Characterization of a KCS-like KASII from <Emphasis Type="Italic">Jessenia bataua</Emphasis> that Elongates Saturated and Monounsaturated Stearic Acids in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

As the world population grows, the demand for food increases. Although vegetable oils provide an affordable and rich source of energy, the supply of vegetable oils available for human consumption is limited by the "fuel vs food" debate. To increase the nutritional value of vegetable oil, metabolic engineering may be used to produce oil crops of desirable fatty acid composition. We have isolated and characterized β-ketoacyl ACP-synthase II (KASII) cDNA from a high-oleic acid palm, Jessenia bataua. Jessenia KASII (JbKASII) encodes a 488-amino acid polypeptide that possesses conserved domains that are necessary for condensing activities. When overexpressed in E. coli, recombinant His-tagged JbKASII was insoluble and non-functional. However, Arabidopsis plants expressing GFP-JbKASII fusions had elevated levels of arachidic acid (C20:0) and erucic acid (C22:1) at the expense of stearic acid (C18:0) and oleic acid (C18:1). Furthermore, JbKASII failed to complement the Arabidopsis KASII mutant, fab1-2. This suggests that the substrate specificity of JbKASII is similar to that of ketoacyl-CoA synthase (KCS), which preferentially elongates stearic and oleic acids, and not palmitic acid. Our results suggest that the KCS-like JbKASII may elongate C18:0 and C18:1 to yield C20:0 and C22:1, respectively. JbKASII may, therefore, be an interesting candidate gene for promoting the production of very long chain fatty acids in transgenic oil crops.     

Production of 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid from olive oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Microbial modification of naturally occurring materials is one of the efficient ways to add new values to them. Hydroxylation of free unsaturated fatty acids by microorganism is a good example of those modifications. Among microbial strains studied for that purpose, a new bacterial isolate Pseudomonas aeruginosa PR3 has been well studied to produce several hydroxy fatty acids from different unsaturated fatty acids. Of those hydroxy fatty acids, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was efficiently produced from oleic acid by strain PR3. However, it was highly plausible to use vegetable oil containing oleic acid rather than free oleic acid as a substrate for DOD production by strain PR3. In this study, we firstly tried to use olive oil containing high content of oleic acid as a substrate for DOD production. DOD production from olive oil was confirmed by structural determination with GC, TLC, and GC/MS analysis. DOD production yield from olive oil was 53.5%. Several important environmental factors were also tested. Galactose and glutamine were optimal carbon and nitrogen sources, and magnesium ion was critically required for DOD production from olive oil. Results from this study demonstrated that natural vegetable oils containing oleic acid could be used as efficient substrate for the production of DOD by strain PR3.     

Production of a value-added hydroxy fatty acid, 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid,from high oleic safflower Oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFAs), originally obtained in small amounts from plant systems, are good examples of structurally modified lipids, and they render special properties such as higher viscosity and reactivity compared to normal fatty acids. Based on these properties, HFAs possess high industrial potential in a wide range of applications. Recently, various microbial strains were tested for the production of HFAs from different unsaturated fatty acids since HFA production is limited to plant systems. Among the microbial strains tested, Pseudomonas aeruginosa PR3 has been well studied for the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) from oleic acid. Previously, we reported that strain PR3 could utilize triolein instead of oleic acid as a substrate for the production of DOD (Appl. Microbiol. Biotechnol. 2007, 74: 301–306). In this study, we focused on utilization of vegetable oil as a substrate for DOD production by PR3. Consequently, strain PR3 efficiently utilized high oleic safflower oil as a substrate for DOD production. Optimal initial medium pH and incubation time were pH 8.0 and 72 h, respectively. Optimal carbon and nitrogen sources were fructose and glutamine, respectively. Results from this study demonstrate that normal vegetable oils could be used as efficient substrates for the production of value-added HFAs by microbial bioconversion.     

Cloning of a Novel Omega-6 Desaturase from Flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) and Its Functional Analysis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The Δ12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Δ12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids—putative membrane-bound Δ12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Δ12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence ‘YNNKL’ was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.     

An evaluation of the diet of <Emphasis Type="Italic">Mysis relicta</Emphasis> using gut contents and fatty acid profiles in lakes with and without the invader <Emphasis Type="Italic">Bythotrephes longimanus</Emphasis> (<Emphasis Type="Italic">Onychopoda,Cercopagidae</Emphasis>)

Diets of Mysis relicta from four lakes in central Ontario that had been invaded by Bythotrephes longimanus and three lakes that had not been invaded were investigated using gut content analysis and fatty acid (FA) composition. Gut content analysis of M. relicta revealed a high incidence of cannibalism in all lakes, and consumption of B. longimanus and native zooplanktivorous midges in the genus Chaoborus in lakes where these were present. Cladocera other than B. longimanus were present in the guts of all M. relicta examined except those from Bernard Lake, the lake with the most B. longimanus. In that lake, B. longimanus was the most frequent diet item. Copepod remains were found in 60–100% of M. relicta guts with the lowest frequency occurring in Bernard Lake. Fatty acids (FA) that contributed strongly to the variation in FA composition in M. relicta, as revealed by a principal component analysis, were C16:0 (palmitic acid), C16:1n7 (palmitoleic acid), C18:1n9c (oleic acid), C20:4n6 (arachidonic acid), C20:5n3 (eicosapentaenoic acid), and C22:6n3 (docosahexaenoic acid). Significant differences in FA amount and composition of M. relicta were found between invaded and non-invaded lakes, and among lakes within these groups. Generally, M. relicta in non-invaded lakes had higher concentrations of C16:0, C18:1n9c, C18:2n6c (linoleic acid), C18:3n3 (α-linolenic acid) and C20:4n6, while M. relicta in invaded lakes had higher concentrations of C22:6n3. Two of the non-invaded lakes had lower water transparency, as measured by Secchi depth, which may be the reason why mysids and abundant populations of Chaoborus spp. could be found in the water column during the day. However, differences in FA profiles and gut contents of M. relicta between invaded and non-invaded lakes are consistent with competition for Cladocera in the presence of the invader rather than pre-existing differences among lakes. We conclude that the diet of M. relicta is affected by the invasion of B. longimanus.     

Overexpression of <Emphasis Type="Italic">SsDGAT2</Emphasis> from <Emphasis Type="Italic">Sapium sebiferum</Emphasis> (L.) Roxb Increases Seed Oleic Acid Level in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Sapium sebiferum (L.) Roxb is one of the most important oil trees in China. Diacylglycerol acyltransferases (DGATs) esterify sn-1, 2-diacylglycerol with a long-chain fatty acyl-CoA, the last step and the rate-limiting step of triacylglycerol (TAG) biosynthesis in prokaryotic and eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the SsDGAT2 gene had not been reported to date. To clarify the function of SsDGAT2, we cloned the CDS (rapid amplification of cDNA end) of SsDGAT2 by RACE technology. The full-length CDS of SsDGAT2 contains 1011 bp and encodes a protein of 336 amino acids. Recombinant SsDGAT2 restored TAG biosynthesis to the yeast strain Saccharomyces cerevisiae H1246 TAG-deficient mutant and preferentially incorporated unsaturated C18 fatty acids into lipids. To investigate the biotechnological potential of SsDGAT2, it was expressed under the control of the 35S promoter in Arabidopsis Col-4. The oleic acid content increased by 50 % in transgenic plants relative to the control. The results indicated that most of the oleic acid increase was at the expense of linolenic acid (18:3) content, which suggests that high-oleic-acid-content seeds can be created by the overexpression of SsDGAT2 in S. sebiferum (L.) Roxb.     

Response of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> roots to lipo-chitooligosaccharide from <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and other chitin-like compounds

Lipo-chitooligosaccharides (LCOs) are bacteria-to-plant signals required for the establishment of rhizobia–legume nitrogen fixing symbioses. The ability of LCO [Nod Bj V (C_18:1, MeFuc)] isolated from B. japonicum (strain 532C), and of oligomers of chitosan (tetramer, pentamer) and chitin (pentamer) to affect the developmental morphology of roots in Arabidopsis thaliana (L.) Heynh ecotype Columbia (Col-0) was assessed using an interactive scanner-based image analysis system. LCOs have been shown to play a role in plant organogenesis at nanomolar concentrations. LCO and the chitin pentamer promoted root growth and development in Arabidopsis at concentrations of 10 nM and 100 μM, respectively. The LCO treated Arabidopsis plants had about 35% longer roots than untreated control plants. Similarly, treatment with 100 μM chitin pentamer (CHIT5) resulted in 26% longer roots than the untreated plants; however, chitosan oligomer (CH4 or CH5) treated plants did not differ from the control plants at either concentration (100 or 1 μM). Both LCOs and the chitin pentamer at higher concentrations increased root surface area, mean root diameter and number of root tips. However, leaf area increase was observed only in plants treated with LCO at 10 nM.     

Sophorolipid production by <Emphasis Type="Italic">Candida bombicola</Emphasis> on oils with a special fatty acid composition and their consequences on cell viability

Sophorolipids production by the yeast Candia bombicola is most favourable when glucose is used as a carbon source in combination with a hydrophobic carbon source such as a common vegetable oil. Most vegetable oils are comprised of C16–C18 fatty acids, an ideal range for sophorolipid production. The use of oils with either shorter or longer fatty acids, such has coconut oil or meadowfoam oil, respectively, was evaluated. Such oils did not contribute to enhanced sophorolipid production when compared to cultures run on glucose as the sole carbon source. Moreover, a toxic effect of medium-chain fatty acids towards stationary C. bombicola cells was demonstrated.     

Efficient accumulation of oleic acid in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> caused by expression of rat elongase 2 gene (<Emphasis Type="Italic">rELO2</Emphasis>) and its contribution to tolerance to alcohols

When the cells of Saccharomyces cerevisiae are exposed to high concentration of ethanol, the content of oleic acid (C18:1n-9) increased as the initial concentration of ethanol increased. Based on this observation, we attempted to confer ethanol tolerance to S. cerevisiae by manipulating fatty acid composition of the cells. Rather than altering OLE1 expression [the desaturase making both C16:1n-7 (palmitoleic acid) and C18:1n-9], we introduced elongase genes. Introduction of rat elongase 1 gene (rELO1) into S. cerevisiae gave cis-vaccenic acid (cis-C18:1n-7) by conversion from C16:1n-7, and the increase in this C18:1 fatty acid did not confer ethanol tolerance to the cells. On the other hand, the introduction of rat elongase 2 gene (rELO2), which elongates C16:0 to C18:0, drastically increased C18:1n-9 content, and the cells acquired ethanol tolerance, emphasizing the specific role of C18:1n-9. Furthermore, the transformant of rELO2 also conferred tolerance to n-butanol, n-propanol, and 2-propanol.     

A novel <Emphasis Type="Italic">FAD2</Emphasis>-<Emphasis Type="Italic">1 A</Emphasis> allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content

The alteration of fatty acid profiles in soybean to improve soybean oil quality has been a long-time goal of soybean researchers. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of soybean oil compared to other oils. In the lipid biosynthetic pathway, the enzyme fatty acid desaturase 2 (FAD2) is responsible for the conversion of oleic acid precursors to linoleic acid precursors in developing soybean seeds. Two genes encoding FAD2-1A and FAD2-1B were identified to be expressed specifically in seeds during embryogenesis and have been considered to hold an important role in controlling the seed oleic acid content. A total of 22 soybean plant introduction (PI) lines identified to have an elevated oleic acid content were characterized for sequence mutations in the FAD 2-1A and FAD2-1B genes. PI 603452 was found to contain a deletion of a nucleotide in the second exon of FAD2-1A. These important SNPs were used in developing molecular marker genotyping assays. The assays appear to be a reliable and accurate tool to identify the FAD 2-1A and FAD2-1B genotype of wild-type and mutant plants. PI 603452 was subsequently crossed with PI 283327, a soybean line that has a mutation in FAD2-1B. Interestingly, soybean lines carrying both homozygous insertion/deletion mutation (indel) FAD2-1A alleles and mutant FAD2-1B alleles have an average of 82–86% oleic acid content, compared to 20% in conventional soybean, and low levels of linoleic and linolenic acids. The newly identified indel mutation in the FAD2-1A gene offers a simple method for the development of high oleic acid commercial soybean varieties.     

Physiological differences in the formation of the glycolipid biosurfactants,mannosylerythritol lipids,between <Emphasis Type="Italic">Pseudozyma antarctica</Emphasis> and <Emphasis Type="Italic">Pseudozyma aphidis</Emphasis>

Vegetable oil is the usual carbon source for the production of biosurfactants (BS), mannosylerythritol lipids (MEL). To simplify the procedures of BS production and recovery, we investigated the extracellular production of MEL from water-soluble carbon sources instead of vegetable oils by using two representative yeast strains. The formation of extracellular MEL from glucose was confirmed by thin layer chromatography (TLC) and HPLC analysis. On glucose cultivation, pure MEL were easily prepared by only solvent extraction of the culture medium, different from the case of soybean oil cultivation. The fatty acid profile of the major MEL produced from glucose was similar to that produced from soybean oil based on GC–MS analysis. The resting cells of Pseudozyma antarctica T-34 produced MEL by feeding of glucose only and gave a yield of 12 g l⁻¹. In contrast, Pseudozyma aphidis ATCC 32657 gave no MEL from glucose. Moreover, the extracellular lipase activities were detected at high levels during the cultivation regardless of the carbon sources. These results indicate that all the biosynthesis pathways for MEL in P. antarctica T-34 should constitutively function. In conclusion, P. antarctica T-34 thus has potential for BS production from glucose.     

Microarray analysis of differentially expressed genes between <Emphasis Type="Italic">Brassica napus</Emphasis> strains with high- and low-oleic acid contents

An increase in oleic acid (C18:1) content is a desirable trait. Despite the critical roles of the two desaturases, FAD2 and FAD3, in the control of fatty acid desaturation, a dispute remains over whether inactivation of their genes alone is sufficient enough to generate the high-oleic trait. To address this question, we employed microarray technology to investigate the difference in gene expression profile between two different Brassica napus strains with high-C18:1 (71.71%) and low-C18:1 (55.6%) contents, respectively. Our study revealed 562 differentially expressed genes, of which 194 genes were up-regulated and 368 down-regulated. Based on the Gene Ontology classification, these genes were classified into 23 functional categories. Three of the up-regulated genes represent B. napus homologs of Arabidopsis genes encoding a cytosolic isoform of pyruvate kinase (AT3G55810), Δ9 acyl-lipid desaturase (AT1G06080, ADS1) and fatty acyl-ACP thioesterase B (AT1G08510), respectively. Conversely, the homologs of two Arabidopsis sequences encoding Δ9 acyl-lipid desaturase (AT2G31360, ADS2) and FAD3 desaturase (AT2G29980) were down-regulated in the high-oleic acid strain. Furthermore, 60 differentially expressed genes were classified as associated with relevant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Collectively, our results suggest that expressing the high-oleic acid trait may require a coordinated regulation of diverse regulatory and metabolic gene networks in addition to inactivation of the FAD2 and FAD3 genes in the oilseed. A set of the differentially expressed genes identified in this study will facilitate our efforts to tap the germplasms with the potential to express the high-oleic acid trait.     

Production of 7, 10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid from triolein via lipase induction by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFA) have gained importance because of their special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. The bacterial isolate Pseudomonas aeruginosa (PR3) was reported to produce mono-, di-, and trihydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced with high yield from oleic acid by PR3. Up to now, the substrates used for microbial HFA production were free fatty acids. However, it is possible to utilize triacylglycerides, specifically triolein containing three oleic groups, as a substrate by microbial enzyme system involved in HFA production from oleic acid. In this study we used triolein as a substrate and firstly report that triolein could be efficiently utilized by PR3 to produce DOD. Triolein was first hydrolyzed into oleic acid by the triolein-induced lipase and then the released oleic acid was converted to DOD by PR3. Results from this study demonstrated that natural vegetable oils, without being intentionally hydrolyzed, could be used as efficient substrates for the microbial production of value-added hydroxy fatty acids.     

Characterization and genetic analysis of a low-temperature-sensitive mutant, <Emphasis Type="Italic">sy</Emphasis>-<Emphasis Type="Italic">2</Emphasis>, in <Emphasis Type="Italic">Capsicum chinense</Emphasis>

A temperature-sensitive mutant of Capsicum chinense, sy-2, shows a normal developmental phenotype when grown above 24°C. However, when grown at 20°C, sy-2 exhibits developmental defects, such as chlorophyll deficiency and shrunken leaves. To understand the underlying mechanism of this temperature-dependent response, phenotypic characterization and genetic analysis were performed. The results revealed abnormal chloroplast structures and cell collapse in leaves of the sy-2 plants grown at 20°C. Moreover, an excessive accumulation of reactive oxygen species (ROS) resulting in cell death was detected in the chlorophyll-deficient sectors of the leaves. However, the expression profile of the ROS scavenging genes did not alter in sy-2 plants grown at 20°C. A further analysis of fatty acid content in the leaves showed the impaired pathway of linoleic acid (18:2) to linolenic acid (18:3). Additionally, the Cafad7 gene was downregulated in sy-2 plants. This change may lead to dramatic physiological disorder and alteration of leaf morphology in sy-2 plants by losing low-temperature tolerance. Genetic analysis of an F₂ population from a cross between C. chinense ‘sy-2’ and wild-type C. chinense ‘No. 3341’ showed that the sy-2 phenotype is controlled by a single recessive gene. Molecular mapping revealed that the sy-2 gene is located at a genomic region of the pepper linkage group 1, corresponding to the 300 kb region of the Ch1_scaffold 00106 in tomato chromosome 1. Candidate genes in this region will reveal the identity of sy-2 and the underlying mechanism of the temperature-dependent plant response.     

Accumulation of a Cocoa-Butter-Like Lipid by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Cultivated on Agro-Industrial Residues

Yarrowia lipolytica was cultivated on mixtures of saturated free fatty acids (an industrial derivative of animal fat called stearin), technical glycerol (the main by-product of bio-diesel production facilities), and glucose. The utilization of technical glycerol and stearin as co-substrates resulted in higher lipid synthesis and increased citric acid production than the combination of glucose and stearin. The lipids produced contained significant amounts of stearic acid (50–70%, wt/wt) and lower ones of palmitic (15–20%, wt/wt), oleic (7–20%, wt/wt), and linoleic (2–7%, wt/wt) acid. Single-cell oil having a composition similar to cocoa-butter up to 3.4 g/L was produced, whereas in some cases relatively increased citric acid quantities (up to 14 g/L) were excreted into the growth medium. The microorganism presented a high specificity for lauric, myristic, and palmitic acid, while a discrimination for the stearic acid was observed. As a conclusion, microbial metabolism could be directed by using mixtures of inexpensive saturated fats, glycerol, and glucose as co-substrates, in order to accumulate lipids with predetermined composition, e.g., cocoa-butter equivalents. Received: 1 April 2002 / Accepted: 4 May 2002     

Overexpression of <Emphasis Type="Italic">FAD2</Emphasis> promotes seed germination and hypocotyl elongation in <Emphasis Type="Italic">Brassica napus</Emphasis>

The enzyme fatty acid desaturase 2 (FAD2) transforms oleic acid (C18:1) to linoleic acid (C18:2) in plants and as such is involved in fatty acid synthesis. It is also involved in plant development and self-defense, such as seed germination, leaf expansion and cold resistance. We have cloned the full coding region of the Brassica napus FAD2 gene and ectopically expressed it in B. napus expressing low levels of FAD2. Overexpression of FAD2 under the control of the CaMV 35S promoter resulted in an up-regulated FAD2 mRNA level in B. napus as expected. Further analysis revealed that the FAD2 transgenic lines varied greatly in terms of their physiological characteristics, such as enhanced seed germination and increased hypocotyl length, compared to non-transgenic plants, suggesting that up-regulated FAD2 can promote seed germination and hypocotyl elongation in B. napus. Our results demonstrate the possible roles of FAD2 in plant development and also provide a platform for further analysis of fatty acid synthesis in plants.     

Characterization of long-chain fatty acid uptake in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

Studies evaluating the uptake of long-chain fatty acids in Caulobacter crescentus are consistent with a protein-mediated process. Using oleic acid (C18:1) as a substrate, fatty acid uptake was linear for up to 15 min. This process was saturable giving apparent V_max and K_m values of 374 pmol oleate transported/min/mg total protein and 61 μM oleate, respectively, consistent with the notion that one or more proteins are likely involved. The rates of fatty acid uptake in C. crescentus were comparable to those defined in Escherichia coli. Uncoupling the electron transport chain inhibited oleic acid uptake, indicating that like the long-chain fatty acid uptake systems defined in other gram-negative bacteria, this process is energy-dependent in C. crescentus. Long-chain acyl CoA synthetase activities were also evaluated to address whether vectorial acylation represented a likely mechanism driving fatty acid uptake in C. crescentus. These gram-negative bacteria have considerable long-chain acyl CoA synthetase activity (940 pmol oleoyl CoA formed/min/mg total protein), consistent with the notion that the formation of acyl CoA is coincident with uptake. These results suggest that long-chain fatty acid uptake in C. crescentus proceeds through a mechanism that is likely to involve one or more proteins.     

Validation of <Emphasis Type="Italic">DGAT1</Emphasis>-<Emphasis Type="Italic">2</Emphasis> polymorphisms associated with oil content and development of functional markers for molecular breeding of high-oil maize

The gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1-2) is a key quantitative trait locus that controls oil content and oleic acid composition in maize kernels. Here we re-sequenced the DGAT1-2 region responsible for oil variation in a maize landrace set and in 155 inbred lines (35 high-oil and 120 normal lines). The high-oil DGAT1-2 allele was present in most Northern Flint and Southern Dent populations but was absent in five of eight Corn Belt Dent open-pollinated populations and in most of the earlier inbred lines. Loss of the high-oil DGAT1-2 allele possibly resulted from genetic drift in the early twentieth century when a few Corn Belt Dent populations were selected for the development of high-grain-yield inbred lines. Association analysis detected significant effects of two PCR-based functional markers (HO06 and DGAT04; developed based on DGAT1-2 polymorphisms) on kernel oil content and oleic acid composition using the 155 inbred lines. Zheng58 and Chang7-2, the parent inbred lines of elite hybrid Zhengdan958, were used to transfer the favorable allele from the high-oil line By804 using marker-assisted backcrossing with the two functional markers. In BC5F2:3 populations, oil content of the three genotypes (−/−, +/−, and +/+) was, respectively, 3.37, 4.20, and 4.61% (Zheng58 recipient line) and 4.14, 4.67, and 5.25% (Chang7-2 recipient line). Oil content of homozygous kernels containing the high-oil DGAT1-2 allele increased by 27–37% compared with recurrent parents. Hence, these functional markers can be used to re-introduce the high-oil DGAT1-2 allele into modern inbred lines for increased oil content through marker-assisted backcrossing.