Prospects of applying a combination of DNA transposition and site-specific recombination in plants: a strategy for gene identification and cloning

The concept of gene identification and cloning using insertional mutagenesis is well established. Many genes have been isolated using T-DNA transformation or transposable elements. Maize transposable elements have been introduced into heterologous plant species for tagging experiments. The behaviour of these elements in heterologous hosts shows many similarities with transposon behaviour in Zea mays. Site-specific recombination systems from lower organisms have also been shown to function efficiently in plant cells. Combining transposon and site-specific recombination systems in plants would create the possibility to induce chromosomal deletions. This transposition-deletion system could allow the screening of large segments of the genome for interesting genes and may also permit the cloning of the DNA corresponding to the deleted material by the same site-specific recombination reaction in vitro. This methodology may provide a unique means to construct libraries of large DNA clones derived from defined parts of the genome, the phenotypic contribution of which is displayed by the mutant carrying the deletion.     

Transposon mutagenesis of nuclear photosynthetic genes in Zea mays

The discovery of a new maize (Zea mays L.) transposon system, Mutator, and the cloning of the 1.4 kilobase transposon, Mul, have made feasible the isolation of nuclear photosynthetic genes which are recognized only by their mutant phenotype. Mutant maize plants which express a high chlorophyll fluorescent (hcf) phenotype due to a defect in the electron transport or photophosphorylation apparatus have been isolated following mutagenesis with an active Mutator stock. The affected genes and their products in these mutants are inaccessible to classical methods of analysis. However, mutagenesis with the Mutator transposon makes it possible to isolate these genes.Although the PSII-deficient mutant hcf3 has been thoroughly studied by classical photo-biological methods, the nature of the lesion which results in the observed phenotype has not been established. A Mutator-induced allele of hcf3 has been isolated. A fragment of genomic DNA has been identified which is homologous to Mul and co-segregates with the mutant phenotype. This fragment is expected to contain a portion of the hcf3 locus which will be used to clone the normal gene. Direct study of the gene can provide insight into the nature and function of its polypeptide product.This approach can be used to study any photosynthetic gene which has been interrupted by a transposon. The isolation of more than 100 different chemically-induced hcf mutants, most of which can not be fully characterized using classical means, indicates the wealth of information which can be obtained using a transposon tagging technique.     

插入诱变在拟南芥基因克隆中的应用

随着各种基因克隆方法的建立 ,克隆的拟南芥基因越来越多 ,其中转座子标签和T DNA插入诱变克隆的拟南芥基因的数目最多 ,插入诱变已成为克隆和鉴定很多重要植物基因的方法。     

Plant tagnology

Transposable elements have been used as an effective mutagen and as a tool to clone tagged genes. Insertion of a transposable element into a gene can lead to loss- or gain-of-function, changes in expression pattern, or can have no effect on gene function at all, depending on whether the insertion took place in coding or non-coding regions of the gene. Cloning transposable elements from different plant species has made them available as a tool for the isolation of tagged genes using homologous or heterologous tagging strategies. Based on these transposons, new elements have been engineered bearing reporter genes that can be used for expression analysis of the tagged gene, or resistance genes that can be used to select for knockout insertions. While many genes have been cloned using transposon tagging following traditional forward genetics strategies, gene cloning has ceased to be the rate-limiting step in the process of determining sequence–function relations in several important plant model species. Large-scale insertion mutagenesis and identification of insertion sites following a reverse genetics strategy appears to be the best method for unravelling the biological role of the thousands of genes with unknown functions identified by genome or expressed sequence tag (EST) sequencing projects. Here we review the progress in forward tagging technologies and discuss reverse genetics strategies and their applications in different model species.     

Development of Ac and Ds transposon tagging lines for gene isolation in diploid potato

For the development of an efficient transposon tagging strategy it is important to generate populations of plants containing unique independent transposon insertions that will mutate genes of interest. To develop such a transposon system in diploid potato the behavior of the autonomous maize transposable element Ac and the mobile Ds element was studied. A GBSS (Waxy) excision assay developed for Ac was used to monitor excision in somatic starch-forming tissue like tubers and pollen. Excision of Ac results in production of amylose starch that stains blue with iodine. The frequency and patterns of blue staining starch granules on tuber slices enabled the identification of transformants with different Ac activity. After excision the GBSS complementation was usually not complete, probably due to the segment of DNA flanking Ac that is left behind in the GBSS gene. Molecular and phenotypic analyses of 40 primary transformants classified into 4 phenotypic classes revealed reproducible patterns. A very high percentage (32.5%) of the primary transformants clearly showed early excision in the first transformed cell as displayed both by the analysis of the GBSS excision marker gene as well as DNA blot analyses. Genotypes useful for tagging strategies were used for crosses and the frequency of independent germinal transpositions was assessed. In crosses to Ds genotypes, excision of Ds was revealed that correlated to the activity of the Ac genotype. A line displaying Ac amplification to multiple copies conferred a high frequency of independent Ds transpositions. The genotypes described here are useful in somatic insertion mutagenesis aimed at the isolation of tagged mutations in diploid potato.     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

Transposable elements in the fungal plant pathogenFusarium oxysporum

The genome of the fungal plant pathogenFusarium oxysporum contains at least six different families of transposable elements. Representatives of both DNA transposons and retrotransposons have been identified, either by cloning of dispersed repetitive sequences (Foret andpalm) or by trapping in the nitrate reductase gene (Fot1, Fot2 Impala andHop).Fot1 andImpala elements are related to theTc1 andmariner class of transposons. These transposable elements can affect gene structure and function in several ways: inactivation of the target gene through insertion, diversification of the nucleotide sequence by imprecise excisions, and probably chromosomal rearrangements as suggested by the extensive karyotype variation observed among field isolates. Comparisons of the distribution of these elements inFusarium populations have improved our understanding of population structure and epidemiology and provided support for horizontal genetic transfer. Also they could be developed as genetic tools for tagging genes, a cloning strategy that is particularly promising in imperfect fungi.     

小麦HMW谷蛋白亚基基因克隆研究进展

高分子量麦谷蛋白亚基 (HMW GS)作为小麦胚乳中的重要贮藏蛋白 ,其组成及含量对小麦面粉的烘烤品质具有重要的决定作用。因此 ,改变小麦中HMW 谷蛋白的组成及含量是小麦品质改良的主要内容。而定向克隆小麦HMW GS基因则为利用基因工程方法改良小麦品质提供新的基因资源 ,从而为优质小麦的发展起到积极的推动作用。综述了近 2 0年来国内外小麦HMW GS基因克隆的研究进展 ,并讨论了近年来发展起来的一些新的基因克隆方法及其在小麦HMW GS基因克隆上的应用前景。     

Tagging genes for blast resistance in rice via linkage to RFLP markers

Summary Both Pi-2(t) and Pi-4(t) genes of rice confer complete resistance to the blast fungal pathogen Pyricularia oryzae Cav. As economically important plant genes, they have been recently characterized phenotypically, yet nothing is known about their classical linkage associations and gene products. We report here the isolation of DNA markers closely linked to these blast resistance genes in rice. The DNA markers were identified by testing 142 mapped rice genomic clones as hybridization probes against Southern blots, consisting of DNA from pairs of nearly isogenic lines (NILs) with or without the target genes. Chromosomal segments introgressed from donor genomes were distinguished by restriction fragment length polymorphisms (RFLPs) between the NILs. Linkage associations of the clones with Pi-2(t) and Pi4(t) were verified using F₃ segregating populations of known blast reaction. Cosegregation of the resistant genotype and donor-derived allele indicated the presence of linkage between the DNA marker and a blast resistance gene. RFLP analysis showed that Pi-2(t) is closely linked to a single-copy DNA clone RG64 on chromosome 6, with a distance of 2.8+1.4(SE) cMorgans. Another blast resistance gene, Pi-4(t), is 15.3+4.2(SE) cMorgans away from a DNA clone RG869 on chromosome 12. These chromosomal regions can now be examined with additional markers to define the precise locations of Pi-2(t) and Pi-4(t). Tightly linked DNA markers may facilitate early selection for blast resistance genes in breeding programs. These markers may also be useful to map new genes for resistance to blast isolates. They may ultimately lead to the cloning of those genes via chromosome walking. The gene tagging approach demonstrated in this paper may apply to other genes of interest for both monogenic and polygenic traits.     

Thetms2 gene as a negative selection marker in rice

A conditional negative selection marker is essential for high throughput insertional mutagenesis with any two-element transposon tagging system. Thetms2 gene encodes indoleacetic acid hydrolase (IAAH) which converts naphthaleneacetamide (NAM) to the potent auxin naphthaleneacetic acid, a phytotoxic derivative. This gene, under the control of the manopine synthase gene 2 promoter fromAgrobacterium tumefaciens and exogenously applied NAM, have been used effectively as a negative selector inAc/Ds insertional mutagenesis ofArabidopsis thaliana (Sundaresan et al., 1995). In this study we show thattms2 can also be used as a negative selector in rice. T₁ transgenic seedlings expressing thistms2 gene under the control of themas2’ promoter showed significant reduction in shoot and root growth in the presence of 5–10 μM NAM under specified growth conditions compared to plants not containing this gene.     

Advances in coffee tissue culture and its practical applications

Genetic improvement of coffee, an important commercial crop, through classical breeding is slow and cumbersome. Biotechnology offers alternative strategies for generating new and improved coffee varieties, including those resistances to environmental extremes, pests, and diseases, low in caffeine, and with uniform fruit maturation. Large improvements in somatic embryogenesis, development of haploids, and scale-up of micropropagation have been accomplished in the last 5 yr. The recent identification of expressed sequence tags (EST) that are differentially expressed during the infestation of coffee plants by coffee leaf miners and the isolation and cloning of the promoter for the N-methyltransferase gene associated with caffeine production open up the possibility of producing varieties of coffee with new traits. This review provides a summary of in vitro biological advances and directions as to how they could be applied to improve the production and quality of coffee.     

Saturation mutagenesis using maize transposons

Transposon mutagenesis facilitates gene discovery by tagging genes for cloning. New genomics projects are now cataloging transposon insertion sites to define all maize genes. Once identified, transposon insertions are 'hot spots' for generating new alleles that are useful in functional studies.     

Transposon Mutagenesis in the Study of Plant Development

Transposon mutagenesis has provided one of the first and most important routes to gene identification and characterization. In the 17 years since the bz1 gene was first tagged with Activator (Ac), more than 60 genes involved in plant development have been cloned using elements such as Supressor-mutator (Spm) and Mutator (Mu) from maize and Tag1 from Arabidopsis. The advantages of transposon-induced alleles in the study of developmental processes go beyond cloning to include sector analysis, generation of new alleles, and conditional expression based on suppression. The laborious technique of directed tagging that led to many of these successes is now being supplanted by systematic projects to produce large collections of transposon insertions that are precharacterized using PCR-based methods and publicly accessible for both forward and reverse genetics. Of the tens of thousands of new genes postulated to exist in Arabidopsis and other species, most are turning out to have no obvious phenotypic effect. The challenge for functional genomics is now to elucidate the apparently subtle actions of genes at a rate commensurate with their discovery. Referee: Dr. Paul Chomet, Monsanto Co., 62 Maritime Dr., Mystic, CT 06355     

Molecular cloning of protease gene(s) from Xanthomanas campestris pv. campestris: Expression in Escherichia coli and role in pathogenicity

Summary The recombinant plasmid pIJ3070 isolated from a genomic library of Xanthomonas campestris pv. campestris constructed in the conjugal cosmid pLAFR3 contains protease gene(s) which can be expressed in Escherichia coli. Tn5 mutagenesis and subcloning revealed that the protease structural gene(s) is(are) located in a ca. 10 kb EcoRI fragment. Several protease-minus mutants of X. c. campestris were obtained by Tn5 mutagenesis of pIJ3070 and marker exchange techniques. Studies of pathogenicity of these Tn5 mutants showed that the protease is not critically important for the pathogenicity of X. c. campestris on turnip plants but may play a minor role in disease development.Abbreviations Gm gentamicin - Km kanamycin - Rif rifampicin - Spc spectinomycin - Sm streptomycin - Tc tetracycline     

Analysis of developmentally interesting genes cloned from higher plants by insertional mutagenesis

The ability of transposable elements to generate gene mutations by excising from one site in the genome and reintegrating into new, different sites elsewhere in the genome has led to the development of procedures whereby the elements can be used to tag specific gene sequences for eventual isolation and analysis through gene cloning. This transposon tagging strategy is particularly useful in those situations where limited knowledge of the biochemistry of the target gene precludes gene cloning by conventional strategies. This approach, in conjunction with the more general insertional mutagenesis approach using T-DNA, has led to the cloning and subsequent analysis of several genes from higher plants involved in particular developmental processes. Studies of this nature should eventually shed light on the precise molecular mechanisms utilized to regulate and control cellular differentiation in plants.     

Trends in comparative genetics and their potential impacts on wheat and barley research

We review some general points about comparative mapping, the evolution of gene families and recent advances in the understanding of angiosperm phylogeny. These are considered in relation to studies of large-genome cereals, particularly barley (Hordeum vulgare) and wheat (Triticum aestivum), with reference to methods of gene isolation. The relative merits of direct map-based cloning in barley and wheat, utilization of the smaller genome of rice (Oryza sativa) and gene homology methods that utilize information from model species such as Arabidopsis thaliana are briefly discussed.     

Chromosomal localization and molecular-marker tagging of the powdery mildew resistance gene (Lv) in tomato

We report the tagging of a powdery mildew [Leveillula taurica (Lév.) Arnaud.] resistance gene (Lv) in tomato using RAPD and RFLP markers. DNA from a resistant (cv Laurica) and a susceptible cultivar were screened with 300 random primers that were used to amplify DNA of resistant and susceptible plants. Four primers yielded fragments that were unique to the resistant line and linked to the resistance gene in an F₂ population. One of these amplified fragments, OP248, with a molecular weight of 0.7 kb, was subsequently mapped to chromosome 12, 1 cM away from CT134. Using RFLP markers located on chromosome 12, it was shown that approximately one half of chromosome 12 (about 42 cM), in the resistant variety is comprised of foreign DNA, presumably introgressed with the resistance gene from the wild species L. chilense. Further analysis of a backcross population revealed that the Lv gene lies in the 5.5-cM interval between RFLP markers, CT211 and CT219. As a prelude to map-based cloning of the Lv gene, we are currently enriching the density of markers in this region by a combination of RAPD primers and other techniques.     

Construction and characterization of a half million clone BAC library of durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>)

Durum wheat (Triticum turgidum ssp. durum, 2n = 4x = 28, genomes AB) is an economically important cereal used as the raw material to make pasta and semolina. In this paper we present the construction and characterization of a bacterial artificial chromosome (BAC) library of tetraploid durum wheat cv. Langdon. This variety was selected because of the availability of substitution lines that facilitate the assignment of BACs to the A and B genome. The selected Langdon line has a 30-cM segment of chromosome 6BS from T. turgidum ssp. dicoccoides carrying a gene for high grain protein content, the target of a positional cloning effort in our laboratory. A total of 516,096 clones were organized in 1,344 384-well plates and blotted on 28 high-density filters. Ninety-eight percent of these clones had wheat DNA inserts (0.3% chloroplast DNA, 1.4% empty clones and 0.3% empty wells). The average insert size of 500 randomly selected BAC clones was 131 kb, resulting in a coverage of 5.1-fold genome equivalents for each of the two genomes, and a 99.4% probability of recovering any gene from each of the two genomes of durum wheat. Six known copy-number probes were used to validate this theoretical coverage and gave an estimated coverage of 5.8-fold genome equivalents. Screening of the library with 11 probes related to grain storage proteins and starch biosynthesis showed that the library contains several clones for each of these genes, confirming the value of the library in characterizing the organization of these important gene families. In addition, characterization of fingerprints from colinear BACs from the A and B genomes showed a large differentiation between the A and B genomes. This library will be a useful tool for evolutionary studies in one of the best characterized polyploid systems and a source of valuable genes for wheat. Clones and high-density filters can be requested at Communicated by P. LangridgeThe first two authors contributed equally to the investigation     

Targeted mutagenesis of a conserved anther‐expressed P450 gene confers male sterility in monocots

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.