I got my fragment analysis results of Microsatellite markers designed using haploid species. I am using a gene marker to analyze the peaks. I read that since it is haploid, i should see only one peak (means one allele per locus?). But i see many for types of markers ( di, tri , ...) . I want to select polymorphic markers and want to score them for population analysis.
Also, gene marker has the option only to analyze plant dinucleotide and plant fragments. So i am not sure what settings should i select for fungal species?
i would appreciate if someone could clear my doubts?
Dear @Chiti Agarwal I refer your statement:
......since it is haploid, i should see only one peak (means one allele per locus?).
Is it true for all haploids? Haploids of polyploids have more than one allele per locus.
@arbind , this is what is confusing me. I am not sure how many peaks am I supposed to see for the haploid species and how to score them?
Hi,
if you used a DNA that belongs to a single sample (for example a single plant of a given variety from which you extracted its DNA) you must find a single allele per locus but if you used a mixture DNA belong to several individuals and obviously from the same species you can find several alleles
As your individuals are haploids you should see one peak (one allele per locus) for each marker (locus) you use. To define genetic profile of each individual you have to use several markers in order to obtain an haplotype. You can define the population structure according to the haplotype you obtain for each sample
Thanks Angela Teani Angela. for marker 1 , I am seeing one peat at 240 , 2nd at 250 and 3rd at 300.. and its is the same for all markers i used. these peaks are very high and clear. So is this okay?
or you mean i should see only 1 peak at one place for example at 240?
Dear @Chiti I'm not shure what do you mean for haploid species. As apparently if you find three different peaks which are clear and high the species should be triploid (three alleles in each marker), if you exclude any contamination of PCR aspecific products.
Did you develop the primers for these loci--that is, do you know what the expected length of the fragment should be? And are these di-, tri- or tetranucleotide repeats?True microsat fragments frequently show stutters (e.g. multiple peaks different by 2 bp for dinucleotide repeats), but "artifacts" which may sometimes be amplfied should NOT show stutter bands. Just to be clear, you are seeing 3 peaks for one individual sample, not across multiple samples? What size standard are you using for the gels?
Theoretically, you should see only a single peak, considering you used a single biological sample. However, stutter peaks are also common so you might have to differentiate between your peaks and stutter peaks.
What fungal species are you working with? Because if you are working with an ascomycete you should see one peak, but if you are working with a basidiomycete or oomycete it is possible to see two peaks.
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