Simple Sequence Repeat (SSR)- a small segment of DNA, usually 2 to 5 bp in length that repeats itself a number of times. Useful SSRs usually repeat the core motif 9-30 times. Some of the major core motifs that we use in the development of SSR markers for soybean include ATT, AT, CTT, and CT.
Polymerase Chain Reaction (PCR)- an in vitro method for producing the large amounts of a specific fragment of DNA necessary for analysis. Basically a reaction that Xeroxes DNA making millions of exact copies of the same fragment. Step over here for more information about PCR.
SSR Marker Development
The first step in the process of creating a useful Simple Sequence Repeat (SSR) DNA marker is the construction of a DNA library in which small pieces of soybean DNA are inserted into a cloning vector. We use the older and quite well-known soybean cultivar ‘Williams’ as the source of soybean DNA for our DNA libraries. The cloning vector we use is a plasmid vector, pBluescript +KS. Each individual plasmid, containing a different piece of soybean DNA, is then "transformed" or inserted into E. coli cells (look here for a graphical explanation of this). The plasmid vector with the inserted soybean DNA multiplies many times within the E. coli cell. The resulting collection of E. coli cells, each containing a plasmid with a different piece of soybean DNA, is referred to as a plasmid library. Once the library is constructed, it is screened for plasmids that contain soybean DNA with a desired SSR motif such as (ATT)n, (AT)n, (CT)n, (CTT)n, etc. Plasmid clones that are determined to contain the desired motif are then isolated so that the DNA sequence of the entire soybean insert can be determined. DNA sequence determination is performed on a Perkin-Elmer ABI 377 Automated DNA Sequencer. The raw sequence data from each plasmid insert is end-trimmed and analyzed using Perkin-Elmer ABI Auto Assembler software. The determination of DNA sequence is important for two reasons 1) it verifies the presence of the SSR in the soybean insert and 2) it provides the exact DNA sequence on either side of the SSR, which is necessary to construct primers. The DNA sequences from each new SSR-containing soybean insert are checked against each other and against all previously sequenced clones to eliminate duplicates. Clones that are unique i.e., that we have not previously identified and that possess an SSR of sufficient length, are advanced to the next step of the SSR marker development process. This is the selection of PCR primers to the regions flanking the SSR.
We use the primer selection software Oligo 5.0 to identify optimal PCR primers, which generally are 15-31 DNA bases in length and produce products ranging in length from 90-300 basepairs (bp), depending on the length of the included SSR. Primers are also selected and optimized for a 47º C annealing temperature, which is the standard annealing temperature for our PCR reactions with soybean SSR markers. Head this way to see our PCR protocols. The PCR primers are then synthesized by a local DNA synthesis firm.
Once the primers are synthesized, a number of additional tests are required before they can be utilized to produce a useful soybean SSR marker. To test their effectiveness, the primers are used in PCR amplifications of both their original plasmid, upon which the DNA sequence was determined, and on Williams soybean DNA. The PCR reactions are 32P radiolabeled and then separated on denaturing polyacrylamide DNA sequencing gels. Primers that perform well, by producing a single clean product with both the plasmid and Williams soybean DNA are advanced for further testing. This second test involves a broader array of soybean DNA that includes 12 genotypes. These include the diverse soybean cultivars Clark, Harosoy, Jackson, Williams, Archer, Amsoy, Fiskeby, Minsoy, Noir 1, Tokyo, the experimental line A81-356022, and G. soja (wild soybean ) PI 468.916. Primers that produce discrete single products that vary in size among the 12 soybean genotypes, i.e. are polymorphic, are considered useful markers and are assigned a name designated with an S followed by the core motif and then the primer number. For example, the 586th soybean SSR primer with an ATT motif would be named Satt586 as shown below. In the past, primer sets that were determined to be polymorphic in one or more mapping populations [USDA/Iowa State (A81-356022 x G. soja PI 468.916), the University of Utah (Minsoy x Noir I), and/or the University of Nebraska (Clark x Harosoy)] were mapped in one or more of these populations by collaborator K. Gordon Lark, Department of Biology, University of Utah or Alex Kahler, Biogenetic Services, Inc., Brookings, SD. Currently new markers are being positioned on the University of Utah, Minsoy x Noir 1 map by K. Gordon Lark and associates.
This image shows an autoradiograph of plasmid clone 339A2 which shows discrete PCR products which are rather polymorphic among the 12 genotypes. As shown, each polymorphism results from a differing ATT SSR repeat length. This locus was designated Satt586.
Another method of determining SSR length polymorphism is to use flourescent tags on the upper, or forward primer. This tag can then be detected when the sample is run on an automated sequencer, as shown below.