Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.     

Non-TIR-NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genes encoding for proteins with nucleotide-binding site and leucine-rich repeat motifs (NBS-LRR) have been suggested to play a general role in plant defence mechanism. In Prunus species, many TIR (Toll / Interleukin-1 Receptor), and only very few non-TIR sequences were identified, which was explained either by the unequal distribution of TIR/non-TIR sequences in the Prunus genome or by the incapability of primers in the amplification of non-TIR RGAs. The objective of this work was to check whether a new semi-nested PCR strategy can be developed for the targeted isolation of non-TIR-NBS-LRR Resistance Gene Analog (RGA) sequences from apricot. Three primers (CUB-P-loop F, CUB-Kin2 F and CUB-HD R) were designed, from which CUB-Kin2 F and CUB-HD R were constructed to anneal selectively to the non-TIR sequences. A colony Polymerase Chain Reaction (PCR) indicated that out of the 96 clones tested 28 showed amplification using the newly developed primers, while no amplification occurred when using the formerly described primers. Half of the 28 positive clones were sequenced and they turned out to represent 11 different non-TIR RGA sequences. A phylogenetic analysis was carried out based on an alignment containing 293 Rosaceae and 21 non-Rosaceaa sequences. A significantly higher ratio (91%) of non-TIR sequences were arranged in multi-genera clades than that of (57%) the TIR groups confirming that non-TIR sequences might be of more ancient origin than TIR sequences.     

Inheritance of resistance to Plum pox virus in apricot (Prunus armeniaca L.)

The inheritance of resistance to Plum pox virus (PPV) has been studied in 1,178 apricot hybrids. Seven hundred and eighteen F1 hybrids, obtained from controlled crosses between the susceptible Greek cultivar “Bebecou” and the resistant PPV cultivars of American origin (“Stark Early Orange,” ‘NJA2,” ‘Veecot,” “Sunglo,” “Harlayne,” and “Orangered”) were evaluated for resistance to the PPV-M (Marcus) strain, 8 years after artificial inoculation. The inheritance of resistance to PPV has been additionally studied for the first time in a BC₁ population of 95 apricot hybrids for four vegetative periods. Reaction of each hybrid to PPV-M was scored through visual symptoms, indexing onto GF-305 and double-antibody sandwich enzyme-linked immunosorbent assay tests. Segregation within the hybrids, determined by Chi-squared analysis, fits a 1:1 ratio (P ≤ 0.05) of the resistant vs susceptible, indicating that resistance to PPV is controlled by a single dominant gene locus and that the above six resistant cultivars are heterozygous for the trait. Plants carrying this gene may initially develop disease symptoms on leaves but eventually recover and no virus can be detected in leaves. Susceptible plants show similar symptoms initially but remain symptomatic. Inheritance of resistance to PPV also has been studied in 365 F1 hybrids by crossing the resistant cultivar “Stella” with the susceptible “Bebecou” and the resistant cultivars “Sunglo” and “NJA2,” for 8 years after inoculation. The segregation ratio was 1:0 (resistant/susceptible) suggesting that “Stella” is homozygous for the resistance trait. The purpose of this work was the enhancement of the knowledge of inheritance of resistance to PPV for the selection of new cultivars.     

Genomic and cDNA microsatellites from apricot (Prunus armeniaca L.)

We developed primers for the amplification of 24 polymorphic nuclear microsatellites in apricot (Prunus armeniaca L.). Thirteen loci originated from three genomic libraries enriched for TC, TG and AAG motifs. Eight loci were developed from three fruit EST (Expressed‐Sequence‐Tag) libraries and three from a leaf cDNA microsatellite‐enriched library. There were up to nine alleles per polymorphic locus in 12 different cultivars. No difference in allele numbers were shown between cDNA and genomic‐source loci. Mean expected heterozygosity was 0.65 (range: 0.15–0.87). Mendelian segregation was confirmed for all loci. These markers should be helpful for diversity studies, genome mapping and cultivar identification in apricot and related species.     

Agrobacterium-mediated transformation of apricot (Prunus armeniaca L.) leaf explants

A protocol for Agrobacterium-mediated stable transformation for scored, whole leaf explants of the apricot (Prunus armeniaca) cultivar Helena was developed. Regenerated shoots were selected using a two-step increased concentrations of paromomycin sulphate. Different factors affecting survival of transformed buds, including possible toxicity of green fluorescent protein (GFP) and time of exposure to high cytokine concentration in the regeneration medium, were examined. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines was 5.6%, when optimal conditions for bud survival were provided. Southern blot analysis on four randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene. This is the first time that stable transformation of an apricot cultivar is reported and constitutes also one of the few reports on the transformation of Prunus cultivars.     

Effective pollination period in apricot (Prunus armeniaca L.) varieties

A study of the effective pollination period (E.P.P.) of six varieties of apricot has shown that in the warm climate of the South Mediterranean this parameter is extremely short. The percentage of fruit set of emasculated flowers diminishes rapidly with time. Temperature plays an important role in this process and is responsible for the rapid degradation of the stigma, which hinders pollen germination.     

An apricot (Prunus armeniaca L.) F2 progeny linkage map based on SSR and AFLP markers,mapping plum pox virus resistance and self-incompatibility traits

A genetic linkage map of apricot ( Prunus armeniaca L.) was constructed using AFLP and SSR markers. The map is based on an F(2) population (76 individuals) derived from self-pollination of an F(1) individual ('Lito') originated from a cross between 'Stark Early Orange' and 'Tyrinthos'. This family, designated as 'Lito' x 'Lito', segregated for two important agronomical traits: plum pox virus resistance (PPV) and self-incompatibility. A total of 211 markers (180 AFLPs, 29 SSRs and two agronomic traits) were assigned to 11 linkage groups covering 602 cM of the apricot genome. The average distance (cM/marker) between adjacent markers is 3.84 cM. The PPV resistance trait was mapped on linkage group G1 and the self-incompatibility trait was mapped on linkage group G6. Twenty two loci held in common with other Prunus maps allowed us to compare and establish homologies among the respective linkage groups.     

Pistil traits and flower fate in apricot (Prunus armeniaca)

Although pollination is essential for both seed and fruit set in most angiosperms, even after an adequate pollination, only a fraction of the flowers develop into fruits. The role played by floral traits on reproductive success is well known, but the possible influence of pistil traits has been overlooked, probably because of the difficulty of non-destructive pistil examination. The aim of this work was to examine the influence of several pistil traits on reproductive success in apricot ( Prunus armeniaca ). For this purpose, in a population of individually labelled flowers, the styles were cut off once the pollen tubes had reached the ovary but prior to the achievement of fertilisation. This approach allowed relating several morphological and physiological pistil parameters in the dissected styles and stigmas to the subsequent set or abscission of the corresponding ovary that remained in the plant. Under the same pollination conditions, the flowers that finally set a fruit show a larger stigmatic area and a higher number of pollen grains, pollen tubes growing along the style and xylem vessels surrounding the transmitting tissue than flowers that abscise before the establishment of fruit set. Furthermore, starch is present in the transmitting tissue of the style in all the flowers that develop into fruits but only in half of the flowers that abscise. The examined pistil traits established prior to fertilisation are related to flower fate, suggesting that the capacity of a flower to become a fruit could be preconditioned at anthesis.     

Stamen development and winter dormancy in apricot (Prunus armeniaca)

Background and Aims

In temperate woody perennials, flower bud development is halted during the winter, when the buds enter dormancy. This dormant period is a prerequisite for adequate flowering, is genetically regulated, and plays a clear role in possibly adapting species and cultivars to climatic areas. However, information on the biological events underpinning dormancy is lacking. Stamen development, with clear differentiated stages, appears as a good framework to put dormancy in a developmental context. Here, stamen developmental changes are characterized in apricot (Prunus armeniaca) and are related to dormancy.

Methods

Stamen development was characterized cytochemically from the end of August to March, over 4 years. Developmental changes were related to dormancy, using the existing empirical information on chilling requirements.

Key Results

Stamen development continued during the autumn, and the flower buds entered dormancy with a fully developed sporogenous tissue. Although no anatomical changes were observed during dormancy, breaking of dormancy occurred following a clear sequence of events. Starch accumulated in particular places, pre-empting further development in those areas. Vascular bundles developed and pollen mother cells underwent meiosis followed by microspore development.

Conclusions

Dormancy appears to mark a boundary between the development of the sporogenous tissue and the occurrence of meiosis for further microspore development. Breaking of dormancy occurs following a clear sequence of events, providing a developmental context in which to study winter dormancy and to evaluate differences in chilling requirements among genotypes.     

Purification and biochemical characteristics of pectinesterase from Malatya apricot (Prunus armeniaca L.)

Pectinesterase (PE) in Malatya apricot pulp (Prunus armeniaca L.) was extracted and purified through (NH(4))(2)SO(4) precipitation, dialysis, and DEAE-Sephadex gel filtration chromatography. The samples obtained from the dialysis procedure, named partially purified enzyme, were used for characterization of the apricot pectinesterase. The effect of various factors such as pH, temperature, heat, and storage stability on the partially purified apricot PE enzyme was investigated. Optimum pH value was 9.0 for PE with 1% pectin in 0.1 N NaCl (w/v). The optimum temperature for apricot PE was found to be 60 degrees C on standard analysis conditions. Heat inactivation studies showed a decrease in enzymatic activity at temperatures above 70 degrees C. Km and V(max) values were 0.77 mM and 1.75 micromol min(-1) mg(-1) for apricot PE. Five inhibitors were tested in the study; the most effective inhibitor was found to be sodium carbonate (100% inhibition). The order of inhibitory effectiveness was: Na(2)CO(3), iodine, lauril sulphate, AgNO(3), EDTA. Thermal inactivation data indicated that apparent activation energy with pectin substrate was 2.96 kcal mol(-1) for the enzyme. Ascorbic acid, CaCl(2), and KCl showed activatory effect on the apricot PE enzyme.     

Ovary starch reserves and flower development in apricot (Prunus armeniaca)

In histerant species where flowering takes place prior to leaf emergence, a flower lifespan occurs in the absence of new photoassimilates and at the expense of pre-stored reserves either in the plant as a whole or in the flower itself. In the present study, the role that the photoassimilates stored in the flowers might play in flower development from anthesis to fertilization in Prunus armeniaca L. (apricot), a histerant species, was explored. Starch content in individual flowers was measured with the help of an image analysis system. Starch content decreased from its highest value at anthesis and disappeared from the ovary 9 days later. This decrease was inversely related to an increase in ovary size and in cell number in the pericarp, suggesting an intraflower, self-supported development. This process is conserved in both pollinated and nonpollinated flowers and therefore seems to be inherent to the flower at anthesis. The onset of fruiting is preceded by the establishment of large differences among ovaries; while some experience continuous growth, others stop growing and eventually drop. Interestingly, large differences in starch content are found among flowers at anthesis. These results are discussed in terms of the possible implications of pre-stored starch in the flower supporting initial flower development.     

Influence of intraovular reserves on ovule fate in apricot (Prunus armeniaca L.)

 In many plant species with multiovulate ovaries, a considerable reduction in the number of ovules takes place. However, the underlying physiological causes are not clear. In Prunus spp., although flowers present two ovules, usually only one seed is produced. We have followed the development and degeneration of the two ovules in apricot (Prunus armeniaca L.) and examined the extent to which carbohydrates within the ovule might be involved in determining the fate of the ovule. While the primary ovule grows in the days following anthesis, growth of the secondary ovule is arrested. Starch distribution along the different ovular tissues exhibits several changes that are different in the two ovules. Primary ovule growth is inversely related to starch content and this growth takes place independently of pollination since it occurs in the same way in pollinated and unpollinated flowers. In the secondary ovule, starch disappears simultaneously from all ovular structures and callose is layered at the chalazal end of the nucellus. The size of the secondary ovule does not change significantly from anthesis to degeneration, and callose starts to accumulate 5 days after anthesis. Likewise, this process occurs independently of pollination. These results are discussed in terms of the implications of the starch content of ovules in fertilization success and ovule fate. Received: 26 August 1997 / Revision accepted: 17 December 1997     

Genetic diversity analysis and population structure in apricot (Prunus armeniaca L.) grown under north-western himalayas using ISSR markers

Investigation of genetic variability and population relationship of 50 accessions of the apricot (Prunus armeniaca L.) was carried out using ISSR markers. The results revealed that the number of alleles per locus varied from 4 to 8 with a mean value of 6.75, and the mean effective number of alleles (Ne) per locus was 1.54. Similarly, the polymorphic information content (PIC) values ranged from 0.464 to 0.424, with a mean value of 0.424. The mean heterozygosity, marker index, resolving power, and effective multiplex ratio (EMR) ranged from 0.001 to 0.002, 0.01–0.06, 1.76–3.84, and 1–4.12. The dendrogram clustered genotypes into two main clades based on their origins. The population structure revealed two sub-populations with some admixtures. The average expected heterozygosity and population differentiation within two sub-populations was 0.1428 and 0.216, respectively. The results outcome reveals that the four ISSR markers comprehensively separated the indigenous germplasm from the exotic germplasm. The genetic divergence within indigenous genotypes and exotic genotypes could allow for future insights into apricot breeding programs.     

Differential expression levels of aroma-related genes during ripening of apricot (Prunus armeniaca L.)

Fruit aroma is a complex trait, particularly in terms of the number of different biosynthetic pathways involved, the complexity of the final metabolites, and their regulation. In order to understand the underlying biochemical processes involved in apricot aroma, four cDNAs (Pa-aat, EU784138; Pa-adh EU395433; Pa-pdc EU395434; and Pa-lox EU439430) encoding an alcohol acyl transferase (AAT), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lipoxygenase (LOX), respectively, were isolated and characterized at four stages of maturity in Prunus armeniaca L. cv. Modesto. We observed a reduction in aldehyde and alcohol production between early-harvested fruit and late-harvest fruit, concomitant with an increase in ester production. qPCR analyses showed that the expression levels of the adh gene and the lox gene stayed constant at all stages. Interestingly, aat levels showed a sharp increase in the late-harvest stages concurrent with the changes observed in ester levels. The significance of these changes in relation to aroma production in apricot is discussed.     

Immobilization of apricot pectinesterase (Prunus armeniaca L.) on porous glass beads and its characterization

Pectinesterase isolated from Malatya apricot pulp was covalently immobilized onto glutaraldehyde-containing amino group functionalized porous glass beads surface by chemical immobilization at pH 8.0. The amount of covalently bound apricot PE was found 1.721 mg/g glass support. The properties of immobilized enzyme were investigated and compared to those of free enzyme. The effect of various parameters such as pH, temperature, activation energy, heat and storage stability on immobilized enzyme were investigated. Optimum pH and temperature were determined to be 8.0 and 50 °C, respectively. The immobilized PE exhibited better thermostability than the free one. Kinetic parameters of the immobilized enzyme (K_m and V_max values) were also evaluated. The K_m was 0.71 mM and the V_max was 0.64 μmol min^?1 mg^?1. No drastic change was observed in the K_m and V_max values. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. Thermal and storage stability experiments were also carried out. It was observed that the immobilized enzyme had longer storage stability and retained 50% of its initial activity during 30 days.     

The protective effects of Prunus armeniaca L (apricot) against methotrexate-induced oxidative damage and apoptosis in rat kidney

This study was conducted to evaluate a possible protective role of apricot in apoptotic cell death induced by methotrexate (MTX) and renal damage by different histological and biochemical parameters. Twenty-eight rats were divided into four groups, control, apricot, methotrexate, and apricot + methotrexate. Methotrexate induced renal failure, as shown by significant serum creatinine and urea elevation. Additionally, the results indicated that methotrexate significantly induced lipid peroxidation and reduced antioxidant activities in rats. In contrast, apricot significantly prevented toxic effects of methotrexate via increased catalase, superoxide dismutase, and glutathione levels but decreased formation of malondialdehyde. Also, it was determined that exposure to methotrexate leads to significant histological damage in kidney tissue such as glomerulosclerosis and apoptosis. On the other hand, these effects can be eliminated with apricot diet. These data indicate that apricot may be useful in preventing undesirable effects of MTX such as nephrotoxicity.     

鼠类对山杏(Prunus armeniaca)种子扩散及存活作用研究

虽然有关鼠类搬运森林种子的证据已很清楚,但这些被移走种子的存活情况却知之甚少.提出了一个新的标记和跟踪种子的方法--标签法,即将种子拴一带有编码的细长金属片,研究了北京东灵山地区山杏(Prunus armeniaca)种子的扩散距离和存活率.于1998年6月19～20日,7月3日和10月23日共在24个样点释放1440粒山杏种子.几乎所有释放的种子在10d内被鼠类取走.夏天释放的种子比秋天释放的种子消失的速度快.大多数种子的扩散距离在20m以内,小于鼠类的活动距离.鼠类吃掉种子的速度很快,但当种子变得稀少时,种子存活率有所提高.山杏种子6、7月份的每日存活率小于其它月份的每日存活率.     

The effect of summer shading on flower bud morphogenesis in apricot (Prunus armeniaca L.)

The aim of this investigation was to assess whether imposed summer shading treatments in apricot (Prunus armeniaca L.) can affect the main phenological phases related to the floral morphogenesis (floral differentiation, xylogenesis), flower bud growth and quality in terms of bud capacity to set fruit. Experimental trials were carried out on fully-grown trees of ‘San Castrese’ and ‘Stark Early Orange’ cultivars characterized by different biological and agronomical traits to which shadings were imposed in July and August. Histological analysis was carried out from summer onwards in order to determine the evolution of floral bud differentiation, and the acropetal progression of primary xylem differentiation along the flower bud axis. Periodical recordings to evaluate the bud drop, blooming time, flowering and fruit set rates were performed also. These shade treatments determined a temporary shutdown of floral differentiation, slowed xylem progression up to the resumption of flower bud growth and a reduced entity of flowering and fruit set. These events were particularly marked in ‘San Castrese’ cultivar, which is well known for its adaptability to different climatic conditions. These findings suggest that adequate light penetration within the canopy during the summer season could be the determining factor when defining the qualitative traits of flower buds and their regular growth, and ultimately to obtain good and constant crops.     

鼠类对山杏（Prunus armeniaca）种子扩散及存活作用研究

虽然有关鼠类搬运森林种子的证据已很清楚,但这些初移走种子的存活情况却知之甚少。提出了一个新的标记和跟踪种子的方法－－标签法,即将种子拴一带有编码的细长金属片,研究了北京东灵山地区山杏（Prunus armeniaca）种子的扩散距离和存活率。于1998年6月19－20日,7月3日和10月23日共在24个样点释放1440粒山杏种子。几科所有释放的种子在10d内被鼠类取走,夏天释放的种子比秋天释放的种子消失的速度快。大多数种子的扩散距离在20m以内,小于鼠类的活动距离,鼠类吃掉种子的速度很快,但当种子变得稀少时,种子存活率有所提高。山杏种子6、7月份的每日存活率小于其它月份的每日存活率。