Background Genome wide association studies (GWAS) are a powerful tool for identifying quantitative trait loci (QTL) and causal single nucleotide polymorphisms (SNPs)/genes associated with various important traits in crop species. Typically, GWAS in crops are performed using a panel of inbred lines, where multiple replicates of the same inbred are measured and the average phenotype is taken as the response variable. Here we describe and evaluate single plant GWAS (sp-GWAS) for performing a GWAS on individual plants, which does not require an association panel of inbreds. Instead sp-GWAS relies on the phenotypes and genotypes from individual plants sampled from a randomly mating population. Importantly, we demonstrate how sp-GWAS can be efficiently combined with a bulk segregant analysis (BSA) experiment to rapidly corroborate evidence for significant SNPs. Results In this study we used the Shoepeg maize landrace, collected as an open pollinating variety from a farm in Southern Missouri in the 1960’s, to evaluate whether sp-GWAS coupled with BSA can efficiently and powerfully used to detect significant association of SNPs for plant height (PH). Plant were grown in 8 locations across two years and in total 768 individuals were genotyped and phenotyped for sp-GWAS. A total of 306 k polymorphic markers in 768 individuals evaluated via association analysis detected 25 significant SNPs (P ≤ 0.00001) for PH. The results from our single-plant GWAS were further validated by bulk segregant analysis (BSA) for PH. BSA sequencing was performed on the same population by selecting tall and short plants as separate bulks. This approach identified 37 genomic regions for plant height. Of the 25 significant SNPs from GWAS, the three most significant SNPs co-localize with regions identified by BSA. Conclusion Overall, this study demonstrates that sp-GWAS coupled with BSA can be a useful tool for detecting significant SNPs and identifying candidate genes. This result is particularly useful for species/populations where association panels are not readily available.

Maize (Zea mays) seeds are a good source of protein, despite being deficient in several essential amino acids. However, eliminating the highly abundant but poorly balanced seed storage proteins has revealed that the regulation of seed amino acids is complex and does not rely on only a handful of proteins. In this study, we used two complementary omics-based approaches to shed light on the genes and biological processes that underlie the regulation of seed amino acid composition. We first conducted a genome-wide association study to identify candidate genes involved in the natural variation of seed protein-bound amino acids. We then used weighted gene correlation network analysis to associate protein expression with seed amino acid composition dynamics during kernel development and maturation. We found that almost half of the proteome was significantly reduced during kernel development and maturation, including several translational machinery components such as ribosomal proteins, which strongly suggests translational reprogramming. The reduction was significantly associated with a decrease in several amino acids, including lysine and methionine, pointing to their role in shaping the seed amino acid composition. When we compared the candidate gene lists generated from both approaches, we found a nonrandom overlap of 80 genes. A functional analysis of these genes showed a tight interconnected cluster dominated by translational machinery genes, especially ribosomal proteins, further supporting the role of translation dynamics in shaping seed amino acid composition. These findings strongly suggest that seed biofortification strategies that target the translation machinery dynamics should be considered and explored further.

MS performed the experiments, wrote the manuscript, and processed and analyzed data, AY wrote the manuscript and carried out metabolic analysis, CB carried out genotyping experiments, YC analyzed data, VS analyzed data, SH carried out genotyping and metabolic analysis, EK performed GLS measurements, CK peformed initial gtr1/2 experiment, AL verified analytical methods and assisted with statistical aid, H N-E provided gtr1/2 mutants and initial analysis, DK provided all the GLS mutants and GLS related measurements from the population, RA conceived the experimental design, supervised the work, provided funding, and wrote the manuscript. All authors have reviewed the final version of the manuscript and approved it and therefore are equally responsible for the integrity and accuracy of its content.

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Free amino acids (FAAs) and protein-bound amino acids (PBAAs) in seeds play an important role in seed desiccation, longevity, and germination. However, the effect that water stress has on these two functional pools, especially when imposed during the crucial seed setting stage is unclear. To better understand these effects, we exposed Arabidopsis plants at the seed setting stage to a range of water limitation and water deprivation conditions and then evaluated physiological, metabolic, and proteomic parameters, with special focus on FAAs and PBAAs. We found that in response to severe water limitation, seed yield decreased, while seed weight, FAA, and PBAA content per seed increased. Nevertheless, the composition of FAAs and PBAAs remained unaltered. In response to severe water deprivation, however, both seed yield and weight were reduced. In addition, major alterations were observed in both FAA and proteome compositions, which indicated that both osmotic adjustment and proteomic reprogramming occurred in these naturally desiccation-tolerant organs. However, despite the major proteomic alteration, the PBAA composition did not change, suggesting that the proteomic reprogramming was followed by a proteomic rebalancing. Proteomic rebalancing has not been observed previously in response to stress, but its occurrence under stress strongly suggests its natural function. Together, our data show that the dry seed PBAA composition plays a key role in seed fitness and therefore is rigorously maintained even under severe water stress, while the FAA composition is more plastic and adaptable to changing environments, and that both functional pools are distinctly regulated.