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

Powdery mildew is one of the most devastating diseases of wheat which significantly decreases yield and quality. Identification of new sources of resistance and their implementation in breeding programs is the most effective way of disease control. Two major powdery mildew resistance loci conferring resistance to all races in seedling and adult plant stages were identified in the emmer wheat landrace GZ1. Their positions, effects, and transferability were verified using two linkage maps (1,510 codominant SNP markers) constructed from two mapping populations (276 lines in total) based on the resistant GZ1 line. The dominant resistance locus QPm.GZ1-7A was located in a 90 cM interval of chromosome 7AL and explains up to 20% of the trait variation. The recessive locus QPm.GZ1-2A, which provides total resistance, explains up to 40% of the trait variation and was located in the distal part of chromosome 2AL. The locus was saturated with 14 PCR-based markers and delimited to a 0.99 cM region which corresponds to 4.3 Mb of the cv. Zavitan reference genome and comprises 55 predicted genes with no apparent candidate for the QPm.GZ1-2A resistance gene. No recessive resistance gene or allele was located at the locus before, suggesting the presence of a new powdery mildew resistance gene in the GZ1. The mapping data and markers could be used for the implementation of the locus in breeding. Moreover, they are an ideal base for cloning and study of host-pathogen interaction pathways determined by the resistance genes.

• Keywords
• GZ1, QTL mapping, emmer, powdery mildew (Blumeria graminis D. C. f. sp. tritici), resistance, wheat,
• Publication type
• Journal Article MeSH

Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.

• Keywords
• DNA amplification, DNA isolation, cell cycle synchronization, gene mapping and cloning, genome sequencing, liquid chromosome suspension, marker development, mitotic metaphase chromosomes, repetitive DNA labelling,
• MeSH
• Cell Cycle MeSH
• Chromosomes, Plant * genetics   MeSH
• Metaphase MeSH
• Flow Cytometry MeSH
• Plants * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Crop breeding for resistance to pathogens largely relies on genes encoding receptors that confer race-specific immunity. Here, we report the identification of the wheat Pm4 race-specific resistance gene to powdery mildew. Pm4 encodes a putative chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions, a unique domain architecture among known resistance proteins. Pm4 undergoes constitutive alternative splicing, generating two isoforms with different protein domain topologies that are both essential for resistance function. Both isoforms interact and localize to the endoplasmatic reticulum when co-expressed. Pm4 reveals additional diversity of immune receptor architecture to be explored for breeding and suggests an endoplasmatic reticulum-based molecular mechanism of Pm4-mediated race-specific resistance.

• MeSH
• Alternative Splicing * MeSH
• Ascomycota immunology   MeSH
• Cloning, Molecular MeSH
• Evolution, Molecular MeSH
• Plant Diseases genetics   MeSH
• Disease Resistance genetics   MeSH
• Protein Kinases genetics   physiology   MeSH
• Triticum enzymology   genetics   microbiology   MeSH
• Recombination, Genetic MeSH
• Genes, Plant MeSH
• Plant Proteins genetics   physiology   MeSH
• Gene Silencing MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Protein Kinases MeSH
• Plant Proteins MeSH

Plasma membrane-associated and intracellular proteins and protein complexes play a pivotal role in pathogen recognition and disease resistance signaling in plants and animals. The two predominant protein families perceiving plant pathogens are receptor-like kinases and nucleotide binding-leucine-rich repeat receptors (NLR), which often confer race-specific resistance. Leaf rust is one of the most prevalent and most devastating wheat diseases. Here, we clone the race-specific leaf rust resistance gene Lr14a from hexaploid wheat. The cloning of Lr14a is aided by the recently published genome assembly of ArinaLrFor, an Lr14a-containing wheat line. Lr14a encodes a membrane-localized protein containing twelve ankyrin (ANK) repeats and structural similarities to Ca2+-permeable non-selective cation channels. Transcriptome analyses reveal an induction of genes associated with calcium ion binding in the presence of Lr14a. Haplotype analyses indicate that Lr14a-containing chromosome segments were introgressed multiple times into the bread wheat gene pool, but we find no variation in the Lr14a coding sequence itself. Our work demonstrates the involvement of an ANK-transmembrane (TM)-like type of gene family in race-specific disease resistance in wheat. This forms the basis to explore ANK-TM-like genes in disease resistance breeding.

• MeSH
• Ankyrin Repeat genetics   MeSH
• Basidiomycota pathogenicity   MeSH
• Gene Pool MeSH
• Haplotypes MeSH
• Membrane Proteins genetics   MeSH
• Mutagenesis MeSH
• Plant Diseases genetics   MeSH
• Disease Resistance genetics   MeSH
• Triticum genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Genes, Plant genetics   MeSH
• Plant Proteins genetics   MeSH
• Plant Breeding MeSH
• Nicotiana genetics   MeSH
• Gene Silencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• Plant Proteins MeSH

Mutations at the LYS3 locus in barley have multiple effects on grain development, including an increase in embryo size and a decrease in endosperm starch content. The gene underlying LYS3 was identified by genetic mapping and mutations in this gene were identified in all four barley lys3 alleles. LYS3 encodes a transcription factor called Prolamin Binding Factor (PBF). Its role in controlling embryo size was confirmed using wheat TILLING mutants. To understand how PBF controls embryo development, we studied its spatial and temporal patterns of expression in developing grains. The PBF gene is expressed in both the endosperm and the embryos, but the timing of expression in these organs differs. PBF expression in wild-type embryos precedes the onset of embryo enlargement in lys3 mutants, suggesting that PBF suppresses embryo growth. We predicted the down-stream target genes of PBF in wheat and found them to be involved in a wide range of biological processes, including organ development and starch metabolism. Our work suggests that PBF may influence embryo size and endosperm starch synthesis via separate gene control networks.

• Keywords
• (DAF), Days after flowering, (DOF), DNA binding with one zinc finger, (PBF), Prolamin-box binding factor, (TILLING), Targeting induced local lesions in genomes, High lysine, Large embryo, PBF, SIFT), Sorting intolerant from tolerant, Shrunken endosperm,
• Publication type
• Journal Article MeSH

Stem rust is an important disease of wheat that can be controlled using resistance genes. The gene SuSr-D1 identified in cultivar 'Canthatch' suppresses stem rust resistance. SuSr-D1 mutants are resistant to several races of stem rust that are virulent on wild-type plants. Here we identify SuSr-D1 by sequencing flow-sorted chromosomes, mutagenesis, and map-based cloning. The gene encodes Med15, a subunit of the Mediator Complex, a conserved protein complex in eukaryotes that regulates expression of protein-coding genes. Nonsense mutations in Med15b.D result in expression of stem rust resistance. Time-course RNAseq analysis show a significant reduction or complete loss of differential gene expression at 24 h post inoculation in med15b.D mutants, suggesting that transcriptional reprogramming at this time point is not required for immunity to stem rust. Suppression is a common phenomenon and this study provides novel insight into suppression of rust resistance in wheat.

• MeSH
• Basidiomycota pathogenicity   MeSH
• Chromosomes, Plant genetics   MeSH
• Gene Duplication MeSH
• Gene Expression MeSH
• Phenotype MeSH
• Plant Immunity genetics   MeSH
• Poaceae classification   genetics   MeSH
• Chromosome Mapping MeSH
• Mediator Complex genetics   MeSH
• Mutation MeSH
• Plant Diseases genetics   immunology   microbiology   MeSH
• Disease Resistance genetics   MeSH
• Triticum genetics   immunology   microbiology   MeSH
• Gene Expression Regulation, Plant MeSH
• Genes, Plant genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Mediator Complex MeSH

The identification of genes of agronomic interest in bread wheat (Triticum aestivum L.) is hampered by its allopolyploid nature (2n = 6x = 42; AABBDD) and its very large genome, which is largely covered by transposable elements. However, owing to this complex structure, aneuploid stocks can be developed in which fragments or entire chromosomes are missing, sometimes resulting in visible phenotypes that help in the cloning of affected genes. In this study, the 2C gametocidal chromosome from Aegilops cylindrica was used to develop a set of 113 deletion lines for chromosome 3D in the reference cultivar Chinese Spring. Eighty-four markers were used to show that the deletions evenly covered chromosome 3D and ranged from 6.5 to 357 Mb. Cytogenetic analyses confirmed that the physical size of the deletions correlated well with the known molecular size deduced from the reference sequence. This new genetic stock will be useful for positional cloning of genes on chromosome 3D, especially for Ph2 affecting homoeologous pairing in bread wheat.

• Keywords
• Ph2, deletion line, gametocidal, homoeologous pairing, wheat,
• Publication type
• Journal Article MeSH

A genetic linkage map of dioecious garden asparagus (Asparagus officinalis L., 2n = 2x = 20) was constructed using F1 population, simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. In total, 1376 SNPs and 27 SSRs were used for genetic mapping. Two resulting parental maps contained 907 and 678 markers spanning 1947 and 1814 cM, for female and male parent, respectively, over ten linkage groups representing ten haploid chromosomes of the species. With the aim to anchor the ten genetic linkage groups to individual chromosomes and develop a tool to facilitate genome analysis and gene cloning, we have optimized a protocol for flow cytometric chromosome analysis and sorting in asparagus. The analysis of DAPI-stained suspensions of intact mitotic chromosomes by flow cytometry resulted in histograms of relative fluorescence intensity (flow karyotypes) comprising eight major peaks. The analysis of chromosome morphology and localization of 5S and 45S rDNA by FISH on flow-sorted chromosomes, revealed that four chromosomes (IV, V, VI, VIII) could be discriminated and sorted. Seventy-two SSR markers were used to characterize chromosome content of individual peaks on the flow karyotype. Out of them, 27 were included in the genetic linkage map and anchored genetic linkage groups to chromosomes. The sex determining locus was located on LG5, which was associated with peak V representing a chromosome with 5S rDNA locus. The results obtained in this study will support asparagus improvement by facilitating targeted marker development and gene isolation using flow-sorted chromosomes.

• Keywords
• Asparagus officinalis, FISH, SNPs, SSRs, flow-sorted chromosomes, genetic map, sex chromosome,
• Publication type
• Journal Article MeSH