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.     

Genetic loci for diet-induced atherosclerotic lesions and plasma lipids in mice

Genetic factors independent of those affecting plasma lipid levels are a major contributor to risk for atherosclerosis in humans, yet the basis for these is poorly understood. This study examined plasma lipids and diet-induced atherosclerosis in 16-month-old female mice of strains C56BL/6J and DBA/2J. Mice of the parental strains, from recombinant inbred strains derived from these (BXD RI), and F₂ progeny were fed an atherogenic diet for 16 weeks, beginning at 1 year of age. This induced atherosclerotic lesion formation in both parental strains, accompanied by increased plasma LDL levels. However, individual BXD RI strains and the BXD F₂ mice demonstrated a range of atherosclerotic lesion formation that was not or at best weakly correlated with plasma lipid levels. Quantitative trait locus (QTL) analysis of the BXD F₂ mice identified a locus with significant linkage (lod 4.5) for aortic lesion size on Chromosome (Chr) 10 that was independent of plasma lipids. Other loci with suggestive or significant linkage for various plasma lipid measures were identified on Chr 2, 3, 4, 5, 6, 7, 11, and 17. In this intercross, the genes primarily influencing atherosclerosis are distinct from those controlling plasma lipid levels.     

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     

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     

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.     

Genetic maps of mouse chromosome 17 including 12 new anonymous DNA loci and 25 anchor loci.

An interspecific backcross between lab mice and Mus spretus was used to construct a multilocus map of Chromosome 17 consisting of 12 new anonymous loci and 9 anchor loci. In addition, 7 anonymous DNA loci were added to the Chr 17 map for the BXD strains. Although we were able to identify readily the most likely gene order in the interspecific backcross, we found no evidence for an unambiguous gene order using the BXD recombinant inbred strains. Comparison of the interspecific backcross map and the BXD RI strain map revealed evidence in the interspecific backcross for a longer total genetic length, enhanced recombination distal to H-2, a segment showing suppressed recombination, and strong interference.     

A linkage map of mouse Chromosome 1 using an interspecific cross segregating for the gld autoimmunity mutation

An interspecific backross was used to define a high resolution linkage map of mouse Chromosome (Chr) 1 and to analyze the segregation of the generalized lymphoproliferative disease (gld) mutation. Mice homozygous for gld have multiple features of autoimmune disease. Analysis of up to 428 progeny from the backcross [(C3H/HeJ-gld x Mus spretus)F₁ x C3H/HeJ-gld] established a map that spans 77.6 cM and includes 56 markers distributed over 34 ordered genetic loci. The gld mutation was mapped to a less than 1 cM segment on distal mouse Chr 1 using 357 gld phenotype-positive backcross mice. A second backcross, between the laboratory strains C57BL/6J and SWR/J, was examined to compare recombination frequency between selected markers on mouse Chr 1. Significant differences in crossover frequency were demonstrated between the interspecific backcross and the inbred laboratory cross for the entire interval studied. Sex difference in meiotic crossover frequency was also significant in the laboratory mouse cross. Two linkage groups known to be conserved between segments of mouse Chr 1 and the long arm of human Chrs 1 and 2 where further defined and a new conserved linkage group was identified that includes markers of distal mouse Chr 1 and human Chr 1, bands q32 to q42.     

QTL influencing baseline hematocrit in the C57BL/6J and DBA/2J lineage: age-related effects

Baseline serum hematocrit varies substantially in the population. While additive genetic factors account for a large part of this variability, little is known about the genetic architecture underlying the trait. Because hematocrit levels vary with age, it is plausible that quantitative trait loci (QTL) that influence the phenotype also show an age-specific profile. To investigate this possibility, hematocrit was measured in three different age cohorts of mice (150, 450, and 750 days) of the C57BL/6J (B6) and the DBA2/J (D2) lineage. QTL were searched in the B6D2F₂ intercross and the BXD recombinant inbred (RI) strains. The effects of these QTL were explored across the different age groups. On the phenotypic level, baseline serum hematocrit declines with age in a sex-specific manner. In the B6D2F₂ intercross, suggestive QTL that influence the phenotype were located on Chromosomes (Chr) 1, 2, 7, 11, 13, and 16. With the exception of the QTL on Chr 2, all of these QTL exerted their largest effect at 750 days. The QTL on Chr 1, 2, 7, 11 and 16 were confirmed in the BXD RIs in a sex- and age-specific manner. Linkage analysis in the BXD RIs revealed an additional significant QTL on Chr 19. Baseline serum hematocrit is influenced by several QTL that appear to vary with the age and sex of the animal. These QTL primarily overlap with QTL that have been shown to regulate hematopoietic stem cell phenotypes.     

A High-Density SNP Map of Sunflower Derived from RAD-Sequencing Facilitating Fine-Mapping of the Rust Resistance Gene R12

A high-resolution genetic map of sunflower was constructed by integrating SNP data from three F₂ mapping populations (HA 89/RHA 464, B-line/RHA 464, and CR 29/RHA 468). The consensus map spanned a total length of 1443.84 cM, and consisted of 5,019 SNP markers derived from RAD tag sequencing and 118 publicly available SSR markers distributed in 17 linkage groups, corresponding to the haploid chromosome number of sunflower. The maximum interval between markers in the consensus map is 12.37 cM and the average distance is 0.28 cM between adjacent markers. Despite a few short-distance inversions in marker order, the consensus map showed high levels of collinearity among individual maps with an average Spearman''s rank correlation coefficient of 0.972 across the genome. The order of the SSR markers on the consensus map was also in agreement with the order of the individual map and with previously published sunflower maps. Three individual and one consensus maps revealed the uneven distribution of markers across the genome. Additionally, we performed fine mapping and marker validation of the rust resistance gene R₁₂, providing closely linked SNP markers for marker-assisted selection of this gene in sunflower breeding programs. This high resolution consensus map will serve as a valuable tool to the sunflower community for studying marker-trait association of important agronomic traits, marker assisted breeding, map-based gene cloning, and comparative mapping.     

Low resolution mapping around the flavivirus resistance locus (Flv) on mouse Chromosome 5

Although the phenomenon of innate resistance to flaviviruses in mice was recognized many years ago, it was only recently that the genetic locus (Flv) controlling this resistance was mapped to mouse Chromosome (Chr) 5. Here we report the fine mapping of the Flv locus, using 12 microsatellite markers which have recently been developed for mouse Chr 5. The new markers were genotyped in 325 backcross mice of both (C3H/HeJxC3H/ RV)F₁xC3H/HeJ and (BALB/cxC3H/RV)F₁xBALB/c backgrounds, relative to Flv. The composite genetic map that has been constructed identifies three novel microsatellite loci, D5Mit68, D5Mit159, and D5Mit242, tightly linked to the Flv locus. One of those loci, D5Mit159, showed no recombinations with Flv in any of the backcross mice analyzed, indicating tight linkage (<0.3 cM). The other two, D5Mit68 and D5Mit242, exhibited two and one recombinations with Flv (0.6 and 0.3 cM) respectively, defining the proximal and distal boundaries of a 0.9-cM segment around this locus. The proximal flanking marker, D5Mit68, maps to a segment on mouse Chr 5 homologous to human Chr 4. This, together with the previous data produced by our group, locates Flv to a region on mouse Chr 5 carrying segments that are conserved on either human Chr 4, 12, or 7, but present knowledge does not allow precise identification of the syntenic element.     

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 MAPPING OF THE LAMINARIA JAPONICA (LAMINARALES,PHAEOPHYTA) USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS1

To establish a molecular‐marker‐assisted system of breeding and genetic study for Laminaria japonica Aresch., amplified fragment length polymorphism (AFLP) was used to construct a genetic linkage map of L. japonica featuring 230 progeny of F₂ cross population. Eighteen primer combinations produced 370 polymorphic loci and 215 polymorphic loci segregated in a 3:1 Mendelian segregation ratio (P ≤ 0.05). Of the 215 segregated loci, 142 were ordered into 27 linkage groups. The length of the linkage groups ranged from 6.7 to 90.3 centimorgans (cM) with an average length of 49.6 cM, and the total length was 1,085.8 cM, which covered 68.4% of the estimated 1,586.9 cM genome. The number of mapped markers on each linkage group ranged from 2 to 12, averaging 5.3 markers per group. The average density of the markers was 1 per 9.4 cM. Based on the marker density and the resolution of the map, the constructed linkage map can satisfy the need for quantitative trait locus (QTL) location and molecular‐marker‐assisted breeding for Laminaria.     

An integrated high-density RFLP-AFLP map of tomato based on two Lycopersicon esculentum×L. pennellii F2 populations

 Two independent F₂ populations of Lycopersicon esculentum×L. pennellii which have previously been investigated in RFLP mapping studies were used for construction of a highly saturated integrated AFLP map. This map spanned 1482 cM and contained 67 RFLP markers, 1078 AFLP markers obtained with 22 EcoRI+MseI primer combinations and 97 AFLP markers obtained with five PstI+MseI primer combinations, 231 AFLP markers being common to both populations. The EcoRI+MseI AFLP markers were not evenly distributed over the chromosomes. Around the centromeric region, 848 EcoRI+ MseI AFLP markers were clustered and covered a genetic distance of 199 cM, corresponding to one EcoRI+ MseI AFLP marker per 0.23 cM; on the distal parts 1283 cM were covered by 230 EcoRI+MseI AFLP markers, corresponding to one marker per 5.6 cM. The PstI/MseI AFLP markers showed a more even distribution with 16 PstI/MseI AFLP markers covering a genetic distance of 199 cM around the centromeric regions and 81 PstI/MseI AFLP markers covering a genetic distance of 1283 cM on the more distal parts, corresponding to one marker per 12 and 16 cM respectively. In both populations a large number of loci showed a significant skewed segregation, but only chromosome 10 loci showed skewness that was similar for both populations. This ultra-dense molecular-marker map provides good perspectives for genetic and breeding purposes and map-based cloning. Received: 3 September 1998 / Accepted: 27 October 1998     

A high-density microsatellite consensus map for bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

A microsatellite consensus map was constructed by joining four independent genetic maps of bread wheat. Three of the maps were F₁-derived, doubled-haploid line populations and the fourth population was Synthetic × Opata, an F₆-derived, recombinant-inbred line population. Microsatellite markers from different research groups including the Wheat Microsatellite Consortium, GWM, GDM, CFA, CFD, and BARC were used in the mapping. A sufficient number of common loci between genetic maps, ranging from 52 to 232 loci, were mapped on different populations to facilitate joining the maps. Four genetic maps were developed using MapMaker V3.0 and JoinMap V3.0. The software CMap, a comparative map viewer, was used to align the four maps and identify potential errors based on consensus. JoinMap V3.0 was used to calculate marker order and recombination distances based on the consensus of the four maps. A total of 1,235 microsatellite loci were mapped, covering 2,569 cM, giving an average interval distance of 2.2 cM. This consensus map represents the highest-density public microsatellite map of wheat and is accompanied by an allele database showing the parent allele sizes for every marker mapped. This enables users to predict allele sizes in new breeding populations and develop molecular breeding and genomics strategies.Electronic Supplementary Material Supplementary material is available for this article at     

Integrated map of AFLP, SSLP and RFLP markers using a recombinant inbred population of rice (Oryza sativa L.)

 A molecular map of rice consisting of 231 amplified fragment length polymorphisms (AFLPs), 212 restriction fragment length polymorphisms (RFLPs), 86 simple-sequence length polymorphisms (SSLPs), five isozyme loci, and two morphological mutant loci [phenol staining of grain (Ph), semi-dwarf habit (sd-1)] has been constructed using an F₁₁ recombinant inbred (RI) population. The mapping population consisted of 164 RI lines and was developed via single-seed descent from an intercross between the genetically divergent parents Milyang 23 (M) (tongil type) and Gihobyeo (G) ( japonica type). A subset of previously mapped RFLP and SSLP markers were used to construct the map framework. The AFLP markers were derived from ten EcoRI(+2) and MseI(+3) primer combinations. All marker types were well distributed throughout the 12 chromosomes. The integrated map covered 1814 cM, with an average interval size of 3.4 cM. The MG map is a cornerstone of the Korean Rice Genome Research Program (KRGRP) and is being continuously refined through the addition of partially sequenced cDNA markers derived from an immature-seed cDNA library developed in Korea, and microsatellite markers developed at Cornell. The population is also being used for quantitative trait locus (QTL) analysis and as the basis for marker-assisted variety development. Received: 24 June 1997 / Accepted: 25 November 1997     

Genetic determinants of weight of fast- and slow-twitch skeletal muscles in old mice

The main goal of the study was to explore the genetic architecture underlying muscle weight in old mice. Weight of soleus, tibialis anterior (TA), extensor digitorum longus (EDL), and gastrocnemius muscles was measured in the C57BL/6J (B6) and DBA/2J (D2) strains and derivative generations: a panel of the BXD recombinant inbred (RI) strains and a B6D2 F₂ intercross at the age of 800 days. The between-strain difference in muscle weight (B6 > D2) ranged between 16% and 38%. Linkage analysis identified suggestive quantitative trait loci (QTL) on Chromosomes (Chr) 2, 6, 7, 8, 19, and X that influenced muscle weight in the 800-day-old group. Comparison of weights at 200, 500, and 800 days revealed a variable effect of age among the four muscles. Linkage analysis in the B6D2 F₂ population combined across the three different age groups identified muscle-, sex-, and age-specific QTL on Chr 1, 2, 3, 5, 6, 8, 9, 11, 13, 17, X, and Y. Genetic factors that influence the rate of weight change (within-strain weight difference at two ages) over the lifespan of BXD RIs were mapped to the markers D2Mit369 and D3Mit130 at the genome-wide p < 0.05 for TA muscle in males (between 200 and 800 days) and females (between 500 and 800 days), respectively. Analysis of all age groups supported previous findings that the genetic effects may be muscle-, age-, and sex-specific.     

An integrated SSR based linkage map for <Emphasis Type="Italic">Zoysia matrella</Emphasis> L. and <Emphasis Type="Italic">Z. japonica</Emphasis> Steud.

Japanese lawngrass (Zoysia japonica) and Manila grass (Z. matrella) are the two most important and commonly used Zoysia species. A consensus based SSR linkage map was developed for the genus by combining maps from each species. This used previously constructed maps for two Z. japonica populations and a new map from Z. matrella. The new SSR linkage map for Z. matrella was based on 86 F₂ individuals and contained 213 loci and covered a map distance of 1,351.2 cM in 32 linkage groups. Comparison of the three linkage maps constructed from populations with different genetic backgrounds indicated that most markers exhibited a consensus order, although some intervals or regions displayed discrepancy in marker orders or positions. The integrated map comprises 507 loci with a mean interval of 4.1 cM, covering a map distance of 2,066.6 cM in 22 linkage groups. The SSR-based map will allow marker-assisted selection and be useful for the mapping and cloning of economically important genes or quantitative trait loci.     

Mapping of the Mod-1 locus on mouse Chromosome 9

A new method for typing the Mod-1 locus on mouse Chromosome (Chr) 9 was developed, based on restriction fragment length polymorphism (RFLP) within a polymerase chain reaction (PCR)-amplified fragment. The new method led us to revise the strain distribution pattern (SDP) of Mod-1 in the BXD (C57BL/6JxDBA/2J) and AKXD (AKR/J x DBA/2J) recombinant inbred (RI) strains. The new SDP eliminates several previously reported examples of double recombination events between Mod-1 and the closest flanking loci in the BXD and AKXD strains. In the BXD strains, the revised SDP of Mod-1 was identical to that of the Mod-1-related D9Rtil locus. Thus, the identity of D9Rtil as a Mod-1-related locus rather than Mod-1 itself is in question. The method was also applied to an interspecific backcross panel between an inbred strain of Mus musculus molossinus (MSM/Ms) and C57BL/6J to map Mod-1 with respect to surrounding microsatellite loci, defining the proximal localization of Mod-1 with respect to D9Mit10 with a genetic distance of 0.6±0.6 cM.     

Identification and mapping of AFLP markers linked to peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) resistance to the aphid vector of groundnut rosette disease

Groundnut rosette disease is the most destructive viral disease of peanut in Africa and can cause serious yield losses under favourable conditions. The development of disease-resistant cultivars is the most effective control strategy. Resistance to the aphid vector, Aphis craccivora, was identified in the breeding line ICG 12991 and is controlled by a single recessive gene. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) analysis were employed to identify DNA markers linked to aphid resistance and for the development of a partial genetic linkage map. A F_2:3 population was developed from a cross using the aphid-resistant parent ICG 12991. Genotyping was carried out in the F₂ generation and phenotyping in the F₃ generation. Results were used to assign individual F₂ lines as homozygous-resistant, homozygous-susceptible or segregating. A total of 308 AFLP (20 EcoRI+3/MseI+3, 144 MluI+3/MseI+3 and 144 PstI+3/MseI+3) primer combinations were used to identify markers associated with aphid resistance in the F_2:3 population. Twenty putative markers were identified, of which 12 mapped to five linkage groups covering a map distance of 139.4 cM. A single recessive gene was mapped on linkage group 1, 3.9 cM from a marker originating from the susceptible parent, that explained 76.1% of the phenotypic variation for aphid resistance. This study represents the first report on the identification of molecular markers closely linked to aphid resistance to groundnut rosette disease and the construction of the first partial genetic linkage map for cultivated peanut.