Centromere-Linkage Analysis and Consolidation of the Zebrafish Genetic Map

The ease of isolating mutations in zebrafish will contribute to an understanding of a variety of processes common to all vertebrates. To facilitate genetic analysis of such mutations, we have identified DNA polymorphisms closely linked to each of the 25 centromeres of zebrafish, placed centromeres on the linkage map, increased the number of mapped PCR-based markers to 652, and consolidated the number of linkage groups to the number of chromosomes. This work makes possible centromere-linkage analysis, a novel, rapid method to assign mutations to a specific linkage group using half-tetrads.     

Genetic Positioning of Centromeres through Half-Tetrad Analysis in Gynogenetic Diploid Families of the Zhikong Scallop (Chlamys farreri)

Centromere mapping is a powerful tool for improving linkage maps, investigating crossover events, and understanding chiasma interference during meiosis. Ninety microsatellite markers selected across all linkage groups (LGs) from a previous Chlamys farreri genetic map were studied in three artificially induced meiogynogenetic families for centromere mapping by half-tetrad analysis. Inheritance analyses showed that all 90 microsatellite loci conformed to Mendelian inheritance in the control crosses, while 4.4 % of the microsatellite loci showed segregation departures from an expected 1:1 ratio of two homozygote classes in meiogynogenetic progeny. The second division segregation frequency (y) of the microsatellites ranged from 0.033 to 0.778 with a mean of 0.332, confirming the occurrence of partial chiasma interference in this species. Heterogeneity of y is observed in one of 42 cases in which markers were typed in more than one family, suggesting variation in gene–centromere recombination among families. Centromere location was mostly in accordance with the C. farreri karyotype, but differences in marker order between linkage and centromere maps occurred. Overall, this study makes the genetic linkage map a more complete and informative tool for genomic studies and it will also facilitate future research of the structure and function of the scallop centromeres.     

A microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) characterized by large sex-specific differences in recombination rates

We constructed a genetic linkage map for a tetraploid derivative species, the rainbow trout (Oncorhynchus mykiss), using 191 microsatellite, 3 RAPD, 7 ESMP, and 7 allozyme markers in three backcross families. The linkage map consists of 29 linkage groups with potential arm displacements in the female map due to male-specific pseudolinkage arrangements. Synteny of duplicated microsatellite markers was used to identify and confirm some previously reported pseudolinkage arrangements based upon allozyme markers. Fifteen centromeric regions (20 chromosome arms) were identified with a half-tetrad analysis using gynogenetic diploids. Female map length is approximately 10 M, but this is a large underestimate as many genotyped segments remain unassigned at a LOD threshold of 3.0. Extreme differences in female:male map distances were observed (ratio F:M, 3.25:1). Females had much lower recombination rates (0.14:1) in telomeric regions than males, while recombination rates were much higher in females within regions proximal to the centromere (F:M, 10:1). Quadrivalent formations that appear almost exclusively in males are postulated to account for the observed differences.     

A meta-analysis of kidney microarray datasets: investigation of cytokine gene detection and correlation with rt-PCR and detection thresholds

Background

Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers.

Results

We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM.

Conclusion

By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.     

Genetic and physical maps of human chromosome 4 based on dinucleotide repeats.

Characterization of inherited variations within tandem arrays of dinucleotide repeats has substantially advanced the construction of genetic maps using linkage approaches over the last several years. Using a backbone of 10 newly identified microsatellite repeats on human chromosome 4 and 6 previously identified short tandem repeat element polymorphisms, we have constructed several genetic maps and a physical map of human chromosome 4. The genetic and physical maps are in complete concordance with each other. The genetic maps include a 15-locus microsatellite-based linkage map, a framework map of high support incorporating a total of 39 independent loci, a 25-locus high-heterozygosity, easily used index map, and a gene-based comprehensive map that provides the best genetic location for 35 genes mapped to chromosome 4. The 16 microsatellite markers are each localized to one of nine regions of chromosome 4, delineated by a panel of somatic cell hybrids. These results demonstrate the utility of PCR-based repeat elements for the construction of genetic maps and provide a valuable resource for continued high-resolution mapping of chromosome 4 and of genetic disorders to this chromosome.     

A genetic linkage map and comparative genome analysis of common carp (Cyprinus carpio L.) using microsatellites and SNPs

A genetic linkage map is a powerful research tool for mapping traits of interest and is essential to understanding genome evolution. The aim of this study is to provide an expanded genetic linkage map of common carp to effectively carry out quantitative trait loci analysis and conduct comparative mapping analysis between lineages. Here, we constructed a genetic linkage map of common carp (Cyprinus carpio L.) using microsatellite and single-nucleotide polymorphism (SNP) markers in a 159 sibling family. A total of 246 microsatellites and 306 SNP polymorphic markers were genotyped in this family. Linkage analysis using JoinMap 4.0 organized 427 markers (186 microsatellites and 241 SNPs) to 50 linkage groups, ranging in size from 1.4 to 130.1 cM. Each group contained 2-30 markers. The linkage map covered a genetic distance of 2,039.2 cM and the average interval for markers within the linkage groups was approximately 6.4 cM. In addition, comparative genome analysis within five model teleost fish revealed a high percentage (74.7%) of conserved loci corresponding to zebrafish chromosomes. In most cases, each zebrafish chromosome comprised two common carp linkage groups. The comparative analysis also revealed independent chromosome rearrangements in common carp and zebrafish. The linkage map will be of great assistance in mapping genes of interest and serve as a reference to approach comparative mapping and enable further insights into the comprehensive investigations of genome evolution of common carp.     

A linkage map of the basidiomycete Coprinus cinereus based on random amplified polymorphic DNAs and restriction fragment length polymorphisms

A genetic linkage map of the basidiomycete Coprinus cinereus was constructed on the basis of the segregation of 219 RAPD markers, 28 RFLP markers and the A and B mating-type loci among 40 random basidiospore progeny from a single cross between a wild-type homokaryon, KF(3)#2, and an AmutBmut strain, #326. Thirteen linkage groups covering a total of 1346cM were identified and correlated to the 13 chromosomes of this fungus by hybridization of RFLP and RAPD marker probes to CHEF blots. These probes also revealed chromosome length polymorphisms (CLP), which could be associated with haplotype plots of the progeny. The average kb/cM ratio in this cross was approximately 27.9kb/cM. The AmutBmut strain undergoes sexual development without mating, because of mutations in both A and B mating-type loci, and has been used to identify mutations affecting developmental processes such as dikaryosis, fruit body morphogenesis, and meiosis. The markers in the map, especially the RAPD ones, would facilitate mapping of genes responsible for such mutations induced in the AmutBmut strain.     

Single linkage group per chromosome genetic linkage map for the horse, based on two three-generation, full-sibling, crossbred horse reference families

A genetic linkage map of the horse consisting of 742 markers, which comprises a single linkage group for each of the autosomes and the X chromosome, is presented. The map has been generated from two three-generation full-sibling reference families, sired by the same stallion, in which there are 61 individuals in the F2 generation. Each linkage group has been assigned to a chromosome and oriented with reference to markers mapped by fluorescence in situ hybridization. The average interval between markers is 3.7 cM and the linkage groups collectively span 2772 cM. The 742 markers comprise 734 microsatellite and 8 gene-based markers. The utility of the microsatellite markers for comparative mapping has been significantly enhanced by comparing their flanking sequences with the human genome sequence; this enabled conserved segments between human and horse to be identified. The new map provides a valuable resource for genetically mapping traits of interest in the horse.     

A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus

Background  

Eucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Quantitative Trait Loci (QTLs) in Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus.     

Microsatellite-centromere mapping in Cynoglossus semilaevis using gynogenetic diploid families produced by the use of homologous and non-homologous sperm

Twenty-one microsatellite markers were studied in three meiogynogenetic families of Cynoglossus semilaevis gunther for centromere mapping using half-tetrad analysis. Among the 13 mapped loci, 10 were estimated to be located in the telomeric region, one in the centromeric region, and two in the intermediate region of the chromosome. This study provides a basis for constructing a linkage map of C. semilaevis .     

A Preliminary Genetic Linkage Map of the Pacific Abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F₁ family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (α = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.     

A preliminary microsatellite genetic map of the ostrich (Struthio camelus)

Molecular genetic maps can provide information for the identification and localization of major genes associated with quantitative traits. However, there are currently no published genetic linkage maps for any ratites. Herein, a preliminary genetic map of ostrich was developed using a two-generation ostrich reference family by linkage analysis of 104 polymorphic microsatellite markers, including 40 novel markers reported in this study. A total of 35 microsatellite markers were placed into 13 linkage groups. Five linkage groups are composed of three or more loci, whereas the remaining eight groups each contained two markers. The sex-averaged map spans 365.4 cM. The marker interval of each linkage group ranges from 5.3 to 25.4 cM, and the average interval distance is 16.61 cM. The male map covers 342.7 cM, with an average intermarker distance of 15.58 cM, whereas the female map is 456.7 cM, with the average intermarker spacing of 20.76 cM. In order to screen the orthologous loci between ostrich and chicken, all of the flanking sequences of the 104 polymorphic loci, nine monomorphic loci and a further 12 reported microsatellite loci for ostrich were screened against the chicken genomic sequence using the BLAST algorithm (Altschul et al., 1990), and corresponding orthologs were found for 13 sequences. The microsatellite loci and genetic map developed in this study will be useful for QTL mapping, population genetics and phylogenetic studies in the ratite. In addition, the 13 orthologous loci identified in this study will be advantageous to the construction of a comparative genetic map between chicken and ostrich.     

Genetic linkage map of the loach <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis> (Teleostei: Cobitidae)

In the present study, the first genetic linkage map of the loach Misgurnus anguillicaudatus was constructed with 164 microsatellite markers and a color locus, and it included 155 newly developed markers. A total of 159 microsatellite markers and a color locus were mapped in 27 linkage groups (LGs). The female map covered 784.5 cM with 153 microsatellite markers and a color locus, whereas the male map covered 662.2 cM with 119 microsatellite markers. The centromeric position in each LG was estimated by marker-centromere mapping based on half-tetrad analysis. In 4 LGs (LG2, LG3, LG4, and LG5), the centromere was estimated at the intermediate region. In LG1, LG11, and LG12, the centromere was estimated to shift from the sub-intermediate region to the end (telomeric). The number of these LGs (7) was identical to the collective number of bi-arm metacentric (5) and sub-metacentric chromosome (2) of the haploid chromosome set (n = 5) of the loach. In the other LGs, the position of the centromere was estimated at the end or outside. These results indicate satisfactory compliance between the linkage map and the chromosome set. Our map would cover approximately almost the entire loach genome because most markers were successfully mapped.     

A genetic linkage map of the model legume Lotus japonicus and strategies for fast mapping of new loci

A genetic map for the model legume Lotus japonicus has been developed. The F(2) mapping population was established from an interspecific cross between L. japonicus and L. filicaulis. A high level of DNA polymorphism between these parents was the source of markers for linkage analysis and the map is based on a framework of amplified fragment length polymorphism (AFLP) markers. Additional markers were generated by restriction fragment length polymorphism (RFLP) and sequence-specific PCR. A total of 524 AFLP markers, 3 RAPD markers, 39 gene-specific markers, 33 microsatellite markers, and six recessive symbiotic mutant loci were mapped. This genetic map consists of six linkage groups corresponding to the six chromosomes in L. japonicus. Fluorescent in situ hybridization (FISH) with selected markers aligned the linkage groups to chromosomes as described in the accompanying article by Pedrosa et al. 2002(this issue). The length of the linkage map is 367 cM and the average marker distance is 0.6 cM. Distorted segregation of markers was found in certain sections of the map and linkage group I could be assembled only by combining colormapping and cytogenetics (FISH). A fast method to position genetic loci employing three AFLP primer combinations yielding 89 markers was developed and evaluated by mapping three symbiotic loci, Ljsym1, Ljsym5, and Ljhar1-3.     

Genetic positioning of centromeres using half-tetrad analysis in a 4x-2x cross population of potato

From biological and genetic standpoints, centromeres play an important role in the delivery of the chromosome complement to the daughter cells at cell division. The positions of the centromeres of potato were determined by half-tetrad analysis in a 4x-2x population where the male parent produced 2n pollen by first-division restitution (FDR). The genetic linkage groups and locations of 95 male parent-derived amplified fragment length polymorphism markers could be determined by comparing their position on a 2x-2x highly saturated linkage map of potato. Ten centromere positions were identified by 100% heterozygosity transmitted from the 2n heterozygous gametes of the paternal parent into the tetraploid offspring. The position of these centromeric marker loci was in accordance with those predicted by the saturated 2x-2x map using the level of marker clustering as a criterion. Two remaining centromere positions could be determined by extrapolation. The frequent observation of transmission of 100% heterozygosity proves that the meiotic restitution mechanism is exclusively based on FDR. Additional investigations on the position of recombination events of three chromosomes with sufficient numbers of markers showed that only one crossover occurred per chromosome arm, proving strong interference of recombination between centromere and telomere.     

Anchoring of canine linkage groups with chromosome-specific markers

A high-resolution genetic map with polymorphic markers spaced frequently throughout the genome is a key resource for identifying genes that control specific traits or diseases. The lack of rigorous selection against genetic disorders has resulted in many breeds of dog suffering from a very high frequency of genetic diseases, which tend to be breed-specific and usually inherited as autosomal recessive or apparently complex genetic traits. Many of these closely resemble human genetic disorders in their clinical and pathologic features and are likely to be caused by mutations in homologous genes. To identify loci important in canine disease genes, as well as traits associated with morphological and behavioral variation, we are developing a genetic map of the canine genome. Here we report on an updated version of the canine linkage map, which includes 341 mapped markers distributed over the X and 37 autosomal linkage groups. The average distance between markers on the map is 9.0 cM, and the linkage groups provide estimated coverage of over 95% of the genome. Fourteen linkage groups contain either gene-associated or anonymous markers localized to cosmids that have been assigned to specific canine chromosomes by FISH. These 14 linkage groups contain 150 microsatellite markers and allow us to assign 40% of the linkage groups to specific canine chromosomes. This new version of the map is of sufficient density and characterization to initiate mapping of traits of interest. Received: 23 February 1999 / Accepted: 28 April 1999     

Molecular mapping and tagging of genes in crop plants

In India, molecular mapping and tagging of agronomically important genes using RFLP and RAPD markers have been carried out in three different crops: rice, mustard and chickpea. In rice, tagging of genes for resistance to gall midge and blast has been accomplished. Molecular mapping of cooking quality traits in rice is in progress. For fingerpringting rice cultivars, suitable probe enzyme combinations have been identified. In mustard, a partial RFLP linkage map has been constructed and one of the yellow seed-coat colour loci has been mapped. Significant associations of RFLP markers with quantitative traits have also been established. Potential use of RAPD markers to identify heterotic groups among mustard accessions has been demonstrated. In chickpea, the occurrence of considerable interspecific DNA polymorphism as revealed by RAPD analysis has facilitated construction of a partial linkage map.     

A first genetic linkage map of mulberry (Morus spp.) using RAPD, ISSR, and SSR markers and pseudotestcross mapping strategy

To lay the foundation for molecular breeding efforts, the first genetic linkage map of mulberry (2n=2x=28) was constructed with 50 F1 full-sib progeny using randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and simple sequence repeat (SSR) markers and two-way pseudotestcross mapping strategy. We selected 100 RAPD, 42 ISSR, and 9 SSR primers that amplified 517 markers, of which 188 (36.36%) showed a test-cross configuration, corresponding to the heterozygous condition in one parent and null in the other. Two separate female and male maps were constructed using 94 each of female- and male-specific testcross markers, containing 12 female linkage groups and 14 male linkage groups. At a minimum logarithm of the odds (LOD) score threshold of 6.0 and at a maximum map distance of 20 cM, the female map covered a 1,196.6-cM distance, with an average distance of 15.75 cM and maximum map distance of 37.9 cM between two loci; the male-specific map covered a 1,351.7-cM distance, with an average distance of 18.78 cM and a maximum map distance between two loci is of 34.7 cM. The markers distributed randomly in all linkage groups without any clustering. All 12 linkage groups in the female-specific map consisted of 4–10 loci ranging in length from 0 to 140.4 cM, and in the male-specific map, the 13 largest linkage groups (except linkage group 12, which contained three loci) consisted of 4–12 loci, ranging in length from 53.9 to 145.9 cM and accounting for 97.22% of the total map distance. When mapping, progeny pass through their juvenile phase and assume their adult characters, mapping morphological markers and identification of quantitative trait loci for adaptive traits will be the primary target. In that sense, our map provides reference information for future molecular breeding work on Morus and its relatives.     

Genetic Linkage Mapping and Analysis of Muscle Fiber-Related QTLs in Common Carp (Cyprinus carpio L.)

A genetic linkage map of common carp (Cyprinus carpio L.) was constructed using Type I and Type II microsatellite markers and a pseudo-testcross mapping strategy. The microsatellite markers were isolated from microsatellite-enriched genomic libraries and tested for their segregation in a full-sib mapping panel containing 92 individuals. A total of 161 microsatellite loci were mapped into 54 linkage groups. The total lengths of the female, male and consensus maps were 2,000, 946, and 1,852?cM, with an average marker spacing of approximately 13, 7, and 11?cM, respectively. Muscle fiber-related traits, including muscle fiber cross-section area and muscle fiber density, were mapped to the genetic map. Three QTLs for muscle fiber cross-section area and two QTLs for muscle fiber density were identified when considering both significant and suggestive QTL effects. The QTLs with largest effects for muscle fiber cross-section area and muscle fiber density were 21.9% and 18.9%, and they were located in LG3, respectively.     

A combined amplified fragment length polymorphism and randomly amplified polymorphism DNA genetic kinkage map of Mycosphaerella graminicola,the septoria tritici leaf blotch pathogen of wheat

An F(1) mapping population of the septoria tritici blotch pathogen of wheat, Mycosphaerella graminicola, was generated by crossing the two Dutch field isolates IPO323 and IPO94269. AFLP and RAPD marker data sets were combined to produce a high-density genetic linkage map. The final map contained 223 AFLP and 57 RAPD markers, plus the biological traits mating type and avirulence, in 23 linkage groups spanning 1216 cM. Many AFLPs and some RAPD markers were clustered. When markers were reduced to 1 per cluster, 229 unique positions were mapped, with an average distance of 5.3 cM between markers. Because M. graminicola probably has 17 or 18 chromosomes, at least 5 of the 23 linkage groups probably will need to be combined with others once additional markers are added to the map. This was confirmed by pulsed-field gel analysis; probes derived from 2 of the smallest linkage groups hybridized to two of the largest chromosome-sized bands, revealing a discrepancy between physical and genetic distance. The utility of the map was demonstrated by identifying molecular markers tightly linked to two genes of biological interest, mating type and avirulence. Bulked segregant analysis was used to identify additional molecular markers closely linked to these traits. This is the first genetic linkage map for any species in the genus Mycosphaerella or the family Mycosphaerellaceae.