Conclusion
Our study provides new insights into the genetic basis of oil biosynthesis in T. grandis and demonstrates that integrating RNA sequencing and TRAS is a powerful strategy to perform association study independent of a reference genome for dissecting important traits in T. grandis.
Methods
We constructed a high integrity reference sequence of expressed regions of the genome in T. grandis and performed transcriptome-referenced association study (TRAS) for 10 fatty acid and oil traits of kernels in the 170 diverse T. grandis landraces. To confirm the TRAS result, we performed functional validation and molecular biology experiments for oil significantly associated genes.
Results
We identified 41 SNPs from 34 transcripts significantly associated with 7 traits by TRAS (-log10 (P) greater than 6.0). Results showed that LOB domain-containing protein 40 (LBD40) and surfeit locus protein 1 (SURF1) may be indirectly involved in the regulation of oil and sciadonic acid biosynthesis, respectively. Moreover, overexpression of TgLBD40 significantly increased seed oil content. The nonsynonymous variant in the TgLBD40 coding region discovered by TRAS could alter the oil content in plants. Pearson's correlation analysis and dual-luciferase assay indicated that TgLBD40 positively enhanced oil accumulation by affecting oil biosynthesis pathway genes, such as TgDGAT1.