I am Exploring the Single nucleotide polymorphisms (SNPs) of waxy (wx) gene in the rice varieties cultivated in Pakistan. I am using Tetra-arms PCR for this study. But I am concerned about some technicalities of this technique. Particularly about the rice waxy gene homozygosity and heterozygosity.

1) I am confused about homozygous and heterozygous alleles. Because when I studied different waxy alleles in the rice varieties, I was very confused about the point that if the rice genome is 2n, then why a particular variety has only one type of allele? Except one paper, I was unable to find the term homozygous and heterozygous alleles for the rice Wx gene. In that paper, the author used heterozygous term for hybrid rice varieties not for the pure lines. Now I just want to confirm that either the homozygous or heterozygous term only applies to the hybrid rice? Let us take the example of Wxa and Wxb alleles. None of the studies indicate that the variety they explored was homozygous for Wxa or homozygous for Wxb, neither they said that the variety they explored had both Wxa and Wxb alleles (i.e., heterozygous).

2) If I perform the PCR of different primers combinations three combinations for one SNP

i) Forward outer + forward inner (fragment amplification

ii) Forward outer + reverse inner (Wxa allele specific)

iii) Reverse outer + forward inner (Wxb allele specific)

for a particular allele in different tubes and load them in separate wells of the gel then, there must be only two bands one from the outer primers (as a quality control) and second from the either of the alleles? That is if I get two bands (of same size) in the same lane it means there is something wrong with my reaction or if I get three bands in three lanes (i.e., one from outer primers and one each from each of the alleles) will show that I am not having the pure lines of rice varieties i.e., the variety is heterogenetic not the heterozygous?

3) Do we carry PCR for all the primers in different tubes and load them in different wells of a gel?

I shall be thankful to you if you guide me and give your expert opinion.