Locating centromeres on genome sequences can be challenging. The high density of repetitive elements in these regions makes sequence assembly problematic, especially when using short-read sequencing technologies. It can also be difficult to distinguish between active and recently extinct centromeres through sequence analysis. An effective solution is to identify genetically active centromeres (functional in meiosis) by half-tetrad analysis. This genetic approach involves detecting heterozygosity along chromosomes in segregating populations derived from gametes (half-tetrads). Unreduced gametes produced by first division restitution mechanisms comprise complete sets of nonsister chromatids. Along these chromatids, heterozygosity is maximal at the centromeres, and homologous recombination events result in homozygosity toward the telomeres. We genotyped populations of half-tetrad-derived individuals (from Brassica interspecific hybrids) using a high-density array of physically anchored SNP markers (Illumina Brassica 60K Infinium array). Mapping the distribution of heterozygosity in these half-tetrad individuals allowed the genetic mapping of all 19 centromeres of the Brassica A and C genomes to the reference Brassica napus genome. Gene and transposable element density across the B. napus genome were also assessed and corresponded well to previously reported genetic map positions. Known centromerespecific sequences were located in the reference genome, but mostly matched unanchored sequences, suggesting that the core centromeric regions may not yet be assembled into the pseudochromosomes of the reference genome. The increasing availability of genetic markers physically anchored to reference genomes greatly simplifies the genetic and physical mapping of centromeres using half-tetrad analysis. We discuss possible applications of this approach, including in species where half-tetrads are currently difficult to isolate.

We present an efficient high-throughput flow cytometric method that builds on previously published methods and permits rapid ploidy discrimination in plants. By using Brassica napus L. microspore-derived plants as an example, we describe how 192 leaf tissue samples may be processed and analyzed comfortably by one operator in 6 h from tissue sampling to ploidy determination. The technique involves placing young leaf samples in two 96-well racks, using a bead-beating procedure to release nuclei into a lysis solution, filtering the samples on 96-well filter plates, staining with propidium iodide, and then rapidly estimating DNA ploidy using a plate loader on a BD FACSCanto II flow cytometer. Throughout the sample preparation process, multichannel pipetting allows faster and less error-prone sample handling. In two 96-well plates of samples, the histogram peaks of DNA content from flow cytometry were wellresolved in 189 of 192 samples tested (98.4%), with CV values ranging from 2.98% to 6.20% with an average CV of 4.35% (SD 5 0.68%). This new method is useful in doubled haploid plant breeding programs where early discrimination of haploid and doubled haploid (i.e., diploid) plantlets can confer significantly improved operational efficiencies. We discuss how this method could be further refined including adapting the method to robotic sample processing. Flow cytometry (FCM) is the predominant method for measuring nuclear DNA content (9). FCM involves staining cells with a DNA-specific fluorescent dye and separating the cells into single file within the liquid core stream of a flow chamber, in which they are intercepted by a high-intensity light source or laser focused on a small region known as the observation point. The laser excites the fluorescent dye that is bound to the DNA from which light scatters and fluorescence emissions are measured (10). FCM of plant cells is made difficult by structural irregularities (e.g., interlinked cells of irregular shape and rigid cell walls) typical of plant tissues. Furthermore, plant cells are frequently larger than the orifices in the flow chamber (50-100 lm diameter) of most flow cytometers (11-13). These problems associated with large and irregular cells may be overcome by isolating the nuclei that are smaller and more regularly shaped (13). In early protocols, nuclear suspensions were prepared from plant single-cell suspensions (protoplasts) using enzymes to digest cell wall materials.

The efficiency of production of doubled haploid plants in canola (Brassica napus L.) breeding programmes is reduced when large numbers of haploid and infertile plants survive until flowering. We used flow cytometry to determine ploidy status and predict subsequent fertility of microspore-derived plantlets from three canola genotypes, with or without colchicine treatment of microspore suspensions. Young leaf tissue was sampled from microspore-derived plantlets within 1 week of transfer to soil, and processed immediately by flow cytometry. The process was repeated on the same plants 3-5 weeks later. Of the 519 plants transferred to soil, 57.2% were consistently haploid at both sample times, 33.5% were consistently diploid at both sample times, and the remainder (9.2%) were uncertain or inconsistent in ploidy status across sampling times. Of the 518 plants that survived to flowering, 32.4% were diploid at both times of sampling and fertile (set seed) and 46.3% were haploid at both sampling times and infertile. Another 10.8% were haploid at both sampling times and fertile, but had low pollen viability and seed set, and some were triploid or of uncertain ploidy level. Colchicine treatment of microspore suspensions significantly increased the proportion of diploid plants from 9.7 to 69.7%, with significant variation among genotypes. Evidence from simple sequence repeat marker loci indicated that diploid and fertile plants from the control treatment (no colchicine) were derived from spontaneously doubled haploid gametes, rather than unreduced gametes or somatic tissue. Flow cytometry at the first sample time was very efficient in detecting diploid plants of which 94.2% were subsequently fertile.

Microspore culture is used extensively in several crop species to generate diverse populations of homozygous, doubled haploid lines for breeding and genetic analyses. In our canola (Brassica napus L.) doubled haploid breeding programme we regularly observe conjoined microspore-derived embryos, most commonly twins, joined either at the base of the hypocotyl or along the length of the hypocotyl axis. The aim of this study was to determine if twinned embryos were genetically identical or non-identical in order to gauge their value for breeding and linkage analysis. Microsatellite marker fingerprinting of 12 pairs of twinned embryos produced by microspore culture of heterozygous F(1) lines revealed that pairs of twins were genetically identical. Based on this finding, we recommend breeders and geneticists using microspore culture technology to retain only one embryo from each pair of twinned embryos.