs of somatic chromosomes. C, Karyogram derived from the image shown in B. D, Idiogram showing the distinctive and diagnostic shapes and lengths of the haploid set of chromosomes. The sites of the two rDNA loci are indicated. E, Light micrograph of two pollen mother cells at metaphase I. Note the five ring bivalents in each. Bars = 10 μm.

Figure 3

Growth habit and anatomy of the diploid B. distachyon ecotype ABR1 grown in glass jars (A) on vermiculite supplemented with 0.5× Hoagland solution (Draper et al., 1988) at 1 week (left jar) and 2 weeks (right jar), and at high density (B) in 30- × 18-cm trays grown on 1:1 mixes of Levington's:gravel. Bar = 5 cm. Flower morphology (C) prior to dehiscence and indicating hairy palea (p) and lemea (l), stigma (s), and anthers (a). Bar = 1 mm. Each plant has 6 to 10 flowering spikes that actually set seed and seed heads (D) have a “brome-like” appearance and typically carry 10 viable seeds. Bar = 4 mm. At maturity (E), ABR1 plants were 15 cm high with short nonrhizomatous roots. Bar = 5 cm. F, B. distachyon ABR1 (8 weeks post-sowing) is compared in size with rice cv IR64 (20 weeks post-sowing). G, 2,000 M1 progeny of γ-irradiated (at the Vienna Atomic Energy Institute) B. distachyon seeds grown under typical temperate greenhouse conditions.

Figure 4

Tissue culture and transformation of B. distachyon. A, B. distachyon ABR1 seed head at 15 d postanthesis (bar = 4 mm) from which (B) isolated seeds have had their paleas and lemmas removed (embryos arrowed, bar = 0.5 mm) or (C) the embryos are isolated from endosperm to indicate embryo development (classes 1–5, bar = 0.5 mm). D, Scanning electron micrograph showing immature embryo structure. Indicated are the coleoptile (col), scutellum (scut), radical (rad), and coleorhiza (chz). Bar = 0.1 mm. E, Embryogenic callus (c) formation around the edge of the scutellum (scut) after 15 d of culture (bar = 0.1 mm), which (F) scanning electron micrography revealed to contain many organized structures with a distinctive embryoid shape (arrowed; coleoptilar pore; bar = 20 μm). G, Propagated embryogenic callus of ABR100 stained for GUS activity 24 h following biolistic bombardment with pACt1GUSHm. Bar = 1 mm. H, Selection of ABR100 transgenic tissue on Hm- (40 μg mL⁻¹) selective media. Bar = 3 mm. I, The second subculture of regenerating plants from bombarded embryogenic callus of ABR100 on selective media. Bar = 5 mm. J, Isolated Hm-resistant plantlet of ABR100 exhibiting tiller formation. Bar = 5 mm. K, Hm-resistant ABR100 shoot stained for GUS activity (bar = 1 cm). L, Rooted T₀ transgenic lines in soil producing viable seeds. M, The PCR amplification of hgh gene internal sequences (516-bp product) from five independent T₁ transgenic lines (1–5). N, Southern blot of BamHI-digested genomic DNA from T₁ transgenic lines (1–5) and wild-type ABR100 probed with hgh sequences. Indicated is the 1.07-kb BamHI internal hgh gene fragment.

Figure 5

Targets for B. distachyon research. Responses to pathogen challenge and grain development. A, The responses of ABR1 to challenge with condiophores of Blumeria graminis f. sp. Triticae. Bar = 0.1 mm. Arrowed are the condiophore (c), appressorial germtube (a) and papillae formation (p) and single cell-death (d) in the host cell. Challenging with Puccinia striformis f. sp. triticae (wheat yellow stripe rust) elicits (B) localized necrotic flecking on ABR105 and (C) yellow uridea (pustule) formation on ABR100 that develop from (D) extensive areas of necrotic tissue. (Uridea forming within the necrotic tissue are arrowed.) E, Variable responses by B. distachyon ecotypes to challenge with M. grisea Guy-11. F, Comparison of mature seed size and morphology of B. distachyon ABR1 with rice and wheat cv Kalyansona). Embryos are arrowed on each seed. Bar = 2 mm.