Identifying loci controlling total starch content of leaf in Nicotiana tabacum through genome-wide association study

Starch is an important primary metabolite in plants, which can provide bioenergy for fuel ethanol production. There are many studies focusing on starch metabolism in Arabidopsis, maize, and rice, but few reports have been made on the starch content of tobacco leaves. Hence, to identify the marker-trait associations and isolate the candidate genes related to starch content of tobacco leaf, the genome-wide association study (GWAS) was performed using a multiparent advanced generation intercross (MAGIC) population consisting of 276 accessions genotyped by a 430 K SNP array. In this study, we detected the leaf starch content of tobacco plants cultivated in two places (Zhucheng and Chenzhou), which showed a wide variation of starch content in the population. A total of 28 and 45 significant single-nucleotide polymorphism (SNP) loci associated with leaf starch content were identified by single-locus and multi-locus GWAS models, respectively, and the phenotypic variance explained by these loci varied from 1.80 to - 14.73%. Furthermore, among these quantitative trait loci (QTLs), one SNP, AX-106011713 located on chromosome 19, was detected repeatedly in multiple models and two environments, which was selected for linkage disequilibrium (LD) analysis to obtain the target candidate region. Through gene annotation, haplotype, and gene expression analysis, two candidate genes encoding E3 ubiquitin-protein ligase (Ntab0823160) and fructose-bisphosphate aldolase (Ntab0375050) were obtained. Results showed that the variety carrying the beneficial alleles of the two candidate genes had higher gene expression level and leaf starch content, suggesting the potential role of candidate genes in enhancing the level of tobacco leaf starch content. Furthermore, silencing of Ntab0823160 in tobacco leaves reduced the content of total starch to 39.41-69.75% of that in the wide type plants. Taken together, our results provide useful resources for further investigation of the starch metabolic pathway and are also beneficial for the creation of eco-friendly cultivars with increased accumulation of leaf starch content.