Multi-omics-driven advances in the understanding of triacylglycerol biosynthesis in oil seeds.

Vegetable oils are rich sources of polyunsaturated fatty acids and energy as well as valuable sources of human food, animal feed, and bioenergy. Triacylglycerols, which are comprised of three fatty acids attached to a glycerol backbone, are the main component of vegetable oils. Here, we review the development and application of multiple-level omics in major oilseeds and emphasize the progress in the analysis of the biological roles of key genes underlying seed oil content and quality in major oilseeds. Finally, we discuss future research directions in functional genomics research based on current omics and oil metabolic engineering strategies that aim to enhance seed oil content and quality, and specific fatty acids components according to either human health needs or industrial requirements.

A reactive oxygen species burst causes haploid induction in maize.

Haploid induction (HI) is an important tool in crop breeding. Phospholipase A1 (ZmPLA1)/NOT LIKE DAD (NLD)/MATRILINEAL (MTL) is a key gene controlling HI in maize; however, the underlying molecular mechanism remains unclear. In this study, to dissect why loss of ZmPLA1 function could mediate HI we performed a comprehensive multiple omics analysis of zmpla1 mutant anthers by integrating transcriptome, metabolome, quantitative proteome, and protein modification data. Functional classes of significantly enriched or differentially abundant molecular entities were found to be associated with the oxidative stress response, suggesting that a reactive oxygen species (ROS) burst plays a critical role in HI. In support of this, we further discovered that a simple chemical treatment of pollen with ROS reagents could lead to HI. Moreover, we identified ZmPOD65, which encodes a sperm-specific peroxidase, as a new gene controlling HI. Taken together, our study revealed a likely mechanism of HI, discovered a new gene controlling HI, and created a new method for HI in maize, indicating the importance of ROS balance in maintaining normal reproduction and providing a potential route to accelerate crop breeding.

Genetic variants and underlying mechanisms influencing variance heterogeneity in maize.

Traditional genetic studies focus on identifying genetic variants associated with the mean difference in a quantitative trait. Because genetic variants also influence phenotypic variation via heterogeneity, we conducted a variance-heterogeneity genome-wide association study to examine the contribution of variance heterogeneity to oil-related quantitative traits. We identified 79 unique variance-controlling single nucleotide polymorphisms (vSNPs) from the sequences of 77 candidate variance-heterogeneity genes for 21 oil-related traits using the Levene test (P < 1.0 × 10-5 ). About 30% of the candidate genes encode enzymes that work in lipid metabolic pathways, most of which define clear expression variance quantitative trait loci. Of the vSNPs specifically associated with the genetic variance heterogeneity of oil concentration, 89% can be explained by additional linked mean-effects genetic variants. Furthermore, we demonstrated that gene × gene interactions play important roles in the formation of variance heterogeneity for fatty acid compositional traits. The interaction pattern was validated for one gene pair (GRMZM2G035341 and GRMZM2G152328) using yeast two-hybrid and bimolecular fluorescent complementation analyses. Our findings have implications for uncovering the genetic basis of hidden additive genetic effects and epistatic interaction effects, and we indicate opportunities to stabilize efficient breeding and selection of high-oil maize (Zea mays L.).

Leveraging GWAS data to identify metabolic pathways and networks involved in maize lipid biosynthesis.

Maize (Zea mays mays) oil is a rich source of polyunsaturated fatty acids (FAs) and energy, making it a valuable resource for human food, animal feed, and bio-energy. Although this trait has been studied via conventional genome-wide association study (GWAS), the single nucleotide polymorphism (SNP)-trait associations generated by GWAS may miss the underlying associations when traits are based on many genes, each with small effects that can be overshadowed by genetic background and environmental variation. Detecting these SNPs statistically is also limited by the levels set for false discovery rate. A complementary pathways analysis that emphasizes the cumulative aspects of SNP-trait associations, rather than just the significance of single SNPs, was performed to understand the balance of lipid metabolism, conversion, and catabolism in this study. This pathway analysis indicated that acyl-lipid pathways, including biosynthesis of wax esters, sphingolipids, phospholipids and flavonoids, along with FA and triacylglycerol (TAG) biosynthesis, were important for increasing oil and FA content. The allelic variation found among the genes involved in many degradation pathways, and many biosynthesis pathways leading from FAs and carbon partitioning pathways, was critical for determining final FA content, changing FA ratios and, ultimately, to final oil content. The pathways and pathway networks identified in this study, and especially the acyl-lipid associated pathways identified beyond what had been found with GWAS alone, provide a real opportunity to precisely and efficiently manipulate high-oil maize genetic improvement.

Single nucleus sequencing reveals spermatid chromosome fragmentation as a possible cause of maize haploid induction.

Production of maternal haploids using a conspecific haploid inducer is routine and highly efficient in maize. However, the underlying mechanism of haploid induction (HI) is unclear. We develop a method to isolate three nuclei from a pollen grain and four microspores from a tetrad for whole-genome sequencing. A high rate of aneuploidy is observed at the three-nucleus stage (6/22 pollens) rather than at the tetrad stage (1/72 microspores) in one HI line CAU5. Frequent aneuploidy is also observed in another two inducer lines, but not in two regular lines, which implies that HI may be associated with pollen aneuploidy. We further sequence the individual embryos and endosperms of 88 maize kernels crossing between regular and inducer lines. Genome-wide elimination of the CAU5-derived chromosome is identified in eight of 81 embryos. Together, these results suggest that continuous chromosome fragmentation occurring post meiosis in the gametophyte may cause haploidy of the embryo.

Broadening Our Portfolio in the Genetic Improvement of Maize Chemical Composition.

The adoption of recombinant inbred line and introgression line populations, as well as the study of association mapping panels, has greatly accelerated our ability to identify the genes underlying plant phenotypic variance. In tandem, the development of metabolomics approaches has greatly enhanced our ability to comprehensively define cellular chemical composition. As a consequence, breeding for chemical composition is being extended beyond our traditional targets of oil and protein to include components such as essential amino acids, vitamins, and antioxidant secondary metabolites with considerable purported consequences for human health. Here, we review the above-mentioned developments paying particular attention to the genetic architecture of metabolic traits as well as updating the perspective for utilizing metabolomics in maize improvement.

Multiple loci not only Rf3 involved in the restoration ability of pollen fertility, anther exsertion and pollen shedding to S type cytoplasmic male sterile in maize.

KEY MESSAGE: Thirty loci for fertility restoration of pollen fertility, anther exsertion and pollen shedding to maize CMS-S were identified by GWAS. S type cytoplasmic male sterile (CMS-S) is the main type of CMS in maize; poor understanding of the genetic architecture of fertility restoration to CMS-S is one of the reasons to impede its utility in hybrid breeding. In this study, genome-wide identification of genetic loci for fertility restoration ability to CMS-S was firstly conducted with a set of testcrossing association mapping panel in three environments. A total of 19, 3 and 8 significant loci (P < 1.8 × 10(-6), α = 1) for pollen fertility, anther exsertion and pollen shedding were identified, respectively, and individual locus explained up to 28.26% of phenotypic variation. Of them, only Rf3, the main restorer-fertility gene of CMS-S, was identified for the three traits simultaneously. In addition, 83 candidate genes within the 100 kb extension regions of these loci were predicted. These results revealed that besides Rf3 multiple genetic loci and mechanisms are involved in the fertility restoration ability to CMS-S. Results in this study would provide important information for understanding the genetic architecture of fertility restoration to CMS-S in maize.

Dissecting meiotic recombination based on tetrad analysis by single-microspore sequencing in maize.

Meiotic recombination drives eukaryotic sexual reproduction and the generation of genome diversity. Tetrad analysis, which examines the four chromatids resulting from a single meiosis, is an ideal method to study the mechanisms of homologous recombination. Here we develop a method to isolate the four microspores from a single tetrad in maize for the purpose of whole-genome sequencing. A high-resolution recombination map reveals that crossovers are unevenly distributed across the genome and are more likely to occur in the genic than intergenic regions, especially common in the 5'- and 3'-end regions of annotated genes. The direct detection of genomic exchanges suggests that conversions likely occur in most crossover tracts. Negative crossover interference and weak chromatid interference are observed at the population level. Overall, our findings further our understanding of meiotic recombination with implications for both basic and applied research.

Genome-wide association study dissects the genetic architecture of oil biosynthesis in maize kernels.

Maize kernel oil is a valuable source of nutrition. Here we extensively examine the genetic architecture of maize oil biosynthesis in a genome-wide association study using 1.03 million SNPs characterized in 368 maize inbred lines, including 'high-oil' lines. We identified 74 loci significantly associated with kernel oil concentration and fatty acid composition (P < 1.8 × 10(-6)), which we subsequently examined using expression quantitative trait loci (QTL) mapping, linkage mapping and coexpression analysis. More than half of the identified loci localized in mapped QTL intervals, and one-third of the candidate genes were annotated as enzymes in the oil metabolic pathway. The 26 loci associated with oil concentration could explain up to 83% of the phenotypic variation using a simple additive model. Our results provide insights into the genetic basis of oil biosynthesis in maize kernels and may facilitate marker-based breeding for oil quantity and quality.

A transposable element insertion within ZmGE2 gene is associated with increase in embryo to endosperm ratio in maize.

Most of the maize kernel oil is located in the embryo while the majority of starch is located in the endosperm. Maize kernel composition and value are affected significantly by the ratio of the embryo size to the endosperm size; however, the genetic regulation of embryo to endosperm ratio (EER) in maize is unknown. Here we identified ZmGE2 gene, which encodes a cytochrome p450 protein, as a gene associated with EER variation in maize. We first expressed rice Giant Embryo (GE) gene driven by oleosin promoter in maize and detected a 23.2 % reduction in EER in transgenic seeds, demonstrating the existence of evolutionarily conserved mechanisms for EER determination in rice and maize. We next identified maize GE2, a homolog of rice GE sharing 70 % identity in amino sequence, as a candidate based on the similar expression pattern and co-localization with a previously detected QTL for EER. Followed by linkage and association mapping, a 247-bp transposable element (TE) insertion in 3'-untranslated region of ZmGE2 gene was identified to be associated with increase in EER and kernel oil content. Expression level of the favorable ZmGE2 allele containing the 247-bp TE insertion was strongly reduced. In addition, the 247-bp TE insertion site was a selection target during the artificial long-term selection for the high EER trait in a high oil population. This is the first report that demonstrates an association of ZmGE2 with EER variation in maize and identifies ZmGE2 gene as a promising target for manipulation of EER and grain composition by either transgenic approach or molecular breeding in maize.

Characterization of QTL for oil content in maize kernel.

Kernel oil content in maize is a complex quantitative trait. Phenotypic variation in kernel oil content can be dissected into its component traits such as oil metabolism and physical characteristics of the kernel, including embryo size and embryo-to-endosperm weight ratio (EEWR). To characterize quantitative trait loci (QTL) for kernel oil content, a recombinant inbred population derived from a cross between normal line B73 and high-oil line By804 was genotyped using 228 molecular markers and phenotyped for kernel oil content and its component traits [embryo oil content, embryo oil concentration, EEWR, embryo volume, embryo width, embryo length, and embryo width-to-length ratio (EWLR)]. A total of 58 QTL were identified for kernel oil content and its component traits in 26 genomic regions across all chromosomes. Eight main-effect QTL were identified for kernel oil content, embryo oil content, embryo oil concentration, EEWR, embryo weight, and EWLR, each accounting for over 10 % of the phenotypic variation in six genomic regions. Over 90 % of QTL identified for kernel oil content co-localized with QTL for component traits, validating their molecular contribution to kernel oil content. On chromosome 1, the QTL that had the largest effect on kernel oil content (qKO1-1) was associated with embryo width; on chromosome 9, the QTL for kernel oil content (qKO9) was related to EEWR (qEEWR9). Embryo oil concentration and embryo width were identified as the most important component traits controlling the second largest QTL for kernel oil content on chromosome 6 (qKO6) and a minor QTL for kernel oil content on chromosome 5 (qKO5-2), respectively. The dissection of kernel oil QTL will facilitate future cloning and/or functional validation of kernel oil content, and help to elucidate the genetic basis of kernel oil content in maize.

A genome-wide survey of maize lipid-related genes: candidate genes mining, digital gene expression profiling and co-location with QTL for maize kernel oil.

Lipids play an important role in plants due to their abundance and their extensive participation in many metabolic processes. Genes involved in lipid metabolism have been extensively studied in Arabidopsis and other plant species. In this study, a total of 1003 maize lipid-related genes were cloned and annotated, including 42 genes with experimental validation, 732 genes with full-length cDNA and protein sequences in public databases and 229 newly cloned genes. Ninety-seven maize lipid-related genes with tissue-preferential expression were discovered by in silico gene expression profiling based on 1984483 maize Expressed Sequence Tags collected from 182 cDNA libraries. Meanwhile, 70 QTL clusters for maize kernel oil were identified, covering 34.5% of the maize genome. Fifty-nine (84%) QTL clusters co-located with at least one lipid-related gene, and the total number of these genes amounted to 147. Interestingly, thirteen genes with kernel-preferential expression profiles fell within QTL clusters for maize kernel oil content. All the maize lipid-related genes identified here may provide good targets for maize kernel oil QTL cloning and thus help us to better understand the molecular mechanism of maize kernel oil accumulation.

Major and minor QTL and epistasis contribute to fatty acid compositions and oil concentration in high-oil maize.

High-oil maize is a useful genetic resource for genomic investigation in plants. To determine the genetic basis of oil concentration and composition in maize grain, a recombinant inbred population derived from a cross between normal line B73 and high-oil line By804 was phenotyped using gas chromatography, and genotyped with 228 molecular markers. A total of 42 individual QTL, associated with fatty acid compositions and oil concentration, were detected in 21 genomic regions. Five major QTL were identified for measured traits, one each of which explained 42.0% of phenotypic variance for palmitic acid, 15.0% for stearic acid, 27.7% for oleic acid, 48.3% for linoleic acid, and 15.7% for oil concentration in the RIL population. Thirty-six loci were involved in 24 molecular marker pairs of epistatic interactions across all traits, which explained phenotypic variances ranging from 0.4 to 6.1%. Seven of 18 mapping candidate genes related to lipid metabolism were localized within or were close to identified individual QTL, explaining 0.7-13.2% of the population variance. These results demonstrated that a few major QTL with large additive effects could play an important role in attending fatty acid compositions and increasing oil concentration in used germplasm. A larger number of minor QTL and a certain number of epistatic QTL, both with additive effects, also contributed to fatty acid compositions and oil concentration.

Comparison of nutritional traits variability in selected eighty-seven inbreds from Chinese maize (Zea mays L.) germplasm.

Among cereals, only maize has not only a high amount of carotenoids, tocopherols, and oil content but also is rich in starch and protein content compared with other major food crops, such as rice and wheat. The present investigation was made primarily to assess the genetic variability for nutritionally important traits in 87 elite maize inbreds representing major heterotic groups in China. Carotenoid and tocopherol fractions were measured by high-performance liquid chromatography (HPLC), whereas oil, starch, and protein contents were detected by a VECTER22/N near-infrared analyzer. Significant interactions between genotypes and years were observed for all the traits. The pooled mean values of beta-carotene, beta-cryptoxanthin, alpha-carotene, lutein, zeaxanthin, and total carotenoids were 0.449, 0.876, 0.121, 5.803, 3.048, and 10.298 microg g (-1), respectively, whereas the combined mean performance of alpha-tocopherol, gamma-tocopherol, delta-tocopherol, and total tocopherols were 23.98, 32.90, 2.189, and 59.55 microg g (-1), respectively. The average protein, starch, and oil contents were observed to be 12.28, 64.51, and 3.55%, respectively. High level of heritability estimates were observed for all the traits and ranged from 65.6% (protein content) to 92.5% (alpha/gamma-tocopherol ratio). Most of the traits studied in this experiment were either significantly positive correlated or independent. The present finding exhibits substantial opportunities to the breeders for improvement of these traits in maize cultivars and also suggests further exploration of a new source of elite breeding stocks containing a high level of these nutritionally important compounds. Finally, these findings may also help in biofortification of maize.