It is hard to find an explanation that fits all of the results but 2 possibilities are

1 samples 6-10 have a pcr inhibitor present in the dna,

2 The upper allele is itself associated with a sequence/base change and that stopped one primer from annealing in expt1 but some small change in pcr conditions caused the pcr to fail in expt1, You could try amplifying a sample from 6 to 10 at dilutions (less dna 1/2, 1/4 and 1/8 at a higher Mg concentration (2,5mM) and a lower annealing temperature (3c lower)

You can check snpdb for snps under your primers and if the amplifications are still confusing design a new primer set which does not have any primer sequence overlap with the original pair

Dear Paul Rutland, Thank you for your suggestions.

I utilized a primer that is known for detecting my specific R-gene-linked marker. The first time I conducted the test, the result (shown in the first picture) matched the phenotypic expression (artificial inoculation data). However, when I tested the same sample with new DNA, the second picture showed a different result.

Saif

Saiful Islam Please check the PCR machine-block. One possible chance is, it could be by tubes (no amplification) may not getting proper conditions during PCR cycles.

Dhananjayan Dhanasooraj Thanks for the suggestions. Actually, I am getting worried about the smear bands shown in the 2nd picture.

Good. The only difference in your experiments seems the added DNA in PCR reaction (I understood in such a way, sorry if not). The the problem could be the issue with added DNA (as suggested by Paul Rutland). If possible check with spectrometry and gel electrophoresis (if not done). - Best wishes

Sorry Saiful Islam I misunderstood the nature of the problem, Please ignore everything that I said. My only thought at the moment is that the primers are depurinating (if they are stored in water not TE) or degrading ( if stored in a frost free or self de icing freezer and the smaller degradation products are annealing all over the place, Where the correct sequence exists there is enough intact primer to dominate the reaction but where the sequence does not exist you are seeing a ladder of non specific product possibly caused by just one primer annealing in 2 orientations and producing a weaker and different length product from the one expected. Was there a long time between doing the 2 assays ?

Paul Rutland Thanks for your note. That might be a cause. Since I used the primer pair, kept it in the lab a long time ago (say 8-10 months), and stored it in 4 degree C refrigerator. However, I ordered a new primer set and let you know the update.

Thank you so much again for your kind advice.

Dhananjayan Dhanasooraj Thanks for your concern. I did you the processes you mentioned.

Thank you again