Transcriptome and Metabolome Analyses of Glucosinolates in Two Broccoli Cultivars Following Jasmonate Treatment for the Induction of Glucosinolate Defense to Trichoplusia ni (Hübner)

Lepidopteran larvae growth is influenced by host plant glucosinolate (GS) concentrations, which are, in turn, influenced by the phytohormone jasmonate (JA). In order to elucidate insect resistance biomarkers to lepidopteran pests, transcriptome and metabolome analyses following JA treatments were conducted with two broccoli cultivars, Green Magic and VI-158, which have differentially induced indole GSs, neoglucobrassicin and glucobrassicin, respectively. To test these two inducible GSs on growth of cabbage looper (Trichoplusia ni), eight neonate cabbage looper larvae were placed onto each of three plants per JA treatments (0, 100, 200, 400 M) three days after treatment. After five days of feeding, weight of larvae and their survival rate was found to decrease with increasing JA concentrations in both broccoli cultivars. JA-inducible GSs were measured by high performance liquid chromatography. Neoglucobrassicin in Green Magic and glucobrassicin in VI-158 leaves were increased in a dose-dependent manner. One or both of these glucosinolates and/or their hydrolysis products showed significant inverse correlations with larval weight and survival (five days after treatment) while being positively correlated with the number of days to pupation. This implies that these two JA-inducible glucosinolates can influence the growth and survival of cabbage looper larvae. Transcriptome profiling supported the observed changes in glucosinolate and their hydrolysis product concentrations following JA treatments. Several genes related to GS metabolism differentiate the two broccoli cultivars in their pattern of transcriptional response to JA treatments. Indicative of the corresponding change in indole GS concentrations, transcripts of the transcription factor MYB122, core structure biosynthesis genes (CYP79B2, UGT74B1, SUR1, SOT16, SOT17, and SOT18), an indole glucosinolate side chain modification gene (IGMT1), and several glucosinolate hydrolysis genes (TGG1, TGG2, and ESM1) were significantly increased in Green Magic (statistically significant in most cases at 400 M) while UGT74B1 and MYB122 were significantly increased in VI-158. Therefore, these metabolite and transcript biomarker results indicate that transcriptome profiling can identify genes associated with the formation of two different indole GS and their hydrolysis products. Therefore, these metabolite and transcript biomarkers could be useful in an effective marker-assisted breeding strategy for resistance to generalist lepidopteran pests in broccoli and potentially other Brassica vegetables

Targeted Metabolomic and Transcriptomic Analyses of “Red Russian” Kale (Brassicae napus var. pabularia) Following Methyl Jasmonate Treatment and Larval Infestation by the Cabbage Looper (Trichoplusia ni Hübner)

Methyl jasmonate (MeJA), synthesized in the jasmonic acid (JA) pathway, has been found to upregulate glucosinolate (GS) biosynthesis in plant species of the Brassicaceae family. Exogenous application of MeJA has shown to increase tissue GS concentrations and the formation of myrosinase-mediated GS hydrolysis products (GSHPs). In vitro and in vivo assays have demonstrated the potential health-promoting effects of certain GSHPs. MeJA is also known to elicit and induce genes associated with defense mechanisms to insect herbivory in Brassica species. To investigate the relationship between MeJA-induced GS biosynthesis and insect defense, three treatments were applied to “Red Russian” kale (Brassicae napus var. pabularia) seedlings: (1) a 250 μM MeJA leaf spray treatment; (2) leaf infestation with larvae of the cabbage looper (Trichoplusia ni (Hübner)); (3) control treatment (neither larval infestation nor MeJA application). Samples of leaf tissue from the three treatments were then assayed for changes in GS and GSHP concentrations, GS gene biosynthesis expression, and myrosinase activity. Major differences were observed between the three treatments in the levels of GS accumulation and GS gene expression. The insect-damaged samples showed significantly lower aliphatic GS accumulation, while both MeJA and T. ni infestation treatments induced greater accumulation of indolyl GS. The gene expression levels of CYP81F4,MYB34, and MYB122 were significantly upregulated in samples treated with MeJA and insects compared to the control group, which explained the increased indolyl GS concentration. The results suggest that the metabolic changes promoted by MeJA application and the insect herbivory response share common mechanisms of induction. This work provides potentially useful information for kale pest control and nutritional quality

Chemopreventive glucosinolate accumulation in various broccoli and collard tissues: Microfluidic-based targeted transcriptomics for by-product valorization

Floret, leaf, and root tissues were harvested from broccoli and collard cultivars and extracted to determine their glucosinolate and hydrolysis product profiles using high performance liquid chromatography and gas chromotography. Quinone reductase inducing bioactivity, an estimate of anti-cancer chemopreventive potential, of the extracts was measured using a hepa1c1c7 murine cell line. Extracts from root tissues were significantly different from other tissues and contained high levels of gluconasturtiin and glucoerucin. Targeted gene expression analysis on glucosinolate biosynthesis revealed that broccoli root tissue has elevated gene expression of AOP2 and low expression of FMOGS-OX homologs, essentially the opposite of what was observed in broccoli florets, which accumulated high levels of glucoraphanin. Broccoli floret tissue has significantly higher nitrile formation (%) and epithionitrile specifier protein gene expression than other tissues. This study provides basic information of the glucosinolate metabolome and transcriptome for various tissues of Brassica oleracea that maybe utilized as potential byproducts for the nutraceutical market

Effect Of Leaf Surface Chemical Properties On Efficacy Of Sanitizer For Rotavirus Inactivation

The use of sanitizers is essential for produce safety. However, little is known about how sanitizer efficacy varies with respect to the chemical surface properties of produce. To answer this question, the disinfection efficacies of an oxidant-based sanitizer and a new surfactant-based sanitizer for porcine rotavirus (PRV) strain OSU were examined. PRV was attached to the leaf surfaces of two kale cultivars with high epicuticular wax contents and one cultivar of endive with a low epicuticular wax content and then treated with each sanitizer. The efficacy of the oxidant-based sanitizer correlated with leaf wax content as evidenced by the 1-log10 PRV disinfection on endive surfaces (low wax content) and 3-log10 disinfection of the cultivars with higher wax contents. In contrast, the surfactant-based sanitizer showed similar PRV disinfection efficacies (up to 3 log10) that were independent of leaf wax content. A statistical difference was observed with the disinfection efficacies of the oxidant-based sanitizer for suspended and attached PRV, while the surfactant-based sanitizer showed similar PRV disinfection efficacies. Significant reductions in the entry and replication of PRV were observed after treatment with either disinfectant. Moreover, the oxidant-based-sanitizer-treated PRV showed sialic acid-specific binding to the host cells, whereas the surfactant-based sanitizer increased the nonspecific binding of PRV to the host cells. These findings suggest that the surface properties of fresh produce may affect the efficacy of virus disinfection, implying that food sanitizers should be carefully selected for the different surface characteristics of fresh produce

Genetic variation in \u3ci\u3eMiscanthus\u3c/i\u3e X \u3ci\u3egiganteus\u3c/i\u3e and the importance of estimating genetic distance thresholds for differentiating clones

Miscanthus x giganteus (Mxg) is an important bioenergy feedstock crop, however, genetic diversity among legacy cultivars may be severely constrained. Only one introduction from Japan to Denmark of this sterile, triploid, vegetatively propagated crop was recorded in the 1930s. We sought to determine if the Mxg cultivars in North America were all synonyms, and if they were derived from the European introduction. We used 64 nuclear and five chloroplast simple sequence repeat (SSR) markers to estimate genetic similarity for 27 Mxg accessions from North America, and compared them with six accessions from Europe, including the species’ type-specimen. A subset of accessions was also evaluated by restriction-site associated DNA sequencing (RAD-seq). In addition, we assessed the potential of new crosses to increase Mxg genetic diversity by comparing eight new triploid Mxg progeny grown from seed, along with samples of the parental species M. sacchariflorus and M. sinensis. Estimates of genotyping error rates were essential for distinguishing between experimental error and true genotypic differences among accessions. Given differences in estimated error rates and costs per marker for SSRs and RAD-seq, the former is currently more cost-effective for determining if two accessions are genetically identical. We concluded that all of the Mxg legacy cultivars were derived via vegetative propagation from a single genet. In contrast with the Mxg legacy cultivars, genetic similarity to the type-specimen of eight new triploid Mxg progeny ranged from 0.46 to 0.56. Though genetic diversity among the Mxg legacy cultivars is critically low, new crosses can provide much-needed variation to growers

A framework genetic map for \u3ci\u3eMiscanthus sinensis\u3c/i\u3e from RNAseq-based markers shows recent tetraploidy

Background: Miscanthus (subtribe Saccharinae, tribe Andropogoneae, family Poaceae) is a genus of temperate perennial C4 grasses whose high biomass production makes it, along with its close relatives sugarcane and sorghum, attractive as a biofuel feedstock. The base chromosome number of Miscanthus (x = 19) is different from that of other Saccharinae and approximately twice that of the related Sorghum bicolor (x = 10), suggesting largescale duplications may have occurred in recent ancestors of Miscanthus. Owing to the complexity of the Miscanthus genome and the complications of self-incompatibility, a complete genetic map with a high density of markers has not yet been developed. Results: We used deep transcriptome sequencing (RNAseq) from two M. sinensis accessions to define 1536 single nucleotide variants (SNVs) for a GoldenGate™ genotyping array, and found that simple sequence repeat (SSR) markers defined in sugarcane are often informative in M. sinensis. A total of 658 SNP and 210 SSR markers were validated via segregation in a full sibling F1 mapping population. Using 221 progeny from this mapping population, we constructed a genetic map for M. sinensis that resolves into 19 linkage groups, the haploid chromosome number expected from cytological evidence. Comparative genomic analysis documents a genomewide duplication in Miscanthus relative to Sorghum bicolor, with subsequent insertional fusion of a pair of chromosomes. The utility of the map is confirmed by the identification of two paralogous C4-pyruvate, phosphate dikinase (C4-PPDK) loci in Miscanthus, at positions syntenic to the single orthologous gene in Sorghum. Conclusions: The genus Miscanthus experienced an ancestral tetraploidy and chromosome fusion prior to its diversification, but after its divergence from the closely related sugarcane clade. The recent timing of this tetraploidy complicates discovery and mapping of genetic markers for Miscanthus species, since alleles and fixed differences between paralogs are comparable. These difficulties can be overcome by careful analysis of segregation patterns in a mapping population and genotyping of doubled haploids. The genetic map for Miscanthus will be useful in biological discovery and breeding efforts to improve this emerging biofuel crop, and also provide a valuable resource for understanding genomic responses to tetraploidy and chromosome fusion

A framework genetic map for Miscanthus sinensis from RNAseq-based markers shows recent tetraploidy

Abstract Background Miscanthus (subtribe Saccharinae, tribe Andropogoneae, family Poaceae) is a genus of temperate perennial C4 grasses whose high biomass production makes it, along with its close relatives sugarcane and sorghum, attractive as a biofuel feedstock. The base chromosome number of Miscanthus (x = 19) is different from that of other Saccharinae and approximately twice that of the related Sorghum bicolor (x = 10), suggesting large-scale duplications may have occurred in recent ancestors of Miscanthus. Owing to the complexity of the Miscanthus genome and the complications of self-incompatibility, a complete genetic map with a high density of markers has not yet been developed. Results We used deep transcriptome sequencing (RNAseq) from two M. sinensis accessions to define 1536 single nucleotide variants (SNVs) for a GoldenGate™ genotyping array, and found that simple sequence repeat (SSR) markers defined in sugarcane are often informative in M. sinensis. A total of 658 SNP and 210 SSR markers were validated via segregation in a full sibling F1 mapping population. Using 221 progeny from this mapping population, we constructed a genetic map for M. sinensis that resolves into 19 linkage groups, the haploid chromosome number expected from cytological evidence. Comparative genomic analysis documents a genome-wide duplication in Miscanthus relative to Sorghum bicolor, with subsequent insertional fusion of a pair of chromosomes. The utility of the map is confirmed by the identification of two paralogous C4-pyruvate, phosphate dikinase (C4-PPDK) loci in Miscanthus, at positions syntenic to the single orthologous gene in Sorghum. Conclusions The genus Miscanthus experienced an ancestral tetraploidy and chromosome fusion prior to its diversification, but after its divergence from the closely related sugarcane clade. The recent timing of this tetraploidy complicates discovery and mapping of genetic markers for Miscanthus species, since alleles and fixed differences between paralogs are comparable. These difficulties can be overcome by careful analysis of segregation patterns in a mapping population and genotyping of doubled haploids. The genetic map for Miscanthus will be useful in biological discovery and breeding efforts to improve this emerging biofuel crop, and also provide a valuable resource for understanding genomic responses to tetraploidy and chromosome fusion

Variation in Broccoli Cultivar Phytochemical Content under Organic and Conventional Management Systems: Implications in Breeding for Nutrition

Organic agriculture requires cultivars that can adapt to organic crop management systems without the use of synthetic pesticides as well as genotypes with improved nutritional value. The aim of this study encompassing 16 experiments was to compare 23 broccoli cultivars for the content of phytochemicals associated with health promotion grown under organic and conventional management in spring and fall plantings in two broccoli growing regions in the US (Oregon and Maine). The phytochemicals quantified included: glucosinolates (glucoraphanin, glucobrassicin, neoglucobrassin), tocopherols (δ-, γ-, α-tocopherol) and carotenoids (lutein, zeaxanthin, β-carotene). For glucoraphanin (17.5%) and lutein (13%), genotype was the major source of total variation; for glucobrassicin, region (36%) and the interaction of location and season (27.5%); and for neoglucobrassicin, both genotype (36.8%) and its interactions (34.4%) with season were important. For δ- and γ-tocopherols, season played the largest role in the total variation followed by location and genotype; for total carotenoids, genotype (8.41–13.03%) was the largest source of variation and its interactions with location and season. Overall, phytochemicals were not significantly influenced by management system. We observed that the cultivars with the highest concentrations of glucoraphanin had the lowest for glucobrassicin and neoglucobrassicin. The genotypes with high concentrations of glucobrassicin and neoglucobrassicin were the same cultivars and were early maturing F₁ hybrids. Cultivars highest in tocopherols and carotenoids were open pollinated or early maturing F₁ hybrids. We identified distinct locations and seasons where phytochemical performance was higher for each compound. Correlations among horticulture traits and phytochemicals demonstrated that glucoraphanin was negatively correlated with the carotenoids and the carotenoids were correlated with one another. Little or no association between phytochemical concentration and date of cultivar release was observed, suggesting that modern breeding has not negatively influenced the level of tested compounds. We found no significant differences among cultivars from different seed companies