Understanding how things should be done often helps us design experiment on how to achieve the objectives more efficiently. Therefore, it is always good to try to understand and know how we should proceed in doing something. In a Q & A session of the AGH635 lecture, one of the student asked me the following question: “If I Want to Do RAPD Marker Analysis in Plants, How Should I Proceed ?”
To be able to work efficiently with RAPD marker, one may have to consider exploring the following steps. Depending on objectives of the study, the steps to follow may be either just step 1-4 or step 1-6. If the objective of the study is just investigating the genetic distances among accessions, then conducting step 1-4 should be enough. However, if the objective of the study was doing linkage analysis or tagging gene controlling certain phenotype with markers, then one needs to complete step 1-6.
The complete steps are as follow:
- Population development,
- Primer screening,
- Analysis data for Diversity,
- Linkage analysis among markers and Gene Tagging.
- Step (1) Population development: This part of activity has been described in a number of previous posting. So, ones should review the previous posting for this subject.
- Step (2) Primer screening: This objective was to select suitable primers for the stated studies. Not all of the available random 10-mer oligo nucleotide primers (subsequently called “primer”) are suitable for specific study, in different plant species, and even in different population of the same plant species. That is why ones need to identify which primers should be used in their studies through primer screening activities.
- Step (3) Data scoring and genotyping: Once electrophoregrams (photograph of the gel electrophoresis results) of RAPD markers are generated, ones need to score the markers for each locus in each plant sample. If necessary, depending on the objective of the study, one need to convert the score data into genotype data (genotyping).
- Step (4) Analysis data for Diversity: The score of RAPD marker data can be used to generate distance matrices and to estimate genetic diversity among plant samples in the studied population. The steps end here if the objective was only to study the genetic distance and genetic diversity. To analyze genetic distance and diversity among individuals, one needs the help of certain software, such as : NTSys, Darwin, and the like.
- Step (5) Phenotyping: If the objective of the study is to associate marker with characters, it was necessary to record the phenotype of each of the plant in the mapping population. The phenotype observation (phenotyping) for the target character need to be as accurate as possible since it will be associated with marker.
- Step (6) Linkage analysis among markers and Gene Tagging: Genotype data based on the RAPD markers (generated in Step 3) and phenotype/characters data (collected in Step 5) are analyzed to construct linkage groups among markers and among markers and characters. To analyze linkage among markers and characters, one needs the help of certain software, such as : MapMaker, QTL Map, Join Map, and the like.