Assembly of Aegilops umbellulata chromosomes

We sequenced and assembled seven chromosomes of Aegilops umbellulata. The assembly with Meraculous resulted in ~ 101k - 191k scaffolds per chromosome with N50 size 2.9kb – 4.0kb. The scaffold sequences were used for development of molecular markers specific for cDNAs sequences mapped on Ae. umbellulata chromosomes Pairwise alignment of wheat cDNA-sequences and the chromosomal scaffolds of Ae. umbellulata identified candidate sequences. In order to analyze the structure and homeology of Aegilops chromosomes, forty-three mapped wheat cDNAs covering all seven chromosome groups were localized by FISH.

Methods
Flow-sorted chromosomes were treated with proteinase K and their DNA was amplified by multiple displacement amplification (MDA) using Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Chalfont St. Giles, United Kingdom) as described by Šimková et al. (2008). Three independent amplifications from each chromosome were pooled to reduce amplification bias and 2µg of amplified DNA were fragmented and used to prepare Illumina sequencing libraries with TruSeq DNA PCR-Free Library Preparation Kit (Illumina, Inc., San Diego, USA). All samples were sequenced on Illumina HiSeq 2000 according manufacturer's instruction to produce 2x100 bp paired-end reads. Each chromosome or chromosome arm was sequenced on a separate lane of the instrument to obtain ~130 millions of pair-end read (~26 Gbp) for each chromosome/arm. De novo assembly was done with MaSuRCA assembler (Zimin et al. 2013) using default parameters. Finally, contigs shorter than 200 bp were removed from the assembly of each chromosome prior further analysis.