Construction of a yeast artificial chromosome library of tomato and identification of cloned segments linked to two disease resistance loci

Summary We have constructed a yeast artificial chromosome (YAC) library of tomato for chromosome walking that contains the equivalent of three haploid genomes (22 000 clones). The source of high molecular weight DNA was leaf protoplasts from the tomato cultivars VFNT cherry and Rio Grande-PtoR, which together contain loci encoding resistance to six pathogens of tomato. Approximately 11 000 YACs have been screened with RFLP markers that cosegregate withTm-2a andPto — loci conferring resistance to tobacco mosaic virus andPseudomonas syringae pv.tomato, respectively. Five YACs were identified that hybridized to the markers and are therefore starting points for chromosome walks to these genes. A subset of the library was characterized for the presence of various repetitive sequences and YACs were identified that carried TGRI, a repeat clustered near the telomeres of most tomato chromosomes, TGRII, an interspersed repeat, and TGRIIl, a repeat that occurs primarily at centromeric sites. Evaluation of the library for organellar sequences revealed that approximately 10% of the clones contain chloroplast sequences. Many of these YAC clones appear to contain the entire 155 kb tomato chloroplast genome. The tomato cultivars used in the library construction, in addition to carrying various disease resistance genes, also contain the wild-type alleles corresponding to most recessive mutations that have been mapped by classical linkage analysis. Thus, in addition to its utility for physical mapping and genome studies, this library should be useful for chromosome walking to genes corresponding to virtually any phenotype that can be scored in a segregating population.     

破色期番茄果实均一化cDNA沉默文库的构建和功能基因筛选模型的初步建立

番茄果实的成熟是由多基因精细调控的一个过程．利用破色期番茄果实,根据复性动力学原理在mRNA水平进行均一化操作使高丰度和低丰度的mRNA丰度接近．然后把均一化之后mRNA反转录得到cDNA,再与基因沉默载体pTRV重组,最后把构建好的载体通过电转化的方法转入到GV3101农杆菌中,从而建立起破色期番茄果实均一化cDNA沉默文库．通过番茄果实中病毒诱导基因沉默技术,对cDNA沉默文库进行初步筛选,从而确定功能基因筛选模型．在模型建立阶段,以番茄红素合成途径相关的PDS基因作为内标基因,在100个混合农杆菌样中,成功筛选到了PDS基因．     

Generation of Arabidopsis Mutants by Heterologous Expression of a Full-Length cDNA Library from Tomato Fruits

Heterologous expression of cDNA library in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with a plant transformation-ready binary vector that contained a higher percentage of full-length cDNAs since synthesized double-stranded cDNA was size-selected using gel electrophoresis, with cDNA sizes of 2–5 kb being gel-purified for ligation onto the binary vector. Sequencing of 81 cDNA clones indicates that 75% (61) are full-length genes, which is similar to sequencing of inserted cDNA in Arabidopsis. The library was used to transform Arabidopsis plants. Among the 7,000 mutants, one was found to be a dwarf due to the expression of an ATP synthase, and another vegetative mutant did not produce flowers even after 7 months. The technique was validated by reintroducing the tomato ribosomal protein L9 gene and can be used in any other plant species as a gene discovery tool.     

Advanced backcross QTL analysis in a cross between an elite processing line of tomato and its wild relative L. pimpinellifolium

Approximately 170 BC2 plants from a cross between an elite processing inbred (recurrent parent) and the wild species Lycopersicon pimpinellifolium LA1589 (donor parent) were analyzed with segregating molecular markers covering the entire tomato genome. Marker data were used to identify QTLs controlling a battery of horticultural traits measured on BC2F1 and BC3 families derived from the BC2 individuals. Despite its overall inferior appearance, L. pimpinellifolium was shown to possess QTL alleles capable of enhancing most traits important in processing tomato production. QTL-NIL lines, containing specific QTLs modifying fruit size and shape, were subsequently constructed and shown to display the transgressive phenotypes predicted from the original BC2 QTL analysis. The potential of exploiting unadapted and wild germplasm via advanced backcross QTL analysis for the enhancement of elite crop varieties is discussed.     

Identification of a pathogenicity locus in Xanthomonas campestris pv. vesicatoria

Summary Mutants of a tomato strain ofXanthomonas campestris pv.vesicatoria (XCV), causal agent of bacterial spot of tomato and pepper, were produced using the transposon Tn5 carried in the suicide plasmid pGS9. One prototrophic mutant, M461, was isolated which caused no visible reaction on tomato or pepper, but maintained the wild-type ability to induce a hypersensitive reaction (HR) on tobacco. This mutant showed similar growth characteristics to the wild-type in culture, but growth in planta was reduced. A genomic library of wild-type XCV was constructed in the broad host range cosmid vector pLAFR3. Clone p6AD4 restored pathogenicity to M461 on tomato and the ability to induce a HR on pepper. This clone contained ca. 22 kb of XCV DNA. The insertion in M461 was in a site corresponding to a 1.1 kbEcoRI fragment of p6AD4.     

Introgression lines of Solanum sitiens,a wild nightshade of the Atacama Desert,in the genome of cultivated tomato

The wild tomato relative Solanum sitiens is a xerophyte endemic to the Atacama Desert of Chile and a potential source of genes for tolerance to drought, salinity and low‐temperature stresses. However, until recently, strong breeding barriers prevented its hybridization and introgression with cultivated tomato, Solanum lycopersicum L. We overcame these barriers using embryo rescue, bridging lines and allopolyploid hybrids, and synthesized a library of introgression lines (ILs) that captures the genome of S. sitiens in the background of cultivated tomato. The IL library consists of 56 overlapping introgressions that together represent about 93% of the S. sitiens genome: 65% in homozygous and 28% in heterozygous (segregating) ILs. The breakpoints of each segment and the gaps in genome coverage were mapped by single nucleotide polymorphism (SNP) genotyping using the SolCAP SNP array. Marker‐assisted selection was used to backcross selected introgressions into tomato, to recover a uniform genetic background, to isolate recombinant sub‐lines with shorter introgressions and to select homozygous genotypes. Each IL contains a single S. sitiens chromosome segment, defined by markers, in the genetic background of cv. NC 84173, a fresh market inbred line. Large differences were observed between the lines for both qualitative and quantitative morphological traits, suggesting that the ILs contain highly divergent allelic variation. Several loci contributing to unilateral incompatibility or hybrid necrosis were mapped with the lines. This IL population will facilitate studies of the S. sitiens genome and expands the range of genetic variation available for tomato breeding and research.     

Isolation and characterization of a fruit-specific cDNA and the corresponding genomic clone from tomato

Differential screening of a cDNA bank constructed from ripe tomato fruit mRNA allowed the isolation of cDNA clone 2A11 which is entirely fruit-specific, is expressed at steadily increasing levels from anthesis to breaker, and accounts for approximately 1% of the messenger RNA in mature tomato fruit. A genomic clone corresponding to the 2A11 cDNA was isolated from a tomato genomic library. Sequence comparison of the cDNA clone with the genomic clone shows they are identical over the shared region with the genomic clone possessing a single large intron near the 5 end of the message.The open reading frame of 2A11 would encode a sulfur-rich polypeptide 96 amino acids in length. The identity of the putative protein is unknown. In situ hybridization shows that the 2A11 message is found throughout the pericarp cells in a tomato fruit. In contrast, in situ hybridization of early ripening stages with a polygalacturonase probe shows higher mRNA levels in cells of the outer pericarp and cells surrounding the vascular regions of the pericarp.     

Molecular cloning of a tomato leaf cDNA encoding an aspartic protease, a systemic wound response protein

A full-length cDNA encoding an aspartic protease (LeAspP) has been cloned from a tomato leaf cDNA library. Using LeAspP cDNA as a probe in gel blots, LeAspP mRNA was shown to be systemically induced in tomato leaves by wounding. Application of methyl jasmonate to leaves of intact tomato plants, or supplying systemin to young tomato plants through their cut stems, induces synthesis of LeAspP mRNA. LeAspP message is regulated in tomato similar to several systemic wound response proteins (swrps) that are part of the defense response in tomato plants directed against herbivore attacks.     

Creation of BAC genomic resources for cocoa (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) for physical mapping of RGA containing BAC clones

We have constructed and validated the first cocoa (Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp (palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.Communicated by J.W. Snape     

Development of a new transformation-competent artificial chromosome (TAC) vector and construction of tomato and rice TAC libraries

Recent research has shown that BIBAC (binary bacterial artificial chromosome) and TAC (transformation-competent artificial chromosome) vector systems are very useful tools for map-based cloning of agronomically important genes in plant species. We have developed a new TAC vector that is suitable for both dicot and monocot transformation. Using this new TAC vector, we constructed large-insert genomic libraries of tomato and rice. The tomato library contains 96,996 clones (28.3-38.5 kb insert size) and has 3.18 haploid genome equivalents. The rice TAC library has 32.7 kb average insert size and has 9.24 haploid genome equivalents. The quality of these two libraries was tested using PCR to verify genome coverage. Individual clones were characterized to confirm insert integrity by Southern analysis, end sequencing and genetic mapping. To investigate the potential application of these TAC libraries in map-based cloning, TAC constructs containing a 45 kb fragment were introduced into the rice genome via Agrobacterium-mediated transformation. Molecular analysis indicates that the 45 kb fragment was successfully transferred into the rice genome. Although rearrangements of the introduced DNA were detected, 50% of regenerated plants contained at least one intact copy of the 45 kb clone and associated vector sequences. These libraries provide us with a valuable resource to rapidly isolate important genes in tomato and rice.     

Construction and characterization of a bacterial artificial chromosome library ofArabidopsis thaliana

We constructed an ordered 3,948-clone arabidopsis BAC library. The library has a combined average insert size of 100 kb (n=54). Assuming a haploid genome size of 100,000 kb, the BAC library contains 3.95 haploid genome equivalents with a 98 percent probability of isolating a specific genomic region. The library was screened with five arabidopsis cDNA probes and one tomato probe; all probes hybridized to at least one (and in most cases three) BAC clones in the library.     

Tomato chromosome 6: a high resolution map of the long arm and construction of a composite integrated marker-order map

Integration of molecular and classical genetic maps is an essential requirement for marker-assisted breeding, quantitative trait locus mapping and map-based cloning. With respects to tomato, such maps are only available for the top part of chromosome 1, for chromosome 3 and for the short arm and the centromere proximal part of the long arm of chromosome 6. Employing an L. esculentum line carrying an L. hirsutum introgression we constructed an integrated linkage map for the telomere proximal part of the long arm of tomato chromosome 6, thereby completing the integrated map published previously. With an average map distance of only 0.6 cM the map provides detailed information on the relative position of molecular markers and several traits of economical importance, such as the fruit color marker B. Furthermore, two additional crosses using lines containing L. pennellii introgressions were performed to address the question as to how the recombination frequency in a marked interval on the long arm of chromosome 6 is affected by introgressed segments from different origins. It is concluded that recombination is not merely affected by the local level of homology but also by surrounding sequences. Combination of all the linkage data generated in various crosses described in this and other studies enabled the construction of the first integrated map of an entire tomato chromosome. This map carries 42 loci and shows the position of 15 classical genes relative to 59 molecular markers.     

Construction,characterization, and screening of a transformation-competent artificial chromosome library of peach

As a genome model of fruit trees, peach (Prunus persica [L.] Batch) has advantages for studying structural and functional genomics. Okubo, a traditional peach variety used as a parent in Asian peach breeding, displays economically valuable agronomic traits. To develop an efficient platform for peach gene cloning and genomic research, a large-insert genomic DNA library of Okubo was constructed in a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, which can accept and stably maintain large genomic DNA fragments in bothEscherichia coli andAgrobacterium tumefaciens. The TAC library contains 41,472 recombinant clones with an average insert size of approximately 42 kb, and it is equivalent to 6 haploid peach genomes. The TAC library was stored in 2 ways: one copy as frozen cultures in 108 pieces of 384-well plates and another copy as bulked pools in 36 pieces of 96-well plates, each well containing 12 individual clones. The lack of hybridization signal to chloroplast and mitochondrial genes indicated that the TAC library had no significant cytoplast organelle DNA contamination. TAC clones were stable inE. coli cells until generation 100 and stable in bothE. coli andA. tumefaciens. Twenty-one clones containing the polygalacturonase-inhibiting protein (PGIP) gene were detected by using pooled PCR in the TAC library. Positive clones can be used for peach PGIP gene cloning and functional analysis. The library is well suited for gene cloning and genetic engineering in peach.     

Construction and characterization of a transformation-competent artificial chromosome (TAC) library ofZizania latifolia (Griseb.)

The tribe Oryzeae consists of 12 genera and 71 species with a world distribution.Zizania latifolia (Griseb.) Turcz. ex Stapf is included in this tribe and possesses numerous traits valuable for rice breeding, such as disease and insect resistance, cold and flood tolerance, and high grain quality. The genetics and breeding ofZ. latifolia are still in their infancy. To facilitate genomic studies ofZizania, a genomic DNA library was constructed using a transformation-competent artificial chromosome (TAC) vector system. The TAC library contains 91, 584 TAC clones with an average insert size of approximately 45 kb, covering six haploidZizania genome equivalents. Very low signals after hybridization with chloroplast and mitochondrial genes indicate that the TAC library is predominantly composed of nuclear DNA. The TAC clones were stable inE. coli for 100 generations. Clones containing thedihydrodipicolinate synthase (DHPS) gene were screened by pooled PCR. The positive clones can be used forZ. latifolia DHPS gene cloning and functional analysis. The library will be useful in studies of genome structure, gene cloning and evolution of rice.     

Nucleotide sequence of cDNA encoding α-luffin,a ribosome-inactivating protein from Luffa cylindrica

A cDNA library from tomato planta macho viroid (TPMV)-infected tomato was constructed. The library was screened at low stringency with a tobacco PR-R cDNA probe. An 832 bp cDNA from a mRNA present only in infected tissue was isolated. Nucleotide sequence showed high homology with the osmotin from both tobacco and tomato (NP24). This cDNA probably corresponds to the AP24 and P23 proteins previously described in tomato and induced upon fungal and viroid infection.     

An ethylene-related cDNA from ripening apples

We report the isolation of a ripening-related apple cDNA which is complementary to a mRNA which may be involved in ethylene production. Poly(A)⁺ RNA was extracted from cortical tissue of ripe apple fruit (Malus domestica Borkh cv. Golden Delicious) and a cDNA library constructed in the plasmid vector pSPORT. The library was screened with pTOM13, a tomato cDNA clone thought to code for ACC oxidase in that fruit. An apple cDNA clone (pAP4) was isolated and sequenced. The 1182 bp cDNA insert includes an open reading frame of 942 bp, and shows strong homology with reported tomato and avocado sequences, both at the nucleic acid and amino acid levels. The polypeptide has a calculated molecular mass of 35.4 kDa and a calculated pI of 5.15. In apple cortical tissue, expression of pAP4-complementary RNA increased with ethylene production by the fruit during ripening. Expression was also enhanced in both ethylene-treated and wounded fruit.     

Tomato TILLING technology: development of a reverse genetics tool for the efficient isolation of mutants from Micro-Tom mutant libraries

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1-SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato.     

Expression of Arabidopsis APETALA1 in tomato reduces its vegetative cycle without affecting plant production

Important agronomic traits such as fruit quality, harvesting efficiency or production largely depend on flowering time. We have analysed the effect of the overexpression of the Arabidopsis APETALA1 MADS-box gene on vegetative and reproductive growth of tomato. Constitutive expression of APETALA1 in tomato plants has major effects on the length of their growth cycle as well as on their growth habit. Transgenic tomato plants initiated flowering after the production of 6 vegetative nodes as compared to 11 nodes for the wild type plants. Most of tomato 35S:AP1 plants also showed determinate growth habit, similar to the phenotype of self pruning tomato mutants, as well as an initial reduction of their axillary growth. Moreover, development and fertility of flowers were not affected in plants expressing AP1. Consequently, fruit formation in transgenic plants grown under greenhouse conditions occurred normally, which permitted a similar fruit yield compared to control plants. Since traits conferred by AP1 expression are dominant, its expression in tomato breeding lines could provide advantages for the development of new hybrid varieties with shorter generation time, determinate growth, and reduced pruning requirements.     

Advanced backcross QTL analysis of a Lycopersicon esculentum ×Lycopersicon parviflorum cross

Lycopersicon parviflorum is a sexually compatible, wild tomato species which has been largely unutilized in tomato breeding. The Advanced Backcross QTL (AB-QTL) strategy was used to explore this genome for QTLs affecting traits of agronomic importance in an interspecific cross between a tomato elite processing inbred, Lycopersicon esculentum E6203, and the wild species L. parviflorum (LA2133). A total of 170 BC2 plants were genotyped by means of 133 genetic markers (131 RFLPs; one PCR-based marker, I-2, and one morphological marker, u, uniform ripening). Approximately 170 BC3 families were grown in replicated field trials, in California, Spain and Israel, and were scored for 30 horticultural traits. Significant putative QTLs were identified for all traits, for a total of 199 QTLs, ranging from 1 to 19 QTLs detected for each trait. For 19 (70%) traits (excluding traits for which effects of either direction are not necessarily favourable or unfavourable) at least one QTL was identified for which the L. parviflorum allele was associated with an agronomically favourable effect, despite the overall inferior phenotype of the wild species. Received: 14 September 1999 / Accepted: 7 October 1999