Somatic hybridization as a tool for tomato breeding

Protoplast fusion can be used to produce somatic hybrids of species that cannot be obtained by sexual hybridization. The possibility to introgress genes from Solanum species into the cultivated tomato species Lycopersicon esculentum, and to obtain novel cytoplasm-nucleus combinations (cybrids) was considered as an important strategy to extend the genetic variation available for tomato breeding. Somatic hybrids between L. esculentum and other Lycopersicon species, as well as between L. esculentum and Solanum or Nicotiana species, have been produced. Specific mutants, genotypes with antibiotic resistances, and metabolic inhibition by iodoacetate or iodoacetamide and irradiation were used for the selection of hybrids. In addition, the improvement of protoplast culture techniques and the use of the favourable tissue culture traits derived from species such as L. peruvianum, which have been introduced into tomato by classical breeding, allowed the efficient recovery of somatic hybrids. However, the occurrence of somatic incongruity in fusion combinations of L. esculentum and Solanum and even more in L. esculentum and Nicotiana, did not allow the production of true cybrids and/or fertile hybrids, indicating the importance of both cytoplasm-nucleus and nucleus-nucleus interactions in somatic incongruity. Another problem with fusions between distantly related species is the strongly reduced fertility of the hybrids and the very limited homoeologous recombination between chromosomes of the parental species. Partial genome transfer from donor to recipient through microprotoplast (+) protoplast fusion, and the production of monosomic or disomic chromosome addition lines, light overcome some of these problems. In symmetric somatic hybrids between L. esculentum and S. tuberosum the occurrence of limited somatic and meiotic recombination was demonstrated. Fertile progeny plants could be obtained, though at a low frequency, when embryo rescue was performed on a large scale after backcrossing hexaploid somatic tomato (+) potato hybrids with a tetraploid potato genotype. The potential value of genomic in situ hybridization (GISH) and RFLPs for the analysis of the genome/chromosome composition of the hybrids has been demonstrated for intergeneric somatic hybrids between Lycopersicon and Solanum.Abbreviations cpDNA chloroplast DNA - mtDNA mitochondrial DNA     

SolGenes — a Solanaceae database

Biological investigations have the potential to produce large volumes of data, much of which is condensed or omitted from publications, and which may not be readily accessible to researchers outside that discipline. Bringing together in a single repository information whose common thread is the Solanaceae family gives researchers a valuable, multidisciplinary tool. The SolGenes (Solanaceae) database project, a part of the USDA Plant Genome Research Program, is meant to be such a tool. The database has facilities for handling genetic and physical maps, DNA sequences, pathologies and germplasm stocks, as well as other types of information. It currently contains tomato, potato and pepper genetic maps based on a common set of probes, tomato germplasm data including wild species and stocks, autoradiogram images of distributed probes and much more. Current efforts are focused on loading images of tomato mutants, expanding the germplasm data, and setting up a gopher server. The database software (ACEDB) provides a user-friendly, graphical interface with links between logically related pieces of information, allowing the user to view data from multiple sources with just a few clicks of the mouse. The database and software are freely available to interested parties.Abbreviations ftp file transfer protocol     

A genomic library of Lycopersicon pennellii in L. esculentum: A tool for fine mapping of genes

The cultivated tomato contains only a small fraction of the genetic variation present in its wild relatives. In order to use the wild germplasm in tomato breeding and genetic studies we developed a new kind of genetic resource which is composed of 50 L. esculentum lines each containing a single introgression from the green fruited species L. pennellii (LA 716). Each of the introgression lines is nearly isogenic to the cultivated tomato; these lines provide complete coverage of the wild species genome. The lines contain on the average an introgression of 33 cM from a total genome size of 1200 cM. The size and identity of the introgressed segments was determined based on RFLP analysis of 350 markers. This resource can be viewed as a genomic library of the wild species in the cultivated background. It covers the entire genome with single independent inserts per line and therefore every phenotypic difference between the introgression lines can be associated with the unique introgressed segment. The development and potential application of this resource are discussed.     

Molecular genetic characterisation of the Asc locus of tomato conferring resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The Alternaria stem canker disease of tomato is caused by the fungal pathogen Alternaria alternata f. sp. lycopersici and its host-selective AAL-toxins. Resistance to the pathogen and insensitivity to the toxins are conferred by the Asc locus on chromosome 3L. Sensitivity to AAL-toxins is a relative character; the toxins inhibit development of all tested tomato tissues but susceptible cultivars are much more sensitive than resistant cultivars. In addition to tomato, some other plant and animal species are sensitive to the toxins as well. The likely mode of action of AAL-toxins is interference with sphingolipid biosynthesis by specific inhibition of ceramide synthase activity. To molecularly isolate Asc, transposon tagging and positional cloning strategies are applied. As a first step, transposon insertions and restriction fragment length polymorphism (RFLP) markers are identified in proximity of the Asc locus. Subsequently, the transposons are used to inactivate Asc by insertion mutagenesis, and the RFLP markers are used to identify yeast artificial chromosomes (YACs) with tomato DNA inserts. Once an Asc-insertion mutant and/or a YAC encompassing Asc has been obtained, physical isolation and characterisation of Asc will be conceivable. Elucidation of the molecular role of Asc will illuminate the specificity of host recognition by Alternaria alternata f. sp. lycopersici.Abbreviations AAL-toxin Alternaria alternata lycopersici-toxin - A. a. lycopersici Alternaria alternata f. sp. lycopersici - Asc Alternaria stem canker - HST host-selective toxin     

Induced mutations – A new paradigm in plant breeding

The use of ionizing radiation, such as X-rays, gamma rays and neutrons and chemical mutagens for inducing variation, is well established. Induced mutations have been used to improve major crops such as wheat, rice, barley,cotton, peanuts, and beans, which are seed propagated. Since the establishment of the Joint FAO/IAEA Division of the Nuclear Techniques in Agriculture, more than 1800 cultivars obtained either as direct mutants or derived from their crosses have been released worldwide in 50 countries. In vegetatively propagated plants, many of mutants were derived from irradiating rooted stem cuttings, detached leaves, and dormant plants. According to the FAO/IAEA database, of the 465 mutants released among the vegetatively propagated plants, most are in the floricultural plants and a few in fruit trees. These include chrysanthemum, Alstroemeria, dahlia, bougainvillea, rose, Achimenes,begonia, carnation, Streptocarpus, and azalea. The irradiation of in vitro cultured date palm, apple, potato, sweet potato and pineapple now provides a means to treat large populations which would not have been possible before. Irradiation of micropropagated plants, axillary and adventitious buds, apical meristems, regenerative callus cultures, anthers and microspores, and somatic embryos provides a miniaturized version of trees and seeds in the Petridish instead of the field. During the last decade, the use of radio-actively labeled probes in recombinant DNA research for cloning and mapping plant genes and transgenesis, particularly for RFLP, micro satellite based DNA fingerprinting, has become a routine procedure. Many homeotic mutants that change floral development have been isolated in Arabidopsis, Petunia, Antirrhinum and Lycopersicon. Mutants of Arabidopsis are being used to analyze genes, which determine response to auxins, cytokinins, gibberellin, abscisic acid and ethylene in plant growth, floral development and senescence, fruit formation and ripening. These mutants are facilitating the isolation, identification and cloning of the genes, which would ultimately help in designing crops with improved yield, increased stress tolerance, longer shelf-life and reduced agronomic inputs. The identification and analysis of mutants by using molecular techniques of DNA fingerprinting and mapping with PCR based markers, such as RAPDs, AFLP and STMS, and mutant tagging shall bring a new dimension in gene technology. Already, mutations can be linked to changes in DNA sequences for some plant traits and to establish molecular maps in structural and functional genomics of crop plants. These in turn would lead to a rapid enhancement of crop yields and quality. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intragenic vectors for gene transfer without foreign DNA

The intragenic vector system involves identifying functional equivalents of vector components from the genome of a specific crop species (or related species to which it can be hybridised) and using these DNA sequences to assemble vectors for transformation of that plant species. This system offers an attractive alternative to current genetic engineering strategies where vectors are based on DNA sequences that usually originate from bacteria. The construction of intragenic vectors enables the well-defined genetic improvement of plants with all transferred DNA originating from within the gene pool already available to plant breeders. In this manner genes can be introgressed into elite cultivars in a single step without linkage drag and without the incorporation of foreign DNA. The resulting plants are non-transgenic, although they are derived using the tools of molecular biology and plant transformation. The use of intragenic vectors for the transfer of genes from within the gene pools of crops may help to alleviate some of the major public concerns over the deployment of GM crops in agriculture, notably the ethical issue associated with the transfer of DNA across wide taxonomic boundaries. This paper reviews the progress toward the development and use of intragenic vectors and the implications of their use for the genetic improvement of crops. Dedicated to the late Hans Sandbrink for his enthusiasm in helping to develop intragenic vectors     

Orientation and integration of the classical and molecular genetic maps of chromosome 11 in rice

Summary The classical genetic map and molecular map of rice chromosome 11 were oriented to facilitate the use of these maps for genetic studies and rice improvement. Three morphological markers (d-27, z-2, and la) were crossed to a rice breeding line, IRBB21, which has the Xa-21 gene for bacterial blight resistance. Three F₂ populations were analyzed with RFLP markers known to be located on chromosome 11. Segregation analysis of molecular markers and morphological markers was used to construct an RFLP map for each population. The recombination frequency between markers varied from population to population although the marker order on the maps was the same for all three populations. Based on a common set of markers mapped in the three populations, an integrated map was generated consisting of both RFLP and morphological markers. The genetic distance between markers on this map was determined by taking a weighted average of the data from the three populations. The oriented map serves as a bridge to understand the relationship between the classical and molecular linkage maps. Based on this information, the location of several genes on the classical map can be approximated with respect to RFLP markers without having to map them directly.     

A comparative analysis of the genetic relationships between rye cultivars using RFLP and RAPD markers

Summary The genetic similarities of eight closely related rye cultivars were estimated using two molecular marking techniques: restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). Cultivars were evaluated for variation by 11 random cDNA and genomic clones used in combination with four restriction enzymes and 40 decamer primers. A total of 53 polymorphic RFLP fragments and 94 polymorphic RAPD fragments were observed. Based on the presence/absence of fragments, two genetic similarity matrices were calculated which were then used in cluster analysis. Differences between pair of cultivars were observed in RFLP and RAPD dendrograms. RFLP analysis produced estimates of genetic relationships more in accordance with the partially known pedigree of the cultivars than did RAPD analysis. The use of bulk samples of DNA in these analyses affected the sensitivity of RAPD assays more strongly. Dendrograms which took into account all fragments produced, either by RFLP or RAPD, reflected better the relationships between cultivars than did dendrograms based on only one type of marker. This reflects the importance of the number of markers used in determining the genetic relationships between genotypes.     

分子标记技术在番茄抗性育种中的应用

介绍了DNA分子标记的各种类型,RFLP、RAPD、SSR、AFLP和SNP标记是植物遗传作图最常用的,介绍了各种类型的使用范围以及优点和缺点。综述了近年来DNA分子标记技术在番茄抗性育种中的应用,包括耐冷性,耐盐性,抗病性,抗虫害等方面的应用,并就今后番茄分子育种主要研究方向进行了讨论。     

A skeletal linkage map of Hordeum bulbosum L. and comparative mapping with barley (H. vulgare L.)

A restriction fragment length polymorphism (RFLP) based linkage map of a cross between two diploid Hordeum bulbosum (2n = 2x = 14) clones, PB1 and PB11, was constructed from 46 recombinant progeny clones. Since both parents are heterozygous, separate and combined parental maps were constructed. All of the RFLP markers screened had previously been mapped in barley (H. vulgare L.) so that comparative maps could be produced. The PB1 linkage map consists of 20 RFLP marker loci assigned to four linkage groups covering 94.3 cM. The PB11 linkage map consists of 27 RFLP marker loci assigned to six linkage groups covering 149.1 cM. Thirteen markers polymorphic in both parents were used as ‘anchors’ to create a combined linkage map consisting of 38 loci assigned to six linkage groups and covering a genetic distance of 198 cM. Marker order was highly conserved in a comparison with the linkage map of H. vulgare (Laurie etal., 1995). However, in contrast, the genetic distances for the same markers were very different being 649 cM and 198 cM respectively, a genetic distance ratio of 1: 3.3. Thus although the map was short, it can be presumed to cover half the genome of H. bulbosum. This study provides further confirmation of the close relationship between the two species and gives a basis for the development of marker mediated introgression through interspecific hybridisation between the two species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular characterization of the progeny of <Emphasis Type="Italic">Solanum etuberosum</Emphasis> identifies a genomic region associated with resistance to potato leafroll virus

Potato leafroll virus (PLRV; Genus Polerovirus; Family Luteoviridae) is one of the most important virus pathogens of potato worldwide and breeders are looking for new sources of resistance. Solanum etuberosum Lindl., a wild potato species native to Chile, was identified as having resistances to PLRV, potato virus Y, potato virus X, and green peach aphid. Barriers to sexual hybridization between S. etuberosum and cultivated potato were overcome through somatic hybridization. Resistance to PLRV has been identified in the BC₁, BC₂ and BC₃ progeny of the somatic hybrids of S. etuberosum (+) S. tuberosum haploid × S. berthaultii Hawkes. In this study, RFLP markers previously mapped in potato, tomato or populations derived from S. palustre (syn S. brevidens) × S. etuberosum and simple sequence repeat (SSR) markers developed from tomato and potato EST sequences were used to characterize S. etuberosum genomic regions associated with resistance to PLRV. The RFLP marker TG443 from tomato linkage group 4 was found to segregate with PLRV resistance. This chromosome region has not previously been associated with PLRV resistance and therefore suggests a unique source of resistance. Synteny groups of molecular markers were constructed using information from published genetic linkage maps of potato, tomato and S. palustre (syn. S. brevidens) × S. etuberosum. Analysis of synteny group transmission over generations confirmed the sequential loss of S. etuberosum chromosomes with each backcross to potato. Marker analyses provided evidence of recombination between the potato and S. etuberosum genomes and/or fragmentation of the S. etuberosum chromosomes.     

Transforming the plastome: genetic markers and DNA delivery systems

Summary Stable chloroplast transformants were first obtained following particle bombardment of tobacco leaves, and later by PEG-mediated uptake of DNA by protoplasts. The transforming DNA in these studies was itself of plastid origin and carried double (streptomycin, spectinomycin) antibiotic resistance which was used to select transformants. Integration was by homologous recombination, and both donor and recipient were Nicotiana species. Recent characterisation of plastid mutants of Solanum nigrum has allowed the extension of this gene replacement approach to include Nicotiana:Solanum combinations.The introduction of functional heterologous genes into the plastome is an alternative approach based on the use of constructs in which a bacterial resistance gene is flanked by sequences homologous to a region of the recipient plastome. Thus homologous recombination in the flanking sequences allows introduction of a foreign gene. A large number of putative transformants can be generated by the method, but this apparent attraction is partly offset by the need for repeated cycles of re-selection to obtain homoplasmic plants. In contrast, homoplasmy can be accomplished in a single selection step using plastome-encoded antibiotic resistance markers.The plastome is an attractive target for the introduction of useful genes into crop plants, as maternal inheritance acts as an insurance against unwanted spread of the foreign gene, and the large plastome copy number ensures immediate gene amplification and may influence levels of expression. Specific characters encoded on the plastid DNA, including components of photosynthesis and other aspects of metabolism, will also become open to manipulation as a consequence of developments in plastid transformation.     

Analysis of the molecular basis of Pseudomonas syringae pv. tomato resistance in tomato

Tomato (Lycopersicon esculentum) cultivars that contain the Pto bacterial resistance locus also exhibit sensitivity to fenthion, an organophosphorous insecticide. Resistance to Pseudomonas syringae pv. tomato (Pst) encoded by the Pto gene and sensitivity to fenthion cosegregate in large F₂ populations and were apparently introgressed together into tomato from the wild species Lycopersion pimpinellifolium. In order to isolate the genes responsible for these two phenotypes and to study their molecular basis, a multistep map-based cloning project was initiated. A closely linked RFLP marker was used to isolate a yeast artificial chromosome (YAC) clone that spanned the Pto region. Transcribed sequences within the Pto region were identified by isolating cDNA clones that hybridized to the YAC clone. Transformation of candidate cDNA clones into a Pst-susceptible, fenthion-insensitive tomato line succeeded in identifying a cDNA conferring Pst resistance and a separate cDNA conferring sensitivity to fenthion. The cDNA clones represent members of a tandemly repeated gene family and encode putative serine-threonine protein kinases. The role of these kinases in recognizing a signal from Pst or the fenthion molecule and in activating the plant response is currently being investigated.Abbreviations Pst Pseudomonas syringae pv. tomato - RAPD random amplified fragment polymorphism - RFLP restriction fragment length polymorphism - YAC yeast artifical chromosome     

The role of selection on the genetic structure of pathogen populations: Evidence from field experiments with Mycosphaerella graminicola on wheat

Summary Coevolution refers to reciprocal genetic changes that occur in two or more ecologically interacting species. In agricultural ecosystems, we are especially concerned with the genetic response of pathogen populations to resistant cultivars produced by plant breeding programs. It would be useful to be able to predict whether disease resistance is likely to be durable or ephemeral before a cultivar is widely grown. Though it may not be possible to predict durability in advance, knowledge of the genetic structure of pathogen populations may prove useful for making predictions about the rate at which pathogens adapt to resistant varieties. Much has been learned about the genetic structure of populations of obligate fungal pathogens such as rusts and mildews, which have become paradigms for plant pathology. We have focused our effort on the population genetics of the less known, non-specialized, necrotrophic pathogens, such as the Septorias of small grains. Our approach has been to use DNA fingerprinting and RFLP analysis to conduct field experiments that elucidate how populations of fungal pathogens adapt in agroecosystems. Our results suggest that mating system may have a greater impact than natural selection on the genetic structure of populations of Mycosphaerella graminicola (anamorph Septoria tritici).     

Resistance to soybean cyst nematode and molecular polymorphism in various sources of Peking soybean

Summary Cultivar Peking has been extensively used as a source of resistance to Race 3 and Race 5 of soybean cyst nematode, Heterodera glycines I., and Peking genes for resistance are present in a wide range of resistant soybean cultivars. Peking is also used as a host differential in the soybean cyst nematode race classification system. Thirteen Peking lines maintained in the USDA Soybean Germplasm Collection and in several breeding programs were surveyed using RFLP and RAPD markers for genetic characterization. Based on the molecular diversity combined with reaction to soybean cyst nematode, Peking genotypes from a common original source were identified. Peking lines PI 297543 (introduction from Hungary), and PI 438496A, PI 438496B and PI 438496C (introductions from Russia) represented unrelated germplasms. Identified molecular polymorphism can be used to validate the genetic purity of Peking lines used as host differentials for soybean cyst nematode classification system as well as utilization of an individual germplasm line in genetic-breeding programs.     

Disease resistance in protected crops and mushrooms

Summary Cultivars of tomatoes, cucumbers, lettuce and peppers have been bred for resistance to one or more pathogens. Some tomato and cucumber cultivars have resistance to a wide range of diseases. Resistance has been transient in many cases and a succession of cultivars with new genes or new combinations of resistance genes has been necessary to maintain control. There has been a number of notable exceptions and these have included durable resistance to such pathogens asFulvia fulva and tomato mosaic virus. With lettuce the resistance situation is complicated by the occurrence of fungicide resistant pathotypes. There are no strains ofAgaricus bisporus purposely bred for disease resistance.In protected flower crops only resistance to Fusarium wilt in carnations has been purposely bred but differences in disease resistance are apparent in cultivars of many ornamental crops. This is particularly so in chrysanthemums where there are cultivars with resistance to many of the major pathogens. Similar situations occur with other flower crops and pot plants. Cultivars of some species have not been systematically investigated for resistance.The need for genetic resistance will increase with the further reduction, in the limits on pesticide use and an increasing public awareness and importance of pesticide pollution.ADAS is an executive agency of the Ministry of Agiculture, Fisheries and Food and the Welsh Office.     

桉树的分子标记技术及遗传图谱构建进展

桉树是世界上的三大类速生树种之一，具有重要经济价值。本文着重论述了RFLP、RAPD、AFLP和SSR等4种DNA分子标记技术在桉树上的应用和推广，讨论了基于上述分子标记构建的桉树遗传连锁图的现状及其应用，提出了当前桉树遗传图谱构建存在的问题，进一步探讨了桉树的分子标记技术和遗传图谱构建的发展趋势。     

The ‘European Apple Genome Mapping Project’-developing a strategy for mapping genes coding for agronomic characters in tree species

Summary A recently initiated collaborative project involving apple breeders in seven European countries is described. The objective is to improve the European apple crop by molecular-aided breeding to increase efficency and reduce the time-scale in breeding for resistance, tree habit and fruit quality. The strategy adopted provides a model for similar studies in fruit, forest and other woody species. The project is based on progenies from a small number of crosses involving many important agronomic genes. Replication of these reference progenies by vegetative propagation will enable studies to be carried out simultaneously in each country. By developing a range of molecular markers, including isozymes, RFLPs and sequence-tagged DNA probes, an integrated molecular map is being constructed for use in a wide range of breeding and genetic studies. Construction of a database recording many mapped molecular markers will enable efficient exploitation of data in future genetic, breeding and physiological studies of apple. Aspects of the adopted strategy, techniques and management are discussed in the context of mapping genes in perennial crop genomes.     

Molecular mapping of a new gene for resistance to rice blast (Pyricularia grisea Sacc.)

A single dominant blast resistance gene conferring resistance to a Korean rice blast isolate was identified in rice variety `Suweon 365'. We report the chromosomal localization and molecular mapping of this blast resistance gene designated as Pi-18, which confers resistance to Korean isolate `KI-313' of the blast pathogen. To know whether there is a relationship among genes conditioning resistance to location-specific isolates of the blast pathogen and thereby to identify linked markers to resistance gene for isolate KI-313 collected in Korea, RFLP markers previously reported to be linked to major blast resistance genes in different rice germplasm and other markers mapped to nearby regions were surveyed for polymorphism between a resistant (`Suweon 365') and a susceptible (`Chucheongbyeo') parent. Linkage associations of the RFLP markers with the resistance gene were verified using an F2 and F3 segregating population of known blast reaction. RFLP analysis showed that Pi-18 was located near the end of chromosome 11, linked to a single copy clone RZ536 at a distance of 5.4 centiMorgans (cM) and that this gene was different from Pi-1(t). An allelism test revealed that this gene was also different from Pi-k. Currently, a combination of RAPD and microsatellite primers is being employed to find additional markers in this region. Tightly linked DNA markers will facilitate selection for resistant genotypes in breeding programs and provide the basis for map based cloning of this new blast resistance gene. This revised version was published online in July 2006 with corrections to the Cover Date.     

The biotechnology of crop legumes

Summary The absence of variety-independent gene transfer methods for major agronomic species has, until now, limited the usefulness of recombinant DNA techniques to crop improvement programs. Until recently, only Solanaceous crops could be used to study fundamental and applied problems in plant sciences. During the past five years rapid advances in cell biology, in combination with the development of novel gene transfer methodology allowed utilization of the tools of plant molecular biology in conventional breeding programs. Cereal and leguminous species were considered to be recalcitrant to genetic manipulation. As a result of the development of direct DNA transfer methodology into organized tissue, we are now in a position to introduce any foreign gene into almost all of the major cereals and legumes. This can be achieved efficiently, often in a variety-independent fashion. The object of this review is to provide a comprehensive account of the state of the art in gene transfer for the cultivated leguminous crops. Important oilseed and feed species primarily in industrialized countries, as well as minor but equally important species for sustaining growth populations in developing countries will be examined. Advantages of the various gene transfer methods that were shown to be useful for specific crops, as well as limitations and problems associated with each crop and gene transfer method will be discussed. Data from field trials of transgenic legumes, where available, will be presented.