Hi,
I have 200++ SSR markers across genome, but I don't have enough resources to test all the SSRs on my samples. And I want to select SSRs that are distributed uniformly in each chromosome. How do I know which is the most suitable?
highly polymorphic marker is good for diversity analysis. If you dont have PIC for the marker then you have to run all the marker. based on chromosome no take 10 marker each chromosome .
Hi Vinay Kumar Jain,
Thanks for the answer. So, I need to run all markers to get PIC then?
by the way, how do I choose the 10 markers in the chromosome? Is it randomly?
Dear Lim
The authors of the below article provided a protocol for determining the number of markers needed for dominant marker (such as ISSR) studies that I hope will help you to answer your question.
There is no definite answer to your question. However, I usually draw my the number of loci based on several reasons as follow. However, the way you do depend on the objective of your study. Use the following as general guidelines only.
1) If you design your SSR primers based on fragment of genomic sequences (or any other sequences in this matter), you may want to evaluate the syntheni (relatif positions) of your sequences to reference genome and explore the possible locations of your sequences to the reference genome.
You may be able to associate and group your SSR containing fragments into different chromosomes. Subsequently, you can select the representative markers based on their relative chromosome positions. You will not want to only use a set of SSR loci that are associated with one or two chromosome, but spread the loci to as many chromosome as possible. Many plant genomes are available at Phytozone website (https://phytozome.jgi.doe.gov/pz/portal.html) and you can select one that is the closest to your plant (or just use Arabidopsis).
2) If you have not previously used the SSR primers, I would suggest you screen your SSR primers using genomic DNA of 2-3 accessions, to get ideas which primers produce amplicons (PCR amplified products) and which are not. You can dismiss the SSR primers producing no amplicon.
3) Subsequently, SSR primers yielding amplicons in step 2 may be tested using 10 or so accessions (select the most diverse 10 accessions) to evaluate the highest possible allele variability for the evaluated SSR marker loci. The more allele variability, the better the SSR primer for the genetic diversity analysis. You can also eliminate any SSR primers yielding monomorphic marker or ones yielding difficult to score alleles. And so on.
4) Once you identified the ideals SSR loci (i.e. yielding amplicons, are polymorphics, and having as many alleles as possible in the tested ten accessions, and distributed to as many chromosomes as possible), then depending on the chromosome number of your studied organism (i.e. 2n=2x=32, haploid chromosome n=16) you will decide how many SSR marker loci you will use. I would suggest to use at least 2-3 x the number of haploid chromosomes (i.e. 2-3 x n). However, the amount of your available funding will be the limitation. If you have no funding limit, then use as many as possible.
I saw some publication use as many as 16-32 SSR marker loci, depending on where they publish the manuscript. Therefore, you may want to consider your target journal as guidance for determining number of loci to use. Check the published manuscript with similar topics as your study and see how many loci their study have use. Try to draw your study using as close number of loci as possible to the published references.
I hope these will help you decide what you want to do in your research. Good luck.
Dear
according my experience it is ideal that use 5 SSR primers per chromosome, butt you can use 2 or 3 primers per chromosome.
review location your SSR before Primers selection.
(cover all chromosome by 2 or 3 primers)
best regards
S. Sodarsono's response is appropriate. If you have a genetic linkage map for your test organism available you can select the representative polymorphic SSRs for each LG or chromosome. As low as 14 SSR have been found to be discriminatory enough for genetic diversity studies in the past. Therefore, using 30 well distributed SSR across the genome of the test organism should be enough. However, if you intend to use them for mapping, you may need a lot more depending on genome size. This helped me long ago.
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