Map-based cloning in crop plants. Tomato as a model system: I. Genetic and physical mapping of jointless

A map-based cloning scheme is being used to isolate the jointless (j) gene of tomato. The jointless locus is defined by a single recessive mutation that completely suppresses the formation of the fruit and flower pedicel and peduncle abscission zone. jointless was mapped in an F₂ population of an interspecific cross between Lycopersicon esculentum and Lycopersicon pennellii to a 7.1 cM interval between two restriction fragment length polymorphism (RFLP) markers TG523 and TG194. Isogenic DNA pools were then constructed from a subset of the mapping population and screened with 800 random decamers for random amplification of polymorphic DNA (RAPD) polymorphisms. Five new RAPD markers were isolated and mapped to chromosome 11, two of which were mapped within the targeted interval. One marker, RPD158, was mapped 1.5 cM to the opposite side of jointless relative to TG523 and thus narrowed the interval between the closest flanking markers to 3.0 cM. Physical mapping by pulse-field gel electrophoresis using TG523 and RPD158 as probes demonstrated that both markers hybridize to a common 600 kb SmaI restriction fragment. This provided an estimate of 200 kb/cM for the relationship between physical and genetic distances in the region of chromosome 11 containing the j locus. The combined results provide evidence for the feasibility of the next step toward isolation of the jointless gene by map-based cloning — a chromosome walk or jump to jointless.     

Map-based cloning in crop plants. Tomato as a model system: I. Genetic and physical mapping of jointless

A map-based cloning scheme is being used to isolate the jointless (j) gene of tomato. The jointless locus is defined by a single recessive mutation that completely suppresses the formation of the fruit and flower pedicel and peduncle abscission zone. jointless was mapped in an F₂ population of an interspecific cross between Lycopersicon esculentum and Lycopersicon pennellii to a 7.1 cM interval between two restriction fragment length polymorphism (RFLP) markers TG523 and TG194. Isogenic DNA pools were then constructed from a subset of the mapping population and screened with 800 random decamers for random amplification of polymorphic DNA (RAPD) polymorphisms. Five new RAPD markers were isolated and mapped to chromosome 11, two of which were mapped within the targeted interval. One marker, RPD158, was mapped 1.5 cM to the opposite side of jointless relative to TG523 and thus narrowed the interval between the closest flanking markers to 3.0 cM. Physical mapping by pulse-field gel electrophoresis using TG523 and RPD158 as probes demonstrated that both markers hybridize to a common 600 kb SmaI restriction fragment. This provided an estimate of 200 kb/cM for the relationship between physical and genetic distances in the region of chromosome 11 containing the j locus. The combined results provide evidence for the feasibility of the next step toward isolation of the jointless gene by map-based cloning — a chromosome walk or jump to jointless.     

Map-based cloning of the tomato genomic region that spans the Sw-5 tospovirus resistance gene in tomato

Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400 kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100 kb, spanned by nine overlapping cosmid clones. Received: 13 March 1997 / Accepted: 11 may 1997     

Map-based cloning of the tomato genomic region that spans the Sw-5 tospovirus resistance gene in tomato

Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400?kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100?kb, spanned by nine overlapping cosmid clones.     

Localization of<Emphasis Type="Italic"> jointless-2</Emphasis> gene in the centromeric region of tomato chromosome 12 based on high resolution genetic and physical mapping

Abstract Abscission is a universal process whereby plants shed their organs, such as flowers, fruit and leaves. In tomato, the non-allelic mutations jointless and jointless-2 have been discovered as recessive mutations that completely suppress the formation of pedicel abscission zones. A high resolution genetic map of jointless-2 was constructed using 1,122 jointless F₂ plants. Restriction fragment length polymorphism (RFLP) marker RPD140 completely co-segregated with the jointless-2 locus and mapped in a 2.4 cM interval between RFLP markers CD22 and TG618. To chromosome walk to jointless-2, all three markers were used to screen a bacterial artificial chromosome (BAC) library and contigs were developed. Intensive efforts to expand and merge the BAC contigs were unsuccessful because of the highly repetitive sequence content on the distal ends of each contig. To determine the physical distance between and the orientation of the three contigs, we used high resolution pachytene fluorescence in situ hybridization (FISH) mapping. The RPD140 contig was positioned in the centromeric region of chromosome 12 between two large pericentric heterochromatin blocks, about 50 Mb from the TG618 contig on the short arm and 10 Mb from the CD22 contig on the long arm, respectively. Based on high resolution genetic and physical mapping, we conclude that the jointless-2 gene is located within or near the chromosome 12 centromere where 1 cM is approximately 25 Mb in length.Communicated by Q. ZhangM.A. Budiman, S-B. Chang and S. Lee contributed equally to the work.     

Genetic mapping of jointless-2 to tomato chromosome 12 using RFLP and RAPD markers

Abscission zones are specialized regions in plants, usually located at the base of most plant parts, such as flowers, fruit and leaves, where organs are shed. Although a great deal of information is known about the physiological and biochemical events that lead to organ shedding, very little is known of the molecular events that lead to the formation of the abscission zone itself. In tomato, two recessive mutations have been discovered that completely suppress the formation of flower and fruit pedicel abscission zones, i.e., jointless (j) and jointless-2 (j-2), both tentatively localized to chromosome 11 about 30 cM apart. Because the study of the control of abscission zone development is important for both basic and applied research we are using a map-based cloning approach to identify the jointless genes. The first step in any positional cloning experiment is to establish segregating mapping populations for the target gene and identify closely linked molecular markers that flank the locus. In this study, bulked segregant analysis was used to identify a RAPD marker associated with the j-2 locus, RPD140. To determine the chromosome location of RPD140, we converted it to an RFLP marker that was then mapped on the Cornell reference tomato map in a marker-dense region of chromosome 12. To verify that the j-2 locus was located on tomato chromosome 12, we used nine chromosome 12 RFLP markers linked with RPD140 to map the j-2 gene in an interspecific F₂ mapping population of 151 plants segregating for j-2. The j-2 gene was localized to a 3.0-cM interval between RPD140 and TG618 on tomato chromosome 12. Received: 29 March 1999 / Accepted: 13 October 1999     

Isolation of a 6.2 kb genomic fragment carrying the Adh1 gene of tomato and its expression in transgenic tobacco

An 11 kb Eco RI genomic fragment containing the alcohol dehydrogenase (Adh1) gene was cloned. Cross-hybridization with three Adh2 cDNA clones suggested that the entire coding region of the Adh1 gene was contained on a 6.2 kb Xba I/Hind III subfragment. Using RFLP linkage analysis, the genomic clone was mapped on chromosome 4 between the markers TG 182 and TG 65 in a position corresponding to the Adh1 locus. To further confirm the Adh1 origin of the genomic clone, tobacco plants were transformed with the 6.2 kb Xba I/Hinb III genomic subfragment. Isozyme analysis demonstrated that in transgenic tobacco plants functional tomato specific ADH-1 homodimers were synthesized as well as heterodimers composed of tobacco and tomato subunits.     

Chromosome walking with YAC clones in Arabidopsis: isolation of 1700 kb of contiguous DNA on chromosome 5, including a 300 kb region containing the flowering-time gene CO

The co mutation of Arabidopsis thaliana causes a late-flowering phenotype that is insensitive to day-ength. The mutation was mapped previously to the upper arm of chromosome 5, approximately 1.6 cM from the chalcone synthase gene (CHS). We were provided with five yeast artificial chromosome (YAC) libraries and used these to perform a chromosome walk from CHS to the CO gene. In this paper we report the isolation of 1700 kb of contiguous Arabidopsis DNA, which represents approximately 1%–2% of the genome, inserted in YACs. This required the detailed analysis of 67 YACs, from which 87 end probes were isolated and examined in hybridisation experiments. This analysis showed that approximately 40% of the YACs presented problems in chromosome walking experiments because they contained repetitive sequence at one of their termini, were chimaeric or because part of the plant DNA was deleted. DNA fragments isolated from YACs were used as restriction fragment length polymorphism (RFLP) markers to localize CO to a 300 kb region within the cloned DNA. We compare the physical distance between CHS and CO with the genetic distance and find that in this region 1 cM is equivalent to approximately 200 kb.     

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F₂ population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08 ₇₅₀ was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).     

Molecular mapping of the Arabidopsis locus RPP11 which conditions isolate-specific hypersensitive resistance against downy mildew in ecotype RLD

Isolate WELA of the plant pathogenic oomycete fungus Peronospora parasitica causes downy mildew in the Arabidopsis thaliana ecotypes Weiningen (Wei-0) and La-er, whereas ecotypes RLD and Col-0 are resistant. Genetic crosses between resistant RLD and susceptible Wei-0 showed that resistance was inherited in a simple Mendelian fashion as a monogenic dominant trait. The interactions between different isolates of P. parasitica and ecotypes of A. thaliana show race-specific variation and fit a gene-for-gene relationship. The RPP11 resistance gene was mapped by following the co-segregation of the resistance phenotype with RFLP markers in a mapping population of 254 F₃ families derived from RLD x Wei-0 F₂ individuals. Linkage analysis using version 1.9 of the MAPMAKER program placed the RPP11 resistance locus on chromosome III between marker m249 (two recombinants) and marker g2534 (six recombinants). Markers g2534 and g4117 are on YAC EG7H1. Marker g4117 and one end probe (N5) generated from YAC EG7H1 showed no recombinants. The YAC end probe N5, which was generated by plasmid rescue, was used to screen clones in the Eric Ward YAC library and a YAC was fished (EW19B12) which also hybridised with m249. Thus, a YAC contig has been established over the region where the resistance locus maps. Because the YACs were made with ecotype Columbia DNA it is necessary to isolate the equivalent region from RLD in order to clone the resistance locus. To this end a phage -DASH genomic library was prepared from RLD and a contig covering the relevant region of the YACs is currently under construction.     

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.     

High-resolution, human–bovine comparative mapping based on a closed YAC contig spanning the bovine mh locus

A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments. Received: 6 August 1998 / Accepted: 28 October 1998     

Direct Complementation ofChlamydomonasMutants with Amplified YAC DNA

We previously described the construction and characterization of aChlamydomonasgenomic library in yeast artificial chromosomes (YACs). Here we describe the isolation and genetic mapping of YACs at the FLA10 locus on theunichromosome as well as isolation of a YAC spanning the PF14 locus on chromosome VI. Genetic mapping of YAC end clones by RFLP analyses in interspecific crosses reveals that YACs with a physical size of 150 kb commonly span genetic intervals defined by one or two recombination events in crosses of approximately 20 tetrads. This promises to make chromosomal walking inChlamydomonasa relatively efficient enterprise. We also describe our development of a method for direct complementation of mutant genes by transformation with amplified wildtype YAC DNA. The use of positional cloning using YACs and this direct functional assay for the presence of a gene in a YAC represent powerful molecular genetic tools enabling the cloning of most anyChlamydomonasgene.     

Correlation of genetic and physical structure in the region surrounding the I2Fusarium oxysporum resistance locus in tomato

Summary The dominant gene I ₂ confers on tomato (Lycopersicon esculentum) resistance against the fungus Fusarium oxysporum f. sp. lycopersici race 2. A restriction fragment length polymorphism (RFLP) marker, TG105, has recently been found to be tightly linked to I ₂. The potential for cloning this gene by a reverse genetics approach prompted us to describe in both genetic and physical detail the region surrounding the I ₂ locus on chromosome 11. We have analyzed patterns of segregation of RFLP markers on chromosome 11 and Fusarium resistance in 140 F₂ plants from a cross between Fusarium-resistant and susceptible parental lines. Marker TG105 mapped 0.4 centi-Morgan (CM) from I ₂. Physical analysis of TG105 and its flanking RFLP markers, TG26 and TG36, by pulsed field gradient gel electrophoresis (PFGE) yielded a restriction map for this region encompassing at least 620 kb of the tomato genome. TG105 and TG26 hybridized to the same 175 kb MluI-NruI restriction fragment. We have therefore linked two genetically distinct RFLP markers. Based on the 4.1 cM distance between them, we have assigned a mean value of 43 kb for each cM recombination distance in the vicinity of I ₂. This local ratio between physical and genetic distances is more than 10-fold below the average for the tomato genome. It should therefore be possible to clone I ₂ by chromosome walking from TG105.     

Construction and characterization of a rice YAC library for physical mapping

Genomic libraries of rice,Oryza sativa L. cv. Nipponbare, in yeast artificial chromosomes were prepared for construction of a rice physical map. High-molecular-weight genomic DNA was extracted from cultured suspension cells embedded in agarose plugs. After size fractionation of theEco RI- andNot I-digested DNA fragments, they were ligated with pYAC4 and pYAC55, respectively, and used to transformSaccharomyces cerevisiae AB1380. A total of 6932 clones were obtained containing on average ca. 350 kb DNA. The YAC library was estimated to contain six haploid genome equivalents. The YACs were examined for their chimerism by mapping both ends on an RFLP linkage map. Most YACs withEco RI fragments below 400 kb were intact colinear clones. About 40% of clones were chimeric. Genetic mapping of end clones from large size YACs revealed that the physical distance corresponding to 1 cM genetic distance varies from 120 to 1000 kb, depending on the chromosome region. To select and order YAC clones for making contig maps, high-density colony hybridization using ECL was applied. With several probes, at least one and at most ten YAC clones could be selected in this library. The library size and clone insert size indicate that this YAC library is suitable for physical map construction and map-based cloning.     

Closing in on the Rieger syndrome gene on 4q25: mapping translocation breakpoints within a 50-kb region.

Rieger syndrome (RGS) is an autosomal dominant disorder of morphogenesis affecting mainly the formation of the anterior eye chamber and of the teeth. RGS has been localized to human chromosome 4q25 by linkage to epidermal growth factor (EGF). We have constructed a detailed physical map and a YAC contig of the genomic region encompassing the EGF locus. Using FISH, several YACs could be shown to cross the breakpoint in two independent RGS patients with balanced 4q translocations. Alu- and LINE-fragmentation of a 2.4-Mb YAC generated a panel of shorter YACs ranging in size from 2.4 Mb to 75 kb. Several fragmentation YACs were subcloned in cosmids, which were mapped to specific subregions of the original YAC by hybridization to the fragmentation panel to further refine the localization of the translocation breakpoints, allowing mapping of the breakpoints to within the most-telomeric 200 kb of the original 2.4-Mb YAC. FiberFISH of cosmids located in this 200-kb region mapped the two translocation breakpoints within a 50-kb region approximately 100-150 kb centromeric to D4S193, significantly narrowing down the candidate region for RGS. The mapping data and resources reported here should facilitate the identification of a gene implicated in Rieger syndrome.     

High-resolution linkage analysis and physical characterization of the EIX-responding locus in tomato

An ethylene-inducing xylanase (EIX) from Tricohoderma viride is a potent elicitor of ethylene biosynthesis, localized cell death and other defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum). Wild species of tomato, such as Lycopersicon cheesmanii and Lycopersicon pennellii, do not respond to EIX treatment. The F₁ progeny of a L. esculentum×L. cheesmanii and a L. esculentum×L. pennellii cross responded to EIX treatment with an increase in ethylene biosynthesis and the induction of localized cell death. The F₂ progeny of the above mentioned crosses segregated 3:1 (responding:non-responding). We mapped the EIX-responding locus (Eix) to the short arm of chromosome 7 using a population of introgression lines (ILs), containing small RFLP-defined chromosome segments of L. pennellii introgressed into L. esculentum. RFLP analysis of 990 F₂ plants that segregated for the introgressed segment mapped the Eix locus 0.1 cM and 0.9 cM from the flanking markers TG61 and TG131, respectively. Using the marker TG61 we isolated a yeast artificial chromosome (YAC) clone that carries 300-kb DNA segments derived from the Eix region. By mapping the ends of this YAC clone we show that it spans the Eix locus. Thus, positional cloning of the Eix locus appears feasible. Received: 20 March 1999 / Accepted: 30 April 1999     

Molecular genetic analysis of theripening-inhibitor andnon-ripening loci of tomato: A first step in genetic map-based cloning of fruit ripening genes

Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato,ripening-inhibitor (rin), andnon-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning therin andnor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning therin locus is estimated to be 200–300 kb/cM, while thenor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked torin andnor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains thenot locus.     

Genetic and physical analysis of a YAC contig spanning the fungal disease resistance locus Asc of tomato (Lycopersicon esculentum)

The Alternaria stem canker disease of tomato is caused by the necrotrophic fungal pathogen Alternaria alternata f. sp. lycopersici (AAL). The fungus produces AAL toxins that kill the plant tissue. Resistance to the fungus segregates as a single locus, called Asc, and has been genetically mapped on chromosome 3 of tomato. We describe here the establishment of a 1383-kb YAC contig covering the Asc locus and a series of plants selected for recombination events around the Asc locus. It was shown that the YAC contig corresponds to a genetic distance of at least 11.2 cM. Thus, the recombination rate in the Asc region is six times higher (123 kb/cM) than the average for the tomato genome. Furthermore, the Asc locus could be localised to a 91-kb fragment, thus paving the way for the cloning and identification of the Asc gene(s) by complementation. Received: 31 July 1998 / Accepted: 6 October 1998     

Construction of a barley (Hordeum vulgare L.) YAC library and isolation of a Hor1-specific clone

We have constructed an EcoRI-based YAC (yeast artificial chromosome) library from barley (Hordeum vulgare L. cv. Franka) using the vector pYAC4. The library consists of approximately 18 000 recombinant YACs with insert sizes ranging between 100 and 1000 kb (average of 160 kb) corresponding to 50% of the barley genome. Size fractionation after ligation resulted in an increased average insert size (av. 370 kb) but also in a substantial decrease in cloning efficiency. Less than 1% of the colonies showed homology to a plastome-specific probe; approximately 50% of the colonies displayed a signal with a dispersed, highly repetitive barley-specific probe. Using a primer combination deduced from the sequence of a member of the small Hor1 gene family coding for the C-hordein storage proteins, the library was screened by polymerase chain reaction and subsequently by the colony hybridization technique. A single YAC, designated Y66C11, with a 120 kb insert was isolated. This DNA fragment represents a coherent stretch from the terminal part of the Hor1 gene region as judged from the correspondence of the restriction patterns between Y66C11 DNA and barley DNA after hybridization with the Hor1-specific probe. Restriction with the isoschizomeric enzymes HpaII/MspI suggests a high degree of methylation of the Hor1 region in mesophyll cells but not in YAC-derived (yeast) DNA.