Despite more than a century of intensive study of mitotic chromosomes, their three-dimensional organization remains enigmatic. The last decade established Hi-C as a method of choice for study of spatial genome-wide interactions. Although its utilization has been focused mainly on studying genomic interactions in interphase nuclei, the method can be also successfully applied to study 3D architecture and genome folding in mitotic chromosomes. However, obtaining sufficient number of mitotic chromosomes as an input material and effective coupling with Hi-C method is challenging in plant species. An elegant way to overcome hindrances with obtaining a pure mitotic chromosome fraction is their isolation via flow cytometric sorting. This chapter presents a protocol describing plant sample preparation for chromosome conformation studies, for flow-sorting of plant mitotic metaphase chromosomes and for the Hi-C procedure.

• Keywords
• 3D architecture, Chromatin interaction, Flow sorting, Hi-C, Plant chromosomes,
• MeSH
• Cell Nucleus genetics   MeSH
• Chromatin * genetics   MeSH
• Chromosomes * genetics   MeSH
• Genomics methods   MeSH
• Molecular Conformation MeSH
• Plants genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin * MeSH

Optical mapping-a technique that visualizes short sequence motives along DNA molecules of hundred kilobases to megabase in size-has found an important place in genome research. It is widely used to facilitate genome sequence assemblies and analyses of genome structural variations. Application of the technique is conditional on availability of highly pure ultra-long high-molecular-weight DNA (uHMW DNA), which is challenging to achieve in plants due to the presence of the cell wall, chloroplasts, and secondary metabolites, just as a high content of polysaccharides and DNA nucleases in some species. These obstacles can be overcome by employment of flow cytometry, enabling a fast and highly efficient purification of cell nuclei or metaphase chromosomes, which are afterward embedded in agarose plugs and used to isolate the uHMW DNA in situ. Here, we provide a detailed protocol for the flow sorting-assisted uHMW DNA preparation that has been successfully used to construct whole-genome as well as chromosomal optical maps for 20 plant species from several plant families.

• Keywords
• Bionano genome map, Chromosomes, Flow cytometry and sorting, HMW DNA preparation, Nuclei, Optical mapping, ultralong high-molecular-weight DNA,
• MeSH
• Chromosomes, Plant * genetics   MeSH
• Genome, Plant MeSH
• Flow Cytometry methods   MeSH
• Restriction Mapping MeSH
• Plants * genetics   MeSH
• Sequence Analysis, DNA methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.

• Keywords
• Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
• MeSH
• Chromosomes, Plant * MeSH
• Chromosomes * MeSH
• Cytogenetics MeSH
• Karyotyping MeSH
• Flow Cytometry methods   MeSH
• Suspensions MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Suspensions MeSH

Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of the missing genes and alleles. Development of alien introgression lines of wheat may be facilitated by the knowledge of DNA sequences of Aegilops chromosomes. As the Aegilops genomes are complex, sequencing relevant Aegilops chromosomes purified by flow cytometric sorting offers an attractive route forward. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some individual chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In the case of Ae. geniculata, fourteen chromosome-populations were discriminated, allowing the separation of nine individual chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug, and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped, revealing clear separation of the GAA-rich Aegilops chromosomes from the GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5% to 96.6% and from 87.8% to 97.7%, respectively. Differences in flow karyotypes between Ae. biuncialis and Ae. geniculata were analyzed and discussed. Chromosome-specific genomic resources will facilitate gene cloning and the development of molecular tools to support alien introgression breeding of wheat.

• Keywords
• Aegilops biuncialis, Aegilops geniculata, chromosome flow sorting, flow karyotyping, genome dissecting,
• Publication type
• Journal Article MeSH

Effective utilization of genetic diversity in wild relatives to improve wheat requires recombination between wheat and alien chromosomes. However, this is suppressed by the Pairing homoeologous gene, Ph1, on the long arm of wheat chromosome 5B. A deletion mutant of the Ph1 locus (ph1b) has been used widely to induce homoeologous recombination in wheat × alien hybrids. However, the original ph1b mutation, developed in Chinese Spring (CS) background has poor agronomic performance. Hence, alien introgression lines are first backcrossed with adapted wheat genotypes and after this step, alien chromosome segments are introduced into breeding lines. In this work, the ph1b mutation was transferred from two CSph1b mutants into winter wheat line Mv9kr1. Homozygous genotypes Mv9kr1 ph1b/ph1b exhibited improved plant and spike morphology compared to Chinese Spring. Flow cytometric chromosome analysis confirmed reduced DNA content of the mutant 5B chromosome in both wheat genotype relative to the wild type chromosome. The ph1b mutation in the Mv9kr1 genotype allowed wheat-alien chromosome pairing in meiosis of Mv9kr1ph1b_K × Aegilops biuncialis F1 hybrids, predominantly with the Mb-genome chromosomes of Aegilops relative to those of the Ub genome. High frequency of wheat-Aegilops chromosome interactions resulted in rearranged chromosomes identified in the new Mv9kr1ph1b × Ae. Biuncialis amphiploids, making these lines valuable sources for alien introgressions. The new Mv9kr1ph1b mutant genotype is a unique resource to support alien introgression breeding of hexaploid wheat.

• Keywords
• Aegilops biuncialis, bread wheat, chromosome flow sorting, homoeologous recombination, in situ hybridization, meiotic chromosome pairing, ph1b mutant,
• Publication type
• Journal Article MeSH