Artificial hybridization of some Abies species

Crossability relationships between six species of the Mediterranean, North American and Asian firs was tested using Abies alba and A. nordmanniana as female parents and A. alba, A. numidica, A. procera, A. grandis, and A. holophylla as pollen parents. An overwhelming majority of the crosses attempted was found to be compatible. In particular, it is true of the A. alba cross with A. numidica and those of A. nordmanniana with A. alba, A. numidica, A. procera, and A. holophylla. The crossing A. nordmanniana × A. grandis was the only exception producing empty seeds. Cytological study revealed the gametophytic incompatibility to be responsible for reproductive isolation of these species. At seedling level, all the interspecific crosses of A. nordmanniana surpassed in height growth self-pollinated control. The cross A. alba × A. numidica was comparable in this respect with control variants from open and self-pollination. Except for height growth, some characteristics of needle stomata are provided for individual crosses. The crosses A. nordmanniana with A. procera and A. holophylla represent unique interspecific combinations whose existence has not been reported yet. Based on needle stomata characteristics, the potential for increased resistance and drought tolerance of the hybrids with A. numidica involved as parental species is discussed.     

Evaluation of koji prepared with various molds for mirin-making

Koji is one of the raw materials used for mirin-making, and it is traditionally prepared with Aspergillus oryzae. To improve productivity and to be available for a variety of mirin, various koji were prepared with A. niger, A. awamori, A. usamii mut. shiro-usamii, and Rhizopus oligosporus and examined for possible to use in mirin-making. The degrees of digestion of the starch and protein in various koji were at its highest with either koji prepared with A. oryzae at pH 7.0 or with A. usamii mut. shiro-usamii at pH 3.0. The degree of digestion of the protein in koji made with A. usamii mut. shiro-usamii was decreased in koji digestion at higher temperature (at over 50°C) compared to that in koji made with A. oryzae. We examined the ratio of koji to steamed glutinous rice to identify the optimum value for mirin production. The optimum ratios for digestion of starch in the mash with koji of A. oryzae and A. usamii mut. shiro-usamii were 0.10 and 0.15, respectively. These values were in agreement with the 0.10–0.25 established by experience. Therefore, of the five strains tested, A. usamii mut. shiro-usamii, among the tested molds was the most suitable strain for preparing koji in mirin-making without decreasing productivity, while lending new qualities to the mirin. The koji prepared with A. oryzae had a good smell but the koji prepared with A. usamii mut. shiro-usamii had a little mold smell. Then if this little mold smell could be improved, this koji can be used in mirin-making. The koji prepared with A. niger contained the most citric acid of all strains but the digestivities of starch and protein in the koji was less than those of koji prepared with A. oryzae and A. usamii mut. shiro-usamii. The digestivities of starch and protein in koji prepared with A. awamori were the lowest values. In 35% alcohol, its productivities in mirin-making seemed to be low. Because of the highest content of lactic acid, the koji prepared with R. oligosporus seemed to be suitable for mirin-making. Then if its productivity could be improved, this koji can be used in mirin-making.     

Identification of a cation-specific channel (TipA) in the cell wall of the gram-positive mycolata Tsukamurella inchonensis: the gene of the channel-forming protein is identical to mspA of Mycobacterium smegmatis and mppA of Mycobacterium phlei

Detergent extracts of whole cells of the Gram-positive bacterium Tsukamurella inchonensis ATCC 700082, which belongs to the mycolata, were studied for the presence of ion-permeable channels using lipid bilayer experiments. One channel with a conductance of about 4.5 nS in 1 M KCl was identified in the extracts. The channel-forming protein was purified to homogeneity by preparative SDS-PAGE. The protein responsible for channel-forming activity had an apparent molecular mass of about 33 kDa as judged by SDS-PAGE. Interestingly, the protein showed cross-reactivity with polyclonal antibodies raised against a polypeptide derived from MspA of Mycobacterium smegmatis similarly as the cell wall channel of Mycobacterium phlei. Primers derived from mspA were used to clone and sequence the gene of the cell wall channels of T. inchonensis (named tipA for T. inchonensis porin A) and M. phlei (named mppA for M. phlei porin A). Surprisingly, both genes, tipA and mppA, were found to be identical to mspA of M. smegmatis, indicating that the genomes of T. inchonensis, M. phlei and M. smegmatis contain the same genes for the major cell wall channel. RT-PCR revealed that tipA is transcribed in T. inchonensis and mppA in M. phlei. The results suggest that despite a certain distance between the three organisms, their genomes contain the same gene coding for the major cell wall channel, with a molecular mass of 22 kDa for the monomer.     

A multigene approach to determine the molecular phylogeography of Argiope mangal and Argiope dang (Araneae: Araneidae) and their genetic relationships with the Argiope aetherea species group

The genus Argiope represents a collection of morphologically diverse orb-weaving spiders with a cosmopolitan distribution. Argiope dang and A. mangal are two Southeast Asian species that share remarkably similar morphological characteristics congeneric to the A. aetherea species group. However, in previous investigations, molecular data have not been used for the taxonomic categorization of A. dang and A. mangal. Additionally, gene flow between populations of A. dang and of A. mangal in their natural habitats has not been adequately studied. In the present study, mitochondrial 16S, COI and COII markers were used in addition to the nuclear H3 gene to elucidate the relationships between A. dang, A. mangal and various congeners within the A. aetherea species group. The haplotype diversity of A. dang and A. mangal was also investigated. Based on our molecular results, the A. aetherea species group was composed of three genetic lineages: (i) [A. dang - (A. radon - A. picta)] - [A. modesta - A. aetherea]; (ii) A. mangal; and (iii) [(A. jinghongensis - A. aetheroides) - A. versicolor]. Haplotype analyses revealed a lack of gene flow between A. mangal populations in Peninsular Malaysia and those in Singapore. Populations of A. dang showed isolation-by-distance throughout the range of the species from Laos to Singapore. In this study for the first time, genetic data of A. mangal were reported for specimens collected from its type locality, Singapore. Four new distribution records were also reported: A. dang in Peninsular Malaysia and Cambodia; A. modesta in Bali, Indonesia; and A. jinghongensis in Peninsular Malaysia.     

Reevaluation of the odd chrysidid genus Atoposega Krombein (Hymenoptera,Chrysididae, Amiseginae)

The south Asian amisegine genus Atoposega Krombein, 1957, is reevaluated. Three new species, A. rufithorax, A. striata and A. thailandica are described from Thailand and the previously described species, A. lineata (Krombein, 1957) from Borneo, A. rieki (Krombein, 1957) from Myanmar and A. simulans Kimsey, 1986 from Malaysia are redescribed. The species, A. decorata Kimsey, 1995, was found to lack the generic characters diagnostic for Atoposega. Atoposega is only known from females.     

Characterization of Brazilian accessions of wild Arachis species of section Arachis (Fabaceae) using heterochromatin detection and fluorescence in situ hybridization (FISH)

The cytogenetic characterization of Arachis species is useful for assessing the genomes present in this genus, for establishing the relationship among their representatives and for understanding the variability in the available germplasm. In this study, we used fluorescence in situ hybridization (FISH) to examine the distribution patterns of heterochromatin and rDNA genes in 12 Brazilian accessions of five species of the taxonomic section Arachis. The heterochromatic pattern varied considerably among the species: complements with centromeric bands in all of the chromosomes (A. hoehnei) and complements completely devoid of heterochromatin (A. gregoryi, A. magna) were observed. The number of 45S rDNA loci ranged from two (A. gregoryi) to eight (A. glandulifera), while the number of 5S rDNA loci was more conserved and varied from two (in most species) to four (A. hoehnei). In some species one pair of 5S rDNA loci was observed adjacent to 45S rDNA loci. The chromosomal markers revealed polymorphism in the three species with more than one accession (A. gregoryi, A. magna and A. valida) that were tested. The previous genome assignment for each of the species studied was confirmed, except for A. hoehnei. The intraspecific variability observed here suggests that an exhaustive cytogenetic and taxonomic analysis is still needed for some Arachis species.     

Homologs of Aflatoxin Biosynthesis Genes and Sequence of aflR in Aspergillus oryzae and Aspergillus sojae

The presence, but not expression, of homologs of three structural genes and a regulatory gene necessary for aflatoxin biosynthesis in Aspergillus parasiticus and A. flavus was shown for A. oryzae and A. sojae. Homologs of the regulatory gene aflR were cloned and sequenced from A. oryzae and A. sojae.     

Some highly proteinaceous Acacia gum exudates of the subseries Juliflorae

Analytical data for the gum exudates from Acacia difficilis, A. dimidiata, A. eriopoda, A. maidenii, A. stipuligera, A. torulosa and A. tumida are presented. Of these, five are highly proteinaceous; they also have high methoxyl contents and very low rhamnose contents. In contrast, A. dimidiata shows no unusual analytical parameters, and A. maidenii gum has alowarabinose content and a high rhamnose content, thus having a sugar composition of the type first observed in the gum from A. saligna. The gum from A. maidenii is also of interest as its analytical data are closely similar to those for A. longifolia, the only other tetramerous member of the subseries Juliflorae to have been studied. The data reported extend even further the unusual ranges of analytical parameters found within the Juliflorae, and confirm its great heterogeneity and chemotaxonomic interest.     

Some Acacia gum exudates of the section Phyllodineae

Australian gum specimens from Acacia saliciformis, A. xanthina, A. rostellifera, A. murrayana (two specimens differing in the mode of initiation of gum exudation), A. georginae, A. cyclops, A. implexa, and an un-named species (Maslin ‘P31’) have been analysed. The first four of these are placed within Bentham's Series 1, subseries 6F, A. georginae within subseries 7E, and the remainder within subseries 7F. These data extend considerably the ranges of the analytical parameters reported previously for phyllodine species. The molecular weights of the gums from A. cyclops and A. implexa are much higher than those reported earlier for South African specimens; this may affect some taxonomic deductions based on their examination. The gum composition of A. saligna can no longer be regarded as atypical of a phyllodinous species; a suggestion that A. saligna should be transferred to the section Juliflorae may require reconsideration. The major difference between the specimens of gum from A. murrayana lies in their nitrogenous content. Data are reported for the amino acid compositions of the gums from A. saliciformis and A. xanthina.     

Acacia gum exudates from species of the series gummiferae

An analytical study of the gum exudates from the African species Acacia ehrenbergiana (three specimens), A.xanthophloea (two specimens), A.hockii and A.sieberana var.villosa, and of the Australian species A.calcigera, has been made. There are now 19 species within the series Gummiferae Benth. for which gum parameters are available; of these, only A. ehrenbergiana gum displays a slightly negative optical rotation. The data for the three gum specimens from A. ehrenbergiana give a further example of the extent to which the gum from different trees of one particular species can vary in composition. The data for A.sieberana var. villosa gum are compared with the values established previously for subsp. sieberana; the differences between varieties of one species are similar in extent to those established for some subspecies. Although A. xanthophloea, A. hockii, A. ehrenbergiana, A. seyal and A. karroo are regarded as being very closely related botanically, the values for some of their analytical parameters differ considerably and strongly support the view that it is correct to retain them as distinct species.     

Effectiveness of ISSR markers for determination of the Aneura pinguis cryptic species and Aneura maxima

Aneura pinguis and Aneura maxima belong to the simple thalloid liverworts. Previous isozyme studies revealed that A. pinguis is a complex of cryptic species difficult to distinguished based on morphology. In the present study four cryptic species of the A. pinguis complex and A. maxima were examined by means of ISSR method to assess genetic variation and to develop species-specific markers. Eight ISSR primers used generated 460 bands, of which 453 were polymorphic. The highest values of resolving power 28.4 and marker index 18.1 were noted for primer 835 (AG)₈-YC, while polymorphism information content for primer 842 (GA)₇-AYG. The total gene diversity (H_T) based on polymorphic loci was 0.284 for A. pinguis and 0.06 for A. maxima. ISSR markers supported existence of cryptic species in A. pinguis and showed genetic isolation between them. Species-specific bands were found for all studied cryptic species of A. pinguis and A. maxima, thus ISSRs can be used for their identification. A. maxima clearly differ from all the A. pinguis cryptic species in each amplified ISSR primer. The AMOVA conducted for the A. pinguis complex showed that most of genetic variation (Φ_PT 0.586) was present among species.     

A New Black Aspergillus Species, A. vadensis, Is a Promising Host for Homologous and Heterologous Protein Production

A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.     

Systematic investigation of Amphioxus (Branchiostoma floridae) microRNAs

Ascaris lumbricoides and Ascaris suum are parasitic nematodes living in the small intestine of humans and pigs, and can cause the disease ascariasis. For long, there has been controversy as to whether the two ascaridoid taxa represent the same species due to their significant resemblances in morphology. However, the complete mitochondrial (mt) genome data have been lacking for A. lumbricoides in spite of human and animal health significance and socio-economic impact globally of these parasites. In the present study, we sequenced the complete mt genomes of A. lumbricoides and A. suum (China isolate), which was 14,303 bp and 14,311 bp in size, respectively. The identity of the mt genomes was 98.1% between A. lumbricoides and A. suum (China isolate), and 98.5% between A. suum (China isolate) and A. suum (USA isolate). Both genomes are circular, and consist of 36 genes, including 12 genes for proteins, 2 genes for rRNA and 22 genes for tRNA, which are consistent with that of all other species of ascaridoid studied to date. All genes are transcribed in the same direction and have a nucleotide composition high in A and T (71.7% for A. lumbricoides and 71.8% for A. suum). The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. Phylogenetic analyses of A. lumbricoides and A. suum using concatenated amino acid sequences of 12 protein-coding genes, with three different computational algorithms (Bayesian analysis, maximum likelihood and maximum parsimony) all clustered in a clade with high statistical support, indicating that A. lumbricoides and A. suum was very closely related. These mt genome data and the results provide some additional genetic evidence that A. lumbricoides and A. suum may represent the same species. The mt genome data presented in this study are also useful novel markers for studying the molecular epidemiology and population genetics of Ascaris.     

Prevalence and Genotypes of Anaplasma Species and Habitat Suitability for Ticks in a Mediterranean Ecosystem

Anaplasma species are tick-transmitted pathogens that impact veterinary and human health. Sicily is one of the locations where these pathogens are endemic. Sicily represents a typical Mediterranean ecosystem to study Anaplasma infection and tick habitat suitability. The aims of this study were (i) to characterize by 16S rRNA and species-specific msp4 gene PCR the prevalence and genotypes of A. marginale, A. phagocytophilum, and A. ovis in the most abundant host species in Sicilian provinces and (ii) to correlate differences between hosts and between western and eastern Sicily with the habitat suitability for ticks in these regions. Differences were found in the prevalence of Anaplasma spp. between different hosts and between western and eastern provinces. The differences in Anaplasma prevalence between different hosts may be explained by pathogen host tropism. The differences between western and eastern provinces correlated with the tick habitat suitability in these regions. The analysis of Anaplasma genotypes suggested a higher host and regional specificity for A. phagocytophilum than for A. marginale and A. ovis strains, a finding probably associated with the broader host range of A. phagocytophilum. The presence of identical A. marginale genotypes in the two regions may reflect cattle movement. The results for A. ovis suggested the possibility of some genotypes being host specific. These results provide information potentially useful for the management of tick-borne diseases caused by Anaplasma spp. in Sicily and other Mediterranean regions and may contribute to the development of models to predict the risks for these tick-borne pathogens.     

Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae)

The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades - Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66 kDa protein mRNA, clone L5-400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%.     

The gum exudates from some closely related Acacia species of the subseries Uninerves racemosae (section phyllodineae)

Australian gum specimens from Acacia aestivalis, A. chrysella, A. jennerae and A. microbotrya (five specimens differing slightly in some morphological characters) have been studied. These species, placed within Bentham's Series 1, subseries 6F (Uninerves racemosae) are closely related, forming part of the recognized A. microbotrya group. The five specimens from A. microbotrya show minor variations, similar in extent to those established previously for gums from other species. The gums from A. chrysella and A. jennerae are similar to those from A. microbotrya in chemical composition. The gum from A. aestivalis differs from those from A. microbotrya, A. chrysella and A. jennerae in two main respects: it is more acidic and has a much higher methoxyl content. Thus significant differences in gum composition can be shown by some species that differ only slightly in morphological characters. Data for the amino acid compositions of the proteinaceous components of the gums from A. aestivalis, A. jennerae and A. microbotrya, differ considerably from those for the gums from other species belonging to the Uninerves racemosae, e.g. A. saliciformis and A. xanthina, which are much more viscous and have higher proteinaceous contents containing much higher proportions of the amino acids commonly involved in linkages with sugars. Of the closely related species studied, A. aestivalis is closer to A. microbotrya than A.jennerae in terms of the amino acid compositions of their gums, a reversal in the relative affinities shown by their polysaccharide parameters. Thus amino acid compositions are of interest chemotaxonomically and also in terms of the tertiary structures of Acacia gum exudates.     

Relationships ofAmaranthus caudatus

Three species ofAmaranthusare cultivated for their edible seeds:A. hypochondriacus L.,A. cruentusL., andA. caudatusL. The first two are native to Mexico and Guatemala, while the third originated in the Andes. Some authors recognize a fourth species,A. MantegazzianusPass. (A. edulisSpeg.), also from South America. Recent interest in amaranths as crops for improving Third World nutrition makes studies of relationships among amaranth species and intraspecific variation important. The weedy speciesA. hybridus L. (A. quitensisHBK) has been suggested as the progenitor ofA. caudatus, and it appears to be the closest wild relative of the crop. However, discovery of semidomesticated, darkseeded amaranths in Ecuador that are referable toA. caudatusraises some questions. The dark-seeded plants might represent a transitional form between the crop and its weedy progenitor, the product of independent selection of special forms ofA. hybridus, the result of introgressive hybridization between the crop and related weed, established escapes from cultivation, or remnants of the ancestor of the crop which may have been simply wildA. caudatusand notA. hybridus. Detailed morphological comparisons have been made among cultivated forms ofA. caudatus, the semidomesticate, andA. hybridus. Genetic data have been considered, and 2 mixed populations includingA. hybridusand the semidomesticate have been examined. Although all the other hypotheses cannot be eliminated, the dark-seededA. caudatusplants seem most likely to represent escapes from cultivation. Separate recognition ofA. Mantegazzianusdoes not seem warranted.     

Development and validation of a new Ppo-A1 marker useful for marker-assisted selection in tetraploid wheats

Polyphenol oxidase (PPO) activity is a major cause of undesirable brown color of semolina. In tetraploid wheat, the Ppo-A1 gene is significantly involved in the phenotypic expression of PPO activity. The main goal of this study was to develop and validate a more efficient marker for Ppo-A1 to facilitate marker-assisted selection for low PPO activity in tetraploid wheat breeding programs. A large tetraploid wheat collection, including durum cultivars, domesticated and wild accessions, was used to evaluate the PPO activity. The heritability values indicated that the phenotypic expression of PPO activity was mainly due to genotypic effect. PPO18, and a new marker named MG18, were used to study the Ppo-A1 allelic variation in a tetraploid wheat collection. PPO18 analysis detected four alleles (Ppo-A1b, Ppo-A1e, Ppo-A1f and Ppo-A1g). The high frequency of Ppo-A1g (no PCR product) detected in the tetraploid wheat collection, led to the development of a new genome-specific Ppo-A1 marker (MG18). MG18 analysis identified the same alleles as PPO18 which were associated with low or high PPO activity. The new MG18 marker was more efficient than PPO18 in detecting the four different alleles of Ppo-A1 in the tetraploid wheat collection. Indeed, the accessions assigned to the Ppo-A1g group, according to PPO18, when tested with MG18, were better classified in the four alleles of the Ppo-A1 gene. The MG18 analysis proved that the PPO18 marker overestimated the number of accessions with Ppo-A1g. Therefore, MG18 can be applied to large-scale marker-assisted selection for PPO activity in durum breeding programs.