Analyses of Biofuel Related miRNAs and Their Targets in Sweet Sorghum with Identification of Relevant Gametes’ DNA Signatures

Binod Gyawali, Tennessee State University


Sweet sorghum (Sorghum bicolor L.) is highly promising feedstock for biofuel production. MicroRNAs (miRNAs) are known as key regulators in different biological processes including sugar accumulation in sweet sorghum plants. In contrast, little is known about the expression of sorghum mature candidate miRNAs and sugar accumulation related biological validation of their targets. In addition, single-cell analysis is still challenging in plants because cell wall hinders lysis to yield the nuclear contents. Through this report, a total of 475 biofuel relevant targets are predicted for the 226 conserved and novel miRNAs generated using the deep sequencing of small RNA libraries of sweet sorghum. Further quantification and expressional studies were conducted on selected 36 candidate mature miRNAs along with their 18 targets obtained from three biological replicates under several growth stages of two sweet sorghum varieties, i.e., Dale and Topper 76-6 through quantitative PCR. Altogether, seven predicted miRNAs and their 10 target mRNAs were found differentially expressed under various growth conditions in both the varieties. This study showed a positive as well as negative correlations between levels of miRNA expressions and that of their target mRNAs. Also, a physical method was developed to isolate 30 microspores per variety towards whole-genome amplification using REPLI-g single cell kit. Microsatellites or simple sequence repeats (SSR) based markers amplified through seven primer pairs were used to analyze parental leaves’ and pre-amplified microspores’ DNA samples. The segregation of parental alleles (SSRs) among microspores of both varieties were observed and checked against the expected 1:1 Mendelian ratio (χ2≥2.1, P˂0.05). Altogether, 18 parental SSR markers for Dale and 12 for Topper 76-6 variety were scored for segregation in their microspores. In Dale variety, 72.2% parental markers showed distorted segregation, while in Topper 76-6, 83.33% showed such segregation. It is noteworthy that, by observing microspores for SSR markers’ segregation, all (100%) pollen can be analyzed for efficient construction of genetic maps and gamete merit assessments while requiring limited space for breeding populations’ maintenance. This study aids in the understanding of biofuel relevant genomics of sweet sorghum through the study of small RNAs expression and single gametes’ genotyping via SSR markers.

Subject Area

Plant sciences|Alternative Energy

Recommended Citation

Binod Gyawali, "Analyses of Biofuel Related miRNAs and Their Targets in Sweet Sorghum with Identification of Relevant Gametes’ DNA Signatures" (2020). ETD Collection for Tennessee State University. Paper AAI28026373.