My thesis concerned the study of the regulation of stem sugar accumulation using cultivars of sorghum as model system, and is divided into five chapters, all of them being published. The first chapter gives an account of sorghum as biofuel crop that was published in Current Opinion in Biotechnology as an invited review article.
The second chapter lays out the concept of my thesis and the discovery of genes that could play a role in the accumulation of stem sugars. The Messing lab was previously involved in the sequence analysis of the sorghum genome as the first example of the Panicoideae, the subfamily of the grasses that includes sugarcane and maize. Sorghum was chosen as reference because it has a smaller genome with 730 MB compared to 2,300 MB of the allotetraploid maize, and the 10,000 MB polyploidy/aneuploid sugarcane genome. Furthermore, sorghum displays outstanding variation for stem soluble sugars, with cultivars known as sweet sorghum harboring stem-sucrose levels comparable to those in sugarcane, and cultivars used for grain production known as grain sorghum harboring low levels of stem-sucrose.
Thus, creation of segregating populations derived from crossing sweet with grain sorghums allows for a genetic approach to study stem sugar accumulation. Therefore, I selected sorghum cultivars that differed in stem sugar levels (measured in Brix degrees) at the time of flowering (when sugar content peaked). To correlate gene expression with stem sugar content I selected grain sorghum cultivar (BTx623) and sweet sorghum cultivar (Rio), and took advantage of a commercial Affymetrix oligonucleotide array designed from 8,224 sugarcane transcripts to determine differential expression of sorghum transcripts from stem tissue. This work demonstrated the efficacy of a comparative hybridization approach between sugarcane and sorghum, two closely related species that shared a common ancestor 8-9 million years ago. Indeed, 70% of sugarcane transcripts present in the array gave a positive signal when hybridized with sorghum RNA, indicating that both species may share the same gene repertoire when it comes to stem sugar accumulation. This work was published in the journal RICE. In a follow up study, I extended the cross-species comparative hybridization experiment to use differences in hybridization intensities of RNAs from grain and sweet sorghum on the sugarcane array as means to identify and validate nucleotide polymorphisms within the transcribed regions of genes to subsequently develop molecular markers that were polymorphic among grain and sweet sorghum cultivars and could be used in mapping experiments. This work was published as a second paper in the journal RICE.
To have a more comprehensive characterization of the stem transcriptome in relation to sugar content from grain and sweet sorghum, I combined next-generation sequencing with bulk segregant analysis (BSA) in order to address differential expression of microRNAs from BTx623 and Rio, and from a pool of selected F2 plants derived from their cross that segregated for sugar content and flowering time. This allowed me to discover that increased expression of microRNA 169 from grain sorghum BTx623 was inherited in selected F2 plants displaying low sugar content. My study represented the first characterization of the small RNA component of the sorghum stem transcriptome in relation to soluble sugar content and led me to pinpoint miR169 as candidate miRNA family involved in my trait of interest. This work was published in BMC Genomics. I extended this finding from an evolutionary point of view, comparing the origin and evolution of miR169 gene copies arranged in tandem in the genomes of five grass species. This work was published in Genome Biology & Evolution.
Download PDF of PhD Thesis here:
Link To Publications:
Genome Biology & Evolution (2013):
Current Opinion in Biotechnology (2012):
BMC Genomics (2011):
GCB Bioenergy (2009):
RICE (2009):
RICE (2008):
Graduate studies at Rutgers University were possible thanks to fellowships from the Waksman Institute of Microbiology and