Experiment
To study the genetic architecture of F1 hybrid sterility inbred strains representing M. m. musculus (PWD/Ph) and M. m. domesticus (C57BL/6J) were used. Hybrid (PWD x C57BL/6J)F1 male mice have low weight of paired testes, no sperm in ductus epidydimis and are sterile. F1 males from the reciprocal cross, (C57BL/6J X PWD/Ph)F1, are affected by only a partial spermatogenic arrest compatible with fertility.
Consomic female mice C57BL/6J-Chr X.1Pwd/Ph/Fore, C57BL/6J-Chr X.2Pwd/Ph/Fore and C57BL/6J-Chr X.3Pwd/Ph/Fore were crossed with PWD/Ph males. The male progeny of C57BL/6J-Chr X.2Pwd/Ph/Fore females displayed the typical F1 hybrid sterility phenotype with small testes and no sperm, while crosses of both C57BL/6J-Chr X.1Pwd/Ph/Fore and C57BL/6J-Chr X.3Pwd/Ph/Fore females yielded fertile males. Based on the known distal PWD border of C57BL/6J-Chr X.1Pwd/Ph/Fore and the proximal border of C57BL/6J-Chr X.3Pwd/Ph/Fore, the region carrying the PWD specific incompatibility locus was delimited to 61.0-94.3 Mb.
To estimate the number and location of hybrid sterility genes the fertility parameters in 254 N2 males generated from a backcross of (PWD/Ph x C57BL/6J)F1 x C57BL/6J mice were analyzed. The information from QTL analysis was complemented by crosses involving C57BL/6J-Chr #PWD/Ph/Fore chromosome substitution strains.
Mice were sacrificed at 9 wks and three quantitative phenotypes were measured for QTL analysis: body weight (BW); wet weight of paired testis (TW); and log transformed sperm count (logSC). Genomic DNA was extracted from liver. A set of 86 SSLP (MIT) markers were used to genotype backcross males. An additional 14 MIT markers were added to regions of interest (position of markers, NCBI build 37 July 2007). R12.1 and its QTL package were used to perform statistical analysis. TW and LogSC were modeled as continuous variables, fertility/subfertility as a binary varaible. Genome wide significance was calculated by 1000 permutations and compared to app=5% threshold. For two-dimensional scans to detect espistasis the scantwo fuction was used.
The joint distribution of TW and logSC were analyzed to model and sort the 254 N2 males into sterile/subfertile and fertile groups, comprising 31 and 223 mice respectively. Single QTL interval mapping with 74 informative SSLPs identified two highly significant QTLs for both TW and SC on Chr 17 and Chr X.
QTL Hstq1 (hybrid sterility QTL 1) mapped to Chromosome 17 with a LOD score of 5.42 at markers D17Mit19 (3.0 cM) and D17Zt634 (8.2 cM). Hstq1 is comprised of individual QTL Hstq1a, a locus linked to testes weight (LOD=4.86) and Hstq2, a locus linked with sperm count (LOD=5.67); all mapping to the same markers with a 1.5 LOD interval between 0.0 and 34.9 Mb. The QTL also overlapped with QTL Hst1 and Prdm9, known to affect hybrid sterility.
QTL Hstx2, (hybrid sterility, X chromosome 2) mapped to Chromosome X with a LOD score of 8.82 at markers DXMit140 (19.0 cM), DXMit76 (20.0 cM) and DXSr62 (26.7 cM). Hstx2 is comprised of individual QTL Hstx2a, a locus linked to testes weight (LOD=10.75) and Hstx2b, a locus linked to sperm count (LOD=14.63); all mapping to the same markers with a 1.5 LOD interval between 35.3 and 88.5 Mb. Hstx2 overlapped QTL Hstx1 responsible for defects in spermatogenesis also on Chr X.
All 23 sterile/subfertile animals carried both the PWD allele at DXSr62 on Chr X and PWD/B6 heterozygosity at the D17Zt634 locus on Chr 17. The simultaneous presence of incompatible genotypes at both chromosomes was a necessary condition for hybrid sterility, a clear epistatic interaction. This incompatibility was not seen in any of the 17 genotyped fertile males. All remaining 214 animals were genotyped at the same two loci and an additional 47 males with the same incompatibility (D-M) were found. 70 of the 254 N2 males displayed the incompatible allelic combination on Chrs 17 and X, of which 31 were sterile or subfertile (44%).
Genome wide genotyping of the 70 mice revealed two additional hybrid sterility loci reaching or exceeding significance:
QTL Hstq2 mapped to Chromosome 14 with a LOD score of 4.58 at markers D14Mit258 (17.0 cM) and D14MIt183 (19.0 cM). Hstq2 is comprised of individual QTL Hstq2a, a locus linked to testes weight (LOD=3.36) and Hstq2b, a locus linked to sperm count (LOD=6.37); all mapping to the same markers with a 1.5 LOD interval between 34.1 and 60.3 Mb. The subfertile phenotype did not completely segregate with PWD/B6 heterozygosity at this locus.
QTL Twq2 (testis weight QTL 2) mapped to Chromosome 13 with a LOD score of 3.03 between markers D13Mit202 and D13Mit35 at 60.0 cM. The 1.5 LOD interval spanned from 60.1 to 120.1 Mb for the testis weight phenotype only. This QTL conferred larger testis for PWD/B6 heterozygotes than B6/B6 homozygotes.
No other interacting loci were identified in the two locus scan. Thus, no other strong loci comparable to those on Chr 17 and Chr X were found. In conclusion the backcross to the B6 parent identified two strong hybrid sterility loci on Chr 17 and Chr X, whose epistatic interaction is obligatory but not sufficient for meiotic arrest in F1 hybrid sterility.
Crosses of consomic female mice, C57BL/6J-Chr XPWD/Ph/Fore, and male mice, C57BL/6J-Chr 17PWD/Ph/Fore, confirmed the indispensable role of the minor hybrid sterility loci on Chrs 14 and 13. The male progeny carried Chr X < PWD> and a PWD/B6 heterosomic pair of Chr 17 alleles, yet produced sperm.
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