I made crosses using a common female parent with several male parents to get F1s. Both parents are Upland cotton (Gossypium hirsutum). I used SSR markers to confirm the crosses. The markers are polymorphic between two parents. The ideal situation is that the F1s should show both the alleles (bands on argarose gel) from male and female parents. However, in some cases, alleles from female parent is missing, and in a few cases, alleles from male parents is missing. I tried different markers, and got the same results. I am wondering whether someone can provide some clue about this phenomenon.
Thanks
Are the markers polymorphic within each parents? Are they wild parents?
If I understand correctly, you are getting the expected heterozygous bands from most SSRs tested for a particular hybrid, but for some SSRs one or the other allele is missing. Thus, you are certain that they are truly hybrids, but you are curious about these odd SSR results. Correct?
One idea-
There could be differential primer binding between the two alleles. If the sequence of either primer isn't an exact match for either of the two parents' primer binding sites, then you may see preferential amplification of the "more exactly matched" allele in the heterozygote. You could test this by running the reaction with a mixture of parental DNA.
If this is the case, you might add excess primers to your reaction with the hybrid to overwhelm the competition.
Oh, you said that you have tried different markers with the same results. In that case, I'd guess you have some selfs or seed contamination among your hybrids.
1. When you did PCR, did you include the parental lines along with the F1 'hybrid' lines? And the parental lines' bands came out ok?
2. Selfing (failure of outcross) can be explained when only the female band was shown (without the male band), as mentioned by Jack.
3. How 'pure' (homozygosity) is the female line ? You mentioned that the parental lines have gone through several generations of selfing. Can it be that this female allele is a rare heterozygous, so in some F1 hybrids, you can only see the male allele band, but no female allele band?
To Jack:
I am getting the expected heterozygous bands for some of hybrids using one or more SSRs, but for some hybrids, I don't get expected bands for some hybrids. These hybrids are from a common female parent crossed with different male parents. I tried different markers for these hybrids, I got nearly the same results, expected bands from female parent are missing. The interesting thing is that I got expected results for hybrids from another common female parent crossed with same male parents. So, I guess there is something to do with the common female parent.
I am sure there is no seed contamination among the hybrids.
Your idea of differential primer binding between the two alleles could be a good explaination.
Thanks
To answer Yuan-Yeu's questions:
1. I did include the parental lines along with the F1 bybrid lines, and the parental liines worked very well. I got expected bands from parental lines.
2. For most cases, the female band were missing, and in a few cases, male parental bands were missing.
3. The female line was selfed more than five generations. I would say it is pretty "pure".
As I mentioned to Jack, the phenomenon occured from the hybrids which were from a common female parent crossed with different males parnents. Hybrids from another common female parent crossed to same set of male parents don't this situation.
Hi, Hui:
Be careful declaring a line "pure". Five selfing generations from an F1 leaves you with approximately 3% heterozygosity. That said, if you've tested multiple markers on a potential hybrid and found the same results, then residual heterozygosity is probably not the problem.
Could there have been an error in planting the females, such that the female that is giving you the problem was replaced by one of your lines intended as a male (or another line altogether)? Is the DNA that you are loading as "female parent" from the exact plant that the hybrid seed with odd results was taken from?
Please don't take this wrong. I'm not accusing you of sloppy work. Accidents happen and are often the explanation for unexpected results.
I would be very surprised if this was a case of heterozygous deletion, especially if you are observing this in more than one hybrid.
In this L X T design missing of male parent allele reveal F1 is self instead of hybrid provided all is perfect with marker data. In case of missing allele of female parent reflect problem with marker data. Female parent would definitely carry allele whether selfed or crossed ?
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