RESUMEN
In the evolution of land plants, the plant immune system has experienced expansion in immune receptor and signaling pathways. Lineage-specific expansions have been observed in diverse gene families that are potentially involved in immunity but lack causal association. Here, we show that Rps8-mediated resistance in barley to the pathogen Puccinia striiformis f. sp. tritici (wheat stripe rust) is conferred by a genetic module: Pur1 and Exo70FX12, which are together necessary and sufficient. Pur1 encodes a leucine-rich repeat receptor kinase and is the ortholog of rice Xa21, and Exo70FX12 belongs to the Poales-specific Exo70FX clade. The Exo70FX clade emerged after the divergence of the Bromeliaceae and Poaceae and comprises from 2 to 75 members in sequenced grasses. These results demonstrate the requirement of a lineage-specific Exo70FX12 in Pur1-mediated immunity and suggest that the Exo70FX clade may have evolved a specialized role in receptor kinase signaling.
RESUMEN
BACKGROUND: Recent improvements in DNA sequencing and genome scaffolding have paved the way to generate high-quality de novo assemblies of pseudomolecules representing complete chromosomes of wheat and its wild relatives. These assemblies form the basis to compare the dynamics of wheat genomes on a megabase scale. RESULTS: Here, we provide a comparative sequence analysis of the 700-megabase chromosome 2D between two bread wheat genotypes-the old landrace Chinese Spring and the elite Swiss spring wheat line 'CH Campala Lr22a'. Both chromosomes were assembled into megabase-sized scaffolds. There is a high degree of sequence conservation between the two chromosomes. Analysis of large structural variations reveals four large indels of more than 100 kb. Based on the molecular signatures at the breakpoints, unequal crossing over and double-strand break repair were identified as the molecular mechanisms that caused these indels. Three of the large indels affect copy number of NLRs, a gene family involved in plant immunity. Analysis of SNP density reveals four haploblocks of 4, 8, 9 and 48 Mb with a 35-fold increased SNP density compared to the rest of the chromosome. Gene content across the two chromosomes was highly conserved. Ninety-nine percent of the genic sequences were present in both genotypes and the fraction of unique genes ranged from 0.4 to 0.7%. CONCLUSIONS: This comparative analysis of two high-quality chromosome assemblies enabled a comprehensive assessment of large structural variations and gene content. The insight obtained from this analysis will form the basis of future wheat pan-genome studies.
Asunto(s)
Cromosomas de las Plantas/genética , Genoma de Planta , Análisis de Secuencia de ADN , Triticum/genética , Emparejamiento Base/genética , Intercambio Genético , Roturas del ADN de Doble Cadena , Variaciones en el Número de Copia de ADN/genética , Reparación del ADN/genética , Flujo Génico , Genes de Plantas , Haplotipos/genética , Familia de Multigenes , Polimorfismo de Nucleótido Simple/genética , Eliminación de Secuencia/genética , Sintenía/genéticaRESUMEN
"Map-based cloning" is a frequently used approach to isolate rust resistance genes. A critical step during map-based cloning is the transition from genetic information, i.e., a genetic map, to physical sequence information. Bacterial artificial chromosome clones are often used to establish sequence information spanning a genetic interval. However, a major limitation of BAC clones consists in their small insert size of 100-200 kb. Targeted chromosome-based cloning via long-range assembly (TACCA) is a method that can replace BAC library screening. This approach involves chromosome flow-sorting and the establishment of a long-range de novo assembly. This chapter provides an overview of TACCA as well as a detailed description of sequence analyses, molecular marker development, and candidate gene identification.
Asunto(s)
Cromosomas de las Plantas/genética , Genes de Plantas , Genómica/métodos , Enfermedades de las Plantas/genética , Triticum/genética , Secuencia de Bases , Basidiomycota/fisiología , Mapeo Cromosómico/métodos , Cromosomas Artificiales Bacterianos/genética , Clonación Molecular/métodos , ADN de Plantas/genética , Resistencia a la Enfermedad , Biblioteca de Genes , Genoma de Planta , Enfermedades de las Plantas/microbiología , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido SimpleRESUMEN
Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging. Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species. A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest. In order to accelerate gene cloning from any cropping line, we report 'targeted chromosome-based cloning via long-range assembly' (TACCA). TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage to assemble complex genomes. We applied TACCA to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months. Using this assembly we cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein.