Kimberly Clemons and Dr. Joshua Udall, Plant and Wildlife Sciences
Introduction
The purpose of the copy number variation (CNV) project was to determine if there is a variation in the DNA content between tissue types of Gossypium hirsutum and Gossympium barbadense. Specifically if the cotton fiber cells contain CNV that contribute to the increased output of cellulose and other proteins that promote its rapid elongation.
Methodology
4 allotetraploid cotton lines, 2 from G. barbadense and 2 from G. hirsutum were going to be re-sequencing to look for CNV. Plants from G. hirsutum were selected to start with as they were blooming more frequently. They were tagged in the green house and when blooms were ready they were pollinated with their own pollen and allowed to grow for 5-8 days. After that point they were contained in a -80 degree freezer until enough pollen could be separated from the seeds for DNA to be extracted. Using Qiagen DNeasy Plant Mini Kits we extracted the DNA from leaf and fiber tissues. The DNA would be quantified using PicoGreen and normalized with 1XTE buffer. The DNA would then be used to create Illumina TruSeq DNA libraries and validated using Aligent DNA 7500 chips. The sequences would be mapped against the genome reference sequence which would show which region of the genome is being amplified in the cotton fibers. We expected that cotton leaf tissue to have a uniform coverage ever but in the cotton fibers due to CNV (Fig 2A and Fig 2B).
Results & Discussion
The project was started in February when the grant was awarded. Cotton and leaf tissue was collected from the green house and extracted once enough fibers were collected. DNA extractions from the young fibers did not yield the quality DNA that was required to make an Illumina library. Due to the time required to collect enough cotton fiber tissue from the same plant it took much of my remaining time at BYU to collect everything. I graduated in April and taught a new technician where the project was and how to proceed. In recent contact with the Udall lab the project hasn’t made progress but Dr. Udall would like to have a technician pick it up again following semester and finish it to completion.
Conclusion
Because of the difficulty in extracting quality DNA from the young fiber tissues the project had not reached a conclusion yet. We still think that there is a CNV event that occurs while fibers are rapidly elongating.
Scholarly Sources
Page JT, Huynh M, Liechty Z, Grupp K, Stelly D, Hulse AM, Ashrafi H, Van Deynze A, Wendel JF, Udall JA. 2013b. Insights into the Evolution of Cotton Diploids and Polyploids from Whole-Genome Re-sequencing. G3: Genes, Genomes, and Genomics. 3:1809-1818.