Targeted Assembly of a Puma Rye (Secale cereale L.) 5RL Chromosomal Translocation (5A:5RL) By Subtraction from a Whole Chromosome Shotgun Assembly of Norstar Wheat (Triticum aestivum L.) Chromosome 5A

Low-temperature tolerance is an important agronomic trait for both spring and winter cereal crops. Winter habit rye displays the greatest low-temperature tolerance amongst temperate cereal crops. Despite the identification of regions conferring low-temperature tolerance in rye and wheat, efforts to exploit the superior low-temperature tolerance of rye for improvement of wheat have been unsuccessful. The cereal group 5 chromosomes, and in particular a region designated as Fr₂ have been implicated in low-temperature tolerance. Using a subtractive approah we have assembled a chromosomal translocation of the long arm of Puma chromosome 5R in order to facilitate detailed characterization of cold related genes located on this translocation which includes the Fr2 region. This approach involved Illumina Hiseq and Miseq sequencing of flow-sorted Norstar wheat 5A chromosomes and Norstar wheat - Puma rye 5A:5RL chromosomes, whole-chromosome assembly of Norstar 5A, subtraction of reads originating from 5A from the 5A:5RL sequences using the 5A assembly and finally, assembly of the 5RL region.