Construction and characterization of a bacterial artificial chromosome library of banana (<Emphasis Type="Italic">Musa acuminata</Emphasis> Colla)

A bacterial artificial chromosome (BAC) library for banana was constructed from leaves of the wild diploid 'Calcutta 4' clone (Musa acuminata subsp. Burmannicoides 2n = 2 x = 22). 'Calcutta 4' is widely used in breeding programs for its resistance to the current major disease of banana and is being used to build a genetic reference map of banana. As banana leaves are particularly rich in polyphenols and polysaccharides a protocol was adapted to isolate intact nuclei and high-molecular-weight (HMW) DNA. A total of 55,152 clones with an average insert size of 100 kb were picked. The frequency of BAC clones carrying inserts derived from chloroplast and mitochondrial DNA was estimated to be 1.5%. The coverage of the library is equivalent to 9.0-times the haploid genome. The BAC library was screened with 13 RFLP probes belonging to the 8 linkage groups of the consensus molecular map of banana. A total of 135 clones were identified giving an average of 10.38 clones for each locus. This BAC library will be a valuable starting tool for many of the goals of the recently emerged International Musa Genomic Consortium. One of our initial objectives will be to develop a banana physical map by BAC-FISH (fluorescent in situ hybridization) viewing the characterization of translocation break points.     

Photosynthetic characteristics of an endangered species <Emphasis Type="Italic">Camellia nitidissima</Emphasis> and its widespread congener <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Saturation (SI) and compensation (CI) irradiances [μmol(photon) m⁻² s⁻¹] were 383.00±18.40 and 12.95±0.42 for wild C. nitidissima (in mid-July) and 691.00±47.39 and 21.91±1.28 for wild C. sinensis, respectively. C. nitidissima is a shade tolerant species, whereas C. sinensis has a wide ecological range of adaptability to irradiance. Both wild and cultivated C. nitidissima demonstrated low maximum net photosynthetic rate, maximum carboxylation rate, maximum electron transfer rate, and SI, which indicated low photosynthesis ability of leaves that were unable to adapt to strong irradiance environment. Both C. nitidissima and C. sinensis demonstrated strong photosynthetic adaptabilty in new environments. Hence proper shading may raise photosynthetic efficiency of cultivated C. nitidissima and promote its growth.     

Construction and characterization of a bacterial artificial chromosome library of marine macroalga<Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Rhodophyta)

A large-DNA-fragment library is necessary for research into thePorphyra genome. In this study, a bacterial artificial chromosome (BAC) library ofPorphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research inP. yezoensis. These authors contributed equally to this work.     

<Emphasis Type="Italic">Paenibacillus camelliae</Emphasis> sp. nov., isolated from fermented leaves of <Emphasis Type="Italic">Camellia sinensis</Emphasis>

A novel bacterium, strain blls-2^T was isolated from Pu’er tea. The isolate was Gram-positive, endospore-forming motile rod that grew at 15∼42°C and pH 6.0∼10.2. The DNA G+C content was 48.3 mol%, the predominant isoprenoid quinone was MK-7, and the predominant cellular fatty acid was anteiso-C15:0 (54.2%) followed by C16:0 (15.5%) and iso-C16:0 (8.2%). The polar lipid pattern of blls-2^T was characterized by the presence of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. Phy-logenetic analysis based on 16S rRNA gene sequence showed that the strain was affiliated within the Paenibacillaceae. The strain was most closely related to Paenibacillus granivorans A30^T, with a similarity of 97.1%. Based on the phylogenetic and phenotypic characteristics of strain blls-2^T, the isolate is thought to represent a novel taxon in the genus Paenibacillus. The name Paenibacillus camelliae sp. nov. is proposed for the fermented tea isolate; the type strain is blls-2^T (= KCTC 13220^T= CECT 7361^T).     

Isolation and characterization of polyphenol oxidase- and peroxidase-producing <Emphasis Type="Italic">Bacillus</Emphasis> strains from fully fermented tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Sixteen aerobic endospore-forming Bacillus spp. were isolated from fully fermented tea leaf samples from 10 tea factories in Lahijan and Langrod cities (Gillan province, Iran). Bacillus spp. isolates were characterized using phenotypic characteristics, antibiotic susceptibility and cellular fatty acid (CFA) patterns. Based on the data obtained, five isolates of tea Bacillus spp. (TB): TB2, TB4, TB6, TB10 and TB12 belonged to the species B. subtilis. Two isolates, TB1 and TB14 were recognized as B. licheniformis. Two Bacillus spp. isolates, TB9 and TB 16 were identified as B. sphaericus. Two isolates, TB5 and TB13 were shown to be B. pumilus. Two isolates, TB7 and TB15 belonged to B. cereus. Amongst the isolates, Bacillus sp. TB3, Bacillus sp. TB8 and Bacillus sp. TB11 showed different phenotypic traits, distinct antibiotic sensitivity and fatty acid profiles, and they may represent novel species. The isolates showed polyphenol oxidase (tyrosinase) and peroxidase activities. The highest polyphenol oxidase and peroxidase activities were observed for Bacillus sp. TB3 and B. licheniformis TB14, respectively, where values of 5.48 and 3.73 units mL⁻¹ were observed.     

Physiological and biochemical responses of <Emphasis Type="Italic">Camellia sinensis</Emphasis> to stress associated with <Emphasis Type="Italic">Empoasca vitis</Emphasis> feeding

The tea green leafhopper, Empoasca vitis, is the most serious pest in plantations of tea, Camellia sinensis. Beyond physical damage to the leaves, tea yields may be affected if feeding stress causes physiological and biochemical changes in the tea plant, which affected the quality and flavor of the tea. Yet the effect of feeding stress, induced by E. vitis, is largely unknown. We measured the injury index and the physiological and biochemical responses of C. sinensis to stress by E. vitis feeding in a series of laboratory trials. Using 2-year-old C. sinensis plants, we tested the effects of leafhopper feeding at different densities—0, 5, 10, and 20 leafhoppers—and different durations of exposure—1, 4, 7, and 10 days—on potential changes in chlorophyll, tea polyphenols, nutrient content, activities of protective enzymes (peroxidase, POD; superoxide dismutase, SOD; and catalase, CAT), and the lipid peroxidation (MDA). We found that the injury indices for tea leaves increased continuously as the density of E. vitis increased in the same day, and simultaneously, as the time of leafhoppers damage increased, the injury indices for tea leaves also increased. Our results also indicated that feeding by E. vitis caused a considerable decline in chlorophyll a, chlorophyll b, total chlorophyll in tea leaves and soluble carbohydrate content, and an increase in tea polyphenols. Soluble protein content showed a direct increasing relationship with the increasing leafhopper density and the duration of exposure. Throughout the period of E. vitis exposure, there was highly significant difference in the activities of protective enzymes and MDA content. Additionally, POD, SOD, and CAT activities in tea leaves were elevated significantly with the increase of leafhopper density. Lipid peroxidation (MDA) content also increased after the exposure to leafhopper feeding. Overall, our results indicate that although C. sinensis displays a certain level of tolerance to E. vitis feeding stress, higher density of leafhoppers, and longer exposure duration, can cause severe damage to tea leaves and also a decline in plant defense of tea, so as to affect the tea quality.     

Asymmetric somatic hybridization between UV-irradiated <Emphasis Type="Italic">Citrus unshiu</Emphasis> and <Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">sinensis</Emphasis>: regeneration and characterization of hybrid shoots

In the present paper, attempts were made to explore the possibility of employing ultraviolet (UV) irradiation in citrus asymmetric fusion for transfer of limited amount of favorable traits from a desirable cultivar to a target one. Exposure of Satsuma mandarin (Citrus unshiu Marc.) embryogenic protoplasts to UV at an intensity of 300 μW cm⁻² led to reduced viability, especially under long irradiation duration. The protoplasts could not grow during culture when they were irradiated for over 30 s. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay revealed extensive DNA fragmentation in the UV-irradiated protoplasts compared with those without UV treatment. Electrofusion between UV-irradiated protoplasts of Satsuma mandarin (donor) with those of Jincheng (C. sinensis Osbeck, recipient), a local cultivar of superior quality, gave rise to regeneration of several lines of shoots, which failed to root despite enormous endeavors. Ploidy analysis via flow cytometry and chromosome counting showed that four selected shoots were either diploid, triploid or tetraploid. Random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) confirmed the shoots, irrespective of their ploidy level, as putative somatic hybrids. Cleaved amplified polymorphism sequences (CAPS) demonstrated that the shoots predominantly got their cytoplasmic components, in terms of chloroplast (cp) and mitochondrion DNA, from Jincheng, along with possible recombination of cpDNA in some shoot lines. The current data indicated that UV-based asymmetric fusion could also be employed in citrus somatic hybridization with the intention of creating novel germplasms, which may provide an alternative approach for cultivar improvement.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Silencing of Caffeine Synthesis through Root Transformation in <Emphasis Type="Italic">Camellia sinensis</Emphasis> L.

Tea [Camellia sinensis (L.) O. Kuntze] is a perennial and most popular non-alcoholic caffeine-containing beverage crop. Tea has several constraints for its genetic improvement such as its high polyphenolic content and woody perennial nature. The development of transgenic tea is very difficult, laborious, and time taking process. In tea, regeneration requires minimum 8–12 months. In view of this, attempt has been made in this article to develop a rapid, efficient, and quite economical Agrobacterium-mediated root transformation system for tea. The feasibility of the developed protocol has been documented through silencing caffeine biosynthesis. For this, one-month-old tea seedlings were exposed to fresh wounding at the elongation zone of roots and were inoculated with Agrobacterium tumefaciens cultures carrying a RNAi construct (pFGC1008-CS). The pFGC1008-CS contained 376 bp of caffeine synthase (CS) cDNA fragment in sense and antisense direction with an intron in between. This has made the RNAi construct to produce a hairpin RNA (ihpRNA). The suppressed expression of CS gene and a marked reduction in caffeine and theobromine contents in young shoots of tea seedlings were obtained after root transformation through Agrobacterium infiltration. Such transformation system could be useful for functional analysis of genes in tea like woody and perennial plants.     

<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.     

Molecular evidence for a natural diploid hybrid between <Emphasis Type="Italic">Miscanthus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis> (Poaceae) and <Emphasis Type="Italic">M. sacchariflorus</Emphasis>

The hybrid origin of Miscanthus purpurascens has previously been proposed, primarily because of its intermediate morphology. In this study, phylogenies based on the DNA sequences from the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS), on the DNA sequences of the trnL intron and trnL-F intergenic spacer of chloroplast DNA, and on amplified fragment length polymorphism (AFLP) fingerprinting confirm that M. purpurascens originated through homoploid hybridization between M. sinensis and M. sacchariflorus. Two different types of ITS sequences were identified from almost all plants of M. purpurascens. One type was found to be closely related to M. sinensis and the other to M. sacchariflorus. Miscanthus purpurascens was found to possess many M. sinensis- and M. sacchariflorus-specific AFLP bands but no band specific to itself. Clustering with the Unweighted Pair Group Method with Arithmetic Mean and principal coordinate analysis based on the AFLP data also demonstrated that M. purpurascens is an approximate intermediate of the two species. In addition, M. purpurascens has the plastid genome of M. sinensis or M. sacchariflorus, suggesting that either species could be its maternal parent. All specimens of M. purpurascens and its coexisting parental species are identified as diploids (2n = 2x = 38). Possible mechanisms of natural hybridization, hybrid status, chloroplast DNA recombination, and evolutionary implications of this hybridization are also discussed.     

Late-acting self-incompatibility in tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

The self-incompatibility of tea plant (Camellia sinensis (L.) O. Kuntze) was studied with the methods of aniline blue fluorescence assay and paraffin sections. The characteristics of pollen tube elongation after hand pollination was analyzed in 4 tea cultivars, including ‘Keemenzhong’, ‘Longjing-changye’, ‘Fuding-dabaicha’ and ‘Yabukita’, under self-pollination and cross-pollination, respectively. Although there were some difference among cultivars, pollen tubes elongated through the style and reach the ovary successfully at 48 h after pollination for both cross- and self-pollen tubes in all the four cultivars of tea. Pollen tubes entered into the ovule micropyles, however, only for cross-pollination, but not for self-pollination. Pollen tubes of selfing plants, failed in fertilizing, seemed have some difficulties to enter the ovule. All of which indicated that the self-incompatibility of tea plant is a late-acting self-incompatibility system (LSI) or an ovarian sterility (OS), in which the self incompatibility was due to none self pollen tube penetrating into the ovule and no fertilization.     

Cloning and expression of selenocysteine methyltransferase cDNA from <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Selenocysteine methyltransferase (SMT), specifically methylates selenocysteine (SeCys) to produce the nonprotein amino acid Se-methyl selenocysteine (SeMSC) and played key role of removing selenium toxic effect at higher levels to the plant. Here we report the cloning of a cDNA encoding selenocysteine methyltransferase from Camellia sinensis (CsSMT) and expression of CsSMT in Escherichia coli. CsSMT isolated by RT-PCR and RACE-PCR reaction. CsSMT is a 1,401 bp cDNA with an open reading frame predicted to encode a 351 amino acid, 40.5 kDa protein; The predicted amino acid sequences of CsSMT shows 74% identity with A. bisulcatus selenocysteine methyltransferase (AbSMT) and 69% identity with Broccoli (Brassica oleracea var. italica) selenocysteine methyltransferase (BoSMT), and shares 53, 73 and 65% identity, respectively, with Arabidopsis thaliana homocysteine S-methyltransferase AtHMT1, AtHMT2, and AtHMT3, and 65% to Zea mays homocysteine S-methyltransferase (ZmHMT2). Analyses of CsSMT showed that it lacks obvious chloroplast or mitochondrial targeting sequences and contains a consensus sequence of GGCC for a possible zinc-binding motif near the C-terminal and a conserved Cys residue upstream of the zinc-binding motif as other related methyltransferases. Expression of CsSMT correlated with the presence of SMT enzyme activity in cell extracts, and bacteria containing recombinant CsSMT plasmid showed much high tolerance to selenate and selenite.     

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">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.     

Construction and characterization of a soybean yeast artificial chromosome library and identification of clones for the<Emphasis Type="Italic"> Rps6</Emphasis> region

We report the construction and characterization of the first soybean yeast artificial chromosome (YAC) library using high-molecular weight DNA isolated from leaf nuclei of the cultivar Conrad 94 that carries Phytophthora resistance genes Rps1-k and Rps6. The quality of this library has been evaluated through analysis of 393 randomly selected YAC clones. The library consists of 36,864 clones, of which 19,956 carry single soybean YACs with an average size of about 285 kb. The library represents approximately five soybean genome equivalents. The probability of finding any soybean sequences from this library is about 0.99. The library was screened for 43 SSR markers representing the whole soybean genome. We were able to identify positive YAC pools for 95% of the SSR markers. Two YAC clones carrying molecular markers linked to the Rps6 gene were identified. The YAC library reported here would be a useful resource for map-based cloning of agronomically important soybean genes and also to complement the effort towards construction of the physical map for the soybean genome.