M. Wirthensohn

Linkage disequilibrium, genetic association mapping and gene localization in crop plants

DNA-based molecular markers have been extensively utilized for a variety of studies in both plant and animal systems. One of the major uses of these markers is the construction of genome-wide molecular maps and the genetic analysis of simple and complex traits. However, these studies are generally based on linkage analysis in mapping populations, thus placing serious limitations in using molecular markers for genetic analysis in a variety of plant populations. Therefore, alternative approach has been suggested, linkage disequilibrium-based association analysis which detects and locates quantitative trait loci (QTL) by the strength of the correlation between a trait and a marker. Although association analysis has already been used for studies on genetics of complex traits in humans, its use in plants has newly started. In the present review, we describe what is known about variation in linkage disequilibrium (LD) and summarize published results on association studies in crop plant species. We give a list of different factors affecting LD, and discuss the current issues of LD research in plants. Later, we also describe the various uses of LD in crop plants research and summarize the present status of LD researches in different plant genomes. Finally, future key issues about the application of these studies on the localization of genes in these crop plants have been also discussed.

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.

Characterising the flavour phenotypes of almond (Prunus dulcis Mill.) kernels

Summary To determine the chemical basis of almond flavour phenotypes, non-bitter, semi-bitter, and bitter kernels from 101 almond trees, all derived from a common maternal parent, were evaluated for flavour using three approaches: (1) sensory analysis (i.e., by taste); (2) amygdalin quantitation (using High Performance Liquid Chromatography); and (3) non-targeted analysis of volatile metabolites released on maceration (by Solid Phase Micro-Extraction, Gas Chromatography-Mass Spectrometry, and Chemical Sensor). Tasting identified three categories of flavour (non-bitter, semi-bitter, and bitter) that were also distinguishable on the basis of chemical analyses, and by a chemical sensor using principal component analysis. Highly significant correlations were found between amygdalin content and the taste panel’s score for ‘marzipan’, and also between the taste panel’s scores for sweet and overall taste preference. Non-targeted metabolite analysis identified benzyl alcohol and 2,3-butanediol as potentially important flavour components of almond, for the first time.

Variability in waxiness of Eucalyptus gunnii foliage for floriculture

Eucalyptus gunnii leaves can appear as green or glaucous phenotypes with the latter more desirable for floriculture. The epicuticular wax from these two types were compared morphologically using environmental scanning electron microscopy, chemically using gas chromatography, and molecular markers were found to distinguish the two types using RAPDs and bulked segregant analysis. Both phenotypes had wax tubes, which in the glaucous type were significantly longer and thicker and the surface area was covered more densely than on the green type. When compared chemically, the glaucous wax seemed to contain a higher percentage of alkanes, alcohols and free fatty acids, but lower percentages of β-diketones and esters than the wax from the green type. The glaucous type had a greater yield of wax per unit area of leaf than the green. Seven molecular markers were found which would distinguish the green and glaucous bulks. No individual marker could totally distinguish all green individuals from all glaucous individuals but this could be achieved using combinations of markers.

Interspecific Hybridization between Banksia hookeriana Meisn. and Banksia prionotes Lindl. (Proteaceae)

Interspecific hybridization of Banksia hookeriana and Banksia prionotes was investigated via pollen tube growth, seed set, morphological measurements, and analysis of RAPD polymorphisms. Pollen tube growth to the ovary was observed following selfing and crossing pollination of each species and following interspecific hybridization to B. hookeriana as the female parent, but not in the reciprocal cross. All crosses resulted in seed set, except for self-pollination of B. prionotes and interspecific pollination to B. prionotes as the female parent. Mortality of hybrid seedlings was high. RAPD analysis of hybrid seedlings from two families showed paternal B. prionotes bands in all 11 tested seedlings. Leaf length or width of nine hybrid seedlings that survived to the 10-leaf stage was intermediate between that of intraspecific seedlings of both parents at the same age. A mature natural interspecific hybrid was fertile following controlled selfing and backcrossing to each parental species, as well as following open pollination, but seed set was lower, than for the parental species. It was concluded that hybridization between B. hookeriana and B. prionotes is unilateral, with interspecific seed set of B. hookeriana comparable to that following intraspecific pollination.

Molecular analyses of evolution and population structure in a worldwide almond [Prunus dulcis (Mill.) D.A. Webb syn. P. amygdalus Batsch] pool assessed by microsatellite markers

A total of 158 almond accessions representative of the diversity of almond across the five continents were included for analysis using 17 microsatellite polymorphic markers. Genetic relationships among genotypes were estimated using cluster analysis, allowing their differentiation in two main groups, one with the domesticated almond cultivars and selections and the other with all wild Prunus species close to almond. The unweighted pair group method average tree drawn from this analysis classified the genotypes according to their geographical origin, confirming the particular evolution of different almond ecotypes. Structure analysis showed a strong subpopulation structure and linkage disequilibrium decaying with increasing genetic linkage distance. Analysis of molecular variance confirmed that most of the genetic variability was within populations. Therefore the connection structure between the different populations and the possible bottlenecks in the expansion of almond cultivars could be established.

Influence of deficit irrigation strategies on fatty acid and tocopherol concentration of almond (Prunus dulcis).

The effects of deficit irrigation on almond fatty acid and tocopherol levels were studied in a field trial. Mature almond trees were subjected to three levels of deficit irrigation (85%, 70% and 55% of potential crop evapotranspiration (ETo), as well as control (100% ETo) and over-irrigation (120% ETo) treatments. Two deficit irrigation strategies were employed: regulated deficit irrigation (RDI) and sustained deficit irrigation (SDI). Moderate deficit irrigation (85% RDI and 85% SDI) had no detrimental impact on almond kernel lipid content, but severe and extreme deficiencies (70% and 55%) influenced lipid content. Unsaturated fatty acid (USFA) and saturated fatty acid (SFA) contents fluctuated under these treatments, the oleic/linoleic ratio increased under moderate water deficiency, but decreased under severe and extreme water deficiency. Almond tocopherols concentration was relatively stable under deficit irrigation. The variation between years indicated climate has an effect on almond fruit development. In conclusion it is feasible to irrigate almond trees using less water than the normal requirement, without significant loss of kernel quality.

Effect of pruning on regrowth of cut foliage stems of seventeen Eucalyptus species.

Summary. Seventeen species of Eucalyptus were subjected to trials investigating their suitability for floriculture and the effect of pruning on cut foliage production. There was variation in leaf colour within and between species, and in the time to phase change and flowering. There were significant differences at 16 months between species for tree height, trunk diameter and lignotuber diameter with E. globulus having the largest dimensions. Following pruning at 19 months, there was an initial significant interaction between species and pruning height in relation to tree height, height increment, and trunk and lignotuber diameter. After 1 year there were significant differences between species, in the length of stems, number of stems and total weight of stems, with E. globulus producing the highest number and weight of stems. There was a positive correlation of trunk diameter and lignotuber diameter at 3 and 6 months after pruning with the number of cut foliage stems produced at 12 months. For E. gunnii pruning to 1.0 m at 25 months produced the most stems at 6 months after pruning.

Effects of commercially produced almond by-products on chemotherapy-induced mucositis in rats

AIM To determine if almond extracts reduce the severity of chemotherapy-induced mucositis as determined through biochemical, histological and behavioural markers. METHODS Intestinal mucositis is a debilitating condition characterized by inflammation and ulceration of the gastrointestinal mucosa experienced by cancer patients undergoing chemotherapy. Certain bioactive plant products have shown promise in accelerating mucosal repair and alleviating clinical symptoms. This study evaluated almond extracts for their potential to reduce the severity of chemotherapy-induced mucositis in Dark Agouti rats. Female Dark Agouti rats were gavaged (days 3-11) with either PBS, almond hull or almond blanched water extract at two doses, and were injected intraperitoneally with 5-fluorouracil (5-FU-150 mg/kg) or saline on day 9 to induce mucositis. Burrowing behavior, histological parameters and myeloperoxidase activity were assessed. RESULTS Bodyweight was significantly reduced in rats that received 5-FU compared to saline-treated controls (P < 0.05). Rats administered 5-FU significantly increased jejunal and ileal MPO levels (1048%; P < 0.001 and 409%; P < 0.001), compared to healthy controls. Almond hull extract caused a pro-inflammatory response in rats with mucositis as evidenced by increased myeloperoxidase activity in the jejunum when compared to 5-FU alone (rise 50%, 1088 ± 96 U/g vs 723 ± 135 U/g, P = 0.02). Other extract-related effects on inflammatory activity were minimal. 5-FU significantly increased histological severity score compared to healthy controls confirming the presence of mucositis (median of 9.75 vs 0; P < 0.001). The extracts had no ameliorating effect on histological severity score in the jejunum or ileum. Burrowing behavior was significantly reduced in all chemotherapy-treated groups (P = 0.001). The extracts failed to normalize burrowing activity to baseline levels. CONCLUSION Almond extracts at these dosages offer little beneficial effect on mucositis severity. Burrowing provides a novel measure of affective state in studies of chemotherapy-induced mucositis.

Molecular modeling of S-RNases involved in almond self-incompatibility

Gametophytic self-incompatibility (GSI) is a mechanism in flowering plants, to prevent inbreeding and promote outcrossing. GSI is under the control of a specific locus, known as the S-locus, which contains at least two genes, the RNase and the SFB. Active S-RNases in the style are essential for rejection of haploid pollen, when the pollen S-allele matches one of two S-alleles of the diploid pistil. However, the nature of their mutual interactions at genetic and biochemical levels remain unclear. Thus, detailed understanding of the protein structure involved in GSI may help in discovering how the proteins involved in GSI may function and how they fulfill their biological roles. To this end, 3D models of the SC (Sf) and two SI (S8 and S23) S-RNases of almond were constructed, using comparative modeling tools. The modeled structures consisted of mixed α and β folds, with six helices and six β-strands. However, the self-compatible (Sf) RNase contained an additional extended loop between the conserved domains RC4 and C5, which may be involved in the manifestation of self-compatibility in almond.