Application of AFLP markers for QTL mapping in the rabbit.

Two rabbit (Oryctolagus cuniculus) inbred strains (AX/JU and IIIVO/JU) have been used for genetic analysis of quantitative traits related to dietary cholesterol susceptibility. Application of the AFLP (amplified fragment length polymorphism) technique with 15 primer combinations revealed 226 polymorphisms between the 2 inbred strains. A total of 57 animals from a backcross progeny (IIIVO/JU x [IIIVO/JU x AX/JU]F1) were available for the genetic analysis. These backcross animals were fed a commercial pelleted diet fortified with 0.3% w/w cholesterol during a test period that lasted five weeks. A male genetic map could be constructed, consisting of 12 linkage groups and 103 AFLP markers. Linkage analysis between the cholesterol-related traits and marker loci revealed a significant LOD score for the relative weight of adrenal glands in males (LOD score = 3.83), whereas suggestive linkages were found for basal serum total cholesterol levels in females (LOD score = 2.69), for serum total cholesterol response (area under the curve) in males (LOD score = 2.21), and for hematocrit in males (LOD score = 3.24).

Chromosomal localization of genes involved in biosynthesis, metabolism or transport of cholesterol in the rat.

Several genes involved in biosynthesis, transport or metabolism of cholesterol have been localized on rat chromosomes by using a radiation hybrid (RH) panel. The genes, coding for squalene epoxidase (Sqle), mevalonate kinase (Mvk), and farnesyl diphosphate farnesyl transferase 1 (Fdft1) which are involved in cholesterol biosynthesis, have been mapped on chromosome 7, 12, and 15, respectively. The genes coding for phospholipid transfer protein (Pltp), sterol carrier protein-2 (Scp2), ATP binding cassette reporter A7 (Abca7), scavenger receptor class B, type 1 (Cd36l1), steroidogenic acute regulatory protein (Star), and lecithin:cholesterol acyl transferase (Lcat), which are involved in the transfer and/or metabolism of cholesterol, have been mapped on chromosome 3, 5, 7, 12, 16, and 19, respectively. Each of the genes Scp2, Sqle and Fdft1 maps close to a QTL for serum total cholesterol in rat, suggesting that these three genes might represent candidate genes for the previously mapped QTLs.

A rat linkage map based on BC x LEW intercross.

A genetic linkage map consisting of 258 polymorphic loci has been constructed on the basis of an F2 intercross between the BC/CpbU and LEW/OlaHsd inbred rat strains. When compared to previously published maps a discrepancy was found for rat chromosome 7. The map spans a sex-averaged genetic length of 1790 cM and has an average marker spacing of 7.7 cM. It was estimated that this genetic map is linked to about 90% of the DNA in the rat genome. Because LEW/OlaHsd and BC/CpbU strains differ for dietary cholesterol susceptibility and hepatic copper content, the map is considered to be a valuable tool for studying the genetic background of these complex traits.

Mapping of rabbit chromosome 1 markers generated from a microsatellite-enriched chromosome-specific library.

A genomic DNA library was produced from flow-sorted rabbit chromosome 1 and enriched for fragments containing CA-repeats. Clones containing CA-repeats were identified and primers for amplification of the microsatellite were developed after sequencing the clone. The degree of polymorphism was tested in rabbits from different breeds. This approach identified 12 microsatellite markers which could be used for studying linkage relationships in the progeny of an F(2)-intercross: (AX/JUxIIIVO/JU) F(2), and two backcrosses: (OS/JxX/J)X/J and (WH/JxX/J)X/J. Seven of these markers were mapped on chromosome 1.

Genetic analysis and mapping of biochemical markers in an F2 intercross of two inbred strains of the rabbit (Oryctolagus cuniculus).

A total of 40 biochemical and four immunological markers found to be polymorphic in the rabbit in previous studies were screened in the AX/JU and IIIVO/JU inbred strains. Although the strains are considered unrelated, only eight (biochemical) markers werefound to be polymorphic between the two strains. These eight markers were analyzed in an F2 intercross population. Linkage was found for Est-5 and C on chromosome 1 and for Es-1, Est-2, Est-4, Est-6 and HP on linkage group VI. Two polymorphic markers, Es-3 and Mhr-1 could not be linked to any of the other markers.

Mapping of a QTL for serum HDL cholesterol in the rabbit using AFLP technology.

The amplified fragment length polymorphism (AFLP) technique is a DNA technology that generates the so-called AFLP markers. These markers are genomic restriction fragments detected after two rounds of polymerase chain reaction (PCR) without prior knowledge of nucleotide sequence. Here we describe the first application of the AFLP technique in the rabbit. We have tested two primer combinations. The results obtained with the DNA from rabbits of different breeds justify the conclusion that AFLP analysis is an effective tool for genetic studies in the rabbit. In addition, we contribute to the linkage map of the rabbit by localizing two AFLP markers on rabbit linkage group VI (LG VI). For this purpose the progeny of a IIIVO/JU x [IIIVO/JU x AX/JU]F(1) backcross were genotyped for 12 AFLP markers and 3 LG VI classical markers [one coat color marker (e) and two biochemical markers (Es-1 and Est-2)]. AX/JU is a dietary cholesterol-susceptible (hyperresponding) inbred strain and IIIVO/JU is a dietary cholesterol resistant (hyporesponding) inbred strain. Moreover, it is possible to evoke dietary cholesterol-induced aorta atherosclerosis in a relatively short time period in AX/JU rabbits, in contrast to IIIVO/JU rabbits. A significant cosegregation was found between basal serum HDL cholesterol level (i.e., the level on a low-cholesterol, control diet) and an AFLP marker on LG VI. It is concluded that one or more genes of LG VI are regulating the basal serum HDL cholesterol level in rabbits. Thus the present study with rabbits clearly illustrates the value of AFLP markers for the construction of linkage maps and mapping of quantitative trait loci (QTL).

Mapping of microsatellite loci and association of aorta atherosclerosis with LG VI markers in the rabbit.

Twenty-three rabbit microsatellites were extracted from the EMBL nucleotide database. Nine of these markers, together with nine earlier published microsatellite markers, were found to be polymorphic between the AX/JU and IIIVO/JU inbred strains. By using an F(2) intercross we could integrate five markers into the rabbit linkage map. One anonymous microsatellite marker could be assigned to chromosome 1, and one microsatellite marker, located within the metallothionein-1 gene, could be added to linkage group VI (LG VI). Three microsatellite markers (one anonymous, one located within the PMP2 gene, and one located within the FABP6 gene) constitute a new linkage group (LG XI). We also measured the degree of dietary cholesterol-induced aorta atherosclerosis in the F(2) animals. A significant cosegregation was found between the degree of aorta atherosclerosis and the allelic variation of the biochemical marker Est-2 on LG VI in male rabbits. This association was not found in female rabbits.

Sequencing and chromosomal assignment of the rat endothelial-derived lipase gene (Lipg).

Part of the nucleotide sequence of the Lipg gene in the rat was established using primers based on the mRNA sequence described in the mouse. The rat intron sequence served as a template for designing primers for the specific amplification of rat Lipg. A rat-hamster radiation hybrid (RH) panel was used for chromosomal assignment of the rat Lipg gene. The Lipg gene was found to be located on rat chromosome 18 in the vicinity of the marker D18Mit11; a region reported to be homologous with both human and mouse chromosome 18.

Genetic analysis of dystrophic cardiac calcification in DBA/2 mice.

A DBA/2 x D2B6F1 backcross was produced in order to study the genetic background of pathological soft tissue calcification in the mouse. Calcification was assessed in the myocardium, kidney and tongue. Significant co-segregation was found with the genotype of microsatellite markers on the proximal end of Chromosome 7. This region contains a candidate gene, Hrc, coding for the histidine-rich calcium binding protein in the sarcoplasmatic reticulum. The results support the hypothesis that the gene previously reported to be responsible for DCC (dystrophic cardiac calcification) in C3H mice (1) causes generalized soft tissue calcification in DBA/2 mice.

Use of AFLP markers for gene mapping and QTL detection in the rat.

The AFLP technique is a new DNA marker technology based on the selective amplification of restriction fragments. Multiple polymorphic markers are simultaneously produced and can be tested in one PCR. No prior information on genomic DNA sequences is needed. In the current study, we contribute 18 AFLP markers to the linkage map of the rat. Seven AFLP markers were assigned to specific chromosomes by analysis of a (BN x ACI)F1 x ACI backcross progeny. Another 11 AFLP markers were mapped by using a panel of the H x B/B x H recombinant inbred (RI) strains. Genotypes of these AFLP markers were also tested for correlations with some blood pressure phenotypes in the RI strains. Suggestive correlation was found between the mean arterial pressure and two closely linked AFLP markers located on chromosome 20. The current study illustrates the value of AFLP markers for the construction of linkage maps and the detection of quantitative trait loci.

Strain-specific response to hypercholesterolaemic diets in the rat.

The cholesterolaemic effect of 2 hypercholesterolaemic diets was tested in 12 rat inbred strains. Diet I is a commercial diet supplemented with 2.0% (w/w) cholesterol and 5.0% (w/w) olive oil; diet II is identical to diet I with addition of 0.5% (w/w) sodium cholate. Strains with the highest plasma cholesterol response after diet I (BN and LEW) also had the highest cholesterol response after diet II (hyperresponders, mean response > 3.5 mmol/l). In the strains DA, SHR, BC, WAG, LOU, PVG and BUF the strain mean cholesterol response remained below 1.3 mmol/l after both diets (hyporesponders). Strains F344 and OM had an intermediate cholesterol response after both diets (normoresponders, mean response between 1.3 and 3.5 mmol/l). Only in the strains LOU, PVG and SHR there appeared to be a significant higher cholesterol response after diet II when compared with the cholesterol response after diet I. In the strain WKY this difference was of a borderline significance (P = 0.052) and this strain turned from a normoresponder after diet I into a hyperresponder after diet II. Liver cholesterol levels as measured after feeding diet II for two weeks also appeared to be strain-specific. No correlation was found between the plasma cholesterol response after diet II and the liver cholesterol levels. Changes in plasma phospholipid and triglyceride levels have been measured for both diet I and diet II. For group means a correlation between the cholesterol response and the change in phospholipid levels was found (r = 0.86 for diet I, P < 0.001 and r = 0.76 for diet II, P < 0.01). No such correlation was found for triglyceride levels.

Polymorphic microsatellite DNA markers in the rabbit (Oryctolagus cuniculus).

By searching the EMBL nucleotide database a total of 157 rabbit nuclear gene microsatellites were obtained (VAN LITH and VAN ZUTPHEN, Animal Genetics 27, 387-395, 1996). Thirteen of these were analysed by PCR to examine the degree of polymorphism of the amplified fragments in rabbits from different breeds. The 13 pairs of primers resulted in polymorphic products with an average of four alleles per microsatellite sequence (ranging between 2-11). There was a positive relationship between the longest repeat unit number in the nucleotide sequence and the number of alleles detected. The results obtained so far justify the conclusion that rabbit microsatellites extracted from the EMBL nucleotide sequence database are sufficiently polymorphic to be useful as Type 1 markers in rabbit genetic studies.

Use of simple sequence length polymorphisms for genetic characterization of rat inbred strains.

Genetic monitoring is an essential component of colony management and for the rat has been accomplished primarily by using immunological and biochemical markers. Here, we report that simple sequence length polymorphisms (SSLPs) are a faster and more economical way of monitoring inbred strains of rats. We characterized 61 inbred strains of rats, using primer pairs for 37 SSLPs. Each of these loci appeared to be highly polymorphic, with the number of alleles per locus ranging between 3 and 14 and, as a result, all the 61 inbred strains tested in this study could be provided with a unique strain profile. These strain profiles are also used for estimating the degree of similarity between strains. This information may provide the rationale in selecting strains for genetic crosses or for other specific purposes.

Immunogenicity testing of diphtheria and tetanus vaccines by using isogenic mice with possible implications for potency testing.

The antitoxin response of isogenic mice (inbred strains and F1 hybrids), immunized with diphtheria and tetanus reference vaccines, was compared with the response of animals from an NIH outbred stock. The variance in antitoxin response was smaller within the groups of isogenic mice than within the group of mice from the outbred strain. The case for estimating the potency of diphtheria and tetanus vaccines by using isogenic mice is discussed. It is concluded that the general introduction of one common inbred strain or F1 hybrid selected on the basis of sensitivity would increase the comparability of data between laboratories and would enable an estimated reduction in the use of animals of about 35% or an improvement in the precision of the estimate of potency.

Linkage of the gene for uncoupling protein to esterase-1,2 and haptoglobin in the rat.

Genetic analysis of a (BN x ACI)ACI backcross for the biochemical markers Es-1 and Es-2 (linkage group V) and a microsatellite marker of Ucp (chromosome 19) revealed linkage between these three loci. This linkage relationship confirms that in the rat linkage group V genes are located on chromosome 19. Genetic analysis of a (BN x LEW)LEW backcross showed that Hp is linked to Ucp, thus indicating that the Hp locus is also located on chromosome 19. These results substantiate the linkage homology of RNO 19, MMU 8, and HSA 16.

Influence of amount of dietary fat and protein on esterase-1 (ES-1) activities of plasma and small intestine in rats.

The objective of the present study was to characterize nutritionally esterase-1 (ES-1). For this purpose, the effects of replacement of dietary carbohydrates by isoenergetic amounts of either fat or protein on ES-1 activities of plasma and small intestine were studied in male rats. Purified diets differing in the amounts of maize starch plus dextrose, casein and various types of fat were used. Plasma and jejunal ES-1 activities were found to be increased with increasing fat intakes. As to the type of fat, increasing plasma ES-1 activities were induced by coconut fat, olive oil, maize oil and medium-chain triacylglycerols, in this order. Maize oil induced higher jejunal ES-1 activities than coconut fat and olive oil, but had similar effects to medium-chain triacylglycerols. Maize oil was more powerful in increasing plasma ES-1 activity than isoenergetic amounts of casein, but with respect to jejunal ES-1 activity these dietary components were equally effective. It is concluded that the amounts of fat and protein in the diet are important determinants of ES-1 activities in plasma and jejunum.

Segregation of genes from donor strain during the production of recombinant congenic strains.

Recombinant congenic strains (RCS) constitute a set of inbred strains which are designed to dissect the genetic control of multigenic traits, such as tumour susceptibility or disease resistance. Each RCS contains a small fraction of the genome of a common donor strain, while the majority of genes stem from a common background strain. We tested at two stages of the inbreeding process in 20 RCS, derived from BALB/cHeA and STS/A, to see whether alleles from the STS/A donor strain are distributed over the RCS in a ratio as would theoretically be expected. Four marker genes (Pep-3; Pgm-1; Gpi-1 and Es-3) located at 4 different chromosomes were selected and the allelic distribution was tested after 3-4 and after 12 generations of inbreeding. The data obtained do not significantly deviate from the expected pattern, thus supporting the validity of the concept of RCS.

Genetic variation within and between lines of diabetes-prone and non-diabetes-prone BB rats; allele distribution of 8 protein markers.

Twenty-four inbred and 2 outbred lines of the BB rat have been genetically characterized by establishing the allele distribution of 8 monogenic protein markers. The marker genes are: plasma alkaline phosphatase-1 (Alp-1), catalase-1 (Cs-1), carboxylesterases (Es-1, Es-2, Es-14), glyoxalase I (Glo-1), group specific component (Gc), and haemoglobin-beta-chain (Hbb). At least 3 linkage groups are represented by this set of markers. Genetic variation was found both within and between lines. Within-line variation was observed in 4 lines, including the 2 outbred lines. The other 22 lines could be subdivided into 4 groups, each representing a unique allele distribution pattern.