Unveiling the evolutionary history of lingonberry (Vaccinium vitis-idaea L.) through genome sequencing and assembly of European and North American subspecies.

Lingonberry (Vaccinium vitis-idaea L.) produces tiny red berries that are tart and nutty in flavor. It grows widely in the circumpolar region, including Scandinavia, northern parts of Eurasia, Alaska, and Canada. Although cultivation is currently limited, the plant has a long history of cultural use among indigenous communities. Given its potential as a food source, genomic resources for lingonberry are significantly lacking. To advance genomic knowledge, the genomes for 2 subspecies of lingonberry (V. vitis-idaea ssp. minus and ssp. vitis-idaea var. 'Red Candy') were sequenced and de novo assembled into contig-level assemblies. The assemblies were scaffolded using the bilberry genome (Vaccinium myrtillus) to generate a chromosome-anchored reference genome consisting of 12 chromosomes each with a total length of 548.07 Mb [contig N50 = 1.17 Mb, BUSCO (C%) = 96.5%] for ssp. vitis-idaea and 518.70 Mb [contig N50 = 1.40 Mb, BUSCO (C%) = 96.9%] for ssp. minus. RNA-seq-based gene annotation identified 27,243 and 25,718 genes on the respective assembly, and transposable element detection methods found that 45.82 and 44.58% of the genome were repeats. Phylogenetic analysis confirmed that lingonberry was most closely related to bilberry and was more closely related to blueberries than cranberries. Estimates of past effective population size suggested a continuous decline over the past 1-3 MYA, possibly due to the impacts of repeated glacial cycles during the Pleistocene leading to frequent population fragmentation. The genomic resource created in this study can be used to identify industry-relevant genes (e.g. anthocyanin production), infer phylogeny, and call sequence-level variants (e.g. SNPs) in future research.

Exploring Genetic and Epigenetic Changes in Lingonberry Using Molecular Markers: Implications for Clonal Propagation.

Lingonberry (Vaccinium vitis-idaea L.) is an important and valuable horticultural crop due to its high antioxidant properties. Plant tissue culture is an advanced propagation system employed in horticultural crops. However, the progeny derived using this technique may not be true-to-type. In order to obtain the maximum return of any agricultural enterprise, uniformity of planting materials is necessary, which sometimes is not achieved due to genetic and epigenetic instabilities under in vitro culture. Therefore, we analyzed morphological traits and genetic and epigenetic variations under tissue-culture and greenhouse conditions in lingonberry using molecular markers. Leaf length and leaf width under greenhouse conditions and shoot number per explant, shoot height and shoot vigor under in vitro conditions were higher in hybrid H1 compared to the cultivar Erntedank. Clonal fidelity study using one expressed sequence tag (EST)-polymerase chain reaction (PCR), five EST-simple sequence repeat (SSR) and six genomic (G)-SSR markers revealed monomorphic bands in micropropagated shoots and plants in lingonberry hybrid H1 and cultivar Erntedank conforming genetic integrity. Epigenetic variation was studied by quantifying cytosine methylation using a methylation-sensitive amplification polymorphism (MSAP) technique. DNA methylation ranged from 32% in greenhouse-grown hybrid H1 to 44% in cultivar Erntedank under a tissue culture system. Although total methylation was higher in in vitro grown shoots, fully methylated bands were observed more in the greenhouse-grown plants. On the contrary, hemimethylated DNA bands were more prominent in tissue culture conditions as compared to the greenhouse-grown plants. The study conclude that lingonberry maintains its genetic integrity but undergoes variable epigenetic changes during in vitro and ex vitro conditions.

Epigenomic insight of lingonberry and health-promoting traits during micropropagation.

Epigenetic variation plays a role in developmental gene regulation and responses to the environment. An efficient interaction of zeatin-induced cytosine methylation and secondary compounds has been displayed for the first time in tissue-culture shoots/plants of lingonberry (Vaccinium vitis-idaea L.) cultivar Erntedank in vitro (NC1, in a liquid medium; NC2, on a semi-solid medium), ex vitro (NC3, node culture-derived plants; LC1, leaf culture-derived plants) and its cutting-propagated (ED) plants. Through methylation-sensitive amplification polymorphism (MSAP) assay, we observed highest methylated sites in leaf regenerants (LC1) from all primer combinations (108 bands), along with the highest secondary metabolites. The four types of tissue culture-derived shoots/plants (NC1, NC2, NC3, LC1) showed higher methylation bands than cutting propagated donor plants (ED) that exhibited 79 bands of methylation, which is comparatively low. Our study showed more methylation in micropropagated shoots/plants than those derived from ED plants. On the contrary, we observed higher secondary metabolites in ED plants but comparatively less in micropropagated shoots (NC1, NC2) and plants (NC3, LC1).

Phenotypic variation and epigenetic insight into tissue culture berry crops.

Berry crops, a nutrient powerhouse for antioxidant properties, have long been enjoyed as a health-promoting delicious food. Significant progress has been achieved for the propagation of berry crops using tissue culture techniques. Although bioreactor micropropagation has been developed as a cost-effective propagation technology for berry crops, genetic stability can be a problem for commercial micropropagation that can be monitored at morphological, biochemical, and molecular levels. Somaclonal variations, both genetic and epigenetic, in tissue culture regenerants are influenced by different factors, such as donor genotype, explant type and origin, chimeral tissues, culture media type, concentration and combination of plant growth regulators, and culture conditions and period. Tissue culture regenerants in berry crops show increased vegetative growth, rhizome production, and berry yield, containing higher antioxidant activity in fruits and leaves that might be due to epigenetic variation. The present review provides an in-depth study on various aspects of phenotypic variation in micropropagated berry plants and the epigenetic effects on these variations along with the role of DNA methylation, to fill the existing gap in literature.

Genetic Diversity of Blueberry Genotypes Estimated by Antioxidant Properties and Molecular Markers.

Blueberries (Vaccinium spp.) have gained much attention worldwide because of their potential health benefits and economic importance. Genetic diversity was estimated in blueberry hybrids, wild clones and cultivars by their antioxidant efficacy, total phenolic and flavonoid contents, and express sequence tag-simple sequence repeat (SSR) (EST-SSR), genomic (G)-SSR and express sequence tag-polymerase chain reaction (EST-PCR) markers. Wide diversity existed among the genotypes for antioxidant properties, with the highest variation for DPPH radical scavenging activity (20-fold), followed by the contents of total flavonoids (16-fold) and phenolics (3.8-fold). Although a group of 11 hybrids generated the maximum diversity for antioxidant activity (15-fold), wild clones collected from Quebec, Canada, had the maximum variation for total phenolic (2.8-fold) and flavonoid contents (6.9-fold). Extensive genetic diversity was evident from Shannon's index (0.34 for EST-SSRs, 0.29 for G-SSR, 0.26 for EST-PCR) and expected heterozygosity (0.23 for EST-SSR, 0.19 for G-SSR, 0.16 for EST-PCR). STRUCTURE analysis separated the genotypes into three groups, which were in agreement with principal coordinate and neighbour-joining analyses. Molecular variance suggested 19% variation among groups and 81% among genotypes within the groups. Clustering based on biochemical data and molecular analysis did not coincide, indicating a random distribution of loci in the blueberry genome, conferring antioxidant properties. However, the stepwise multiple regression analysis (SMRA) revealed that 17 EST-SSR, G-SSR and EST-PCR markers were associated with antioxidant properties. The study is valuable to breeding and germplasm conservation programs.

Tissue culture-induced DNA methylation in crop plants: a review.

Plant tissue culture techniques have been extensively employed in commercial micropropagation to provide year-round production. Tissue culture regenerants are not always genotypically and phenotypically similar. Due to the changes in the tissue culture microenvironment, plant cells are exposed to additional stress which induces genetic and epigenetic instabilities in the regenerants. These changes lead to tissue culture-induced variations (TCIV) which are also known as somaclonal variations to categorically specify the inducing environment. TCIV includes molecular and phenotypic changes persuaded in the in vitro culture due to continuous sub-culturing and tissue culture-derived stress. Epigenetic variations such as altered DNA methylation pattern are induced due to the above-mentioned factors. Reportedly, alteration in DNA methylation pattern is much more frequent in the plant genome during the tissue culture process. DNA methylation plays an important role in gene expression and regulation of plant development. Variants originated in tissue culture process due to heritable methylation changes, can contribute to intra-species phenotypic variation. Several molecular techniques are available to detect DNA methylation at different stages of in vitro culture. Here, we review the aspects of TCIV with respect to DNA methylation and its effect on crop improvement programs. It is anticipated that a precise and comprehensive knowledge of molecular basis of in vitro-derived DNA methylation will help to design strategies to overcome the bottlenecks of micropropagation system and maintain the clonal fidelity of the regenerants.

Inhibition of Inflammatory Cytokine Expression Prevents High-Fat Diet-Induced Kidney Injury: Role of Lingonberry Supplementation.

Chronic low-grade inflammation is a major stimulus for progression of chronic kidney disease (CKD) in individuals consuming high-fat diet. Currently, there are limited treatment options for CKD other than controlling the progression rate and its associated complications. Lingonberry (Vaccinium vitis-idaea L.) is rich in anthocyanins with demonstrated anti-inflammatory effect. In the current study, we investigated the potential renal protective effect of lingonberry and its anthocyanin (cyanidin-3-glucoside) in high-fat diet fed obese mice and in human proximal tubular cells. Prolonged consumption of high-fat diets is strongly associated with obesity, abnormal lipid and glucose metabolism. Mice (C57BL/6J) fed a high-fat diet (62% kcal fat) for 12 weeks developed renal injury as indicated by an elevation of blood urea nitrogen (BUN) level as well as an increase in renal kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and renin expression. Those mice displayed an activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and increased expression of inflammatory cytokines-monocyte chemoattractant-1 (MCP-1), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) in the kidneys. Mice fed a high-fat diet also had a significant elevation of inflammatory cytokine levels in the plasma. Dietary supplementation of lingonberry for 12 weeks not only attenuated high-fat diet-induced renal inflammatory response but also reduced kidney injury. Such a treatment improved plasma lipid and glucose profiles, reduced plasma inflammatory cytokine levels but did not affect body weight gain induced by high-fat diet feeding. Lingonberry extract or its active component cyanidin-3-glucoside effectively inhibited palmitic acid-induced NF-κB activation and inflammatory cytokine expression in proximal tubular cells. These results suggest that lingonberry supplementation can reduce inflammatory response and prevent chronic kidney injury. Such a renal protective effect by lingonberry and its active component may be mediated, in part, through NF-κB signaling pathway.

In Vitro Propagation and Variation of Antioxidant Properties in Micropropagated Vaccinium Berry Plants-A Review.

The berry crops in genus Vacciniun L. are the richest sources of antioxidant metabolites which have high potential to reduce the incidence of several degenerative diseases. In vitro propagation or micropropagation has been attractive to researchers for its incredible potential for mass production of a selected genotype in a short time, all year round. Propagation techniques affect the antioxidant activity in fruits and leaves. Total antioxidant activity was higher in the fruit of in vitro propagated plants compare to the plants grown ex vivo. This review provides critical information for better understanding the micropropagation and conventional propagation methods, and their effects on antioxidant properties and morphological differentiation in Vaccinium species, and fills an existing gap in the literature.

Antioxidant properties and structured biodiversity in a diverse set of wild cranberry clones.

Wild germplasm with elevated antioxidants are a useful resource for using directly and in a breeding program. In a study with 136 wild clones and two cranberry cultivars, phenolic, flavonoid and anthocyanin contents varied 2.79, 2.70 and 17.46 times, respectively. The antioxidant activity ranged from 1.17 ± 0.01 to 2.53 ± 0.05 mg/g and varied 2.16 times. Seventy-five of wild clones and the cultivar Franklin were grouped into five distinct classes by molecular structure analysis using inter simple sequence repeat (ISSR), expressed sequence tag-simple sequence repeat (EST-SSR) and EST-polymerase chain reaction (PCR) markers. Grouping with DNA markers did not coincide with that of based on antioxidant properties. Present study indicates that genetic diversity analysis combined with antioxidant properties of wild germplasm play a significant role for conservation and in selecting diverse genotypes for future berry crop improvement.

Thidiazuron-induced somatic embryogenesis and changes of antioxidant properties in tissue cultures of half-high blueberry plants.

An efficient protocol of somatic embryogenesis (SE) has been developed for the first time in four half-high blueberry (Vaccinium corymbosum L. × V. angustifolium Ait.) cultivars. Thidiazuron (TDZ), a plant growth regulator with potential activities for shoot regeneration and shoot proliferation, was found most effective for somatic embryo formation when added to a nutrient medium at high concentration (9 µM). Although TDZ was also best for embryo germination at low concentration (2.3 µM), it was followed by zeatin at 4.6 µM for the same. Plantlets developed from SE were removed from the nutrient medium and transferred on a peat: perlite medium where 100% survival rate was acquired following the acclimatization process in a greenhouse. The concentrations of total phenolic and flavonoid contents were higher in greenhouse-grown conventionally cutting-propagated donor mother plants than those of respective SE plants for 'St. Cloud', 'Patriot' and 'Northblue' but not for 'Chippewa'. The effect of propagation method and/or the older age of donor mother plants were clearly visible exclusively as the 15-year-old donor plants showed higher level of 2,2-diphenyl-1-picrylhydrazyl scavenging activity than the eight-weeks-old SE plants in all four cultivars.

Supplementing diet with Manitoba lingonberry juice reduces kidney ischemia-reperfusion injury.

BACKGROUND: Lingonberry (Vaccinium vitis-idaea L.) contains high levels of anthocyanins which are bioavailable in the kidney and may be protective against ischemia-reperfusion (IR)-induced acute kidney injury. This study investigated the effect of lingonberry juice on the IR-induced stress-activated signalling pathway and inflammatory response in the kidney. RESULTS: Sprague-Dawley rats subjected to kidney IR had significantly impaired kidney function, with increased activation of the JNK signalling pathway and increased inflammatory response, measured using a multiplex panel containing an extensive array of inflammatory biomarkers. In rats fed 1 mL lingonberry juice daily for 3 weeks prior to IR, kidney function was protected and attenuation of inflammatory response and JNK signalling was reflected in the reduction of the measured biomarkers. In vitro results in cultured HK-2 cells confirmed that lingonberry anthocyanins reduced JNK signalling and inflammatory gene expression after IR. CONCLUSION: This study shows, for the first time, that daily supplementation with lingonberry juice may protect against loss of kidney function induced by IR injury by modulating JNK signalling and inhibiting the subsequent inflammatory response. © 2017 Her Majesty the Queen in Right of Canada. Journal of the Science of Food and Agriculture © 2017 Society of Chemical Industry.

Phenolics of Selected Cranberry Genotypes (Vaccinium macrocarpon Ait.) and Their Antioxidant Efficacy.

Free, esterified, and bound phenolic fractions of berries from five different cranberry genotypes and two market samples were evaluated for their total phenolic, flavonoid, and monomeric anthocyanin contents as well as their antioxidant efficacy using TEAC, ORAC, DPPH radical, reducing power, and ferrous ion chelation capacity assays. HPLC-MS/MS analysis was performed for two of the rich sources (Pilgrim and wild clone NL2) of phenolics and high antioxidant activity. Among the genotypes, Pilgrim showed the highest phenolic and flavonoid contents and wild clones NL3 and NL2 showed the highest monomeric anthocyanin and proanthocyanidin content, respectively. Protocatechuic and syringic acids were detected only in Pilgrim, whereas luteolin 7-O-glucoside, quercetin 3-O-rhamnoside, quercetin 3-O-galactoside, proanthocyanidin B-type, and myricetin 3-O-galactoside were found in wild clone NL3 genotype. Moreover, proanthocyanin trimer A-type and dimer B-type predominated in the wild clone NL2, whereas proanthocyanidin dimer B and trimer A were predominant in Pilgrim.

Manitoba Lingonberry (Vaccinium vitis-idaea) Bioactivities in Ischemia-Reperfusion Injury.

Evidence for the efficacy of dietary interventions in protecting against cardiovascular disease has grown significantly, with flavonoids and anthocyanins receiving special attention. Lingonberry (Vaccinium vitis-idaea L.) is a good source of these compounds, and this study examined the protective effects of wild lingonberry found in Manitoba, Canada, against ischemia-reperfusion (IR) injury. Manitoba lingonberry contained 3793 ± 27 mg gallic acid equiv, 120,501 ± 7651 µmol trolox equiv, and 575 ± 20 mg cyanidin-3-glucoside equiv per 100 g dry weight, which correspond with high total phenolic content, antioxidant activity, and anthocyanin content, respectively. A complete methanolic extract and both anthocyanin-rich and phenolic-rich fractions inhibited apoptosis in H9c2 cells during simulated IR. Lingonberry extract and fractions significantly inhibited several markers of apoptosis induced by IR, including nuclei condensation, caspase-3 activation, and MAP kinase signaling. These results provide the first analysis of Manitoba lingonberry and highlight the mechanistic importance of dietary berry compounds for cardiovascular health.