Nejvíce citovaný článek - PubMed ID 11102393
Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.)
Wheat and its close relatives have large and complex genomes, making gene cloning difficult. Nevertheless, developments in genomics over the past decade have made it more feasible. The large and complex genomes of cereals, especially bread wheat, have always been a challenge for gene mapping and cloning. Nevertheless, recent advances in genomics have led to significant progress in this field. Currently, high-quality reference sequences are available for major wheat species and their relatives. New high-throughput genotyping platforms and next-generation sequencing technologies combined with genome complexity reduction techniques and mutagenesis have opened new avenues for gene cloning. In this review, we provide a comprehensive overview of the genes cloned in wheat so far and discuss the strategies used for cloning these genes. We highlight the advantages and drawbacks of individual approaches and show how particular genomic progress contributed to wheat gene cloning. A wide range of new resources and approaches have led to a significant increase in the number of successful cloning projects over the past decade, demonstrating that it is now feasible to perform rapid gene cloning of agronomically important genes, even in a genome as large and complex as that of wheat.
Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.
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.
- Klíčová slova
- Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
- MeSH
- chromozomy rostlin * MeSH
- chromozomy * MeSH
- cytogenetika MeSH
- karyotypizace MeSH
- průtoková cytometrie metody MeSH
- suspenze MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- suspenze MeSH
To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool1. Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance. We cloned the stem rust resistance gene Sr43, which was crossed into bread wheat from the wild grass Thinopyrum elongatum2,3. Sr43 encodes an active protein kinase fused to two domains of unknown function. The gene, which is unique to the Triticeae, appears to have arisen through a gene fusion event 6.7 to 11.6 million years ago. Transgenic expression of Sr43 in wheat conferred high levels of resistance to a wide range of isolates of the pathogen causing stem rust, highlighting the potential value of Sr43 in resistance breeding and engineering.
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.
- Klíčová slova
- Aegilops biuncialis, Aegilops geniculata, chromosome flow sorting, flow karyotyping, genome dissecting,
- Publikační typ
- časopisecké články MeSH
Rye (Secale cereale) is a climate-resilient cereal grown extensively as grain or forage crop in Northern and Eastern Europe. In addition to being an important crop, it has been used to improve wheat through introgression of genomic regions for improved yield and disease resistance. Understanding the genomic diversity of rye will assist both the improvement of this crop and facilitate the introgression of more valuable traits into wheat. Here, we isolated and sequenced the short arm of rye chromosome 7 (7RS) from Triticale 380SD using flow cytometry and compared it to the public Lo7 rye whole genome reference assembly. We identify 2747 Lo7 genes present on the isolated chromosome arm and two clusters containing seven and sixty-five genes that are present on Triticale 380SD 7RS, but absent from Lo7 7RS. We identified 29 genes that are not assigned to chromosomal locations in the Lo7 assembly but are present on Triticale 380SD 7RS, suggesting a chromosome arm location for these genes. Our study supports the Lo7 reference assembly and provides a repertoire of genes on Triticale 7RS.
- Klíčová slova
- isolated chromosome arm sequencing, presence-absence variation, rye,
- MeSH
- chromozomy rostlin genetika MeSH
- jedlá semena genetika MeSH
- odolnost vůči nemocem genetika MeSH
- pšenice genetika MeSH
- triticale * genetika MeSH
- žito * genetika MeSH
- Publikační typ
- časopisecké články 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.
- Klíčová slova
- GZ1, QTL mapping, emmer, powdery mildew (Blumeria graminis D. C. f. sp. tritici), resistance, wheat,
- Publikační typ
- časopisecké články MeSH
The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. Here, we develop a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and use positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62, which has also been transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines show high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance.
The VERNALIZATION1 (VRN1) gene encodes a MADS-box transcription factor and plays an important role in the cold-induced transition from the vegetative to reproductive stage. Allelic variability of VRN1 homoeologs has been associated with large differences in flowering time. The aim of this study was to investigate the genetic variability of VRN1 homoeologs (VRN-A1, VRN-B1 and VRN-D1). We performed an in-depth sequence analysis of VRN1 homoeologs in a panel of 105 winter and spring varieties of hexaploid wheat. We describe the novel allele Vrn-B1f with an 836 bp insertion within intron 1 and show its specific expression pattern associated with reduced heading time. We further provide the complete sequence of the Vrn-A1b allele, revealing a 177 bp insertion in intron 1, which is transcribed into an alternative splice variant. Copy number variation (CNV) analysis of VRN1 homoeologs showed that VRN-B1 and VRN-D1 are present in only one copy. The copy number of recessive vrn-A1 ranged from one to four, while that of dominant Vrn-A1 was one or two. Different numbers of Vrn-A1a copies in the spring cultivars Branisovicka IX/49 and Bastion did not significantly affect heading time. We also report on the deletion of secondary structures (G-quadruplex) in promoter sequences of cultivars with more vrn-A1 copies.
- Klíčová slova
- CNV, VRN1, allelic variation, alternative splice variants, next generation sequencing, wheat,
- MeSH
- alely * MeSH
- alternativní sestřih MeSH
- chléb MeSH
- genetická variace * MeSH
- genová dávka * MeSH
- inzerční mutageneze MeSH
- polyploidie * MeSH
- pšenice genetika MeSH
- represorové proteiny genetika MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- represorové proteiny MeSH
Breeding of agricultural crops adapted to climate change and resistant to diseases and pests is hindered by a limited gene pool because of domestication and thousands of years of human selection. One way to increase genetic variation is chromosome-mediated gene transfer from wild relatives by cross hybridization. In the case of wheat (Triticum aestivum), the species of genus Aegilops are a particularly attractive source of new genes and alleles. However, during the evolution of the Aegilops and Triticum genera, diversification of the D-genome lineage resulted in the formation of diploid C, M, and U genomes of Aegilops. The extent of structural genome alterations, which accompanied their evolution and speciation, and the shortage of molecular tools to detect Aegilops chromatin hamper gene transfer into wheat. To investigate the chromosome structure and help develop molecular markers with a known physical position that could improve the efficiency of the selection of desired introgressions, we developed single-gene fluorescence in situ hybridization (FISH) maps for M- and U-genome progenitors, Aegilops comosa and Aegilops umbellulata, respectively. Forty-three ortholog genes were located on 47 loci in Ae. comosa and on 52 loci in Ae. umbellulata using wheat cDNA probes. The results obtained showed that M-genome chromosomes preserved collinearity with those of wheat, excluding 2 and 6M containing an intrachromosomal rearrangement and paracentric inversion of 6ML, respectively. While Ae. umbellulata chromosomes 1, 3, and 5U maintained collinearity with wheat, structural reorganizations in 2, 4, 6, and 7U suggested a similarity with the C genome of Aegilops markgrafii. To develop molecular markers with exact physical positions on chromosomes of Aegilops, the single-gene FISH data were validated in silico using DNA sequence assemblies from flow-sorted M- and U-genome chromosomes. The sequence similarity search of cDNA sequences confirmed 44 out of the 47 single-gene loci in Ae. comosa and 40 of the 52 map positions in Ae. umbellulata. Polymorphic regions, thus, identified enabled the development of molecular markers, which were PCR validated using wheat-Aegilops disomic chromosome addition lines. The single-gene FISH-based approach allowed the development of PCR markers specific for cytogenetically mapped positions on Aegilops chromosomes, substituting as yet unavailable segregating map. The new knowledge and resources will support the efforts for the introgression of Aegilops genes into wheat and their cloning.
