A novel resource for genomics of Triticeae: BAC library specific for the short arm of rye (Secale cereale L.) chromosome 1R (1RS).

BackgroundGenomics of rye (Secale cereale L.) is impeded by its large nuclear genome (1C approximately 7,900 Mbp) with prevalence of DNA repeats (> 90%). An attractive possibility is to dissect the genome to small parts after flow sorting particular chromosomes and chromosome arms. To test this approach, we have chosen 1RS chromosome arm, which represents only 5.6% of the total rye genome. The 1RS arm is an attractive target as it carries many important genes and because it became part of the wheat gene pool as the 1BL.1RS translocation.ResultsWe demonstrate that it is possible to sort 1RS arm from wheat-rye ditelosomic addition line. Using this approach, we isolated over 10 million of 1RS arms using flow sorting and used their DNA to construct a 1RS-specific BAC library, which comprises 103,680 clones with average insert size of 73 kb. The library comprises two sublibraries constructed using HindIII and EcoRI and provides a deep coverage of about 14-fold of the 1RS arm (442 Mbp). We present preliminary results obtained during positional cloning of the stem rust resistance gene SrR, which confirm a potential of the library to speed up isolation of agronomically important genes by map-based cloning.ConclusionWe present a strategy that enables sorting short arms of several chromosomes of rye. Using flow-sorted chromosomes, we have constructed a deep coverage BAC library specific for the short arm of chromosome 1R (1RS). This is the first subgenomic BAC library available for rye and we demonstrate its potential for positional gene cloning. We expect that the library will facilitate development of a physical contig map of 1RS and comparative genomics of the homoeologous chromosome group 1 of wheat, barley and rye

Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly

Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging. Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species. A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest. In order to accelerate gene cloning from any cropping line, we report 'targeted chromosome-based cloning via long-range assembly' (TACCA). TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage to assemble complex genomes. We applied TACCA to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months. Using this assembly we cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein