Genome-Wide MicroRNAs and Single Gamete Based Genetic Profiling of Sweet Sorghum

Pakistan-US Science and Technology Cooperation Program
Phase 6 (2015 Deadline)

Genome-Wide MicroRNAs and Single Gamete Based Genetic Profiling of Sweet Sorghum Varieties for Biofuel Production
US partner: Ahmad Aziz, Tennessee State University
Pak partner: Muhammad Younas Barozai, University of Balochistan, Quetta

Progress Reports

2016: Tennessee State University (TSU) and University of Baluchistan (UOB) initiated a joint project titled: “Genome-Wide microRNAs and Single Gamete Based Genetic Profiling of Sweet Sorghum Varieties for Biofuel Production” at US and Pakistan campuses, respectively. This international relationship between these two universities was a well- received initiative, as both campuses serve the underserved communities at their locations. To foster this mutual understanding, TSU campus Project Investigator visited UOB campus during summer 2016 while introducing project activities at a couple of other Pakistani institutes. The innovative protocols for single gamete isolation as well as microRNA were standardized for contemporary research on sorghum as biofuel plant. Several educational and training opportunities were also created at TSU and UOB campuses that involved both college students as well as pre-college students.

2017: Tennessee State University (TSU) and University of Baluchistan (UoB) continued a joint project titled: “Genome-Wide microRNAs and Single Gamete Based Genetic Profiling of Sweet Sorghum Varieties for Biofuel Production” at US and Pakistan campuses, respectively. Through mutual international relationship between these two campuses, the UoB Project Investigator visited TSU during summer 2017 while engaging in inter-campus project’s educational and research activities. The single gamete genotyping as well as microRNA analyses on biofuel relevant traits from US and Pakistani sweet sorghum varieties are ongoing after standardization of the innovative protocols. Several educational and training opportunities were also created at both campuses to involve pre-college, undergraduate and graduate students through project’s research activities. Multi-level research and educational activities were shared at different forums to widen the circle of project’s stakeholders.

2018: At Tennessee State University (TSU) through three biological and two technical replicates, two USA sweet sorghum (Sorghum bicolor) varieties (Dale and Topper 76-6) as well as two Pakistani sweet sorghum varieties (Dasht Local and Achi Turi) were subjected to small RNAs isolation, libraries construction and deep-sequencing using Illumina HiSeq platform at pre-flowering and flowering stages. To catalogue the potential biofuel relevant miRNAs, numerous sorghum genes sequences (searched with terms: sugar, sucrose, glucose, fructose, fatty acids, lipid, alcohol, cellulose, starch and cell wall) were download from the Phytozome- v12.1 and as a potential target of conserved and novel miRNAs were identified and subjected to psRNATarget. All the RNAseq files were submitted to NCBI-Genbank Sequence Read Archive (SRA) database with accession number SUB4324853. The analyses produced 276 miRNAs common in three tissues at vegetative and reproductive stages for all the four varieties of; while 490 were found specific to only vegetative stage and 951 were detected at only reproductive stage. A total of 450 biofuel related targets were also identified for these miRNAs while these key genes belonged to plant productivity relevant traits such as sucrose transporter (SUT), sucrose phosphate synthase (SPS) and sugar transporter SWEET (sugars will eventually be exported transporter) families. A total of 36 miRNAs and their 18 targets were randomly selected and validated through quantitative DNA amplifications. At University of Baluchistan (UoB) research focus was based on in-silico approach, and a total of 25 miRNAs were thereby identified in Sorghum as biofuel usage relevance. For these gene expression influencing small RNAs, 4247 potential target genes were also predicted; which belonged to the 142 Gene Ontology (GO) enrichment terms of biological processes, molecular functions and cellular components. These GO enrichment terms appear to be involved in physiological processes like, stress related, disease related, growth and development, metabolism, transcription factors and others. The findings of this in-silico research will enable better devising of Sorghum plants regrading biofuel relevant qualities and traits. The research results of these studies were published as a manuscript in Pakistan Journal of Botany.

Undergraduate and graduate students remained engaged at TSU campus for project’s individual gamete genotyping related research activities while making award winning presentations. The projects’ results were shared with stakeholders at the North American Community 2017 Interdisciplinary Education Conference (Ottawa-Gatineau, Canada. September 28-30, 2017), Plant and Animal Genome XXVI Conference (PAG, January 12-17, 2018, San Diego, CA, USA), Posters at the TN Capitol Event (February 19, 2018, Nashville, TN), 15th TLSAMP Annual Research Conference, Tennessee Technological University, February 22-23. 2018, Chattanooga, TN), 40th (April 2-6, 2018) TSU Annual University-Wide Research Symposium, Nashville, TN (USA), MANRRS Research Symposium, Greensboro, NC, USA. April 4-7, 2018) and July 19, 2018 Small Farm EXPO Sorghum Mini Course of Tennessee State University (Nashville, TN). Similarly, M.Sc./M.Phil./PhD students at UoB were trained for the skills of in-silico based miRNAs studies and their importance with the biofuel related target genes.