Skip to content

Advertisement

You're viewing the new version of our site. Please leave us feedback.

Learn more

BMC Genomics

Open Access

Efficient single nucleotide polymorphism discovery in laboratory rat strains using wild rat-derived SNP candidates

  • Bart MG Smits1,
  • Victor Guryev1,
  • Dimphy Zeegers1,
  • Dirk Wedekind2,
  • Hans J Hedrich2 and
  • Edwin Cuppen1Email author
BMC Genomics20056:170

https://doi.org/10.1186/1471-2164-6-170

Received: 23 September 2005

Accepted: 29 November 2005

Published: 29 November 2005

Abstract

Background

The laboratory rat (Rattus norvegicus) is an important model for studying many aspects of human health and disease. Detailed knowledge on genetic variation between strains is important from a biomedical, particularly pharmacogenetic point of view and useful for marker selection for genetic cloning and association studies.

Results

We show that Single Nucleotide Polymorphisms (SNPs) in commonly used rat strains are surprisingly well represented in wild rat isolates. Shotgun sequencing of 814 Kbp in one wild rat resulted in the identification of 485 SNPs as compared with the Brown Norway genome sequence. Genotyping 36 commonly used inbred rat strains showed that 84% of these alleles are also polymorphic in a representative set of laboratory rat strains.

Conclusion

We postulate that shotgun sequencing in a wild rat sample and subsequent genotyping in multiple laboratory or domesticated strains rather than direct shotgun sequencing of multiple strains, could be the most efficient SNP discovery approach. For the rat, laboratory strains still harbor a large portion of the haplotypes present in wild isolates, suggesting a relatively recent common origin and supporting the idea that rat inbred strains, in contrast to mouse inbred strains, originate from a single species, R. norvegicus.

Background

Genetic variation exists between individuals (or strains) of all organisms and it makes up the genetic basis for phenotypic differences between individuals. In addition, genetic variation functions as a valuable resource for mapping phenotypic traits in model organisms. Single Nucleotide Polymorphisms (SNPs) are the most abundant form of genetic variation and therefore dominate high-resolution genetic mapping strategies. Moreover, numerous well-performing high-throughout SNP detection technologies have been developed, like oligonucleotide array-based technology, mass-spectrometry-based technology (MALDI-TOF), and sequence-based technology (pyrosequencing, DHPLC) [1], which makes automated SNP detection favored above the more labor-intensive detection of microsatellite markers [2].

Since the availability of its genome, the laboratory rat is gaining influence as a genetic model organism [3]. In addition, over 200 well-characterized inbred strains that are models for a wide variety of human diseases are available [4, 5]. However, the availability of genetic tools, like a dense genome-wide SNP marker set, is still subordinate compared to other commonly used model organisms. This is illustrated by the number of entries in dbSNP, the central SNP repository of NCBI [6]: the amount of human (>10,000,000), chicken (>3,000,000), and mouse (>500,000) entries surpass the amount of rat entries (>43,000) spectacularly. In search for rat SNPs, experimental [7, 8] and computational [9] approaches have been employed, but these efforts primarily resulted in SNPs associated with coding regions. For genetic mapping purposes, a much denser marker set, preferentially equally distributed over the genome, is required.

Laboratory rat strains are thought to be established from a limited number of founder animals originating from a domesticated wild population [10, 11]. The value of inbred strains emanates from the close genetic uniformity that facilitates phenotyping and genotyping. In principle, inbred strains are selectively bred for certain traits from a genetically diverse pool, comprising diverse genetic information about the trait. However, since many of the current rat strains were derived from common ancestral stocks and simply inbred to increase genetic uniformity, inbred strains clearly share alleles [12]. Although such simplified models are essential for biomedical research, modulating effects on the clinical manifestation of a trait resulting from genetic heterogeneity in a population can only be studied to a limited extent in F1 hybrids. The use of a carefully chosen selection of inbred strains may address this issue, but the choice depends on knowledge on the relationship between the strains and hence the degree of genetic variation. Alternatively, wild-derived strains may be good alternatives to introduce sufficient genetic variation in laboratory experiments [13, 14].

Based on a preliminary observation that alleles from laboratory rat strains are frequently detected in wild-derived samples, we developed a wild rat-based SNP discovery approach. The method consists of shotgun sequencing of a wild rat-derived genomic library followed by comparison with the published rat genome (strain Brown Norway). Genotyping commonly used rat strains for newly identified SNPs revealed that 84% of SNP-alleles (and 87% of all genetic variation) occurring between BN and a single wild individual is also represented in one or more laboratory strains. A user-friendly webtool allows exploration of the genetic variation between any arbitrary combinations of two strains that were used in this study, making all information directly available for experimental use.

Results

Wild rat-based SNP discovery

It is generally believed that commonly used rat strains originate from a wild-derived founder population of limited size [10]. To examine whether polymorphisms found in laboratory strains are still represented in individuals of the wild population, we typed two wild-derived samples for confirmed SNPs of the CASCAD database [9]. Interestingly, about 53% of alleles (n = 147), which were confirmed to exist in laboratory strains, were also represented in wild 1, wild 2 or both ( not shown). Hence, a preselection of highly likely candidate SNPs could potentially be made by genotyping wild individuals and comparing the sequences to the rat genome sequence (Brown Norway).

Accordingly, we performed random shotgun sequencing on a genomic library of a wild rat (wild 1). We generated shotgun traces (814 Kbp) by bidirectional sequencing of about 1,600 colonies (Table 1). 85.5% of the reads (2545/2975; Table 1) could be mapped to a unique location in the Brown Norway rat genome using BLAT [15], resulting in the automated identification of nearly 5,000 ambiguous nucleotide positions (potential polymorphisms). Manual inspection of the sequencing reads reduced this set of potential polymorphisms to a set of 746 real SNPs and 122 indels. The average SNP rate between BN (BN/SsNMcw; genome sequencing project) and this single wild rat is estimated to be about 1 per 900 bp and, hence, discovery of a novel SNP can be expected every second shotgun read. A subset of the discovered SNPs was verified and genotyped in 36 commonly used strains (including BN). To this end, we designed primers for 451 SNP-containing amplicons (about 300 bp) of which 416 (92.2%) were successfully read by unidirectional sequencing of the PCR products, resulting in roughly 119 Kbp high quality sequence per strain or individual (Table 1).
Table 1

Statistics on shotgun sequencing of the wild rat-derived genomic library

picked colonies

1632

readable sequence reads/sequenced bases

2975/814,440

uniquely mapped (BLAT) reads/bases

2545/768,683

ambiguous positions

4902

strong candidates after manual inspection

868 (746 SNPs + 122 indels)

successfully read/amplicons designed*

416/451 (~1.65 candidate SNP/amplicon) (92.2%)

amplified bases per strain or wild individual

118,971

* Amplicons are designed for the 746 SNP candidates.

Wild rat-derived SNP characteristics

The verification of 746 candidate SNPs by amplicon-based resequencing in 36 inbred rat strains and three wild-derived samples (wild 1, 2, and 3) revealed 960 polymorphisms, consisting of 90 indels, seven 2-bp substitutions, one 3-bp substitution, one 5-bp substitution, and 861 SNPs, of which only one was tri-allelic. The amplicons are randomly distributed over the genome (Fig. 1). We observed heterozygous positions in the outbred strains, but unexpectedly some were also found in the inbred strains (for detailed information: [see Additional file 1] or [6]). For our analysis, we considered these loci to be polymorphic as compared to the BN genome sequence.
Figure 1

Distribution of amplicons (451 loci) designed for verification and subsequent genotyping of candidate shotgun-based SNPs in 36 commonly used inbred strains.

From the 746 shotgun-based candidate SNPs, 685 were located in the 416 PCR amplicons that worked, and 485 (71%) were reconfirmed by resequencing (shotgun-based; Table 2). Strikingly, for 408 (84%) of the confirmed SNPs, the wild rat allele is also present in one or more commonly used strains, with only 36 (7.4%) being specific to BN (Table 2). Of the remaining 77 (16%) SNPs, wild rat alleles are not present in any of the 36 selected strains and could be considered wild rat-specific. These results illustrate that shotgun sequencing one wild individual efficiently identifies shared polymorphisms among commonly used rat strains.
Table 2

SNP discovery results

 

shotgun- based

genotyping-based (only wild 1*)

genotyping-based (wild 1, 2, 3*)

total (only wild 1*)

total (wild 1, 2, 3*)

BN specific

36 (7.4 %)

9 (2.5 %)

7 (1.9 %)

45 (5.3 %)

43 (5.0 %)

wild specific

77 (15.9 %)

12 (3.4 %)

30 (8.0 %)

89 (10.6 %)

107 (12.4 %)

in 35 strains, not in wild

0

204 (57.0 %)

156 (41.5 %)

204 (24.2 %)

156 (18.1 %)

in 35 strains, shared with wild

372 (76.7 %)

133 (37.1 %)

183 (48.7 %)

505 (59.9 %)

555 (64.5 %)

total

485 (100 %)

358 (100 %)

376 (100 %)

843 (100 %)

861 (100 %)

* By genotyping two other wild individuals (wild 2 and 3), additional polymorphisms were identified, which could not have been found by shotgun sequencing only wild 1.

While genotyping by resequencing, 358 novel SNPs were discovered that were not identified in the shotgun sequencing experiment (genotyping-based; Table 2). About 39% (139) of this set can be accounted for by differences in the sequence coverage between the shotgun reads and the resequencing genotyping reads (Table 2), whereas the remaining part of this set is strongly biased towards SNPs that are not polymorphic between BN and wild rat 1 and thus could not have been discovered in the shotgun experiment. Interestingly, about 37% of the newly discovered SNPs are polymorphic between the shotgun sequenced wild rat and any of the inbred strains (Table 2). When considering all SNPs that are polymorphic in the set of 36 commonly used laboratory strains, of the majority (66%) the wild rat allele is found back in one of the strains (total; Table 2) and this percentage increases only slightly (70%) when two other wild individuals (wild 2 and 3) are included in the analysis. This indicates that wild rat-based SNP discovery is already highly efficient using a single wild sample.

Based on the genotyping results, the SNP rate between BN and the shotgun sequenced wild rat (wild 1) is 1 SNP per 190 bp (626 SNPs/119 Kbp). The SNP rate within the 36 rat strains, including BN, is 1 in 158 (Table 2; 45+204+505 SNPs/119 Kbp) and the SNP rate in the entire experiment, including the wild rat (wild 1), BN, and the other strains is 1 in 141 bp (Table 2; 843 SNPs/119 Kbp). To compare wild rat inter-individual variation with the inter-strain variation for commonly used inbred strains, we calculated the number of SNPs that are polymorphic when comparing arbitrary combinations of 3 strains. Genotyping of 861 SNP positions in the three wild rats resulted in 438 polymorphic positions, whereas the most polymorphic combination of inbred strains in this experiment (BN, BH, and SHR) yielded 427 SNPs. This indicates that three random, but potentially related, Dutch wild rats are about equally polymorphic as three carefully selected inbred strains. Inclusion of wild isolates from other locations worldwide may increase the efficiency of the SNP discovery approach.

Intraspecific phylogenetic network

Relationships among different rat strains have been determined previously by phylogenetic tree reconstruction based on microsatellite markers [16, 17]. However, intraspecific relationships for laboratory strains are often very challenging to determine, due to small genetic distances and complex gene flow. The resulting multitude of plausible trees is best expressed by a network, which displays alternative potential evolutionary paths in the form of cycles [18]. We used Network software (v4.111 Reduced-Joining, [19]) to construct a spatial network, based on 861 SNP markers in 36 rat strains and three wild rat individuals (Fig. 2). The three wild individuals are grouped together, possibly due to the geographic and possibly genetic relation between the samples, but in accordance with the last paragraph of the previous section, they appear relatively unrelated as compared to the set of inbred strains.
Figure 2

Strain relationships in a network structure. End nodes (yellow dots) represent strains. Some end nodes are double-size, meaning that they are supported by two samples. Interconnecting nodes where lines come together, represent a possible precursor.

The majority of the SNPs (485 of 861) was selected for being polymorphic between wild 1 and BN. As a result, different BN substrains (BN/Ztm, BN/Crl), depicted as a double-sized end node because of high similarity, and different wild rat individuals (wild 1, wild 2, and wild 3) are grouped together as the outliers. Several strains that are known to be closely related (source RGD-strains: [20]) are also grouped together, like DA and COP or SS and SR. Interestingly, WKY is also an outlier, indicating that besides BN, this strain can be utilized as an alternative mapping strain. WKY is already commonly used as a normotensive control strain in genetic mapping of blood pressure quantitative trait loci [21]. WKY is known to be closely related to SHR and these strains are indeed grouped together (Fig. 2). Additionally, BDII and BDIX are related and BDE is an RI strain from E3. These strain combinations are also grouped together. Wistar is contributing to a large subset of these strains, like WKY, WC, BDII, MWF, LEW, and WF, which contributes to the complexity of the network structure.

Data availability

The use of genetic markers for mapping traits in rat strains has been exploited for long time already. Current marker sets in rats are mostly limited to microsatellites [22, 23], which are not abundantly available and are commonly detected in a more laborious way than SNPs. In this study, we have determined a total of about 35,000 genotypes (about 960 loci in 36 inbred strains), out of which the vast majority are SNPs. This data is accessible via a versatile webtool [24]. Pairs of strains of interest can be selected and explored on presence of verified genetic variation. Besides a graphical representation of the location of the SNPs on a genome map, primer sequences that were successfully used in our experiments are also provided. In a pairwise comparison matrix (Table 3), we plotted the absolute number of polymorphic positions for each of the (sub-)strains or individuals used. Interestingly, for some strains different alleles are observed in substrains (e.g. BN/Crl differs from BN/Ztm at 4 positions), in line with previous observations [8].
Table 3

Absolute number of polymorphic positions between strains in a pairwise comparison.

 

ACI

AO

AUG

BDE

BDII

BDIX

BDV

BH

BN

BN2

BS

BUF

COP

DA

DA2

E3

F344

LEW

LE

LOU

LUDW

MWF

MNS

NAR

OM

PAR

PVG

R33

RP

SD

SD2

SHR

SR

SS

WAG

WC

WF

WIST

WKY

wild3

wild2

wild1

ACI

x

                                         

AO

131

x

                                        

AUG

111

167

x

                                       

BDE

145

178

158

x

                                      

BDII

130

166

143

148

x

                                     

BDIX

129

196

191

159

115

x

                                    

BDV

109

131

145

137

78

142

x

                                   

BH

145

189

192

212

156

177

167

x

                                  

BN

225

263

279

266

206

258

244

274

x

                                 

BN2

227

270

285

268

222

271

251

274

4

x

                                

BS

159

167

163

178

166

185

143

180

246

251

x

                               

BUF

148

158

181

171

168

190

151

166

250

263

158

x

                              

COP

84

194

187

166

155

147

155

191

262

275

203

192

x

                             

DA

62

107

116

134

115

130

117

130

210

216

150

126

116

x

                            

DA2

76

141

144

157

149

167

127

176

261

269

193

167

151

2

x

                           

E3

136

189

170

86

154

180

144

204

244

253

172

182

171

148

180

x

                          

F344

132

165

178

178

152

150

138

144

170

176

141

156

166

121

165

168

x

                         

LEW

156

178

213

197

160

163

141

169

213

222

166

160

191

133

181

186

16

x

                        

LE

131

144

136

155

148

152

122

142

221

224

157

127

147

110

140

167

142

149

x

                       

LOU

145

146

165

191

153

203

120

178

242

250

138

171

212

111

142

192

126

150

149

x

                      

LUDW

153

175

186

198

161

169

153

195

252

263

183

177

189

123

163

215

115

125

133

161

x

                     

MWF

148

147

172

166

136

166

111

167

209

222

135

148

185

133

164

158

115

134

148

136

163

x

                    

MNS

151

167

178

173

158

186

122

169

239

250

155

194

210

128

176

166

123

132

156

141

159

137

x

                   

NAR

147

169

184

212

168

177

145

177

233

249

146

155

197

134

193

188

137

166

122

170

153

164

151

x

                  

OM

127

161

153

170

120

158

143

150

216

222

154

138

176

125

147

183

125

156

127

156

139

143

144

147

x

                 

PAR

140

182

168

166

149

158

133

175

225

227

155

138

167

136

159

169

128

136

127

151

159

133

160

150

162

x

                

PVG

95

164

152

153

153

181

129

184

252

263

170

151

164

110

150

148

142

175

145

143

196

146

161

172

160

147

x

               

R33

155

198

183

213

169

198

186

177

261

257

173

210

204

142

184

223

183

209

164

185

196

191

187

182

159

173

189

x

              

RP

146

159

171

171

132

164

109

186

216

230

113

161

176

138

175

153

119

141

134

141

166

108

157

136

147

132

139

166

x

             

SD

121

154

156

177

153

160

129

118

233

247

149

134

174

100

148

184

131

138

135

125

145

144

141

149

103

133

130

147

138

x

            

SD2

95

116

134

150

117

130

122

85

219

220

117

96

145

104

115

168

109

115

92

99

110

118

121

107

90

121

109

126

109

16

x

           

SHR

159

212

166

186

179

175

168

204

264

275

188

178

180

135

189

205

156

176

173

202

188

180

200

184

182

177

192

207

187

185

139

x

          

SR

129

171

172

170

160

161

147

138

235

244

163

131

170

120

172

174

146

169

156

157

175

150

160

164

136

117

134

166

160

60

64

184

x

         

SS

114

145

167

183

142

150

139

111

249

253

149

152

175

117

161

186

136

161

121

134

156

146

153

128

130

143

145

145

144

69

46

191

83

x

        

WAG

120

105

156

147

143

158

115

160

197

200

96

125

164

94

136

151

110

132

127

96

145

120

121

129

140

126

128

154

108

118

84

181

119

110

x

       

WC

140

164

158

155

129

180

92

188

214

232

151

177

177

125

158

157

131

160

156

145

171

87

157

179

152

158

138

213

126

153

122

195

170

157

126

x

      

WF

155

183

195

211

162

164

152

183

266

274

176

173

193

129

175

224

120

123

133

156

50

180

158

148

141

155

196

189

157

141

97

201

179

135

149

186

x

     

WIST

101

116

116

113

98

101

112

93

160

162

107

91

119

83

114

133

82

86

82

110

95

89

94

85

105

101

96

115

100

83

51

112

71

67

76

114

91

x

    

WKY

169

208

198

213

164

188

168

208

264

276

208

181

205

128

170

228

180

210

165

229

206

200

220

195

189

197

208

205

194

196

148

115

196

189

183

216

203

101

x

   

wild3

137

181

153

177

162

164

134

149

175

194

156

173

153

111

172

169

162

182

156

184

173

171

160

149

134

140

161

154

165

161

120

152

160

152

140

158

178

108

163

x

  

wild2

197

233

207

223

182

213

194

187

256

268

203

221

211

181

227

218

204

243

180

234

220

213

210

203

177

198

219

202

214

210

163

190

208

205

208

213

210

141

210

52

x

 

wild1

334

414

368

406

329

405

339

372

520

551

369

400

395

315

392

397

386

446

338

415

410

413

391

403

331

352

377

371

387

373

321

334

375

388

372

404

410

280

357

134

157

x

The matrix is built from genotyping data of 960 polymorphisms in 36 strains and three wild individuals. Two inbred strains are represented by two substrains (BN and DA) and outbred SD is represented by two individuals from different stocks. Sets of polymorphisms, including a graphical representation, can be retrieved from [24].

Simulation experiment wild rat-based SNP discovery

To get insight in the benefits of using wild rats in SNP discovery studies, we simulated larger scale experiments based on the results obtained in the experiments described above. Shotgun sequencing of 814 Kbp resulted in the identification of 485 SNPs. For 408 of those, the wild rat allele was also represented in laboratory rat strains and hence of interest for research purposes. The maximum amount of SNPs that can be discovered by fully sequencing this single rat is calculated by multiplying the SNP frequency (408/814,440) with the rat genome size (2,48 Gbp), which is 1,252,911 SNPs. Since none of our shotgun reads were overlapping, we can calculate the relation between shotgun sequencing reads of the wild rat and the amount of SNPs that will be found by scaling up this methodology, assuming random distribution of 400 bp shotgun reads over the genome (Fig. 3a). One million shotgun reads of a single wild rat would already result in the discovery of 200,000 novel SNPs that are polymorphic in commonly used rat strains. This simulation indicates that a relatively small sequencing effort could potentially result in a vast expansion of the amount of genetic variation for the rat.
Figure 3

a) Simulation of wild rat-based SNP discovery experiment. Simulation is based on the discovery of 485 SNPs between wild 1 and BN in 814 Kbp of shotgun sequence. For 408 of those, the wild rat alleles is found back in one or more inbred strains. The relation between generation of randomly distributed 400 bp shotgun reads and estimated number of newly discovered SNPs is plotted. b) Simulation of SNP discovery experiment, using carefully selected (most polymorphic compared to BN) rat strains (SHR, AUG, and WF) or all rat strains, in comparison with wild rat-based SNP discovery. Simulation is based on 539, 304, 292, 287, and 754 SNPs for wild 1, AUG, SHR, WF, and all strains respectively, in 119 Kbp of genotyped sequence.

Because shotgun sequencing was only done in the wild rat 1, we cannot make a direct comparison between wild rat-based SNP discovery and SNP discovery based on rat strains separately. However, a similar simulation experiment can be performed by treating the genotyping resequencing as shotgun reads. For wild 1, this would result in the identification of 577 SNPs as compared to the BN genome sequence. For 539 of those, the wild rat allele is found back in one of the inbred strains. For the combination of three strains most polymorphic as compared to BN in this experiment, the latter number would be 304, 292, and 287 for AUG, SHR, and WF, respectively. Simulations based on these numbers show that it requires nearly two times as much shotgun sequencing in different inbred strains separately to discover the same amount of SNPs that can be found using the wild rat shotgun sequencing approach. It should be mentioned that parallel shotgun sequencing of all 36 inbred strains until saturation has the potential to yield 1.6 times as many SNPs as compared to the wild-derived approach (Fig. 3b). An advantage of using inbred strains for SNP discovery is that the genotype of the strain is immediately known. Nevertheless, reconfirmation of the SNP or genotyping of other strains of interest may be necessary anyway, minimizing the relevance of this advantage.

Discussion

An increase in the amount of documented genetic variation for the rat will be essential to allow for high-resolution genetic mapping of the many inherited traits that have now been described for a wide variety of rat inbred strains. In addition, insight into genetic variation between rat strains provides valuable information on genetic relationships between strains, which can be instrumental to dissect the genetic basis of phenotypic differences. The wild rat-based shotgun sequencing method described here provides an efficient approach to generate such a dense map of genetic variation. To be able to benefit from haplotype-based mapping approaches [2528] a high marker density is needed to first reliably define haplotype blocks in strains of interest [29]. For the mouse, it has recently been announced that 15 inbred strains will be fully resequenced to achieve this goal [30]. With extreme dense genotype maps, it may even become possible to clone traits by haplotype-based in silico mapping [25], but to achieve this, it is estimated that complete sequences of over 50 strains are needed [29]. Although densities needed for these approaches are not reached, we do show here that wild rat-based SNP discovery is potentially much more effective than shotgun sequencing different inbred strains. We propose that the most effective SNP discovery strategy for the rat would be one based on shotgun sequencing of a single wild-derived sample and subsequent low-cost high-throughput genotyping of the resulting candidates in the laboratory strains of interest. Many other model organisms are currently undergoing full coverage sequencing and SNP discovery in these organisms will become increasingly important, especially for those organisms that are selectively bred for specific traits, such as cow and pig. Pilot experiments using for example wild-derived swine samples could be performed to test whether it is eligible to efficiently transfer the wild isolate-based SNP discovery strategy to other organisms.

Our results do provide insight in the genetic descent of the laboratory rat. It is generally accepted that current rat strains underwent two major genetic bottlenecks. First, they originate from a small founder population of domesticated wild rats and second, they were selectively inbred to obtain homogeneity [11]. The three Dutch wild rats used in this study are potentially relatively closely related as compared to wild rats from different parts of the world, but the genetic variation between them is mostly larger than or sporadically equal to any combination of three inbred strains, indeed suggesting the existence a common genetic bottleneck for laboratory strains. In addition, the laboratory rat does not show an extensive polymorphism rate in the MHC (major histocompatibilty complex) as compared to other species [31], like human, cattle etc. Cramer et al. has analyzed the MHC of wild rats and compared the data with those from inbred strains [32]. In line with our observation, there were not many new haplotypes.

We observed that wild rat genetic variation is to a large extent represented in the inbred strains, which is in sharp contrast to genetic variation in wild-derived mouse strains that is mostly unique [33]. Contrary to classical mouse inbred strains, where multiple subspecies contribute to the genetic make-up [13, 34] and recent mouse strains, derived from different Mus species [35], laboratory rat strains are most likely descending from a single rat species, Rattus norvegicus [10].

An independent study using 42 microsatellites in German and Japanese wild-derived samples showed that the genetic profiles were quite divergent, partially owing to different geographic locations [36]. Our study involved only Dutch wild rats, suggesting that the inclusion of wild rats from different parts of the world could result in even more efficient SNP discovery, although it also remains to be demonstrated what proportion of the additional discovered alleles is present in the inbred strains and if a geographic bias for this exists.

When multiple SNPs are present per locus/amplicon, independent haplotypes can be discerned. The genetic variation identified here is mostly organized in a limited amount of haplotypes per locus (Table 4). Theoretically, an amplicon containing two or three SNPs can be represented by four and eight haplotypes, respectively, but in our dataset the vast majority of amplicons harboring multiple SNPs is represented by only two or three haplotypes (Table 4). Again, these observations suggest the existence of a common and small founding population with very limited haplotype diversity and/or a very narrow genetic bottleneck before inbred strain selection. The observed small genetic basis in a wide selection of laboratory rat strains does not mimic genetic variation in the human population and as a result, studies and pharmacological tests in rat models neglect potential modulatory effects caused by genetic variation. Although the use of F1 crosses and mosaic populations [37] could address this issue, our data suggests that wild-derived rats may be very useful to this end, since a large amount of all genetic variation present in a large selection of inbred strains, is already represented in a limited number of individuals. Therefore, it would be very interesting to investigate genetic variation in recently domesticated inbred [38] and outbred rats such as wild-type Groningen rats (WTG) [39]. Alternatively, careful selection of inbred strains based on genotyping data and subsequent random breeding may also expose the wild side of laboratory rats.
Table 4

Haplotype analysis in 36 strains for all SNP-containing amplicons

number of haplotypes

 

2

3

4

5

6

7

8

9

10

11

12

 

2 SNPs

46

57

8

         

3 SNPs

11

26

8

3

0

0

0

     

4 SNPs

4

11

5

3

1

1

0

0

0

0

0

 

5 SNPs

1

3

3

2

0

0

0

0

0

0

0

 

6 SNPs

1

1

1

1

1

0

0

0

0

0

0

 

7 SNPs

1

0

1

0

2

0

0

0

0

0

0

 

8 SNPs

0

0

0

0

0

0

0

0

0

0

0

 

9 SNPs

0

0

0

0

0

0

0

0

0

0

1

 

10 SNPs

0

0

0

0

0

0

0

0

0

0

0

 

11 SNPs

0

0

0

0

1

0

0

0

0

0

0

 

total

64

97

27

9

5

1

0

0

0

0

1

204*

*) Total number of amplicons that contains at least two SNPs. Amplicons containing no SNPs or only indels were excluded from this analysis. Amplicons containing 1 SNP are also excluded, since two-state SNPs always give rise to two haplotypes.

Conclusion

We describe a SNP discovery platform for the rat that is based on two steps. First, candidate SNPs are discovered by shotgun sequencing a wild rat, followed by genotyping laboratory strains of interest. We show that 84% of alleles in wild rats as compared to the sequenced Brown Norway rat genome are also represented in a set of 36 laboratory strains. Hence, the approach described here would be an efficient strategy for the discovery of novel informative SNPs in the laboratory rat. Inclusion of other wild samples, preferably from different locations in the world could result in an even more effective SNP discovery platform, as the three wild rats in our study, caught in relative close vicinity to each other, were already more polymorphic than the most polymorphic combination of carefully selected inbred strains. Based on the more than 34,000 genotyping datapoints obtained in this study, we postulate two things. First, laboratory rats originate from a single rat species, and inbred stains are relatively closely related with a limited number of haplotypes, reflecting known genetic bottlenecks in strain establishment. Second, wild rats have the potential to represent the degrees of genetic variation as present in the human population much more efficiently than a random selection of inbred strains. This makes them or wild-derived strains potentially well-suited for studying modulatory effects of genetic background variation on specific phenotypes, such as behavior or responses to drug treatment.

Methods

Genomic DNA isolation, shotgun library construction

Wild rat 1 (Rattus norvegicus) was caught in the canals of Utrecht and kindly provided by the Pest Control Service of the City of Utrecht (Utrecht, The Netherlands). Wild rat 2 was trapped in Gassel, a village located approximately 100 km south-east of Utrecht and was kindly provided by Tien Derks (Gassel, The Netherlands). Wild rat 3 was caught in a basement in Amsterdam, located 50 km north of Utrecht and kindly provided Romke Koch (Amsterdam, The Netherlands). Rat strains BN/Crl and Crl:Wistar (outbred) were obtained from Charles River The Netherlands. Liver samples of commonly used rat strains ACI/Ztm, BDE/Ztm, BDII/Ztm, BDIX/Ztm, BDV/Ztm, BH/Ztm, BN/Ztm, BS/Ztm, DA/Ztm, E3/Ztm, F344/Ztm, LE/Ztm, LEW/Ztm, LOU/CZtm, MNS/Ztm, MWF/Ztm, NAR/Ztm, OM/Ztm, PAR/Ztm, R33/Ztm, WC/Ztm, WF/Ztm, WKY/Ztm were provided by D.W. (Hannover Medical School, Germany) and liver samples of strains AO/OlaHsd, AUG/OlaHsd, BUF/SimRijHsd, COP/Hsd, DA/OlaHsd, LUDW/OlaHsd, PVG/OlaHsd, RP/AEurRijHsd, SHR/NHsd, SR/JrHsd, SS/JrHsd, WAG/RijHsd and 2 individuals of Hsd:SD (outbred) were kindly provided by Harlan (Horst, The Netherlands). Samples were lysed overnight in 20 ml lysis buffer, containing 100 mM Tris (pH 8.5), 200 mM of NaCl, 0.2% of SDS, 5 mM of EDTA, and 100 μg/ml of freshly added Proteinase K at 55°C under continuous rotation. Tissue debris was spinned down for 20 min at 10,000 × g and supernatant was transferred to a fresh tube. DNA was purified by phenol-chloroform extraction and precipitated by adding an equal volume of isopropanol, mixing and centrifugation for 20 min, 10,000 × g at 4°C. The supernatant was removed by gently inverting the tube and the pellets were washed with 70% ethanol and dissolved in 1000 μl water. The concentration was measured by Optical Densitometry at 260 nm.

Wild rat-derived genomic library construction and shotgun sequencing

Sheared wild rat-derived genomic DNA of approximately 1–2 Kbp in size was cloned into the Sma I-site of pUC19. Fractions of the glycerol stock of the transformed library (E. coli DH10B) were plated on LB-plates containing 50 μg/ml ampicilin, 200 μg/ml IPTG, and 0.01% X-gal for standard blue/white screening on inserts. White colonies were picked in 20 μl water. Lysis occurred at 95°C for 10 min. 5 μl of 5× diluted lysate was used for the PCR reaction. For PCR, universal M13 primers were used, namely M13F: TGTAAAACGACGGCCAGT, M13R: AGGAAACAGCTATGACCAT. PCR, sequencing and cycling conditions were similar as for strain genotyping, described below. Sequencing was performed using universal M13 primers.

PCR conditions for strain genotyping

PCR was carried out using a touchdown thermocycling program (92°C for 60 sec; 12 cycles of 92°C for 20 sec, 65°C for 20 sec with a decrement of 0.6°C per cycle, 72°C for 30 sec; followed by 20 cycles of 92°C for 20 sec, 58°C for 20 sec and 72°C for 30 sec; 72°C for 180 sec; GeneAmp9700, Applied Biosystems) and contained 30–50 ng genomic DNA, 0.2 μM of each forward primer and 0.2 μM of each reverse primer, 400 μM of each dNTP, 25 mM Tricine, 7.0% Glycerol (w/v), 1.6% DMSO (w/v), 2 mM MgCl2, 85 mM Ammonium acetate pH 8.7 and 0.2 U Taq Polymerase in a total volume of 10 μl.

Sequencing reactions, purification, and analysis

PCR products were diluted with 25 μl water and 1 μl was directly used as template for the sequencing reactions. Sequencing reactions, containing 0.25 μl BigDYE (v3.1; Applied Biosystems, Foster City, CA, USA), 3.75 μl 2.5× dilution buffer (Applied Biosystems) and 0.4 μM universal M13 primer in a total volume of 10 μl, were performed using cycling conditions recommended by the manufacturer (40 cycles of 92°C for 10 sec, 50°C for 5 sec and 60°C for 120 sec). Of sequencing products, 5 μl was purified by ethanol precipitation in the presence of 40 mM sodium-acetate and analyzed on 96-capillary 3730XL DNA analyzers (Applied Biosystems), using the standard RapidSeq protocol. Sequences were analyzed for presence of heterozygous mutations using PolyPhred [40], followed by manual inspection of the polymorphic positions.

Automation

All PCR and sequencing reactions were set up on a Tecan Genesis RSP200 liquid handling workstation, with a robotic and an 8-channel pipetting arm, an integrated 96-channel pipetting head (TEMO96, Tecan), and four integrated dual-384 well PCR blocks (Applied Biosystems).

Mapping of shotgun reads and SNP discovery

Shotgun reads were assigned to positions in the RGSC 3.1 rat genome assembly using blat search [15]. Shotgun reads that complied with our mapping criteria, namely those having at least 80 identical bp for the best hit and no more than 60 identical bp for second blat hit were retained for further analysis. Blast nucleotide sequence alignments between shotgun read and corresponding genomic segment were used for discovery of single base variations (including single base indels). A site was treated as polymorphic only in the case when it has identical 5'- and 3'-flanks of at least 5 bp. A custom designed web-application was employed for manual chromatogram inspection and confirmation of a correct shotgun base-call for every polymorphic SNP locus. Primer design for resequencing was performed using a local web-interface [41] to the PRIMER3 program [42].

Simulation model for wild rat-based SNP discovery

To estimate the number of SNPs to be discovered by the wild rat resequencing approach we performed computer simulations using the observed sample-specific polymorphism frequencies and the rat genome size of 2.48 Gbp as an input. We used a Monte-Carlo method for the placement of N 400-bp shotgun reads to the genome and calculated the total size of genome covered by N shotgun reads. To obtain a conservative estimate by assuming low heterozygosity in wild-derived strain the estimate of number of SNPs is given by product of covered genome size and polymorphism rate.

Declarations

Acknowledgements

We thank Harlan (Horst – Netherlands), the Pest Control Service of the City of Utrecht (Utrecht, The Netherlands), Romke Koch (Amsterdam, The Netherlands), and Tien Derks (Gassel, The Netherlands) for kindly providing rat tissue samples. This work was supported by the Dutch Ministry of Economic Affairs through the Innovation Oriented Research Program on Genomics.

Authors’ Affiliations

(1)
Hubrecht Laboratory, Functional Genomics Group
(2)
Institute for Laboratory Animal Science, Hannover Medical School

References

  1. Kristensen VN, Kelefiotis D, Kristensen T, Borresen-Dale AL: High-throughput methods for detection of genetic variation. Biotechniques. 2001, 30 (2): 318-322.PubMedGoogle Scholar
  2. Sellick GS, Longman C, Tolmie J, Newbury-Ecob R, Geenhalgh L, Hughes S, Whiteford M, Garrett C, Houlston RS: Genomewide linkage searches for Mendelian disease loci can be efficiently conducted using high-density SNP genotyping arrays. Nucleic Acids Res. 2004, 32 (20): e164-10.1093/nar/gnh163.PubMedPubMed CentralView ArticleGoogle Scholar
  3. Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, Scherer S, Scott G, Steffen D, Worley KC, Burch PE, al. : Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature. 2004, 428 (6982): 493-521. 10.1038/nature02426.PubMedView ArticleGoogle Scholar
  4. Greenhouse DD, Festing MFW, Hasan S, Cohen AL: Catalogue of Inbred Strains of Rats. Genetic Monitoring of Inbred Strains of Rats. Edited by: Hedrich HJ. 1990, Stuttgard , Gustav Fisher, 410-480.Google Scholar
  5. Hedrich HJ: Taxonomy and stocks and strains. The Laboratory Rat. 2005, Academic Press, 71-91, in press.Google Scholar
  6. NCBI dbSNP. [http://www.ncbi.nlm.nih.gov/SNP/]
  7. Zimdahl H, Nyakatura G, Brandt P, Schulz H, Hummel O, Fartmann B, Brett D, Droege M, Monti J, Lee YA, Sun Y, Zhao S, Winter EE, Ponting CP, Chen Y, Kasprzyk A, Birney E, Ganten D, Hubner N: A SNP map of the rat genome generated from cDNA sequences. Science. 2004, 303 (5659): 807-10.1126/science.1092427.PubMedView ArticleGoogle Scholar
  8. Smits BM, van Zutphen BF, Plasterk RH, Cuppen E: Genetic variation in coding regions between and within commonly used inbred rat strains. Genome Res. 2004, 14 (7): 1285-1290. 10.1101/gr.2155004.PubMedPubMed CentralView ArticleGoogle Scholar
  9. Guryev V, Berezikov E, Malik R, Plasterk RH, Cuppen E: Single nucleotide polymorphisms associated with rat expressed sequences. Genome Res. 2004, 14 (7): 1438-1443. 10.1101/gr.2154304.PubMedPubMed CentralView ArticleGoogle Scholar
  10. Lindsey JR: Historical Foundations. The Laboratory Rat. Edited by: Baker HJ, Lindsey JR, Weisbroth SH. 1979, New York , Academic Press, 1: 1-36.View ArticleGoogle Scholar
  11. Hedrich HJ: History, Strains and Models. The laboratory Rat. Edited by: Krinke GJ. 2000, London , Academic PressGoogle Scholar
  12. Koch LG, Britton SL: Strains. The Behavior of the Laboratory Rat. Edited by: Whishaw IQ, Kolb B. 2005, Oxford, UK , Oxford University PressGoogle Scholar
  13. Ideraabdullah FY, de la Casa-Esperon E, Bell TA, Detwiler DA, Magnuson T, Sapienza C, de Villena FP: Genetic and haplotype diversity among wild-derived mouse inbred strains. Genome Res. 2004, 14 (10A): 1880-1887. 10.1101/gr.2519704.PubMedPubMed CentralView ArticleGoogle Scholar
  14. Yalcin B, Fullerton J, Miller S, Keays DA, Brady S, Bhomra A, Jefferson A, Volpi E, Copley RR, Flint J, Mott R: Unexpected complexity in the haplotypes of commonly used inbred strains of laboratory mice. Proc Natl Acad Sci U S A. 2004, 101 (26): 9734-9739. 10.1073/pnas.0401189101.PubMedPubMed CentralView ArticleGoogle Scholar
  15. Kent WJ: BLAT--the BLAST-like alignment tool. Genome Res. 2002, 12 (4): 656-664. 10.1101/gr.229202. Article published online before March 2002.PubMedPubMed CentralView ArticleGoogle Scholar
  16. Canzian F: Phylogenetics of the laboratory rat Rattus norvegicus. Genome Res. 1997, 7 (3): 262-267.PubMedView ArticleGoogle Scholar
  17. Thomas MA, Chen CF, Jensen-Seaman MI, Tonellato PJ, Twigger SN: Phylogenetics of rat inbred strains. Mamm Genome. 2003, 14 (1): 61-64. 10.1007/s00335-002-2204-5.PubMedView ArticleGoogle Scholar
  18. Bandelt HJ, Forster P, Rohl A: Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol. 1999, 16 (1): 37-48.PubMedView ArticleGoogle Scholar
  19. Homepage Fluxus Engineering. [http://fluxus-engineering.com]
  20. Rat Genome Database (RGD) strain information. [http://rgd.mcw.edu/strains]
  21. Rapp JP: Genetic analysis of inherited hypertension in the rat. Physiol Rev. 2000, 80 (1): 135-172.PubMedGoogle Scholar
  22. Kwitek AE, Gullings-Handley J, Yu J, Carlos DC, Orlebeke K, Nie J, Eckert J, Lemke A, Andrae JW, Bromberg S, al. : High-density rat radiation hybrid maps containing over 24,000 SSLPs, genes, and ESTs provide a direct link to the rat genome sequence. Genome Res. 2004, 14 (4): 750-757. 10.1101/gr.1968704.PubMedPubMed CentralView ArticleGoogle Scholar
  23. Steen RG, Kwitek-Black AE, Glenn C, Gullings-Handley J, Van Etten W, Atkinson OS, Appel D, Twigger S, Muir M, Mull T, al. : A high-density integrated genetic linkage and radiation hybrid map of the laboratory rat. Genome Res. 1999, 9 (6): AP1-8.PubMedGoogle Scholar
  24. CASCAD SNPview. [http://cascad.niob.knaw.nl/snpview]
  25. Grupe A, Germer S, Usuka J, Aud D, Belknap JK, Klein RF, Ahluwalia MK, Higuchi R, Peltz G: In silico mapping of complex disease-related traits in mice. Science. 2001, 292 (5523): 1915-1918. 10.1126/science.1058889.PubMedView ArticleGoogle Scholar
  26. Liao G, Wang J, Guo J, Allard J, Cheng J, Ng A, Shafer S, Puech A, McPherson JD, Foernzler D, Peltz G, Usuka J: In silico genetics: identification of a functional element regulating H2-Ealpha gene expression. Science. 2004, 306 (5696): 690-695. 10.1126/science.1100636.PubMedView ArticleGoogle Scholar
  27. Pletcher MT, McClurg P, Batalov S, Su AI, Barnes SW, Lagler E, Korstanje R, Wang X, Nusskern D, Bogue MA, Mural RJ, Paigen B, Wiltshire T: Use of a dense single nucleotide polymorphism map for in silico mapping in the mouse. PLoS Biol. 2004, 2 (12): e393-10.1371/journal.pbio.0020393.PubMedPubMed CentralView ArticleGoogle Scholar
  28. Wang X, Korstanje R, Higgins D, Paigen B: Haplotype analysis in multiple crosses to identify a QTL gene. Genome Res. 2004, 14 (9): 1767-1772. 10.1101/gr.2668204.PubMedPubMed CentralView ArticleGoogle Scholar
  29. Flint J, Valdar W, Shifman S, Mott R: Strategies for mapping and cloning quantitative trait genes in rodents. Nat Rev Genet. 2005, 6 (4): 271-286. 10.1038/nrg1576.PubMedView ArticleGoogle Scholar
  30. Pearson H: Mouse sequencing plan aims to boost models. Nature. 2004, 432 (7013): 5-10.1038/432005a.PubMedView ArticleGoogle Scholar
  31. Gunther E, Walter L: The major histocompatibility complex of the rat (Rattus norvegicus). Immunogenetics. 2001, 53 (7): 520-542. 10.1007/s002510100361.PubMedView ArticleGoogle Scholar
  32. Cramer DV, Chakravarti A, Arenas O, Humprieres J, Mowery PA: Genetic diversity within and between natural populations of Rattus norvegicus. J Hered. 1988, 79 (5): 319-324.PubMedGoogle Scholar
  33. Campino S, Behrschmidt C, Bagot S, Guenet JL, Cazenave PA, Holmberg D, Penha-Goncalves C: Unique genetic variation revealed by a microsatellite polymorphism survey in ten wild-derived inbred strains. Genomics. 2002, 79 (5): 618-620. 10.1006/geno.2002.6570.PubMedView ArticleGoogle Scholar
  34. Abe K, Noguchi H, Tagawa K, Yuzuriha M, Toyoda A, Kojima T, Ezawa K, Saitou N, Hattori M, Sakaki Y, Moriwaki K, Shiroishi T: Contribution of Asian mouse subspecies Mus musculus molossinus to genomic constitution of strain C57BL/6J, as defined by BAC-end sequence-SNP analysis. Genome Res. 2004, 14 (12): 2439-2447. 10.1101/gr.2899304.PubMedPubMed CentralView ArticleGoogle Scholar
  35. Guenet JL, Bonhomme F: Wild mice: an ever-increasing contribution to a popular mammalian model. Trends Genet. 2003, 19 (1): 24-31. 10.1016/S0168-9525(02)00007-0.PubMedView ArticleGoogle Scholar
  36. Voigt B, Kitada K, Kloting I, Serikawa T: Genetic comparison between laboratory rats and Japanese and German wild rats. Mamm Genome. 2000, 11 (9): 789-790. 10.1007/s003350010137.PubMedView ArticleGoogle Scholar
  37. Cholnoky E, Fischer J, Varga M, Gyorffy G: Aspects of genetically defined populations in toxicity testing. II. Genotypic differences in sensitivity to a toxic dextran preparation. Z Versuchstierkd. 1974, 16 (1): 43-48.PubMedGoogle Scholar
  38. Ohno K, Niwa Y, Kato S, Kondo K, Oda S, Inouye M, Yamamura H: Establishment of new inbred strains derived from Japanese wild rats (Rattus norvegicus). Jikken Dobutsu. 1994, 43 (2): 251-255.PubMedGoogle Scholar
  39. de Boer SF, Lesourd M, Mocaer E, Koolhaas JM: Selective antiaggressive effects of alnespirone in resident-intruder test are mediated via 5-hydroxytryptamine1A receptors: A comparative pharmacological study with 8-hydroxy-2-dipropylaminotetralin, ipsapirone, buspirone, eltoprazine, and WAY-100635. J Pharmacol Exp Ther. 1999, 288 (3): 1125-1133.PubMedGoogle Scholar
  40. Nickerson DA, Tobe VO, Taylor SL: PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res. 1997, 25 (14): 2745-2751. 10.1093/nar/25.14.2745.PubMedPubMed CentralView ArticleGoogle Scholar
  41. Local PRIMER3 interface. [http://primers.niob.knaw.nl]
  42. Rozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000, 132: 365-386.PubMedGoogle Scholar

Copyright

© Smits et al; licensee BioMed Central Ltd. 2005

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement