QTL and candidate gene mapping for polyphenolic composition in apple fruit.

BACKGROUND: The polyphenolic products of the phenylpropanoid pathway, including proanthocyanidins, anthocyanins and flavonols, possess antioxidant properties that may provide health benefits. To investigate the genetic architecture of control of their biosynthesis in apple fruit, various polyphenolic compounds were quantified in progeny from a 'Royal Gala' × 'Braeburn' apple population segregating for antioxidant content, using ultra high performance liquid chromatography of extracts derived from fruit cortex and skin. RESULTS: Construction of genetic maps for 'Royal Gala' and 'Braeburn' enabled detection of 79 quantitative trait loci (QTL) for content of 17 fruit polyphenolic compounds. Seven QTL clusters were stable across two years of harvest and included QTLs for content of flavanols, flavonols, anthocyanins and hydroxycinnamic acids. Alignment of the parental genetic maps with the apple whole genome sequence in silico enabled screening for co-segregation with the QTLs of a range of candidate genes coding for enzymes in the polyphenolic biosynthetic pathway. This co-location was confirmed by genetic mapping of markers derived from the gene sequences. Leucoanthocyanidin reductase (LAR1) co-located with a QTL cluster for the fruit flavanols catechin, epicatechin, procyanidin dimer and five unknown procyanidin oligomers identified near the top of linkage group (LG) 16, while hydroxy cinnamate/quinate transferase (HCT/HQT) co-located with a QTL for chlorogenic acid concentration mapping near the bottom of LG 17. CONCLUSION: We conclude that LAR1 and HCT/HQT are likely to influence the concentration of these compounds in apple fruit and provide useful allele-specific markers for marker assisted selection of trees bearing fruit with healthy attributes.

Creating a neurogenic environment: the role of BDNF and FGF2.

Regional environmental cues present in the adult brain determine the fate of adult neural progenitor cells. To determine whether the growth factors BDNF or FGF2 can create a neurogenic environment outside the SVZ, we used AAV(1/2)-mediated gene transfer to produce ectopic BDNF or FGF2 expression in the normal adult rat striatum and transplanted SVZ-derived progenitor cells into this region. We observed that ectopic expression of BDNF in the striatum promoted neuronal differentiation of transplanted adult neural progenitor cells, while FGF2 expression supported the survival and proliferation of transplanted progenitor cells in the adult striatum. However, region-specific neuronal differentiation of transplanted progenitor cells was not observed in the adult striatum, suggesting ectopic BDNF or FGF2 expression was insufficient for the generation of mature neuronal phenotypes. This study provides direct in vivo evidence that ectopic striatal expression of either BDNF or FGF2 can induce neurogenesis in non-neurogenic regions of the adult brain.

AAV-mediated delivery of BDNF augments neurogenesis in the normal and quinolinic acid-lesioned adult rat brain.

Brain-derived neurotrophic factor (BDNF) plays a major role in regulating the survival and fate of progenitor cells in the adult brain. In order to extend previous observations in the normal adult brain and advance our knowledge regarding the effect of BDNF on neurogenesis in the injured brain, this study directly compared the effect of BDNF on basal and injury-induced neurogenesis in relation to progenitor cell distribution and levels of neuronal differentiation and survival. BDNF was overexpressed in the subventricular zone (SVZ) via recombinant adeno-associated virus (AAV(1/2)) delivery, and newly generated cells were identified using bromodeoxyuridine (BrdU) labelling. Selective striatal cell loss was induced in a subgroup of rats by unilateral striatal injection of quinolinic acid (QA) 21 days after AAV(1/2) injection. In the normal brain, BDNF overexpression significantly increased BrdU-positive cell numbers in the rostral migratory stream, indicating enhanced progenitor cell migration. Following QA lesioning, we observed a reduction in BrdU immunoreactivity in the SVZ. Overexpression of BDNF restored BrdU-positive cell numbers in the QA-lesioned SVZ to that observed in the normal brain. Most significantly, BDNF enhanced the recruitment of progenitor cells to the QA-lesioned striatum and promoted neuronal differentiation in both the normal and QA-lesioned striatum. Our findings indicate that BDNF augments the recruitment, neuronal differentiation and survival of progenitor cells in both neurogenic and non-neurogenic regions of the normal or QA-lesioned brain. Enhanced expression of BDNF may therefore be a viable strategy for augmenting neurogenesis from endogenous progenitor cells.

Verification of functional AAV-mediated neurotrophic and anti-apoptotic factor expression.

The use of viral vectors for gene delivery offer many advantages for both basic research and therapeutic application through the continuous expression of a gene product within a target region. It is vital however that any gene product is correctly expressed in a biologically active form, and this should be confirmed prior to large scale in vivo studies. Using adeno-associated viral (AAV) vectors to direct the expression of either a neurotrophic factor or an anti-apoptotic protein, we have developed a range of in vitro assays to verify functional transgenic protein expression. Brain-derived neurotropic factor (BDNF) activity was confirmed by demonstrating enhanced generation of GABAergic neurons in embryonic (E15) striatal cultures and AAV-mediated glial-derived neurotrophic factor (GDNF) function using an assay for dopaminergic differentiation of embryonic (E14) ventral mesencephalic cultures. To assess functional anti-apoptotic factor expression we designed cell-survival assays, using embryonic cortical cultures to confirm Bcl-x(L) activity and the HT1080 cell-line for X-linked inhibitor of apoptosis protein (XIAP) activity following AAV-mediated expression. This study demonstrates that the use of functional assays provides valuable confirmation of desired biotherapeutic expression prior to extensive investigation with new gene delivery vectors.