Multiplex microsatellite marker panels for genetic monitoring of common rat strains

Abstract

Because microsatellite markers have a high degree of genetic variability, they are an effective tool for genetic monitoring. We have developed a genotyping panel containing 87 microsatellite markers that are polymorphic among commonly used inbred rat strains, including ACI, Fischer 344, Lewis, Brown Norway, Wistar-Furth, and Wistar-Kyoto. The markers are located at approximately 15- to 20-cM intervals along each of the 20 autosomes. By using fluorescently labeled primers and multiplex PCR analysis, the entire genome can be assayed with only 8 reactions. The resulting amplicons from these reactions can be distinguished from one another by both their size and the fluorescent dye associated with them. Amplicons are analyzed and allele sizes are determined by using automated capillary-based instrumentation. These multiplex panels provide a cost-effective and rapid method for genetic monitoring for applications ranging from assessing genetic contamination in a rat colony to moving mutations from one genetic background to another by using a speed congenic approach.

Figure 1.

Overview of analysis process. Primer sets for the microsatellite markers are multiplexed into 8 PCR reactions. The products from the PCR reactions are mixed with a size standard and separated by capillary electrophoresis using an ABI 3100 Prism Genetic Analyzer. Specialized software is used to determine the allele sizes for each microsatellite marker.

Figure 2.

Distribution of microsatellite markers across the genome. The chromosomes are drawn to scale based on the data from the current rat genome sequence (RGSC 3.4). The physical position of each marker on the 20 rat autosomes is shown. Each segment (black or gray boxes) represents 20 Mb. The average distance between adjacent markers on each chromosome is 30 Mb (19 cM).

Figure 3.

Representative data output for analysis of LEW/SsNHsd DNA with markers D2Rat3 and D3Rat53. The 2 markers were amplified in a single PCR reaction and separated by capillary electrophoresis. The upper panel depicts the alleles for D3Rat53, and the lower panel depicts the alleles for D2Rat3. The alleles are represented by the peaks. The sizes in basepairs (bp) are indicated on the top x axis, such that smaller alleles appear to the left, and larger alleles appear to the right. The values on the y axis indicate fluorescent signal intensity given in relative fluorescent units (RFU) and vary for each sample. In this example, both primer sets were labeled with the same fluorescent dye (6FAM), but amplicons are readily distinguished due to the differences in the allele sizes: 162 bp for D3Rat53 and 121 bp for D2Rat3. As expected, this inbred strain is homozygous for the alleles depicted.

Figure 4.

Representative data output for rat marker D11Arb4. Each row represents the results for a single DNA sample: LEW/SsNHsd (LEW), F344/NHsd (F344), BN/RijHsd (BN), ACI/N (ACI), WF/NHsd (WF), or WKY/NHsd (WKY). The alleles are represented by the peaks. Many markers have a characteristic ‘stutter’ pattern (multiple peaks), as seen here. The arrow indicates the actual allele peak. The sizes in basepairs (bp) are indicated on the top x axis, such that smaller alleles appear to the left and larger alleles appear to the right. The values on the y axis indicate fluorescent signal intensity given in relative fluorescent units (RFU) and vary for each sample. In this example, there are 5 different allele sizes, with ACI/N and WF/NHsd sharing similarly sized alleles (monomorphic).