A Transgenic Mutant of<i>Lactuca sativa</i>(Lettuce) with a T-DNA Tightly Linked to Loss of Downy Mildew Resistance

Okubara, Patricia A.; Arroyo-García, Rosa; Shen, Katherine A.; Mazier, Marianne; Meyers, Blake C.; Ochoa, O.; Kim, Shinje; Yang, Chin-Ying; Michelmore, Richard W.

doi:10.1094/mpmi.1997.10.8.970

Cited by 23 publications

(19 citation statements)

References 28 publications

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“…Second, these types of transposable elements transpose by a cut-and-paste mechanism, causing unstable mutations or imprecise excisions that are difficult to detect. In lettuce, for instance, imprecise excision of a Ds element produced mutants that were not tagged with the element (Okubara et al, 1997). Because they transpose by a copy-and-paste mechanism, no such problems can occur with retrotransposons, making them especially suitable for obtaining flanking plant sequences as well as for facilitating PCR-based reversegenetics screening of DNA pools.…”

Section: Discussionmentioning

confidence: 99%

Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from Tobacco

Mazier¹,

Botton²,

Flamain³

et al. 2007

Plant Physiol.

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The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3b-hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome.

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Section: Discussionmentioning

confidence: 99%

Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from Tobacco

Mazier¹,

Botton²,

Flamain³

et al. 2007

Plant Physiol.

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“…In addition, even if no transgenic variety has been commercialized yet, L. sativa has already been engineered for many traits such as herbicide resistance (McCabe et al, 1999;Nagata et al, 2000), pathogen resistance (Okubara et al, 1997) and other agronomic traits (Curtis et al, 1999;Goto et al, 2000;Pileggi et al, 2001). Although we did not investigate crop-wild lettuce hybridization using transgenic varieties, our results have clear implications for the risks assessment of their cultivation.…”

Section: Hybrid Characteristics and Relevance For Ge Lettuce Cultivationmentioning

confidence: 99%

Hybridization rates between lettuce (Lactuca sativa) and its wild relative (L. serriola) under field conditions

D’Andrea

Felber

Guadagnuolo

2008

Environ. Biosafety Res.

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. Progeny was screened morphologically for detecting natural hybrids. Prior to the experiment, specific RAPD markers were used to confirm that morphological characters were reliable for hybrid identification. Hybridization occurred up to the maximal distance tested (40 m), and hybridization rates varied between 0 to 26%, decreasing with distance. More than 80% of the wild plants produced at least one hybrid (incidence of hybridization, IH) at 0 m and 1 m. It equaled 4 to 5% at 40 m. In sympatric crop-wild populations, cross-pollination between cultivated lettuce and its wild relative has to be seen as the rule rather than the exception for short distances.

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“…None of these markers was missing in all of the deletion mutants, indicating that we had not saturated the region, the markers were duplicated, or the closest breakpoints overlapped within the Dm3 gene and no region was missing in all the mutants. Subsequently, two markers, microsatellite MSAT15-34 and the hybridization marker IPCR 800 , were identified from the sequence flanking a dm3 T-DNA insertion mutant that indicated a region was missing in all the mutants (Okubara et al, 1997). Both of these markers are duplicated within the Dm3 region; one copy of each marker was missing in all mutants.…”

Section: Localization Of Additional Molecular Markers On the Deletionmentioning

confidence: 99%

“…Consequently, 500 more RAPD primers and 648 AFLP primer pairs were screened against a panel of DNA samples from four fast neutron-induced Dm mutants. The largest deletion mutant, dm3r1608 (Okubara et al, 1997), was used to identify markers missing in the Dm3 region. Markers missing in dm3r1608 were then mapped on the complete panel of nine Dm3 deletion mutants.…”

Section: Localization Of Additional Molecular Markers On the Deletionmentioning

confidence: 99%

“…(A) Diagram of a typical RGC2 gene. Markers AM14 and AC15 800 (Anderson et al, 1996), IPCR 800 and MSAT15-34 (Okubara et al, 1997), and MSATE6, SCINT2, and NBS2B were used to map the deletion breakpoints and BAC groups ( Figure 2). (B) The 1.4-kb region in the 5Ј end and AC15 800 in the middle of the coding region were sequenced for phylogenetic analysis and to confirm the identity of the sequences contained on the BACs.…”

Section: Estimation Of the Size Of The Dm3 Regionmentioning

confidence: 99%

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The Major Resistance Gene Cluster in Lettuce Is Highly Duplicated and Spans Several Megabases

Meyers¹,

Chin²,

Shen³

et al. 1998

The Plant Cell

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102

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At least 10 Dm genes conferring resistance to the oomycete downy mildew fungus Bremia lactucae map to the major resistance cluster in lettuce. We investigated the structure of this cluster in the lettuce cultivar Diana, which contains Dm3. A deletion breakpoint map of the chromosomal region flanking Dm3 was saturated with a variety of molecular markers. Several of these markers are components of a family of resistance gene candidates ( RGC2 ) that encode a nucleotide binding site and a leucine-rich repeat region. These motifs are characteristic of plant disease resistance genes. Bacterial artificial chromosome clones were identified by using duplicated restriction fragment length polymorphism markers from the region, including the nucleotide binding site-encoding region of RGC2. Twenty-two distinct members of the RGC2 family were characterized from the bacterial artificial chromosomes; at least two additional family members exist. The RGC2 family is highly divergent; the nucleotide identity was as low as 53% between the most distantly related copies. These RGC2 genes span at least 3.5 Mb. Eighteen members were mapped on the deletion breakpoint map. A comparison between the phylogenetic and physical relationships of these sequences demonstrated that closely related copies are physically separated from one another and indicated that complex rearrangements have shaped this region. Analysis of low-copy genomic sequences detected no genes, including RGC2 , in the Dm3 region, other than sequences related to retrotransposons and transposable elements. The related but divergent family of RGC2 genes may act as a resource for the generation of new resistance phenotypes through infrequent recombination or unequal crossing over. INTRODUCTIONDisease resistance genes frequently occur in tightly linked clusters (Pryor, 1987; Crute and Pink, 1996;Michelmore and Meyers, 1998). Clusters of plant resistance genes were first established by use of classic genetic techniques; detailed molecular analyses are now beginning to unravel the complexity of these loci and the underlying mechanisms determining their structure (Parniske et al., 1997;Song et al., 1997). It is becoming increasingly apparent that such clusters may be both common and complex genomic regions in plants.Clusters of resistance genes have been identified in diverse plant species. More than 30 different resistance specificities to the single fungal pathogen responsible for flax rust disease, Melampsora lini , have been mapped to five linkage groups (Flor, 1971;Islam and Shepherd, 1991). These loci exemplify two possible genetic arrangements that may exist for clusters of resistance genes: the flax L locus contains at least 13 allelic rust resistance specificities, and the more complex M locus exists as a tandem array of at least seven genes (Islam and Shepherd, 1991). In maize, multiple Rp genes, both linked and allelic, have been observed to mediate resistance to the rust fungus Puccinia sorghi ; 16 genetically separable loci were mapped to a single cluster known as th...

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A Transgenic Mutant ofLactuca sativa(Lettuce) with a T-DNA Tightly Linked to Loss of Downy Mildew Resistance

Cited by 23 publications

References 28 publications

Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from Tobacco

Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from Tobacco

Hybridization rates between lettuce (Lactuca sativa) and its wild relative (L. serriola) under field conditions

The Major Resistance Gene Cluster in Lettuce Is Highly Duplicated and Spans Several Megabases

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