The distribution of RFLP markers on chromosome 2(2H) of barley in relation to the physical and genetic location of 5S rDNA

The 5S rDNA locus on the long arm of barley chromosome 2(2H) was genetically mapped in two crosses in relation to 30 other RFLP loci. Comparison of the genetic maps with the previously published physical position of the 5S rDNA, determined by in-situ hybridization, showed that there was a marked discrepancy between physical and genetic distance in both crosses, with recombination being less frequent in the proximal part of the arm. Pooled information from the present study and other published genetic maps showed that at least 26 of the 44 (59%) RFLPs that have been mapped on 2(2H)L lie distal to the 5S rDNA locus even though this region is only 27% of the physical length of the arm. The distribution of RFLP markers is significantly different from expected (P < 0.01), implying that the low-copy sequences used for RFLP analysis occur more frequently in distal regions of the arm and, or, that sequences in distal regions are more polymorphic.     

Factors affecting polymorphism at microsatellite loci in bread wheat [<Emphasis Type="Italic">Triticum aestivum</Emphasis> (L.) Thell]: effects of mutation processes and physical distance from the centromere

The effects of factors known to influence the level of polymorphism at microsatellite loci were studied using 99 markers and seven lines of bread wheat. Mutational factors as well as indirect selective events shape diversity at these loci. Theory predicts that the selection of favorable alleles should reduce polymorphism at neutral neighboring loci in genomic areas with low recombination rates. In wheat, local recombination rate is positively correlated with physical distance from the centromere. Seventy four loci among the 99 used could be physically located on the chromosome. We studied how the following affected the diversity among a set of inbred lines: the length of the alleles, the motif (CA versus CT), the structure of the loci (perfect versus imperfect) and the chromosomal position of the loci. For each locus, we determined whether the polymorphism observed at a locus was compatible with the Stepwise Mutation Model (SMM) or the Two-Phase Model (TPM). Both the mutation rate and the compatibility with the SMM or the TPM were shown to be variable between loci. Wheat microsatellite loci were found to be more variable when segregating alleles were perfect and had long motifs (composed of many repetitions). Diversity observed at 19 loci was not compatible with the SMM. Loci located in distal regions, with presumably high recombination rates, had longer allele sizes and were more polymorphic than loci located in proximal regions. We conclude that both mutation factors and indirect selective events vary according to the local recombination rate and therefore jointly influence the level of polymorphism at microsatellite loci in wheat.Communicated by J. Dvorak     

RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat

A population of single chromosome recombinant lines was developed from the cross between a frost-sensitive, vernalization-insensitive substitution line, ‘Chinese Spring’ (Triticum spelta 5A) and a frost-tolerant, vernalization-sensitive line, ‘Chinese Spring’ (‘Cheyenne’ 5A), and used to map the genes Vrn1 and Fr1 controlling vernalization requirement and frost tolerance, respectively, relative to RFLP markers located on this chromosome. The Vrn1 and Fr1 loci were located closely linked on the distal portion of the long arm of 5AL, but contrary to previous observations, recombination between them was found. Three RFLP markers, Xpsr426, Xcdo504 and Xwg644 were tightly linked to both. The location of Vrn1 suggests that it is homoeologous to other spring habit genes in related species, particularly the Sh2 locus on chromosome 7 (5H) of barley and the Sp1 locus on chromosome 5R of rye.     

Highly recombinogenic regions at seed storage protein loci on chromosome 1DS of Aegilops tauschii, the D-genome donor of wheat

A detailed RFLP map was constructed of the distal end of the short arm of chromosome 1D of Aegilops tauschii, the diploid D-genome donor species of hexaploid wheat. Ae. tauschii was used to overcome some of the limitations commonly associated with molecular studies of wheat such as low levels of DNA polymorphism. Detection of multiple loci by most RFLP probes suggests that gene duplication events have occurred throughout this chromosomal region. Large DNA fragments isolated from a BAC library of Ae. tauschii were used to determine the relationship between physical and genetic distance at seed storage protein loci located at the distal end of chromosome 1DS. Highly recombinogenic regions were identified where the ratio of physical to genetic distance was estimated to be <20 kb/cM. These results are discussed in relation to the genome-wide estimate of the relationship between physical and genetic distance.     

Genetic analysis of eight loci tightly linked to neurofibromatosis 1

The genetic locus for neurofibromatosis 1 (NF1) has recently been mapped to the pericentromeric region of chromosome 17. We have genotyped eight previously identified RFLP probes on 50 NF1 families to determine the placement of the NF1 locus relative to the RFLP loci. Thirty-eight recombination events in the pericentromeric region were identified, eight involving crossovers between NF1 and loci on either chromosomal arm. Multipoint linkage analysis resulted in the unique placement of six loci at odds greater than 100:1 in the order of pter-A10-41-EW301-NF1-EW207-CRI-L581-CRI-L946 -qter. Owing to insufficient crossovers, three loci--D17Z1, EW206, and EW203--could not be uniquely localized. In this region female recombination rates were significantly higher than those of males. These data were part of a joint study aimed at the localization of both NF1 and tightly linked pericentromeric markers for chromosome 17.     

Identification of an RFLP interval containing Pch2 on chromosome 7AL in wheat.

The gene Pch2 in 'Cappelle Desprez' is one of two genes found in hexaploid wheat known to confer resistance to eyespot disease. This study was conducted to develop an RFLP linkage map of the distal portion of wheat chromosome 7AL, and to locate and identify markers closely associated with Pch2 for use in marker-assisted selection. Ten loci in addition to Pch2 were mapped on chromosome 7AL, using segregation data from 102 homozygous chromosome 7A recombinant substitution lines derived from 'Chinese Spring' x 'Chinese Spring' ('Cappelle Desprez' 7A). The Pch2 locus was bracketed by two RFLP markers, one 11.0 cM distal to Xcdo347 and the other 18.8 cM proximal to Xwg380. The position of Pch2 on chromosome 7AL is similar to that of Pch1 on chromosome 7DL, suggesting that these resistance genes are homoeoloci. Although no single marker was closely linked to Pch2, simultaneous selection of the flanking RFLP markers Xcdo347 and Xwg380 could be used for selecting Pch2, since double recombination occurred in only 3% of the recombinant population. The use of the flanking RFLP markers to select for Pch2, in combination with previously identified Pch1-linked markers, would facilitate the development of cultivars carrying two genes for resistance to eyespot.     

RFLP maps of potato and their alignment with the homoeologous tomato genome

Summary An RFLP linkage map of the potato is presented which comprises 304 loci derived from 230 DNA probes and one morphological marker (tuber skin color). The self-incompatibility locus of potato was mapped to chromosome I, which is homoeologous to tomato chromosome I. By mapping chromosome-specific tomato RFLP markers in potato and, vice versa, potato markers in tomato, the different potato and tomato RFLP maps were aligned to each other and the similarity of the potato and tomato genome was confirmed. The numbers given to the 12 potato chromosomes are now in accordance with the established tomato nomenclature. Comparisons between potato RFLP maps derived from different genetic backgrounds revealed conservation of marker order but differences in chromosome and total map length. In particular, significant reduction of map length was observed in interspecific compared to intraspecific crosses. The distribution of regions with distorted segregation ratios in the genome was analyzed for four potato parents. The most prominent distortion of recombination was found to be caused by the self-incompatibility locus.     

Size Variation of the Nonrecombining Region on the Mating-Type Chromosomes in the Fungal Podospora anserina Species Complex

Sex chromosomes often carry large nonrecombining regions that can extend progressively over time, generating evolutionary strata of sequence divergence. However, some sex chromosomes display an incomplete suppression of recombination. Large genomic regions without recombination and evolutionary strata have also been documented around fungal mating-type loci, but have been studied in only a few fungal systems. In the model fungus Podospora anserina (Ascomycota, Sordariomycetes), the reference S strain lacks recombination across a 0.8-Mb region around the mating-type locus. The lack of recombination in this region ensures that nuclei of opposite mating types are packaged into a single ascospore (pseudohomothallic lifecycle). We found evidence for a lack of recombination around the mating-type locus in the genomes of ten P. anserina strains and six closely related pseudohomothallic Podospora species. Importantly, the size of the nonrecombining region differed between strains and species, as indicated by the heterozygosity levels around the mating-type locus and experimental selfing. The nonrecombining region is probably labile and polymorphic, differing in size and precise location within and between species, resulting in occasional, but infrequent, recombination at a given base pair. This view is also supported by the low divergence between mating types, and the lack of strong linkage disequilibrium, chromosomal rearrangements, transspecific polymorphism and genomic degeneration. We found a pattern suggestive of evolutionary strata in P. pseudocomata. The observed heterozygosity levels indicate low but nonnull outcrossing rates in nature in these pseudohomothallic fungi. This study adds to our understanding of mating-type chromosome evolution and its relationship to mating systems.     

Meiotic Recombination, Noncoding DNA and Genomic Organization in Caenorhabditis Elegans

The genetic map of each Caenorhabditis elegans chromosome has a central gene cluster (less pronounced on the X chromosome) that contains most of the mutationally defined genes. Many linkage group termini also have clusters, though involving fewer loci. We examine the factors shaping the genetic map by analyzing the rate of recombination and gene density across the genome using the positions of cloned genes and random cDNA clones from the physical map. Each chromosome has a central gene-dense region (more diffuse on the X) with discrete boundaries, flanked by gene-poor regions. Only autosomes have reduced rates of recombination in these gene-dense regions. Cluster boundaries appear discrete also by recombination rate, and the boundaries defined by recombination rate and gene density mostly, but not always, coincide. Terminal clusters have greater gene densities than the adjoining arm but similar recombination rates. Thus, unlike in other species, most exchange in C. elegans occurs in gene-poor regions. The recombination rate across each cluster is constant and similar; and cluster size and gene number per chromosome are independent of the physical size of chromosomes. We propose a model of how this genome organization arose.     

High-resolution mapping of the S and Z loci of Phalaris coerulescens.

Self incompatibility (SI) in Phalaris coerulescens is gametophytically determined by two unlinked multi allelic loci (S and Z). Neither the S nor Z genes have yet been cloned. As part of a map-based cloning strategy, high-resolution maps of the S and Z regions were generated from distorted segregating populations using RFLP probes from wheat, barley, oat, and Phalaris. The S locus was delimited to 0.26 cM with two boundary markers (Xwg811 and Xpsr168) and cosegregated with Xbm2 and Xbcd762. Xbcd266 was the closest marker linked to Z (0.9 cM). A high level of colinearity in the S and Z regions was found in both self-incompatible and -compatible species. The S locus was localized to the subcentromere region of chromosome 1 and the Z locus to the long arm end of chromosome 2. Several rice BAC clones orthologous to the S and Z locus regions were identified. This opens the possibility of using the rice genome sequence data to generate more closely linked markers and identify SI candidate genes. These results add further support to the conservation of gene order in the S and Z regions of the grass genomes.     

Linkage analysis of the human dopamine beta-hydroxylase gene

The human gene for dopamine beta-hydroxylase (D beta H) has been mapped to chromosome 9q34. Using polymerase chain reaction amplification of exon 11 of the D beta H gene followed by digestion of the reaction products with FnuDII (BstUI), we detected a low-frequency restriction fragment length polymorphism (RFLP). The CEPH panel of family DNAs was genotyped for this RFLP, enabling us to determine the linkage relationships between D beta H and four other loci previously mapped to human chromosome 9q. We obtained two-point recombination frequencies (theta) between D beta H and arginosuccinate synthetase (theta = 0, LOD = 7.37), the ABO blood group locus (theta = 0, LOD = 4.5), CRI-P111 (theta = 0, LOD = 2.1), and D9S31 (theta = .06, LOD = 2.81).     

Identification of Quantitative Trait Loci Influencing Wood Specific Gravity in an Outbred Pedigree of Loblolly Pine

We report the identification of quantitative trait loci (QTL) influencing wood specific gravity (WSG) in an outbred pedigree of loblolly pine (Pinus taeda L.). QTL mapping in an outcrossing species is complicated by the presence of multiple alleles (>2) at QTL and marker loci. Multiple alleles at QTL allow the examination of interaction among alleles at QTL (deviation from additive gene action). Restriction fragment length polymorphism (RFLP) marker genotypes and wood specific gravity phenotypes were determined for 177 progeny. Two RFLP linkage maps were constructed, representing maternal and paternal parent gamete segregations as inferred from diploid progeny RFLP genotypes. RFLP loci segregating for multiple alleles were vital for aligning the two maps. Each RFLP locus was assayed for cosegregation with WSG QTL using analysis of variance (ANOVA). Five regions of the genome contained one or more RFLP loci showing differences in mean WSG at or below the P = 0.05 level for progeny as grouped by RFLP genotype. One region contained a marker locus (S6a) whose QTL-associated effects were highly significant (P > 0.0002). Marker S6a segregated for multiple alleles, a prerequisite for determining the number of alleles segregating at the linked QTL and analyzing the interactions among QTL alleles. The QTL associated with marker S6a appeared to be segregating for multiple alleles which interacted with each other and with environments. No evidence for digenic epistasis was found among the five QTL.     

Homologous recombination between direct repeat sequences yields P-glycoprotein containing amplicons in arsenite resistant Leishmania.

The protozoan parasite Leishmania often responds to drug pressure by amplifying part of its genome. At least two loci derived from the same 800 kb chromosome were amplified either as extrachromosomal circles or linear fragments after sodium arsenite selection. A 50 kb linear amplicon was detected in six independent arsenite mutants and revertants grown in absence of arsenite rapidly lost the amplicon and part of their resistance. The circular extrachromosomal amplicons, all derived from the H locus of Leishmania, were characterized more extensively. In all cases, direct repeated sequences appeared to be involved in the formation of circular amplicons. Most amplicons were generated after homologous recombination between two linked P-glycoprotein genes. This recombination event was, in two cases, associated with the loss of one allele of the chromosomal copy. A novel rearrangement point was found in a mutant where the amplicon was created by recombination between two 541 bp direct repeats surrounding the P-glycoprotein gene present at the H locus. It is also at one of these repeats that an H circle with large inverted duplications was formed. We propose that the presence of repeated sequences in the H locus facilitates the amplification of the drug resistance genes concentrated in this locus.     

A molecular genetic map of the long arm of chromosome 6R of rye incorporating the cereal cyst nematode resistance gene, CreR

 A genetic map of the long arm of chromosome 6R of rye was constructed using eight homoeologous group-6 RFLP clones and five PCR markers derived from the rye-specific dispersed repetitive DNA family, R173. The map was developed using a novel test-cross F₁ (TC-F₁) population segregating for resistance to the cereal cyst nematode. Comparisons were made between the map generated with other rye and wheat group-6 chromosome maps by the inclusion of RFLP clones previously mapped in those species. Co-linearity was observed for common loci. This comparison confirmed a dramatic reduction in recombination for chromosome 6R in the TC-F₁ population. The CreR locus was included in the linkage map via progeny testing of informative TC-F₁ individuals. CreR mapped 3.7 cM distal from the RFLP locus, XksuF37. Comparative mapping should allow the identification of additional RFLP markers more closely linked to the CreR locus. Received: 14 April 1998 / Accepted: 29 April 1998     

Identification of RFLP markers closely linked to the H1 gene conferring resistance to Globodera rostochiensis in potato

Summary Resistance to the root cyst nematode Globodera rostochiensis is an agronomic trait that is at present incorporated into most new potato varieties. Major dominant genes are available that originate from wild and cultivated Solanum species closely related to the cultivated European potato (Solanum tuberosum ssp. tuberosum). One of those genes, H1, from S. Tuberosum ssp. andigena, was mapped to a distal position on potato chromosome V using restriction fragment length polymorphism (RFLP) markers. The H1 locus segregates independently from Gro1, a second dominant gene presumably from S. Spegazzinii that confers resistance to G. Rostochiensis and which has been mapped to chromosome VII. One marker, CP113, was linked without recombination to the H1 locus.     

RFLP-based genetic map of the homoeologous group 3 chromosomes of wheat and rye

Summary Genetic maps of chromosomes 3A, 3B and 3D of wheat and 3R of rye were developed using 22 DNA probes and two isozyme marker systems. Analysis of the 49 loci mapped showed extreme clustering around the centromere in all four maps, with large gaps in the distal chromosome regions, which is interpreted as being due to strong localisation of recombination towards the ends of the wheat and rye chromosomes. In the centromeric regions gene orders are highly conserved between the three wheat genomes and the rye genome. However, the unpredictable behaviour of the DNA clones that map in distal chromosome locations may indicate that the genomes are diverging most rapidly in the regions of higher recombination. A comparison of cDNA and genomic probes showed the latter to be much more efficient for revealing RFLP. Some classes of gDNA clones, i.e. chromosome-specific sequences and those hybridizing in a non-homoeologous manner, were seen to be most polymorphic. Correlations between map locations and RFLP levels showed no clear relationship. In addition to anonymous DNA clones, the locations of known function clones, sedoheptulose-1,7-bisphosphatase (XSbp), carboxypeptidase I (XCxp1) and a bZIP protein (XEmbp), were ascertained along with those for two isozyme loci, Mal-1 and Est-5.     

Identification of YAC clones containing the mutable slender glume locus slg in rice (Oryza sativa L.).

A mutable slender glume gene slg, which often reverts to the wild-type state, was induced by gamma-ray irradiation of seeds of the japonica rice cultivar 'Gimbozu'. The final goal was to understand whether the slender glume mutation was associated with the insertion of a transposable element, utilizing map-based cloning techniques. The RFLP (restriction fragment length polymorphism) analysis revealed that the slg locus was located between two RFLP loci, XNpb33 and R1440, on chromosome 7 with recombination values of 3.1% and 1.0%, respectively. Using these two RFLP loci as probes, five YAC (yeast artificial chromosome) clones containing either of these two loci were selected from a YAC library. Subsequently, both end fragments of these YAC clones, amplified by the inverse PCR (IPCR) method, were used to select new YAC clones more closely located to the slg locus. After repeating such a procedure, we successfully constructed a 6-cM YAC contig, and identified four overlapping YAC clones, Y1774, Y3356, Y5124, and Y5762, covering the slg locus. The chromosomal location of the slg was narrowed down to the region with a physical distance of less than 280 kb between the right-end fragments of Y1774 and Y3356.     

Cosmid walking and chromosome jumping in the region of PKD1 reveal a locus duplication and three CpG islands.

The locus responsible for the most common form of autosomal dominant polycystic kidney disease (PKD1) is located on chromosome 16p13.3. Genetic mapping studies indicate that PKD1 is flanked on the proximal side by the DNA marker 26.6 (D16S125). Here we show that 26.6 has undergone a locus duplication and that the two loci are less than 150kb apart. One of the two loci contains a polymorphic TaqI site that has been used in genetic studies and represents the proximal boundary for the PKD1 locus. We demonstrate that the polymorphic locus is the more proximal of the two 26.6-hybridizing loci. Therefore, four cosmids isolated from the distal 26.6-hybridizing locus contain candidate sequences for the PKD1 gene. These cosmids were found to contain two CpG islands that are likely markers for transcribed regions. A third CpG island was detected and cloned by directional chromosome jumping.     

RFLP mapping of the genes controlling hybrid breakdown in rice (Oryza sativa L.)

 Complementary recessive genes hwd1 and hwd2 controlling hybrid breakdown (weakness of F₂ and later generations) were mapped in rice using RFLP markers. These genes produce a plant that is shorter and has fewer tillers than normal plants when the two loci have only one or no dominant allele at both loci. A cultivar with two dominant alleles at the hwd1 locus and a cultivar with two dominant alleles at the hwd2 locus were crossed with a double recessive tester line. Linkage analysis was carried out for each gene independently in two F₂ populations derived from these crosses. hwd1 was mapped on the distal region of rice genetic linkage map for chromosome 10, flanked by RFLP markers C701 and R2309 at a distance of 0.9 centiMorgans (cM) and 0.6 cM, respectively. hwd2 was mapped in the central region of rice genetic linkage map for chromosome 7, tightly linked with 4 RFLP markers without detectable recombination. The usefulness of RFLP mapping and map information for the genes controlling reproductive barriers are discussed in the context of breeding using diverse rice germplasm, especially gene introduction by marker-aided selection.     

Physical mapping of restriction fragment length polymorphism (RFLP) markers in homoeologous groups 1 and 3 chromosomes of wheat by in situ hybridization.

Using wheat ditelosomic lines and in situ hybridization of biotin-labelled DNA probes, 18 restriction fragment length polymorphism (RFLP) markers were physically located on homoeologous groups 1 and 3 chromosomes of wheat. Most of the markers hybridized to chromosome arms in a physical order concordant with the genetic maps. A majority of the markers studied were clustered in non-C-banded, distal euchromatic areas, indicating the presence of recombination hot spots and cold spots in those regions. However, on IBS the markers were well dispersed, which could be due to the abundance of heterochromatin throughout the arm. An inversion between Xpsr653 and Xpsr953 was observed on 1AL. One new Xpsr688 locus, approximately 20-26% from the centromere, was found on 1AS and 1BS. The physical location of Xpsr170 on group 3 chromosomes probably represents an alternative to the loci on the genetic map. Finally, Xpsr313 was mapped to two physical loci on IDL. Five markers were located to bins consistent with the deletion-based physical maps.