title: Excision of plastid marker genes using directly repeated DNA sequences.
abstract: Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required, and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes. Release of selection allows the accumulation of marker-free plastid genomes generated by marker excision, which is spontaneous, random, and a unidirectional process. Positive selection is provided by linking marker excision to restoration of the coding region of an herbicide resistance gene from two overlapping but incomplete coding regions. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat-mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastid genomes during growth, development and flowering of T0 plants allows the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, UK.
authors.forename: Elisabeth A
authors.initials: EA
authors.lastname: Mudd
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Madesis
authors.forename: Elena Martin
authors.initials: EM
authors.lastname: Avila
authors.forename: Anil
authors.initials: A
authors.lastname: Day
mesh_terms: D002696:Chlamydomonas / Q000235:genetics; D002736:Chloroplasts / Q000235:genetics*; D004254:DNA Nucleotidyltransferases; D018742:DNA, Chloroplast / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics*; D005819:Genetic Markers; D006540:Herbicides / Q000494:pharmacology; D018545:Lactuca / Q000235:genetics; D019684:Magnoliopsida / Q000235:genetics* / Q000502:physiology; D030821:Plants, Genetically Modified / Q000235:genetics; D011938:Rec A Recombinases / Q000235:genetics; D011995:Recombination, Genetic; D012639:Seeds / Q000235:genetics / Q000502:physiology; D056916:Segmental Duplications, Genomic / Q000235:genetics*; D013025:Glycine max / Q000235:genetics; D014026:Nicotiana / Q000235:genetics
chemical_list: D018742:DNA, Chloroplast; D005819:Genetic Markers; D006540:Herbicides; D004254:DNA Nucleotidyltransferases; D011938:Rec A Recombinases; C077570:Site-specific recombinase