Abstract A quantitative trait locus (QTL) for blood pressure was previously detected by linkage analysis in a region of rat chromosome 2 using segregating populations derived from crosses of the Dahl salt-sensitive (Dahl S) rat with rats of the Wistar-Kyoto (WKY) strain or Milan normotensive strain (MNS). Two congenic strains, S.WKY-D2N35/Nep and S.MNS-Adh/D2Mit5, have been constructed by replacing a region of chromosome 2 of the Dahl S rat with the homologous region (ie, low blood pressure QTL allele) of either the WKY or MNS rat, respectively. Systolic pressures of congenic strains S.WKY-D2N35/Nep and S.MNS-Adh/D2Mit5 fed a 2% NaCl diet for 24 days were 44±4.6 and 29±4.5 mm Hg lower, respectively, than that of the comparably treated Dahl S rats. The differences between congenic and Dahl S rats in blood pressure were highly significant (P<.001) and were corroborated by significantly (P<.001) lower ratio of heart weight to body weight in the congenic strains compared with Dahl S rats. The data from two congenic strains combined unequivocally establish the existence of a blood pressure QTL on rat chromosome 2.
Genetic linkage mapping using segregating populations has identified chromosome regions potentially harboring QTLs for BP. One such region is on rat chromosome 2, ie, the region encompassed by D2N35 and Nep markers.1 2 The QTL allele for high BP is from the Dahl S rat and the QTL allele for low BP is from either the WKY or MNS rat.2 Linkage analysis for the existence of a QTL is problematic unless it is supported by very highly significant probability levels on the order of 10−4 to 10−5.3 4 These levels of significance were achieved in our previous work on combining the data from two F2 populations.2 To prove that a QTL exists, however, it is necessary to follow up the linkage analysis with the construction of a congenic strain in which a chromosomal fragment from one strain is introgressed into another strain; the strains are then compared for BP differences.5 In the present case, the chromosome 2 region containing a low BP QTL allele is placed on the genetic background of the Dahl S rat. The experiment was duplicated using chromosomal regions donated from WKY and MNS rats.
Inbred hypertensive Dahl S rats were developed6 and bred in our own colonies. Normotensive WKY were obtained originally from Harlan Sprague Dawley (Indianapolis, Ind), and MNS were from Veterinary Resources Branch, National Institutes of Health, Bethesda, Md. Strains were maintained by brother-sister mating in our colony.
Designation of Congenic Strains
Two congenic strains were produced in the current study. They are designated as S.WKY-D2N35/neutral endopeptidase, abbreviated S.WKY-D2N35/Nep, and S.MNS-alcohol dehydrogenase/D2Mit5, abbreviated S.MNS-Adh/D2Mit5. In each of the above designated congenic strains, the recipient strain, Dahl S, goes first, followed by a period and the donor strain. After the hyphen, two chromosome markers separated by a slash represent the chromosome region of the recipient Dahl S strain being replaced by that of the donor normotensive strain.
Construction of Congenic Strains
The basic breeding scheme in our studies was to put a specific region of chromosome 2 of a normotensive strain (ie, WKY or MNS) onto the genetic background of the Dahl S strain using a standard breeding protocol.5 7 Dahl S rats were initially crossed to rats of a normotensive strain. F1 progeny were backcrossed to Dahl S rats to produce the first backcross generation (BC1). BC1 heterozygous for the chromosome region of interest were then backcrossed again to Dahl S rats to produce BC2. This process was repeated for a total of eight backcrosses, each time choosing rats heterozygous throughout the region of interest as breeders for the next backcross. At BC8, two heterozygotes were intercrossed to generate homozygotes containing two alleles of the normotensive strain throughout the entire chromosomal region of interest, thus fixing the region of chromosome 2 from the normotensive strain on the background of the S strain. In selecting rats on the basis of their genotypes throughout the region of interest on chromosome 2, the following markers were used: (1) for the S.WKY-D2N35/Nep strain, markers D2N35, Camk, Nakα1, Gca, and Nep; (2) for the S.MNS-Adh/D2Mit5 strain, markers Adh, D2N35, Camk, Nakα1, Gca, Nep, At1b, and D2Mit5.
Strain BP comparisons were done with pairs of strains and using only male rats. For example, Dahl S and S.WKY-D2N35/Nep rats were bred at the same time, and pups were weaned at 30 days of age. Rats were caged four per cage, two Dahl S and two S.WKY-D2N35/Nep rats. At 40 days of age, they were fed a 2% NaCl diet (Teklad diet 94217, Harlan Teklad) with free access to water. After 24 days on the diet, BP was taken once daily on each rat for 4 days by the tail-cuff method8 with rats in the conscious, restrained state using equipment made by IITC Inc. Rats were warmed to 28°C for BP determination. At least three consistent BP readings at a given daily session were obtained and averaged as the reading for that session. The four BP sessions for each rat were performed by two different operators, who did not know the identity of the rats; each operator performed two sessions on each rat. The readings from all four sessions were averaged as the BP of the rat. Rats were killed by an overdose of sodium pentobarbital, and body weight and heart weight were obtained.
Genotyping and Linkage Maps
Genotyping of genetic markers followed the same methods reported previously.1 2 9 10 11 12 Simple sequence repeats for each marker were amplified by the polymerase chain reaction (PCR) and analyzed by agarose or polyacrylamide gel electrophoresis. Linkage maps were constructed using the MAPMAKER/EXP program.13 14
Two congenic strains were produced: (1) S.WKY-D2N35/Nep rats were produced by substituting the chromosome 2 region from D2N35 to Nep of the Dahl S rat with the homologous region of the WKY; (2) S.MNS-Adh/D2Mit5 rats were produced by replacing the chromosome region Adh to D2Mit5 of the Dahl S rat with the homologous region of the MNS rat (Figure⇓). The entire region substituted in the S.WKY-D2N35/Nep strain is homozygous for the WKY allele (ie, WW) for all the markers located within the region (Figure⇓, left). The entire region substituted in the S.MNS-Adh/D2Mit5 strain is homozygous for the MNS allele (ie, MM) for all the markers located within the region (Figure⇓, right).
If there is truly a BP QTL present in the chromosome 2 regions in question, the BP of the rats for both congenic strains should be significantly lower than that of Dahl S rats.1 2 The Table⇓ shows pairwise strain comparisons of the congenic rats with Dahl S rats. BPs of congenic S.WKY-D2N35/Nep rats and congenic S.MNS-Adh/D2Mit5 rats were 44±4.6 and 29± 4.5 mm Hg lower than that of Dahl S rats, respectively. These differences are highly statistically significant (P<.001). The ratios of heart weight to body weight of congenic rats were also highly significantly lower than those of Dahl S rats (Table⇓).
In our other linkage studies, an additional BP QTL was also detected in F2 S×WKY and F2 S×MNS populations. This QTL is located in a region on rat chromosome 10.1 12 To verify that this region on chromosome 10 does not contain any residual genome from WKY or MNS in the two congenic strains developed for chromosome 2, chromosome markers located in the chromosome 10 region of interest were genotyped for the rats of the S.WKY-D2N35/Nep and S.MNS-Adh/D2Mit5 congenic strains. The results showed that the entire chromosome 10 region in question in each congenic strain is homozygous SS. The chromosome 10 QTL is the only other QTL we know about in crosses of Dahl S with MNS or WKY and is the only other region tested.
The present data establish a major BP QTL in the rat chromosome 2 region between markers D2N35 and Nep. This is the region shared by both congenic S.WKY-D2N35/Nep and S.MNS-Adh/D2Mit5 strains (Figure⇑). After eight backcrosses used in the production of the congenic strains, the genetic background of the congenic strain outside of the selected region is expected on average to be 99.6% Dahl S genes and 0.4% residual (unwanted) genomic regions of the normotensive donor strain (WKY or MNS). Because the two congenic strains were derived independently, it is very unlikely after eight backcrosses that both strains would by chance contain genomic regions of residual donor-strain genetic material in their genetic backgrounds that would affect BP. The chromosome 10 region, which was implicated as containing a BP QTL in our other linkage studies involving WKY and MNS strains,1 12 was shown to be homozygous for the Dahl S rat allele in both congenic strains. Therefore, the BP effect observed for the S.WKY-D2N35/Nep and S.MNS-Adh/D2Mit5 congenic strains (Table⇑) can be attributed to the chromosome 2 region between the D2N35 and Nep markers.
BP differences between congenic and Dahl S rats were corroborated by differences in heart weight–body weight ratios. It is noteworthy that the only valid comparisons are within experiments, not between experiments. Thus although, for example, BP was consistent in the Dahl S strain between experiments (205.8±3.91 versus 209.8±4.11 mm Hg), heart weight–body weight ratio (3.80±0.047 versus 4.23±0.054) was not (Table⇑). The latter is attributed to different people dissecting the hearts between experiments. In our studies, we use heart weight–body weight ratio as an index for corroborating chronic BP differences. In all our linkage and congenic studies so far carried out, heart weight and BP correlate very well (Reference 1717 ; Table⇑, current study; and unpublished data, 1996). It is also likely, however, that heart weight is influenced by loci that act independently of BP QTL.18 19 20 21
Although there are differences in the precise protocol used for genotyping, BP effects of congenic strains in the present study are consistent with those of cosegregation studies using F2 populations. In both F2 WKY×S and F2 MNS×S populations, the rats homozygous for the WKY or MNS QTL allele had BP lower than the rats homozygous for the Dahl S QTL allele.2 Likewise, the BPs of both congenic strains were also lower than that of Dahl S rats (Table⇑).
Theoretically, there could be more than one BP QTL located in the chromosome 2 region in question. Consequently, the BP effect observed in each congenic strain could come from the effects of these separate QTL. This possibility of having multiple QTL in the congenic region will be resolved by constructing congenic substrains, each one containing a smaller substituted segment within the broad chromosome 2 region under study.
The BP reduction of the congenic S.WKY-D2N35/Nep strain is about 15 mm Hg more than that of the congenic S.MNS-Adh/D2Mit5 strain (Table⇑). This difference could arise if the QTL allele of the WKY rat was different from that of the MNS rat; differing QTL alleles could have distinctive BP effects. Another possibility is that the WKY and MNS rats could have the same QTL allele in the D2N35/Nep region but that the larger substitution in the congenic S.MNS-Adh/D2Mit5 strain might contain other genes modifying its effect. The construction of S.MNS-Adh/D2Mit5 rats with a larger region of chromosome 2 was done because the putative QTL-containing region was poorly defined (larger) in the linkage analysis of the F2 S×MNS population.2 Also, there was evidence that a second QTL around At1b might exist,2 11 22 although At1b itself as the QTL was not supported by DNA sequence determinations of coding regions.11 On the basis of previous data, one would expect the longer segment in S.MNS-Adh/D2Mit5 to lower BP more than the shorter segment of S.WKY-D2N35/Nep. The opposite was the case. Thus, the difference in BP between the congenic strains is unexplained, although each strain obviously had a major effect on BP.
The region between D2N35 and Nep on chromosome 2 harbors several genes that could be construed as candidates for the BP QTL. These include Camk, Nakα1, Gca, and Nep (Figure⇑). Because the substituted region is large (41 cM), speculation as to the identity of the actual QTL is premature.
Selected Abbreviations and Acronyms
|Dahl S||=||inbred Dahl salt-sensitive rat(s) (SS/Jr)|
|MNS||=||Milan normotensive rat(s)|
|QTL||=||quantitative trait locus|
This work was supported by a Grant-in-Aid from the American Heart Association, National Center, and a Grant-in-Aid from the American Heart Association, Ohio Affiliate, to A.Y. Deng, and by grants from the National Institutes of Health and by the Helen and Harold McMaster Chair in Biochemistry and Molecular Biology to J.P. Rapp.
- Received October 11, 1996.
- Revision received November 5, 1996.
- Accepted January 7, 1997.
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