Tez özetleri Astronomi ve Uzay Bilimleri Anabilim Dalı

Determination of Retrotransposon Movements and Products in

Barley (Hordeum vulgare L.) Tissue Culture
In this study, the insertional polymorphism deriving from BAGY2 retrotransposon movement in barley (Hordeum vulgare L. cv. Golden Promise) calli and shoots due to culturing duration was investigated.
In the study, 45- and 90-day-old calli deriving from the same embryo and their regenerants were used as tissue culture samples. These samples were considered as study a group and all molecular studies were replicated with 3 study groups. At the same time, uncultured embryo (Control 1), seedling leaves germinated between filter papers (Control 2) and seedling leaves germinated on MS medium (Control 3) were used as control in the study. The experiments were replicated with 4 individuals in each control to determine if there was any naturally occurring BAGY2 polymorphism between the individuals. IRAP marker technique was employed to detect BAGY2 polymorphism. To verify the IRAP results, copy number alterations of BAGY2 internal domains between individuals were analyzed by qPCR. In addition, to confirm that all the BAGY2 internal domains were present as a complete copy, internal domains of Control 1 samples were sequenced. The obtained sequences and BAGY2 protein sequences were compared by tblastn and the similarity rates were determined.
IRAP-PCR results with control samples showed no natural BAGY2 insertional polymorphism between individuals. However, the detection of 8% polymorphism between Control 1/Control 2 and Control 1/Control 3 samples showed that the BAGY2 insertional profile might vary between different organs. 0-20% polymorphism was detected between tissue culture samples. The lower polymorphism rate in the shoots compared to the calli samples showed that the cells with more stable genome had an elevated chance to regenerate. By qPCR analyses, it was determined that the copy number of BAGY2 internal domains in calli and shoots showed alterations. These results confirmed that the polymorphic bands detected by IRAP technique could result from the increase in BAGY2 copy number. The sequencing results of BAGY2 internal domains revealed similarity rates by GAG 90%, PR 86%, RT 92%, RH 94%, and INT 92% with BAGY2 protein sequence. These results proved that all the BAGY2 internal domains were present as a complete copy in the barley variety used in the study.
The results obtained from this study indicated that tissue culture duration lead to insertional polymorphism due to the movement of BAGY2 retrotransposon and that the retrotransposons could be the source of somaclonal variation. These results might contribute to the detection of the tissue culture duration effects on insertional movements of retrotransposons. Besides, it could be a model to define the effects of tissue culture duration on retrotransposon movements in animal stem cells studies. The studies for the detection of polymorphisms derived from insertional movements of retrotransposons could provide a basis to understand the retrotransposon-based diseases.