Integrating genetic linkage maps with pachytene chromosome structure in maize

Genetic linkage maps reveal the order of markers based on the frequency of recombination between markers during meiosis. Because the rate of recombination varies along chromosomes, it has been difficult to relate linkage maps to chromosome structure. Here we use cytological maps of crossing over based on recombination nodules (RNs) to predict the physical position of genetic markers on each of the 10 chromosomes of maize. This is possible because (1). all 10 maize chromosomes can be individually identified from spreads of synaptonemal complexes, (2). each RN corresponds to one crossover, and (3). the frequency of RNs on defined chromosomal segments can be converted to centimorgan values. We tested our predictions for chromosome 9 using seven genetically mapped, single-copy markers that were independently mapped on pachytene chromosomes using in situ hybridization. The correlation between predicted and observed locations was very strong (r(2) = 0.996), indicating a virtual 1:1 correspondence. Thus, this new, high-resolution, cytogenetic map enables one to predict the chromosomal location of any genetically mapped marker in maize with a high degree of accuracy. This novel approach can be applied to other organisms as well.     

Evidence of cryptic introgression in tomato (Solanum lycopersicum L.) based on wild tomato species alleles

ABSTRACT: BACKGROUND: Many beneficial traits (e.g. disease or abiotic stress resistance) have been transferred into crops through crosses with their wild relatives. The 13 recognized species of tomato (Solanum section Lycopersicon) are closely related to each other and wild species genes have been extensively used for improvement of the crop, Solanum lycopersicum L. In addition, the lack of geographical barriers has permitted natural hybridization between S. lycopersicum and its closest wild relative Solanum pimpinellifolium in Ecuador, Peru and northern Chile. In order to better understand patterns of S. lycopersicum diversity, we sequenced 47 markers ranging in length from 130 to 1200 bp (total of 24 kb) in genotypes of S. lycopersicum and wild tomato species S. pimpinellifolium, Solanum arcanum, Solanum peruvianum, Solanum pennellii and Solanum habrochaites. Several of the markers had previously been hypothesized as carrying wild species alleles within S. lycopersicum, i.e., cryptic introgressions. RESULTS: Each marker was mapped with high confidence (e < 1 x 10-30) to a single genomic location using BLASTN against tomato whole genome shotgun chromosomes (SL2.40) database. Neighbor-joining trees showed high mean bootstrap support (86.8 plus or minus 2.34%) for distinguishing red-fruited from green-fruited taxa for 38 of the markers. Hybridization and parsimony splits networks, genomic map positions of markers relative to documented introgressions, and historical origins of accessions were used to interpret evolutionary patterns at nine markers with putatively introgressed alleles. CONCLUSION: Of the 47 genetic markers surveyed in this study, four were involved in linkage drag on chromosome 9 during introgression breeding, while alleles at five markers apparently originated from natural hybridization with S. pimpinellifolium and were associated with primitive genotypes of S. lycopersicum. The positive identification of introgressed genes within crop species such as S. lycopersicum will help inform conservation and utilization of crop germplasm diversity, for example, facilitating the purging of undesirable linkage drag or the exploitation of novel, favorable alleles.     

Chromosomal rearrangements between tomato and Solanum chilense hamper mapping and breeding of the TYLCV resistance gene Ty-1

Tomato yellow leaf curl disease, a devastating disease of Solanum lycopersicum (tomato), is caused by a complex of begomoviruses generally referred to as Tomato yellow leaf curl virus (TYLCV). Almost all breeding for TYLCV resistance has been based on the introgression of the Ty-1 resistance locus derived from Solanum chilense LA1969. Knowledge about the exact location of Ty-1 on tomato chromosome 6 will help in understanding the genomic organization of the Ty-1 locus. In this study, we analyze the chromosomal rearrangement and recombination behavior of the chromosomal region where Ty-1 is introgressed. Nineteen markers on tomato chromosome 6 were used in F(2) populations obtained from two commercial hybrids, and showed the presence of a large introgression in both. Fluorescence in situ hybridization (FISH) analysis revealed two chromosomal rearrangements between S. lycopersicum and S. chilense LA1969 in the Ty-1 introgression. Furthermore, a large-scale recombinant screening in the two F(2) populations was performed, and 30 recombinants in the Ty-1 introgression were identified. All recombination events were located on the long arm beyond the inversions, showing that recombination in the inverted region was absent. Disease tests on progenies of informative recombinants with TYLCV mapped Ty-1 to the long arm between markers MSc05732-4 and MSc05732-14, an interval overlapping with the reported Ty-3 region, which led to the indication that Ty-1 and Ty-3 may be allelic. With this study we prove that FISH can be used as a diagnostic tool to aid in the accurate mapping of genes that were introgressed from wild species into cultivated tomato.     

Integration of cytogenetic and genetic linkage maps unveils the physical architecture of tomato chromosome 2

We report the integration of the linkage map of tomato chromosome 2 with a high-density bacterial artificial chromosome fluorescence in situ hybridization (BAC-FISH)-based cytogenetic map. The euchromatic block of chromosome 2 resides between 13 and 142 cM and has a physical length of 48.12 microm, with 1 microm equivalent to 540 kb. BAC-FISH resolved a pair of loci that were 3.7-3.9 Mb apart and were not resolved on the linkage map. Most of the regions had crossover densities close to the mean of approximately 200 kb/cM. Relatively hot and cold spots of recombination were unevenly distributed along the chromosome. The distribution of centimorgan/micrometer values was similar to the previously reported recombination nodule distribution along the pachytene chromosome. FISH-based physical maps will play an important role in advanced genomics research for tomato, including map-based cloning of agronomically important traits and whole-genome sequencing.     

Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique.

The primary objective of this study was to elucidate gene organization and to integrate the genetic linkage map for barley (Hordeum vulgare L.) with a physical map using ultrasensitive fluorescence in situ hybridization (FISH) techniques for detecting signals from restriction fragment length polymorphism (RFLP) clones. In the process, a single landmark plasmid, p18S5Shor, was constructed that identified and oriented all seven of the chromosome pairs. Plasmid p18S5Shor was used in all hybridizations. Fourteen cDNA probes selected from the linkage map for barley H. vulgare 'Steptoe' x H. vulgare 'Morex' (Kleinhofs et al. 1993) were mapped using an indirect tyramide signal amplification technique and assigned to a physical location on one or more chromosomes. The haploid barley genome is large and a complete physical map of the genome is not yet available; however, it was possible to integrate the linkage map and the physical locations of these cDNAs. An estimate of the ratio of base pairs to centimorgans was an average of 1.5 Mb/cM in the distal portions of the chromosome arms and 89 Mb/cM near the centromere. Furthermore, while it appears that the current linkage maps are well covered with markers along the length of each arm, the physical map showed that there are large areas of the genome that have yet to be mapped.     

Localization of the Huntington's disease gene to a small segment of chromosome 4 flanked by D4S10 and the telomere

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of late onset, characterized by progressive motor disturbance, psychological manifestations, and intellectual deterioration. The HD gene has been genetically mapped by linkage to the DNA marker D4S10, but the exact physical location of the HD defect has remained uncertain. To delineate critical recombination events revealing the physical position of the HD gene, we have identified restriction fragment length polymorphisms for two recently mapped chromosome 4 loci, RAF2 and D4S62, and determined the pattern of segregation of these markers in both reference and HD pedigrees. Multipoint linkage analysis of the new markers with D4S10 and HD establishes that the HD gene is located in a very small physical region at the tip of the chromosome, bordered by D4S10 and the telomere. A crossover within the D4S10 locus orients this segment on the chromosome, providing the necessary information for efficient application of directional cloning strategies for progressing toward, and eventually isolating, the HD gene.     

Radiation hybrid mapping of 70 rat genes from a data set of differentially expressed genes

The spontaneously hypertensive rat (SHR) is a model of human essential hypertension. Increased blood pressure in SHR is associated with other risk factors associated with cardiovascular disease, including insulin resistance and dyslipidemia. DNA microarray studies identified over 200 differentially expressed genes and ESTs between SHR and normotensive control rats. These clones represent candidate genes that may underlie previously detected QTLs in SHR. This study made use of the publication of two whole-genome maps to identify positional QTL candidates. Radiation hybrid (RH) mapping was used to determine the chromosomal locations of 70 rat genes and ESTs from this dataset. Most of the locations are novel, but in five cases we identified a definitive map location for genes previously mapped by somatic cell hybrids and/or linkage analysis. Genes for which the mouse genome map location was already determined mapped to syntenic segments in the rat genome map, except for two rat genes whose map locations confirmed previous findings. Where synteny comparisons could be made only with the human, 74% of the genes mapped in this study lay in a conserved syntenic segment. Chromosomal localisation of these mouse and human orthologs to syntenic segments produces a high level of confidence in the data presented in this study. The data provide new map locations for rat genes and will aid efforts to advance the rat genome map. The data may also be used to prioritize candidate QTL genes in SHR and other rat strains on the basis of their map location.     

High-resolution chromosome mapping of BACs using multi-colour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6

Within the framework of the International Solanaceae Genome Project, the genome of tomato (Solanum lycopersicum) is currently being sequenced. We follow a 'BAC-by-BAC' approach that aims to deliver high-quality sequences of the euchromatin part of the tomato genome. BACs are selected from various libraries of the tomato genome on the basis of markers from the F2.2000 linkage map. Prior to sequencing, we validated the precise physical location of the selected BACs on the chromosomes by five-colour high-resolution fluorescent in situ hybridization (FISH) mapping. This paper describes the strategies and results of cytogenetic mapping for chromosome 6 using 75 seed BACs for FISH on pachytene complements. The cytogenetic map obtained showed discrepancies between the actual chromosomal positions of these BACs and their markers on the linkage group. These discrepancies were most notable in the pericentromere heterochromatin, thus confirming previously described suppression of cross-over recombination in that region. In a so called pooled-BAC FISH, we hybridized all seed BACs simultaneously and found a few large gaps in the euchromatin parts of the long arm that are still devoid of seed BACs and are too large for coverage by expanding BAC contigs. Combining FISH with pooled BACs and newly recruited seed BACs will thus aid in efficient targeting of novel seed BACs into these areas. Finally, we established the occurrence of repetitive DNA in heterochromatin/euchromatin borders by combining BAC FISH with hybridization of a labelled repetitive DNA fraction (Cot-100). This strategy provides an excellent means to establish the borders between euchromatin and heterochromatin in this chromosome.     

Comparative linkage map of the Solanum lycopersicoides and S. sitiens genomes and their differentiation from tomato.

The wild nightshades Solanum lycopersicoides and Solanum sitiens are closely affiliated with the tomatoes (Lycopersicon spp.). Intergeneric hybridization with cultivated tomato (Lycopersicon esculentum) is impeded by strong reproductive barriers including hybrid sterility and suppressed recombination. Conservation of genome structure between these nightshades and tomato was studied by construction of a genetic map from F2 S. sitiens x S. lycopersicoides and comparison with existing maps of tomato. Owing to self-incompatibility of the F1, two hybrid plants were crossed to obtain a population of 82 F2 individuals. Using 166 previously mapped RFLP markers and 5 restriction enzymes, 101 loci polymorphic in the S. sitiens x S. lycopersicoides population were identified. Analysis of linkage between the markers resulted in a map with 12 linkage groups covering 1192 cM and one unlinked marker. Recombination rates were similar to those observed in tomato; however, significant segregation distortion was observed for markers on 7 out of the 12 chromosomes. All chromosomes were colinear with the tomato map, except for chromosome 10, where a paracentric inversion on the long arm was detected. In this region, S. sitiens and S. lycopersicoides share the same chromosomal configuration previously reported for potato (S. tuberosum) and pepper (Capsicum), suggesting that of tomato is derived. The 10L inversion explains the lack of recombination detected among homeologous chromosomes of intergeneric hybrids in this region. On this basis, we recognize two principle genomes, designated L for the Lycopersicon spp., and S for S. lycopersicoides and S. sitiens, the first examples of structural differentiation between tomato and its cross-compatible wild relatives.     

RFLP Mapping in Soybean: Association between Marker Loci and Variation in Quantitative Traits

Quantitative trait loci (QTLs) have been mapped to small intervals along the chromosomes of tomato (Lycopersicon esculentum), by a method we call substitution mapping. The size of the interval to which a QTL can be mapped is determined primarily by the number and spacing of previously mapped genetic markers in the region surrounding the QTL. We demonstrate the method using tomato genotypes carrying chromosomal segments from Lycopersicon chmielewskii, a wild relative of tomato with high soluble solids concentration but small fruit and low yield. Different L. chmielewskii chromosomal segments carrying a common restriction fragment length polymorphism were identified, and their regions of overlap determined using all available genetic markers. The effect of these chromosomal segments on soluble solids concentration, fruit mass, yield, and pH, was determined in the field. Many overlapping chromosomal segments had very different phenotypic effects, indicating QTLs affecting the phenotype(s) to lie in intervals of as little as 3 cM by which the segments differed. Some associations between different traits were attributed to close linkage between two or more QTLs, rather than pleiotropic effects of a single QTL: in such cases, recombination should separate desirable QTLs from genes with undesirable effects. The prominence of such trait associations in wide crosses appears partly due to infrequent reciprocal recombination between heterozygous chromosomal segments flanked by homozygous regions. Substitution mapping is particularly applicable to gene introgression from wild to domestic species, and generally useful in narrowing the gap between linkage mapping and physical mapping of QTLs.     

Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome

Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analysis, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analysis was used to construct a physical and genetic map of the S. kunkelii CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal endonuclease double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kb. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.     

Identification of crossovers in Wilson disease families as reference points for a genetic localization of the gene

Summary Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. A minimum recombinant analysis using D13S22, ESD, RB1, D13S31, D13S55, D13S26, D13S39, and D13S12, all localized at 13q14-q22, has been carried out in 20WD families of Northwest-European origin. No inconsistencies have been observed with respect to locus order or location of the WD locus (WND) compared with previous linkage studies. D13S31 was mapped as the closest marker proximal to WND, whereas D13S55 and D13S26 were mapped as the closest markers distal to WND. We have identified a crossover between WND and D13S31 in one family and a crossover between WND and D13S55 in another. These crossover sites can be used as reference points for new chromosome 13q14-q21 markers, and are therefore important for a more accurate mapping of the WD locus.     

Long-range physical maps of two loci (Aps-1 and GP79) flanking the root-knot nematode resistance gene (Mi) near the centromere of tomato chromosome 6

The root knot nematode resistance gene Mi in tomato has been mapped in the pericentromeric region of chromosome 6. With the objective of isolating Mi through a map-based cloning approach, we have previously identified and ordered into a high-resolution genetic linkage map a variety of tightly linked molecular markers. Using pulsed-field gelelectrophoresis and various rarely cutting restriction enzymes in single, double and partial digestions, we now report long-range physical maps of the two closest flanking markers, acid phosphatase-1 (Aps-1) and GP79, which span over 400 and 800 kb, respectively. It is concluded that the physical distance between both markers is larger than predicted on the basis of genetic linkage analysis. Furthermore, two RFLP markers (H3F8 and H4H10) which map genetically to the same locus as Aps-1 do not show physical linkage, indicating severe suppression of recombination in this region of the chromosome. Finally, no evidence was obtained showing the presence of a CpG island near Aps-1.     

Genetic mapping of the dentinogenesis imperfecta type II locus.

Dentinogenesis imperfecta type II (DGI-II) is an autosomal dominant disorder of dentin formation, which has previously been mapped to chromosome 4q12-21. In the current study, six novel short tandem-repeat polymorphisms (STRPs) have been isolated, five of which show significant evidence of linkage to DGI-II. To determine the order of the STRPs and define the genetic distance between them, nine loci (including polymorphisms for two known genes) were mapped through the CEPH reference pedigrees. The resulting genetic map encompasses 16.3 cM on the sex-averaged map. To combine this map with a physical map of the region, all of the STRPs were mapped through a somatic cell hybrid panel. The most likely location for the DGI-II locus within the fixed marker map is in the D4S2691-D4S2692 interval of 6.6 cM. The presence of a marker that shows no recombination with the DGI-II phenotype between the flanking markers provides an important anchor point for the creation of physical continuity across the DGI-II candidate region.     

A physical map with yeast artificial chromosome (YAC) clones covering 63% of the 12 rice chromosomes.

A new YAC (yeast artificial chromosome) physical map of the 12 rice chromosomes was constructed utilizing the latest molecular linkage map. The 1439 DNA markers on the rice genetic map selected a total of 1892 YACs from a YAC library. A total of 675 distinct YACs were assigned to specific chromosomal locations. In all chromosomes, 297 YAC contigs and 142 YAC islands were formed. The total physical length of these contigs and islands was estimated to 270 Mb which corresponds to approximately 63% of the entire rice genome (430 Mb). Because the physical length of each YAC contig has been measured, we could then estimate the physical distance between genetic markers more precisely than previously. In the course of constructing the new physical map, the DNA markers mapped at 0.0-cM intervals were ordered accurately and the presence of potentially duplicated regions among the chromosomes was detected. The physical map combined with the genetic map will form the basis for elucidation of the rice genome structure, map-based cloning of agronomically important genes, and genome sequencing.     

A comparative location database (CompLDB): map integration within and between species

We have adapted the Location Database (LDB) map-integration strategy of Morton et al. [Ann Hum Genet 56:223–232] (1992) as above to create an integrated map for each of several species for which fully annotated genome sequences are not yet available (sheep, cattle, pig, wallaby), using all types of partial maps for that species, including cytogenetic, linkage, somatic-cell hybrid, and radiation hybrid maps. An integrated map provides not only predictions of the kilobase location of every locus, but also predicts locations (in cM) and cytogenetic band locations for every locus. In this way a comprehensive linkage map and a comprehensive cytogenetic map are created, including all loci, irrespective of whether they have ever been linkage mapped or physically mapped, respectively. High-resolution physical maps from annotated sequenced species have also been placed alongside the integrated maps. This has created a powerful tool for comparative genomics. The LDB map-integration strategy has been extended to make use of zoo-FISH comparative information. It has also been extended to enable the creation of a “virtual” map for each species not yet sequenced by using mapping data from fully sequenced species. All of the partial maps, together with the integrated map, for each species have been placed in a database called Comparative Location Database (CompLDB), which is available for querying, browsing, or download in tabular form at . Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A high-density rice genetic linkage map with 2275 markers using a single F2 population.

A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using > 130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.     

A Primary Genetic Linkage Map of 14 Polymorphic Loci for the Short Arm of Human Chromosome 8

A genetic linkage map of markers for the short arm of human chromosome 8 has been constructed with 14 polymorphic DNA markers on the basis of genotypes obtained in 40 CEPH reference families. This unbroken map spans 45 cM in males and 79 cM in females. The 14 markers include three genes, MSR, LPL, and NEFL, and one anonymous DNA segment that were previously assigned to chromosome 8. The other 10 marker had been isolated from a chromosome 8-specific cosmid library and physically localized to chromosomal bands by fluorescence in situ hybridization. The order of loci determined by genetic linkage was consistent with their physical locations. This map will facilitate efficient linkage studies of human genetic diseases that may be segregating on chromosome 8p and will provide anchor points for development of high-resolution maps for this chromosomal region.     

Localization of the murine macrophage colony-stimulating factor gene to chromosome 3 using interspecific backcross analysis

The chromosomal location of the murine macrophage colony-stimulating factor (Csfm) gene was determined by interspecific backcross analysis. We mapped Csfm to mouse chromosome 3, 2.5 cM distal to Ngfb and Nras and 1.3 cM proximal to Amy-2. CSFM maps to human chromosome 5q, while AMY2, NGFB, and NRAS map to human chromosome 1p. The chromosomal location of Csfm thus disrupts a previously identified conserved linkage group between mouse chromosome 3 and human chromosome 1. The location of Csfm also identifies yet another mouse chromosome that shares synteny with human chromosome 5q, a region involved in several different types of myeloid disease.