Papers by Jaroslav Dolezel
The presence of three homoeologous genomes and large genome size (1C~17,000 Mbp) hamper physical ... more The presence of three homoeologous genomes and large genome size (1C~17,000 Mbp) hamper physical mapping and positional cloning in hexaploid wheat (Triticum aestivum L., 2n=6x=42). An attractive approach to reduce the complexity of these accomplishments is to dissect the genome to smaller parts such as chromosomes and chromosome arms. We have shown previously that laser flow cytometry is a suitable method to achieve this goal in a number of species. This approach involves a preparation of aqueous suspensions of intact mitotic chromosomes. The chromosomes in suspension are stained by a DNAspecific fluorochrome and classified according to relative fluorescence intensity. Any chromosome, which differs in relative DNA content from other chromosomes, can be discriminated and sorted at high speed.
Chromosome genomics uncovers plant genome organization and function
Biotechnology Advances, 2021
The identification of causal genomic loci and their interactions underlying various traits in pla... more The identification of causal genomic loci and their interactions underlying various traits in plants has been greatly aided by progress in understanding the organization of the nuclear genome. This provides clues to the responses of plants to environmental stimuli at the molecular level. Apart from other uses, these insights are needed to fully explore the potential of new breeding techniques that rely on genome editing. However, genome analysis and sequencing is not straightforward in the many agricultural crops and their wild relatives that possess large and complex genomes. Chromosome genomics streamlines this task by dissecting the genome to single chromosomes whose DNA is then used instead of nuclear DNA. This results in a massive and lossless reduction in DNA sample complexity, reduces the time and cost of the experiment, and simplifies data interpretation. Flow cytometric sorting of condensed mitotic chromosomes makes it possible to purify single chromosomes in large quantities, and as the DNA remains intact this process can be coupled successfully with many techniques in molecular biology and genomics. Since the first experiments with flow cytometric sorting in the late 1980s, numerous applications have been developed, and chromosome genomics has been having a significant impact in many areas of research, including the sequencing of complex genomes of important crops and gene cloning. This review discusses these applications, describes their contribution to advancements in plant genome analysis and gene cloning, and outlines future directions.
Towards the construction of a high density genetic linkage map of wheat chromosome 5A
*) CRA-GPG Genomic Research Centre, Via S. Protaso 302, 29017 Fiorenzuola d'Arda (PC), Italy ... more *) CRA-GPG Genomic Research Centre, Via S. Protaso 302, 29017 Fiorenzuola d'Arda (PC), Italy **) CRA – Cereal Research Centre S.S. 16, km 675 I-71100 Foggia, Italy ***) Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, CZ-77200, Olomouc, Czech Republic ****) Diversity Arrays Technology Pty Ltd, Building 1 CSIRO Forestry, 1 Wilf Crane Crescent, PO Box 7141, ACT 2600, Yarralumla, Australia *****) Department of Agricultural and Food Sciences, University of Modena and Reggio Emilia Via Amendola 2 I-42100 Reggio Emilia, Italy ******) Genomics Platform, Parco Tecnologico Padano Via Einstein I-26900 Località Cascina Codazza (LO), Italy *******) Department of Environmental and Agro-Biology and Agro-Chemistry, University of Tuscia Via San Camillo de Lellis s.n.c I-01100 Viterbo, Italy ********) Department of Environmental and Agro-Forestry Biology and Chemistry, University of Bari Via Amendola 165-A I-70126 Bari, Italy *********) ENEA BIOTEC-GEN Casaccia Research Centre Via Anguillarese 301 I-00123 Roma, Italy
Theoretical and Applied Genetics, Dec 7, 2018
A segment of Triticum militinae chromosome 7G harbors a gene(s) conferring powdery mildew resista... more A segment of Triticum militinae chromosome 7G harbors a gene(s) conferring powdery mildew resistance which is effective at both the seedling and the adult plant stages when transferred into bread wheat (T. aestivum). The introgressed segment replaces a piece of wheat chromosome arm 4AL. An analysis of segregating materials generated to positionally clone the gene highlighted that in a plant heterozygous for the introgression segment, only limited recombination occurs between the introgressed region and bread wheat 4A. Nevertheless, 75 genetic markers were successfully placed within the region, thereby confining the gene to a 0.012 cM window along the 4AL arm. In a background lacking the Ph1 locus, the localized rate of recombination was raised 33-fold, enabling the reduction in the length of the region containing the resistance gene to a 480 kbp stretch harboring 12 predicted genes. The substituted segment in the reference sequence of bread wheat cv. Chinese Spring is longer (640 kbp) and harbors 16 genes. A comparison of the segments' sequences revealed a high degree of divergence with respect to both their gene content and nucleotide sequence. Of the 12 T. militinae genes, only four have a homolog in cv. Chinese Spring. Possible candidate genes for the resistance have been identified based on function predicted from their sequence.
Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. ... more Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to Fusarium oxysporum f. sp. cubense (Foc), the causal agent of this disease, is genetically dissected in this study using two Musa acuminata ssp. malaccensis segregating populations segregating for Foc Tropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on Chromosome 3 of ‘DH-Pahang’ reference assembly v4. Within this region, there is a cluster of pattern recognition receptors, namely leucine rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell wall associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant but not in suscept...
If Mendel Was Using CRISPR: Genome Editing Meets Non‐Mendelian Inheritance
Advanced Functional Materials
On July 20 this year, 200th anniversary of the birth of Gregor Mendel, an Augustinian monk who la... more On July 20 this year, 200th anniversary of the birth of Gregor Mendel, an Augustinian monk who laid the foundations of genetics, is celebrated. His findings provided the stepping stone for rational crop improvement based on cross breeding and selection. While this approach contributed to the development of superior varieties, its potential is limited by genetic variability available within a species and its relatives. Even though physical and chemical mutagenesis enabled the induction of new genetic variability on a large scale, random mutagenesis did not allow targeting particular genome loci. This became feasible only after the development of genome editing tools in the beginning of the 21st century and the ability to precisely edit plant genomes opened a new era in plant research and crop breeding. Mendel probably did not consider a possibility of directly modifying genetic information and the advantages which genetic engineering can bring to society. However, the basic principle...
Africrop/fonio_smcpp: fonio_smcpp
Fine structure and transcription dynamics of bread wheat ribosomal DNA loci deciphered by a multi‐omics approach
The Plant Genome, 2022
Three out of four RNA components of ribosomes are encoded by 45S ribosomal DNA (rDNA) loci, which... more Three out of four RNA components of ribosomes are encoded by 45S ribosomal DNA (rDNA) loci, which are organized as long head‐to‐tail tandem arrays of nearly identical units, spanning several megabases of sequence. Due to this structure, the rDNA loci are the major sources of gaps in genome assemblies, and gene copy number, sequence composition, and expression status of particular arrays remain elusive, especially in complex genomes harboring multiple loci. Here we conducted a multi‐omics study to decipher the 45S rDNA loci in hexaploid bread wheat. Coupling chromosomal genomics with optical mapping, we reconstructed individual rDNA arrays, enabling locus‐specific analyses of transcription activity and methylation status from RNA‐ and bisulfite‐sequencing data. We estimated a total of 6,650 rDNA units in the bread wheat genome, with approximately 2,321, 3,910, 253, and 50 gene copies located in short arms of chromosomes 1B, 6B, 5D, and 1A, respectively. Only 1B and 6B loci contributed substantially to rRNA transcription at a roughly 2:1 ratio. The ratio varied among five tissues analyzed (embryo, coleoptile, root tip, primary leaf, mature leaf), being the highest (2.64:1) in mature leaf and lowest (1.72:1) in coleoptile. Cytosine methylation was considerably higher in CHG context in the silenced 5D locus as compared with the active 1B and 6B loci. In conclusion, a fine genomic organization and tissue‐specific expression of rDNA loci were deciphered, for the first time, in a complex polyploid species. The results are discussed in the context of wheat evolution and transcription regulation.
Pea (Pisum sativum L.) was the original model organism for Mendel´s discovery of the laws of inhe... more Pea (Pisum sativum L.) was the original model organism for Mendel´s discovery of the laws of inheritance and kept this model status until the advent of molecular biology at the end of the 20th century. Pea is also one of the world’s oldest domesticated crops. It is currently the third most widely grown pulse crop, as its seeds serve as a protein-rich food for humans and livestock alike. While several legume species genome's draft sequences have been produced, progress in pea genomics has lagged behind largely as a consequence of its complex and large genome size. The pea genome is large (ca 4.45 Gb), probably resulting from recent expansion of retrotransposons followed by sequence diversification. The Pea Genome International Project has undertaken several complementary strategies in order to produce a high-quality draft sequence of the species. We will present how this draft sequence opens the way to renew strategies in pea breeding.
Next Generation Breeding of East African Highland Bananas – The Main Staple of East African Great Lakes
Genomic breeding approaches for East African bananas
The polyploidy nature of banana is a limiting factor in the implementation of strategies such as ... more The polyploidy nature of banana is a limiting factor in the implementation of strategies such as marker assisted selection (MAS) or genome wide association mapping (GWAS). The triploid nature of cultivated varieties complicates conventional breeding strategies and improved varieties can take up to 20 years before they can be released to the public, which necessitates the use of efficient molecular tools to more rapidly respond to abiotic and biotic stresses and to address the needs of growers and consumers. In addition, the high cost of phenotyping perennial large-stature plants such as banana, and the rapidly decreasing cost of genotyping, makes the use of predictive genomic selection models using single nucleotide polymorphism (SNP) markers extremely attractive to banana breeders. A Genomic Selection (GS) training population consisting of 307 banana genotypes was developed for initial analysis with ploidy levels of the plant material ranging from diploids to tetraploids. Plants were genotyped using the genotyping by sequencing (GBS) approach (Elshire et al., 2011) with PstI as the sole restriction enzyme. Sequence data was processed through a bioinformatics workflow and single nucleotide polymorphisms (SNPs) were called using the genomic analysis tool kit (GATK). Data was filtered for quality and for loci with >50% missing data. Phenotypic data for 25 traits are being collected from two locations since 2012. Yield-related traits (fruit pulp diameter, bunch weight, number of suckers, etc.) are collected at flowering and harvest Analysis of GBS data resulted in 11201 SNP loci. The results of multiple prediction models are discussed and compared
454 sequence survey of wheat chromosome 4A reveals its structure and is a new resource for high-throughput development of chromosome-specific markers
Comunicacion presentada en el IWGSC workshop el 4 de septiembre de 2011 en la ciudad de Mejico. E... more Comunicacion presentada en el IWGSC workshop el 4 de septiembre de 2011 en la ciudad de Mejico. El IWGSC workshop se celebro junto al 21th International Triticeae Mapping Initiative (ITMI).-- Hernandez, Pilar et al.
Molecular Biology Reports, 2020
Diploid A genome wheat species harbor immense genetic variability which has been targeted and pro... more Diploid A genome wheat species harbor immense genetic variability which has been targeted and proven useful in wheat improvement. Development and deployment of sequence-based markers has opened avenues for comparative analysis, gene transfer and marker assisted selection (MAS) using high throughput cost effective genotyping techniques. Chromosome 2A of wheat is known to harbor several economically important genes. The present study aimed at identification of genic sequences corresponding to full length cDNAs and mining of SSRs and ISBPs from 2A draft sequence assembly of hexaploid wheat cv. Chinese Spring for marker development. In total, 1029 primer pairs including 478 gene derived, 501 SSRs and 50 ISBPs were amplified in diploid A genome species Triticum monococcum and T. boeoticum identifying 221 polymorphic loci. Out of these, 119 markers were mapped onto a pre-existing chromosome 2A genetic map consisting of 42 mapped markers. The enriched genetic map constituted 161 mapped markers with final map length of 549.6 cM. Further, 2A genetic map of T. monococcum was anchored to the physical map of 2A of cv. Chinese Spring which revealed several rearrangements between the two species. The present study generated a highly saturated genetic map of 2A and physical anchoring of genetically mapped markers revealed a complex genetic architecture of chromosome 2A that needs to be investigated further. Sequence Submission: 513 genic sequences (partial sequences) of T. monococcum and T. boeoticum submitted to GenBank (KP240087-KP240600).
Genetic Resources and Crop Evolution, 2018
Bananas (Musa spp.), including dessert and cooking types, are of major importance in the tropics.... more Bananas (Musa spp.), including dessert and cooking types, are of major importance in the tropics. Due to extremely high levels of sterility, the diversity of cultivated bananas is fixed over long periods of time to the existing genotypes. This pattern puts banana-based agrosystems at risk. Therefore, assessing the extent of wild and cultivated banana diversity, conserving it and making it available for further use is a priority. We report here the collection of new wild and cultivated banana germplasm in the Autonomous Region of Bougainville, Papua New Guinea. In total, 61 accessions were collected and their names and uses were recorded when possible. Classification was also provided based on the observations made in the field. Three wild specimens were collected. Among the 58 cultivated accessions, we noted that eight were used as ornamental plants, seven were edible varieties of the Fe'i type and two were natural tetraploids from the Musa section. The ploidy was then checked by flow cytometry and the accessions were genotyped with a set of 19 SSR markers. The genotyping results were merged to the dataset from Christelova ´et al. (Biodivers Conserv 26:801-824, 2017). This joint analysis helped refine Electronic supplementary material The online version of this article () contains supplementary material, which is available to authorized users.
The field verification activity: a cooperative approach to the management of the globalMusain vitro collection at the International Transit Centre
Acta Horticulturae, 2016
The International Transit Centre (ITC) (Bioversity International, KULeuven, Belgium) currently ho... more The International Transit Centre (ITC) (Bioversity International, KULeuven, Belgium) currently holds the largest ex situ collection of Musa germplasm, partly available to researchers, breeders and the larger Musa community via the online ordering system in the Musa Germplasm Information System (MGIS). It is thus extremely important that somaclonal variants and mislabeled accessions are removed from the collection to ensure that users have access to a valid and reliable source of Musa genetic resources. Through Bioversity's Field Verification (FV) activity, a combination of morphological and molecular characterization along with a validation by a panel of experts is used to assess the genetic integrity of the Musa accessions held at ITC. The FV includes ploidy determination and SSR and DArT fingerprinting data (produced by the Musa Genotyping Centre, Institute of Experimental Botany, Czech Republic and DArT P/L, Australia) combined with minimum descriptors and standard photographs of accessions in partners' field collections. The compiled molecular and morphological data are sent to a panel of ten Musa taxonomists in charge of the validation of the accessions' genetic integrity. This workflow allows the quality control of the germplasm available to users while adding value to the content of the ITC by producing standardized characterization data. Status has been determined for 370 of 832 accessions sent to field partners, and the first round will be completed when all virus-free accessions are validated. Results thus far indicate that most of the in vitro collection is genetically stable, supporting the use of the tissue culture protocols; however, questions have arisen regarding the optimal duration after which accessions should be genetically verified; how the work can be better organized and communicated, and the implications of the type of somaclonal variations observed. A large body of information has been generated and is accessible to the public via MGIS, including documentation (data and photographs) for 540 accessions, covering 63% of the collection currently available for distribution. This information adds significant value to the ITC collection and is a clear asset to the Musa community
Acta Horticulturae, 2016
Major diseases, including Fusarium wilt tropical race 4, threaten banana production systems world... more Major diseases, including Fusarium wilt tropical race 4, threaten banana production systems worldwide. New sources of genetic resistance are considered necessary in the fight against such diseases. The triangular region of Indonesia taking in Sulawesi, the Maluku Islands and Lesser Sunda Islands was prioritized by the Global Musa Genetic Resources Network, MusaNet for exploration and collecting. It is just east of the 'Wallace Line', which is recognized as a transition zone for flora in southeast Asia, and had been little explored. Bioversity International funded a team of scientists from Indonesia and Australia to make collecting missions in the triangle in October 2012 and February 2013. Suckers and seeds of 35 promising new accessions were collected. About 90% of these are either wild species or diploid cultivars of more direct use to breeding programs. These were morphologically characterized during the collecting missions and included a set of photographs recommended by Bioversity's Taxonomic Advisory Group. Cigar leaf samples were also collected and sent as fresh samples to the International Banana Genotyping Centre in the Czech Republic. Ploidy and DNA (SSR) genotyping determinations from these samples have been invaluable in quickly interpreting and better appreciating what has been discovered. The new accessions have been grown on at Solok field collection, West Sumatra and will be made available by Indonesia to the international community, including breeding programs, for evaluation and utilization. Information on wild Eumusa prompts a rethinking of the phytogeography of Musa acuminata. The variation within the Australimusa species M. lolodensis highlights the need for broader study of this Musa section. French Plantain-like edible AAs and prospects for the generation of African plantains in the region were identified. The mission indicated existence of local edible ABs in eastern Indonesia in association with balbisiana hybrids origins in the region. Further explorations in the region should add to Musa diversity knowledge.
Both male and female meiosis contribute to non‐Mendelian inheritance of parental chromosomes in interspecific plant hybrids (Lolium × Festuca)
New Phytologist
Summary Some interspecific plant hybrids show unequal transmission of chromosomes from parental g... more Summary Some interspecific plant hybrids show unequal transmission of chromosomes from parental genomes to the successive generations. It has been suggested that this is due to a differential behavior of parental chromosomes during meiosis. However, underlying mechanism is unknown. We analyzed chromosome composition of the F2 generation of Festuca × Lolium hybrids and reciprocal backcrosses to elucidate effects of male and female meiosis on the shift in parental genome composition. We studied male meiosis, including the attachment of chromosomes to the karyokinetic spindle and gene expression profiling of the kinetochore genes. We found that Lolium and Festuca homoeologues were transmitted differently to the F2 generation. Female meiosis led to the replacement of Festuca chromosomes by their Lolium counterparts. In male meiosis, Festuca univalents were attached less frequently to microtubules than Lolium univalents, lagged in divisions and formed micronuclei, which were subsequently...
Venn diagram showing wheat read distribution with a significant similarity to Brachypodium, rice and sorghum
PLOS ONE, 2011
Frontiers in Genetics, Aug 18, 2020
Structural variations (SVs) such as copy number and presence-absence variations are polymorphisms... more Structural variations (SVs) such as copy number and presence-absence variations are polymorphisms that are known to impact genome composition at the species level and are associated with phenotypic variations. In the absence of a reference genome sequence, their study has long been hampered in wheat. The recent production of new wheat genomic resources has led to a paradigm shift, making possible to investigate the extent of SVs among cultivated and wild accessions. We assessed SVs affecting genes and transposable elements (TEs) in a Triticeae diversity panel of 45 accessions from seven tetraploid and hexaploid species using high-coverage shotgun sequencing of sorted chromosome 3B DNA and dedicated bioinformatics approaches. We showed that 23% of the genes are variable within this panel, and we also identified 330 genes absent from the reference accession Chinese Spring. In addition, 60% of the TEderived reference markers were absent in at least one accession, revealing a high level of intraspecific and interspecific variability affecting the TE space. Chromosome extremities are the regions where we observed most of the variability, confirming previous hypotheses made when comparing wheat with the other grasses. This study provides deeper insights into the genomic variability affecting the complex Triticeae genomes at the intraspecific and interspecific levels and suggests a phylogeny with independent hybridization events leading to different hexaploid species.
Across domains of biological research using genome sequence data, high-quality reference genome s... more Across domains of biological research using genome sequence data, high-quality reference genome sequences are essential for characterizing genetic variation and understanding the genetic basis of phenotypes. However, the construction of genome assemblies for various species is often hampered by complexities of genome organization, especially repetitive and complex sequences, leading to mis-assembly and missing regions. Here, we describe a high-throughput gold standard genome assembly workflow using a largescale bacterial artificial chromosome (BAC) library with a refined two-step pooling strategy and the Lamp assembler algorithm. This strategy minimizes the laborious processes of physical map construction and clone-by-clone sequencing, enabling inexpensive sequencing of several thousand BAC clones. By applying this strategy with a minimum tiling path BAC clone library for the short arm of chromosome 2D (2DS) of bread wheat, 98% of BAC sequences, covering 92.7% of the 2DS chromosome, were assembled correctly for this species with a highly complex and repetitive genome. We also identified 48 large misassemblies in the reference wheat genome assembly (IWGSC RefSeq v1.0) and corrected these large mis-assemblies in addition to filling 92.2% of the gaps in RefSeq v1.0. Our 2DS assembly represents a new benchmark for the assembly of complex genomes with both high accuracy and efficiency. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Papers by Jaroslav Dolezel