I have done crosses in three populations, selfed the F1s to F2s now wanted to check whether there is foreign pollen. I bulked the F2 seeds and genotyped both parents with the F2s. I used 12 SSR panels with approximately 90 markers in total. Out of the 90 only 36 worked, as in worked in all the samples. For one population only 6 SSRs worked and I could derive the F2 alleles properly. However, in the other population only 18 markers worked and only two markers indicated alleles not present in the parental lines. In the last population, 28 out of 36 worked whereby only 3 SSRs indicated alleles that couldn't be traced to the parental lines. I want to know how best to analyse these results. This is a QC attempt to see whether the bulked F2 seed is not contaminated. To bulk, I planted out 20 seeds and mixed the leaf discs when tissue harvesting.
You must be sure that your SSR markers are single locus markers. The problem with SSRs is that they often are repeated over the genome and depending on the detection technique you use you can not see all alleles. For example in agarose or polyacrylamide gels you may be not able to distinguish SSrs that differ in a small number of nucleotides. I would rather use a set of SSrs that are validated and works for various populations and that were sequenced and mapped to ensure their uniqe position in the genome rather than the set of 90 unrelible markers. Be also aware that SSR artefacts are often seen as a result of Taq polymerase nature as well as they are not conserved.
Hi Rafal thanks for the answer. single locus markers you mean they should be linked to the trait of interest? I am doing whole genome profiling and have used them in the past to sort our material into heterotic groupings which our breeders were happy with
Here is a paper that our group recently published doing this in Pennisetum.
http://www.scirp.org/journal/AJPS/
it is volume 4, number 5, may 2013. we used EST-SSRs to confirm interspecific hybrids, and the markers had been confirmed and mapped to another species genome prior to confirmation. check out the marker development in the materials and methods. hopefully this will clarify your question
you're welcome!! if you check out my research gate page you can download it there also. 'confirmation of pearl millet-napiergrass hybrids using EST-derived SSRs'
Is it possible there to use fragment analyser to track small variations? As Rafai said, they may not belong to the region of interest since SSRs repeat frequently in the genome specially when you are doing the whole genome...
No the markers do not need to be linked to any trait. The fact that they are mapped suggest that there is no segregating distortions and is a good indication (not proof) that the markesr are singl locus. Of course it may happen that two or more SSR sequences are close one to another and they differ in length, so you may obtain complex picture even in such case. However if you have sequence data , maybe you can search if the marker is repeated in the genome.
to check the purity, you must have the 2 parental male and female for this cross. Each one have the band and SSR as a co-dominant markers can check this as it has a specific band for checking the purity.
- Badges
- Science topic