Abstract
Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genotype, except R570, suggesting that the peaks may represent ancestral Saccharum sub genomes. The ability to flow sort Saccharum chromosomes will allow us to isolate and analyse chromosomes of interest and further examine the structure and evolution of the sugarcane genome.
Highlights
Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production
Modern sugarcane cultivars are derived from crosses between S. officinarum and S. spontaneum initially made by early sugarcane breeders in Java and India at the end of the nineteenth century[11]
Mitotic cells are accumulated at metaphase using a mitotic spindle inhibitor, most commonly amiprophos methyl (APM)
Summary
Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. We have successfully modified the protocol of Vrána et al.[19] to classify isolated mitotic metaphase chromosomes according to their relative DNA content and generate the first flow karyotypes for sugarcane. At a concentration of 0.5% Triton-X100 in the fixation and the homogeniser speed set to 16,000 rpm the peaks in the flow karyotype was the clearest and there were mostly well-fixed chromosomes on the slides (Table 2 and Supplementary Fig. S3).
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