Hello, why u dont try with the [other] Arabidopsis Col0. Ask for the other labo. We will see it's u methode or u plants  

It can be just SNP (single nucleotide polymorphism) at those sites. SNP is a common phenomenon in many organisms, including human beings. They may or may not cause phenotypic change.

Hi,

Have you checked what kind of mutations are those? If they are in the CDS and are silent mutations, they could be as suggested, just SNPs. Also, the sequence of the Arabidopsis Col-0 is not necessarily perfect because sequencing methods have also a chance for error (now and then I have found divergences from my cloned sequences), so as long as the base change is not doing something clear, your clones might as well be right. If not, I agree, I would try with another Col-0 from a different lab (that's why when you ask for a transgenic line, it is highly recommended to ask also for its wild-type background, because mutations can be fixed in labs, like it occurs in nature).

Cheers,

Ruben

Hi, I have checked these mutations. They indeed change amino acids. They are in exon of  CDS. However, I don't know whether these are silent mutations. How to make sure about this? 

Hi,

If the change of aminoacid is to a similar one it could be less of an issue in terms of protein functionatily. Other possibility is that STOP codons are generated and the protein interrupted.

I would do as Fan Su suggest and try with a Col0 from a different lab, if you want to use the reference Col0. Otherwise, if they are indeed SNPs as suggested by Yuan-Yeu you could just have a variant of the protein that occurs naturally, so you could go ahead. Just make sure that in the publication you submit the sequence you cloned and describe the base changes.

Cheers,

Ruben

Hi actually all,

there are a few things mixed up in the previous answers and in the question as well.

1) The Col-0 sequence is one of the highest quality of all genome sequences available across all species. Nevertheless, there will be few errors and therefore, any segment checked by detailed Sanger sequencing could identify such an sequencing error.

2) However, these have to be distinguished from SNPs: if there are true SNPs (since there are more as you describe, I rather conclude that these are true differences and not sequenicing errors), then you are no longer dealing with Col-0 (not as classified above as a Col with SNP: this is misleading statement, then it is no longer Col!), but with another accession or contamination. As suggested above, just get Col-0 from the stock center, from another lab, ...

3) How to check whether the mutations are silent (this has nothing to do whether it is Col or another accessions: if there are true mutations, then it wil not be Col anymore, although indeed every generation is accumulating mutations as hs been measured; nevertheless, it is unlikely that you stumble upon those)? In fact, you already checked: since the mutations lead to changes in amino acid, they are not silent (mutation without  changing amino acids is meaning a silent mutation).

Hope this clarifies a few issues,

Good luck,

Tony Schaeffner

Munich,Germany