Time of pruning affects fruit abscission, stem carbohydrates and yield of macadamia.

Macadamia (Macadamia integrifolia Maiden and Betche, M. tetraphylla Johnson and hybrids) orchards in Australia are typically hedged around anthesis (September). Such hedging reduces yields, largely through competition for carbohydrates between early fruit set and the post-pruning vegetative flush, but also through a reduction in photosynthetic capacity caused by the loss of canopy. We examined whether hedging at other times might mitigate yield losses. Hedging time was found to affect yields across four cultivars: 'A4', 'A38', '344' and '816'. Yield losses were lower for trees hedged in November-December than for trees hedged in September. Yields for trees hedged in June were higher than for trees hedged in September in one experiment, but were similar in a second experiment. Yield losses for September and October hedging were similar. Hedging time changed the pattern of fluctuations in stem water-soluble carbohydrates (WSC). WSC declined shortly after hedging in September, October or November, and the declines preceded increases in fruit abscission relative to unpruned control trees. The increase in fruit abscission was less pronounced for the trees hedged in November, consistent with the idea that fruit become less sensitive to carbon limitation as they mature.

Post-pruning shoot growth increases fruit abscission and reduces stem carbohydrates and yield in macadamia.

BACKGROUND AND AIMS: There is good evidence for deciduous trees that competition for carbohydrates from shoot growth accentuates early fruit abscission and reduces yield but the effect for evergreen trees is not well defined. Here, whole-tree tip-pruning at anthesis is used to examine the effect of post-pruning shoot development on fruit abscission in the evergreen subtropical tree macadamia (Macadamia integrifolia, M. integrifolia × tetraphylla). Partial-tree tip-pruning is also used to test the localization of the effect. METHODS: In the first experiment (2005/2006), all branches on trees were tip-pruned at anthesis, some trees were allowed to re-shoot (R treatment) and shoots were removed from others (NR treatment). Fruit set and stem total non-structural carbohydrates (TNSC) over time, and yield were measured. In the second experiment (2006/2007), upper branches of trees were tip-pruned at anthesis, some trees were allowed to re-shoot (R) and shoots were removed from others (NR). Fruit set and yield were measured separately for upper (pruned) and lower (unpruned) branches. KEY RESULTS: In the first experiment, R trees set far fewer fruit and had lower yield than NR trees. TNSC fell and rose in all treatments but the decline in R trees occurred earlier than in NR trees and coincided with early shoot growth and the increase in fruit abscission relative to the other treatments. In the second experiment, fruit abscission on upper branches of R trees increased relative to the other treatments but there was little difference in fruit abscission between treatments on lower branches. CONCLUSIONS: This study is the first to demonstrate an increase in fruit abscission in an evergreen tree in response to pruning. The effect appeared to be related to competition for carbohydrates between post-pruning shoot growth and fruit development and was local, with shoot growth on pruned branches having no effect on fruit abscission on unpruned branches.

Construction of an almond linkage map in an Australian population Nonpareil x Lauranne.

BACKGROUND: Despite a high genetic similarity to peach, almonds (Prunus dulcis) have a fleshless fruit and edible kernel, produced as a crop for human consumption. While the release of peach genome v1.0 provides an excellent opportunity for almond genetic and genomic studies, well-assessed segregating populations and the respective saturated genetic linkage maps lay the foundation for such studies to be completed in almond. RESULTS: Using an almond intraspecific cross between 'Nonpareil' and 'Lauranne' (N x L), we constructed a moderately saturated map with SSRs, SNPs, ISSRs and RAPDs. The N x L map covered 591.4 cM of the genome with 157 loci. The average marker distance of the map was 4.0 cM. The map displayed high synteny and colinearity with the Prunus T x E reference map in all eight linkage groups (G1-G8). The positions of 14 mapped gene-anchored SNPs corresponded approximately with the positions of homologous sequences in the peach genome v1.0. Analysis of Mendelian segregation ratios showed that 17.9% of markers had significantly skewed genotype ratios at the level of P < 0.05. Due to the large number of skewed markers in the linkage group 7, the potential existence of deleterious gene(s) was assessed in the group. Integrated maps produced by two different mapping methods using JoinMap® 3 were compared, and their high degree of similarity was evident despite the positional inconsistency of a few markers. CONCLUSIONS: We presented a moderately saturated Australian almond map, which is highly syntenic and collinear with the Prunus reference map and peach genome V1.0. Therefore, the well-assessed almond population reported here can be used to investigate the traits of interest under Australian growing conditions, and provides more information on the almond genome for the international community.

Morphological, genetic, and pathogenic characterization of Colletotrichum acutatum, the cause of anthracnose of almond in Australia.

Almond anthracnose was reported for the first time in Australia in 1998 and has since been observed in all of the major almond-growing regions. The organism causing anthracnose was confirmed as Colletotrichum acutatum using taxon-specific polymerase chain reaction (PCR). Three main morphotypes of C. acutatum from almond in Australia were identified (namely, pink, orange, and cream colony color) and the optimum temperature for mycelial growth of representative isolates was 25 degrees C. Australian isolates of C. acutatum were more similar morphologically to the pink subpopulation of C. acutatum from California than to the gray Californian subpopulation and the isolates of Colletotrichum from Israel. Inter-simple-sequence-repeat (ISSR) PCR analysis revealed that the majority of Australian isolates shared an identical banding pattern whereas Australian isolates of C. acutatum from almond were distinct from isolates of the pink and gray subpopulations of C. acutatum from almond in California and of Colletotrichum spp. from almond in Israel. Sequence analysis of the internally transcribed spacer (ITS1-2) ribosomal DNA region of representative isolates differed from the results of ISSR-PCR in that polymorphisms were revealed among isolates, indicating that some genetic variation may be present. Pathogenicity experiments on detached leaves and fruit revealed pathogenic variation among representative isolates of C. acutatum from almond in Australia, California, and Israel; however, all isolates tested caused disease. Distinct subgroups among Australian isolates of C. acutatum from almond were not supported on the basis of morphology, mycelial growth rates, ISSR-PCR, and pathogenicity.

Mapping SNP-anchored genes using high-resolution melting analysis in almond.

Peach and almond have been considered as model species for the family Rosaceae and other woody plants. Consequently, mapping and characterisation of genes in these species has important implications. High-resolution melting (HRM) analysis is a recent development in the detection of SNPs and other markers, and proved to be an efficient and cost-effective approach. In this study, we aimed to map genes corresponding to known proteins in other species using the HRM approach. Prunus unigenes were searched and compared with known proteins in the public databases. We developed single-nucleotide polymorphism (SNP) markers, polymorphic in a mapping population produced from a cross between the cloned cultivars Nonpareil and Lauranne. A total of 12 SNP-anchored putative genes were genotyped in the population using HRM, and mapped to an existing linkage map. These genes were mapped on six linkage groups, and the predicted proteins were compared to putative orthologs in other species. Amongst those genes, four were abiotic stress-responsive genes, which can provide a starting point for construction of an abiotic resistance map. Two allergy and detoxification related genes, respectively, were also mapped and analysed. Most of the investigated genes had high similarities to sequences from closely related species such as apricot, apple and other eudicots, and these are putatively orthologous. In addition, it was shown that HRM can be an effective means of genotyping populations for the purpose of constructing a linkage map. Our work provides basic genomic information for the 12 genes, which can be used for further genetic and functional studies.

High resolution melting analysis of almond SNPs derived from ESTs.

High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.

Regulation of floral initiation in horticultural trees.

The intention of this review is to discuss floral initiation of horticultural trees. Floral initiation is best understood for herbaceous species, especially at the molecular level, so a brief overview of the control of floral initiation of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) precedes the discussion of trees. Four major pathways to flowering have been characterized in Arabidopsis, including environmental induction through photoperiod and temperature, autonomous floral initiation, and regulation by gibberellins. Tropical trees are generally induced to flower through environmental cues, whereas floral initiation of temperate deciduous trees is often autonomous. In the tropical evergreen tree mango, Mangifera indica L., cool temperature is the only factor known to induce flowering, but does not ensure floral initiation will occur because there are important interactions with vegetative growth. The temperate deciduous tree apple, Malus domestica Borkh., flowers autonomously, with floral initiation dependent on aspects of vegetative development in the growing season before anthesis, although with respect to the floral initiation of trees in general: the effect of the environment, interactions with vegetative growth, the roles of plant growth regulators and carbohydrates, and recent advances in molecular biology, are discussed.

A seed coat cyanohydrin glucosyltransferase is associated with bitterness in almond (Prunus dulcis) kernels.

The secondary metabolite amygdalin is a cyanogenic diglucoside that at high concentrations is associated with intense bitterness in seeds of the Rosaceae, including kernels of almond (Prunus dulcis (Mill.), syn. Prunus amygdalus D. A. Webb Batsch). Amygdalin is a glucoside of prunasin, itself a glucoside of R-mandelonitrile (a cyanohydrin). Here we report the isolation of an almond enzyme (UGT85A19) that stereo-selectively glucosylates R-mandelonitrile to produce prunasin. In a survey of developing kernels from seven bitter and 11 non-bitter genotypes with polyclonal antibody raised to UGT85A19, the enzyme was found to accumulate to higher levels in the bitter types in later development. This differential accumulation of UGT85A19 is associated with more than three-fold greater mandelonitrile glucosyltransferase activity in bitter kernels compared with non-bitter types, and transcriptional regulation was demonstrated using quantitative-PCR analysis. UGT85A19 and its encoding transcript were most concentrated in the testa (seed coat) of the kernel compared with the embryo, and prunasin and amygdalin were differentially compartmentalised in these tissues. Prunasin was confined to the testa and amygdalin was confined to the embryo. These results are consistent with the seed coat being an important site of synthesis of prunasin as a precursor of amygdalin accumulation in the kernel. The presence of UGT85A19 in the kernel and other tissues of both bitter and non-bitter types indicates that its expression is unlikely to be a control point for amygdalin accumulation and suggests additional roles for the enzyme in almond metabolism.

Erratum to: A seed coat cyanohydrin glucosyltransferase is associated with bitterness in almond (Prunus dulcis) kernels.

The secondary metabolite amygdalin is a cyanogenic diglucoside that at high concentrations is associated with intense bitterness in seeds of the Rosaceae, including kernels of almond (Prunus dulcis (Mill.), syn. Prunus amygdalus D. A. Webb Batsch). Amygdalin is a glucoside of prunasin, itself a glucoside of R-mandelonitrile (a cyanohydrin). Here we report the isolation of an almond enzyme (UGT85A19) that stereo-selectively glucosylates R-mandelonitrile to produce prunasin. In a survey of developing kernels from seven bitter and 11 non-bitter genotypes with polyclonal antibody raised to UGT85A19, the enzyme was found to accumulate to higher levels in the bitter types in later development. This differential accumulation of UGT85A19 is associated with more than three-fold greater mandelonitrile glucosyltransferase activity in bitter kernels compared with non-bitter types, and transcriptional regulation was demonstrated using quantitative-PCR analysis. UGT85A19 and its encoding transcript were most concentrated in the testa (seed coat) of the kernel compared with the embryo, and prunasin and amygdalin were differentially compartmentalised in these tissues. Prunasin was confined to the testa and amygdalin was confined to the embryo. These results are consistent with the seed coat being an important site of synthesis of prunasin as a precursor of amygdalin accumulation in the kernel. The presence of UGT85A19 in the kernel and other tissues of both bitter and non-bitter types indicates that its expression is unlikely to be a control point for amygdalin accumulation and suggests additional roles for the enzyme in almond metabolism.

Increased tomato yield through pollination by native Australian Amegilla chlorocyanea (Hymenoptera: Anthophoridae).

Amegilla spp. (Hymenoptera: Anthophoridae) have been suggested as potential native Australian alternative to overseas used bumblebees (Bombus spp.) for pollination of tomato in greenhouses. In this study, we investigate the effectiveness of Amegilla chlorocyanea Cockerell as a greenhouse pollinator of tomato, Lycopersicon esculentum Mill. We show that (1) a single buzz by a female increases tomato weight by 11% compared with pollination by using an industrial pollination wand, (2) multiple buzzes increase tomato weight compared with a single buzz, and (3) unlimited flower visits lead to an increase in fruit weight of 21% compared with wand pollination. These results are comparable with those achieved by bumblebee pollination and demonstrate that A. chlorocyanea is a valid alternative to bumblebees for greenhouse tomato pollination in Australia.

Improved methods in Agrobacterium-mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols.

A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 muM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.

Paternity analysis using microsatellite markers to identify pollen donors in an olive grove.

Olive (Olea europaea L.) is a wind-pollinated, allogamous species that is generally not considered to be self-compatible. In addition, cross-incompatibilities exist between cultivars that can result in low fruit set if compatible pollinisers are not planted nearby. In this study, microsatellite markers were used to identify 17 genotypes that were potential pollen donors in a commercial olive orchard. DNA typing with the same primers was also applied to 800 olive embryos collected from five cultivars in the grove over 2 years of study. Pollen donors for the cultivars Barnea, Corregiola, Kalamata, Koroneiki, and Mission were estimated by paternity analysis, based on the parental contribution of alleles in the genotypes of the embryos. The exclusion probability for the marker set was 0.998 and paternity was assigned on the basis of the 'most likely method'. Different pollen donors were identified for each of the maternal cultivars indicating that cross-compatibilities and incompatibilities varied between the genotypes studied. Cross-pollination was the principal method of fertilization, as selfing was only observed in two of the embryos studied and both of these were from the cultivar Mission. This is the first report where these techniques have been applied to survey the pollination patterns in an olive grove. The results indicate that careful planning in orchard design is required for efficient pollination between olive cultivars.

A molecular linkage map of olive (Olea europaea L) based on RAPD, microsatellite, and SCAR markers.

An integrated molecular linkage map of olive (Olea europaea L.) was constructed based on randomly amplified polymorphic DNA (RAPD), sequence characterized amplified region (SCAR), and microsatellite markers using the pseudo-testcross strategy. A mapping population of 104 individuals was generated from an F1 full-sib family of a cross between 'Frantoio' and 'Kalamata'. The hybridity of the mapping population was confirmed by genetic similarity and nonmetric multidimensional scaling. Twenty-three linkage groups were mapped for 'Kalamata', covering 759 cM of the genome with 89 loci and an average distance between loci of 11.5 cM. Twenty-seven linkage groups were mapped for 'Frantoio', covering 798 cM of the genome with 92 loci and an average distance between loci of 12.3 cM. For the integrated map, 15 linkage groups covered 879 cM of the genome with 101 loci and an average distance between loci of 10.2 cM. The size of the genomic DNA was estimated to be around 3000 cM. A sequence characterized amplified region marker linked to olive peacock disease resistance was mapped to linkage group 2 of the integrated map. These maps will be the starting point for studies on the structure, evolution, and function of the olive genome. When the mapping progeny pass through their juvenile phase and assume their adult characters, mapping morphological markers and identification of quantitative trait loci for adaptive traits will be the primary targets.

Interaction between Vitis vinifera and grape phylloxera: changes in root tissue during nodosity formation.

BACKGROUND AND AIMS: The interaction between the gall-forming grapevine parasite, phylloxera, and the susceptible grapevine species Vitis vinifera was investigated. METHODS: Phylloxera and grapevines were cocultivated using both potted and micropropagated grapevines. Development of nodosities on primary roots was studied by microscopy and histochemistry, and nodosities were analysed for biochemical changes and changes in gene expression. KEY RESULTS: Within a nodosity, phylloxera fed at a site in the root cortex. Nodosity development was characterized by swelling of the root tissue distal to the feeding site, lack of development of a suberized endodermis, and starch and amino acid accumulation, and was eventually followed by root necrosis. No evidence of a defence response was observed in pre-necrotic nodosities, but defence-type responses were observed in tissue adjacent to necrotic regions. Changes in gene expression were not detected by northern hybridization using DNA probes encoding a range of V. vinifera transcripts. CONCLUSIONS: Nodosities on V. vinifera potentially function as nutrient reservoirs, and defence responses to phylloxera attack were not detected.

Comparison of ELISA and RT-PCR for the detection of Prunus necrotic ring spot virus and prune dwarf virus in almond (Prunus dulcis).

A technique based on the reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect the presence of Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) simultaneously in almond. This paper presents the results of a 3-year study comparing both enzyme-linked immunosorbent assay (ELISA) and RT-PCR for the detection of PNRSV and PDV using 175 almond leaf samples. Multiplex RT-PCR was found to be more sensitive than ELISA, especially when followed by nested PCR for the detection of PDV. The RT-PCR technique has the added advantage that plant material can be tested at any time throughout the growing season.

Purification of anthocyanins from species of Banksia and Acacia using high-voltage paper electrophoresis.

A new method has been developed for the isolation and rapid identification of anthocyanins from two floricultural crops based on the use of high-voltage paper electrophoresis with bisulphite buffer. Using this method, anthocyanin pigments were successfully purified as their negatively charged bisulphite-addition compounds from crude extracts of plant tissue. In conjunction with liquid chromatography-electrospray mass spectrometry, the method enabled the anthocyanins from the flowers of two Banksia species and the leaves of two Acacia species to be identified. The Banksia flowers contained both cyanidin and peonidin-based pigments, while the Acacia leaves contained cyanidin and delphinidin derivatives.

Structural basis of the rind disorder oleocellosis in Washington navel orange (Citrus sinensis L. Osbeck).

Oleocellosis, a physiological rind disorder of citrus fruit, is an unattractive surface blemish caused by phytotoxic effects of released rind oils. The development of oleocellosis in Washington navel orange (Citrus sinensis L. Osbeck) was examined by following a time sequence of surface symptoms and microscopic rind changes. The two natural causes of oleocellosis were simulated: mechanical damage to the fruit and transfer of rind oil between fruit. Mechanical fruit injury resulted in rupture of the epidermis above oil glands. Released surface oil appeared to infiltrate the rind via the ruptured epidermis resulting in rapid degeneration of cortical, but not epidermal, cell contents. Oil application to the rind surface produced a more severe blemish than did mechanical damage. The oil appeared to diffuse through the cuticle causing degeneration of the contents of all cell layers, including the epidermis. Loss of membrane integrity was detected within 30 min, followed by cell content degeneration and cell collapse. The resulting blemish, characterized by rind collapse and darkening, developed substantially within 3 d and was attributed to the cellular damage.