Barriers to interspecific hybridization between Juglans nigra L. and J. regia L species

Juglans nigra and Juglans regia are phylogenetically divergent species. Despite the economic interest in Juglans?×?intermedia (J. nigra?×? J. regia), walnut hybridization is rare under natural conditions and still difficult using controlled pollination. Here, we evaluated some reproductive mechanisms that may prevent successful natural hybridization. The study of flowering phenology of 11 J. nigra and 50 J. regia trees growing in a plantation provided information regarding the opportunity for interspecific crosses. Variation in flower size, pollen quality of putative donors, and variation in seed yield and rate of hybrid production among putative maternal trees were examined. DNA fingerprinting and parentage analyses based on nine microsatellites permitted the identification of hybrids and hybridogenic parent. Our data indicated that overlap occurred between the staminate flowering of protogynous J. regia and the beginning of pistillate flowering of protogynous J. nigra. Differences in floral size were computed between walnut species. Only three hybrids among 422 offspring of eleven J. nigra progenies were identified. Interspecific hybridization involving pollination of one early-flowering-protogynous J. nigra by three protogynous J. regia trees was detected. The correct development of J. regia male gametophytes, high pollen viability (86.5 %), and germination (57.6 %) ruled out the possibility that low pollen quality contributed to depressed hybrid production. Our findings indicated that these two species tended to remain reproductively isolated. The substantial disjunction in flowering time and additional prezygotic barriers such as differences in floral size and conspecific pollen advance may affect interspecific gene flow between J. regia and J. nigra.     

Effects of desiccation and controlled rehydration on germination in vitro of pollen of walnut (Juglans spp.)

Abstract Moisture content and germination percentages on agar-solidified medium were determined for pollen of Juglans regia and J. nigra at the time of release from anther and at 24-h intervals until germination fell to zero. Moisture content of fresh J. regia pollen ranged from 4.6 to 12.1%, and from 3.9 to 4.0% for J. nigra pollen; in vitro germination percentages were uniformly high throughout these ranges. Germination declined as pollen moisture fell below 5% in J. regia with regression analyses predicting 50% germination at 3.9–4.4% moisture. After 4 d at ambient laboratory conditions, all pollen had lost the ability to germinate when incubated directly on medium. This pollen was incubated in chambers where humidity was maintained at saturation and 33% saturation, and retested. Pollen up to 22 d old and having moisture contents as low as 2.6% regained the ability to germinate in vitro when it was incubated in a water-saturated environment before being placed on the agar-solidified medium.     

Microsatellite markers for <Emphasis Type="Italic">Juglans cinerea</Emphasis> L. and their utility in other Juglandaceae species

Ten polymorphic microsatellite markers were found to amplify in butternut (Juglans cinerea; Juglandaceae). These microsatellite loci were found to amplify across most of nine other species and five hybrids examined. Loci were highly polymorphic, with 18 to 32 alleles per locus across species. These nuclear microsatellite markers will be useful in examining genetic diversity within and among populations of butternut, and in distinguishing butternut from interspecific hybrids.     

DNA markers identify hybrids between butternut (Juglans cinerea L.) and Japanese walnut (Juglans ailantifolia Carr.)

Butternut (Juglans cinerea L.) is a temperate deciduous hardwood native to the eastern USA and southern Canada valued for its nuts and wood. Butternut’s survival is threatened by butternut canker, a disease caused by the exotic fungus Sirococcus clavigignenti-juglandacearum Nair, Kostichka & Kuntz. Field observations indicate that trees commonly called buartnut (a hybrid of butternut and its close congener Japanese walnut (Juglans ailantifolia × J. cinerea)) may be more resistant to butternut canker than is either parental species. Hybrids are difficult to distinguish morphologically from butternuts, and scientists have expressed concern over the possibility of range-wide genetic invasion by Japanese walnut via hybridization with butternut. We used pair-wise combinations of 40 random primers to screen bulked DNA pools of butternut, Japanese walnut, and buartnuts to identify genomic regions unique to Japanese walnut. We ultimately identified one ITS region marker, one chloroplast marker, one mitochondrial marker, and six nuclear markers. The utility of the markers for identifying hybrids was tested and verified using more than 190 genotypes. The markers will be used to identify buartnut hybrids based on the presence of introgressed genomic fragments inherited from Japanese walnut. We confirmed that hybrids have a complex genetic history and present features of the parental species in all possible combinations. These results will assist in the identification and testing of (non-hybrid) butternut for breeding and reintroduction of the species to its former habitats.     

Beiträge zur Morphologie der Pollenkörner vonJuglans regia undJ. nigra

Zusammenfassung Die Pollenkörner vonJuglans regia undJ. nigra sind, entgegen anderen Angaben, im gequollenen Zustand annähernd kugelig-rund und im trockenen Zustand kugelig-eingedellt. Trockene Pollenkörner runden sich, sobald man sie in eine Flüssigkeit gibt, blitzartig ab. Gleichzeitig mit der Form der Pollenkörner ändert sich auch die Beschaffenheit des Pollenkitts, der auf den Pollenkörnern gelagert ist; auf gequollenen Pollenkörnern bildet er kleinere und größere, ölige Kügelchen; auf Trockenpollen erscheint er als eine ± zähflüssige, formlose Masse. Das Sporoderm besteht aus drei Schichten: einer punctitegillaten Sexine, einer strukturlosen Nexine und einer dünnen Intine, die im Bereich der Keimporcn linsenförmige Verdickungen, sog. Zwischenkörper, bildet. Die Zwischenkörper sind meist voneinander isoliert und bestehen in der Hauptmasse aus Pektinverbindungen. In der Regel sind 12–16 Keimporen ausgebildet, die beiJuglans regia kreisrund und beiJ. nigra elliptisch sind.Die AngabenSchanderls über die ölreiche Endoexine und den Ölauspreßmechanismus beiJuglans regia wurden widerlegt. Es konnte gezeigt werden, daß die Pollenkörner vonJuglans regia undJ. nigra einen ähnlichen Bau besitzen wie viele andere Vertreter windblütiger Familien.     

Response of liquid flow resistance to soil drying in seedlings of four deciduous angiosperms

Summary Soil-leaf resistance to liquid water flow (R) in moist and drying soil was compared in three-month-old seedlings of two drought tolerant (white [Quercus alba L.], post oak [Q. stellata Wangenh.]) and two drought sensitive forest species (sugar maple [Acer saccharum Marsh.], black walnut [Juglans nigra L.]). At high soil moisture (_s–0.3 MPa), R was higher in J. nigra than in the other species, and as soil water was depleted R increased most in this species. In contrast, the lowest resistance at all levels of soil moisture was observed in Q. stellata. At _s of –1.5 MPa, R of drought-sensitive J. nigra and A. saccharum was about twice as high as that of the two drought-tolerant Quercus species. The difference in R between the two Quercus species was much smaller than that between this pair and the other two species. These differences among species in flow resistance may be attributable to: 1) variation in the balance between root surface area and leaf area, 2) variation in the inherent absorption capacity of the root systems and in xylem water conducting systems or 3) differences in root permeability, shrinkage and mortality in severely stressed seedlings. As the soil dried, seedlings of all species exhibited pronounced reductions in transpiration rate, which prevented development of large water potential gradients between leaves and the soil. Reduction in transpiration in J. nigra was especially pronounced, resulting in a decrease in the soil-to-leaf water potential gradient in dry soil despite high flow resistance. The observed differences among species in flow resistance are correlated with natural distribution patterns.     

Die Bildung von Serotonin in Juglans regia L.

Summary The serotonin content of growing fruits and of germinating seeds of Juglans regia has been studied. In the embryo 0.4–0.6 mg serotonin/g FW were found; in contrast no serotonin was detectable in the fleshy pericarp and in the seed coat. Serotonin was also not detectable in leaves, stems and roots of the adult plant. Most of the serotonin found in the embryo is formed after abscission of the seeds. During the synthesis of serotonin there are no dramatic changes in the chemical composition of the seeds (Tables 3–5).The formation of serotonin could be followed in isolated cotyledons and under sterile conditions. This serotonin formation is stimulated by exogenous tryptophan (Fig. 2). That tryptophan acts as a precursor of serotonin could be demonstrated with labelled DL-tryptophan (benzene ring ¹⁴C) (U). The possibility of stimulating serotonin formation in isolated cotyledons by the addition of tryptophan is limited to a certain stage of development and cannot be observed with material from fully matured seeds (Fig. 3).No serotonin was found in callus tissue and adventitious roots formed by isolated cotyledons; all the serotonin remained in the cotyledons. This was also the case in young seedlings, in which only the cotyledons showed the characteristic high serotonin content, whereas leaves, stems and roots contained no serotonin (Table 6).From these data we conclude that serotonin formation in the embryo of Juglans regia is not a special type of nitrogen storage but a way of ammonia detoxification in which ammonia from protein amino acid degradation is incorporated into serotonin via tryptophan.     

Pollen competition among Populus deltoides Marsh., P. nigra L. and P. maximowiczii Henry in fertilizing P. deltoides ovules and siring its seed crop

Summary Interspecific pollen competition among Populus deltoides, P. nigra and P. maximowiczii in fertilizing P. deltoides ovules was studied by using a pollen mixture technique, allozymes and leaf morphology. The frequencies of F₁ seedlings of different paternities in pollen-mix crosses showed highly significant (P<0.01) departures from the 11 ratio expected if pollen selection was random. P. deltoides pollen was the most competitive. The mean percentages of F₁ seedlings of P. deltoides paternity in crosses with pollen mixes P. deltodes + P. nigra, P. deltoides + P. maximowiczii, and P. deltoides + P. nigra + P. maximowiczii were 95.0, 92.5, and 84.8, respectively. P. maximowiczii pollen was more competitive than P. nigra pollen, which was at a selective disadvantage. An average of 83.6% of F₁ progenies of the eight crosses with P. nigra + P. maximowiczii pollen showed P. maximowiczii paternity. Also, in four crosses with P. deltoides + P. nigra + P. maximowiczii pollen, the relative proportion of P. deltoides × P. maximowiczii seedlings (13.4%) was higher than that of P. deltoides × P. nigra seedlings (1.8%). Pollen proportions in the pollen mixes and pollen size did not significantly affect the competitive ability of the pollen. The relative pollen competitive ability indicated reproductive affinities among the species.     

Pollen-pistil interactions in Populus: β-galactosidase activity associated with pollen-tube growth in the crosses P. nigra x P. nigra and P. nigra x P. alba

Summary In order to understand the nature of interspecific barriers in Populus, we have explored pollen/pistil interactions in intra- and interspecific crosses Populus nigra x P. nigra and P. nigra x P. alba. The kinetics of pollen-tube growth demonstrated that P. nigra and P. alba pollen tubes have distinct behavioral patterns inside P. nigra pistils. P. alba pollen tubes exhibit an unique S-shaped growth curve and an arrested growth site near the sylodium. P. nigra pollen tubes exhibit two growth phases, in the stigmatic tissues and in the ovarian cavity respectively. P. nigra and P. alba curves diverge 5 h after controlled pollination and could be related to a change in the physiology of the P. nigra pollen tube, which shifts from an autotrophic to a heterotrophic type of nutrition. Protein analysis of pollinated stigmatic extracts (0,6 and 20 h after pollination) revealed qualitative and quantitative differences that are related to the presence of either P. nigra or P. alba pollen tubes inside the stigmatic tissues. Increasing numbers of protein bands were detectable from 0 to 20 h after pollination only in intraspecific cross. Glycoproteins were detected, and the differences observed were dependent of the cross. -Galactosidase activity was found in pollinated stigmas, but an increase in its activity (one isozyme of pHi 4.2) between 6 h and 20 h after pollination was detected only in the intraspecific cross. This enzyme could play a role in heterotrophic pollen-tube nitrition, and its activity could be the final result of a series of interactions started by the initial pollen-stigma dialog.     

Nachweis und Lokalisation von Tryptophan-Synthetase in Samen von Juglans regia L.

Summary Cell-free extracts from seeds of Juglans regia synthesize tryptophan from L-serine and indole. Tryptophan synthetase has maximal activity in the range between pH 7 and 8. The enzyme is associated with a particulate fraction (density 1,210 g/ml) which is separated from the mitochondria (density 1,191 g/ml) after isopycnic density centrifugation on a continuous sucrose gradient.     

Inheritance of isozymes in Brassica campestris L. and genetic divergence among different species of Brassiceae

Electrophoretic investigations of Brassica campestris revealed 8 polymorphic loci that had not been described earlier. The inheritance of 7 of them was determined by crosses between different accessions of B. campestris. A phylogeny of B. campestris, B. nigra, B. alboglabra, Sinapis arvensis and S. alba was estimated from the analysis of 15 isozyme loci. The phylogeny showed a close relationship between B. nigra and S. arvensis and a less close relationship between B. campestris and B. oleracea. S. alba was remotely-related to all of the other species.     

Chloroplast DNA inheritance in Populus

Summary The inheritance of chloroplast (cp) DNA was examined in F₁ hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen enzymes and five heterologous cpDNA probes were used to screen restriction fragment polymorphisms among the parents. The mode of cpDNA inheritance was demonstrated in progenies of P. deltoides × P. nigra crosses with 26 restriction fragment polymorphisms of cpDNA differentiating P. deltoides from P. nigra, as revealed by 12 enzyme-probe combinations, and in progenies of P. deltoides × P. maximowiczii crosses with 12 restriction fragment polymorphisms separating P. deltoides from P. maximowiczii, as revealed by 7 restriction enzyme-probe combinations. In all cases, F₁ offspring of P. deltoides × P. nigra and P. deltoides × P. maximowiczii crosses had cpDNA restriction fragments of only their maternal P. deltoides parent. The results clearly demonstrated uniparental-maternal inheritance of the chloroplast genome in interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii. Intraspecific P. deltoides hybrids also had the same cpDNA restriction fragments as their maternal parent. Maternal inheritance of the chloroplast genome in Populus is in agreement with what has been observed for most other angiosperms.     

The influence of culture conditions on mycorrhiza formation between the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings

Interactions between an isolate of the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings were studied at the structural and ultrastructural levels. Several different conditions were tested with or without sugar and in a sterile or nonsterile medium. In the growth cabinet, the A. africana/Pisolithus sp. interactions did not produce ectomycorrhizas. A fungal sheath was formed but no Hartig net, and an unusual host epidermal cell wall was observed. Hyphae of Pisolithus sp. induced modifications of epidermal cells of 15-day-old A. africana seedlings indicative of non-mycorrhizal interactions, such as wall thickening, wall ingrowth, papillae formation, degraded host wall material and the presence of intracellular hyphae. Wall ingrowth consisted of depositions of host cell wall materials giving a positive reaction for polysaccharides; however, wall thickenings and papillae showed no homogeneous reactions for polysaccharides. In glasshouse conditions, inocula of Pisolithus sp. in the form of spores or mycelia entrapped in peat-vermiculite added to sterilized soil produced typical ectomycorrhizae only with 6-month-old A. africana seedlings. Under these conditions, no conspicuous cell wall reactions occurred on A. africana roots. The results demonstrate that the establishment of an association between an ectomycorrhizal fungus and a potential host plant is strongly influenced by seedling age and/or environmental conditions. Therefore, in vitro synthesis is not a conclusive demonstration of a symbiotic relationship.     

Chloroplast DNA variation in Populus. II. Interspecific restriction fragment polymorphisms and genetic relationships among Populus deltoides,P. nigra,P. maximowiczii,and P. x canadensis

Restriction fragment analysis was conducted to determine interspecific chloroplast DNA (cpDNA) variation and genetic relationships among Populus deltoides, P. nigra, P. x canadensis (P. deltoides x P. nigra), and P. maximowiczii. Total cellular DNAs of these poplars were digested with 16 restriction endonucleases, and Southern blots of the restriction digests were probed with six different cloned cpDNA fragments from Petunia. P. deltoides, P. nigra, and P. maximowiczii each had a distinct chloroplast genome, separated by many restriction-site and restriction-fragment-length mutations, predominantly in the large single-copy region of the genome. P. x canadensis shared the same cpDNA restriction fragment patterns as P. deltoides var. deltoides. P. nigra was most diverged from P. deltoides, and P. deltoides showed close cpDNA relationships to P. maximowiczii. Nucleotide substitutions per site in cpDNA were 0.0036 between P. deltoides and P. maximowiczii, 0.0071 between P. nigra and P. maximowiczii, and 0.0077 between P. deltoides and P. nigra. We suggest that P. nigra should be classified in a new separate section, the Nigrae.     

RETICULATE THICKENINGS IN SOME SPECIES OF JUGLANS

Reticulate thickenings in Juglans nigra, J. major, J. microcarpa, and J. californica (including J. hindsii) appear, under the transmitted light microscope, to be a cluster of very large or “gash-like” vessel-ray pits. The use of scanning electron microscopy shows that these so-called pits are thickenings on the inside wall of the vessel.     

Genetic linkage maps of Populus nigra L. including AFLPs, SSRs, SNPs, and sex trait

Black poplar (Populus nigra L.) is a tree of ecological and economic interest. A better knowledge of P. nigra genome is needed for an effective protection and use of its genetic resources. The main objective of this study is the construction of a highly informative genetic map of P. nigra species including genes of adaptive and economic interest. Two genotypes originated from contrasted natural Italian populations were crossed to generate a F₁ mapping pedigree of 165 individuals. Amplification fragment length polymorphism (AFLP), simple sequence repeat (SSR), and single nucleotide polymorphism (SNP) markers were used to genotype 92 F₁ individuals, and the pseudo-test-cross strategy was applied for linkage analysis. The female parent map included 368 markers (274 AFLPs, 91 SSRs, and 3 SNPs) and spanned 2,104 cM with 20 linkage groups, and the male parent map, including 317 markers (205 AFLPs, 106 SSRs, 5 SNPs, and sex trait), spanned 2,453 cM with 23 main linkage groups. The sex, as morphological trait, was mapped on the linkage group XIX of the male parent map. The generated maps are among the most informative in SSRs when compared to the Populus maps published so far and allow a complete alignment with the 19 haploid chromosomes of Populus sequence genome. These genetic maps provide informative tools for a better understanding of P. nigra genome structure and genetic improvement of this ecologically and economically important European tree species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Application of genetic markers to the discrimination of European Black Poplar (Populus nigra) from American Black Poplar (P. deltoides) and Hybrid Poplars (P. x canadensis) in Switzerland

European Black Poplar (Populus nigra) is considered a rare and endangered tree species because of severe reduction of its natural riverine habitat and potential hybridisation with the related non-indigenous taxa P. deltoides and P. x canadensis. As it is difficult to distinguish these taxa solely based on their morphology, we applied a PCR-based assay with an easy-to-use and robust molecular marker set (cpDNA trnL-trnF/RsaI RFLP, nDNA win3 and nDNA POPX/MspI RFLP) in order to identify pure P. nigra. Different plant tissues could be used for fast and standardised DNA extraction. The application of the three marker types was tested on a number of different Populus taxa, and they were also used for the verification of pure P. nigra in a sample of 304 putative P. nigra individuals from Switzerland. Cross-checking of the DNA data with those using a traditional allozyme approach resulted in complete agreement. The availability of molecular identification methods is an important prerequisite for the conservation of European Black Poplar, because pure, non-introgressed plant material can then be used in restoration projects of European floodplains.     

Allergenicity of Acroptilon repens and Juglans regia pollen in rats

Pollen proteins that are located in the cytoplasm or on the surface of the exine can function as allergens and evoke immune system responses in sensitive patients, leading to allergic rhinitis and asthma. In this research, the pollen allergenicity and ability to induce IgE response of the pollen of two plant species were studied in rats. Acroptilon repens is an herbaceous, invasive plant with entomophilous pollen, while Juglans regia which is a tree crop produces anemophilous pollen. Immunoblot analysis using sera of sensitised rats revealed IgE reactivity to three protein bands including the 70, 41 and 25.12 kDa bands present in the A. repens pollen extract, while only one single immunogenic band of 11 kDa was detected in J. regia pollen extract. Both pollen extracts increased the eosinophil content and caused some clinical signs of allergy in treated rats. The results showed that both entomophilous and anemophilous pollen can be allergenic.     

Thirteen nuclear microsatellite loci for butternut (Juglans cinerea L.)

Butternut (Juglans cinerea L.) is an eastern North American forest tree severely threatened by an exotic fungal pathogen, Sirococcus clavigignenti‐juglandacearum. We report here 13 nuclear microsatellites for genetic evaluation of the remaining natural populations. Summary statistics are reported for individuals from a population of butternuts in central Kentucky (N = 63). All markers were polymorphic, with an average of 13.7 alleles per locus observed. Four loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05).