EST-SSR markers reveal synonymies, homonymies and relationships inconsistent with putative pedigrees in chestnut cultivars

Over the last two centuries, chestnut breeding programs in Europe and Asia have generated an array of chestnut interspecific hybrids, primarily of European (Castanea sativa), Japanese (C. crenata) and Chinese (C. mollissima) ancestry. During this same period, Europeans colonizing North America imported hybrid chestnuts and made interspecific hybrids with native chestnuts, primarily American chestnut (C. dentata). The importation of Chinese chestnut into the United States in the late 19th century also introduced chestnut blight, which triggered an additional interspecific hybridization effort in an attempt to develop blight resistant American chestnuts. Chestnut cultivars used for nut production in the United States and Canada have arisen against this background of non-native introductions and extensive hybridizing. The development of regionally adapted nut producing trees with dependable crops of high quality nuts requires sorting out the identities of existing cultivars. We chose 11 EST-SSR markers from C. mollissima for the initial task of genotyping 65 chestnut cultivars that grow well in the central United States. Many of these cultivars have interspecific pedigrees involving two or more species. We found extensive homonymies and synonymies, genetic groups inconsistent with published pedigrees, contradictory pedigrees and evidence for incorrect species assignments. Accurate inference of the interspecific ancestries of cultivars grown in the United States and Canada will require genotyping of species reference sets for C. sativa, C. crenata, C. mollissima, C. dentata and possibly C. pumila (the Ozark and Allegheny chinquapins).     

Characterization of American hazelnut (Corylus americana) accessions and Corylus americana?×?Corylus avellana hybrids using microsatellite markers

The American hazelnut (Corylus americana Marshall) is native to the eastern United States and Southern Canada. In the early decades of the last century, breeding work by several individuals attempted to combine the cold hardiness and disease resistance of the American hazelnut with the larger nut size of the European hazelnut (C. avellana L.). Many hybrid selections grown today trace back to these early efforts. Over the past three decades, representatives of C. americana were collected and are preserved in Corvallis, Oregon. Seeds were collected along roadsides, in hedgerows, in woodland clearings, and in gardens. The seeds were germinated and the best of the resulting seedlings were preserved for later use. In this study, genetic diversity was studied in 87 American and 67 hybrid hazelnut selections using 21 microsatellite loci. The total number of alleles was 266 in the 162 accessions examined, of which 229 were present in C. americana accessions, but only 168 in the Arbor Day Farm hybrids, and 116 in the hybrids of C. americana ‘Rush’. In the American accessions, a high level of genetic diversity was observed (H _e ?=?0.74, H _o ?=?0.68). A genetic similarity matrix of the American and hybrid accessions, and a few European cultivars, was constructed and the resulting dendrogram revealed seven major groups: European cultivars and ‘Rush’ hybrids, two groups of Arbor Day Farm hybrids, three groups of C. americana accessions, and a mixed group of hybrid and American accessions. Analysis confirmed the reported parentage of 10 hybrids. The genetic diversity among the American hazelnut accessions, untapped to date, will be useful in breeding hybrids that will allow expansion of hazelnut production into eastern North America.     

Polyphenolic profiles and antioxidant activities of heartnut (Juglans ailanthifolia Var. cordiformis) and Persian walnut (Juglans regia L.)

The polyphenolic compositions of three heartnut (Juglans ailanthifolia var. cordiformis) varieties (Imshu, Campbell CW1, and Campbell CW3) were examined and compared with those of two Persian walnut (Juglans regia L.) varieties (Combe and Lake). The nuts were defatted, extracted, and separated into three different fractions, the free phenolic acid (FPA), acid-hydrolyzable phenolic acid (AHPA), and bound phenolic acid (BPA) fractions. The total phenolic contents (TPCs) in both FPA and AHPA of the Persian walnuts were significantly higher (P < 0.001) than those of the heartnuts, but not in the BPA (P = 0.20). LC-ESI-MS(n)() studies revealed that except for the FPA fraction, the major polyphenolics in both heartnut and Persian walnut were ellagic acid and valoneic acid dilactone. Persian walnuts contained an average of 0.29 and 1.31 mg of ellagic acid/g nut in the 80% methanol extractable fractions FPA and AHPA, respectively. Heartnuts contained an average of 0.16 and 0.60 mg of ellagic acid/g nut in the respective fractions. Bound ellagic acid in the residue was 0.93 and 0.70 mg/g of nut in the Persian walnut and in the heartnut, respectively. Valoneic acid dilactone was tentatively identified and quantified as milligrams of ellagic acid equivalent per gram of nut. These components were found to contribute to the strong total antioxidant activities measured using ferric reducing antioxidant power and photochemiluminescence methods.     

Phytochemical inhibition of aflatoxigenicity in Aspergillus flavus by constituents of walnut (Juglans regia)

Tulare walnut, a cultivar highly resistant to aflatoxin formation, was investigated for endogenous phytochemical constituents capable of inhibiting aflatoxigenesis in Aspergillus flavus. The activity, located entirely in the pellicle (seed coat), was extractable to various degrees with polar solvents, although some activity remained unextractable, indicating that the bioactivity resided in a complex of hydrolyzable tannins. These tannins can be hydrolyzed by a fungal tannase present in A. flavus, yielding gallic acid and ellagic acid, testing of which showed that only gallic acid had potent inhibitory activity toward aflatoxin biosynthesis. Comparison of the gallic and ellagic acid content in the pellicle of Tulare and Chico cultivars, over the 2002 and 2003 growing seasons, showed that the gallic acid content increased rapidly with maturation of the nut and was 1.5-2 times higher in Tulare than in Chico. Gallic acid content in the pellicle at maturity of a series of commercial English walnut cultivars, and two black walnut species, was determined as an indicator of potential for inhibition of aflatoxigenesis. Regulation of gallic acid levels in the hydrolyzable tannins of walnuts by conventional breeding or genetic manipulation has the potential to provide new cultivars with high resistance to aflatoxigenesis.     

Detecting hybridization in wild (Coturnix c. coturnix) and domesticated (Coturnix c. japonica) quail populations

Two subspecies of Coturnix coturnix have allopatric distributions in Europe, Maghreb and western Asia (the Common quail C. c. coturnix), and in eastern Asia (the Japanese quail C. c. japonica), except for sympatric breeding areas in the Baikal (Russia) and Kentei (Mongolia) regions, where they could hybridize. Japanese quails have been reproduced in captivity for centuries and domesticated. Massive releasing of Japanese domesticated quails, or hybrids, in west European countries might threaten with hybridization the gene pool of wild quail populations. Here, we used mitochondrial (mtDNA) and nuclear DNA markers (microsatellites), and Bayesian admixture analyses aiming to assess subspecies distinction and identify hybrids in wild and captive-reared quails. A phylogenetic tree identified two major mtDNA clades, corresponding to the two subspecies, and allowed detecting 12 hybrids in wild Common quails sampled in Spain. Additional hybrids were identified in wild quails sampled in Spain, Italy and Senegal using microsatellite markers. Wild quails sampled in Mongolia showed japonica mtDNAs, but their microsatellite genotypes were admixed, suggesting that Common and Japanese quails can hybridize in nature. Some captive-reared stocks of Japanese quails were also admixed. Introgression of domesticated Japanese quail genes in wild Common quail populations might affect the phenotypic expression of functional traits, as body size, feather colours, sexual calls and migratory behaviours. Hence, restocking with captive-reared non-native quails should be banned.     

Jug r 4, a legumin group food allergen from walnut (Juglans regia Cv. Chandler)

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.     

Oxidative stability of tree nut oils

The oxidative stability of selected tree nut oils was examined. The oils of almond, Brazil nut, hazelnut, pecan, pine nut, pistachio, and walnut were extracted using two solvent extraction systems, namely, hexane and chloroform/methanol. The chloroform/methanol system afforded a higher oil yield for each tree nut type examined (pine nut had the highest oil content, whereas almond had the lowest). The fatty acid compositions of tree nut oils were analyzed using gas chromatography, showing that oleic acid was the predominant fatty acid in all samples except pine nut and walnut oils, which contained high amounts of linoleic acid. The tocopherol compositions were analyzed using high-performance liquid chromatography, showing that alpha- and gamma-tocopherols were the predominant tocopherol homologues present; however delta- and beta-tocopherols were also detected in some samples. The oxidative stability of nonstripped and stripped tree nut oils was examined under two conditions, namely, accelerated autoxidation and photooxidation. Progression of oxidation was monitored using tests for conjugated dienes, peroxide value, p-anisidine value, and headspace volatiles. Primary products of oxidation persisted in the earlier stages of oxidation, whereas secondary oxidation product levels increased dramatically during the later stages of oxidation. Hexanal was the major headspace aldehyde formed in all oxidized samples except walnut oil, which contained primarily propanal. Results showed that chloroform/methanol-extracted oils were more stable than hexane-extracted oils in both the accelerated autoxidation and photooxidation studies. Oils of pecan and pistachio were the most stable, whereas oils of pine nut and walnut were the least stable.     

Differential Responses of Jatropha Species on Growth and Physiological Parameters to Salinity Stress at Seedling Plant Stage

The effect of salinity on Jatropha curcas (J. curcas), native to nonsaline humid areas, and Jatropha cinerea (J. cinerea), native to saline dry areas, was compared to assess the potential of cultivating J. curcas for biodiesel production in saline soils that are not suitable for food production. Growth parameters, water relations, chlorophyll content, and stomatal conductance of both species under salinity were measured. Dry weight of both species decreased with increasing concentrations of salt; however, both species can grow at salinities up to ?100 mM sodium chloride (NaCl). Decline of stomatal conductance was one of the main factors causing reduction in growth of Jatropha spp. Growth of J. curcas was inhibited more than that of J. cinerea by decline in growth parameters and chlorophyll content, but J. curcas shows sufficient hardiness to be cultivated in moderately saline soils with more favorable water relations.     

基于SSR标记的我国主栽日本栗品种(系)遗传结构分析和指纹图谱构建

为解析我国主栽日本栗品种资源的遗传多样性并构建其指纹图谱,本研究通过毛细管电泳和高质量的17个SSR分子标记对日本栗品种资源进行位点检测。结果显示,日本栗与茅栗具有较近的种间亲缘关系。在59份日本栗品种(系)中共检测到131个等位位点,每个标记平均有7.706个等位位点;多态信息含量(PIC)变幅为0.375~0.815,平均值为0.605;供试日本栗品种表现出高的遗传多样性。系统发育树、群体结构和主坐标分析结果一致,支持中国境内的部分日本栗品种(系)独立形成一个分支,且大多品种为杂交资源。此外,本研究根据多位点匹配分析确定了CmSI0922、CmSI00702和CmSI0658为核心引物,并利用这3个核心引物构建了59份日本栗品种(系)的指纹图谱。综上所述,相比其他栗属植物,日本栗分布范围较狭窄,但其具有丰富的遗传多样性,且品种资源间存在广泛的基因流。本研究成功构建了59份日本栗品种(系)资源的具有唯一对应关系的指纹图谱,为栗属植物的资源鉴定和保护利用提供了有力支撑。     

Hybridisation with introduced chukars (Alectoris chukar) threatens the gene pool integrity of native rock (A. graeca) and red-legged (A. rufa) partridge populations

The decline of over-hunted red-legged (Alectoris rufa) and rock (A. graeca) partridge populations has been contrasted with massive releases of captive-reared birds, often hybrids with non-indigenous A. chukar. Released interspecific hybrids raise the risks of introgressive hybridisation, and can contribute to further depress the fitness of native populations. Aiming to assess the extent of hybridisation, we genotyped the mtDNA control-region and eight nuclear microsatellites in 671 red-legged, rock and chukar partridges and hybrids, identified by phenotypic traits. Results reveal a diffuse occurrence of hybridisation: (1) 39 samples (6.2%) show mtDNA haplotypes discordant with their phenotypes, indicating red-legged and chukar mtDNA introgression in native rock partridges; (2) admixture analyses of the microsatellite genotypes identified 32 additional rock partridges (5.1%) hybridised mainly with chukars. We analysed also 39 samples collected from a presumed natural red-legged x rock partridge hybrid zone in the French Alps. Surprisingly, 28% birds showed typical chukar mtDNAs, indicating hybridisation with introduced chukars or hybrids. This hybrid zone led to an introgression cline of chukar alleles into neighbouring Alpine rock partridges detectable up to 100 km, which was shorter than expected by neutral genetic theory, and that suggested natural selection against hybrids. These findings indicate that introgressive hybridisation may disrupt local adaptations in natural red-legged partridge and rock partridge populations, and call for strict control of farming and restocking operations.     

Effects of nonindigenous tadpoles on native tadpoles in Florida: evidence of competition

The impacts of nonindigenous species on native ecosystems can be severe, sometimes leading to the extinction of native taxa. Interspecific competition is a potential mechanism of negative impact of invasive species, but few studies have conclusively demonstrated competition between native and nonindigenous taxa. In this study I used experimental manipulations to examine the competitive effects of the larvae of two widely introduced anurans, the cane toad, Bufo marinus, and the Cuban treefrog, Osteopilus septentrionalis, on the growth and development of the larvae of two native anurans (the southern toad, Bufo terrestris, and the green treefrog, Hyla cinerea). The presence of O. septentrionalis larvae consistently impacted growth and development of native larvae, resulting in reduced growth rates and delayed metamorphosis of both native species and smaller mass at metamorphosis of B. terrestris. Hyla cinerea larvae transformed at greater body masses when reared with the rapidly transforming nonindigenous species as a result of competitive release. The negative effects of O. septentrionalis on native larvae were generally significant whether native tadpoles were exposed to O. septentrionalis alone or in combination with B. marinus. In contrast, B. marinus tadpoles did not significantly impact the growth or development of either native species. Neither nonindigenous species significantly decreased the survivorship of native larvae, although a trend toward decreased survivorship was evident for H. cinerea. These results suggest that nonindigenous larval anurans may adversely impact native tadpole communities as a result of interspecific competition.     

Cytogenetic analysis of Kengyilia mutica (Keng) J. L. Yang, Yen and Baum (Poaceae: Triticeae)

Kengyilia mutica (Keng) Yang, Yen et Baum is a hexaploid perennial grass of the tribe Triticeae native to western central China. The analyzer species with known genomic constitution used to produce interspecific hybrids with the target taxon were Roegneria kamoji Ohwi (StStHHYY), K. hirsuta (Keng) J. L. Yang, Yen et Baum (PPStStYY) and K. rigidula (Keng) J. L. Yang, Yen et Baum (PPStStYY). Analysis of metaphase I pairing configurations in the F₁ hybrids indicates that K. mutica possesses the P, St and Y genomes, with only minor structural rearrangements. Chromosome pairing in hybrids supports the inclusion of K. mutica in the genus Kengyilia.     

Soil reinforcement by the roots of six dominant species on eroded mountainous marly slopes (Southern Alps,France)

In marly catchments of the French Southern Alps, the development of plant root systems is essential to increase slope stability and mitigate soil erosion, prevalent in this area. In a context of land restoration, it is important to be able to evaluate plant efficiency for soil reinforcement. This paper presents the results of investigations carried out on six dominant species from marly gullies. It aims to compare the additional soil cohesion they provide at the early stages of their development. The six following species were collected: two tree species, Pinus nigra and Quercus pubescens, two shrubby species, Genista cinerea and Thymus serpyllum, and two herbaceous species, Achnatherum calamagrostis and Aphyllantes monspeliensis. For each of them, we measured root tensile strength and root area ratio in order to calculate the potential root reinforcement and to compare species suitability to prevent shallow mass movements. Results showed significant differences between species. The herbaceous species A. monspeliensis and the shrubby species G. cinerea provided the highest increase in soil shear strength while the tree species, P. nigra and Q. pubescens were the least efficient. These results, along with the knowledge on vegetation dynamics and species response to erosive constraint, allow us to better evaluate land vulnerability to erosion and the efficiency of restoration actions in eroded marly lands.     

Non-native anecic earthworms (Lumbricus terrestris L.) reduce seed germination and seedling survival of temperate and boreal trees species

Recent studies have shown that the introduction of non-native earthworms in previously earthworm-free soils may have negative impacts on the recruitment of certain understory plant species in northern temperate forests. There is a need, therefore, to understand the mechanisms that may underlie this phenomenon. A microcosm study was conducted to test the effects of the anecic earthworm, Lumbricus terrestris L., on the number of days for germination, % seed germination, seedling survival and seedling biomass of 14 tree species native to southern Quebec (Canada). Seeds of these species were germinated and grown in the presence or absence of L. terrestris. The presence of earthworms significantly reduced % seed germination of seven tree species, as well as seedling survival of three tree species. The germination date of three tree species was significantly affected, either positively or negatively, by the presence of earthworms. Earthworms had no effect on seedling biomass. Results suggest that the introduction of L. terrestris into forested ecosystems of southern Québec may potentially alter overstory composition through several mechanisms that differentially affect the recruitment of various tree species in the understory.     

Feeding preferences of native terrestrial isopod species (Oniscoidea,Isopoda) for native and introduced leaf litter

Due to current predictions for Central Europe that forecast higher frequencies of hot and dry summers, Mediterranean drought-tolerant oak species are being evaluated as future forest trees for German forest sites that are becoming increasingly damaged by water deficit. As a result of planting foreign tree species, the leaf litter composition and thus the food resources of native saprophagous macroarthropods will change, possibly altering primary decomposition processes. Therefore, experiments concerning the acceptance and palatability of introduced versus native litter for native isopods were undertaken. Consumption rates of four native isopod species (Porcellio scaber, Oniscus asellus, Trachelipus rathkii, Trachelipus ratzeburgii) were investigated in laboratory choice tests with introduced (Quercus pubescens, Quercus frainetto, Quercus ilex) and comparable native (Fagus sylvatica, Quercus robur) leaf litter. Litter was characterized by measurement of C/N-ratios and lignin content. Although species-specific preferences of isopods could be observed in the experiments, Mediterranean oak litter was consumed by all investigated species. Furthermore, two isopod species even preferred the leaf litter of the introduced Q. ilex. Compared to native beech or oak litter, litter from these introduced tree species thus apparently do not negatively influence the consumption rates of terrestrial isopods. Possible reasons for the determined preferences are discussed.     

Diversity of the European indigenous wild apple (Malus sylvestris (L.) Mill.) in the East Ore Mountains (Osterzgebirge), Germany: II. Genetic characterization

In the present study genetic diversity and hybridization with cultivars were investigated in a population of the endangered European wild apple species Malus sylvestris (L.) Mill. with the aim to establish a basis for the implementation of conservation activities and to ensure its long-term preservation. A total of 284 putative M. sylvestris trees located in the East Ore Mountains were investigated along with a standard set of reference apple genotypes proposed by the European Cooperative Program for Plant Genetic Resources (ECPGR) and 13 old apple cultivars often cultivated in Saxony. The genetic analysis was performed using 12 microsatellite markers also recommend by the ECPGR. To differentiate ‘true type’ M. sylvestris individuals, hybrids and apple cultivars (Malus × domestica Borkh.) a model-based cluster analysis was performed using STRUCTURE. Two clusters were identified consisting of M. sylvestris and M. × domestica genotypes. About 40 % of the putative M. sylvestris showed an admixture of the species-specific allele frequencies and were defined as hybrids. The genetic diversity of the ‘true type’ M. sylvestris population was still high but slightly lower than in the apple cultivars especially since some SSR loci were fixed on one or few alleles in the M. sylvestris population. The differentiation parameters between ‘true type’ wild apple and cultivars indicated a clear discrimination between the wild and cultivated apple individuals. This fact confirms our expectation of the existence of ‘true type’ M. sylvestris individuals in the East Ore Mountains and argues for the realization of preservation measures in this area.     

Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan

Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.     

Phenolic acids, syringaldehyde, and juglone in fruits of different cultivars of Juglans regia L

Phenolic acids (chlorogenic, caffeic, p-coumaric, ferulic, sinapic, ellagic, and syringic acid) as well as syringaldehyde and juglone were identified in ripe fruits of 10 walnut cultivars: Adams, Cisco, Chandler, Franquette, Lara, Fernor, Fernette, Alsoszentivani 117 (A-117), Rasna, and Elit. Analyses were done using a high-performance liquid chromatograph equipped with a diode array detector. Significant differences in the contents of identified phenolics were observed among cultivars. Phenolics were determined separately in the kernel and in the thin skin of the walnut, termed the pellicle. Not only in the kernel but also in the pellicle did syringic acid, juglone, and ellagic acid predominate (average values of 33.83, 11.75, and 5.90 mg/100 g of kernel; and 1003.24, 317.90, and 128.98 mg/100 g of pellicle, respectively), and the contents of ferulic and sinapic acid (average values of 0.06 and 0.05 mg/100 g of kernel and 2.93 and 2.17 mg/100 g of pellicle, respectively) were the lowest in all cultivars. The highest differences in the sum of all identified phenolics were observed between Rasna and Fernette fruits; in Rasna there were >2-fold higher contents of identified phenolics in both kernel and pellicle. It was found that the walnut pellicle is the most important source of walnut phenolics. The ratio between the contents in pellicle and kernel varied by at least 14.8-fold for caffeic acid (cv. Adams) and by up to 752.0-fold for p-coumaric acid (cv. Elit).