Chromosome landing at the barley Rar1 locus

The barley Rar1 gene is an essential component of the race-specific, Mla-12-specified powdery mildew resistance reaction. As part of a map-based cloning strategy designed to isolate Rar1, five barley yeast artificial chromosomes (YACs) have been identified, ranging in size from 300 to 1100?kb. PCR-based YAC end-specific markers have been established and were employed to construct a local YAC contig. Four out of five YAC clones were found to be non-colinear with the source DNA. High-resolution genetic mapping of the YAC ends demonstrated that the set of five overlapping YAC clones encompasses the barley Rar1 gene.     

Chromosome landing at the barley Rar1 locus

The barley Rar1 gene is an essential component of the race-specific, Mla-12-specified powdery mildew resistance reaction. As part of a map-based cloning strategy designed to isolate Rar1, five barley yeast artificial chromosomes (YACs) have been identified, ranging in size from 300 to 1100 kb. PCR-based YAC end-specific markers have been established and were employed to construct a local YAC contig. Four out of five YAC clones were found to be non-colinear with the source DNA. High-resolution genetic mapping of the YAC ends demonstrated that the set of five overlapping YAC clones encompasses the barley Rar1 gene. Received: 9 June 1998 / Accepted: 15 July 1998     

Chromosome landing at the Arabidopsis TORNADO1 locus using an AFLP-based strategy

The Arabidopsis tornado1 (trn1) mutation causes severe dwarfism combined with twisted growth of all organs. We present a chromosome landing strategy, using amplified restriction fragment length polymorphism (AFLP) marker technology, for the isolation of the TRN1 gene. The recessive trn1 mutation was identified in a C24 transgenic line and is located 5?cM from a T-DNA insertion. We mapped the TRN1 locus to the bottom half of chromosome 5 relative to visible and restriction fragment length polymorphism (RFLP) markers. Recombinant classes within a 3-cM region around TRN1 were used to build a high-resolution map in this region, using the AFLP technique. Approximately 300 primer combinations have been used to test about 26?000 fragments for polymorphisms. Seventeen of these AFLP markers were identified in the 3-cM region around TRN1. These markers were mapped within this region using individual recombinants. Four of these AFLP markers co-segregate with TRN1 whereas one maps at one recombinant below TRN1. We isolated and cloned three of these AFLP markers. These markers identified two yeast artificial chromosome (YAC) clones, containing the RFLP marker above and the AFLP marker below TRN1, demonstrating that these YACs span the TRN1 locus and that chromosome landing has been achieved, using an AFLP-based strategy.     

Chromosome landing at the Arabidopsis TORNADO1 locus using an AFLP-based strategy

 The Arabidopsis tornado1 (trn1) mutation causes severe dwarfism combined with twisted growth of all organs. We present a chromosome landing strategy, using amplified restriction fragment length polymorphism (AFLP) marker technology, for the isolation of the TRN1 gene. The recessive trn1 mutation was identified in a C24 transgenic line and is located 5 cM from a T-DNA insertion. We mapped the TRN1 locus to the bottom half of chromosome 5 relative to visible and restriction fragment length polymorphism (RFLP) markers. Recombinant classes within a 3-cM region around TRN1 were used to build a high-resolution map in this region, using the AFLP technique. Approximately 300 primer combinations have been used to test about 26 000 fragments for polymorphisms. Seventeen of these AFLP markers were identified in the 3-cM region around TRN1. These markers were mapped within this region using individual recombinants. Four of these AFLP markers co-segregate with TRN1 whereas one maps at one recombinant below TRN1. We isolated and cloned three of these AFLP markers. These markers identified two yeast artificial chromosome (YAC) clones, containing the RFLP marker above and the AFLP marker below TRN1, demonstrating that these YACs span the TRN1 locus and that chromosome landing has been achieved, using an AFLP-based strategy. Received: 25 April 1996 / Accepted: 26 June 1996     

Genetic mapping and functional analysis of the tomato Bs4 locus governing recognition of the Xanthomonas campestris pv. vesicatoria AvrBs4 protein

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper (Capsicum spp.) and tomato (Lycopersicon spp.). Analysis of 17 different Lycopersicon accessions with avrBs4-expressing X. campestris pv. vesicatoria strains identified 15 resistant and two susceptible tomato genotypes. Genetic analysis revealed that AvrBs4 recognition in tomato is governed by a single locus, designated Bs4 (bacterial spot resistance locus no. 4). Amplified fragment length polymorphism and bulked DNA templates from resistant and susceptible plants were used to define a 2.6-cM interval containing the Bs4 locus. A standard tomato mapping population was employed to localize Bs4-linked markers on the short arm of chromosome 5. Investigation of X. campestris pv. vesicatoria hrp mutant strains revealed that AvrBs4 secretion and avirulence activity are hrp dependent. Agrobacterium-based delivery of the avrBs4 gene into tomato triggered a plant response that phenotypically resembled the hypersensitive response induced by avrBs4-expressing X. campestris pv. vesicatoria strains, suggesting symplastic perception of the avirulence protein. Mutations in the avrBs4 C-terminal nuclear localization signals (NLSs) showed that NLSs are dispensable for Bs4-mediated recognition. Our data suggest that tomato Bs4 and pepper Bs3 employ different recognition modes for detection of the highly homologous X. campestris pv. vesicatoria avirulence proteins AvrBs4 and AvrBs3.     

Chromosome landing at the Mla locus in barley (Hordeum vulgare L.) by means of high-resolution mapping with AFLP markers

 The complex Mla locus of barley determines resistance to the powdery mildew pathogen Erysiphe graminis f. sp. hordei. With a view towards gene isolation, a population consisting of 950 F₂ individuals derived from a cross between the near-isogenic lines ‘P01’ (Mla1) and ‘P10’ (Mla12) was used to construct a high-resolution map of the Mla region. A fluorescence-based AFLP technique and bulked segregant analysis were applied to screen for polymorphic, tightly linked AFLP markers. Three AFLP markers were selected as suitable for a chromosome-landing strategy. One of these AFLP markers and a closely linked RFLP marker were converted into sequence-specific PCR markers. PCR-based screening of approximately 70 000 yeast artificial chromosome (YAC) clones revealed three identical YACs harbouring the Mla locus. Terminal insert sequences were obtained using inverse PCR. The derived STS marker from the right YAC end-clone was mapped distal to the Mla locus. Received: 17 July 1998 / Accepted: 9 August 1998     

Chromosome landing at the bacterial blight resistance gene<Emphasis Type="Italic"> Xa4</Emphasis> locus using a deep coverage rice BAC library

Xa4 is a dominantly inherited rice gene that confers resistance to Philippine race 1 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae in rice. In order to isolate the gene by positional cloning, a bacterial artificial chromosome (BAC) library was constructed from genomic DNA isolated from an Xa4-harboring accession, IRBB56. The library contains 55,296 clones with an average insert size of 132 kb, providing 14 rice genome equivalents. Three DNA markers closely linked to Xa4 were used to screen the library. The marker RS13, a resistance gene analogue that co-segregates with Xa4, identified 18 clones, of which four and six, respectively, were simultaneously detected by the other two markers, G181 and L1044. Fingerprinting and Southern analysis indicated that these clones overlapped and define an interval spanning 420 kb. In an F2 population derived from an indica variety, IR24, and its Xa4-containing near isogenic line (NIL), IRBB4, the susceptible plants were screened in order to map the Xa4 gene genetically and physically. Out of 24 insert ends isolated from the BACs in the contig, three revealed polymorphisms between IR24 and IRBB4. Two insert ends, 56M22F and 26D24R, flanked Xa4 on each side. Based on the overlap of the BACs, six overlapping clones were considered to include the Xa4 allele, one of which, 106P13, was chosen for further investigation.     

Genetic variation at the tomato Cf-4/Cf-9 locus induced by EMS mutagenesis and intralocus recombination

The interaction between tomato (Lycopersicon esculentum) and the leaf mold pathogen Cladosporium fulvum is an excellent model for investigating disease resistance gene evolution. The interaction is controlled in a gene-for-gene manner by Cf genes that encode type I transmembrane extracellular leucine-rich repeat glycoproteins that recognize their cognate fungal avirulence (Avr) proteins. Cf-4 from L. hirsutum and Cf-9 from L. pimpinellifolium are located at the same locus on the short arm of tomato chromosome 1 in an array of five paralogs. Molecular analysis has shown that one mechanism for generating sequence variation in Cf genes is intragenic sequence exchange through unequal crossing over or gene conversion. To investigate this we used a facile genetic selection to identify novel haplotypes in the progeny of Cf-4/Cf-9 trans-heterozygotes that lacked Cf-4 and Cf-9. This selection is based on the ability of Avr4 and Avr9 to induce Cf-4- or Cf-9-dependent seedling death. The crossovers were localized to the same intergenic region defining a recombination hotspot in this cross. As part of a structure-function analysis of Cf-9 and Cf-4, nine EMS-induced mutant alleles have been characterized. Most mutations result in single-amino-acid substitutions in their C terminus at residues that are conserved in other Cf proteins.     

Mapping of a novel MEN-like syndrome locus to rat Chromosome 4

Multiple endocrine neoplasia-like syndrome (MENX) is a hereditary cancer syndrome in the rat characterized by inborn cataract and multiple tumors affecting the neuroendocrine system developed within the first year of life. The spectrum of affected organs is intermediate between MEN type 1 (MEN1) and MEN type 2 (MEN2) syndromes in human, but, in contrast to them, MENX is inherited in a recessive fashion. Here we report the mapping of the MENX locus to rat Chromosome (Chr) 4 by a genome-wide linkage analysis. This analysis was done in 41 animals obtained from a (Wistar/Nhg × SD^we) × SD^we interstrain backcross, where SD^we (Sprague-Dawley white eye) indicates the affected animals. The MENX disease locus was ultimately mapped to a ~22-cM interval on Chr 4 that includes the rat homolog of the human RET proto-oncogene. As activating point mutations of RET are known to be responsible for MEN2 in human, we analyzed several markers located in the proximity of Ret for linkage to the disease phenotype. Our data exclude Ret involvement in MENX and establish that a second gene, playing a role in endocrine tumor formation, lies within the distal part of rat Chr 4. Although heritable human endocrine tumors are quite rare, sporadic tumors of MEN-affected tissues occur at a much higher frequency, and their pathogenesis is poorly understood. The identification of the MENX gene should contribute to our understanding of the genetic mechanisms of neuroendocrine tissue tumorigenesis and may assist in developing new and more appropriate therapeutic strategies for these diseases.     

Chromosome 5 allele loss at the glucocorticoid receptor locus in human colorectal carcinomas

Matched normal/tumor DNA pairs from sporadic colon carcinoma patients were examined for chromosome 5 allele loss using a probe for a functional gene (glucocorticoid receptor = GRL) locus. This locus maps (5q11-q13) close to one of two alternative sites recently reported for a constitutional deletion in a familial adenomatous polyposis (FAP) patient. Tumor-specific allele loss of at least 27% at GRL supports the hypothesis that both hereditary and sporadic forms of colon cancer result from mutations of the same gene. The proximity of the GRL locus to the region of 5q affected in FAP and the observed tumor-specific allele loss at this locus suggest that further research is needed regarding whether genetic alterations in the glucocorticoid receptor may be associated with colon carcinogenesis.     

A congenic mouse and candidate gene at the Chromosome 13 locus regulating bone density

Previously, we identified two significant quantitative trait loci (QTLs) specifying the peak relative bone mass (bone mass corrected for bone size) on chromosomes (Chrs) 11 and 13 by interval mapping in two mouse strains, SAMP2 and SAMP6. The latter strain is an established murine model of senile osteoporosis and exhibits a significantly lower peak relative bone mass than SAMP2 mice. We recently designated the Chr 13 locus as Pbd2 (Peak bone density 2) and constructed a congenic strain, P6.P2-Pbd2(b), which carried a single genomic interval from the Chr 13 of SAMP2 on a SAMP6-derived osteoporotic background. In this study, we have constructed a congenic strain, P2.P6-Pbd2(a), carrying a SAMP6-derived susceptible interval on a SAMP2-derived resistance background. This congenic strain had a lower bone density than the background strain, SAMP2, based on three measurement methods, each utilizing a different principle for evaluating bone density: MD, DXA, and pQCT. Next, a candidate gene approach was used to find polymorphisms of Bmp6 (bone morphogenetic protein 6). The CAG trinucleotide repeat numbers in exon 1 of this gene differ among SAM strains. We found an association of CAG repeat length with relative peak bone mass in mice.     

Evidence for a revertant at the La locus in regenerating hypocotyl segments in tomato

Summary Leafy and leafless phenotypes were regenerated in vitro from hypocotyl segments of leafless forms (reduced and modified) of the homozygous lanceolate (La) mutant in tomato. Segregation of progeny of leafy regenerates into homozygous. mutant (La La), heterozygote (La La ⁺) and normal (La ⁺ La ⁺) indicates that cells forming the shoot apical meristems undergo a genetic reversion, and that the nutrient medium might be selecting for the heterozygote. Among the progeny of the regenerates is a true breeding, unlobed variant. Leaves of the variant are pinnately compound and the margins are entire. Opposite cotyledons followed in development by two simple leaves before the appearance of a pinnately compound leaf with an occasional lanceolate-shaped leaflet suggests that the unlobed variant is morphologically intermediate between La La ⁺ and La ⁺ La ⁺.     

A second gene at the tomato Cf-4 locus confers resistance to cladosporium fulvum through recognition of a novel avirulence determinant

The tomato Cf-4 and Cf-9 genes confer resistance to the leaf mould pathogen Cladosporium fulvum and map at a complex locus on the short arm of chromosome 1. It was previously shown that the gene encoding Cf-4, which recognizes the Avr4 avirulence determinant, is one of five tandemly duplicated homologous genes (Hcr9-4s) at this locus. Cf-4 was identified by molecular analysis of rare Cf-4/Cf-9 disease-sensitive recombinants and by complementation analysis. The analysis did not exclude the possibility that an additional gene(s) located distal to Cf-4 may also confer resistance to C. fulvum. We demonstrate that a number of Dissociation-tagged Cf-4 mutants, identified on the basis of their insensitivity to Avr4, are still resistant to infection by C. fulvum race 5. Molecular analysis of 16 Cf-4 mutants, most of which have small chromosomal deletions in this region, suggested the additional resistance specificity is encoded by Hcr9-4E. Hcr9-4E recognizes a novel C. fulvum avirulence determinant that we have designated Avr4E.     

Genetic characterization of the polycotyledon locus in tomato

Developmental mutants serve as a useful material to unravel the mechanisms necessary for organ development. The polycotyledon (poc) mutant of tomato, with multiple cotyledons in the seedling and varied phenotypic effects in the adult plant is one such mutant. Studies using physiological and anatomical methods in our lab suggest that POC is involved in the negative regulation of polar auxin transport, which is likely the reason for the pleiotropic phenotype in the mutant. Because of the physiological significance of the polycotyledon mutant described in this paper and also being first of its kind in tomato and also other plant species, we are using a map-based cloning approach to map the polycotyledon gene. Molecular mapping of this locus using segregating interspecific F₂ mapping population localized polycotyledon gene close to TG424 marker on the long arm of chromosome 9. The closest marker mapped was a PCR marker identified in this study, E8A2 at a distance of 7.4 cM from the poc locus. The absence of tightly linked RAPD markers and the non-availability of more mapped markers in this region led us to initiate chromosome walk to polycotyledon gene. Both the flanking markers TG248 and E8A2 were used to screen the BAC library and a contig was developed for TG248 marker. The BAC-end sequences were analyzed for their use as RFLP markers to enrich this region for markers. Analysis of the BAC-end sequences revealed that poc is localized in the region surrounded by copia-like retrotransposon elements explaining the absence of markers in the euchromatin region on long arm of chromosome 9. Further studies identified two BAC-end sequences which mapped around the poc locus and also indicated very low physical versus genetic distance ratio in this region. The double mutant analyses of poc with the other two known polycotyledon mutants of tomato, pct and dem revealed allelism with pct; therefore, the poc mutant was named as pct1-2, and also the original pct mutant was renamed as pct1-1.     

Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids

The phenomenon of segregation of gene expression has been examined in intraspecific somatic cell hybrids. Specifically, segregation at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus has been studied in hybrids of Chinese hamster cell lines. The role of chromosome segregation, or other chromosomal events has been assessed by detailed comparison of karyotypes in the 6-thioguanine resistant segregants with those of the parental hybrid lines. The results clearly demonstrate that loss of an entire X chromosome is the primary event responsible for segregation at the HPRT locus, while deletion of a portion of the short arm of an X chromosome was also a frequent event. The results provide the first direct evidence for the assignment of the mapping of this locus to the distal region of the short arm. Analysis of chromosome number distributions in the hybrids and segregants suggests that in selecting chromosomal segregants one may also select for hybrid lines with reduced chromosome stability.