Linked and Unlinked Transposition of a Genetically Marked Dissociation Element in Transgenic Tomato

We have introduced a genetically marked Dissociation transposable element (Ds(neo)) into tomato. In the presence of Ac transposase, Ds(neo) excised from an integrated T-DNA and reinserted at numerous new sites in the tomato genome. The marker genes of Ds(neo) (NPTII) and the T-DNA (HPT) facilitated identification of plants bearing transposon excisions and insertions. To explore the feasibility of gene tagging strategies in tomato using Ds(neo), we examined the genomic distribution of Ds(neo) receptor sites, relative to the location of the donor T-DNA locus. Restriction fragment length polymorphism mapping of transposed Ds(neo) elements was conducted in two tomato families, derived from independent primary transformants each bearing Ds(neo) within a T-DNA at a unique position in the genome. Transposition of Ds(neo) generated clusters of insertions that were positioned on several different tomato chromosomes. Ds(neo) insertions were often located on the same chromosome as the T-DNA donor site. However, no insertion showed tight linkage to the T-DNA. We consider the frequency and distance of Ds(neo) transposition observed in tomato to be well suited for transposon mutagenesis. Our study made use of a novel, stable allele of Ac (Ac3) that we discovered in transgenic tomato. We determined that the Ac3 element bears a deletion of the outermost 5 base pairs of the 5'-terminal inverted repeat. Though incapable of transposition itself, Ac3 retained the ability to mobilize Ds(neo). We conclude that a dual element system, composed of the stable Ac3 trans-activator in combination with Ds(neo), is an effective tool for transposon tagging experiments in tomato.     

Distribution of Unlinked Transpositions of a Ds Element from a T-DNA Locus on Tomato Chromosome 4

In maize, receptor sites for unlinked transpositions of Activator (Ac) elements are not distributed randomly. To test whether the same is true in tomato, the receptor sites for a Dissociation (Ds) element derived from Ac, were mapped for 26 transpositions unlinked to a donor T-DNA locus on chromosome 4. Four independent transposed Dss mapped to sites on chromosome 4 genetically unlinked to the donor T-DNA, consistent with a preference for transposition to unlinked sites on the same chromosome as opposed to sites on other chromosomes. There was little preference among the nondonor chromosomes, except perhaps for chromosome 2, which carried seven transposed Dss, but these could not be proven to be independent. However, these data, when combined with those from other studies in tomato examining the distribution of transposed Acs or Dss among nondonor chromosomes, suggest there may be absolute preferences for transposition irrespective of the chromosomal location of the donor site. If true, transposition to nondonor chromosomes in tomato would differ from that in maize, where the preference seems to be determined by the spatial arrangement of chromosomes in the interphase nucleus. The tomato lines carrying Ds elements at known locations are available for targeted transposon tagging experiments.     

Germinal Excision and Reinsertion Frequencies of the Mobile Element Ds Transposed from Two Unlinked T-DNA Loci in Tomato

Acceptor sites of unlinked transposed Ds element from two T-DNA loci in tomato were mapped. Experimental data obtained from TC₁ progeny testing were employed for estimation of germinal excision frequency (GEF) of Ds element and frequency of its reinsertion (FR). The donor T-DNAs 1481J and 1601D, containing a 35S:NPT transformation marker, a 35S:BAR or nos:BAR excision marker conferring phosphinothricine resistance and a Ds element in the 5 untranslated leader of the nos (or 35S): BAR gene, were located on chromosome 7 and 8, respectively. Ds transposition was induced by 105121 T-DNA carrying stabilized Ac (sAc) which provides a source of transposase and 2:GUS marker conferring -glucuronidase activity. Tomato plants harbouring the Ds in 1481J or 1601D locus and sAc were crossed and F₁D, were crossed individually as seed parents to wild-type plants to generate TC₁ progenies. TC₁ seed was germinated on phosphinothricine (Basta)-containing medium, and individual seedlings carrying a transposed Ds and lacking sAc were identified by PCR (to detect the Ds) on phosphinothricine resistant individuals that lacked -glucuronidase activity. From segregation ratio in TC₁ the germinal excision and reinsertion frequencies of the Ds element were estimated for individual F₁ plants. A total of 14560 TC₁ seedlings of 1481J and 16195 TC₁ seedlings of 1601D was analyzed. We observed high variation between individual plants as regards both GEF and FR despite of donor locus (1481J or 1601D), however, the average germinal excision frequencies as well as average frequencies of reinsertion were very similar for both donor loci: GEF_1481J = 24 %, GEF_1501D = 25 %, FR_1481J = 42 %, FR_1601D = 46 %.     

Germinal Transpositions of the Maize Element Dissociation from T-DNA Loci in Tomato

We have analyzed the pattern of germinal transpositions of artificial Dissociation (Ds) transposons in tomato. T-DNA constructs carrying Ds were transformed into tomato, and the elements were trans-activated by crossing to lines transformed with a stabilized Activator (sAc) that expressed the transposase gene. The sAc T-DNA carried a GUS gene to monitor its segregation. The Ds elements were inserted in a marker gene so that excision from the T-DNA could be monitored. The Ds elements also carried a genetic marker that was intended to be used for reinsertion selection of the elements after excision. Unfortunately, this gene was irreversibly inactivated on crossing to sAc. Germinal excision frequencies of Ds averaged 15-40%, but there was large variation between and within plants. Southern hybridization analysis of stable transposed Ds elements indicated that although unique transpositions predominate, early transposition events can lead to large clonal sectors in the germline of developing plants and to sibling offspring carrying the same transposition event. Multiple germinal transpositions from three different loci were examined for uniqueness, and 15 different transpositions were identified from each of three T-DNA loci that carried a single independent Ds. These were mapped relative to the donor T-DNA loci, and for each locus 70-80% of the transposed elements were closely linked to the donor site.     

Transposition of Ac from the P Locus of Maize into Unreplicated Chromosomal Sites

We have analyzed donor and target sites of the mobile element Activator (Ac) that are altered as a result of somatic transposition from the P locus in maize. Previous genetic analysis has indicated that the two mitotic daughter lineages which result from Ac transposition from P differ in their Ac constitution at the P locus. Both lineages, however, usually contain transposed Ac elements which map to the same genetic position. Using methylation-sensitive restriction enzymes and genomic blot analysis, we identified Ac elements at both the donor P locus and Ac target sites and used this assay to clone the P locus and to identify transposed Ac elements. Daughter lineages were shown to be mitotic descendants from a single transposition event. When both lineages contained Ac genetic activity, they both contained a transposed Ac element on identical genomic fragments independent of the genetic position of the target site. This indicates that in the majority of cases, Ac transposition takes place after replication of the donor locus but before completion of replication at the target site.     

OM Mutations in DROSOPHILA ANANASSAE Are Linked to Insertions of a Transposable Element

It has been hypothesized that Om mutability in Drosophila ananassae (involving spontaneous mutation at 20 loci, resulting in semidominant, nonpleiotropic eye morphology defects) was due to insertion of a transposable element, tom. One particularly unstable X-linked Om allele produced several derivatives, one of which has a more extreme Om phenotype and was accompanied by a singed bristle mutant, sn ^9g. DNA probes from the sn locus of D. melanogaster were used to clone the homologous region of D. ananassae. Analysis of sn^9g DNA detected a 6.5-kb insert. Genomic Southern blotting and in situ hybridization techniques showed that this insert is repetitive and dispersed. The existence of the tom element is supported by genetic mapping that established homology between the 6.5-kb sn^9g insert and Om mutants at the four X-linked loci tested.     

Distribution of Unlinked Receptor Sites for Transposed Ac Elements from the Bz-M2(ac) Allele in Maize

We have shown before that the Ac element from the maize bz-m2(Ac) allele, located in the short arm of chromosome 9 (9S), transposes preferentially to sites that are linked to the bz donor locus. Yet, about half of the Ac transpositions recovered from bz-m2(Ac) are in receptor sites not linked to the donor locus. In this study, we have analyzed the distribution of those unlinked receptor sites. Thirty-seven transposed Ac (trAc) elements that recombined independently of the bz locus were mapped using a set of wx reciprocal translocations. We found that the distribution of unlinked receptor sites for trAs was not random. Ten trAcs mapped to 9L, i.e., Ac had transposed to sites physically, if not genetically, linked to the donor site. Among chromosomes other than 9, the Ac element of bz-m2(Ac) appeared to have transposed preferentially to certain chromosomes, such as 5 and 7, but infrequently to others, such as 1, the longest chromosome in the maize genome. The seven trAc elements in chromosome 5 were mapped relative to markers in 5S and 5L and localized to both arms of 5. We also investigated the transposition of Ac to the homolog of the donor chromosome. We found that Ac rarely transposes from bz-m2(Ac) to the homologous chromosome 9. The clustering of Ac receptor sites around the donor locus has been taken to mean that a physical association between the donor site and nearby receptor sites occurs during transposition. The preferential occurrence of 9L among chromosomes harboring unlinked receptor sites would be expected according to this model, since sites in 9L would tend to be physically closer to 9S than sites in other chromosomes. The nonrandom pattern seen among the remaining chromosomes could reflect an underlying nuclear architecture, i.e., an ordering of the chromosomes in the interphase nucleus, as suggested from previous cytological observations.     

Transposition Pattern of the Maize Element Ac from the Bz-M2(ac) Allele

The pattern of transposition of Ac from the mutable allele bz-m2(Ac) has been investigated. Stable (bz-s) and finely spotted (bz-m(F)) exceptions were selected from coarsely spotted bz-m2(Ac) ears. The presence or absence of a transposed Ac (trAc) in the genome was determined and, when present, the location of the trAc was mapped relative to the flanking markers sh and wx. The salient general features of Ac transposition to sites linked to bz are that the receptor sites tend to be clustered on either side of the bz donor site and that transposition is bidirectional and nonpolar. Thus, the symmetrical clustering in the distribution of receptor sites adjacent to bz differs from the asymmetrical clustering reported in 1984 for the P locus by I. M. GREENBLATT. Though Ac tends to transpose preferentially to closely linked sites, an appreciable fraction of Ac transpositions from bz-m2(Ac) is to unlinked sites: 41% among bz-s derivatives and 59% among bz-m(F) derivatives. Many transposition events among the bz-m(F) selections result in kernels carrying a genetically noncorresponding embryo. These can be interpreted as twin sectors arising at one of the megagametophytic mitoses. The bz locus data fit the earlier (1962) model of I. M. GREENBLATT and R. A. BRINK in which transposition takes place from a replicated donor site to either an unreplicated or replicated receptor site.     

Genetically Based Location from Triploid Populations and Gene Ontology of a 3.3-Mb Genome Region Linked to Alternaria Brown Spot Resistance in Citrus Reveal Clusters of Resistance Genes

Genetic analysis of phenotypical traits and marker-trait association in polyploid species is generally considered as a challenge. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from second division restitution (SDR) to map a genome region linked to Alternaria brown spot (ABS) resistance in triploid citrus progeny. ABS in citrus is a serious disease caused by the tangerine pathotype of the fungus Alternaria alternata. This pathogen produces ACT-toxin, which induces necrotic lesions on fruit and young leaves, defoliation and fruit drop in susceptible genotypes. It is a strong concern for triploid breeding programs aiming to produce seedless mandarin cultivars. The monolocus dominant inheritance of susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. Bulk segregant analysis coupled with genome scan using a large set of genetically mapped SNP markers and targeted genetic mapping by half tetrad analysis, using SSR and SNP markers, allowed locating a 3.3 Mb genomic region linked to ABS resistance near the centromere of chromosome III. Clusters of resistance genes were identified by gene ontology analysis of this genomic region. Some of these genes are good candidates to control the dominant susceptibility to the ACT-toxin. SSR and SNP markers were developed for efficient early marker-assisted selection of ABS resistant hybrids.     

Transposition patterns of unlinked transposed Ds elements from two T-DNA loci on tomato chromosomes 7 and 8

We have previously reported that unlinked transposed Ds elements originating from chromosome 4 of tomato preferentially inserted in chromosome 2. This observation, together with data from other studies, suggested that there may be absolute preferences for transposition, irrespective of the chromosomal location of the donor site. The aim of the present work was to verify whether the distribution of transposed Ds elements on chromosome 2 was non-random and thus whether, unlike the case in maize, unlinked transpositions in tomato are not distributed randomly. To do this, unlinked acceptor sites of Ds elements originating from two donor T-DNA loci lying on chromosomes 7 and 8 were mapped. Receptor sites for tr Ds elements transposed from the 1601D locus on chromosome 8 exhibited a non-random distribution (P<0.01). Eleven out of 46 independent transpositions mapped to chromosome 2 and, as this was statistically significant (P<0.01), proves that receptor sites for this element are not randomly distribution on the chromosomes. In addition, deviation of the observed number from the expected number of tr Dss was close to being significant for chromosome 4 (P=0.05-0.1). In contrast, the distribution of unlinked receptor sites for tr Dss derived from the 1481J locus on chromosome 7 was random. Chi(2)tests were performed for each chromosome, and for chromosome 4 the difference between the observed and the expected number of tr Dss was very high but statistically non-significant (P=0.05-0.1). For chromosome 2 the difference was statistically negligible. Therefore, we conclude that chromosome 2 does not serve as a preferential receptor for the transposition of Ds elements independently of the location of the donor site.     

Red-Mediated Transposition and Final Release of the Mini-F Vector of a Cloned Infectious Herpesvirus Genome

Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via synchronous Red-recombination in E. coli in an en passant mutagenesis-based procedure. Using this technique, the mini-F elements of a cloned infectious varicella zoster virus (VZV) genome were specifically transposed into novel positions distributed over the viral DNA to generate six different BAC variants. In comparison to the other constructs, a BAC variant with mini-F sequences directly inserted into the junction of the genomic termini resulted in highly efficient viral DNA replication-mediated spontaneous vector excision upon virus reconstitution in transfected VZV-permissive eukaryotic cells. Moreover, the derived vector-free recombinant progeny exhibited virtually indistinguishable genome properties and replication kinetics to the wild-type virus. Thus, a sequence-independent, efficient, and easy-to-apply mini-F vector transposition procedure eliminates the last hurdle to perform virtually any kind of imaginable targeted BAC modifications in E. coli. The herpesviral terminal genomic junction was identified as an optimal mini-F vector integration site for the construction of an infectious BAC, which allows the rapid generation of mutant virus without any unwanted secondary genome alterations. The novel mini-F transposition technique can be a valuable tool to optimize, repair or restructure other established BACs as well and may facilitate the development of gene therapy or vaccine vectors.     

Transposition of the maize transposable element Ac in barley (Hordeum vulgare L.)

Transposition of the maize autonomous element Ac (Activator) was investigated in barley (Hordeum vulgare L.) with the aim of developing a transposon tagging system for the latter. The Ac element was introduced into meristematic tissue of barley by microprojectile bombardment. Transposon activity was then examined in the resulting transgenic plants. Multiple excision events were detected in leaf tissue of all plant lines. The mobile elements generated empty donor sites with small DNA sequence alterations, similar to those found in maize. Reintegration of Ac at independent genomic loci in somatic tissue was demonstrated by isolation of new element-flanking regions by AIMS-PCR (amplification of insertion-mutagenized sites). In addition, transmission of transposed Ac elements to progeny plants was confirmed. The results indicate that the introduced Ac element is able to transpose in barley. This is a first step towards the establishment of a transposon tagging system in this economically important crop.     

P Element Transposition in Drosophila Melanogaster: An Analysis of Sister-Chromatid Pairs and the Formation of Intragenic Secondary Insertions during Meiosis

The gap-repair model proposes that P elements move via a conservative, ``cut-and-paste' mechanism followed by double-strand gap repair, using either the sister chromatid or homolog as the repair template. We have tested this model by examining meiotic purturbations of an X-linked ry(+) transposon during the meiotic cycle of males, employing the mei-S332 mutation, which induces high frequency equational nondisjunction. This system permits the capture of both sister-X chromatids in a single patroclinous daughter. In the presence of P-transposase, transpositions within the immediate proximity of the original site are quite frequent. These are readily detectible among the patroclinous daughters, thereby allowing the combined analysis of the transposed element, the donor site and the putative sister-strand template. Molecular analysis of 22 meiotic transposition events provide results that support the gap-repair model of P element transposition. Prior to this investigation, it was not known whether transposition events were exclusively or predominantly premeiotic. The results of our genetic analysis revealed that P elements mobilize at relatively high frequencies during meiosis. We estimated that approximately 4% of the dysgenic male gametes have transposon perturbations of meiotic origin; the proportion of gametes containing lesions of premeiotic origin was estimated at 32%.     

Ac／Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(Ac TPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ae×Ds的杂交组合354个。检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性。结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4％。检测到T-DNA可插入到编码蛋白的基因中。在Ac×Ds的F2代中,Ds因子的转座频率为22.7％。对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制——转座和小完全切离等现象。获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。     

Identification and Analysis of Resistance‐like Genes in the Tomato Genome

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in the world. However, the tomato production is severely affected by many diseases. The use of host resistance is believed to be the most effective approach to control the pathogens. In this study, a total of 1003 resistance‐like genes were identified from the tomato genome using individual full‐length search and conserved domain verification approach. Of the predicted resistance genes, serine/threonine protein kinase was the largest class with 384 genes followed by 212 genes encoding receptor‐like kinase, 107 genes encoding receptor‐like proteins, 68 genes encoding coiled‐coil–nucleotide‐binding site (NBS)–leucine‐rich repeat (LRR) and 19 genes encoding Toll interleukin‐1 receptor domain‐NBS‐LRR. Physical map positions established for all predicted genes using the tomato WGS chromosomes SL2.40 information indicated that most resistance‐like genes clustered on certain chromosomal regions. Comparisons of the sequences from the same resistance‐like genes in S. pimpinellifolium and S. lycopersicum showed that 93.5% genes contained single nucleotide polymorphisms and 19.7% genes contained insertion/deletion. The data obtained here will facilitate isolation and characterization of new resistance genes as well as marker‐assisted selection for disease resistance breeding in tomato.     

Ac/Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(AcTPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ac×Ds的杂交组合354个.检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性.结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4%.检测到T-DNA可插入到编码蛋白的基因中.在Ac×Ds的F2代中,Ds因子的转座频率为22.7%.对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制--转座和不完全切离等现象.获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录.探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略.