Most cited article - PubMed ID 27795210
Rapid gene isolation in barley and wheat by mutant chromosome sequencing
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.
- MeSH
- Genome, Plant MeSH
- Genomics MeSH
- Cloning, Molecular * methods MeSH
- Chromosome Mapping MeSH
- Triticum * genetics MeSH
- Genes, Plant * MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Nucleotide-binding leucine-rich repeat (NLR) disease resistance genes typically confer resistance against races of a single pathogen. Here, we report that Yr87/Lr85, an NLR gene from Aegilops sharonensis and Aegilops longissima, confers resistance against both P. striiformis tritici (Pst) and Puccinia triticina (Pt) that cause stripe and leaf rust, respectively. Yr87/Lr85 confers resistance against Pst and Pt in wheat introgression as well as transgenic lines. Comparative analysis of Yr87/Lr85 and the cloned Triticeae NLR disease resistance genes shows that Yr87/Lr85 contains two distinct LRR domains and that the gene is only found in Ae. sharonensis and Ae. longissima. Allele mining and phylogenetic analysis indicate multiple events of Yr87/Lr85 gene flow between the two species and presence/absence variation explaining the majority of resistance to wheat leaf rust in both species. The confinement of Yr87/Lr85 to Ae. sharonensis and Ae. longissima and the resistance in wheat against Pst and Pt highlight the potential of these species as valuable sources of disease resistance genes for wheat improvement.
- MeSH
- Aegilops genetics microbiology MeSH
- Alleles MeSH
- Basidiomycota * pathogenicity physiology MeSH
- Phylogeny * MeSH
- Plants, Genetically Modified genetics MeSH
- Plant Leaves * microbiology genetics MeSH
- Plant Diseases * microbiology genetics immunology MeSH
- NLR Proteins * genetics MeSH
- Disease Resistance * genetics MeSH
- Triticum * genetics microbiology immunology MeSH
- Puccinia * pathogenicity MeSH
- Genes, Plant MeSH
- Plant Proteins * genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- NLR Proteins * MeSH
- Plant Proteins * MeSH
Cultivated and wild species of the genus rye (Secale) are important but underexploited gene sources for increasing the genetic diversity of bread wheat. Gene transfer is possible via bridge genetic materials derived from intergeneric hybrids. During this process, it is essential to precisely identify the rye chromatin in the wheat genetic background. In the present study, backcross generation BC2F8 from a cross between Triticum aestivum (Mv9kr1) and S. cereanum ('Kriszta,' a cultivar from the artificial hybrid of S. cereale and S. strictum) was screened using in-situ hybridization (GISH and FISH) and analyzed by DArTseq genotyping in order to select potentially agronomically useful genotypes for prebreeding purposes. Of the 329,267 high-quality short sequence reads generated, 27,822 SilicoDArT and 8,842 SNP markers specific to S. cereanum 1R-7R chromosomes were identified. Heatmaps of the marker densities along the 'Lo7' rye reference pseudomolecules revealed subtle differences between the FISH- and DArTseq-based results. This study demonstrates that the "exotic" rye chromatin of S. cereanum introgressed into wheat can be reliably identified by high-throughput DArTseq genotyping. The Mv9kr1-'Kriszta' addition and translocation lines presented here may serve as valuable prebreeding genetic materials for the development of stress-tolerant or disease-resistant wheat varieties.
- Keywords
- DArTseq markers, Secale cereanum, Triticum aestivum, chromosome rearrangements, genotyping, heatmap, introgression lines,
- Publication type
- Journal Article MeSH
Characterisation and genetic mapping of a key gene defining root morphology in bread wheat. Root morphology is central to plants for the efficient uptake up of soil water and mineral nutrients. Here we describe a conditional mutant of hexaploid wheat (Triticum aestivum L.) that when grown in soil with high Ca2+ develops a larger rhizosheath accompanied with shorter roots than the wild type. In wheat, rhizosheath size is a reliable surrogate for root hair length and this was verified in the mutant which possessed longer root hairs than the wild type when grown in high Ca2+ soil. We named the mutant Stumpy and showed it to be due to a single semi-dominant mutation. The short root phenotype at high Ca2+ was due to reduced cellular elongation which might also explain the long root hair phenotype. Analysis of root cell walls showed that the polysaccharide composition of Stumpy roots is remodelled when grown at non-permissive (high) Ca2+ concentrations. The mutation mapped to chromosome 7B and sequencing of the 7B chromosomes in both wild type and Stumpy identified a candidate gene underlying the Stumpy mutation. As part of the process to determine whether the candidate gene was causative, we identified wheat lines in a Cadenza TILLING population with large rhizosheaths but accompanied with normal root length. This finding illustrates the potential of manipulating the gene to disconnect root length from root hair length as a means of developing wheat lines with improved efficiency of nutrient and water uptake. The Stumpy mutant will be valuable for understanding the mechanisms that regulate root morphology in wheat.
- MeSH
- Plant Roots genetics MeSH
- Chromosome Mapping MeSH
- Mutation MeSH
- Triticum * metabolism MeSH
- Soil * MeSH
- Water metabolism MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Soil * MeSH
- Water MeSH
UNLABELLED: Tiller number is a key component of wheat plant architecture having a direct impact on grain yield. Because of their viability, biotic resistance, and abiotic stress tolerance, wild relative species are a valuable gene source for increasing wheat genetic diversity, including yield potential. Agropyron glael, a perennial hybrid of Thinopyrum intermedium and Th. ponticum, was created in the 1930s. Recent genome analyses identified five evolutionarily distinct subgenomes (J, Jst, Jvs, Jr, and St), making A. glael an important gene source for transferring useful agronomical traits into wheat. During a bread wheat × A. glael crossing program, a genetically stable translocation line, WT153397, was developed. Sequential in situ hybridizations (McGISH) with J-, St-, and D-genomic DNA probes and pSc119.2, Afa family, pTa71, and (GAA)7 DNA repeats, as well as molecular markers specific for the wheat 6D chromosome, revealed the presence of a 6DS.6Jvs Robertsonian translocation in the genetic line. Field trials in low-input and high-input breeding nurseries over four growing seasons demonstrated the Agropyron chromosome arm's high compensating ability for the missing 6DL, as spike morphology and fertility of WT153397 did not differ significantly from those of wheat parents, Mv9kr1 and 'Mv Karizma.' Moreover, the introgressed 6Jvs chromosome arm significantly increased the number of productive tillers, resulting in a significantly higher grain yield potential compared to the parental wheat cultivars. The translocated chromosome could be highly purified by flow cytometric sorting due to the intense fluorescent labeling of (GAA)7 clusters on the Thinopyrum chromosome arm, providing an opportunity to use chromosome genomics to identify Agropyron gene variant(s) responsible for the tillering capacity. The translocation line WT153397 is an important genetic stock for functional genetic studies of tiller formation and useful breeding material for increasing wheat yield potential. The study also discusses the use of the translocation line in wheat breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-024-01439-y.
- Keywords
- Agropyron glael, FISH, Flow cytometric sorting, GISH, Tillering, Yield potential,
- Publication type
- Journal Article MeSH
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.
- MeSH
- Cloning, Molecular MeSH
- Chromosome Mapping MeSH
- Multigene Family MeSH
- Triticum * genetics MeSH
- Genes, Plant * genetics MeSH
- Publication type
- Journal Article MeSH
Leaf rust, caused by Puccinia hordei, is one of the most widespread and damaging foliar diseases affecting barley. The barley leaf rust resistance locus Rph7 has been shown to have unusually high sequence and haplotype divergence. In this study, we isolate the Rph7 gene using a fine mapping and RNA-Seq approach that is confirmed by mutational analysis and transgenic complementation. Rph7 is a pathogen-induced, non-canonical resistance gene encoding a protein that is distinct from other known plant disease resistance proteins in the Triticeae. Structural analysis using an AlphaFold2 protein model suggests that Rph7 encodes a putative NAC transcription factor with a zinc-finger BED domain with structural similarity to the N-terminal DNA-binding domain of the NAC transcription factor (ANAC019) from Arabidopsis. A global gene expression analysis suggests Rph7 mediates the activation and strength of the basal defence response. The isolation of Rph7 highlights the diversification of resistance mechanisms available for engineering disease control in crops.
- MeSH
- Arabidopsis * genetics MeSH
- Basidiomycota * MeSH
- Eczema * MeSH
- Hordeum * genetics MeSH
- Poaceae MeSH
- Plant Diseases genetics MeSH
- Gene Expression Regulation MeSH
- Plant Proteins genetics MeSH
- Transcription Factors genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Plant Proteins MeSH
- Transcription Factors 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
The introgression of chromosome segments from wild relatives is an established strategy to enrich crop germplasm with disease-resistance genes1. Here we use mutagenesis and transcriptome sequencing to clone the leaf rust resistance gene Lr9, which was introduced into bread wheat from the wild grass species Aegilops umbellulata2. We established that Lr9 encodes an unusual tandem kinase fusion protein. Long-read sequencing of a wheat Lr9 introgression line and the putative Ae. umbellulata Lr9 donor enabled us to assemble the ~28.4-Mb Lr9 translocation and to identify the translocation breakpoint. We likewise cloned Lr58, which was reportedly introgressed from Aegilops triuncialis3, but has an identical coding sequence compared to Lr9. Cytogenetic and haplotype analyses corroborate that the two genes originate from the same translocation event. Our work sheds light on the emerging role of kinase fusion proteins in wheat disease resistance, expanding the repertoire of disease-resistance genes for breeding.
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.
