Fine-scale clonal structure of the lingonberry Vaccinium vitis-idaea under the nurse plant Pinus pumila vegetation in an alpine region, Mt. Norikura.

The nurse effect is a positive interaction in which a nurse plant improves the abiotic environment for another species (beneficiary plant) and facilitates its establishment. The evergreen shrub Vaccinium vitis-idaea (a beneficiary plant) grows mainly under the dwarf shrub Pinus pumila (a nurse plant) in the alpine regions of central Japan. However, whether V. vitis-idaea shrubs under various P. pumila shrubs spread through clonal growth and/or seeds remains unclear. We investigated the clonal structure of V. vitis-idaea under the nurse plant P. pumila in Japanese alpine regions. MIG-seq analysis was conducted to clarify the clonal diversity of V. vitis-idaea in isolated and patchy P. pumila plots on a ridge (PATs), and in a plot covered by dense P. pumila on a slope adjacent to the ridge (MAT) on Mt. Norikura, Japan. We detected 28 multilocus genotypes in 319 ramets of V. vitis-idaea across 11 PATs and MAT. Three genets expanded to more than 10 m in the MAT. Some genets were shared among neighboring PATs or among PATs and MAT. These findings suggest that the clonal growth of V. vitis-idaea plays an important role in the sustainability of populations. The clonal diversity of V. vitis-idaea was positively related with the spatial size of PATs and was higher in MAT than in PATs at a small scale. Therefore, the spatial spread of the nurse plant P. pumila might facilitate the nurse effect on the genetic diversity of beneficiary plants, leading to the sustainability of beneficiary populations.

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.

Ice holes microrefugia harbor genetically and functionally distinct populations of Vaccinium vitis-idaea (Ericaceae).

In the mountain terrain, ice holes are little depressions between rock boulders that are characterized by the exit of cold air able to cool down the rock surface even in summer. This cold air creates cold microrefugia in warmer surroundings that preserve plant species probably over thousands of years under extra-zonal climatic conditions. We hypothesized that ice hole populations of the model species Vaccinium vitis-idaea (Ericaceae) show genetic differentiation from nearby zonal subalpine populations, and high functional trait distinctiveness, in agreement with genetic patterns. We genotyped almost 30,000 single nucleotide polymorphisms using restriction site-associated DNA sequencing and measured eight functional traits indicative of individual performance and ecological strategies. Genetic results showed high differentiation among the six populations suggesting isolation. On siliceous bedrock, ice hole individuals exhibited higher levels of admixture than those from subalpine populations which could have experienced more bottlenecks during demographic fluctuations related to glacial cycles. Ice hole and subalpine calcareous populations clearly separated from siliceous populations, indicating a possible effect of bedrock in shaping genetic patterns. Trait analysis reflected the bedrock effect on populations' differentiation. The significant correlation between trait and genetic distances suggests the genetic contribution in shaping intraspecific functional differentiation. In conclusion, extra-zonal populations reveal a prominent genetic and phenotypic differentiation determined by history and ecological contingency. Therefore, microrefugia populations can contribute to the overall variability of the species and lead to intraspecific-driven responses to upcoming environmental changes.

Genetic variation associated with healthy traits and environmental conditions in Vaccinium vitis-idaea.

BACKGROUND: Lingonberry (Vaccinium vitis-idaea L.), one of the least studied fruit crops in the Ericaceae family, has a dramatically increased worldwide demand due to its numerous health benefits. Genetic markers can facilitate the selection of berries with desirable climatic adaptations, agronomic and nutritious characteristics to improve cultivation programs. However, no genomic resources are available for this species. RESULTS: We used Genotyping-by-Sequencing (GBS) to analyze the genetic variation of 56 lingonberry samples from across Newfoundland and Labrador, Canada. To elucidate a potential adaptation to environmental conditions we searched for genotype-environment associations by applying three distinct approaches to screen the identified single nucleotide polymorphisms (SNPs) for correlation with six environmental variables. We also searched for an association between the identified SNPs and two phenotypic traits: the total phenolic content (TPC) and antioxidant capacity (AC) of fruit. We identified 1586 high-quality putative SNPs using the UNEAK pipeline available in TASSEL. We found 132 SNPs likely associated with at least one of the environmental or phenotypic variables. To obtain insights on the function of the genomic sequences containing the SNPs likely to be associated with the environmental or phenotypic variables, we performed a sequence-based functional annotation and identified homologous protein-coding sequences with functional roles related to abiotic stress response, pathogen defense, RNA metabolism, and, most interestingly, phenolic compound biosynthesis. CONCLUSIONS: The putative SNPs discovered are the first genomic resource for lingonberry. This resource might prove useful in high-density quantitative trait locus analysis, and association mapping. The identified candidate genes containing the SNPs need further studies on their potential role in local adaptation of lingonberry. Altogether, the present study provides new resources that can be used to breed for desirable traits in lingonberry.

Persistent history of the bird-dispersed arctic-alpine plant Vaccinium vitis-idaea L. (Ericaceae) in Japan.

Arctic-alpine plants have expanded and contracted their ranges in response to the Pleistocene climate oscillations. Today, many arctic-alpine plants have vast distributions in the circumarctic region as well as marginal, isolated occurrences in high mountains at lower latitudes. These marginal populations may represent relict, long-standing populations that have persisted for several cycles of cold and warm climate during the Pleistocene, or recent occurrences that either result from southward step-wise migration during the last glacial period or from recent long-distance dispersal. In light of these hypotheses, we investigated the biogeographic history of the marginal Japanese populations of the widespread arctic-alpine plant Vaccinium vitis-idaea (Ericaceae), which is bird-dispersed, potentially over long distances. We sequenced three nuclear loci and one plastid DNA region in 130 individuals from 65 localities covering its entire geographic range, with a focus on its marginal populations in Japan. We found a homogenous genetic pattern across its enormous range based on the loci analysed, in contrast to the geographically structured variation found in a previous study of amplified fragment length polymorphisms in this species. However, we found several unique haplotypes in the Japanese populations, excluding the possibility that these marginal populations result from recent southward migration. Thus, even though V. vitis-idaea is efficiently dispersed via berries, our study suggests that its isolated populations in Japan have persisted during several cycles of cold and warm climate during the Pleistocene.

A C-repeat binding factor transcriptional activator (CBF/DREB1) from European bilberry (Vaccinium myrtillus) induces freezing tolerance when expressed in Arabidopsis thaliana.

Freezing stress affects all plants from temperate zones to the poles. Global climate change means such freezing events are becoming less predictable. This in turn reduces the ability of plants to predict the approaching low temperatures and cold acclimate. This has consequences for crop yields and distribution of wild plant species. C-repeat binding factors (CBFs) are transcription factors previously shown to play a vital role in the acclimation process of Arabidopsis thaliana, controlling the expression of hundreds of genes whose products are necessary for freezing tolerance. Work in other plant species cements CBFs as key determinants in the trait of freezing tolerance in higher plants. To test the function of CBFs from highly freezing tolerant plants species we cloned and sequenced CBF transcription factors from three Vaccinium species (Vaccinium myrtillus, Vaccinium uliginosum and Vaccinium vitis-idaea) which we collected in the Arctic. We tested the activity of CBF transcription factors from the three Vaccinium species by producing transgenic Arabidopsis lines overexpressing them. Only the Vaccinium myrtillus CBF was able to substantially activate COR (CBF-target) gene expression in the absence of cold. Correspondingly, only the lines expressing the Vaccinium myrtillus CBF were constitutively freezing tolerant. The basis for the differences in potency of the three Vaccinium CBFs was tested by observing cellular localisation and protein levels. All three CBFs were correctly targeted to the nucleus, but Vaccinium uliginosum CBF appeared to be relatively unstable. The reasons for lack of potency for Vaccinium vitis-idaea CBF were not due to stability or targeting, and we speculate that this was due to altered transcription factor function.

Expression of anatomical leaf traits in homoploid hybrids between deciduous and evergreen species of Vaccinium.

We investigated the anatomical expression of leaf traits in hybrids between evergreen Vaccinium vitis-idaea and deciduous V. myrtillus. We compared parents from four populations with their respective F1 hybrids and tested whether (i) transgression can be the source of novel anatomical traits in hybrids; (ii) expression of transgressive traits is more probable for traits with similar values in parents and intermediate for more distinct values, as predicted by theory; and (iii) independent origin of hybrids leads to identical trait expression profiles among populations. We found that anatomical leaf traits can be divided into four categories based on their similarity to parents: intermediate, parental-like, transgressive and non-significant. Contrary to the common view, parental-like trait values were equally important in shaping the hybrid profile, as were intermediate traits. Transgression was revealed in 17/144 cases and concerned mainly cell and tissue sizes. As predicted by theory, we observed transgressive segregation more often when there was little phenotypic divergence, but intermediate values when parental traits were differentiated. It is likely that cell and tissue sizes are phylogenetically more conserved due to stabilising selection, whereas traits such as leaf thickness and volume fraction of the intercellular spaces, showing a consistent intermediate pattern across populations, are more susceptible to directional selection. Hybrid populations showed little similarity in expression profile, with only three traits identically expressed across all populations. Thus local adaptation of parental species and specific genetic background may be of importance.

Identification of phenolic compounds from lingonberry (Vaccinium vitis-idaea L.), bilberry (Vaccinium myrtillus L.) and hybrid bilberry (Vaccinium x intermedium Ruthe L.) leaves.

Phenolic compounds from leaves of lingonberry (Vaccinium vitis-idaea L.), bilberry (Vaccinium myrtillus L.), and the natural hybrid of bilberry and lingonberry (Vaccinium x intermedium Ruthe L., hybrid bilberry) were identified using LC/TOF-MS and LC/MS/MS after extraction from the plant material in methanol in an ultrasonicator. The phenolic profiles in the plants were compared using the LC/TOF-MS responses. This is the first thorough report of phenolic compounds in hybrid bilberry. In total, 51 different phenolic compounds were identified, including flavan-3-ols, proanthocyanidins, flavonols and their glycosides, and various phenolic acid conjugates. Of the identified compounds, 35 were detected in bilberry, 36 in lingonberry, and 46 in the hybrid. To our knowledge, seven compounds were previously unreported in Vaccinium genus and many of the compounds are reported for the first time from bilberry and lingonberry.