Agrobacterium-mediated transformation and inbred line development in the model grass Brachypodium distachyon

Abstract

Brachypodium distachyon (Brachypodium) has been proposed as a model temperate grass because its physical, genetic, and genome attributes (small stature, simple growth requirements, small genome size, availability of diploid ecotypes, annual lifecycle and self fertility) are suitable for a model plant system. Two additional requirements that are necessary before Brachypodium can be widely accepted as a model system are an efficient transformation system and homogeneous inbred reference genotypes. Here we describe the development of inbred lines from 27 accessions of Brachypodium. Determination of c-values indicated that five of the source accessions were diploid. These diploid lines exhibit variation for a variety of morphological traits. Conditions were identified that allow generation times as fast as two months in the diploids. An Agrobacterium-mediated transformation protocol was developed and used to successfully transform 10 of the 19 lines tested with efficiencies ranging from 0.4% to 15%. The diploid accession Bd21 was readily transformed. Segregation of transgenes in the T 1 generation indicated that most of the lines contained an insertion at a single genetic locus. The new resources and methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.

Genetic Architecture of Flowering-Time Variation in Brachypodium distachyon

“…In this study we developed a RIL population between a rapid (Bd21) and a delayed flowering (Bd1-1) accession of B. distachyon (Ream et al, 2014;Vogel et al, 2006) and used it to evaluate the genetic architecture of flowering time under a range of environmental conditions. For this study, we developed a high-resolution genetic map containing 1693 SNP markers using genotyping by sequencing.…”

“…Like wheat and barley, B. distachyon accessions exhibit a considerable amount of natural variation in flowering responses (Ream et al, 2014;Tyler et al, 2014). However, unlike wheat and barley, which fall into two broad categories for flowering requirements (winter and spring varieties), most of the B. distachyon accessions studied to date are likely to be some form of winter annual in their native environments because they all require vernalization to flower rapidly when grown in native photoperiods in growth chambers: under artificial 20-h-long d, accessions such as Bd21 will flower quite rapidly without vernalization (Vogel et al, 2006;Ream et al, 2014); however, when grown under 14 h d to 15 h d, Bd21 requires a very short (2 to 3 weeks) period of vernalization to flower rapidly (Ream et al, 2014). In contrast, many B. distachyon accessions such as Bd1-1 are delayed in flowering even under artificially long d and require an extended period of cold (at least 6 weeks) to saturate their vernalization requirement (Ream et al, 2014).…”

A Flowering Locus C Homolog Is a Vernalization-Regulated Repressor in Brachypodium and Is Cold Regulated in Wheat

“…Seeds of these accessions were obtained from the USDA germplasm, Dr. David Garvin, or Dr. Luis Mur after which seeds were propagated in house (Filiz et al, 2009;Mur et al, 2011;Vogel et al, 2006). Seeds were first incubated during 24 h at 23°C in the dark on a petri dish with filter paper and 2 mL of distilled water to ensure synchronous germination.…”

Interaction of Photoperiod and Vernalization Determines Flowering Time of Brachypodium distachyon

“…Brachypodium spp. is a useful model grass because of its small, completely sequenced diploid genome (International Brachypodium Initiative, 2010), simple growth requirements, large collection of accessions, inbreeding nature, and high rate of recombination (Draper et al, 2001;Vogel et al, 2006Vogel et al, , 2009Opanowicz et al, 2008;Filiz et al, 2009;Brkljacic et al, 2011;Huo et al, 2011;Mur et al, 2011). All of these characteristics make Brachypodium spp.…”