Isolation and Sequencing of a Single Copy of an Introgressed Chromosome From a Complex Genome for Gene and SNP Identification

The large complex genomes of many crops constrain the use of new technologies for genome-assisted selection and genetic improvement. One method to simplify a genome is to break it into individual chromosomes by flow cytometry, however, in many crop species most chromosomes cannot be isolated individually. Flow sorting of a single copy of a chromosome has been developed in wheat and here we demonstrate its use to identify markers of interest in an Erianthus/Sacchurum hybrid. Erianthus/Saccharum hybrids are of interest because Erianthus is known to be highly resistant to soil borne diseases which cause extensive sugarcane yield losses in Australia. Sugarcane (Saccharum) cultivars are autopolyploids with a highly complex genome and over 100 chromosomes. Flow cytometry for sugarcane, as in most crops, does not resolve individual chromosomes to a karyotype peak for sorting. To isolate a single chromosome, we used genomic in situ hybridisation (GISH) to identify the flow karyotype region containing the Erianthus chromosomes, flow sorted single chromosomes from this region, PCR screened for the Erianthus chromosomes and sequenced them. One Erianthus chromosome amplified and sequenced well, and from this data we could identify 57 resistant type genes and SNPs in nearly half of these genes. We developed KASP SNP assays and demonstrated that the identified SNP markers segregated as expected in a small introgression population. The pipeline we developed here to flow sort and sequence single chromosomes could be used in any crop with a large complex genome to rapidly discover and develop markers to important loci.

Flow cytometric characterisation of the complex polyploid genome of Saccharum officinarum and modern sugarcane cultivars

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.

Isolating Chromosomes of the Komodo Dragon: New Tools for Comparative Mapping and Sequence Assembly

We developed new tools to build a high-quality chromosomal map of the Komodo dragon (Varanus komodoensis) available for cross-species phylogenomic analyses. First, we isolated chromosomes by flow sorting and determined the chromosome content of each flow karyotype peak by FISH. We then isolated additional Komodo dragon chromosomes by microdissection and amplified chromosome-specific DNA pools. The chromosome-specific DNA pools can be sequenced, assembled, and mapped by next-generation sequencing technology. The chromosome-specific paint probes can be used to investigate karyotype evolution through cross-species chromosome painting. Overall, the set of chromosome-specific DNA pools of V. komodoensis provides new tools for detailed phylogenomic analyses of Varanidae and squamates in general.

Flow Sorting of Mitotic Chromosomes in Common Wheat (Triticum aestivum L.)

The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.

Cell synchronization and isolation of metaphase chromosomes from maize (Zea mays L.) root tips for flow cytometric analysis and sorting

Accumulation of cells containing metaphase chromosomes is an important step in cytological analyses and chromosome sorting procedures. The goal of this research was to optimize treatment parameters to synchronize the cell cycle of maize root tip meristem cells. Levels of hydroxyurea, a DNA synthesis inhibitor, were assessed for their utility in accumulating cells at the G1 phase of the cell cycle. Trifluralin, amiprophos-methyl, and colchicine were used to accumulate cells containing metaphase chromosomes upon release from hydroxyurea inhibition. Optimal mitotic indices were achieved by treating seedlings with 5 mM hydroxyurea for 18 h, incubating for 1 h without chemical treatment to release the hydroxyurea block, and then treating emerging roots with 1 μM trifluralin for 4 h. The mitotic index of synchronized maize root tips was over 70%. Uniformity of synchronization depended upon selection of seeds with emerging radicles that were similar in length at the time of treatment. Suspensions of intact chromosomes were prepared by a simple slicing procedure. The chromosome preparations were found to be suitable for flow cytometric characterization and sorting. Chromosome peaks of the observed flow karyotype resembled the predicted flow karyotype calculated on the basis of maize chromosome size. Key words : flow karyotype, hydroxyurea, plant chromosome sorting, trifluralin.