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 ...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.展开更多
基金supported by the Israel Science Foundation,grant numbers 2289/16,1366/18,and 2342/18,and the United States–Israel Binational Science Foundation (2019654)C.J.P.was supported by the Genome Canada–funded project 4D Wheat.I.M.and J.D.were supported by the ERDF project Plants as a Tool for Sustainable Global Development (no.CZ.02.1.01/0.0/0.0/16_019/0000827)+1 种基金supported by the United States National Science Foundation (1937855)the United States Department of Agriculture (2020-67013-32577)。
文摘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.
