1. ♦We now know that our PCR protocol is working. ♦From here, we will work on obtaining a larger sample size from local woodlots. ♦If indeed we find only P. maniculatus in local woodlots, it will bring into question the sympatry of the two species in Wisconsin. [Mg ++ ]Well Set 1Well Set 2 3.5 mmLane 1 3.0 mmLane 2 2.5 mmLane 3 2.0 mmLane 4 1.5 mmLane 5 ♦Tail tissue samples of P. maniculatus and P. leucopus were obtained from the Peromyscus Genetic Stock Center. ♦Approximately 3 mm of tissue were clipped from each tail. DNA P REP : ♦Samples were ground on ice in 0.5 ml of normal saline using a micro-pestle in a 1.5 ml microfuge tube ♦0.5 ml 10% Chelex was added ♦Samples were incubated in a boiling water bath for 10 minutes ♦Tissue debris and Chelex were pelleted by a 30 second spin down in an Eppendorf microcentrifuge at 14,000 rpm ♦The supernatant (ca 0.5 ml) was saved and the pellet discarded. M ULTIPLEX PCR P ROTOCOL : ♦Reactions were run using puReTaq Ready-to-Go PCR Beads yielding a final concentration of: 2.5 U Taq DNA polymerase 0.2 mM dNTP 10 mM Tris HCL 50mM KCl 1.5 mM MgCl 2 ([Mg ++ ] was adjusted as noted in Table 1) ♦Primers (P.mani-F-9197, P.leuco-F-9263, and H9375) were used at 0.5 mM. PCR C YCLES ♦94 o C for 120 seconds ♦30 cycles of: 94 o C for 60 seconds (Denature) 56 o C for 90 seconds (Annealing) 72 o C for 90 seconds (Polymerization) ♦72 o C for 10 minutes ♦4 o C Soak DNA A NALYZED : ♦2% Agarose gel in TAE ♦Run at 100V Sample lanes run with Xyline Cyanol as loading dye only Lanker lanes run with Bromophenol Blue as lead dye) Special Thanks to: ♦Dr. George Clokey for his time and patience ♦The Peromyscus Genetic Stock Center of the University of South Carolina ♦Dr. Nathalie Tessier of the University of Montreal, Department of Biological Science ♦The Undergraduate Research Program of UW-Whitewater Figure 1: DNA Analysis. PCR amplified DNA from P. leucopus and P. maniculatus tissue were run as described. Lanes 1 & 6, Loading dye with Xylene Cyanol and Bromophenol blue; lanes 2 & 5, pGEM DNA markers (Promega, Madison, WI); lane 3, P. leucopus PCR amplified DNA and lane 4, P. maniculatus PCR amplified DNA. Figure 2: Varying [Mg ++ ]. PCR amplified DNA from P. maniculatus in Well Set 1 and P. leucopus in Well Set 2. See table 1 for [Mg ++ ] and lane number. Lane 6 for both Well Sets contains loading dye with Xylene Cyanol. Table 1: [Mg ++ ] for the PCR reactions are as noted. Lane numbers to the agarose gel shown in Figure 2. R ESOURCE P ARTITIONING : ♦A strategy developed to reduce competition for resources between two species with similar ecologic niches that occupy the same area. ♦Previous studies suggest that resource partitioning does occur between Peromyscus maniculatus (deer mouse), and Peromyscus leucopus (white-footed mouse). Based on tail morphology which can be problematic due to the two species being almost identical (see images below). R E - EXAMINING THE QUESTION : ♦Our lab decided to re-examine the question using new identification techniques based on PCR amplified mtDNA rather morphology. ♦Both species are supposedly sympatric in Southeastern Wisconsin. ♦Preliminary data showed only the presence of P. maniculatus in both arboreal and ground-layer habitats. A number of samples yielded no DNA fragments when amplified by PCR. ♦Several possibilities could explain the lack of P. leucopus DNA: We do not have a sufficient number of samples. The PCR protocol we are using is not amplifying the DNA P. leucopus is not found in our area. ♦Solutions to these issues: Determine whether or not the PCR protocol was working by using know tissue samples of both species. Optimize [Mg ++ ]. Range of P. maniculatus: mouse occupies both habitats Range of P. leucopus: mouse occupies both habitats Overlapping Range: mice show resource partitioning with P. maniculatus occupying the trees and P. leucopus occupying the ground Serenity Mutchler Kara Kamps Simon Schmidt Mentor: Dr. George Clokey