Identification of QTLs Involved in the Development of Amygdala Kindling in the Rat

Amygdala kindling is useful for modeling human epilepsy development. It has been known that genetic factors are involved in the development of amygdala kindling. The purpose of this study was to identify the loci that are responsible for the development of amygdala kindling. To achieve this, rat strains from a LEXF/FXLE recombinant inbred (RI) strain panel were used. The phenotypes of amygdala kindling-related parameters for seven RI strains and parental LE/Stm and F344/Stm strains were determined. They included the afterdischarge threshold (ADT), the afterdischarge duration (ADD), and the kindling rate, an incidence of development of kindling. Quantitative trait loci (QTL) analysis was performed to identify linkage relationships between these phenotypes and 1,033 SNP markers. Although no significant differences in pre-kindling ADT and ADD were observed, a significant difference in the kindling rate was found for the LEXF/FXLE RI strain. Two QTLs for the amygdala kindling rate (Agkr1 and Agkr2) were identified on rat chromosome 2. These findings clearly prove the existence of genetic influences that are involved in kindling development and suggest that substantial genetic components contribute to the progression of partial seizures into generalized seizures.     

Quantitative trait loci determining weight reduction of testes and pituitary by diethylstilbesterol in LEXF and FXLE recombinant inbred strain rats.

Increasing exposure to environmental endocrine disruptor, xeno-estrogen, is a serious hazard to male reproductive activity. To explore possible genetic control in susceptibility to xeno-estrogen, the weight reduction of testes induced by the continuous administration of a synthetic estrogen, diethylstilbesterol, were investigated by quantitative trait analysis in LEXF and FXLE recombinant inbred strain rats, consisting of 21 independent strains, 9 of their substrains, parental F344/Stm and LE/Stm strains, and (F344 x LE)F1. For the weight of testes, one highly significant quantitative trait locus (QTL) and one significant QTL were mapped on chromosomes 7 and 1, respectively. The QTL on chromosome 7 is closely associated with c-myc. Pituitary weight and serum prolactin were also variable among recombinant inbred strains, but no QTL was detected for them in this study.     

Genetic Models in Applied Physiology. HXB/BXH rat recombinant inbred strain platform: a newly enhanced tool for cardiovascular, behavioral, and developmental genetics and genomics.

This review deals with the largest set of rat recombinant inbred (RI) strains and summarizes past and recent accomplishments with this platform for genetic mapping and analyses of divergent and complex traits. This strain, derived by crossing the spontaneously hypertensive rat, SHR/Ola, with a Brown Norway congenic, BN-Lx, carrying polydactyly-luxate syndrome, is referred to as HXB/BXH. The RI strain set has been used for linkage and association studies to identify quantitative trait loci for numerous cardiovascular phenotypes, including arterial pressure, stress-elicited heart rate, and pressor response, and metabolic traits, including insulin resistance, dyslipidemia and glucose handling, and left ventricular hypertrophy. The strain's utility has been enhanced with development of a new framework marker-based map and strain distribution patterns of polymorphic markers. Quantitative trait loci for behavioral traits mapped include loci for startle motor response and habituation, anxiety and locomotion traits associated with elevated plus maze, and conditioned taste aversion. The polydactyly-luxate syndrome Lx mutation has allowed the study of alleles important to limb development and malformation phenotypes as well as teratogens. The RI strains have guided development of numerous congenic strains to test locus assignments and to study the effect of genetic background. Although these strains were originally developed to aid in studies of rat genetic hypertension and morphogenetic abnormalities, this rodent platform has been shown to be equally powerful for a wide spectrum of traits and endophenotypes. These strains provide a ready and available vehicle for many physiological and pharmacological studies.     

The AXB and BXA set of recombinant inbred mouse strains

The recombinant inbred (RI) set of strains, AXB and BXA, derived from C57BL/6J and A/J, originally constructed and maintained at the University of California/San Diego, have been imported into The Jackson Laboratory and are now in the 29th to 59th generation of brother-sister matings. Genetic quality control testing with 45 proviral and 11 biochemical markers previously typed in this RI set indicated that five strains had been genetically contaminated sometime in the past, so these strains have been discarded. The correct and complete strain distribution patterns for 56 genetic markers are reported for the remaining RI strain set, which consists of 31 living strains and 8 extinct strains for which DNA is available. Two additional strains, AXB 12 and BXA 17, are living and may be added to the set pending further tests of genetic purity. The progenitors of this RI set differ in susceptibility to 27 infectious diseases as well as atherosclerosis, obesity, diabetes, cancer, cleft palate, and hydrocephalus. Thus, the AXB and BXA set of RI strains will be useful in the genetic analysis of several complex diseases.     

Use of recombinant inbred strains to map genes of aging

Recombinant inbred strains have been used in a number of organisms for segregation and linkage analysis of quantitative traits. One major advantage of the recombinant inbred (RI) methodology is that the genetic identity of individuals within a strain permits replicate measures of the same recombinant genotype. Such replicability is important for traits such as aging inDrosophila, where phenotypic expression is highly influenced by different environmental conditions. RI strain methodology has an added advantage for DNA marker-based linkage analysis of traits measured over the lifespan of the organism. The DNA can be extracted from individuals of the same genotype as those measured in a longevity study. In this paper an argument is presented for the use of a set of recombinant inbred strains to map the quantitative trait loci involved in the aging process inDrosophila. A unique use of a set of stable, transposable moleular markers to trace the quantitative trait loci involved is suggested.     

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.     

Genetic characterization of a new set of recombinant inbred lines (LGXSM) formed from the intercross of SM/J and LG/J inbred mouse strains

A new set of LGXSM recombinant inbred (RI) strains is presented. The RI strain panel consists of 18 remaining strains of the original 55 founding strains. Strain characterization is based on 506 polymorphic microsatellites and 4289 single nucleotide polymorphisms (SNPs) distributed across the genome. Average microsatellite intermarker distance is 4.80 ± 4.84 Mb or 2.91 ± 3.21 F₂ cM. SNPs are more densely spaced at 0.57 ± 1.27 Mb. Ninety-five percent of all microsatellite intermarker intervals are separated by less than 15.00 Mb or 8.50 F₂ cM, while 95% of the SNPs are less than 0.95 Mb apart. Strains show expected low levels of nonsyntenic association among loci and complete genomic independence. During inbreeding, the RI strains went through strong natural selection on the agouti locus on Chromosome 2, especially when the epistatically interacting tyrosinase locus on Chromosome 7 carried the wild-type allele. The LG/J and SM/J strains differ in a large number of biomedically important traits, and they and their intercross progeny have been used in multiple mapping studies. The LG×SM RI strain panel provides a powerful new resource for mapping the genetic bases of complex traits and should prove to be of great biomedical utility in modeling complex human diseases such as obesity and diabetes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

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.

     

Gene mapping of sperm quality parameters in recombinant inbred strains of mice

The aim of this study was to map chromosomal regions containing hypothetical genes responsible for the following parameters of mouse semen quality: (1) the percentage of sperm with abnormal head morphology, (2) the level of dead spermatozoa, (3) the percentage of sperm tails with residual cytoplasmic droplets, and (4) the percentage of sperm with impaired sperm tail membrane integrity. We also analyzed any possible correlations between these parameters. The most appropriate animal model for mapping genes controlling quantitative traits (QTL, quantitative trait locus) is a set of recombinant inbred (RI) strains. The set of RI strains used in this study was derived from crosses between two inbred mouse strains, KE and CBA/Kw, which differ significantly in fertility parameters and gamete quality. We analyzed the four parameters of sperm quality in male mice from two parental strains and from 12 RI strains. The strain distribution pattern (SDP) of 187 polymorphic microsatellite markers was prepared for 20 chromosomes of the mouse genome in 12 RI strains. We correlated the SDP of these markers with the values of sperm quality parameters, using MapManager QTX software (ver. b18). The mapping procedure indicated that the percentage of sperm with abnormal head morphology is controlled by gene(s) located in chromosomal regions 11q24, 11q31 and 6q15.6. There was also a strong correlation between male body weight and the hypothetical gene(s) in chromosomal region 18q47. A detailed analysis of the genes located in these regions enabled us to prepare a list of candidate genes. We discuss the basis of the correlation between the measured parameters.     

Analysis of oocyte quality in recombinant inbred mouse strains developed from KE and CBA strains that differ in fertilization efficiency

Recombinant inbred (RI) mouse strains were developed from reciprocal crosses between two inbred strains differing in the proportion of fertilized ova (CBA, 100%; KE, 77%), to analyse the underlying factors. A correlation (r = 0.83, P < 0.01) between fertilization efficiency within 22 RI strains and after mating RI females with KE males proved that oocyte quality was involved. The following oocyte parameters were analysed in RI and progenitor strains: time of meiotic maturation, rapidity of enzymatic removal of egg investments, and proportion of fertilized ova with supplementary spermatozoa in the perivitelline space. Among the RI strains, high incidence of supplementary spermatozoa was correlated with lower efficiency of fertilization (r = -0.58, P < 0.05) and with slow meiotic maturation (r = -64, P < 0.01), suggesting that delayed maturation may affect oocyte ability of being fertilized by the first penetrating spermatozoon. However, significant correlations were also found between characters which coexist within the progenitor strains, but are not likely to be physiologically related; this suggests that RI strains have inherited large blocks of progenitor genomes, not disrupted by recombination. The strain distribution pattern (SDP) of the analysed traits revealed CBA-like, KE-like, and intermediate phenotypes, indicating that they are polygenically determined. No linkages were found between the studied traits and 12 enzymatic markers. However, the SDP for fertilization efficiency showed a preponderance of non-matching strains (15/19) in relation to agouti locus; the known instability of this chromosome region makes it possible that a putative linkage was disrupted by recombination when RI strains were created.     

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.     

A new framework marker-based linkage map and SDPs for the Rat HXB/BXH strain set

A new contiguous genetic linkage map of the HXB/BXH set of rat recombinant inbred (RI) strains was constructed to enhance QTL mapping power and precision, and thereby make the RI strain set a better genomics resource. The HXB/BXH rat RI strains were developed from a cross between the hypertensive SHR/OlaIpcv and normotensive BN-Lx/Cub rat strains and have been shown useful for identifying quantitative trait loci (QTL) for a variety of cardiovascular, metabolic, and behavioral phenotypes. In the current analysis, the DNAs from 31 existing strains, 1 substrain, and 4 extinct strains were genotyped for a selection of polymorphic microsatellite marker loci, predominantly polymorphic framework markers from high-density integrated rat genome maps. The resulting linkage map consists of 245 microsatellite markers spanning a total length of 1789 cM with an average inter-marker distance of ~8.0 cM. This map covers the rat genome contiguously and completely with the exception of two locations on Chromosomes (Chrs) 11 and 16. The new genotypic information obtained also permitted further genetic characterization of the RI strain set including strain independence, genetic similarity among the individual strains, and non-syntenic associations between loci.     

Susceptibility/resistance to mouse hepatitis virus strain 3 and macrophage procoagulant activity are genetically linked and controlled by two non-H-2-linked genes

The mode of inheritance of susceptibility/resistance to mouse hepatitis strain 3 (MHV-3) was determined by typing the set of AXB/BXA recombinant inbred (RI) strains derived from resistant A/J (A) and susceptible C57BL/6J (B) progenitors for susceptibility to infection as determined by the severity of liver pathology. The strain distribution pattern for susceptibility showed a discontinuous variation: one strain was fully resistant (A-like), four strains were fully susceptible (B-like), and 16 strains showed an intermediate degree of susceptibility. The fully susceptible strains developed fulminant hepatitis and died; the fully resistant strain developed no liver disease, whereas a range of disease ranging from mild focal hepatitis to widespread hepatocellular necrosis was seen in the semisusceptible strains. This SDP best fits the two-recessive-gene model of inheritance, and neither of these two loci is linked to the H-2 complex. Macrophage procoagulant activity (PCA) segregated among the RI strains in a strain distribution pattern identical to that of susceptibility/resistance. PCA levels were greater than sevenfold elevated in fully susceptible RI mice and fourfold elevated in semisusceptible mice with no increase in resistant mice. These observations suggest genetic linkage of susceptibility/resistance to MHV-3 infection and macrophage PCA.     

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.     

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     

The LEXF: A new set of rat recombinant inbred strains between LE/Stm and F344

A new set of rat RI strains consisting of 11 independent strains and 13 of their substrains was established by inbreeding F₂ rats between F344/DuCrj and LE/Stm. The strain distribution pattern was examined for 66 microsatellite loci, 8 biochemical genetic markers, 2 histocompatibility loci, and 2 coat color genes. A rat salivary protein gene Spe1 was newly mapped on Chr 1. Received: 13 August 1996 / Accepted: 23 December 1996     

Genetic randomization reveals functional relationships among morphologic and tissue-quality traits that contribute to bone strength and fragility

We examined femora from adult AXB/BXA recombinant inbred (RI) mouse strains to identify skeletal traits that are functionally related and to determine how functional interactions among these traits contribute to genetic variability in whole-bone stiffness, strength, and toughness. Randomization of A/J and C57BL/6J genomic regions resulted in each adult male and female RI strain building mechanically functional femora by assembling unique sets of morphologic and tissue-quality traits. A correlation analysis was conducted using the mean trait values for each RI strain. A third of the 66 correlations examined were significant, indicating that many bone traits covaried or were functionally related. Path analysis revealed important functional interactions among bone slenderness, cortical thickness, and tissue mineral density. The path coefficients describing these functional relations were similar for both sexes. The causal relationship among these three traits suggested that cellular processes during growth simultaneously regulate bone slenderness, cortical thickness, and tissue mineral density so that the combination of traits is sufficiently stiff and strong to satisfy daily loading demands. A disadvantage of these functional interactions was that increases in tissue mineral density also deleteriously affected tissue ductility. Consequently, slender bones with high mineral density may be stiff and strong but they are also brittle. Thus, genetically randomized mouse strains revealed a basic biological paradigm that allows for flexibility in building bones that are functional for daily activities but that creates preferred sets of traits under extreme loading conditions. Genetic or environmental perturbations that alter these functional interactions during growth would be expected to lead to loss of function and suboptimal adult bone quality.     

Limitation of Number of Strains and Persistence of False Positive Loci in QTL Mapping Using Recombinant Inbred Strains

While the identification of causal genes of quantitative trait loci (QTL) remains a difficult problem in the post-genome era, the number of QTL continues to accumulate, mainly identified using the recombinant inbred (RI) strains. Over the last decade, hundreds of publications have reported nearly a thousand QTL identified from RI strains. We hypothesized that the inaccuracy of most of these QTL makes it difficult to identify causal genes. Using data from RI strains derived from C57BL/6J (B6) X DBA/2J (D2), we tested the possibility of detection of reliable QTL with different numbers of strains in the same trait in five different traits. Our results indicated that studies using RI strains of less than 30 in general have a higher probability of failing to detect reliable QTL. Errors in many studies could include false positive loci, switches between QTL with small and major effects, and missing the real major loci. The similar data was obtained from a RI strain population derived from a different pair of parents and a RI strain population of rat. Thus, thousands of reported QTL from studies of RI strains may need to be double-checked for accuracy before proceeding to causal gene identification.     

Parental genetic contributions in the AXB and BXA recombinant inbred mouse strains

Recombinant inbred (RI) strains are a valuable tool in mouse genetics to rapidly map the location of a new locus. Because RI strains have been typed for hundreds of genetic markers, the genotypes of individual strains within an RI set can be examined to identify specific strain(s) containing the desired region(s) of interest (e.g., one or more quantitative trait loci, QTLs) for subsequent phenotype testing. Specific RI strains might also be identified for use as progenitors in the construction of consomic (chromosome substitution strains or CSSs) or congenic lines or for use in the RI strain test (RIST). To quickly identify the genetic contributions of the parental A/J (A) and C57BL/6J (B) strains, we have generated chromosome maps for each commercially available AXB and BXA RI strain, in which the genetic loci are colorcoded to signify the parent of origin. To further assist in strain selection for further breeding schemes, the percentages of A and B parental contributions were calculated, based on the total number of typed markers in the database for each strain. With these data, one can rapidly select the RI strain(s) carrying the desired donor and recipient strain region(s). Because points of recombination are known, starting with RI mice to generate CSSs or congenic lines immediately reduces genomewide screening to those donor-strain regions not already homozygous in the recipient strain. Two examples are presented to demonstrate potential uses of the generated chromosome maps: to select RI strains to construct congenic lines and to perform an RIST forAliq1, a QTL linked to ozone-induced acute lung injury survival.     

Simulation of the distribution of parental strains’ genomes in RC strains of mice

Recombinant Congenic strains (RC strains) were developed to facilitate mapping of genes influencing complex traits controlled by multiple genes. They were produced by inbreeding of the progeny derived from a second backcross from a common `donor' inbred strain to a common `background' inbred strain. Each RC strain contains a random subset of approximately 12.5% of genes from the donor strain and 87.5% of genes from the background strain. In this way the genetic control of a complex disease may be dissected into its individual components. We simulated the production of the RC strains to study to what extent they have to be characterized in order to obtain sufficient information about the distribution of the parental strains' genomes in these strains and to acquire insight into parameters influencing their effectiveness in mapping quantitative trait loci (QTLs). The donor strain genome in the RC strains is fragmented into many segments. Genetic characterization of these strains with one polymorphic marker per 3.3 centiMorgans (cM) is needed to detect 95% of the donor strain genome. The probability of a donor strain segment being located entirely in between two markers of background strain origin that are 3 cM apart (and hence escaping detection) is 0.003. Although the donor strain genome in the RC strains is split into many segments, the largest part still occurs in relatively long stretches that are mostly concentrated in fewer than 13 autosomes, the median being 9 autosomes. Thus, in mapping QTLs, the use of RC strains facilitates the detection of linkage. Received: 20 December 1996 / Accepted: 23 July 1997