The Distribution of Transgene Insertion Sites in Barley Determined by Physical and Genetic Mapping

The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome.

[1]  P. Quail,et al.  Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants , 1996, Transgenic Research.

[2]  J. Snape,et al.  The effect of DNA/gold particle preparation technique, and particle bombardment device, on the transformation of barley (Hordeum vulgare) , 2000, Euphytica.

[3]  Yunzhou Dong,et al.  Epigenetic interactions between Arabidopsis transgenes: characterization in light of transgene integration sites , 2003, Plant Molecular Biology.

[4]  B. Gill,et al.  Identification and high-density mapping of gene-rich regions in chromosome group 1 of wheat. , 1996, Genetics.

[5]  G. Bernardi,et al.  Distribution of genes in the genome of Arabidopsis thaliana and its implications for the genome organization of plants. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA. , 1999, BioTechniques.

[7]  P. Morris,et al.  Chromosomal localization of cotransformed transgenes in the hexaploid cultivated oat Avena sativa L. using fluorescence in situ hybridization , 2000, Heredity.

[8]  Haroldo Salvo-Garrido,et al.  An efficient method for the physical mapping of transgenes in barley using in situ hybridization , 2001 .

[9]  D. Becker,et al.  Localization of introduced genes on the chromosomes of transgenic barley, wheat and triticale by fluorescence in situ hybridization , 1997, Theoretical and Applied Genetics.

[10]  Daniel Schubert,et al.  A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome , 2003, Plant Molecular Biology.

[11]  P. Ouwerkerk,et al.  Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics , 2003, Theoretical and Applied Genetics.

[12]  D. Laurie,et al.  The distribution of RFLP markers on chromosome 2(2H) of barley in relation to the physical and genetic location of 5S rDNA , 1993, Theoretical and Applied Genetics.

[13]  M. Heun,et al.  Barley microsatellites: allele variation and mapping , 1995, Plant Molecular Biology.

[14]  J. Snape,et al.  Use of the firefly luciferase gene in a barley (Hordeum vulgare) transformation system , 2002, Plant Cell Reports.

[15]  L. Luo,et al.  Generation and flanking sequence analysis of a rice T-DNA tagged population , 2004, Theoretical and Applied Genetics.

[16]  M. Morgante,et al.  A simple sequence repeat-based linkage map of barley. , 2000, Genetics.

[17]  G. Bernardi,et al.  The distribution of genes in the genomes of Gramineae. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[18]  P. Lemaux,et al.  Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley (Hordeum vulgare L.) , 2002, Theoretical and Applied Genetics.

[19]  D. Laurie,et al.  RFLP mapping of five major genes and eight quantitative trait loci controlling flowering time in a winter x spring barley (Hordeum vulgare L.) cross. , 1995, Genome.

[20]  B. Gill,et al.  A chromosome region-specific mapping strategy reveals gene-rich telomeric ends in wheat , 1993, Chromosoma.

[21]  D. Kudrna,et al.  A molecular, isozyme and morphological map of the barley (Hordeum vulgare) genome , 1993, Theoretical and Applied Genetics.

[22]  M. Maroof,et al.  Development of simple sequence repeat DNA markers and their integration into a barley linkage map , 1996, Theoretical and Applied Genetics.

[23]  D. Lockhart,et al.  Functional Genomics , 1999, Springer Netherlands.

[24]  R. Kalla,et al.  Agrobacterium tumefaciens‐mediated barley transformation , 1997 .

[25]  D. Somers,et al.  Association of transgene integration sites with chromosome rearrangements in hexaploid oat , 2000, Theoretical and Applied Genetics.

[26]  G. Bernardi,et al.  The distribution of T‐DNA in the genomes of transgenic Arabidopsis and rice , 2000, FEBS letters.

[27]  R. Michelmore,et al.  Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[28]  J. S. Heslop-Harrison,et al.  Physical mapping of four sites of 5S rDNA sequences and one site of the α-amylase-2 gene in barley (Hordeum vulgare). , 1993, Genome.

[29]  X. Deng,et al.  Obtaining and analysis of flanking sequences from T-DNA transformants of Arabidopsis , 2003 .