title: Transgene integration and organization in cotton (Gossypium hirsutum L.) genome.
abstract: While genetically modified upland cotton (Gossypium hirsutum L.) varieties are ranked among the most successful genetically modified organisms (GMO), there is little knowledge on transgene integration in the cotton genome, partly because of the difficulty in obtaining large numbers of transgenic plants. In this study, we analyzed 139 independently derived T0 transgenic cotton plants transformed by Agrobacterium tumefaciens strain AGL1 carrying a binary plasmid pPZP-GFP. It was found by PCR that as many as 31% of the plants had integration of vector backbone sequences. Of the 110 plants with good genomic Southern blot results, 37% had integration of a single T-DNA, 24% had two T-DNA copies and 39% had three or more copies. Multiple copies of the T-DNA existed either as repeats in complex loci or unlinked loci. Our further analysis of two T1 populations showed that segregants with a single T-DNA and no vector sequence could be obtained from T0 plants having multiple T-DNA copies and vector sequence. Out of the 57 T-DNA/T-DNA junctions cloned from complex loci, 27 had canonical T-DNA tandem repeats, the rest (30) had deletions to T-DNAs or had inclusion of vector sequences. Overlapping micro-homology was present for most of the T-DNA/T-DNA junctions (38/57). Right border (RB) ends of the T-DNA were precise while most left border (LB) ends (64%) had truncations to internal border sequences. Sequencing of collinear vector integration outside LB in 33 plants gave evidence that collinear vector sequence was determined in agrobacterium culture. Among the 130 plants with characterized flanking sequences, 12% had the transgene integrated into coding sequences, 12% into repetitive sequences, 7% into rDNAs. Interestingly, 7% had the transgene integrated into chloroplast derived sequences. Nucleotide sequence comparison of target sites in cotton genome before and after T-DNA integration revealed overlapping microhomology between target sites and the T-DNA (8/8), deletions to cotton genome in most cases studied (7/8) and some also had filler sequences (3/8). This information on T-DNA integration in cotton will facilitate functional genomic studies and further crop improvement.
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604, Singapore.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Lin
authors.initials: L
authors.lastname: Cai
authors.forename: Jiaqin
authors.initials: J
authors.lastname: Cheng
authors.forename: Huizhu
authors.initials: H
authors.lastname: Mao
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Fan
authors.forename: Zhaohong
authors.initials: Z
authors.lastname: Meng
authors.forename: Ka Man
authors.initials: KM
authors.lastname: Chan
authors.forename: Huijun
authors.initials: H
authors.lastname: Zhang
authors.forename: Jianfei
authors.initials: J
authors.lastname: Qi
authors.forename: Lianghui
authors.initials: L
authors.lastname: Ji
authors.forename: Yan
authors.initials: Y
authors.lastname: Hong
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D005822:Genetic Vectors; D018745:Genome, Plant; D003368:Gossypium / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic; D019076:Transgenes / Q000502:physiology*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D018744:DNA, Plant; D012333:RNA, Messenger; C036483:T-DNA