A 3.9-centimorgan-resolution human single-nucleotide polymorphism linkage map and screening set

Recent advances in technologies for high-throughout single-nucleotide polymorphism (SNP)-based genotyping have improved efficiency and cost so that it is now becoming reasonable to consider the use of SNPs for genomewide linkage analysis. However, a suitable screening set of SNPs and a corresponding linkage map have yet to be described. The SNP maps described here fill this void and provide a resource for fast genome scanning for disease genes. We have evaluated 6,297 SNPs in a diversity panel composed of European Americans, African Americans, and Asians. The markers were assessed for assay robustness, suitable allele frequencies, and informativeness of multi-SNP clusters. Individuals from 56 Centre d'Etude du Polymorphisme Humain pedigrees, with >770 potentially informative meioses altogether, were genotyped with a subset of 2,988 SNPs, for map construction. Extensive genotyping-error analysis was performed, and the resulting SNP linkage map has an average map resolution of 3.9 cM, with map positions containing either a single SNP or several tightly linked SNPs. The order of markers on this map compares favorably with several other linkage and physical maps. We compared map distances between the SNP linkage map and the interpolated SNP linkage map constructed by the deCode Genetics group. We also evaluated cM/Mb distance ratios in females and males, along each chromosome, showing broadly defined regions of increased and decreased rates of recombination. Evaluations indicate that this SNP screening set is more informative than the Marshfield Clinic's commonly used microsatellite-based screening set.     

Markers for mapping by admixture linkage disequilibrium in African American and Hispanic populations

Population linkage disequilibrium occurs as a consequence of mutation, selection, genetic drift, and population substructure produced by admixture of genetically distinct ethnic populations. African American and Hispanic ethnic groups have a history of significant gene flow among parent groups, which can be of value in affecting genome scans for disease-gene discovery in the case-control and transmission/disequilibrium test designs. Disease-gene discovery using mapping by admixture linkage disequilibrium (MALD) requires a map of polymorphic markers that differentiate between the founding populations, along with differences in disease-gene allele frequencies. We describe markers appropriate for MALD mapping by assessing allele frequencies of 744 short tandem repeats (STRs) in African Americans, Hispanics, European Americans, and Asians, by choosing STR markers that have large differences in composite delta, log-likelihood ratios, and/or I*(2) for MALD. Additional markers can be added to this MALD map by utilization of the rapidly growing single-nucleotide-polymorphism databases and the literature, to achieve a 3-10-cM scanning scale. The map will be useful for studies of diseases, including prostate and breast cancer, diabetes, hypertension, and end-stage renal disease, that have large differences in incidence between the founding populations of either Hispanics or African Americans.     

An integrated map of Oryza sativa L. chromosome 5

The developments of molecular marker-based genetic linkage maps are now routine. Physical maps based on contigs of large insert genomic clones have been established in several plant species. However, integration of genetic, physical, and cytological maps is still a challenge for most plant species. Here we present an integrated map of rice (Oryza sativa L.) chromosome 5, developed by fluorescence in situ hybridization mapping of 18 bacterial artificial chromosome (BAC) clones or PI-derived artificial chromosome (PAC) clones on meiotic pachytene chromosomes. Each BAC/PAC clone was anchored by a restriction fragment length polymorphism marker mapped to the rice genetic linkage map. This molecular cytogenetic map shows the genetic recombination and sequence information of a physical map, correlated to the cytological features of rice chromosome 5. Detailed comparisons of the distances between markers on genetic, cytological, and physical maps, revealed the distributions of recombination events and molecular organization of the chromosomal features of rice chromosome 5 at the pachytene stage. Discordance of distances between the markers was found among the different maps. Our results revealed that neither the recombination events nor the degree of chromatin condensation were evenly distributed along the entire length of chromosome 5. Detailed comparisons of the correlative positions of markers on the genetic, cytological, and physical maps of rice chromosome 5 provide insight into the molecular architecture of rice chromosome 5, in relation to its cytological features and recombination events on the genetic map. The prospective applications of such an integrated cytogenetic map are discussed.     

Chromosome-level homeology in paleopolyploid soybean (Glycine max) revealed through integration of genetic and chromosome maps

Soybean has 20 chromosome pairs that are derived from at least two rounds of genomewide duplication or polyploidy events although, cytogenetically, soybean behaves like a diploid and has disomic inheritance for most loci. Genetically anchored genomic clones were used as probes for fluorescence in situ hybridization (FISH) to determine the level of postpolyploid chromosomal rearrangements and to integrate the genetic and physical maps to (1) assign linkage groups to specific chromosomes, (2) assess chromosomal structure, and (3) determine the distribution of recombination along the length of a chromosome. FISH mapping of seven putatively gene-rich BACs from linkage group L (chromosome 19) revealed that most of the genetic map correlates to the highly euchromatic long arm and that there is extensive homeology with another chromosome pair, although colinearity of some loci does appear to be disrupted. Moreover, mapping of BACs containing high-copy sequences revealed sequestration of high-copy repeats to the pericentromeric regions of this chromosome. Taken together, these data present a model of chromosome structure in a highly duplicated but diploidized eukaryote, soybean.     

Corticotropin releasing hormone (CRH) gene variation: comprehensive resequencing for variant and molecular haplotype discovery in monosomic hybrid cell lines.

Candidate gene association studies have met with mixed success due to many reasons including incomplete surveys of genetic variation and differences in patterns of genetic variation among study populations. We present the results of comprehensive variant discovery for the corticotropin releasing hormone gene (CRH on chromosome 8) encoding a neuropeptide that is central to many physiologic pathways. Mouse-human hybrid cell lines were constructed that are monosomic for human chromosome 8 for resequencing of separated CRH alleles to identify variants and directly determine their chromosomal phase for three major ethnic groups including African Americans (AA), Mexican Americans (MA) and European Americans (EA). We also resequenced diploid individuals to evaluate single nucleotide polymorphism (SNP) discovery in the limited numbers of monosomic hybrid cell lines. Our results show that CRH variation is very different in AA, yielding larger numbers of variants and haplotypes compared to MA and EA. Analysis of LD structure found three haplotype blocks in AA and two blocks in EA. Comparisons between AA and EA groups yielded extremely high measures of genetic differentiation (Wright's F(ST)>0.6), likely reflecting disruptive selection in CRH evolution. Network analysis showed that AA have retained an ancestral CRH haplotype, while the most common EA haplotype is derived from a single recombination event.     

A cytogenetically based physical map of chromosome 1B in common wheat.

We have constructed a cytogenetically based physical map of chromosome 1B in common wheat by utilizing a total of 18 homozygous deletion stocks. It was possible to divide chromosome 1B into 17 subregions. Nineteen genetic markers are physically mapped to nine subregions of chromosome 1B. Comparison of the cytological map of chromosome 1B with an RFLP-based genetic linkage map of Triticum tauschii revealed that the linear order of the genetic markers was maintained between chromosome 1B of hexaploid wheat and 1D of T. tauschii. Striking differences were observed between the physical and genetic maps in relation to the relative distances between the genetic markers. The genetic markers clustered in the middle of the genetic map were physically located in the distal regions of both arms of chromosome 1B. It is unclear whether the increased recombination in the distal regions of chromosome 1B is due to specific regions of increased recombination or a more broadly distributed increase in recombination in the distal regions of Triticeae chromosomes.     

The use of multiple alien chromosome addition aneuploids facilitates genetic linkage mapping of the Gossypium G genome.

Primary germplasm pools represent the most accessible source of new alleles for crop improvement, but not all effective alleles are available in the primary germplasm pool, and breeders must sometimes confront the difficulties of introgressing genes from the secondary and tertiary germplasm pools in cotton by using synthetic polyploids as introgression bridges. Two parental Gossypium nelsonii x Gossypium australe AFLP genetic linkage maps were used to identify G genome chromosome-specific molecular markers, which in turn were used to track the fidelity and frequency of G. australe chromosome transmission in a Gossypium hirsutum x G. australe hexaploid bridging family. Conversely, when homoeologous recombination is low, first generation aneuploids are useful adjuncts to genetic linkage mapping. Although locus ordering was not possible, the distribution of AFLP markers among 18 multiple chromosome addition aneuploids identified mapping errors among the G. australe and G. nelsonii linkage groups and assigned non-segregating G. australe AFLPs to linkage groups. Four putatively recombined G. australe chromosomes were identified in 5 of the 18 aneuploids. The G. australe and G. nelsonii genetic linkage maps presented here represent the first AFLP genetic linkage maps for the Gossypium G genome.     

Racial admixture and its impact on BMI and blood pressure in African and Mexican Americans

Admixed populations such as African Americans and Hispanic Americans present both challenges and opportunities in genetic epidemiologic research. Because of variation in admixture levels among individuals, case-control association studies may be subject to stratification bias. On the other hand, admixed populations also present special opportunities both for examining the role of genetic and environmental factors for observed racial/ethnic differences, and for possibly mapping alleles that contribute to such differences. Here we examined the distribution and relationship of individual admixture (IA) estimates with BMI and three measures of blood pressure in two admixed populations in the NHLBI Family Blood Pressure Program (FBPP): African Americans and Mexican Americans. For the African Americans, we observed modest but significant differences in average African IA among four recruitment sites. We observed a slight excess of African IA among hypertensives compared to normotensives, and a positive (non-significant) regression of African IA on blood pressure in untreated participants. Within Mexican Americans, we found no difference in average IA between hypertensives and normotensives, but a positive (marginally significant) regression of African IA on diastolic blood pressure. We also observed a significant positive regression of Caucasian IA (and negative regression of Native American IA) on BMI. Our results are suggestive of genetic differences between Africans and non-Africans that influence blood pressure, but such effects are likely to be modest compared to environmental ones. Excess obesity among Native Americans compared to whites is not consistent with a simple genetic explanation.     

Generation of a Restriction Fragment Length Polymorphism Linkage Map for Toxoplasma Gondii

We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.     

Male mouse recombination maps for each autosome identified by chromosome painting

Linkage maps constructed from genetic analysis of gene order and crossover frequency provide few clues to the basis of genomewide distribution of meiotic recombination, such as chromosome structure, that influences meiotic recombination. To bridge this gap, we have generated the first cytological recombination map that identifies individual autosomes in the male mouse. We prepared meiotic chromosome (synaptonemal complex [SC]) spreads from 110 mouse spermatocytes, identified each autosome by multicolor fluorescence in situ hybridization of chromosome-specific DNA libraries, and mapped >2,000 sites of recombination along individual autosomes, using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites. We show that SC length is strongly correlated with crossover frequency and distribution. Although the length of most SCs corresponds to that predicted from their mitotic chromosome length rank, several SCs are longer or shorter than expected, with corresponding increases and decreases in MLH1 frequency. Although all bivalents share certain general recombination features, such as few crossovers near the centromeres and a high rate of distal recombination, individual bivalents have unique patterns of crossover distribution along their length. In addition to SC length, other, as-yet-unidentified, factors influence crossover distribution leading to hot regions on individual chromosomes, with recombination frequencies as much as six times higher than average, as well as cold spots with no recombination. By reprobing the SC spreads with genetically mapped BACs, we demonstrate a robust strategy for integrating genetic linkage and physical contig maps with mitotic and meiotic chromosome structure.     

Evidence of a novel quantitative-trait locus for obesity on chromosome 4p in Mexican Americans

Although several genomewide scans have identified quantitative-trait loci influencing several obesity-related traits in humans, genes influencing normal variation in obesity phenotypes have not yet been identified. We therefore performed a genome scan of body mass index (BMI) on Mexican Americans, a population prone to obesity and diabetes, using a variance-components linkage analysis to identify loci that influence BMI. We used phenotypic data from 430 individuals (26% diabetics, 59% females, mean age +/- SD = 43 +/- 17 years, mean BMI +/- SD = 30.0 +/- 6.7, mean leptin (ng/ml) +/- SD = 22.1 +/- 17.1) distributed across 27 low-income Mexican American pedigrees who participated in the San Antonio Family Diabetes Study (SAFDS) for whom a 10-15-cM map is available. In this genomewide search, after accounting for the covariate effects of age, sex, diabetes, and leptin, we identified a genetic region exhibiting the most highly significant evidence for linkage (LOD 4.5) with BMI on chromosome 4p (4p15.1) at 42 cM, near marker D4S2912. This linkage result has been confirmed in an independent linkage study of severe obesity in Utah pedigrees. Two strong positional candidates, the human peroxisome proliferator-activated receptor gamma coactivator 1 (PPARGC1) and cholecystokinin A receptor (CCKAR) with major roles in the development of obesity, are located in this region. In conclusion, we identified a major genetic locus influencing BMI on chromosome 4p in Mexican Americans.     

A physical map of human chromosome 10 and a comparison with an existing genetic map.

A physical map for 13 loci on chromosome 10 was developed by determining the dosage of the corresponding DNA sequences in cell lines with unbalanced chromosome 10 rearrangements. Nine of the sequences were assigned to a smaller segment of the chromosome than previously and four sublocalizations were confirmed. The physical map covers most of chromosome 10, from 10p13 to 10q23. The linear order of loci within the physical map agrees with existing linkage maps of chromosome 10. A comparison between the physical map and existing genetic maps indicate an uneven distribution of recombination for chromosome 10. There appear to be hot spots of recombination in the regions defined by q21.1 and q22-q23. In addition, there is a suppression of recombination in the pericentromeric region in males which is not evident in females.     

Genome-wide linkage scan for the metabolic syndrome: the GENNID study

In the United States, the metabolic syndrome (MetS) constitutes a major public health problem with over 47 million persons meeting clinical criteria for MetS. Numerous studies have suggested genetic susceptibility to MetS. The goals of this study were (i) to identify susceptibility loci for MetS in well-characterized families with type 2 diabetes (T2D) in four ethnic groups and (ii) to determine whether evidence for linkage varies across the four groups. The GENNID study (Genetics of NIDDM) is a multicenter study established by the American Diabetes Association in 1993 and comprises a comprehensive, well-characterized resource of T2D families from four ethnic groups (whites, Mexican Americans, African Americans, and Japanese Americans). Principal component factor analysis (PCFA) was used to define quantitative phenotypes of the MetS. Variance components linkage analysis was conducted using microsatellite markers from a 10-cM genome-wide linkage scan, separately in each of the four ethnic groups. Three quantitative MetS factors were identified by PCFA and used as phenotypes for MetS: (i) a weight/waist factor, (ii) a blood pressure factor, and (iii) a lipid factor. Evidence for linkage to each of these factors was observed. For each ethnic group, our results suggest that several regions harbor susceptibility genes for the MetS. The strongest evidence for linkage for MetS phenotypes was observed on chromosome 2 (2q12.1-2q13) in the white sample and on chromosome 3 (3q26.1-3q29) in the Mexican-American sample. In conclusion, the results suggest that several regions harbor MetS susceptibility genes and that heterogeneity may exist across groups.     

Genome-wide analysis of the frequency and distribution of crossovers at male and female meiosis in Sinapis alba L. (white mustard)

We present the first genetic linkage maps of Sinapis alba (white mustard) and a rigorous analysis of sex effects on the frequency and distribution of crossovers at meiosis in this species. Sex-averaged maps representing recombination in two highly heterozygous parents were aligned to give a consensus map consisting of 382 loci defined by restriction fragment length polymorphisms and arranged in 12 linkage groups with no unlinked markers. The loci were distributed in a near-random manner across the genome, and there was little evidence of segregation distortion. From these dense maps, a subset of spaced informative markers was used to establish recombination frequencies assayed separately in male and female gametes and derived from two distinct genetic backgrounds. Analyses of 746 gametes indicated that recombination frequencies were greater in male gametes, with the greatest differences near the ends of linkage groups. Genetic background had a lesser effect on recombination frequencies, with no discernible pattern in the distribution of such differences. The possible causes of sex differences in recombination frequency and the implications for plant breeding are discussed.     

A comparative analysis of the rainbow trout genome with 2 other species of fish (Arctic charr and Atlantic salmon) within the tetraploid derivative Salmonidae family (subfamily: Salmoninae).

We updated the genetic map of rainbow trout (Oncorhynchus mykiss) for 2 outcrossed mapping panels, and used this map to assess the putative chromosome structure and recombination rate differences among linkage groups. We then used the rainbow trout sex-specific maps to make comparisons with 2 other ancestrally polyploid species of salmonid fishes, Arctic charr (Salvelinus alpinus) and Atlantic salmon (Salmo salar) to identify homeologous chromosome affinities within each species and ascertain homologous chromosome relationships among the species. Salmonid fishes exhibit a wide range of sex-specific differences in recombination rate, with some species having the largest differences for any vertebrate species studied to date. Our current estimate of female:male recombination rates in rainbow trout is 4.31:1. Chromosome structure and (or) size is associated with recombination rate differences between the sexes in rainbow trout. Linkage groups derived from presumptive acrocentric type chromosomes were observed to have much lower sex-specific differences in recombination rate than metacentric type linkage groups. Arctic charr is karyotypically the least derived species (i.e., possessing a high number of acrocentric chromosomes) and Atlantic salmon is the most derived (i.e., possessing a number of whole-arm fusions). Atlantic salmon have the largest female:male recombination ratio difference (i.e., 16.81:1) compared with rainbow trout, and Arctic charr (1.69:1). Comparisons of recombination rates between homologous segments of linkage groups among species indicated that when significant experiment-wise differences were detected (7/24 tests), recombination rates were generally higher in the species with a less-derived chromosome structure (6/7 significant comparisons). Greater similarity in linkage group syntenies were observed between Atlantic salmon and rainbow trout, suggesting their closer phylogenetic affinities, and most interspecific linkage group comparisons support a model that suggests whole chromosome arm translocations have occurred in the evolution of this group. However, some possible exceptions were detected and these findings are discussed in relation to their influence on segregation distortion patterns. We also report unusual meiotic segregation patterns in a female parent involving the duplicated (homeologous) linkage group pair 12/16 and discuss several models that may account for these patterns.     

A comparison of linkage disequilibrium patterns and estimated population recombination rates across multiple populations

Large-scale studies of linkage disequilibrium (LD) have shown considerable variation in the extent and distribution of pairwise LD within and between populations. Taken at face value, these results suggest that genomewide LD maps for one population may not be generalizable to other populations. However, at least part of this diversity is due to some undesirable features of pairwise LD measures, which are well documented for the D' and r2 measures. In this report, we compare patterns of LD derived from pairwise measures with statistical estimates of population recombination rates ( rho ) along a 10-Mb stretch of chromosome 20 in four population samples, comprising East Asians, African Americans, and U.K. and U.S. individuals of western European descent. The results reveal the expected variability of D' within and between populations but show better concordance in estimates of r2 for the same markers across the population samples. Estimates of rho correlate well across populations, but there is still evidence of population-specific spikes and troughs in rho values. We conclude that it is unlikely that a single haplotype map will provide a definitive guide for association studies of many populations; rather, multiple maps will need to be constructed to provide the best-possible guides for gene mapping.     

Twelve loci form a continuous linkage map for human chromosome 18

We have constructed a primary genetic map of human chromosome 18 consisting of 11 DNA markers and one serological marker (JK). Two of these loci define highly polymorphic VNTR systems. The markers define a continuous genetic linkage map of 97 cM in males and 205 cM in females; female genetic distances in a panel of 59 three-generation families were consistently about twice those observed in males. The high odds in support of the linear order of the markers on this recombination map, and the extent of coverage of chromosome 18, indicate that this map will permit efficient linkage studies of human genetic diseases that may be segregating on chromosome 18 and will provide anchor points for development of high-resolution maps for this chromosome.     

Linkage maps of microsatellite DNA markers for the Pacific oyster Crassostrea gigas

We constructed male and female consensus linkage maps for the Pacific oyster Crassostrea gigas, using a total of 102 microsatellite DNA markers typed in 11-day-old larvae from three families. We identified 11 and 12 linkage groups in the male and female consensus maps, respectively. Alignment of these separate maps, however, suggests 10 linkage groups, which agrees with the haploid chromosome number. The male linkage map comprises 88 loci and spans 616.1 cM, while the female map comprises 86 loci and spans 770.5 cM. The male and the female maps share 74 loci; 2 markers remain unlinked. The estimated coverages for the consensus linkage maps are 79% for the male and 70-75% for the female, on the basis of two estimates of genome length. Ninety-five percent of the genome is expected to lie within 16 and 21 cM of markers on the male and female maps, respectively, while 95% of simulated minimum distances to the male and female maps are within 10.1 and 13.6 cM, respectively. Females have significantly more recombination than males, across 118 pairs of linked markers in common to the parents of the three families. Significant differences in recombination and orders of markers are also evident among same-sex parents of different families as well as sibling parents of opposite sex. These observations suggest that polymorphism for chromosomal rearrangements may exist in natural populations, which could have profound implications for interpreting the evolutionary genetics of the oyster. These are the first linkage maps for a bivalve mollusc that use microsatellite DNA markers, which should enable them to be transferred to other families and to be useful for further genetic analyses such as QTL mapping.     

Assignment of rainbow trout linkage groups to specific chromosomes

The rainbow trout genetic linkage groups have been assigned to specific chromosomes in the OSU (2N=60) strain using fluorescence in situ hybridization (FISH) with BAC probes containing genes mapped to each linkage group. There was a rough correlation between chromosome size and size of the genetic linkage map in centimorgans for the genetic maps based on recombination from the female parent. Chromosome size and structure have a major impact on the female:male recombination ratio, which is much higher (up to 10:1 near the centromeres) on the larger metacentric chromosomes compared to smaller acrocentric chromosomes. Eighty percent of the BAC clones containing duplicate genes mapped to a single chromosomal location, suggesting that diploidization resulted in substantial divergence of intergenic regions. The BAC clones that hybridized to both duplicate loci were usually located in the distal portion of the chromosome. Duplicate genes were almost always found at a similar location on the chromosome arm of two different chromosome pairs, suggesting that most of the chromosome rearrangements following tetraploidization were centric fusions and did not involve homeologous chromosomes. The set of BACs compiled for this research will be especially useful in construction of genome maps and identification of QTL for important traits in other salmonid fishes.     

A linkage map of Atlantic salmon (Salmo salar) reveals an uncommonly large difference in recombination rate between the sexes

A genetic linkage map of the Atlantic salmon (Salmo salar) was constructed, using 54 microsatellites and 473 amplified fragment length polymorphism (AFLP) markers. The mapping population consisted of two full-sib families within one paternal half-sib family from the Norwegian breeding population. A mapping strategy was developed that facilitated the construction of separate male and female maps, while retaining all the information contributed by the dominant AFLP markers. By using this strategy, we were able to map a significant number of the AFLP markers for which all informative offspring had two heterozygous parents; these markers then served as bridges between the male and female maps. The female map spanned 901 cM and had 33 linkage groups, while the male spanned 103 cM and had 31 linkage groups. Twenty-five linkage groups were common between the two maps. The construction of the genetic map revealed a large difference in recombination rate between females and males. The ratio of female recombination rate vs. male recombination rate was 8.26, the highest ratio reported for any vertebrate. This map constitutes the first linkage map of Atlantic salmon, one of the most important aquaculture species worldwide.