Identification and Cloning of a Cotton Reference Gene for Increasing Efficiency of Molecular Analysis

The use of an appropriate reference gene for assessment of DNA and genome quality and analysis of transgenic crops is very important. In this research, we have evaluated the SadI gene as a potential reference gene in cotton. First, DNA was extracted, and then a PCR reaction was done using appropriative primers designed for Vector NTI. The PCR products were 623 and 544 bp fragments. The fragments were cloned in pTZ57R/T plasmid. Recombinant plasmids were identified by PCR colony hybridization with appropriative primers and M13 primers and then were sequenced. The sequencing results showed that these two fragments are related to two different copies of the SadI gene that in one of copies a 79 bp deletion had occurred in the second interon. This research is the first report from sequencing of a reference gene showing the presence of two different copies of the SadI gene, and affirming that it is a suitable reference gene for different studies.