Abstract
BackgroundGenomics of rye (Secale cereale L.) is impeded by its large nuclear genome (1C~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.
Highlights
Genomics of rye (Secale cereale L.) is impeded by its large nuclear genome (1C~7,900 Mbp) with prevalence of DNA repeats (> 90%)
The chromosome is characterized by a secondary constriction separating the satellite from the short arm and the 5S rDNA locus on the satellite, and its identity in the sorted fraction was confirmed by Fluorescence in situ hybridization (FISH) with probes for 5S rDNA and pSc119.2 (Figure 1, insert)
Chromosome 1R could be sorted, we considered 1,000 Mbp still too large fraction of the rye genome (~12.7%)
Summary
Genomics of rye (Secale cereale L.) is impeded by its large nuclear genome (1C~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. Other translocations were developed later, involving wheat 1AL and 1DL chromosome arms instead of the 1BL [37,46]. These translocations conferred resistance to wheat against diseases, such as powdery mildew, leaf rust, stem rust, yellow rust, and against insects such as green bug and wheat curl mite, the latter being a vector of wheat streak mosaic virus [4,28,37,46]. A better knowledge of 1RS at the molecular level is needed to clone genes underlying the resistance phenotypes, support further improvement of existing wheat cultivars, and eliminate some negative effects of the translocation on bread making quality [15,23,33]
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