Nuclear genetic variation of <Emphasis Type="Italic">Rosa odorata</Emphasis> var. <Emphasis Type="Italic">gigantea</Emphasis> (Rosaceae): population structure and conservation implications

Rosa odorata var. gigantea is one of the most important ancestors of modern roses, which owns many merit traits including large flower, early flowering, and tea scent. Due to habitat loss and fragmentation, it has been listed as a rare and endangered species in China. In this study, a total of 424 accessions from 27 locations across its major distribution range were sampled. Its genetic diversity and population structure were assessed using a combination of seven nuclear microsatellite markers and one single-copy nuclear gene. Moderate to high within-population genetic diversity and moderate differentiation among populations were revealed despite its narrow distribution. The sampled 27 populations were resolved into two genetic clusters with limited contemporary and historical gene flows. The Red River Fault Zone was inferred to be a physical or geographical barrier to gene flow between these two genetic clusters. Genetic distances were significantly associated with geographic distances, indicating the isolation-by-distance model. Our ecological niche modeling indicated that R. odorata var. gigantea had high current potential areas in the central Yunnan province and substantial losses of high potential distribution areas in the western Yunnan in the future. Two detected clusters showed significant genetic differentiation and represented two separate evolutionary lineages, which should be recognized as two evolutionary significant units (ESUs) for conservation concerns. These results could be applied for the decision-making and conservation planning for this important species.     

Stable <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

London plane tree (Platanus acerifolia Willd.) is an important tree in urban landscaping but it suffers from a number of negative traits which genetic engineering could be used to address. As with many woody species, P. acerifolia has appeared recalcitrant to genetic transformation. However, the recent development of a method for regenerating shoots from P. acerifolia leaf explants suggests that such material could be a target for gene-transfer. Using an Agrobacterium tumefaciens strain in which the T-DNA carries the histochemically detected reporter gene β-glucuronidase (GUS), we have followed the transfer of genes from Agrobacterium to leaf explants of Platanus acerifolia. Using this system, we have identified a set of inoculation and co-cultivation conditions (notably: the pre-treatment of leaf explants with 0.4 M mannitol, an inoculation period of 10 min, a bacterial OD₆₀₀ of 0.8–1.0 and a co-cultivation period of 5 days) that permit a good frequency and reliability of transient gene-transfer. Optimum levels of antibiotics for bacterial elimination and kanamycin-resistant shoot regeneration were also established. By applying these parameters, we recovered eight independent stably transformed shoots that were kanamycin-resistant and contained the nptII T-DNA gene, as confirmed by PCR analysis. Furthermore, Southern blot analysis confirmed that, in at least five of these lines, the transgene was associated with high molecular weight DNA, so indicating integration into the plant genome.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

Analysis of alien introgression in coffee tree (<Emphasis Type="Italic">Coffea</Emphasis><Emphasis Type="Italic">arabica</Emphasis> L.)

The transfer of desired traits from related wild diploid Coffea species into the cultivated allotetraploid C. arabica is essential in coffee breeding to develop pest/disease-resistant cultivars. The present work is an attempt to gain insights into alien introgression in C. arabica. An F2 population derived from a cross between T5296 and Et6 was analysed with simple sequence repeat (SSR; microsatellite) and amplified fragment length polymorphism (AFLP) molecular markers. The T5296 is a derivative of an interspecific hybrid introgressed by the diploid C. canephora species and Et6 is a wild Ethiopian accession of C. arabica. The origin of the revealed polymorphism was determined by comparisons using representative accessions from C. arabica and its two diploid parental species, C. eugenioides and C. canephora. The number and mode of inheritance of canephora-introgressed segments were investigated, as well as their sub-genome localisation and rate of recombination. The results suggested that the transfer of desirable genes into C. arabica from C. canephora is not limited by the ploidy level differences or the suppression of recombination between the different genomes.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the wild carrot <Emphasis Type="Italic">Daucus carota</Emphasis> L. subsp. <Emphasis Type="Italic">halophilus</Emphasis> (Brot.) A. Pujadas for conservation purposes

Daucus carota subsp. halophilus, is a wild crop relative of domestic carrot. It is an aromatic plant widely used in folk medicine due to recognized therapeutic properties of its essential oils. Experiments were carried out to evaluate the potential of in vitro propagation techniques to the conservation of this endemic and endangered taxon. The results showed that shoot tips of in vitro germinated seeds were able to proliferate in the presence of benzyladenine, with the best results being achieved using 4.4 μM, both in the first and second cultures. Shoots rooted after being transferred to 1/2-Murashige and Skoog basal medium. The results indicated that the concentration of benzyladenine used during the multiplication phase did not interfere with the rate of root formation. The obtained plantlets were morphologically and anatomically identical to those obtained by seeds. Some of the in vitro produced shoots developed flowers that produced viable pollen. Plant regeneration was also achieved by somatic embryogenesis induction in cotyledons and root segments cultured in the presence of 4.5 μM 2,4-dichlorophenoxyacetic acid. Somatic embryos converted into plantlets in a medium without growth regulators. Plants obtained either by shoot proliferation or somatic embryogenesis were acclimatized and are now growing at the Coimbra Botanical Garden. The first attempts to reintroduce these plants in the original habitat were successful. It can be concluded that the protocols developed are a useful approach to the conservation of this endemic species.     

Factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Hybanthus</Emphasis><Emphasis Type="Italic">enneaspermus</Emphasis> (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD₆₀₀, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.     

Fine-scale spatial genetic structure and within population male-biased gene-flow in the grasshopper <Emphasis Type="Italic">Mioscirtus wagneri</Emphasis>

Dispersal is a life history trait that plays a key role in population dynamics, determining gene flow and influencing the size, structure and persistence of populations. For these reasons, the study of the genetic consequences of dispersal can be considered a central topic in both conservation and population genetics. In this study we examine the patterns of fine-scale genetic structure within two populations of the grasshopper Mioscirtus wagneri (Orhoptera: Acrididae). For this purpose, we have used seven species-specific microsatellite markers to type 266 individuals from two populations (Peña Hueca and El Salobral) located in Central Spain. We have found subtle genetic differentiation between some sampling patches and significant kinship structures up to 25 m distance which were particularly patent for females. In Peña Hueca locality, patterns of isolation-by-distance at both the patch scale and the individual level have also revealed an association between genetic differentiation/similarity and geographical distance in females but not in males. Overall, these data suggest a fine-scale spatial genetic substructure in the studied populations which seems to be mainly driven by female philopatry. Such pattern of within population genetic structure together with the inferred restricted dispersal distances is likely to contribute to reduce effective population sizes and inter-population gene flow. This can erode genetic variability and limit the colonization ability of this orthoptera, factors which can ultimately compromise the long-term persistence of their small size and isolated populations.     

Fine-scale population genetic structure in Alaskan Pacific halibut (<Emphasis Type="Italic">Hippoglossus stenolepis</Emphasis>)

Pacific halibut collected in the Aleutian Islands, Bering Sea and Gulf of Alaska were used to test the hypothesis of genetic panmixia for this species in Alaskan marine waters. Nine microsatellite loci and sequence data from the mitochondrial (mtDNA) control region were analyzed. Eighteen unique mtDNA haplotypes were found with no evidence of geographic population structure. Using nine microsatellite loci, significant heterogeneity was detected between Aleutian Island Pacific halibut and fish from the other two regions (F _ST range = 0.007–0.008). Significant F _ST values represent the first genetic evidence of divergent groups of halibut in the central and western Aleutian Archipelago. No significant genetic differences were found between Pacific halibut in the Gulf of Alaska and the Bering Sea leading to questions about factors contributing to separation of Aleutian halibut. Previous studies have reported Aleutian oceanographic conditions at deep inter-island passes leading to ecological discontinuity and unique community structure east and west of Aleutian passes. Aleutian Pacific halibut genetic structure may result from oceanographic transport mechanisms acting as partial barriers to gene flow with fish from other Alaskan waters.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Festuca arundinacea</Emphasis> (Schreb.) and <Emphasis Type="Italic">Lolium multiflorum</Emphasis> (Lam.)

Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and beta-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media. Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil. All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS. Three GUS-negative transgenic L. multiflorum and the two F. arundinacea plants were vernalised and allowed to flower. All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed. Progeny analysis of L. multiflorum showed a 24-68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission. However, significant differences were noted between the paternal and maternal expression of hygromycin resistance.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Fine-scale genetic structure of natural <Emphasis Type="Italic">Tuber aestivum</Emphasis> sites in southern Germany

Although the Burgundy truffle (Tuber aestivum) is an ectomycorrhizal fungus of important economic value, its subterranean life cycle and population biology are still poorly understood. Here, we determine mating type and simple sequence repeat (SSR) maternal genotypes of mapped fruiting bodies to assess their genetic structure within two naturally colonized forest sites in southern Germany. Forty-one genotypes were identified from 112 fruiting bodies. According to their mating types, the maternal genotypes were aggregated only in one population. Genotypic diversity of individuals that mostly were small and occurred in 1 out of 2 years of sampling was high. Although these results suggested a ruderal colonization strategy, some genets spread several hundred meters. This result indicates that, besides sexual spore dispersal, vegetative growth or spreading by mycelial propagules contributes to dissemination. In one site, fewer individuals with a tendency to expand genets belonging to only one genetic group were observed. In the second site, numerous small individuals were found and were grouped into two clearly differentiated genetic groups that were spatially intermingled. Forest characteristics and disturbances are possible reasons for the observed genetic patterns. Our findings contribute to a better understanding of the biology of one of the most widespread and commercially important truffle species. This knowledge is critical for establishing and maintaining sustainable long-term truffle cultivations.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Functional conservation of the human <Emphasis Type="Italic">EXT1</Emphasis> tumor suppressor gene and its <Emphasis Type="Italic">Drosophila</Emphasis> homolog <Emphasis Type="Italic">tout</Emphasis><Emphasis Type="Italic">velu</Emphasis>

Heparan sulfate proteoglycans play a vital role in signaling of various growth factors in both Drosophila and vertebrates. In Drosophila, mutations in the tout velu (ttv) gene, a homolog of the mammalian EXT1 tumor suppressor gene, leads to abrogation of glycosaminoglycan (GAG) biosynthesis. This impairs distribution and signaling activities of various morphogens such as Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp). Mutations in members of the exostosin (EXT) gene family lead to hereditary multiple exostosis in humans leading to bone outgrowths and tumors. In this study, we provide genetic and biochemical evidence that the human EXT1 (hEXT1) gene is conserved through species and can functionally complement the ttv mutation in Drosophila. The hEXT1 gene was able to rescue a ttv null mutant to adulthood and restore GAG biosynthesis.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Isolation and <Emphasis Type="Italic">S</Emphasis>-genotyping application of <Emphasis Type="Italic">S</Emphasis>-allelic polymorphic <Emphasis Type="Italic">MdSLFB</Emphasis>s in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.)

Apple (Malus domestica Borkh.) possesses gametophytic self-incompatibility (GSI) which is controlled by S-RNase in the pistil as well as a pollen S-determinant that has not been well characterized. The identification of S-locus F-box brother (SFBB) genes, which are good candidates for the pollen S-determinant in apple and pear, indicated the presence of multiple S-allelic polymorphic F-box genes at the S-locus. In apple, two SFBB gene groups have been described, while there are at least three groups in pear. In this report, we identified five MdSLFB (S-RNase-linked F-box) genes from four different S-genotypes of apple. These genes showed pollen- and S-allele-specific expression with a high polymorphism among S-alleles. The phylogenetic tree suggested that some of them belong to SFBBα or β groups as described previously, while others appear to be different from SFBBs. In particular, the presence of MdSLFB3 and MdSLFB9 suggested that there are more S-allelic polymorphic F-box gene groups in the S-locus besides α and β. Based on the sequence polymorphism of MdSLFBs, we developed an S-genotyping system for apple cultivars. In addition, we isolated twelve MdSLFB-like genes, which showed pollen-specific expression without S-allelic polymorphism.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.