Genetic analysis of alcohol intake in recombinant inbred and congenic strains derived from A/J and C57BL/6J progenitors

The objective of the present study was to map quantitative trait loci (QTL) for alcohol intake using A × B/B × A recombinant inbred (RI) and AcB/BcA recombinant congenic (RC) strains of mice that were independently derived from the A/J and C57BL/6J progenitors. Mice were screened for levels of alcohol consumption with four days of forced exposure to alcohol, followed by three weeks of free choice between water and a 10% alcohol solution. Alcohol consumption data previously collected for 27 A × B/B × A RI strains were reanalyzed using a larger marker set and composite interval mapping. The reanalysis found markers on Chromosome 2 (D2Mit74, 107 cM) (males and females) and on Chromosome 11 (Pmv22, 8 cM) (females only) that exceeded the threshold for significant loci, and found suggestive loci (in males) on Chromosomes 10 (D10 Mit126, 21 cM), 12 (D12Mit37, 1 cM), 15 (Pdgfb, 46.8 cM), and 16 (D16Mit125, 29 cM). An additional suggestive locus was identified in female RI mice on Chromosome 11 (D11Mit120, 47.5 cM). Composite interval mapping (CIM) analysis indicated that there was a significant association between loci at Pdgfb and D2Mit74 in both males and females. Analysis of the AcB/BcA RC strains identified 11 QTL on Chromosomes 2, 3, 5,6, 7, 8, 9, 10, 12, 13, and 15. QTL on Chromosomes 7, 10, 12, and 15 were identified in both the A × B/B × A RI and AcB/BcA RC strains of mice. Additional QTLs identified on Chromosomes 2, 3, 7, 11, and 15 overlap with those previously identified in the literature using strains of mice with a C57BL/6J progenitor.     

Genetics of body weight in the LXS recombinant inbred mouse strains

This is the first phenotypic analysis of 75 new recombinant inbred (RI) strains derived from ILS and ISS progenitors. We analyzed body weight in two independent cohorts of female mice at various ages and in males at 60 days. Body weight is a complex trait which has been mapped in numerous crosses in rodents. The LXS RI strains displayed a large range of weights, transgressing those of the inbred progenitors, supporting the utility of this large panel for mapping traits not selected in the progenitors. Numerous QTLs for body weight mapped in single- and multilocus scans. We assessed replication between these and previously reported QTLs based on overlapping confidence intervals of published QTLs for body weight at 60 days and used meta-analyses to determine combined p values for three QTL regions located on Chromosomes 4, 5, and 11. Strain distribution patterns of microsatellite marker genotypes, weight, and other phenotypes are available on WebQTL () and allow genetic mapping of any heritable quantitative phenotype measured in these strains. We report one such analysis, correlating brain and body weights. Large reference panels of RI strains, such as the LXS, are invaluable for identifying genetic correlations, GXE (Gene X Environment) interactions, and replicating previously identified QTLs. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Genetic structure of the LXS panel of recombinant inbred mouse strains: a powerful resource for complex trait analysis

The set of LXS recombinant inbred (RI) strains is a new and exceptionally large mapping panel that is suitable for the analysis of complex traits with comparatively high power. This panel consists of 77 strains—more than twice the size of other RI sets— and will typically provide sufficient statistical power (=0.8) to map quantitative trait loci (QTLs) that account for 25% of genetic variance with a genomewide p < 0.05. To characterize the genetic architecture of this new set of RI strains, we genotyped 330 MIT microsatellite markers distributed on all autosomes and the X Chromosome and assembled error-checked meiotic recombination maps that have an average F₂-adjusted marker spacing of 4 cM. The LXS panel has a genetic structure consistent with random segregation and subsequent fixation of alleles, the expected 3–4 × map expansion, a low level of nonsyntenic association among loci, and complete independence among all 77 strains. Although the parental inbred strains—Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS)—were derived originally by selection from an 8-way heterogeneous stock selected for differential sensitivity to sedative effects of ethanol, the LXS panel is also segregating for many other traits. Thus, the LXS panel provides a powerful new resource for mapping complex traits across many systems and disciplines and should prove to be of great utility in modeling the genetics of complex diseases in human populations.(Robert W. Williams and Beth Bennett)These authors contributed equally to this work.     

Establishment of consomic strains derived from A/J and SM/J mice for genetic analysis of complex traits

Consomic strains, in which one chromosome is derived from a donor strain and the other chromosomes are derived from the recipient strain, provide a powerful tool for the dissection of complex genetic traits. In this study we established ten consomic strains (A-2^SM, A-6^SM, A-11^SM, A-12^SM, A-13^SM, A-15^SM, A-17^SM, A-18^SM, A-19^SM, A-Y^SM) using the SM/J strain as the donor and the A/J strain as the recipient; these are the parental strains of a set of SMXA recombinant inbred (RI) strains that we had developed previously. We analyzed body weights and blood lipid levels in the consomic and parental strains. The mean values for each trait showed a continuous range of variation in the consomic strains suggesting that they are controlled by multiple genes. We previously identified suggestive QTLs for body weight on chromosome 6 in SMXA RI strains and (SM/J?×?A/J)F₂ mice. The observation that the A-6^SM consomic strain had a significantly lower mean body weight than the A/J strain supports the presence of this QTL on chromosome 6. Similarly, the higher blood triglyceride level in the A-11^SM strain shows the existence of a previously mapped QTL on chromosome 11, and the A-12^SM strain provides evidence of a QTL for blood total cholesterol level on chromosome 12. These consomic strains, along with the previously developed set of SMXA RI strains from A/J and SM/J mice, offer an invaluable and powerful resource for the analysis of complex genetic traits in mice.     

Establishment of consomic strains derived from A/J and SM/J mice for genetic analysis of complex traits

Consomic strains, in which one chromosome is derived from a donor strain and the other chromosomes are derived from the recipient strain, provide a powerful tool for the dissection of complex genetic traits. In this study we established ten consomic strains (A-2^SM, A-6^SM, A-11^SM, A-12^SM, A-13^SM, A-15^SM, A-17^SM, A-18^SM, A-19^SM, A-Y^SM) using the SM/J strain as the donor and the A/J strain as the recipient; these are the parental strains of a set of SMXA recombinant inbred (RI) strains that we had developed previously. We analyzed body weights and blood lipid levels in the consomic and parental strains. The mean values for each trait showed a continuous range of variation in the consomic strains suggesting that they are controlled by multiple genes. We previously identified suggestive QTLs for body weight on chromosome 6 in SMXA RI strains and (SM/J × A/J)F₂ mice. The observation that the A-6^SM consomic strain had a significantly lower mean body weight than the A/J strain supports the presence of this QTL on chromosome 6. Similarly, the higher blood triglyceride level in the A-11^SM strain shows the existence of a previously mapped QTL on chromosome 11, and the A-12^SM strain provides evidence of a QTL for blood total cholesterol level on chromosome 12. These consomic strains, along with the previously developed set of SMXA RI strains from A/J and SM/J mice, offer an invaluable and powerful resource for the analysis of complex genetic traits in mice.

     

Genotyping new BXD recombinant inbred mouse strains and comparison of BXD and consensus maps

Nine additional BXD recombinant inbred (RI) strains have been developed from the F₂ cross of C57BL/6J and DBA/2J mouse strains. A tenth line stopped breeding in the F₁₂ generation. F₂₀ generation breeding pairs from the nine surviving strains and an F₁₂ pair from the extinct line were genotyped at 319 genetic markers (primarily microsatellites) spanning most of the genome. Where typing data were lacking, the established set of 26 BXD strains also were genotyped at these same loci. The availability of these additional nine strains enhances the value of the BXD RI set for analysis of complex phenotypic traits. The proportion of loci still segregating at the F₂₀ generation was found to closely approximate expectation, suggesting that selection favoring the retention of heterozygosity is not a strong factor. However, the number of crossovers between adjacent markers was frequently less than predicted from consensus map distances. A significant deficiency of recombinants was observed on Chrs 3, 4, 14, and X. On Chr 14, the estimated cumulative BXD map distance between the most proximal and distal markers was only 30.2 cM, compared with a distance of 60.0 cM in the consensus map. On the X Chr, the estimated and predicted cumulative distances were 38.8 and 69.5 cM, respectively. Over all chromosomes, the BXD RI map is 14.5% shorter than predicted from the consensus map. It is suggested that distances in some of the consensus maps are inflated. Alternatively, recombinant genotypes could be selected against during inbreeding owing to allelic interactions affecting fitness. The latter interpretation implies that relatively strong intrachromosomal epistasis is common. Received: 2 October 1998 / Accepted: 15 December 1998     

Characterization of the endogenous nonecotropic murine leukemia viruses of NZB/BINJ and SM/J inbred strains

We characterized 84 endogenous nonecotropic proviruses of NZB/B1NJ and SM/J inbred strains by examining proviral junction fragment segregation in recombinant inbred (RI) and backcross mice. Forty-five proviruses were shared with other laboratory strains, but 28 were unique to NZB/BINJ or SM/J. Proviral loci were located on 17 of the 19 mouse autosomes and on both sex chromosomes. These markers will facilitate gene mapping in the NXSM RI set and contribute to the pursuit of a more complete map of the mouse genome.     

Fine mapping of QTL for prepulse inhibition in LG/J and SM/J mice using F2 and advanced intercross lines

Prepulse inhibition (PPI) of the startle response is a measure of sensorimotor gating, a process that filters out extraneous sensory, motor and cognitive information. Humans with neurological and psychiatric disorders, including schizophrenia, obsessive‐compulsive disorder and Huntington's disease, exhibit a reduction in PPI. Habituation of the startle response is also disrupted in schizophrenic patients. In order to elucidate the genes involved in sensorimotor gating, we phenotyped 472 mice from an F₂ cross between LG/J × SM/J for PPI and genotyped these mice genome‐wide using 162 single nucleotide polymorphism (SNP) markers. We used prepulse intensity levels that were 3, 6 and 12 dB above background (PPI3, PPI6 and PPI12, respectively). We identified a significant quantitative trait locus (QTL) on chromosome 12 for all three prepulse intensities as well as a significant QTL for both PPI6 and PPI12 on chromosome 11. We identified QTLs on chromosomes 7 and 17 for the startle response when sex was included as an interactive covariate and found a QTL for habituation of the startle response on chromosome 4. We also phenotyped 135 mice from an F₃₄ advanced intercross line (AIL) between LG/J × SM/J for PPI and genotyped them at more than 3000 SNP markers. Inclusions of data from the AIL mice reduced the size of several of these QTLs to less than 5 cM. These results will be useful for identifying genes that influence sensorimotor gaiting and show the power of AIL for fine mapping of QTLs.     

Genetic dissection of testicular weight in the mouse with the BXD recombinant inbred strains

Testicular weights were studied in the mouse BXD recombinant inbred (RI) strains. These strains were derived from DBA/2J and C57BL/6J progenitors that differ significantly in their testicular weights (0.224 g ± 0.015 vs. 0.161 g ± 0.03, P < 0.0001). The heritability of testicular weights was calculated to be 0.53, and the minimum number of responsible effective factors was estimated to be 5.7. The total genome scanning of the BXD RI strains with over 1000 markers revealed a quantitative trait locus (QTL) on mouse Chromosome (Chr) 13 near the D13Mit3 marker (LOD score 6.9). This QTL region was designated Twq1 and associated with over 75% of genetic variability. Received: 23 January 1998 / Accepted: 16 March 1998     

Linkage map and congenic strains to localize blood pressure QTL on rat Chromosome 10

Our purposes were to develop a linkage map for rat Chromosome (Chr) 10, using chromosome-sorted DNA, and to construct congenic strains to localize blood pressure quantitative trait loci (QTL) on Chr 10 with the map. The linkage mapping panel consisted of three F₂ populations totaling 418 rats. Thirty-two new and 29 known microsatellite markers were placed on the map, which spanned 88.9 centiMorgans (cM). The average distance between markers was 1.46 cM. No markers were separated by more than 6.8 cM. Four congenic strains were constructed by introgressing various segments of Chr 10 from the Milan normotensive strain (MNS) onto the background of the Dahl salt-sensitive (S) strain. A blood pressure QTL with a strong effect on blood pressure (35–42 mm Hg) when expressed on the S background was localized to a 31-cM region between D10Mco6 and D10Mcol. The region does not include the locus for inducible nitric oxide synthase (Nos2), which had been considered to be a candidate locus for the QTL. Received: 25 September 1996 / Accepted: 9 November 1996     

Mapping diabetes QTL in an intercross derived from a congenic strain of the Brown Norway and Goto-Kakizaki rats

Genetic studies in experimental crosses derived from the inbred Goto-Kakizaki (GK) rat model of spontaneous diabetes mellitus have identified quantitative trait loci (QTL) for diabetes phenotypes in a large region of rat Chromosome (RNO) 1. To test the impact of GK variants on QTL statistical and biological features, we combined genetic and physiologic studies in a cohort of F₂ hybrids derived from a QTL substitution congenic strain (QTLSCS) carrying a 110-cM GK haplotype of RNO1 introgressed onto the genetic background of the Brown Norway (BN) strain. Glucose intolerance and altered insulin secretion in QTLSCS rats when compared with BN controls were consistent with original QTL features in a GK × BN F₂ cross. Segregating GK alleles in the QTLSCS F₂ cross account for most of these phenotypic differences between QTLSCS and BN rats. However, significant QTL for diabetes traits in both the QTLSCS and GK × BN F₂ cohorts account for a similar small proportion of their variance. Comparing results from these experimental systems provides indirect estimates of the contribution of genetic interactions and environmental factors to QTL architecture as well as locus and biological targets for future post-QTL mapping studies in congenic substrains. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Stephan C. Collins and Robert H. Wallis contributed equally to this work.     

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.     

High resolution mapping of chromosomal regions controlling resistance to gastrointestinal nematode infections in an advanced intercross line of mice

Fine mapping of quantitative trait loci (QTL) associated with resistance to the gastrointestinal parasite Heligmosomoides polygyrus was achieved on F₆/F₇ offspring (1076 mice) from resistant (SWR) and susceptible (CBA) mouse strains by selective genotyping (top and bottom 20% selected on total worm count in week 6). Fecal egg counts were recorded at weeks 2, 4, and 6, and the average was also analyzed. Blood packed cell volume in weeks 3 and 6 and five immunological traits (mucosal mast cell protease 1, granuloma score, IgG1 against adult worm, IgG1, and IgE to L4 antigen) were also recorded. On Chromosome 1 single-trait analyses identified a QTL with effects on eight traits located at about 24 cM on the F₂ mouse genome database (MGD) linkage map, with a 95% confidence interval (CI) of 20-32 cM established from a multitrait analysis. On Chromosome 17 a QTL with effects on nine traits was located at about 18 cM on the MGD map (CI 17.9-18.4 cM). Strong candidate genes for the QTL position on Chromosome 1 include genes known to be involved in regulating immune responses and on Chromosome 17 genes within the MHC, notably the Class II molecules and tumor necrosis factor.     

Diabetic modifier QTLs identified in F2 intercrosses between Akita and A/J mice

To identify novel genetic modifiers of type 2 diabetes (T2D), we performed quantitative trait loci (QTL) analysis on F₂ progeny of hypoinsulinemic diabetic Akita mice, heterozygous for the Ins2 gene Cys96Tyr mutation, and nondiabetic A/J mice. We generated 625 heterozygous (F₂-Hetero) and 338 wild-type (F₂-Wild) mice with regard to the Ins2 mutation in F₂ intercross progeny. We measured quantitative traits, including plasma glucose and insulin concentrations during the intraperitoneal glucose tolerance test (IPGTT), and body weight (BW). We observed three significant QTLs in hypoinsulinemic hyperglycemic male F₂-Hetero mice, designated Dbm1, Dbm3, and Dbm4 on Chromosomes 6, 14, and 15, respectively. They showed linkage to plasma glucose concentrations, with significant maximum logarithm of odds (LOD) scores of 4.12, 4.17, and 6.17, respectively, all exceeding threshold values by permutation tests. In normoinsulinemic normoglycemic male F₂-Wild mice, Dbm1 on Chromosome 6 showed linkage to both plasma insulin concentrations and BW, and Dbm2 on Chromosome 11 showed linkage to plasma glucose concentrations only, with LOD scores of 4.52 and 6.32, and 5.78, respectively. Based on these results, we concluded that Dbm1, Dbm2, Dbm3, and Dbm4 represent four major modifier QTLs specifically affecting T2D-related traits and that these diabetic modifier QTLs are conditional on the heterozygous Ins2 gene mutation and sex to exert their modifier functions. Identification of the genes responsible for these QTLs would provide new drug development targets for human T2D. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

PLEIOTROPIC EFFECTS OF INDIVIDUAL GENE LOCI ON MANDIBULAR MORPHOLOGY

The genotypic basis of morphological variation is largely unknown. In this study we examine patterns of pleiotropic effects on mandibular morphology at individual gene loci to determine whether the pleiotropic effects of individual genes are restricted to functionally and developmentally related traits. Mandibular measurements were obtained from 480 mice from the F₂ generation of an intercross between the LG/J and SM/J mouse strains. DNA was also extracted from these animals, and 76 microsatellite loci covering the autosomes were scored. Interval mapping was used to detect chromosomal locations with significant effects on various mandibular measurements. Sets of traits mapping to a common chromosomal region were considered as being affected by a single quantitative trait locus (QTL) for mandibular morphology. Thirty-seven such chromosomal regions were identified spread throughout the autosomes. Gene effects were small to moderate with the allele derived from the LG/J strain typically leading to larger size. When dominance was present, the LG/J allele was typically dominant to the SM/J allele. Most loci affected restricted functional and developmental regions of the mandible. Of the 26 chromosomal regions affecting more than two traits, 50% affect the muscular processes of the ascending ramus, 27% affect the alveolar processes carrying the teeth, and 23% affect the whole mandible. Four additional locations affecting two traits had effects significantly associated with alveolar regions. Pleiotropic effects are typically restricted to morphologically integrated complexes.     

Recombinant congenic strains derived from A/J and C57BL/6J: a tool for genetic dissection of complex traits

Complex genetic traits can be dissected in mice, using well-defined sets of recombinant inbred strains, congenic strains, and recombinant congenic strains (RCS). We report the creation of a series of 37 independent RCS derived from the commonly used inbred strains of laboratory mouse A/J (A) and C57BL/6J (B6). These RCS were derived by systematic inbreeding of independent pairs of animals from a (F1 x A) x A and a (F1 x B) x B double backcross (N3), to create AcB and BcA strains, respectively. Fifteen AcB strains and 22 BcA strains at between 18 and 30 generations of inbreeding have been generated, are healthy, and show stable breeding performance. These strains have been genotyped for a total of 625 informative microsatellite DNA markers covering the entire genome, with an average spacing of 2.6 cM. Haplotype analyses indicate that on average, AcB and BcA strains contain 13.25% of the donor genome, a value close to the 12.5% expected from the breeding scheme used in their creation. In the AcB set, approximately 79% of the B6 genome has been transferred in independent strains, while in the BcA set approximately 84% of the A genome is represented on the B6 background. This represents an excellent coverage of congenic segments from both parental genomes in the two sets of strains, which can now be used to map simple and complex traits in a genome-wide fashion. As an example of the power of AcB/BcA strains as a mapping tool, the 37 strains were typed for susceptibility to infection with Legionella pneumophila, a monogenic trait controlled by the Lgn1 locus on Chromosome 13. Analysis of the strain distribution pattern of L. pneumophila susceptibility allowed direct mapping of Lgn1 to a 3-cM interval. The AcB/BcA set should prove a useful tool with which to investigate the complex genetic basis of known interstrain differences between A and B6 for many important diseases.     

Fine mapping of Ath6, a quantitative trait locus for atherosclerosis in mice

Ath6 is a novel quantitative trait locus associated with differences in susceptibility to atherosclerosis between C57BL/6J (B6) and C57BLKS/J (BKS) inbred mouse strains. Combining data from an intercross and a backcross (1593 meioses) between mice from B6 and BKS strains and from The Jackson Laboratory interspecific backcross panels, (C57BL/6J ×Mus spretus) F₁× C57BL/6J and (C57BL/6J × SPRET/Ei) F₁× SPRET/Ei, we constructed a consensus genetic map and narrowed Ath6 to a 1.07 ± 0.26 cM interval between the anonymous DNA marker D12Pgn4 and the gene Nmyc1. This region is near the proximal end of murine Chromosome (Chr) 12, which is homologous to the human chromosomal region 2p24-p25. Marker order in the Ath6 region was concordant among the two crosses and The Jackson Laboratory interspecific backcross panels. This high resolution map rules out candidate genes encoding apolipoprotein B, syndecan 1, and Adam17. The two Ath6 crosses have a combined potential resolution of 0.06 cM. Received: 12 September 2000 / Accepted: 22 February 2001     

The genetic structure of recombinant inbred mice: High-resolution consensus maps for complex trait analysis

Background

Recombinant inbred (RI) strains of mice are an important resource used to map and analyze complex traits. They have proved particularly effective in multidisciplinary genetic studies. Widespread use of RI strains has been hampered by their modest numbers and by the difficulty of combining results derived from different RI sets.

Results

We have increased the density of typed microsatellite markers 2- to 5-fold in each of several major RI sets that share C57BL/6 as a parental strain (AXB, BXA, BXD, BXH, and CXB). A common set of 490 markers was genotyped in just over 100 RI strains. Genotypes of another ~1100 microsatellites were generated, collected, and error checked in one or more RI sets. Consensus RI maps that integrate genotypes of ~1600 microsatellite loci were assembled. The genomes of individual strains typically incorporate 45-55 recombination breakpoints. The collected RI set - termed the BXN set - contains approximately 5000 breakpoints. The distribution of recombinations approximates a Poisson distribution and distances between breakpoints average about 0.5 cM. Locations of most breakpoints have been defined with a precision of < 2 cM. Genotypes deviate from Hardy-Weinberg equilibrium in only a small number of intervals.

Conclusions

Consensus maps derived from RI strains conform almost precisely with theoretical expectation and are close to the length predicted by the Haldane-Waddington equation (X3.6 for a 2-3 cM interval between markers). Non-syntenic associations among different chromosomes introduce predictable distortions in QTL data sets that can be partly corrected using two-locus correlation matrices.