Sequence analysis of rDNA intergenic spacer (IGS) of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

The intergenic spacer region (IGS) has been used for the first time to analyze the genetic variability of Porphyra haitanensis from different areas. In order to determine that whether the IGS sequences could be used for classification and identification in intraspecies of Porphyra, the partial IGS sequences of cultivated strains of P. haitanensis (isolated from Putian-Fujian Province, Shantou-Guangdong Province and Ningbo-Zhejiang Province), were amplified, sequenced and analyzed. The sequence analysis indicated that the partial IGS sequences from the three stains were the external transcribed spacers (ETS) of 3′ end of the IGS gene. In the three stains, the length of IGS sequences ranged from 1,085 to 1,100 bp and the G + C content varied from 50.88% to 51.27%. There were 55 variable sites which occupied approximately 5% of the ETS sequences. Similarity analysis and multisequencing alignment of sequences indicated that the partial IGS sequences of the three stains of P. haitanensis had notable variabilities. Therefore, the IGS sequence could be used as the critical genetic marker in intraspecies of P. haitanensis. Furthermore, IGS sequence analysis will be a powerful tool for genetic diversity and classification in intraspecies of other Porphyra species.     

Structure and organization of the rDNA intergenic spacer region in <Emphasis Type="Italic">Pythium ultimum</Emphasis>

Nucleotide sequences of the rDNA intergenic spacer (IGS) region in Pythium ultimum were determined in 16 clones obtained from three isolates differing in production of sexual organs. Several sequences with different lengths were detected in each isolate, showing heterogeneity in the IGS region. In addition, several tandem repeat regions were detected in all the clones. The sequences, length, and number of each copy largely varied among repeat regions. Length heterogeneity arose from the complex combination of the number of copy within the repeat regions. Furthermore, the nucleotide sequence of each copy and the number of repetition varied not only between isolates but also between clones from an isolate. Based on the sequence similarity and the number of copies in repeat regions, specific patterns different between homothallic P. ultimum and the Pythium group HS (hyphal swellings) were recognized in a few regions. These results suggest that these two groups have slight genetic differences in the IGS region, although the differences in most of the repeat regions were not enough to identify each group.     

<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.     

Comparative mapping between<Emphasis Type="Italic"> Medicago sativa</Emphasis> and<Emphasis Type="Italic"> Pisum sativum</Emphasis>

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by W. R. McCombie     

Phylogenetic relationships in the genera<Emphasis Type="Italic"> Zostera</Emphasis> and<Emphasis Type="Italic"> Heterozostera</Emphasis> (Zosteraceae) based on<Emphasis Type="Italic"> matK</Emphasis> sequence data

Phylogenetic analysis of the plastid (chloroplast) DNA matK gene of Zosteraceae species was undertaken. A molecular phylogenetic tree based on matK sequence data showed the monophyly of Heterozostera tasmanica and subgenus Zosterella and did not support the separation of Heterozostera from the genus Zostera. The tree based on matK supported the monophyly of the subgenus Zostera, and showed that Zosteraceae consist of three main groups: Phyllospadix, which is clearly defined by being dioecious; the subgenus Zosterella and Heterozostera; and the subgenus Zostera. Character-state reconstruction of chromosome number and geographic distribution for our molecular phylogenetic tree showed that 2n=12 is a plesiomorphic character for Zostera and Heterozostera, that the chromosome number was doubled or tripled in two lineages, and that the initial speciation of Zostera and Heterozostera occurred in the Northern Hemisphere. The matK tree showed the close affinity of Z. noltii and Z. japonica, which have disjunct distributions. Zostera marina, which is the only widely distributed species in the subgenus Zostera, also occurring in the northern Atlantic, was shown to be embedded within other subgenus Zostera species.     

Chromosome analysis and sorting in <Emphasis Type="Italic">Vicia sativa</Emphasis> using flow cytometry

Procedures were developed for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) of common vetch (Vicia sativa L., 2n=12). Suspensions of intact chromosomes were prepared from root tips after cell cycle synchronization, formaldehyde fixation, and mechanical homogenization. On average, 3 × 10⁵ morphologically intact chromosomes could be isolated from 25 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing four peaks, representing particular chromosomes and/or pairs of chromosomes with similar relative DNA content. Peaks I and II were assigned to chromosomes 6 and 5, respectively. These chromosomes could be sorted with a purity exceeding 90 %. The two remaining peaks on the flow karyotype were composite, each of them representing a pair of chromosomes. Chromosomes 1 and 3 were assigned to composite peak III while chromosomes 2 and 4 were assigned to composite peak IV. The chromosomes could be sorted with a purity of 99 % from both composite peaks. Bivariate flow karyotyping after simultaneous staining of chromosomes with DAPI and mithramycin was not found helpful in discriminating additional chromosomes. This study extends the number of legume species for which flow cytogenetics is available and provides a new tool for targeted and effective analysis and mapping of common vetch genome.     

Purification and properties of aminoaldehyde dehydrogenase from <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K _m values for APAL, ABAL, and GBAL were 1.5×10^–6, 2.2×10^–6, and 1.3×10^–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH. Electronic Publication     

Taxonomic notes on<Emphasis Type="Italic">Linaria</Emphasis> mill. (<Emphasis Type="Italic">Scrophulariaceae</Emphasis>) for Flora Iberica

Taxonomic considerations and nomenclatural adjustments as a part of a revision ofLinaria Mill. undertaken within the “Flora iberica” project are presented. Data on morphology, seed coat surface, chorology, ecology, synonymy and variability of 11 accepted taxa are reported. The following new combinations are proposed:Linaria aeruginea subsp. cardonica (Font Quer)L. Sáez etM. Sainz,Linaria depauperata subsp.ilergabona (M.B. Crespo etV.J. Arán)L. Sáez, andLinaria saturejoides subsp.angustealata (Willmott)L. Sáez etM.B. Crespo.     

<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">In vitro</Emphasis> direct organogenesis and regeneration of <Emphasis Type="Italic">Medicago sativa</Emphasis>

A rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes was developed via direct organogenesis. Through a successive excision of the newly developed apical and axillary shoots, a lot of adventitious buds were directly induced from the cotyledonary nodes when hypocotyl of explants were vertically inserted into modified Murashige and Skoog (MS) medium supplemented with 0.025 mg dm⁻³ thidiazuron (TDZ) and 3 mg dm⁻³ AgNO₃. When the lower part of shoots excised from explants were immersed into the liquid medium with 1.0 mg dm⁻³ α-naphthaleneacetic acid (NAA) for 2 min, and then transferred to hormone free half-strength MS medium, over 83.3 % of the shoots developed roots, and all plantlets could acclimatize and establish in soil. The protocol has been successfully applied to eight genotypes, with regeneration frequencies ranging from 63.8 to 82.5 %.     

Composition of<Emphasis Type="Italic">Casuarina</Emphasis> leaf litter and its influence on<Emphasis Type="Italic">Frankia-Casuarina</Emphasis> symbiosis in soil

Plant needles ofCasuarina equisetifolia were collected and analyzed in parallel with soil analysis. In three strains ofFrankia—symbionts ofCasuarina—their infectivity and plant performance was determinedin vitro after soil amendment with different leaf litter concentrations. Only one strain was able to nodulate the plant at all litter concentrations (0.5, 3 and 5%) although the nodules were very small. However, all treated plants grew poorly; their growth was reduced by approximately 90% (for 5% litter concentration) compared to plants grown on untreated soil, on the basis of total dry mass. Inhibition of nodulation can be attributed to high concentrations of some elements and compounds that were either found inC. equisetifolia litter or originally found in soil (i.e. chloride, cyanide, copper, manganese and phenols). In general, plant growth decreased as more litter was added. Plant total nitrogen content was also reduced after increasing the litter concentration. The inhibitory effect of high litter concentrations was mainly on plant growth and to a lesser extent on plant nodulation byFrankia strains.     

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.     

Genome-wide comparative analysis of the <Emphasis Type="Italic">IQD</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Background  

Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues. Stimulus-specific generation of intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into cellular responses are integral modules of the transduction process. Several hundred proteins with functions in calcium signaling circuits have been identified, and the number of downstream targets of calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here, we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in Arabidopsis and rice.     

<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.     

A novel <Emphasis Type="Italic">TaSST</Emphasis> gene from wheat contributes to enhanced resistance to salt stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

In this research, through the analyzing of the Triticum aestivum salt-tolerant mutant gene expression profile, under salt stress. A brand new gene with unknown functions induced by salt was cloned. The cloned gene was named Triticum aestivum salt stress protein (TaSST). GenBank accession number of TaSST is ACH97119. Quantitative polymerase chain reaction (qPCR) results exhibited that the expression TaSST was induced by salt, abscisic acid (ABA), and polyethylene glycol (PEG). TaSST could improve salt tolerance of Arabidopsis-overexpressed TaSST. After salt stress, physiological indexes of transgenic Arabidopsis were better compared with WT (wild-type) plants. TaSST was mainly located in the cytomembrane. qPCR analyzed the expression levels of nine tolerance-related genes of Arabidopsis in TaSST-overexpressing Arabidopsis. Results showed that the expression levels of SOS3, SOS2, KIN2, and COR15a significantly increased, whereas the expression of the five other genes showed no obvious change. OsI_01272, the homologous gene of TaSST in rice, was interfered using RNA interference (RNAi) technique. RNAi plants became more sensitive to salt than control plants. Thus, we speculate that TaSST can improve plant salt tolerance.     

Biosynthesis of <Emphasis Type="Italic">Veratrum californicum</Emphasis> specialty chemicals in <Emphasis Type="Italic">Camelina sativa</Emphasis> seed

Economically feasible systems for heterologous production of complex secondary metabolites originating from difficult to cultivate species are in demand since Escherichia coli and Saccharomyces cerevisiae are not always suitable for expression of plant and animal genes. An emerging oilseed crop, Camelina sativa, has recently been engineered to produce novel oil profiles, jet fuel precursors, and small molecules of industrial interest. To establish C. sativa as a system for the production of medicinally relevant compounds, we introduced four genes from Veratrum californicum involved in steroid alkaloid biosynthesis. Together, these four genes produce verazine, the hypothesized precursor to cyclopamine, a medicinally relevant steroid alkaloid whose analogs are currently being tested for cancer therapy in clinical trials. The future supply of this potential cancer treatment is uncertain as V. californicum is slow-growing and not amendable to cultivation. Moreover, the complex stereochemistry of cyclopamine results in low-yield syntheses. Herein, we successfully engineered C. sativa to synthesize verazine, as well as other V. californicum secondary metabolites, in seed. In addition, we have clarified the stereochemistry of verazine and related V. californicum metabolites.     

Genetic analysis of ryanodine receptor function in<Emphasis Type="Italic"> Caenorhabditis elegans</Emphasis> based on<Emphasis Type="Italic"> unc-68</Emphasis> revertants

The Caenorhabditis elegans ryanodine receptor is encoded by the unc-68 gene, and functions as a Ca²⁺-induced Ca²⁺ release channel during muscle contraction. To investigate the factors that suppress calcium release and identify molecules that interact with the ryanodine receptor, we isolated revertants from two unc-68 mutants. Three of the revertants obtained from the null allele unc-68(e540), which displayed normal motility, had intragenic mutations that resulted in failure to splice out intron 21. The other two, kh53 and kh55, had amino acid insertions in the third of the four RyR domains. The brood size and the egg laying rate remain abnormal in these revertants. This suggests the third RyR domain may be required for egg laying and embryogenesis, although we can not determine a molecular mechanism. Five ketamine sensitive revertants recovered from the missense mutant unc-68(kh30) showed altered responses to caffeine, ryanodine, levamisole and ouabain relative to those of the unc-68(kh30) animals. These may carry second-site suppressor mutations, which may define genes for proteins that regulate the Ca²⁺ concentration in body-wall muscle. One of these mutants, kh52 , shows lower motility and higher sensitivity to drugs, and this mutation was mapped to chromosome X. These observations provide a basis for the study of ryanodine receptor functions in embryogenesis and in calcium-mediated regulation of muscle contraction in C. elegans. This is the first study to show that the conserved RyR domain of the receptor acts in egg laying and embryogenesis.Communicated by C. P. Hollenberg     

Identification of a novel <Emphasis Type="Italic">SPLIT</Emphasis>-<Emphasis Type="Italic">HULL</Emphasis> (<Emphasis Type="Italic">SPH</Emphasis>) gene associated with hull splitting in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Key message

The split-hull phenotype caused by reduced lemma width and low lignin content is under control of SPH encoding a type-2 13-lipoxygenase and contributes to high dehulling efficiency.

Abstract

Rice hulls consist of two bract-like structures, the lemma and palea. The hull is an important organ that helps to protect seeds from environmental stress, determines seed shape, and ensures grain filling. Achieving optimal hull size and morphology is beneficial for seed development. We characterized the split-hull (sph) mutant in rice, which exhibits hull splitting in the interlocking part between lemma and palea and/or the folded part of the lemma during the grain filling stage. Morphological and chemical analysis revealed that reduction in the width of the lemma and lignin content of the hull in the sph mutant might be the cause of hull splitting. Genetic analysis indicated that the mutant phenotype was controlled by a single recessive gene, sph (Os04g0447100), which encodes a type-2 13-lipoxygenase. SPH knockout and knockdown transgenic plants displayed the same split-hull phenotype as in the mutant. The sph mutant showed significantly higher linoleic and linolenic acid (substrates of lipoxygenase) contents in spikelets compared to the wild type. It is probably due to the genetic defect of SPH and subsequent decrease in lipoxygenase activity. In dehulling experiment, the sph mutant showed high dehulling efficiency even by a weak tearing force in a dehulling machine. Collectively, the results provide a basis for understanding of the functional role of lipoxygenase in structure and maintenance of hulls, and would facilitate breeding of easy-dehulling rice.

     

Comparative mapping between<Emphasis Type="Italic"> Quercus</Emphasis> and<Emphasis Type="Italic"> Castanea</Emphasis> using simple-sequence repeats (SSRs)

Simple sequence repeat (SSR) markers from Quercus and Castanea were used for comparative mapping between Quercus robur (L.) and Castanea sativa (Mill.). We tested the transferability of SSRs developed in Quercus to Castanea and vice-versa. In total, 47% (25) of the Quercus SSRs and 63% (19) of the Castanea SSRs showed a strong amplification product in the non-source species. From these 44 putative comparative anchor tags, 19 (15 from Quercus and 4 from Castanea) were integrated in two previously established genetic linkage maps for the two genera. SSR loci were sequenced to confirm the orthology of the markers. The combined information from both genetic mapping and sequence analysis were used to determine the homeology between seven linkage groups, aligned on the basis of pairs or triplets of common markers, while two additional groups were matched using a single microsatellite marker. Orthologous loci identified between Q. robur and C. sativa will be useful as anchor loci for comparative mapping studies within the Fagaceae family.Communicated by D.B. NealeThis paper is dedicated to the memory of Paulo Costa