Genetic analysis of glucosinolate variability in broccoli florets using genome-anchored single nucleotide polymorphisms.

KEY MESSAGE: The identification of genetic factors influencing the accumulation of individual glucosinolates in broccoli florets provides novel insight into the regulation of glucosinolate levels in Brassica vegetables and will accelerate the development of vegetables with glucosinolate profiles tailored to promote human health. Quantitative trait loci analysis of glucosinolate (GSL) variability was conducted with a B. oleracea (broccoli) mapping population, saturated with single nucleotide polymorphism markers from a high-density array designed for rapeseed (Brassica napus). In 4 years of analysis, 14 QTLs were associated with the accumulation of aliphatic, indolic, or aromatic GSLs in floret tissue. The accumulation of 3-carbon aliphatic GSLs (2-propenyl and 3-methylsulfinylpropyl) was primarily associated with a single QTL on C05, but common regulation of 4-carbon aliphatic GSLs was not observed. A single locus on C09, associated with up to 40 % of the phenotypic variability of 2-hydroxy-3-butenyl GSL over multiple years, was not associated with the variability of precursor compounds. Similarly, QTLs on C02, C04, and C09 were associated with 4-methylsulfinylbutyl GSL concentration over multiple years but were not significantly associated with downstream compounds. Genome-specific SNP markers were used to identify candidate genes that co-localized to marker intervals and previously sequenced Brassica oleracea BAC clones containing known GSL genes (GSL-ALK, GSL-PRO, and GSL-ELONG) were aligned to the genomic sequence, providing support that at least three of our 14 QTLs likely correspond to previously identified GSL loci. The results demonstrate that previously identified loci do not fully explain GSL variation in broccoli. The identification of additional genetic factors influencing the accumulation of GSL in broccoli florets provides novel insight into the regulation of GSL levels in Brassicaceae and will accelerate development of vegetables with modified or enhanced GSL profiles.

High-density single nucleotide polymorphism (SNP) array mapping in Brassica oleracea: identification of QTL associated with carotenoid variation in broccoli florets.

KEY MESSAGE: A high-resolution genetic linkage map of B. oleracea was developed from a B. napus SNP array. The work will facilitate genetic and evolutionary studies in Brassicaceae. A broccoli population, VI-158 × BNC, consisting of 150 F2:3 families was used to create a saturated Brassica oleracea (diploid: CC) linkage map using a recently developed rapeseed (Brassica napus) (tetraploid: AACC) Illumina Infinium single nucleotide polymorphism (SNP) array. The map consisted of 547 non-redundant SNP markers spanning 948.1 cM across nine chromosomes with an average interval size of 1.7 cM. As the SNPs are anchored to the genomic reference sequence of the rapid cycling B. oleracea TO1000, we were able to estimate that the map provides 96 % coverage of the diploid genome. Carotenoid analysis of 2 years data identified 3 QTLs on two chromosomes that are associated with up to half of the phenotypic variation associated with the accumulation of total or individual compounds. By searching the genome sequences of the two related diploid species (B. oleracea and B. rapa), we further identified putative carotenoid candidate genes in the region of these QTLs. This is the first description of the use of a B. napus SNP array to rapidly construct high-density genetic linkage maps of one of the constituent diploid species. The unambiguous nature of these markers with regard to genomic sequences provides evidence to the nature of genes underlying the QTL, and demonstrates the value and impact this resource will have on Brassica research.

Concentrating immunoprotective phytoactive compounds from fruits and vegetables into shelf-stable protein-rich ingredients.

Co-delivery of edible proteins with health-protective fruit (muscadine grape) and vegetable (kale) phytoactive compounds was accomplished in a biofortified ingredient for use in convenient, portable food formulations. Polyphenolics were concentrated (10-42 mg/g range) in dry muscadine-protein matrices. Kale-fortified protein matrices also captured polyphenolics (8 mg/g), carotenoids (69 µg/g) and glucosinolates (7 µmol/g). Neither total phenolics nor glucosinolates were significantly diminished even after long term (6 months) storage at 4, 20, or 37 °C, whereas carotenoids degraded over time, particularly at higher temperatures. Dry biofortified phytoactive-protein ingredients allowed delivery of immunoprotective compounds from fruits and vegetables in a stable, lightweight matrix.

Genetic Mapping, Candidate Gene Identification and Marker Validation for Host Plant Resistance to the Race 4 of Fusarium oxysporum f. sp. cubense Using Musa acuminata ssp. malaccensis.

Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to Fusarium oxysporum f. sp. Cubense (Foc), the causal agent of this disease, is genetically dissected in this study using two Musa acuminata ssp. Malaccensis segregating populations, segregating for Foc Tropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on chromosome 3 of 'DH-Pahang' reference assembly v4. Within this region, there was a cluster of pattern recognition receptors, namely leucine-rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell-wall-associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins, positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant progenies but not in the susceptible F2 progenies at the onset of infection. This suggests that one or several of these genes may control resistance at this locus. To confirm the segregation of single-gene resistance, we generated an inter-cross between the resistant parent 'Ma850' and a susceptible line 'Ma848', to show that the STR4 resistance co-segregated with marker '28820' at this locus. Finally, an informative SNP marker 29730 allowed the locus-specific resistance to be assessed in a collection of diploid and polyploid banana plants. Of the 60 lines screened, 22 lines were predicted to carry resistance at this locus, including lines known to be TR4-resistant, such as 'Pahang', 'SH-3362', 'SH-3217', 'Ma-ITC0250', and 'DH-Pahang/CIRAD 930'. Additional screening in the International Institute for Tropical Agriculture's collection suggests that the dominant allele is common among the elite 'Matooke' NARITA hybrids, as well as in other triploid or tetraploid hybrids derived from East African highland bananas. Fine mapping and candidate gene identification will allow characterization of molecular mechanisms underlying the TR4 resistance. The markers developed in this study can now aid the marker-assisted selection of TR4 resistance in breeding programs around the world.

Variation in anthocyanin profiles of 27 genotypes of red cabbage over two growing seasons.

Acylated anthocyanins, such as those found in red cabbage, are more heat-, light-, and alkaline pH-stable than non-acylated anthocyanins, making them attractive for a variety of commercial applications. A UPLC-DAD-MSE method with an optimized chromatographic strategy was used to identify 29 red cabbage anthocyanins, predominantly acylated and glucosylated cyanidin derivatives. Anthocyanin profiles of 27 red cabbage genotypes harvested in consecutive growing seasons were measured and assessed for variation. Three unique anthocyanin profile fingerprints were identified through hierarchical clustering analysis. PCA analysis identified anthocyanin accumulation traits and genotypes with high diversity which can be utilized in future investigations into the genetic and molecular basis for anthocyanin production, acylation, and diversity.

A high-throughput, simultaneous analysis of carotenoids, chlorophylls and tocopherol using sub two micron core shell technology columns.

A high-throughput, robust and reliable method for simultaneous analysis of five carotenoids, four chlorophylls and one tocopherol was developed for rapid screening large sample populations to facilitate molecular biology and plant breeding. Separation was achieved for 10 known analytes and four unknown carotenoids in a significantly reduced run time of 10min. Identity of the 10 analytes was confirmed by their UV-Vis absorption spectras. Quantification of tocopherol, carotenoids and chlorophylls was performed at 290nm, 460nm and 650nm respectively. In this report, two sub two micron particle core-shell columns, Kinetex from Phenomenex (1.7µm particle size, 12% carbon load) and Cortecs from Waters (1.6µm particle size, 6.6% carbon load) were investigated and their separation efficiencies were evaluated. The peak resolutions were >1.5 for all analytes except for chlorophyll-a' with Cortecs column. The ruggedness of this method was evaluated in two identical but separate instruments that produced CV<2 in peak retentions for nine out of 10 analytes separated.

RNA-Seq analysis and annotation of a draft blueberry genome assembly identifies candidate genes involved in fruit ripening, biosynthesis of bioactive compounds, and stage-specific alternative splicing.

BACKGROUND: Blueberries are a rich source of antioxidants and other beneficial compounds that can protect against disease. Identifying genes involved in synthesis of bioactive compounds could enable the breeding of berry varieties with enhanced health benefits. RESULTS: Toward this end, we annotated a previously sequenced draft blueberry genome assembly using RNA-Seq data from five stages of berry fruit development and ripening. Genome-guided assembly of RNA-Seq read alignments combined with output from ab initio gene finders produced around 60,000 gene models, of which more than half were similar to proteins from other species, typically the grape Vitis vinifera. Comparison of gene models to the PlantCyc database of metabolic pathway enzymes identified candidate genes involved in synthesis of bioactive compounds, including bixin, an apocarotenoid with potential disease-fighting properties, and defense-related cyanogenic glycosides, which are toxic. Cyanogenic glycoside (CG) biosynthetic enzymes were highly expressed in green fruit, and a candidate CG detoxification enzyme was up-regulated during fruit ripening. Candidate genes for ethylene, anthocyanin, and 400 other biosynthetic pathways were also identified. Homology-based annotation using Blast2GO and InterPro assigned Gene Ontology terms to around 15,000 genes. RNA-Seq expression profiling showed that blueberry growth, maturation, and ripening involve dynamic gene expression changes, including coordinated up- and down-regulation of metabolic pathway enzymes and transcriptional regulators. Analysis of RNA-seq alignments identified developmentally regulated alternative splicing, promoter use, and 3' end formation. CONCLUSIONS: We report genome sequence, gene models, functional annotations, and RNA-Seq expression data that provide an important new resource enabling high throughput studies in blueberry.

Efficient quantification of the health-relevant anthocyanin and phenolic acid profiles in commercial cultivars and breeding selections of blueberries ( Vaccinium spp.).

Anthocyanins and phenolic acids are major secondary metabolites in blueberry with important implications for human health maintenance. An improved protocol was developed for the accurate, efficient, and rapid comparative screening for large blueberry sample sets. Triplicates of six commercial cultivars and four breeding selections were analyzed using the new method. The compound recoveries ranged from 94.2 to 97.5 ± 5.3% when samples were spiked with commercial standards prior to extraction. Eighteen anthocyanins and 4 phenolic acids were quantified in frozen and freeze-dried fruits. Large variations for individual and total anthocyanins, ranging from 201.4 to 402.8 mg/100 g, were assayed in frozen fruits. The total phenolic acid content ranged from 23.6 to 61.7 mg/100 g in frozen fruits. Across all genotypes, freeze-drying resulted in minor reductions in anthocyanin concentration (3.9%) compared to anthocyanins in frozen fruits. However, phenolic acids increased by an average of 1.9-fold (±0.3) in the freeze-dried fruit. Different genotypes frequently had comparable overall levels of total anthocyanins and phenolic acids, but differed dramatically in individual profiles of compounds. Three of the genotypes contained markedly higher concentrations of delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, and malvidin 3-O-glucoside, which have previously been implicated as bioactive principles in this fruit. The implications of these findings for human health benefits are discussed.

Simultaneous extraction and quantitation of carotenoids, chlorophylls, and tocopherols in Brassica vegetables.

Brassica oleracea vegetables, such as broccoli (B. oleracea L. var. italica) and cauliflower (B. oleracea L. var. botrytis), are known to contain bioactive compounds associated with health, including three classes of photosynthetic lipid-soluble compounds: carotenoids, chlorophylls, and tocopherols. Carotenoids and chlorophylls are photosynthetic pigments. Tocopherols have vitamin E activity. Due to genetic and environmental variables, the amounts present in vegetables are not constant. To aid breeders in the development of Brassica cultivars with higher provitamin A and vitamin E contents and antioxidant activity, a more efficient method was developed to quantitate carotenoids, chlorophylls, and tocopherols in the edible portions of broccoli and cauliflower. The novel UPLC method separated five carotenoids, two chlorophylls, and two tocopherols in a single 30 min run, reducing the run time by half compared to previously published protocols. The objective of the study was to develop a faster, more effective extraction and quantitation methodology to screen large populations of Brassica germplasm, thus aiding breeders in producing superior vegetables with enhanced phytonutrient profiles.