Acknowledgements
We thank Vince De Vera for mouse care and Alma Islas-Trejo, Kerri Morimoto, Gonzalo Rincon, Ricardo Verdugo, Karina Guevara, James Chitwood, Lee Nguyen, Galen Williams and Emily Farber for assistance with mouse phenotyping. We also thank Scott Taylor and Dr. Ed DePeters for assistance and use of the UCD Animal Science Nutrition Laboratory for carcass composition analysis. This work was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2005–35205–15453. CRF was supported by the Austin Eugene Lyons fellowship.

Figures and Tables
Figure 1  Outline of the speed congenic approach used to capture genome-wide growth and obesity QTL. Details of the congenic matings are outlined in "Methods". The matings used to construct the MMU2 speed congenic panel are listed on the left of the figure, along with the expected and observed % heterozygosity at each generation. % heterozygosity is defined as the number of markers heterozygous B6 (or HG)/CAST as a percent of the total number of markers typed (N = 79). The right side of the illustration lists the matings used to construct the MMU1, 5, 8, 9, 11 and 17 speed congenic strains, along with the expected and observed % heterozygosity at each generation.
Figure 2  Genome-wide speed congenic strains and summary of QTL effects. White bars indicate the boundaries of CAST donor regions and hatched bars indicate intervals of unknown genotype. The minimum physical intervals (Mbp) of each donor region are listed in Table 1. To provide a general summary of the results, MMU2 is divided into five chromosomal regions (I, II, III, IV and V). Phenotypic differences for male and female (+/+ and hg/hg) mice relative to the appropriate controls are listed. For congenics outside of MMU2, a general summary of QTL effects is listed to the right of each donor region. Abbreviations: WT, body weight; GR, growth rate; T, tail length; NA, nasal-anal body length; TF, total weight of four fat pads; AI, adiposity index and BMI, body mass index.
Figure 3  Growth curves for MMU2 B6.CAST and HG.CAST speed congenic strains. Body weight LSMEANS ± SEM are plotted as a function of time (weeks). Top plot, growth curves in male B6 (+/+) and HG (hg/hg) speed congenic strains; bottom plot, growth curves in female B6 (+/+) and HG (hg/hg) speed congenic strains.
Figure 4  Significant three-way donor region by HG genotype by sex interaction for AI in the 2M donor region. AI plot illustrates the effect of the hg deletion on altering fat deposition between the sexes. The interaction is significant at P = 0.0004.
Figure 5  GenMAPP growth hormone (Gh) signaling pathway. Genes known to be or potentially involved in Gh signaling were mined from primary literature, reviews and book chapters. Genes are organized broadly based on functionality. MMU genomic position for each gene is to the right of each symbol and genes located on MMU2, 9, 11 and 17 are highlighted in yellow, purple, orange and green, respectively. Genes located on MMUX are labeled in white. Forty-four of the genes overlapping QTL intervals were selected for sequencing in the CAST strain.
Table 1  B6.CAST and HG.CAST speed congenic strains developed to isolate and characterize genome-wide QTL affecting growth and obesity
QTLa  MMU  Peak LOD cM (Mbp)b  Full congenic named  Abbreviation  Min. interval (Mbp)e
Wg1  2  31 (52)  B6.CAST-(D2Mit1-D2Mit160)N(6)  B62P  3.8–84.8
HG.CAST-(D2Mit1-D2Mit160)N(6)  HG2P  3.8–84.8
B6.CAST-(D2Mit322-D2Mit439)N(6)  B62PM  42.9–92.0
HG.CAST-(D2Mit322-D2Mit439)N(6)  HG2PM  42.9–92.0
Wg2  2  59–83c  B6.CAST-(D2Mit329-D2Mit490)N(6)  B62M  74.9–138.6
Carp1   (115–150)  HG.CAST-(D2Mit329-D2Mit490)N(6)  HG2M  74.9–138.6
Cara1    B6.CAST-(D2Mit329-D2Mit457)N(6)  B62D  74.9–181.8
Feml1    HG.CAST-(D2Mit329-D2Mit457)N(6)  HG2D  74.9–181.8
Q1Ucd1 -w26  1  16 (35)  HG.CAST-(D1Mit118-D1Mit250)N(6)  HG1  4.0–70.6
Carfhg1  5  38 (100)  HG.CAST-(D5Mit229-D5Mit158)N(6)  HG5  30.7–114.2
Wg3  8  45 (98)  HG.CAST-(D8Mit9-D8Mit198)N(6)  HG8  70.2–104.1
Carfhg2  9  10 (30)  HG.CAST-(D9Mit249-D9Mit133)N(6)  HG9  9.1–84.2
Feml2  9  20 (48)  HG.CAST-(D9Mit249-D9Mit133)N(6)  HG9  9.1–84.2
Carp2   Wg4  Cara2  11  46–50 (80–90)  HG.CAST-(D11Mit260-D11Mit255)N(6)  HG11  61.9–114.3
Feml3   Carp3  Cara3  17  46–48 (70–76)  HG.CAST-(D17Mit213-D17Mit142)N(6)  HG17  14.8–77.3
QTL, quantitative trait locus; MMU, mouse chromosome; LOD, log of the odds
a QTL MGI [59] nomenclature. Q1Ucd1-w26 never received an official MGI name, because it only reached a suggestive level of significance; hg modifier QTL are in bold [24].
b Mbp position according to the August 2005 mm7 UCSC [28] genome assembly (NCBI Build 35), peak LOD from [24].
c In the linkage analysis performed in [24], the peak location for Wg2, Carp1, Cara1 and Feml1 was 59–63 cM (115–120 Mbp) in hg/hg F2 mice and 78–83 cM (135–150 Mbp) in +/+ F2 mice.
d Full congenic name consists of three parts: 1. B6.CAST, indicates the recipient strain is B6 and the donor strain is CAST; HG.CAST, indicates the recipient strain is HG and the donor strain is CAST; 2. markers defining the minimal congenic interval; 3. the number of backcrosses used for speed congenic development.
e Represents the minimum genomic region spanned by CAST donor alleles.
Table 2  Body length and adiposity phenotypes of B6.CAST and HG.CAST MMU2 speed congenic strains
Strain  Sex  N  NA (cm)  Tail (cm)  GFP (g)  FFP (g)  MFP (g)  RFP (g)  TF (g)  AI (%)  BMI
B6C  M  29  9.1 ± 0.04  7.9 ± 0.04  0.344 ± 0.008  0.229 ± 0.006  0.183 ± 0.006  0.066 ± 0.002  0.823 ± 0.018  3.1 ± 0.1  31.6 ± 0.3
B62P  M  20  9.2 ± 0.05  7.9 ± 0.04   0.309 ± 0.010  0.235 ± 0.007  0.178 ± 0.007   0.045 ± 0.003  0.768 ± 0.023  2.9 ± 0.1  30.8 ± 0.4
B62PM  M  24  9.1 ± 0.05  7.8 ± 0.05   0.304 ± 0.010  0.222 ± 0.008  0.173 ± 0.007   0.046 ± 0.003  0.746 ± 0.024  2.9 ± 0.1  31.3 ± 0.4
B62M  M  27  8.9 ± 0.07   7.4 ± 0.06   0.294 ± 0.014  0.239 ± 0.010   0.214 ± 0.009  0.066 ± 0.004  0.813 ± 0.032  3.2 ± 0.1  31.4 ± 0.5
B62D  M  21  9.2 ± 0.06   7.1 ± 0.06   0.259 ± 0.013   0.177 ± 0.009  0.154 ± 0.009   0.038 ± 0.004   0.629 ± 0.030   2.6 ± 0.1   28.9 ± 0.5
B6C  F  39  8.6 ± 0.03  7.6 ± 0.03  0.166 ± 0.007  0.202 ± 0.005  0.141 ± 0.005  0.027 ± 0.002  0.536 ± 0.016  2.6 ± 0.1  27.7 ± 0.3
B62P  F  23  8.6 ± 0.05  7.8 ± 0.04  0.134 ± 0.010  0.224 ± 0.007  0.148 ± 0.007  0.023 ± 0.003  0.528 ± 0.022  2.7 ± 0.1   26.1 ± 0.4
B62PM  F  20   8.4 ± 0.05   7.4 ± 0.05  0.139 ± 0.011  0.214 ± 0.008  0.154 ± 0.007  0.023 ± 0.003  0.530 ± 0.025  2.8 ± 0.1  26.9 ± 0.4
B62M  F  32   8.4 ± 0.05   7.3 ± 0.05  0.153 ± 0.010  0.204 ± 0.007   0.165 ± 0.007  0.029 ± 0.003  0.551 ± 0.023  2.8 ± 0.1  27.9 ± 0.4
B62D  F  23  8.7 ± 0.06   7.0 ± 0.06   0.110 ± 0.013   0.157 ± 0.009  0.135 ± 0.009  0.019 ± 0.004   0.420 ± 0.029   2.2 ± 0.1   25.0 ± 0.5
HGC  M  31  10.2 ± 0.05  8.0 ± 0.05  0.477 ± 0.013  0.317 ± 0.008  0.297 ± 0.010  0.110 ± 0.005  1.201 ± 0.030  3.3 ± 0.1  35.0 ± 0.4
HG2P  M  18  10.1 ± 0.07   8.2 ± 0.07  0.428 ± 0.019  0.326 ± 0.011   0.227 ± 0.014   0.069 ± 0.007   1.051 ± 0.043  3.1 ± 0.1   32.8 ± 0.5
HG2PM  M  29  10.1 ± 0.05  8.0 ± 0.05  0.439 ± 0.014  0.303 ± 0.008   0.244 ± 0.010   0.082 ± 0.005   1.068 ± 0.031  3.1 ± 0.1  33.7 ± 0.4
HG2M  M  31  10.1 ± 0.05   7.4 ± 0.05  0.469 ± 0.014  0.346 ± 0.008  0.306 ± 0.010   0.136 ± 0.005  1.260 ± 0.032  3.6 ± 0.1  33.9 ± 0.4
HGC  F  23  9.6 ± 0.06  7.8 ± 0.06  0.369 ± 0.015  0.305 ± 0.009  0.268 ± 0.011  0.076 ± 0.005  1.020 ± 0.035  3.9 ± 0.1  28.2 ± 0.4
HG2P  F  16   9.2 ± 0.07  7.8 ± 0.07   0.248 ± 0.018  0.311 ± 0.011   0.200 ± 0.013   0.044 ± 0.006   0.804 ± 0.041   3.4 ± 0.1  28.3 ± 0.5
HG2PM  F  36   9.4 ± 0.04  7.7 ± 0.04   0.277 ± 0.012   0.265 ± 0.007   0.208 ± 0.009   0.046 ± 0.004   0.797 ± 0.027   3.2 ± 0.1  27.8 ± 0.3
HG2M  F  28  9.6 ± 0.05   7.2 ± 0.05  0.329 ± 0.014   0.269 ± 0.009  0.247 ± 0.010  0.064 ± 0.005  0.909 ± 0.033   3.4 ± 0.1  29.3 ± 0.4
NA, nasal-anal body length; Tail, tail length; GFP, gonadal fat pad; FFP, femoral fat pad; MFP, mesenteric fat pad; RFP, retroperitoneal fat pad; TF, summed weight of four fat pads; AI, adiposity index (TF/body weight*100) and BMI, body mass index (body weight at sacrifice/NA2 *100). Values are expressed as LSMEANS ± SEM. LSMEANS in bold are significantly different than the respective control after Bonferroni correction (B6 (critical P < 0.00625) or HG (P < 0.00833)) within each sex.
Table 3  Significance levels for the main effects, two-way and three way interactions of MMU2 speed congenic donor region (DR), HG genotype (HG) (+/+ or hg/hg) and sex for selected traits
Donor region (DR)a  6WK  9WK  G26  G29  NA  TF  AI
Proximal (2P)
DR   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001  0.0079
HG   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001
Sex   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001  0.9840
DR*HG   <.0001  0.0094   <.0001  0.0181   <.0001   0.0004  0.0183
DR*sex  0.0287  0.4783  0.0328  0.5938  0.0081  0.8780  0.5218
DR*HG*Sex  0.4092  0.4206  0.6957  0.0882  0.1563  0.1618  0.0219
Proximal middle (2PM)
DR   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001
HG   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001
Sex   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001  0.7248
DR*HG  0.7101  0.4667  0.1277  0.1110  0.7375   0.0026   0.0065
DR*sex  0.1441  0.3947  0.1095  0.4198  0.0104  0.9323  0.6994
DR*HG*Sex  0.9437  0.2294  0.5748  0.0780  0.7697   0.0060   0.0002
Middle (2M)
DR   0.007   <.0001  0.0234   0.0002   0.0006  0.1703  0.9707
HG   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001
Sex   <.0001   <.0001   <.0001   <.0001   <.0001   <.0001   0.0064
DR*HG   0.0057  0.0563  0.1875  0.6072  0.4329  0.3437  0.0256
DR*sex  0.6968  0.0089  0.8356  0.0207  0.5162  0.1460  0.0437
DR*HG*Sex  0.6205  0.1309  0.6500  0.0656  0.6704   0.0035   0.0004
6WK, weight at 6 weeks of age; 9WK, weight at 9 weeks of age; G26, weight gain from 2 to 6 weeks of age; G29, weight gain from 2 to 9 weeks of age; NA, nasal-anal body length; TF, total fat pad weight and AI, adiposity index. Significant effects after a Bonferroni correction (critical P < 0.0071) are in bold.
a The distal (2D) donor region was not analyzed.
Table 4  Body size, growth rate and length phenotypes of HG.CAST speed congenic strains
Strain  Sex  N  2WK (g)  3WK (g)  6WK (g)  9WK (g)  G26 (g)  G29 (g)  NA (cm)  Tail (cm)
HGC  M  31  7.4 ± 0.2  10.1 ± 0.3  30.4 ± 0.4  36.4 ± 0.4  23.1 ± 0.3  29.0 ± 0.4  10.2 ± 0.05  8.0 ± 0.04
HG1  M  19  6.9 ± 0.3  9.6 ± 0.4  28.8 ± 0.6  34.7 ± 0.6  21.9 ± 0.5  27.8 ± 0.5   9.9 ± 0.07  7.8 ± 0.06
HG8  M  15  6.7 ± 0.4  9.2 ± 0.5  29.7 ± 0.8  35.2 ± 0.8  23.1 ± 0.6  28.5 ± 0.7  10.2 ± 0.09  7.9 ± 0.08
HG9  M  29  6.8 ± 0.2   8.8 ± 0.3   27.6 ± 0.5  34.7 ± 0.5   20.7 ± 0.4  27.9 ± 0.4   9.9 ± 0.05   7.8 ± 0.05
HG11  M  23  7.8 ± 0.3  11.1 ± 0.4  31.7 ± 0.6  38.3 ± 0.6  23.8 ± 0.5  30.4 ± 0.5  10.4 ± 0.07   8.4 ± 0.06
HG17  M  10  8.4 ± 0.4  11.4 ± 0.6  29.0 ± 0.9  34.0 ± 1.0   20.7 ± 0.7   25.7 ± 0.8  9.9 ± 0.11   7.5 ± 0.09
HGC  F  23  7.4 ± 0.2  10.1 ± 0.3  23.3 ± 0.5  25.9 ± 0.5  15.9 ± 0.4  18.5 ± 0.4  9.6 ± 0.06  7.8 ± 0.05
HG1  F  31   6.3 ± 0.2   8.2 ± 0.4   21.1 ± 0.5  24.7 ± 0.6  14.8 ± 0.4  18.3 ± 0.5   9.4 ± 0.06   7.5 ± 0.05
HG8  F  22  6.5 ± 0.4  8.7 ± 0.5  23.1 ± 0.8  26.0 ± 0.8  16.5 ± 0.6  19.5 ± 0.7  9.6 ± 0.09  7.6 ± 0.08
HG9  F  27  7.5 ± 0.2  9.7 ± 0.3  22.6 ± 0.4  25.7 ± 0.5  15.1 ± 0.3  18.2 ± 0.4   9.3 ± 0.05  7.6 ± 0.05
HG11  F  24  7.2 ± 0.2  9.6 ± 0.3  22.1 ± 0.5  24.8 ± 0.5  14.8 ± 0.4  17.5 ± 0.5   9.3 ± 0.06  8.0 ± 0.05
HG17  F  17   8.4 ± 0.3  11.0 ± 0.4  22.0 ± 0.6  25.2 ± 0.7   13.6 ± 0.5  16.8 ± 0.5   9.3 ± 0.07   7.4 ± 0.06
2WK, weight at 2 weeks of age; 3WK, weight at 3 weeks of age; 6WK, weight at 6 weeks of age; 9WK, weight at 9 weeks of age; G26, weight gain from 2 to 6 weeks of age; G29, weight gain from 2 to 9 weeks of age; NA, nasal-anal body length; Tail, tail length. Values are expressed as least square means (LSMEANS) ± SEM. LSMEANS in bold are significantly different than the control (HGC) (critical P < 0.005) after Bonferroni correction within each sex.
Table 5  Adiposity phenotypes of HG.CAST speed congenic strains
Strain  Sex  N  GFP (g)  FFP (g)  MFP (g)  RFP (g)  TF (g)  AI (%)  BMI
HGC  M  31  0.477 ± 0.020  0.317 ± 0.011  0.298 ± 0.010  0.110 ± 0.006  1.202 ± 0.042  3.3 ± 0.1  35.0 ± 0.3
HG1  M  19  0.473 ± 0.029  0.353 ± 0.016  0.258 ± 0.014  0.106 ± 0.009  1.190 ± 0.061  3.5 ± 0.2  35.2 ± 0.5
HG8  M  15  0.418 ± 0.038  0.340 ± 0.021   0.230 ± 0.018  0.099 ± 0.012  1.087 ± 0.080  3.0 ± 0.2  33.8 ± 0.6
HG9  M  29   0.615 ± 0.021   0.453 ± 0.012  0.283 ± 0.010   0.166 ± 0.007   1.518 ± 0.045   4.3 ± 0.1  35.6 ± 0.4
HG11  M  23  0.516 ± 0.027  0.312 ± 0.015  0.281 ± 0.013  0.120 ± 0.009  1.229 ± 0.058  3.2 ± 0.2  35.5 ± 0.5
HG17  M  10  0.540 ± 0.043  0.344 ± 0.024  0.278 ± 0.021  0.124 ± 0.014  1.285 ± 0.091  3.8 ± 0.3  35.1 ± 0.8
HGC  F  23  0.370 ± 0.023  0.305 ± 0.013  0.269 ± 0.011  0.076 ± 0.007  1.020 ± 0.048  3.9 ± 0.1  28.1 ± 0.4
HG1  F  31  0.336 ± 0.025  0.328 ± 0.14   0.205 ± 0.012  0.055 ± 0.008  0.924 ± 0.053  3.8 ± 0.2  27.9 ± 0.4
HG8  F  22   0.247 ± 0.037  0.310 ± 0.021   0.193 ± 0.018  0.043 ± 0.012  0.793 ± 0.079   3.1 ± 0.2  27.8 ± 0.6
HG9  F  27   0.624 ± 0.021   0.518 ± 0.012  0.288 ± 0.010   0.152 ± 0.007   1.581 ± 0.045   6.1 ± 0.1   29.9 ± 0.4
HG11  F  24  0.436 ± 0.024  0.332 ± 0.014  0.269 ± 0.012  0.087 ± 0.008  1.124 ± 0.052   4.5 ± 0.2  28.5 ± 0.4
HG17  F  17  0.410 ± 0.029  0.350 ± 0.016  0.262 ± 0.014  0.086 ± 0.009  1.107 ± 0.062  4.4 ± 0.2  28.9 ± 0.5
GFP, gonadal fat pad; FFP, femoral fat pad; MFP, mesenteric fat pad; RFP, retroperitoneal fat pad; TF, summed weight of four fat pads; AI, adiposity index (TF/body weight*100) and BMI, body mass index (body weight at sacrifice/NA2 *100). Values are expressed as least square means (LSMEANS) ± SEM. LSMEANS in bold are significantly different than the control (HGC) (critical P < 0.005) after Bonferroni correction within each sex.
Table 6  Carcass composition phenotypes of HG.CAST speed congenic strains
Line  Sex  N  H2O (g)  %H2O  FAT (g)  %FAT  ASH (g)  %ASH  PROT (g)  %PROT
HGC  M  31  16.12 ± 0.30  57.74 ± 0.52  2.69 ± 0.11  9.77 ± 0.47  0.86 ± 0.01  3.08 ± 0.03  8.19 ± 0.08  29.41 ± 0.29
HG11  M  22  17.28 ± 0.35  58.62 ± 0.62  2.76 ± 0.13  9.43 ± 0.56  0.85 ± 0.01   2.89 ± 0.03  8.51 ± 0.10  29.06 ± 0.30
HG17  M  10   14.27 ± 0.55  55.32 ± 0.97  2.87 ± 0.21  11.34 ± 0.88   0.78 ± 0.02  3.02 ± 0.05  7.80 ± 0.15  30.33 ± 0.35
HGC  F  23  10.08 ± 0.32  51.76 ± 0.56  2.66 ± 0.12  13.83 ± 0.51  0.68 ± 0.01  3.50 ± 0.03  5.95 ± 0.09  30.91 ± 0.27
HG11  F  22  8.95 ± 0.33   48.96 ± 0.58  2.94 ± 0.12   16.02 ± 0.53   0.58 ± 0.01   3.20 ± 0.03  5.80 ± 0.09  31.82 ± 0.32
HG17  F  17  8.88 ± 0.38   48.91 ± 0.67  2.84 ± 0.14  15.69 ± 0.61   0.60 ± 0.02   3.31 ± 0.04  5.80 ± 0.11   32.09 ± 0.50
H2O, carcass water; %H2O, water as a percent of carcass weight; FAT, carcass fat; %FAT, fat as a percent of carcass weight; ASH, carcass ash; %ASH, ash as a percent of carcass weight; PROT, carcass protein, %PROT, protein as a percent of carcass weight. Values are expressed as least square means (LSMEANS) ± SEM. LSMEANS in bold are significantly different than the control (HGC) (critical P < 0.0125) after Bonferroni correction within each sex.
Table 7  Nonsynonymous SNP in MMU2, 9 and 17 hg modifier candidate genes predicted by SIFT and Polyphen to alter protein function
Gene  MMU  Mbp  AAÄa  B6/CASTb Polyphen  CAST/B6 Polyphen  B6/CAST SIFT  CAST/B6 SIFT
Nmi  2  52.0  R243C  B  B  T  D
Mtx2  2  74.7  T1099M  PSD  PSD  T  D
Ubr1  2  120.6  A1371T  B  B  D  T
Ptpns1  2  129.3  H224N  PBD  PBD  T  T
Ubce7ip5  2  130.3  A314P  PSD  PSD  T  T
2   I366T  B  B  T  D
Plcg1  2  160.5  T1165A  B  B  T  D
Mmp9  2  164.7  A514P  B  B  D  D
2   P639L  PBD  PBD  T  T
Tyk2  9  21.0  R234H  PSD  PSD  T  T
C831S  PSD  PSD  T  T
T1099M  PSD  PSD  T  D
Vav  17  55.5  Q29H  PSD  PBD  T  T
Q53H  PBD  PSD  T  T
Q65H  PBD  PSD  T  D
MMU, mouse chromosome; AA, amino acid; PBD, probably damaging, PSD, possibly damaging; B, Benign; T, tolerated and D, deleterious.
a Amino acid change, first residue listed corresponds to the CAST allele and second corresponds to the B6 allele.
b Indicates which allele was designated as "mutant", i.e. B6/CAST, B6 allele is mutant and CAST is wild type.