Biomedical subjects

S D Tanksley

Publications and source records attributed to S D Tanksley.

At least 19 recordsLinked to original sources

PubMed1996

Molecular mapping of the centromeres of tomato chromosomes 7 and 9.

The centromeres of two tomato chromosomes have been precisely localized on the molecular linkage map through dosage analysis of trisomic stocks. To map the centromeres of chromosomes 7 and 9, complementary telo-, secondary, and tertiary trisomic stocks were used to assign DNA markers to their respective chromosome arms and thus to localize the centromere at the junction of the short and long arms. It was found that both centromeres are situated within a cluster of cosegregating markers. In an attempt to order the markers within the centric clusters, genetic maps of the centromeric regions of chromosomes 7 and 9 were constructed from F2 populations of 1620 Lycopersicon esculentum x L. pennellii (E x P) plants and 1640 L. esculentum x L. pimpinellifolium (E x PM) plants. Despite the large number of plants analyzed, very few recombination events were detected in the centric regions, indicating a significant suppression of recombination at this region of the chromosome. The fact that recombination suppression is equally strong in crosses between closely related (E x PM) and remotely related (E x P) parents suggests that centromeric suppression is not due to DNA sequence mismatches but to some other mechanism. The greatest number of centromeric markers was resolved in the L. esculentum x L. pennellii F2 population. The centromere of chromosome 7 is surrounded by eight cosegregating markers: three on the short arm, five on the long arm. Similarly, the centric region of chromosome 9 contains ten cosegregating markers including one short arm marker and nine long arm markers. The localization of centromeres to precise intervals on the molecular linkage map represents the first step towards the characterization and ultimate isolation of tomato centromeres.

Centromere↗

PubMed1996

Characterization and genetic mapping of simple repeat sequences in the tomato genome.

Tomato genomic libraries were screened for the presence of simple sequence repeats (SSRs) with seventeen synthetic oligonucleotide probes, consisting of 2- to 5-basepair motifs repeated in tandem. GAn and GTn sequences were found to occur most frequently in the tomato genome (every 1.2 Mb), followed by ATTn and GCCn (every 1.4 Mb and 1.5 Mb, respectively). In contrast, only ATn and GAn microsatellites (n > 7) were found to be frequent in the GenBank database, suggesting that other motifs may be preferentially located away from genes. Polymorphism of microstellites was measured by PCR amplification of individual loci of by Southern hybridization, using a set of ten tomato cultivars. Surprisingly, only two of the nine microsatellite clones surveyed (five GTn, three GAn and one ATTn), showed length variation among these accessions. Polymorphism was also very limited between Lycopersicon esculentum and L. pennelli, two distant species. Southern analysis using the seventeen oligonucleotide probes identified GATAn and GAAAn as useful motifs for the detection of multiple polymorphic fragments among tomato cultivars. To determine the structure of microsatellite loci, a GAn probe was used for hybridization at low stringency on a small insert genomic library, and randomly selected clones were analyzed. GAn based motifs of increasing complexity were found, indicating that simple dinucleotide sequences may have evolved into larger tandem repeats such as minisatellites as a result of basepair substitution, replication slippage, and possibly unequal crossing-over. Finally, we genetically mapped loci corresponding to two amplified microsatellites, as well as nine large hypervariable fragments detected by Southern hybridization with a GATA8 probe. All loci are located around putative tomato centromeres. This may contribute to understanding of the structure of centromeric regions in tomato.

Base Sequence↗

PubMed1995

Molecular genetic analysis of the ripening-inhibitor and non-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), and non-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 the rin and nor 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 the rin locus is estimated to be 200-300 kb/cM, while the nor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked to rin and nor, 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 the nor locus.

Base Sequence↗

PubMed1995

Abundance, variability and chromosomal location of microsatellites in wheat.

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)n blocks was estimated to be 3.6 x 10(4) and the number of (GT)n blocks to be 2.3 x 10(4) per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.

Base Sequence↗

PubMed1995

Chromosome landing: a paradigm for map-based gene cloning in plants with large genomes.

The original concept behind map-based or positional cloning was to find a DNA marker linked to a gene of interest, and then to 'walk' to the gene via overlapping clones (e.g. cosmids or YACs). While chromosome walking is straightforward in organisms with small genomes, it is difficult to apply in most plant species, which typically have large, complex genomes. The strategy of chromosome walking is based on the assumption that it is difficult and time consuming to find DNA markers that are physically close to a gene of interest. Recent technological developments invalidate this assumption for many species. As a result, the mapping paradigm has now changed such that one first isolates one or more DNA marker(s) at a physical distance from the targeted gene that is less than the average insert size of the genomic library being used for clone isolation. The DNA marker is then used to screen the library and isolate (or 'land' on) the clone containing the gene, without any need for chromosome walking and its associated problems. Chromosome landing, together with the technology that has made it possible, is likely to become the main strategy by which map-based cloning is applied to isolate both major genes and genes underlying quantitative traits in plant species.

Chromosome Mapping↗

PubMed1995

Dominance is the major genetic basis of heterosis in rice as revealed by QTL analysis using molecular markers.

A set of 194 F7 lines derived from a subspecific rice cross showing strong F1 heterosis was backcrossed to the two parents. The materials (388 BC1F7 lines, 194 F8 lines, two parents, F1) were phenotyped for 12 quantitative traits. A total of 37 significant QTLs (LOD > or = 2.0) was detected through 141 RFLP markers in the BC1F7 populations. Twenty-seven (73%) quantitative trait loci (QTLs) were detected in only one of the BC1F7 populations. In 82% of these cases, the heterozygotes were superior to the respective homozygotes. The remaining 10 (27%) QTLs were detected in both BC1F7 populations, and the heterozygote had a phenotype falling between those of the two homozygotes and in no instances were the heterozygotes found to be superior to both homozygotes. These results suggest that dominance complementation is the major genetic basis of heterosis in rice. This conclusion was strengthened by the finding that there was no correlation between most traits and overall genome heterozygosity and that there were some recombinant inbred lines in the F8 population having phenotypic values superior to the F1 for all of the traits evaluated--a result not expected if overdominance was a major contributor to heterosis. Digenic epistasis was not evident.

Genes, Dominant↗

PubMed1995

The tomato Never-ripe locus regulates ethylene-inducible gene expression and is linked to a homolog of the Arabidopsis ETR1 gene.

Fruit ripening represents a complex system of genetic and hormonal regulation of eukaryotic development unique to plants. We are using tomato ripening mutants as tools to elucidate genetic components of ripening regulation and have recently demonstrated that the Never-ripe (Nr) mutant is insensitive to the plant growth regulator ethylene (M.B. Lanahan, H.-C. Yen, J.J. Giovannoni, H.J. Klee [1994] Plant Cell 6:521-530). We report here ethylene sensitivity over a range of concentrations in normal and Nr tomato seedlings and show that the Nr mutant retains residual sensitivity to as little as 1 part per million of ethylene. Analysis of ripening-related gene expression in normal and mutant ethylene-treated fruit demonstrates that Nr exerts its influence on development at least in part at the level of ethylene-inducible gene expression. We have additionally used cloned tomato and Arabidopsis sequences known to influence ethylene perception as restriction fragment length polymorphism probes, and have identified a tomato locus linked to Nr that hybridizes to the Arabidopsis ETR1 gene at low stringency, suggesting the possibility that Nr may be homologous to ETR1.

Arabidopsis↗

PubMed1995

Pulsed-field gel analysis of 5S and satellite DNA in barley.

Pulsed-field gel electrophoresis was used to study the variability of clustered tandemly repeated sequences in barley. Twelve spring barley cultivars were investigated with a heterologous 5S DNA probe and the 118 base pair barley satellite DNA probe HVT01. On a per fragment basis, the 5S probe was 5 times and the barley satellite probe 6.7 times more variable than single- or low-copy RFLP markers, demonstrating their usefulness for cultivar distinction.

Base Sequence↗

PubMed1994

Map-based cloning in crop plants: tomato as a model system II. Isolation and characterization of a set of overlapping yeast artificial chromosomes encompassing the jointless locus.

A map-based cloning technique for crop plants is being developed using tomato as a model system. The target gene jointless is a recessive mutation that completely suppresses the formation of flower and fruit pedicel abscission zones. Previously, the jointless locus was mapped to a 3 cM interval between the two molecular markers TG523 and RPD158. Physical mapping of the jointless region by pulsed-field gel electrophoresis demonstrated that TG523 and RPD158 reside on a 600 kb SmaI fragment. In this study, TG523 was used as a probe to screen a tomato yeast artificial chromosome (YAC) library. Six tomato YAC (TY) clones were isolated, ranging from 220 to 380 kb in size. Genetic mapping of YAC ends demonstrated that this set of overlapping YACs encompasses the jointless locus. Two YAC ends, TY159L (L indicates left end) and TY143R (R indicates right end), cosegregate with the jointless locus. Only one of the six YACs (TY142) contained single-copy DNA sequences at both ends that could be mapped. The two ends of TY142 were mapped to either side of the jointless locus, indicating that TY142 contains a contiguous 285 kb tomato DNA fragment that probably includes the jointless locus. Physical mapping of the TY142 clone revealed that TY159L and TY143R reside on a 55 kb SalI fragment. Southern blot hybridization analysis of the DNAs of tomato lines nearly isogenic for the jointless mutation has allowed localization of the target locus to a region of less than 50 kb within the TY142 clone.

Base Sequence↗

PubMed1994

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.

Base Sequence↗

PubMed1994

Chromosome 2-specific DNA clones from flow-sorted chromosomes of tomato.

We obtained DNA clones specific to tomato chromosome 2 from a small number of chromosomes collected by flow sorting. Suspensions of metaphase chromosomes were prepared from 3-month-old tomato cell cultures of Lycopersicon pennellii. Isolated chromosomes stained with chromomycin A3 and Hoechst 33258 were analyzed on an EPICS 753 flow cytometer using a UV laser to excite Hoechst fluorescence and a 458 nm laser to excite chromomycin A3 fluorescence. Chromosomes from well-resolved peaks on a bivariate flow karyotype were sorted directly onto membrane filters for spot-blot analysis. The filters were processed and hybridized with chromosome-specific repetitive DNA probes. In this way tomato chromosome 1 and chromosome 2 were assigned to peaks in the bivariate flow karyotypes. One thousand copies of the putative chromosome 2 were flow-sorted directly into microfuge tubes. DNA specific to chromosome 2 was amplified by a polymerase chain reaction (PCR) technique using universal 22mer degenerate oligonucleotide primers (DOP) sequences. DOP-PCR yields a smear of fragments of various sizes from 250 to 1600 bp. Amplified products were cloned into the Bluescript plasmid vector. Approximately 11% of the clones contained sequences with highly repetitive elements, and 85% contained only low-copy-number sequences. Eleven clones containing low-copy-number sequences that detect restriction fragment length polymorphisms were placed on the molecular linkage map of tomato. All showed linkage to chromosome 2.

Amino Acid Sequence↗

PubMed1994

A member of the tomato Pto gene family confers sensitivity to fenthion resulting in rapid cell death.

Leaves of tomato cultivars that contain the Pto bacterial resistance locus develop small necrotic lesions within 24 hr after exposure to fenthion, an organophosphorous insecticide. Recently, the Pto gene was isolated and shown to be a putative serine/threonine protein kinase. Pto is one member of a multigene family that is clustered within a 400-kb region on chromosome 5. Here, we report that another member of this gene family, termed Fen, is responsible for the sensitivity to fenthion. Fen was isolated by map-based cloning using closely linked DNA markers to identify a yeast artificial chromosome clone that spanned the Pto region. After transformation with the Fen gene under control of the cauliflower mosaic virus (CaMV) 35S promoter, tomato plants that are normally insensitive to fenthion rapidly developed extensive necrotic lesions upon exposure to fenthion. Two related insecticides, fensulfothion and fenitrothion, also elicited necrotic lesions specifically on Fen-transformed plants. Transgenic tomato plants harboring integrated copies of the Pto gene under control of the CaMV 35S promoter displayed sensitivity to fenthion but to a lesser extent than did wild-type fenthion-sensitive plants. The Fen protein shares 80% identity (87% similarity) with Pto but does not confer resistance to Pseudomonas syringae pv tomato. These results suggest that Pto and Fen participate in the same signal transduction pathway.

Amino Acid Sequence↗

PubMed1993

Map-based cloning of a protein kinase gene conferring disease resistance in tomato.

The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.

Amino Acid Sequence↗

PubMed1993

Comparative linkage maps of the rice and maize genomes.

Genetic linkage maps have been constructed for the rice and maize genomes on the basis of orthologous loci detected with a common set of cDNA clones. Conserved linkage groups could be identified, which together account for more than two-thirds of both genomes. In some instances, entire chromosomes or chromosome arms are nearly identical with respect to gene order and gene content. The results also reveal that most of the genes (> 72%) duplicated during ancient polyploidization are still present in the maize genome in duplicate copy. The comparative maps of rice and maize provide a basis for interpreting molecular, genetic, and breeding information between these two important species and establish a framework for ultimately connecting the genetics of all grass species.

Biological Evolution↗

PubMed1993

Pooled-sampling makes high-resolution mapping practical with DNA markers.

A pooled-sample approach to the construction of high-resolution genetic maps is described. The strategy depends on the existence of an easily selectable target locus and the ability to produce large segregating populations. If these requirements are met, the pooled-sample mapping approach allows tightly linked markers (e.g., restriction fragment length polymorphisms) to be mapped relative to the target with a great economy of effort. The recombination fractions among loci can be estimated by the maximum likelihood method and a simple approximate estimator is derived. The order of loci is deduced using a Bayesian statistical framework to yield posterior probabilities for all possible orderings of a marker set. Optimal pooling strategies and the effects of misclassification of selected individuals are discussed and studied by computer simulation. The feasibility of this method is demonstrated by the high-resolution mapping of a region on chromosome 5 of tomato that contains a gene regulating fruit ripening.

Animals↗

PubMed1993

Organisation of the tomato polyphenol oxidase gene family.

We report the isolation and characterization of seven nuclear genes encoding polyphenol oxidase (PPO) in tomato (Lycopersicon esculentum cv. VFNT Cherry). The seven genes (PPOs A, A', B, C, D, E and F) fall into three structural classes (I, II, and III) based on Eco RI and Hind III restriction fragment length polymorphisms (RFLP). RFLP mapping and PFGE analysis demonstrated that the genes reside on chromosome 8, and may be clustered within a 165 kb region. Phage insert mapping demonstrated PPO E and PPO F (both class III), and PPOs B, D and A (classes I, II and I respectively) are grouped within separate 12.4 kb clusters. The complete nucleotide sequence was determined for each gene. Comparison to cDNAs revealed that the PPOs lack introns. A transcript of about 2 kb is expected for each PPO. Each PPO possesses a region encoding a transit peptide characteristic of polypeptides targeted to the thylakoid lumen. Predicted precursor polypeptides range in mass from 66 to 71 kDa and predicted mature polypeptides range from 57 to 62 kDa. All the PPOs encode two putative copper-binding sites characteristic of bacterial, fungal and mammalian tyrosinases. Five of the seven PPOs possess divergent DNA sequences in their 5' promoter regions. These flanking sequence differences may regulate the differential expression of PPO genes.

Amino Acid Sequence↗

PubMed1993

Genetic and physical mapping of telomeres and macrosatellites of rice.

Telomeres and telomere-associated satellites of rice were genetically and physically analyzed by pulsed-field gel electrophoresis (PFGE) using Arabidopsis telomeric DNA and rice satellite sequences as probes. We demonstrate that Arabidopsis telomeric sequences hybridize to rice telomeres under the conditions of high stringency. Using the Arabidopsis probe, multiple, discrete telomeric fragments could be identified on pulsed-field gel blots of rice DNAs digested with rare-cutting restriction enzymes. Most of the telomeric bands larger than 300 kb are physically linked with satellite bands as revealed by PFGE. Some of the telomeric and satellite bands segregate in a Mendelian fashion and are highly reproducible. Three such telomeric bands have been mapped to the distal ends of RFLP linkage groups: Telsm-1 on chromosome 8, Telsa-1 on chromosome 9 and Telsm-3 on chromosome 11. One segregating satellite band was mapped to an internal region of chromosome 10. Telomeric fragments were shown not only to be genetically linked to but also physically linked (based on PFGE) to the terminal RFLP markers. The physical distance from telomeric sequences to a distal RFLP marker, r45s gene, on chromosome 9, is 200 kb while the distance from telomeric sequences to RG98, a terminal RFLP marker on chromosome 11, is 260 kb. Physical maps of the telomere regions of chromosome 9 and chromosome 11 are presented.

Arabidopsis↗

PubMed1993

Characterization of tomato DNA clones with sequence similarity to human minisatellites 33.6 and 33.15.

A tomato lambda genomic library was screened with the human minisatellites 33.6 and 33.15. Similar tomato sequences are estimated to occur on average every 4000 kb. In thirteen hybridizing clones characterized, the size of minisatellite arrays varied between 100 bp and 3 kb. The structure of the repetitive elements is complex as the human core sequence is interspersed with other elements. In three cases, sequences similar to the human minisatellites were part of a higher-order tandem repeat. The chromosomal position of these sequences was established by ascertaining linkage to previously mapped RFLP markers. In contrast to the human genome, no clustering of minisatellite loci was observed in tomato. The fingerprints generated by hybridizing tomato minisatellites to genomic DNA of a set of cultivars were, in two cases, more variable than those obtained with 33.6 or 33.15. Two of the characterized probes detected 4-8 alleles of a single locus, which displayed 10-15 times more polymorphism than random RFLP clones. Some minisatellites contain di- and tri-nucleotide microsatellite repeated motifs which may account for the high level of polymorphism detected with these clones.

Base Sequence↗