I deal with wheat variety identification by using SSR genotyping and for some markers more alleles appear than expected. I optimized PCR conditions and e.g. different time of final extension shows different ratio of intensity for detected allels (fig.1 DuPw217 repeat AAG). How to determine the real allele, could it be stutter peaks? Is there any RFU ratio for evaluating what is a heterozygote or what is a stutter (fig.2 DuPw167 repeat AAGCAT)?
Could anyone hepl me to score this? Thank you in advance.
This may not be the direct answer to your question, sometimes it will help to compare the allelic patern among the parent and the segregated progenies, which may have homozygous and heterozygous genotype constitutions.
By comparing the parent and offspring peak patterns, one may identify which is the homozygous allele pattern and which is heterozygous.
Generally, all SSR markers have product size (like ~150 bp to 300 bp) so you focus on that band only.
Dear Jitka Klempova,
Greetings, I would like to thank you for your interesting question. Each SSR or microsatellite marker have expected Fragment size by bp. According to the tested genotypes you could find the repeated allele per your genotypes and it real one.
with best regards
Khaled
Very nicely answered by Khaled. Actually you may bump into some reports of RFU ratio for heterozygote and slutters, but it may not suit your study and many a times (especially for wheat) it is highly misleading. This is because the wild variety of wheat is often a mixture of tetraploid and hexaploid variety and we do not know the ratio.
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