# Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule
## 4 Materials and Methods
### Statistical Analysis  
Quantitative variables are expressed as the arithmetic means \(\pm\) standard deviation (SD) or standard error (SE) as specified. A two-tailed paired Student \(t\)-test was used to measure the significance of difference between transgenic lines and wildtype, as presented by \(p\)-value. Multivariate analysis was performed to explore the relationships of precursor and intermediate compounds involved in natural rubber and squalene biosynthesis across all control and transgenic lines with different tissue. Measurements were pre-treated by auto-scaling. [PERSON]'s correlation-based hierarchical cluster analysis was used to group variants by metabolites and individual plant lines (tissue and genotype), respectively. [PERSON]'s correlation analysis was also used to explore univariate associations between each pair of individual studied metabolites. Correlation coefficients with \(p\)-values \(<\) 0.05 were considered significant.  
**Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/metabo12040303/s1](https://www.mdpi.com/article/10.3390/metabo12040303/s1)]([https://www.mdpi.com/article/10.3390/metabo12040303/s1](https://www.mdpi.com/article/10.3390/metabo12040303/s1)), Figure S1: Isoprenoid pathway schematic with squalene synthase downregulation; Figure S2: Growth phenotypes of WT guayule and SQS transgenic lines; Figure S3: Correlation matrix heatmap of precursor and intermediate compounds involved in natural rubber and squalene biosynthesis; Table S1: Metabolites content (\(\mathrm{\SIUnitSymbolMicro g}/\mathrm{\SIUnitSymbolDegree}\)) of 5-month-old guayule tissues.  
Conceptualization, M.W., N.D. and C.M.; methodology, N.D., D.P., B.A., T.K. and E.E.K.B.; formal analysis, D.P., B.A., C.D., G.P., E.E.K.B. and C.M; investigation, N.D., D.P., B.A., T.K. and E.E.K.B.; data curation, C.D.; writing--original draft preparation, D.P.; writing--review and editing, C.D., G.P., E.E.K.B. and C.M.; project administration, C.M. All authors have read and agreed to the published version of the manuscript.  
Research was supported by the United States Department of Agriculture, Agricultural Research Service. This work was also supported by the Joint BioEnergy Institute, which is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research under contract no. DE-AC02-05 CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.  
Not applicable.  
All data presented in this study is available in the article and supplementary material. In addition, the analysis code that supports the findings of this study is available at GitHub [[https://github.com/chenchen-dong/Squalene_Plots](https://github.com/chenchen-dong/Squalene_Plots)]([https://github.com/chenchen-dong/Squalene_Plots](https://github.com/chenchen-dong/Squalene_Plots)) (accessed on 28 March 2022).  
The authors are grateful for the skilled technical assistance of [PERSON], USDA-ARS, for the care of plants and cultures. Technical support by [PERSON] and [PERSON], both of USDA-ARS, is also appreciated.  
The authors declare no conflict of interest.