8(R)-Hydroxyeicosapentaenoic acid (8R-HEPE) induces transcription of cholesterol efflux receptors via activation of liver X receptor in macrophages.

Dyslipidemia is a risk factor for the development of atherosclerotic cardiovascular disease. 8-Hydroxyeicosapentaenoic acid (8-HEPE) from North Pacific krill (Euphausia pacifica) is known to reduce plasma low-density lipoprotein (LDL) cholesterol levels and increase plasma high-density lipoprotein cholesterol levels in LDL receptor knock-out mice fed a western diet. Moreover, 8-HEPE also reduces the area of aortic atherosclerosis in apoE knock-out mice fed the same diet. In this study, we examined the stereochemical-specific activity of 8-HEPE for inducing expression of cholesterol efflux receptors (Abca1 and Abcg1) in J774.1 cells. Our findings show 8R-HEPE induces the expression of Abca1 and Abcg1 via activation of liver X receptor, whereas 8S-HEPE elicits no such activity. These results suggest that 8R-HEPE derived from North Pacific krill may have beneficial effects against dyslipidemia.

Conserved fungal effector suppresses PAMP-triggered immunity by targeting plant immune kinases.

Plant pathogens have optimized their own effector sets to adapt to their hosts. However, certain effectors, regarded as core effectors, are conserved among various pathogens, and may therefore play an important and common role in pathogen virulence. We report here that the widely distributed fungal effector NIS1 targets host immune components that transmit signaling from pattern recognition receptors (PRRs) in plants. NIS1 from two Colletotrichum spp. suppressed the hypersensitive response and oxidative burst, both of which are induced by pathogen-derived molecules, in Nicotiana benthamianaMagnaporthe oryzae NIS1 also suppressed the two defense responses, although this pathogen likely acquired the NIS1 gene via horizontal transfer from Basidiomycota. Interestingly, the root endophyte Colletotrichum tofieldiae also possesses a NIS1 homolog that can suppress the oxidative burst in N. benthamiana We show that NIS1 of multiple pathogens commonly interacts with the PRR-associated kinases BAK1 and BIK1, thereby inhibiting their kinase activities and the BIK1-NADPH oxidase interaction. Furthermore, mutations in the NIS1-targeting proteins, i.e., BAK1 and BIK1, in Arabidopsis thaliana also resulted in reduced immunity to Colletotrichum fungi. Finally, M. oryzae lacking NIS1 displayed significantly reduced virulence on rice and barley, its hosts. Our study therefore reveals that a broad range of filamentous fungi maintain and utilize the core effector NIS1 to establish infection in their host plants and perhaps also beneficial interactions, by targeting conserved and central PRR-associated kinases that are also known to be targeted by bacterial effectors.

A nuclear protein NsiA from Epichloë festucae interacts with a MAP kinase MpkB and regulates the expression of genes required for symbiotic infection and hyphal cell fusion.

The endophytic fungus Epichloë festucae systemically colonizes the intercellular spaces of cool-season grasses to establish a mutualistic symbiosis. Hyphal growth of the endophyte within the host plant is tightly regulated and synchronized with the growth of the host plant. A genetic screen to identify symbiotic genes identified mutant FR405 that had an antagonistic interaction with the host plant. Perennial ryegrass infected with the FR405 mutant were stunted and underwent premature senescence and death. The disrupted gene in FR405 encodes a nuclear-localized protein, designated as NsiA for nuclear protein for symbiotic infection. Like previously isolated symbiotic mutants the nsiA mutant is defective in hyphal cell fusion. NsiA interacts with Ste12, a C2H2 zinc-finger transcription factor, and a MAP kinase MpkB. Both are known as essential components for cell fusion in other fungal species. In E. festucae, MpkB, but not Ste12, is essential for cell fusion. Expression of several genes required for cell fusion and symbiosis, including proA/adv-1, pro41/ham-6, ham7, ham8, and ham9 were downregulated in the nsiA mutant. However, the NsiA ortholog in Neurospora crassa was not essential for hyphal cell fusion. These results demonstrate that the roles of NsiA and Ste12 orthologs in hyphal cell fusion are distinctive between fungal species.

Lineage-specific gene acquisition or loss is involved in interspecific hybrid sterility in rice.

Understanding the genetic basis of reproductive barriers between species has been a central issue in evolutionary biology. The S1 locus in rice causes hybrid sterility and is a major reproductive barrier between two rice species, Oryza sativa and Oryza glaberrima The O. glaberrima-derived allele (denoted S1g) on the S1 locus causes preferential abortion of gametes with its allelic alternative (denoted S1s) in S1g/S1s heterozygotes. Here, we used mutagenesis and screening of fertile hybrid plants to isolate a mutant with an allele, S1mut, which does not confer sterility in the S1mut/S1g and S1mut/S1s hybrids. We found that the causal mutation of the S1mut allele was a deletion in the peptidase-coding gene (denoted "SSP") in the S1 locus of O. glaberrima No orthologous genes of SSP were found in the O. sativa genome. Transformation experiments indicated that the introduction of SSP in carriers of the S1s allele did not induce sterility. In S1mut/S1s heterozygotes, the insertion of SSP led to sterility, suggesting that SSP complemented the loss of the functional phenotype of the mutant and that multiple factors are involved in the phenomenon. The polymorphisms caused by the lineage-specific acquisition or loss of the SSP gene were implicated in the generation of hybrid sterility. Our results demonstrated that artificial disruption of a single gene for the reproductive barrier creates a "neutral" allele, which facilitates interspecific hybridization for breeding programs.

Sat-BSA: an NGS-based method using local de novo assembly of long reads for rapid identification of genomic structural variations associated with agronomic traits.

Advances in next generation sequencing (NGS)-based methodologies have accelerated the identifications of simple genetic variants such as point mutations and small insertions/deletions (InDels). Structural variants (SVs) including large InDels and rearrangements provide vital sources of genetic diversity for plant breeding. However, their analysis remains a challenge due to their complex nature. Consequently, novel NGS-based approaches are needed to rapidly and accurately identify SVs. Here, we present an NGS-based bulked-segregant analysis (BSA) technique called Sat-BSA (SVs associated with traits) for identifying SVs controlling traits of interest in crops. Sat-BSA targets allele frequencies at all SNP positions to first identify candidate genomic regions associated with a trait, which is then reconstructed by long reads-based local de novo assembly. Finally, the association between SVs, RNA-seq-based gene expression patterns and trait is evaluated for multiple cultivars to narrow down the candidate genes. We applied Sat-BSA to segregating F2 progeny obtained from crosses between turnip cultivars with different tuber colors and successfully isolated two genes harboring SVs that are responsible for tuber phenotypes. The current study demonstrates the utility of Sat-BSA for the identification of SVs associated with traits of interest in species with large and heterozygous genomes.

Euphausia pacifica as a source of 8(R)-hydroxy-eicosapentaenoic acid (8R-HEPE), 8(R)-hydroxy-eicosatetraenoic acid (8R-HETE) and 10(R)-hydroxy-docosahexaenoic acid (10R-HDHA).

Although not fully investigated, 8-HEPE, 8-HETE, and 10-HDHA have potentially beneficial effects for human health. Euphausia pacifica (North Pacific krill) is unique in containing several ppm level of 8R-HEPE, and sub-ppm levels of 8R-HETE and 10R-HDHA. Obtaining sufficient quantities of these compounds is a major bottleneck for conducting in vivo experiments to evaluate their biological activities. In this study, we examined an efficient way of obtaining 8R-HEPE, 8R-HETE, and 10R-HDHA by enzymatic production in E. pacifica. We devised a novel method to purify 199.4 mg of 8R-HEPE, 2.1 mg of 8R-HETE and 5.6 mg of 10R-HDHA from 1 kg of E. pacifica. We identified the stereochemistry of the hydroxy group at C-8 of HEPE and HETE and C-10 of HDHA as the R configuration by chiral column chromatography analysis using LC/QTOFMS.Abbreviations: 8-HEPE: 8-hydroxy-eicosapentaenoic acid; 8-HETE: 8-hydroxy-eicosatetraenoic acid; 10-HDHA: 10-hydroxy-docosahexaenoic acid; EPA: eicosapentaenoic acid; TLC-FID, thin layer chromatograph-Flame Ionization Detector; LC/QTOFMS: liquid chromatography/hybrid quadrupole time of flight mass spectrometry.

Effect of fatty acids on melanogenesis and tumor cell growth in melanoma cells.

Fatty acids have various physiological effects on melanoma. For example, palmitic acid (PA) increases melanin levels; linoleic acid and DHA decrease melanin levels; and DHA suppresses tumor growth. In this study, we focused on the relationship between the structure of fatty acids and their physiological effects in melanoma to examine the likely mechanisms of action. We showed that saturated fatty acids and PUFAs display opposing effects on melanin content in melanoma cells. Likewise, PA and EPA have opposing effects in terms of actin polymerization. Our findings suggest that PA and EPA change melanin content in melanoma to alter melanosome trafficking by modulating actin polymerization. Here, we also examined the mechanism of the anti-tumor effect of DHA. We found that DHA interacts with receptor for activated C kinase 1 and represses melanoma cell proliferation by suppressing protein kinase C signaling. Our results suggest a new mechanism to explain the physiological effects of fatty acids.

Next-generation sequencing-based bulked segregant analysis for QTL mapping in the heterozygous species Brassica rapa.

KEY MESSAGE: An improved protocol of QTL-seq, an NGS-based method for bulked segregant analysis we previously developed in rice, allowed successful mapping of QTLs of interest in the highly heterozygous genome of B. rapa, demonstrating the power of this elegant method for genetic analyses in heterozygous species of economic importance. Recent advances in next-generation sequencing (NGS) and the various NGS-based methods developed for rapidly identifying candidate genes of interest have accelerated genetic analysis mainly in the model plants rice and Arabidopsis. Brassica rapa includes several economically important crops such as Chinese cabbage, turnip and various leafy vegetables. The application of NGS-based approaches for the analysis of B. rapa has been limited mainly due to its highly heterozygous genome and poor quality of the reference genome sequence currently available for this species. In this study, we have improved QTL-seq, a method for NGS-based bulked segregant analysis we previously developed in rice, extending its applicability for accelerating the genetic analysis and molecular breeding of B. rapa. Addition of new filters to the original QTL-seq pipeline allowed removal of spurious single-nucleotide polymorphisms caused by alignment/sequencing errors and variability between parents, significantly improving accuracy of the analysis. As proof of principle, we successfully applied the new approach to identify candidate genomic regions controlling flowering and trichome formation using segregating F2 progeny obtained from crosses made between cultivars of B. rapa showing contrasting phenotypes for these traits. We strongly believe that the improved QTL-seq method reported here will extend the applicability of NGS-based genetic analysis not only to B. rapa but also to other plant species of economic importance with heterozygous genomes.

Genome sequencing of the staple food crop white Guinea yam enables the development of a molecular marker for sex determination.

BACKGROUND: Root and tuber crops are a major food source in tropical Africa. Among these crops are several species in the monocotyledonous genus Dioscorea collectively known as yam, a staple tuber crop that contributes enormously to the subsistence and socio-cultural lives of millions of people, principally in West and Central Africa. Yam cultivation is constrained by several factors, and yam can be considered a neglected "orphan" crop that would benefit from crop improvement efforts. However, the lack of genetic and genomic tools has impeded the improvement of this staple crop. RESULTS: To accelerate marker-assisted breeding of yam, we performed genome analysis of white Guinea yam (Dioscorea rotundata) and assembled a 594-Mb genome, 76.4% of which was distributed among 21 linkage groups. In total, we predicted 26,198 genes. Phylogenetic analyses with 2381 conserved genes revealed that Dioscorea is a unique lineage of monocotyledons distinct from the Poales (rice), Arecales (palm), and Zingiberales (banana). The entire Dioscorea genus is characterized by the occurrence of separate male and female plants (dioecy), a feature that has limited efficient yam breeding. To infer the genetics of sex determination, we performed whole-genome resequencing of bulked segregants (quantitative trait locus sequencing [QTL-seq]) in F1 progeny segregating for male and female plants and identified a genomic region associated with female heterogametic (male = ZZ, female = ZW) sex determination. We further delineated the W locus and used it to develop a molecular marker for sex identification of Guinea yam plants at the seedling stage. CONCLUSIONS: Guinea yam belongs to a unique and highly differentiated clade of monocotyledons. The genome analyses and sex-linked marker development performed in this study should greatly accelerate marker-assisted breeding of Guinea yam. In addition, our QTL-seq approach can be utilized in genetic studies of other outcrossing crops and organisms with highly heterozygous genomes. Genomic analysis of orphan crops such as yam promotes efforts to improve food security and the sustainability of tropical agriculture.

Colletotrichum orbiculare Secretes Virulence Effectors to a Biotrophic Interface at the Primary Hyphal Neck via Exocytosis Coupled with SEC22-Mediated Traffic.

The hemibiotrophic pathogen Colletotrichum orbiculare develops biotrophic hyphae inside cucumber (Cucumis sativus) cells via appressorial penetration; later, the pathogen switches to necrotrophy. C. orbiculare also expresses specific effectors at different stages. Here, we found that virulence-related effectors of C. orbiculare accumulate in a pathogen-host biotrophic interface. Fluorescence-tagged effectors accumulated in a ring-like region around the neck of the biotrophic primary hyphae. Fluorescence imaging of cellular components and transmission electron microscopy showed that the ring-like signals of the effectors localized at the pathogen-plant interface. Effector accumulation at the interface required induction of its expression during the early biotrophic phase, suggesting that transcriptional regulation may link to effector localization. We also investigated the route of effector secretion to the interface. An exocytosis-related component, the Rab GTPase SEC4, localized to the necks of biotrophic primary hyphae adjacent to the interface, thereby suggesting focal effector secretion. Disruption of SEC4 in C. orbiculare reduced virulence and impaired effector delivery to the ring signal interface. Disruption of the v-SNARE SEC22 also reduced effector delivery. These findings suggest that biotrophy-expressed effectors are secreted, via the endoplasmic reticulum-to-Golgi route and subsequent exocytosis, toward the interface generated between C. orbiculare and the host cell.

QTL-seq analysis identifies two genomic regions determining the heading date of foxtail millet, Setaria italica (L.) P.Beauv.

Heading date is an important event to ensure successful seed production. Although foxtail millet (Setaria italica (L.) P.Beauv.) is an important foodstuff in semiarid regions around the world, the genetic basis determining heading date is unclear. To identify genomic regions regulating days to heading (DTH), we conducted a QTL-seq analysis based on combining whole-genome re-sequencing and bulked-segregant analysis of an F2 population derived from crosses between the middle-heading cultivar Shinanotsubuhime and the early-heading cultivar Yuikogane. Under field conditions, transgressive segregation of DTH toward late heading was observed in the F2 population. We made three types of bulk samples: Y-bulk (early-heading), S-bulk (late-heading) and L-bulk (extremely late-heading). By genome-wide comparison of SNPs in the Y-bulk vs. the S-bulk and the Y-bulk vs. the L-bulk, we identified two QTLs associated with DTH. The first QTL, qDTH2, was detected on chromosome 2 from the Y-bulk and S-bulk comparison. The second QTL, qDTH7, was detected on chromosome 7 from the Y-bulk and L-bulk comparison. The Shinanotsubuhime allele for qDTH2 caused late heading in the F2 population, whereas the Yuikogane allele for qDTH7 led to extremely late heading. These results suggest that allelic differences in both qDTH2 and qDTH7 determine regional adaptability in S. italica.

A chloroplast-localized protein LESION AND LAMINA BENDING affects defence and growth responses in rice.

Understanding how plants allocate their resources to growth or defence is of long-term importance to the development of new and improved varieties of different crops. Using molecular genetics, plant physiology, hormone analysis and Next-Generation Sequencing (NGS)-based transcript profiling, we have isolated and characterized the rice (Oryza sativa) LESION AND LAMINA BENDING (LLB) gene that encodes a chloroplast-targeted putative leucine carboxyl methyltransferase. Loss of LLB function results in reduced growth and yield, hypersensitive response (HR)-like lesions, accumulation of the antimicrobial compounds momilactones and phytocassanes, and constitutive expression of pathogenesis-related genes. Consistent with these defence-associated responses, llb shows enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae). The lesion and resistance phenotypes are likely to be caused by the over-accumulation of jasmonates (JAs) in the llb mutant including the JA precursor 12-oxo-phytodienoic acid. Additionally, llb shows an increased lamina inclination and enhanced early seedling growth due to elevated brassinosteroid (BR) synthesis and/or signalling. These findings show that LLB functions in the chloroplast to either directly or indirectly repress both JA- and BR-mediated responses, revealing a possible mechanism for controlling how plants allocate resources for defence and growth.

A cytochrome P450, OsDSS1, is involved in growth and drought stress responses in rice (Oryza sativa L.).

Cytochrome P450s are among the largest protein coding gene families in plant genomes. However, majority of the genes remain uncharacterized. Here, we report the characterization of dss1, a rice mutant showing dwarfism and reduced grain size. The dss1 phenotype is caused by a non-synonymous point mutation we identified in DSS1, which is member of a P450 gene cluster located on rice chromosome 3 and corresponds to the previously reported CYP96B4/SD37 gene. Phenotypes of several dwarf mutants characterized in rice are associated with defects in the biosynthesis or perception of the phytohormones gibberellins (GAs) and brassinosteroids (BRs). However, both GA and BR failed to rescue the dss1 phenotype. Hormone profiling revealed the accumulation of abscisic acid (ABA) and ABA metabolites, as well as significant reductions in GA19 and GA53 levels, precursors of the bioactive GA1, in the mutant. The dss1 contents of cytokinin and auxins were not significantly different from wild-type plants. Consistent with the accumulation of ABA and metabolites, germination and early growth was delayed in dss1, which also exhibited an enhanced tolerance to drought. Additionally, expressions of members of the DSS1/CYP96B gene cluster were regulated by drought stress and exogenous ABA. RNA-seq-based transcriptome profiling revealed, among others, that cell wall-related genes and genes involved in lipid metabolism were up- and down-regulated in dss1, respectively. Taken together, these findings suggest that DSS1 mediates growth and stress responses in rice by fine-tuning GA-to-ABA balance, and might as well play a role in lipid metabolism.

The Draft Genome of Hop (Humulus lupulus), an Essence for Brewing.

The female flower of hop (Humulus lupulus var. lupulus) is an essential ingredient that gives characteristic aroma, bitterness and durability/stability to beer. However, the molecular genetic basis for identifying DNA markers in hop for breeding and to study its domestication has been poorly established. Here, we provide draft genomes for two hop cultivars [cv. Saazer (SZ) and cv. Shinshu Wase (SW)] and a Japanese wild hop [H. lupulus var. cordifolius; also known as Karahanasou (KR)]. Sequencing and de novo assembly of genomic DNA from heterozygous SW plants generated scaffolds with a total size of 2.05 Gb, corresponding to approximately 80% of the estimated genome size of hop (2.57 Gb). The scaffolds contained 41,228 putative protein-encoding genes. The genome sequences for SZ and KR were constructed by aligning their short sequence reads to the SW reference genome and then replacing the nucleotides at single nucleotide polymorphism (SNP) sites. De novo RNA sequencing (RNA-Seq) analysis of SW revealed the developmental regulation of genes involved in specialized metabolic processes that impact taste and flavor in beer. Application of a novel bioinformatics tool, phylogenetic comparative RNA-Seq (PCP-Seq), which is based on read depth of genomic DNAs and RNAs, enabled the identification of genes related to the biosynthesis of aromas and flavors that are enriched in SW compared to KR. Our results not only suggest the significance of historical human selection process for enhancing aroma and bitterness biosyntheses in hop cultivars, but also serve as crucial information for breeding varieties with high quality and yield.

QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations.

The majority of agronomically important crop traits are quantitative, meaning that they are controlled by multiple genes each with a small effect (quantitative trait loci, QTLs). Mapping and isolation of QTLs is important for efficient crop breeding by marker-assisted selection (MAS) and for a better understanding of the molecular mechanisms underlying the traits. However, since it requires the development and selection of DNA markers for linkage analysis, QTL analysis has been time-consuming and labor-intensive. Here we report the rapid identification of plant QTLs by whole-genome resequencing of DNAs from two populations each composed of 20-50 individuals showing extreme opposite trait values for a given phenotype in a segregating progeny. We propose to name this approach QTL-seq as applied to plant species. We applied QTL-seq to rice recombinant inbred lines and F2 populations and successfully identified QTLs for important agronomic traits, such as partial resistance to the fungal rice blast disease and seedling vigor. Simulation study showed that QTL-seq is able to detect QTLs over wide ranges of experimental variables, and the method can be generally applied in population genomics studies to rapidly identify genomic regions that underwent artificial or natural selective sweeps.

Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells.

Genome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host-plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non-bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV-based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR-Pik and AVR-Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR-Pik and AVR-Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR-Pik reduced and delayed the hypersensitive response triggered by B. glumae in the non-host plant Nicotiana benthamiana, indicative of an immunosuppressive virulence activity. AVR proteins fused with fluorescent protein and nuclear localization signal were delivered by B. glumae T3SS and observed in the nuclei of infected cells in rice, wheat, barley and N. benthamiana. Our bacterial T3SS-enabled eukaryotic effector delivery and subcellular localization assays provide a useful method for identifying and studying effector functions in monocot plants.

MutMap-Gap: whole-genome resequencing of mutant F2 progeny bulk combined with de novo assembly of gap regions identifies the rice blast resistance gene Pii.

Next-generation sequencing allows the identification of mutations responsible for mutant phenotypes by whole-genome resequencing and alignment to a reference genome. However, when the resequenced cultivar/line displays significant structural variation from the reference genome, mutations in the genome regions missing from the reference (gaps) cannot be identified by simple alignment. Here we report on a method called 'MutMap-Gap', which involves delineating a candidate region harboring a mutation of interest using the recently reported MutMap method, followed by de novo assembly, alignment, and identification of the mutation within genome gaps. We applied MutMap-Gap to isolate the blast resistant gene Pii from the rice cv Hitomebore using mutant lines that have lost Pii function. MutMap-Gap should prove useful for cloning genes that exhibit significant structural variations such as disease resistance genes of the nucleotide-binding site-leucine rich repeat (NBS-LRR) class.

OsGA20ox1, a candidate gene for a major QTL controlling seedling vigor in rice.

Seedling vigor is among the major determinants of stable stand establishment in direct-seeded rice (Oryza sativa L.) in temperate regions. Quantitative trait loci (QTL) for seedling vigor were identified using 250 recombinant inbred lines (RILs) derived from a cross between two japonica rice cultivars Kakehashi and Dunghan Shali. Seedling heights measured at 14 days after sowing were 20.3 and 29.4 cm for Kakehashi and Dunghan Shali, respectively. For the RILs, the height ranged from 14.1 to 31.7 cm. Four putative QTLs associated with seedling height were detected. qPHS3-2, the major QTL that was located on the long arm of chromosome 3, accounted for 26.2 % of the phenotypic variance. Using progeny of the near isogenic lines (NILs) produced by the backcross introduction of a chromosome segment carrying this major QTL into an elite cultivar Iwatekko, we fine-mapped qPHS3-2 to a 81-kb interval between two markers, ID_CAPS_01 and RM16227. Within this mapped region, we identified the gene OsGA20ox1, which is related to gibberellin (GA) biosynthesis. The relative expression levels of GA20ox1 in seedlings of Dunghan Shali and NILs were higher than that of Iwatekko. Concomitantly, the amount of endogenous active GA was higher in Dunghan Shali and the NILs compared to the level detected in Iwatekko. These results indicate that OsGA20ox1 is a strong candidate gene for major QTL controlling seedling vigor in rice.

Identification of a novel enzyme from E. pacifica that acts as an eicosapentaenoic 8R-LOX and docosahexaenoic 10R-LOX.

North Pacific krill (Euphausia pacifica) contain 8R-hydroxy-eicosapentaenoic acid (8R-HEPE), 8R-hydroxy-eicosatetraenoic acid (8R-HETE) and 10R-hydroxy-docosahexaenoic acid (10R-HDHA). These findings indicate that E. pacifica must possess an R type lipoxygenase, although no such enzyme has been identified in krill. We analyzed E. pacifica cDNA sequence using next generation sequencing and identified two lipoxygenase genes (PK-LOX1 and 2). PK-LOX1 and PK-LOX2 encode proteins of 691 and 686 amino acids, respectively. Recombinant PK-LOX1 was generated in Sf9 cells using a baculovirus expression system. PK-LOX1 metabolizes eicosapentaenoic acid (EPA) to 8R-HEPE, arachidonic acid (ARA) to 8R-HETE and docosahexaenoic acid (DHA) to 10R-HDHA. Moreover, PK-LOX1 had higher activity for EPA than ARA and DHA. In addition, PK-LOX1 also metabolizes 17S-HDHA to 10R,17S-dihydroxy-docosahexaenoic acid (10R,17S-DiHDHA). PK-LOX1 is a novel lipoxygenase that acts as an 8R-lipoxygenase for EPA and 10R-lipoxygenase for DHA and 17S-HDHA. Our findings show PK-LOX1 facilitates the enzymatic production of hydroxy fatty acids, which are of value to the healthcare sector.

Identification of candidate flowering and sex genes in white Guinea yam (D. rotundata Poir.) by SuperSAGE transcriptome profiling.

Dioecy (distinct male and female individuals) and scarce to non-flowering are common features of cultivated yam (Dioscorea spp.). However, the molecular mechanisms underlying flowering and sex determination in Dioscorea are largely unknown. We conducted SuperSAGE transcriptome profiling of male, female and monoecious individuals to identify flowering and sex-related genes in white Guinea yam (D. rotundata), generating 20,236 unique tags. Of these, 13,901 were represented by a minimum of 10 tags. A total 88 tags were significantly differentially expressed in male, female and monoecious plants, of which 18 corresponded to genes previously implicated in flower development and sex determination in multiple plant species. We validated the SuperSAGE data with quantitative real-time PCR (qRT-PCR)-based analysis of the expression of three candidate genes. We further investigated the flowering patterns of 1938 D. rotundata accessions representing diverse geographical origins over two consecutive years. Over 85% of accessions were either male or non-flowering, less than 15% were female, while monoecious plants were rare. Intensity of flowering varied between male and female plants, with the former flowering more abundantly than the latter. Candidate genes identified in this study can be targeted for further validation and to induce regular flowering in poor to non-flowering cultivars. Findings of the study provide important inputs for further studies aiming to overcome the challenge of flowering in yams and to improve efficiency of yam breeding.