Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA

Abstract

Integration of transgenic DNA into the plant genome was investigated in 13 transgenic oat (Avena sativa L.) lines produced using microprojectile bombardment with one or two cotransformed plasmids. In all transformation events, the transgenic DNA integrated into the plant genome consisted of intact transgene copies that were accompanied by multiple, rearranged, and/or truncated transgene fragments. All fragments of transgenic DNA cosegregated, indicating that they were integrated at single gene loci. Analysis of the structure of the transgenic loci indicated that the transgenic DNA was interspersed by the host genomic DNA. The number of insertions of transgenic DNA within the transgene loci varied from 2 to 12 among the 13 lines. Restriction endonucleases that do not cleave the introduced plasmids produced restriction fragments ranging from 3.6 to about 60 kb in length hybridizing to a probe comprising the introduced plasmids. Although the size of the interspersing host DNA within the transgene locus is unknown, the sizes of the transgene-hybridizing restriction fragments indicated that the entire transgene locus must be at least from 35-280 kb. The observation that all transgenic lines analyzed exhibited genomic interspersion of multiple clustered transgenes suggests a predominating integration mechanism. We propose that transgene integration at multiple clustered DNA replication forks could account for the observed interspersion of transgenic DNA with host genomic DNA within transgenic loci.

Figure 1

Restriction maps of plasmids used for oat transformation. Positions of probes used in Southern blot analyses are indicated by black bars underneath the appropriate sequences. Not drawn to scale. 35S pro, Cauliflower Mosaic Virus (CaMV) 35S promoter; Adh1 pro, promoter of the maize Adh1 gene; Adh1 i, first intron of the maize Adh1 gene; cp, barley yellow dwarf virus coat protein gene; NOS, nos gene termination sequence. pUC8 and pGEM-3z are plasmid backbones. Restriction sites: A, AccI; B, BamHI; E, EcoRI; H, HindIII; K, KpnI; X, XbaI.

Figure 2

Transgene integration patterns in transgenic oat lines. DNA was restricted with BamHI and EcoRI (A) or HindIII (B) and probed with gusA. BamHI and EcoRI release from pBARGUS a 3.6-kb fragment containing the gusA coding region (see Fig. 1). HindIII has a single restriction site on pBARGUS.

Figure 3

Cosegregation of transgene restriction fragments in a sample of 13 T₂ progeny plants of line 300. DNA was restricted with BamHI and EcoRI and probed with gusA. Plants in lanes 1–9 exhibit all transgene-hybridizing restriction fragments detected in this line. Plants in lanes 10–13 show no transgene-hybridizing restriction fragments.

Figure 4

Multiple transgene integration sites in single-locus transgenic oat lines. DNA was separated by PFGE, Southern blotted, and probed with a mixed probe containing all plasmids used for plant transformation (pBARGUS, pH24, pMAV, pPAV, and pRPV). The plasmid pBARGUS is 9.2 kb (∗).