Identification of 129S6/SvEvTac-Specific Polymorphisms on Mouse Chromosome 11

Abstract

Polymorphisms such as single-nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) can be associated with phenotypic traits and be used as markers for disease diagnosis. Identification of these genetic variations within laboratory mice is crucial to improve our understanding of the genetic background of the mice used for research. As part of a positional cloning project, we sequenced six genes (Mettl16, Evi2a, Psmd11, Cct6d, Rffl, and Ap2b1) within a 6.8-Mb domain of mmu chr 11 in the C57BL/6J and 129S6/SvEvTac inbred strains. Although 129S6/SvEvTac is widely used in the mouse community, there is very little current (or projected future) sequence information available for this strain. We identified 6 Indels and 21 novel SNPs and confirmed genotype information for 114 additional SNPs in these 6 genes. Mettl16 and Ap2b1 contained the largest numbers of variants between the C57BL/6J and 129S6/SvEvTac strains. In addition, we found five new SNPs between 129S6/SvEvTac and 129S1/SvImJ within the Ap2b1 locus. Although we did not detect differences between C57BL/6J and 129S6/SvEvTac within Evi2a, this locus contains a relatively high SNP density compared with the surrounding sequence. Our study highlights the genetic differences among three inbred mouse strains (C57BL/6J, 129S6/SvEvTac, and 129S1/SvImJ) and provides valuable sequence information that can be used to track alleles in genomics-based studies.

Introduction

T he laboratory mouse is the major animal model used for human disease research because of its high relevance to human biology and striking genetic similarities. Understanding the polymorphisms in the laboratory mouse will provide insights into the evolutionary history of the species and increase knowledge of the relationship between genotypic and phenotypic differences (Guenet, 2005). Polymorphisms are genetic variants that arise from spontaneous mutations and by random genetic drift that occurs with a population frequency greater than 1%, eventually reaching a steady state in a population (Schork et al., 2000). Several types of polymorphisms are found within mammalian genomes, including single-nucleotide polymorphisms (SNPs), insertions and deletions (Indels), variable number tandem repeats, copy number variants, and microsatellite repeats.

Different types of polymorphisms are generally created by different kinds of alteration events. For example, SNPs, the most common types of polymorphisms, result from a single base substitution. The most common SNP in the mammalian genome is a C to T transition, whereby a methylated cytosine is spontaneously deaminated to form a thymine base (Miller et al., 2001). As this change is not recognized by DNA repair enzymes, the SNP segregates within the population unless it is under selection, is lost, or by chance randomly fixes. SNP variation in protein-coding genes and in other functionally constrained regions of the genome, such as promoters, enhancers, miRNAs, and other noncoding sequences, contributes significantly to phenotypic variation (Morin, 2004; Sethupathy et al., 2007; Bartel, 2009).

Indels are often, but not always, present in highly repetitive sequences (Chen et al., 2009). They are abundantly distributed across the genome, but not as common as SNPs (Vali et al., 2008). Both of these types of genetic variations can be used as tools in genotyping as well as discovery of disease- or trait-related genes by directly sequencing SNPs or identifying Indels based on size separation or sequence.

Many mouse polymorphism databases are available, such as the Mouse Genome Informatics (MGI) strains, SNPs and Polymorphisms database (www.informatics.jax.org/strains_SNPs.shtml) (Blake et al., 2011), and the Mouse Phenome Database (MPD) SNP collection (http://phenome.jax.org/SNP/) (Bogue et al., 2009). The MGI database provides reference SNP and Indel information as well as the corresponding genotyping assays. SNP queries can be based on strain, map position, marker range, or associated genes. Currently, there are data from 86 mouse strains, but many holes exist in these databases and more assays are needed to fill in the blanks.

While conducting a positional cloning project to identify induced variants from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we detected several 129S6/SvEvTac polymorphisms in potential candidate genes. This ENU mutagenesis screen was performed on C57BL/6J males, which were bred to 129S6/SvEvTac animals carrying an inversion chromosome for the targeted region (Kile et al., 2003; Hentges et al., 2006). The mouse reference sequence is based on C57BL/6J, which is one of the most widely used inbred mouse strains for the generation and analysis of transgenic mice and disease models (Al-Hasani et al., 2004; Tang et al., 2008). 129S6/SvEvTac and 129S1/SvImJ have been used frequently for gene knockout studies (Hibma et al., 2007; Kuo et al., 2010). 129S1/ImJ is one of the 17 additional mouse strains being sequenced by the Sanger Center (Turner et al., 2009). However, there is very little polymorphism data for 129S6/SvEvTac. As a result, it is important to develop SNP and other polymorphism detection assays to help identify 129S6/SvEvTac alleles to facilitate genotyping. Here we provide SNP and Indel information for six genes located on chromosome 11 between these three commonly used laboratory mouse strains.

Materials and Methods

We isolated genomic DNA from mouse tail biopsies from 129S6/SvEvTac and C57BL/6J animals; 129S1/SvImJ DNA was purchased from the Jackson Laboratory. Exons from each gene were sequenced individually. Each 600–800-bp polymerase chain reaction (PCR) amplicon contained one or more exons plus upstream and downstream flanking sequence. Exons larger than 800 bp were sequenced by multiple overlapping sequencing reactions. All amplicons were sequenced in both directions using Big Dye v3.1 chemistry (Applied Biosystems, Foster City, CA). Primers are listed in Table 1.

Table 1.

Primers Used for This Study

Gene-exon	Forward sequence (5′-3′)	Reverse sequence (5′-3′)	Annealing temperature (°C)	Amplicon size
Mettl16-E1F	TTCTCCATCCGCTAACCTAAGAAG	TCAACAGATGTCTCCCACCTAGAC	60	624
Mettl16-E2F	GGCACTAAATATGCTGGTTTCCTC	AATCACACAAAGCACAGTCCTAGC	60	468
Mettl16-E3F	GAACGCTGTACCATGGAGCTATAC	ACTTCTCAATGATACCTGCAGCC	60	1081
Mettl16-E4F	GTGTATCTGCTGTTGTGCTGAATG	TTCTTCCTTCCACGCTTCTCTAAC	60	553
Mettl16-E5/6F	CTCTGAGTTATGTGTGAAATGGGC	CCCAAATCTCAGGGCAATCTATAC	60	731
Mettl16-E7F	TCCAACTCTCCTTCATGGTTTATG	CTGAACAACTTGTGCTAACACTGC	60	616
Mettl16-E8/9F	GATAAACTTGATGCGTCTCCATTG	CTCCGGAAGCCTTAATAACTGAAC	60	504
Mettl16-E10F	ACCATGGGCTAATCCAAGTTAATG	CAGAACTAGGTGGAGGTCTTCGAG	60	490
Mettl16-E11F	CTCCCACTGGCTGTGTGTTC	CTGAGATGGCTGGTAGTCTGTTTC	60	599
Mettl16-E12F	GGAAATTTGAAGAGCTGGGTATTG	TTCTAACCTATTTGGGAGGTGGC	60	577
Mettl16-E13F	CCTATACTGCAGTTGGAAATACGC	ACATGGTATACACACATATACACAGGC	60	425
Mettl16-E14F	CTCTACATGCCTTCAGGGTCTATG	AAGCTGCTATACCCAAATTTCGAC	60	928
Mettl16-E15F	AAAGAGCAACAGGAGTTAGCAGC	GGTCTACCGAAGCTTATCACACAA	60	353
Mettl16-E16F	TCAATAAAGGCCTGAGATTCCTTC	CAACAGCAGTGGGCTTAGTGTTAG	60	552
Evi2aE1F	CACTAAAGATGCCATCATGTAATTTCC	TCAAGACCTTCCTCATCCTGATTT	62	626
Evi2aE2aF	CCCTGACTGTCTCAGCCTTCTAAT	TTAACAGGCTGCACTTGAGATAGC	62	866
Evi2aE2bF	CCATGTTCTCCTTTCCTTCAAGAT	GGTGTTCTCCTCACAGACTTCCTT	62	825
Evi2aE2cF	GCCTCAGGTTCCAGTAATCAGAAT	ATTCAGACAGAGGTCATTCACTGC	62	845
Evi2aE2dF	AGAGAAAGCACGAAGAAGGAACTG	GTGTATGCCTAGTTCCCAAGGAAG	62	887
Pmsd11E1F	ACGGGGCAGAGAGACTACAACT	ACAGTGAGTCAGCAGCACCATAG	62	342
Pmsd11E2F	TTCATTTTCTTTTCTTAGTGAAACGTG	AAAACAGGACTTTCCCTCACCTAT	62	320
Pmsd11E3F	CCATATGCTTGTGTGGAGCTTAAA	CATCCTTCCACACACTACCACAGT	62	401
Pmsd11E4F	CTATGGACTGTGTGAACAACATGG	ACACTCCCATAACTGGAAGCACTA	62	314
Pmsd11E5F	TCAGTTGTTGTGTTGAACTTGATGT	AGCAGGACTTTACCGTGACAAAT	62	375
Pmsd11E6F	GGGCTGGGTATGACAGCTCA	GAGGTGAGCAGTAAGAAAGCATGTC	62	400
Pmsd11E7F	TAGGAACTGGCTTGTGACTTGTG	CTCTGACCACCACACTCTGTATCA	62	450
Pmsd11E8F	TTTTGTTTTGCAGCCCAGAAG	ATTAAGGGAGGAAAACAGGGATG	62	200
Pmsd11E9F	TGATGGTGAGGACAGGCTGTTTAT	CCTCGAGCTCAGGGATCTGC	62	311
Pmsd11E10F	AGCTTGGTCATGAGAGAGTTTCCT	GGCCTCTTACCAGACAGACATACC	62	430
Pmsd11E11/12F	AGGCAGAGCAGAGGACAGTAAGAT	CATCTCTCTCCACTCCCTTACACA	62	574
Pmsd11E13F	TAAATAGAAAGGCGTGCTGTGAAA	CCAACTCTGCAAGAGAAGACAGAG	62	376
Pmsd11E14F	AGAAGCTGACATAGGTGAGTGCTG	CAATTCTGAAGAGGAGGAGAGGTG	62	500
Cct6bE1F	TCAGTTCTTCCTGATTCACCACTC	CGCACAAGGCTAACAGTTTCTATT	62	545
Cct6bE2F	TAAGGTCATCCGGGAGAACTAATC	GCACTGCATCTAATACCTGAATCC	62	415
Cct6bE3F	GAAGCAAAGACTTTCATACAATGTTCA	ACGGGAGCTTTCTCTTTAACCTTT	62	552
Cct6bE4F	ACACATTTATGTTAGTGCCGTTGC	TGAATTGTAAATGCTTGCCTTGTT	62	689
Cct6bE5F	TATATTGGTGTTCCTCAGCTCTGC	AGGGAGATCTTCAAGTCAGCATTT	62	587
Cct6bE6F	TTTGAGCCGTGTAACTTAGAGAATAA	ACTCATGCACATACACACTCACAA	62	440
Cct6bE7F	TTACAAATACGAGCCATCATGTCC	CTTACAAATGTCCACACAGTGCAA	62	549
Cct6bE8F	GACTTTAGACCTCCCTTGCTTCAG	CCCTGACTGTCCTAGAACTCACAA	62	552
Cct6bE9F	ATAACCAAAGTGAGCATGGCATAA	AAACTGTTCTTCATGGAAACACTCAC	62	158
Cct6bE10F	TTGAACTGCCTTTGTAACTCAACC	GGCAGCCTACCTTATCTCTCCTCT	60	621
Cct6bE11F	GAAGAGGGCATTACATCCCATTAC	CAAGTCCTTCATTTCCCACAAGTA	62	658
Cct6bE12F	TAGGTTGGGAATGGGATTCTTCTA	CTTGATGGTAATGGACTGAACCTG	62	469
Cct6bE13F	GGAGAAAGGAGCCAATAGAGAAAG	TACACAGAGGCTGCTGTTACTCCT	60	574
Cct6bE14F	GGTGAGGGTGCTTCCTTGATAATA	AAGCTGGAATTGACTGGGATACTC	62	535
RfflE1F	CCACACCTGTCCCATGAAAGT	GGCCCTACTTCCCTCTGAGTCT	62	362
RfflE2F	TGGTACTAACAGATTATCACTAACTTGC	AGGTGTGTGCTTTAGCTACTCG	62	393
RfflE3F	CGATATTTGGAACACTCACTCTGG	GGGTCTATGCTGCTGTCTGAGTT	62	529
RfflE4F	CTGTGTGTATCATCTCTGGCTGTG	ACTGGACTCAAGGACTTCAGGACT	62	412
RfflE5F	CTGTGTGTATCATCTCTGGCTGTG	ACTGGACTCAAGGACTTCAGGACT	62	412
RfflE6F	GTGTGTGGCTGTCTGCATAAAGTA	AACTGTGTATTCAATGCCTGTTCC	62	484
RfflE7pcrF	CTCCACATGACTTCTCTGAACACA	GCAAGGGCAATACCTGACTCTACT	62	332
RfflE8aF	TGATCTGAGAGTACCAGACTCAAACC	GGATCTTAGGACAGGAGAGACTGG	62	663
RfflE8bpcrF	ACGCATGAACGAATGTCCTATCT	GATCCAAACTCTGACCTGAGGAAT	62	586
RfflE8cpcrF	CCAGTCTCTCCTGTCCTAAGATCC	AGGACCAGAAGACAAAGACAAAGC	62	488
RfflE8dpcrF	GGCCAATAGTATTCCTCAGGTCAG	TTCTCAGCTGTGTCAGTGGTGTAA	62	569
Ap2b1-E1/2	GGGCTTCTTAACCTTACAAGCAAA	ATTTACAGCCTTGCCCAAACTAGA	62	1144
Ap2b1-E3	CCTTGAAGCAAGTTGCAGTTCTTA	CCTTAGTTTGTAAGCCCACACTCA	62	570
Ap2b1-E4	TAAAGTAGGGTCTGTGTGGCCTTT	TCTCAAGACATTGGTGCTATGTTG	60	500
Ap2b1-E5	GGAAGCCAACACTGTCATCTGTAT	ACAGCATGTCCACTCTCTATCAGC	62	527
Ap2b1-E6	AAAGAATGCCATGAGAGATTGGAT	TACCCTGTAGTCGTGTCTCCAAGA	62	479
Ap2b1-E7	CTTGGAAGCTGTCTATGCTGTCAT	AATACGCTAGGGATAGCAATGTCC	62	513
Ap2b1-E8F	CAAGAAGGAAACTGGTGTGAGTGT	AGGGCACCAGTCTATAAAGCAATC	62	590
Ap2b1-E9/10F	CAGGCAGAAAGACTGACTTGATTT	CTTCTGGCCTACACAGGTAGTTGA	62	810
Ap2b1-E11F	TAGCAGAGTCAGTGCCTGTCTGA	TTCAAAGTGCCTTACTTATACGTTTCC	62	292
Ap2b1-E12F	CTTGGAATCCCTTGTGTATGGTTT	TAAGCGCTGCAGCTTTGTTATCTA	62	429
Ap2b1-E13AF	GCATGGTCAATGATTGCTTTGTA	CTGCAACAGATCAAGAAGAGGTGT	62	950
Ap2b1-E13BF	TTCTCAAGAAGCCATCAGAAACAC	AGTCACCCACAGATACCAGATTCA	62	992
Ap2b1-E13CF	GCCTCAAAGCAGATTCTGTTCAAT	TTGAGATTTCAGGCCTGTGCTA	62	1000
Ap2b1-E13DF	ATACTCTGACAAGCGACTTCAGGA	TGAGAGCTCAGAACTGGCAAA	62	945
Ap2b1-E13D2F	CCTTTCATAACAGGTCACAACCAG	TGAGAGCTCAGAACTGGCAAA	62	558
Ap2b1-E14R	CCCTGCCCATATCAATCTATGTTT	AAAGAAGCTCTTCAGAGCAGCAAG	62	596
Ap2b1-E15R	TCTCCTTCCATGTGTAGATTCTGG	CGGCCAACATGATAAACCTATACA	62	564
Ap2b1-E16R	TTCACTCCCACTGCTCAGTTC	AGTCAAGACTGGAGGTTCCATTTC	62	507
Ap2b1-E17R	GACAAAGCCTCTAAAGCACGTGTA	ACGAGGCCCAAACTCAAATAGTTA	62	540
Ap2b1-E18R	AAAGAAGCCAATTGCTCATAGACTTT	TTTGTCCTGTTCTCCAGAGTCAAG	62	541
Ap2b1-E19R	TAGATTGTGTTGGAGCAGGTCAA	CTACCACAATTCTCCGCATGATAC	62	504
Ap2b1-E20R	ACCTACAGACTGAGGATCATTGG	TGAGATTTGAGGTGGCAGAGTAAG	62	527
Ap2b1-E21R	CTCTTAACTGCATGCTTTCTTCCA	TAGGGCTGTGCCTTAGAGAGATTT	62	427
Ap2b1-E22R	CTGCCTGTATCAGAAGAAGCTCTG	GGGAAACCTATAGGGTGGAGACTT	58	1187
Ap2b1-E22AR	AGTCCTGTGTTGCAATCATGAAAT	GAGGCAGGGTCTCAACTATGAAGT	62	287
Ap2b1-E23AR	GCCACACAAGCTGTAGGTAGACAT	AAGACTGTGTTTGATGAGGGTCAG	60	756
Ap2b1-E23BR	TCTTGTGCTGACATTAGCATTCAC	ACCAGTCTCAGCTCTGCTACTCAA	62	853
Ap2b1-E23CR	GAACACTGAGTAGAAAGGGCGACT	CAGTGAGCTGACAGACAGTGAGAA	62	833
Ap2b1-E23DR	TGAGTAGCAGAGCTGAGACTGGTT	GAACTTGGTCAACTTGTCTTTCCA	62	804
Ap2b1-E23ER	TGTGAACACATTGGTGTGAGTCAT	CCAGCTATGTATTGTCCTGGGAAC	62	1100

We analyzed the data using Sequencher 4.9 (Gene Codes, Ann Arbor, MI) to identify polymorphisms among the various strains used in this study. The sequences from different samples were assembled automatically and then entered into a BLAT search against the UCSC genome database (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start) (Kent, 2002) (NCBI Build 37 (Mm11_95772_37)) to obtain the cDNA and genomic DNA sequences. All sequences (including cDNA and genomic regions) were assembled with our sequencing data into a contig that spanned one individual exon. The contig was scanned for any polymorphisms using features of the Sequencher program. Once a variation was identified, we checked the chromatogram manually to distinguish true polymorphisms from sequencing errors. All SNPs and other polymorphisms were compared with SNP information from the MGI database (dbSNP Build 128) to pinpoint SNPs within the contig and identify unique polymorphisms. Exon or intron positions were determined using the UCSC genome database (http://genome.ucsc.edu/) (Fujita et al., 2011). We compiled all polymorphisms into tables containing gene location, nucleotide number, local sequence, and existing SNP ID using the C57BL/6J as the reference strain.

Results

We analyzed Mettl16, Evi2a, Psmd11, Cct6d, Rffl, and Ap2b1 on mouse chromosome 11. Chromatographs for representative SNPs within each locus are shown (Fig. 1). We only detected differences between C57BL/6J and 129S6/SvEvTac in Mettl16 and Ap2b1. However, we summarized SNP information for sequenced genes.

FIG. 1.

Representative chromatographs for Mettl16 (A), Evi2a (B), Psmd11 (C), Cct6b (D), Rffl (E), and Ap2b1 (F). The SNP in each sequence is marked by a black box. We detected no polymorphisms among 129S6/SvEvTac, 129S1/SvImJ, and C57BL/6J in Evi2a, Psmd11, Cct6b, or Rffl. These sites contain known polymorphisms within different inbred mouse strains. SNP, single-nucleotide polymorphism.

The RefSeq annotated transcript for Methyltransferase like 16 (Mettl16), the most proximal gene in the group, is expressed from the Watson strand and contains 10 exons; 4 additional exons are predicted based on Ensembl annotations. We sequenced all 14 exons and identified 18 variants (Table 2). Although C57BL/6J is the mouse reference sequence, the dbSNP database lacks significant C57BL/6J information. Gray lettering denotes the variant, gray boxes indicate new dbSNP information, and bold lettering indicates novel sequence variants between C57BL/6J and 129S6/SvEvTac; all new variants have been deposited into the dbSNP database.

Table 2.

Mettl16 Polymorphisms

Column 1 denotes the relative number based on chromosome position and column 2 indicates the actual nucleotide that is changed based on NCBI Build 37 at the UCSC and Ensembl genome browsers. Location of the variant within the locus (i.e., upstream, 5′ and 3′ UTR, exonic and intronic alleles) is indicated in column 3, for example, intron 8 is denoted I8. We provide upstream and downstream sequence information in column 4, and columns 5 and 6 summarize sequencing data from this study. dbSNP ID number is presented in column 7, and dbSNP genotyping data are shown in columns 8–10; variants without dbSNP IDs are novel alleles. Gray shade indicates no data available in current database. Dark shading indicates distinction between C57BL/6J and 129S6/SvEvTac. Boldface indicates new SNP or indel. Gray letters indicate new information for 129S1/SvlmJ. Gray-colored sequence denotes the variant.

SNP, single nucleotide polymorphism; indel, insertions/deletions polymorphisms; E, Exon; I, Intron; US, upstream; DS, downstream; 3′UTR, 3′ untranslated region; 5′UTR, 5′ untranslated region S, silent variant; M, missense change.

We identified two new SNPs (10 and 18) and Indels (11 and 14) within Mettl16. SNP 10 is a T to G transversion in intron 5, whereas SNP 18 is a G to C transversion located in intron 8. Indel 11 is a 3 bp insertion located within a string of Ts, and it is therefore impossible to determine the exact insertion site. Indel 14 is a 12 bp deletion in 129S6/SvEvTac. We confirmed the C57BL/6J genotype for polymorphisms 13, 16, and 17 and updated the dbSNP database for 13 additional C57BL/6J and all 16 129S6/SvEvTac alleles. Within the Mettl16 locus, 129S6/SvEvTac and 129S6/SvImJ are concordant for all variants tested with both strains. Notably, six SNPs (10, 13, and 15–18) and two Indels (11 and 14) differ between 129S6/SvEvTac and C57BL/6J.

Ecotropic viral integration site 2A (Evi2a) is expressed from the Crick strand and contains two exons (Table 3). Table 3 is organized the same as Table 2, with the following exceptions: amino acid changes are found in column 3 and the remaining column numbers are shifted. We designed five amplicons for sequencing Evi2a, as exon 2 spans 2058 bp. We confirmed the 16 SNPs in the region and did not identify any additional alleles. However, we added genotyping information for C57BL/6J for all SNPs. Three SNPs (4–6) are located in exon 2; SNPs 4 and 5 cause missense changes (M), whereas SNP 6 is a silent variant (S).

Table 3.

Evi2a Polymorphisms

Column 1 denotes the relative number based on chromosome position and column 2 indicates the actual nucleotide that is changed based on NCBI Build 37 at the UCSC and Ensembl genome browsers. Amino acid changes are listed in column 3. Location of the variant within the locus (i.e., upstream, 5′ and 3′ UTR, exonic and intronic alleles) is indicated in column 4, for example, intron 8 is denoted I8. We provide upstream and downstream sequence information in column 5, and columns 6 and 7 summarize sequencing data from this study. dbSNP ID number is presented in column 8, and dbSNP genotyping data are shown in columns 9–11; variants without dbSNP IDs are novel alleles. Gray shade indicates no data available in current database. Blue letters indicate new information for 129S1/SvlmJ.

Proteasome 26S non-ATPase subunit 11 (Psmd11) codes from the Watson strand and contains 14 exons (Table 4). Table 4 has the same organization as Table 3. We designed 13 amplicons for sequencing. Exons 11 and 12 were amplified from one PCR product. We confirmed the four SNPs in the region and did not identify any additional alleles. However, we added genotyping information for C57BL/6J for all SNPs. SNP 2 causes a silent variant in exon 3.

Table 4.

Psmd11 Polymorphisms

Column 1 denotes the relative number based on chromosome position and column 2 indicates the actual nucleotide that is changed based on NCBI Build 37 at the UCSC and Ensembl genome browsers. Amino acid changes are listed in column 3. Location of the variant within the locus (i.e., upstream, 5′ and 3′ UTR, exonic and intronic alleles) is indicated in column 4, for example, intron 8 is denoted I8. We provide upstream and downstream sequence information in column 5, and columns 6 and 7 summarize sequencing data from this study. dbSNP ID number is presented in column 8, and dbSNP genotyping data are shown in columns 9–11; variants without dbSNP IDs are novel alleles. Gray shade indicates no data available in current database.

Chaperonin containing Tcp1, subunit 6b (zeta) (Cct6b) is expressed from the Crick strand and contains 14 exons (Table 5). Table 5 has the same organization as Table 2. We sequenced each exon and confirmed nine SNPs within this locus. SNP 1 is located downstream to Cct6b, whereas SNPs 2 and 3 are within the 3′UTR and should be identified from mRNA. Although we did not identify any additional alleles, we added genotyping information for C57BL/6J for nine SNPs.

Table 5.

Cct6b Polymorphisms

Ring finger and FYVE like domain containing (Rffl) is expressed from the Crick strand, contains 11 exons, and produces 5 alternatively spliced products (Table 6). Table 6 is organized exactly the same as Table 3. We designed 13 amplicons for sequence analysis of Rffl; 3 span exon 11. To reduce complexity, we incorporated all splice variants into a single transcript. RefSeq NM_026097.3 contains exons 5–7 and 9–11. RefSeq NM_001164570.1 contains exons 2, 4, and 6–11. RefSeq NM_001007465.3 contains exons 1 and 6–11. RefSeq NM_001164569.1 contains exons 1, 2, and 6–11. RefSeq NM_001164571.1 contains exons 1, 6, 7, and 9–11. We confirmed 10 SNPs for this locus and found no variants between C56BL/6J and 129S6/SvEvTac. SNPs 9 and 10 are within the 5′UTR of exons 4 and 2, respectively. SNP 8 is a missense variant in exon 6, whereas SNPs 6 and 4 are silent changes in exons 11 and 8, respectively. SNPs 1–3 are located in the 3′UTR of Rffl. SNP 8 causes an Ala to Thr missense change at amino acid positions 90 (NM_001164569.1), 69 (NM_001164570.1), and 55 (NM_026097.3, NM_001007465.3 and NM_001164571.1) depending upon the RefSeq.

Table 6.

Rffl Polymorphism

Column 1 denotes the relative number based on chromosome position and column 2 indicates the actual nucleotide that is changed based on NCBI Build 37 at the UCSC and Ensembl genome browsers. Amino acid changes are listed in column 3. Location of the variant within the locus (i.e., upstream, 5′ and 3′ UTR, exonic and intronic alleles) is indicated in column 4, for example, intron 8 is denoted I8. We provide upstream and downstream sequence information in column 5, and columns 6 and 7 summarize sequencing data from this study. dbSNP ID number is presented in column 8, and dbSNP genotyping data are shown in columns 9–11; variants without dbSNP IDs are novel alleles. Gray shade indicates no data available in current database. Blue letters indicate new information for 129S1/SvlmJ.

Adaptor-related protein complex 2, beta 1 (Ap2b1) is expressed from the Watson strand and contains 22 exons (RefSeq NM_001035854.2) (Table 7). Table 7 is organized exactly the same as Table 3. We designed 30 amplicons: 6 are necessary to span exon 22, 5 are necessary to span exon 12, and exons 8 and 9 are amplified from a single PCR reaction. We identified 80 SNPs and 4 Indels; 19 of the SNPs (28–32, 36, 37, 41–48, 50, 65, 66, and 84) and all Indels (4, 12, 27, and 80) are novel. SNPs 1–3 are located upstream of the transcriptional start site of the two RefSeq genes (NM_001035854.2 and NM_027915.3), but within the 5′UTR of the predicted Ensembl transcripts (ENSMUST00000018875). Variant 4 (a 6 bp insertion) is located within the 5′UTR of exon 1. The remaining three Indels (12, 27, and 80) are located in intron 3, intron 10, and the 3′UTR, respectively. SNP 32 causes a silent change in exon 11. The remaining novel SNPs are located within introns and the 3′UTR. Previously identified SNPs (33 and 56) lead to missense variants (F490L and A602T) in exons 11 and 14, respectively. Additionally expressed polymorphisms include exons 4, 7, 12, 13, 14, 17, and 20. We identified 5 variants between 129S6/SvEvTac and 129S1/SvImJ (blue boxes, 16, 33, 53, 60, and 82) and 46 differences between 129S6/SvEvTac and C57BL/6J (yellow boxes). Notably, SNP 33 leads to a missense variant between 129S6/SvEvTac and 129S1/SvImJ that could account for differences between these two distinct 129 strains.

Table 7.

Ap2b1 Polymorphisms

Column 1 denotes the relative number based on chromosome position and column 2 indicates the actual nucleotide that is changed based on NCBI Build 37 at the UCSC and Ensembl genome browsers. Location of the variant within the locus (i.e., upstream, 5′ and 3′ UTR, exonic and intronic alleles) is indicated in column 3, for example, intron 8 is denoted I8. We provide upstream and downstream sequence information in column 4, and columns 5 and 6 summarize sequencing data from this study. dbSNP ID number is presented in column 7, and dbSNP genotyping data are shown in columns 8–10; variants without dbSNP IDs are novel alleles. Gray shade indicates no data available in current database. Yellow shade indicates distinction between C57BL/6J and 129S6/SvEvTac. Boldface indicates new SNP or indel. Blue shade indicates distinction between 129S6/SvlmJ and 129S6/SvEvTac. Blue letters indicate new information for 129S1/SvlmJ.

The polymorphisms located in these six genes between 129S6/SvEvTac and C57BL/6J strain are summarized in Table 8. In total, we identified 6 Indels and 135 SNPs in all 6 genes; we are the first to report 21 of these SNPs and all Indels. Regarding our analyses, it becomes clear that the major distinctions between the 129S6/SvEvTac and C57BL/6J strains lie within Ap2b1 and Mettl16, whereas the variations between 129S6/SvEvTac and 129S1/SvImJ lie in Ap2b1. This suggests that the remaining four genes might be more highly conserved among the 129S6/SvEvTac, 129S1/SvImJ, and C57BL/6J strains.

Table 8.

Summary of the Polymorphism Analysis

Gene	Name	Chromosome	Location	Genomic size	Number of exons	Express direction	Identified SNP	Novel SNP	SNP in exon	SNP in intron	UTR SNP ^a	Indel	Distinction between 129S6/SvEvTac and C57BL/6J ^b	Distinction between 129S6/SvEvTac and 129S1/SvlmJ ^c	Distinction between C57BL/6J and 129S1/SvlmJ ^d
Mettl16	Methyltransferase-like 16	11	74584365–74632194	47830	10	Forward	16	2	0	15	1	2	5	0	5
Evi2a	Ecotropic viral integration site 2A	11	79340063–79344111	4049	2	Reverse	16	0	14	2	0	0	0	0	0
Psmd11	Proteasome 26S non-ATPase subunit 11	11	80242117–80285635	43519	14	Forward	4	0	1	3	0	0	0	0	0
Cct6b	Chaperonin subunit 6b (zeta)	11	82532752–82577808	45057	14	Reverse	9	0	3	4	2	0	0	0	0
Rffl	Ring finger and FYVE-like domain containing	11	82617324–82684246	66923	8	Reverse	10	0	8	2	0	0	0	0	0
Ap2b1	Adaptor-related protein complex 2, beta 1	11	83116199–83218535	102337	22	Forward	80	19	12	57	11	4	46	5	44
Total							135	21	38	83	14	6	51	5	49

UTR SNP indicates the number of SNPs within the untranslated regions.

The distinction indicates the number of differences in SNPs or indel between 129S6/SvEvTac and C57BL/6J.

The distinction indicates the number of differences in SNPs or indel between 129S6/SvEvTac and 129S1/SvImJ.

The distinction indicates the number of differences in SNPs or indel between C57BL/6J and 129S1/SvImJ.

We converted the current dbSNP information within this region into a heatmap that represents the polymorphism density across the region (Fig. 2). Dark areas contain more variants than light gray segments. It is clear that the six target genes all map to regions that contain high or intermediate numbers of SNPs. Interestingly, there are two blocks with a low SNP density. The first lies between Mettl16 and Evi2a and the second falls between Evi2a and Psmd11. Based on the SNP density, we predict that these two regions contain elements under selective pressure in the two mouse strains or harbor important functional domains.

FIG. 2.

Density map of polymorphisms located in the domain of study. The heatmap represents current dbSNP information across this region. Dark gray indicates highest amount of SNPs and light gray stands for lowest amount. The six genes in this study are marked on the heatmap based on their chromosome locations and gene sizes. Genetic variations including SNPs and insertion/deletion polymorphisms identified in this study are shown by black lines. Each black line correlates with the number of polymorphisms within a 10 kb domain; longer lines signify increased polymorphism densities.

Discussion

The 6 genes in this study span a 6.8 Mb domain that contains a total of 154 genes, 9 miRNAs, and 5 snoRNAs. We concentrated our efforts on genes that are expressed during pre-implantation development or in gametes. The Evi2a locus, which is adjacent to Evi2b, resides within intron 33 of the Nf1 gene. Although the function of Evi2a is unknown, the locus demarcates the boundary between the two conserved regions, as it contains a high number of polymorphisms in a relatively small genomic region. We identified 16 genetic variants within the 4 kb Evi2a locus, whereas the average number of variants identified in this study is 6 within 10 kb. Although C57BL/6J, 129S6/SvEvTac, and 129S1/SvImJ are concordant for all variants of Evi2a, this gene could be crucial for the divergence of other mouse strains. Alternatively, polymorphisms within this intron could be important for Nf1 gene regulation. Consistent with this hypothesis, EVI2A is upregulated in malignant peripheral nerve sheath tumors in neurofibramatosis patients harboring microdeletions within the NF1 locus (Pasmant et al., 2011). The dbSNP database indicates that high levels of allelic variants are present in the distal end of the 6.8-Mb domain that overlaps with Cct6b, Rffl, and Ap2b1. This is consistent with our results, as we sequenced 84 variants within this locus.

The 129 strain is commonly used for knockout and other genetic manipulation studies because of the efficiency of obtaining embryonic stem (ES) cells that colonize to the germline (te Riele et al., 1992). This strain originated in 1928 and has diverged into more than 15 substrains. Different breeding strategies used in the past resulted in phenotypic and genetic diversity among these substrains (Simpson et al., 1997). In this report, we sequenced the 129S6/SvEvTac strain that has been maintained since 1992 at Taconic. According to Simpson et al. (1997), the origin of 129S6/SvEvTac can be traced back to the breeding of the Steel substrains, which resulted from the outcross of the 129/Sv strain to C3HeB/FeJ followed by 12–14 generations of backcrossing to the parental 129/Sv line. The resulting mice, termed 129/SvEv, were distributed to Martin Evans and were maintained by selection for the Steel^J allele. The 129/SvEv strains underwent further genetic manipulations to produce 129/SvEvBrd and 129/SvEv-Gpil ^c. Taconic crossed 129/SvEvBrd and 129/SvEv-Gpil ^c to generate 129S6/SvEvTac. This line has been bred as a separate inbred line at Taconic since 1992.

129S1/SvImJ was developed as a control-inbred strain for many of the Steel-derived strains (Petkov et al., 2004). Therefore, we included this strain as reference to compare the genetic variation between 129S1/SvImJ and 129S6/SvEvTac. Ap2b1, which has 84 polymorphisms across the locus, is the only gene in this study to contain variants between the 129S1/SvImJ and 129S6/SvEvTac inbred strains. There are 46 variants between C57BL/6J and 129S6/SvEvTac, 44 alterations between C57BL/6J and 129S1/SvImJ, and 5 SNPs between 129S6/SvEvTac and 129S1/SvImJ across the gene. Polymorphism number 33 (rs28210244) is a C1571G transversion in exon 11 that causes an F469L amino acid substitution. The remaining variants between the two 129 strains lie within introns or in the 3′UTR.

Ap2b1 encodes one of the two large chain components of the clathrin assembly protein complex 2. Ap2b1 protein is found on the intracellular domain of transmembrane-coated vesicles and is important for protein transport (Schmid et al., 2006). It is unclear why Ap2b1 contains so many polymorphisms and why there are differences between 129S6/SvEvTac and 129S1/SvImJ. It is possible that the genetic variations lead to alterations in gene regulation and/or protein structure changes that respond to strain-specific factors. However, it is equally likely that the high number of polymorphisms between 129S6/SvEvTac and 129S1/SvImJ is simply due to random silent mutations. Most of the variances within the Ap2b1 locus (82/84) lead to silent changes or are located within nonprotein-coding regions of the gene (i.e., introns or UTR), which is consistent with the latter explanation.

Although there are many variants between C57BL/6J and the 129-derived lines in this region of MMU 11, all of the polymorphisms between 129S6/SvEvTac and 129S1/SvImJ are located within a single gene. The domain proximal to Ap2b1 contains no variants between the two 129 strains. Regions of conservation suggest sequences critical for function, that is, gene variation is not functionally tolerated. Phylogenetic studies of nine different 129 strains indicates that the 129S6/SvEvTac and 129S1/SvImJ strains are highly related (Threadgill et al., 1997). Both were derived from a common ancestor bearing the Steel allele of the Kit ligand, although 129S1/SvImJ has been maintained at The Jackson Laboratories and 129S6/SvEvTac is maintained by Taconic. As six of the commonly used mouse ES cell lines have been derived from these two 129 strains, it is important to document the variants between these two 129 strains for future gene targeting and genotyping studies.

Conclusions

In this study, we sequenced 6 genes within a 6.8 Mb domain of mmu 11 and identified a total of 21 new SNPs and 6 Indels as well as confirmed genotype information for 114 additional SNPs among the C57BL/6J, 129S6/SvEvTac, and 129S1/SvImJ inbred strains. Most of these cluster at the two flanking loci, Mettl16 and Ap2b1. Ap2b1 is especially polymorphic among these three strains. These studies highlight the genetic variations among the three inbred strains and provide important information for understanding the evolutionary context of inbred strain divergence in mice.

Footnotes

Acknowledgments

The authors thank Lei Xing and Marianne Luu for excellent technical assistance, Drs. William Muir and Christopher Bidwell for critical reading of the manuscript, and Jason Fields for expert animal care. This project was funded by grants from the Purdue Research Foundation and generous start-up funds from the Department of Animal Sciences and the College of Agriculture at Purdue University.

Disclosure Statement

No competing financial interests exist.

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