Experiment
Previous genome-wide quantitative trait locus (QTL) analyses in an intersubspecific backcross population between C57BL/6JJcl (B6) and wild Mus musculus castaneus mice revealed a major growth QTL (Pbwg1, postnatal body weight growth 1) on a proximal region of mouse chromosome 2.
Using an intersubspecific congenic strain, B6.Cg-(D2Mit33-D2Mit38)/Nga (B6.Cg-Pbwg1), 12 closely linked QTLs for body weight and body composition traits, on an approximately 44.1-Mb wild-derived congenic region, were identified. Among the linked loci, several had unique QTL effects and were located on the distal half of the congenic region. The wild derived allele at Pbwg1.12 and Pbwg1.3 QTL increased body weight and total body length, respectively, despite the fact that the wild mice have a smaller body size than the B6 mice. In contrast, the allele at QTL Pbwg1.5 decreased abdominal white fat weight and prevented obesity in mice fed both standard and high fat diets.
In the current study, the genomic regions harboring three (Pbwg1.12, Pbwg1.3 and Pbwg1.5) of the 12 linked QTLs were fine mapped by phenotypic analysis of F2 mice obtained from intersubspecific subcongenic intercrosses descended from B6.Cg-Pbwg1. Previously constructed subcongenic strains SR1 (B6.Cg-(rs13476521-D2Mit38)/Nga); SR2 (B6.Cg-(rs13476521-D2Mit123)/Nga); SR12 (B6.Cg-(D2Mit205-D2Mit56)/Nga) and newly constructed strain SR21 (B6.Cg-(rs13476521-rs48690987)/Nga) were intercrossed with B6 to create overlapping and non overlapping introgressed regions. [Fig 1].
In total the following F2 individual mice were produced: 273 (138 males and 135 females) for B6 x SR1; 236 (113 males and 123 females) for B6 x SR2; 132 (58 males and 74 females) for B6 x SR12; and 291 (151 males and 140 females) for B6 x SR21.
All mice were weaned at 3 weeks of age and fed standard chow. Genomic DNA was extracted from ear clips of F2 mice. Body weights were measured at 1, 3, 6, 10 and 14 weeks of age. Weight gains were calculated. After overnight fasting mice were sacrificed and total body lengths were immediately measured. Blood samples were taken and the weights of fat pads were recorded. Body weight, weight gain and body composition data for the F2 populations was analyzed with a linear mixed model of the statistical discovery software JMP version 11.1.1.
Table 4:
Taking all results together, a growth QTL, the wild-derived allele of which increased body weight and body weight gain, was localized to an interval between D2Mit433 (57.3 Mb) and D2Mit205 (65.3), In a previous study using congenic strains [J:171917], the growth QTL Pbwg1.12 was physically localized to a maximum interval between D2Mit472 (61.5) and D2Mit327 (69.5) [Fig1] that overlapped with the interval of the growth QTL identified in the current study. The wild-derived allele at Pbwg1.12 increased body weight in the previous study, which was exactly the same allelic effect of the growth QTL identified in the current study. Therefore, the current study succeeded in confirming the presence of Pbwg1.12 and in narrowing it down to a 3.8-Mb interval between D2Mit472 (61.5) and D2Mit205 (65.3).
Likewise, the presence of the Pbwg1.3 QTL affecting total body length that was previously revealed by interval mapping with an F2 intercross population between the B6.Cg-Pbwg1 original congenic and B6 strains [J:121981] was confirmed. Pbwg1.3 was physically defined to an 8.0-Mb interval between D2Mit433 (57.3) and D2Mit205 (65.3).
In the B6 x SR1 intercross, a QTL for which the wild-derived allele decreased inguinal and gonadal fat pad weights was clearly identified. In the B6 x SR21 intercross, however, the presence of the obesity QTL was ambiguous, because P values for diplotype comparisons marginally exceeded the nominal 5% level but did not reach the Bonferroni-corrected 5% level. Previously, the Pbwg1.5 QTL for resistance to obesity was physically mapped to an interval between D2Mit270 (52.9) and D2Mit472 (61.5) [J:153372, Fig 1]. Therefore, the presence of Pbwg1.5 in the current study was confirmed and localized to a 2.1-Mb interval between D2Mit123 (59.4) and D2Mit472 (61.5).
To search for possible candidate genes of the QTL, exome sequencing of genes on the congenic regions was performed and candidate genes were prioritized using bioinformatics analysis. [Sequence data was not available for the wild M. m. castaneus mice captured in the Philippines, in contrast to the CAST/EiJ inbred strain derived from wild M. m. castaneus mice trapped in Thailand, for which the whole genome had been already sequenced.]
Exome sequencing and candidate gene prioritization suggested that Gcg and Grb14 were putative candidate genes for Pbwg1.12 and that Ly75 and Itgb6 were putative candidate genes for Pbwg1.5. These genes had nonsynonymous SNPs, but the SNPs were predicted to be not harmful to protein functions. These results provide information helpful to identify wild-derived quantitative trait genes causing enhanced growth and resistance to obesity.
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