Characterization of multiple nestin isoforms in the goldfish brain.

Nestin is an intermediate filament protein involved in neurogenesis in fish, mice, and humans. In this study we used rapid amplification of cDNA ends PCR to isolate goldfish nestin (nes). PCR analysis and sequencing revealed three different nes transcripts of 4003, 2446, and 2126 nucleotides, which are predicted to generate proteins of 860, 274, and 344 amino acids in length. Sequence analysis suggests that these nes transcripts are likely a result of alternative splicing. We next applied a multiple-antigenic peptide strategy to generate a goldfish-specific nestin antibody. Western blotting with this antibody together with mass spectrometry verified the presence of major nestin protein isoforms with differing molecular weights (~70, 40 and 30kDa). We further examined expression patterns of these nestin protein isoforms in different parts of the goldfish brain and pituitary and found the telencephalon to express all three isoforms at a distinct level and abundance. We report that multiple nestin isoforms are present indicating another level of complexity for the regulation of intermediate filaments in comparison to mammals. Studying the differential roles and regulation of these nestins could lead to a better understanding of cellular remodeling during neurogenesis and the unparalleled regenerative abilities after damage in the teleost CNS.

Diversity within the genus Elymus (Poaceae: Triticeae) II: analyses of variation within 5S nrDNA restrict membership in the genus to species with StH genomes.

The genus Elymus is a repository for a large number of species that have been difficult to classify by traditional techniques due to their remarkable levels of polymorphism. Following the genome analyses of Yen and Yang (Genus Elymus 5:58-362, 2013), we used sequences of the nr5SDNA to investigate diversity within those 24 species having St and H haplomes (Baum et al. Mol Genet Genomics 290:329-42, 2015) and for which the genome status was known. The present work extends this analysis to include eight species for which there was no information on genomic status. Our results show that these eight have nr5SDNA sequences that can be assigned to unit classes of orthologous sequences found in St and H haplomes, suggesting that the presence of St and H haplomes is characteristic of the genus. We then carried out a set of canonical discriminant analyses based on 247 DNA new sequences from these 8 species plus the 1054 sequences previously identified from 24 Elymus species. Sequences were analyzed to answer the following questions: Do the species integrate or are they different? Are the tetraploids different from the higher-ploid species? Are the species united within sections, or the same within regions? How do the species fare when divided according to sections? The main results of the canonical discriminant analyses are that the species are united within the tetraploids and within the hexaploids, within each region and within each section. In addition, a series of classificatory discriminant analyses showed that the identification tests are different, although not sufficiently useful for the discrimination of all the species. We also demonstrate the power of our approach by showing that the voucher for Elymus mobilis is not Elymus at all, but Leymus.

Phylogenetic analysis of the genus Avena based on chloroplast intergenic spacer psbA-trnH and single-copy nuclear gene Acc1.

Two uncorrelated nucleotide sequences, chloroplast intergenic spacer psbA-trnH and acetyl CoA carboxylase gene (Acc1), were used to perform phylogenetic analyses in 75 accessions of the genus Avena, representing 13 diploids, seven tetraploid, and four hexaploids by maximum parsimony and Bayesian inference. Phylogenic analyses based on the chloroplast intergenic spacer psbA-trnH confirmed that the A genome diploid might be the maternal donor of species of the genus Avena. Two haplotypes of the Acc1 gene region were obtained from the AB genome tetraploids, indicating an allopolyploid origin for the tetraploid species. Among the AB genome species, both gene trees revealed differences between Avena agadiriana and the other species, suggesting that an AS genome diploid might be the A genome donor and the other genome diploid donor might be the Ac genome diploid Avena canariensis or the Ad genome diploid Avena damascena. Three haplotypes of the Acc1 gene have been detected among the ACD genome hexaploid species. The haplotype that seems to represent the D genome clustered with the tetraploid species Avena murphyi and Avena maroccana, which supported the CD genomic designation instead of AC for A. murphyi and A. maroccana.

Contrasting patterns of evolution of 45S and 5S rDNA families uncover new aspects in the genome constitution of the agronomically important grass Thinopyrum intermedium (Triticeae).

We employed sequencing of clones and in situ hybridization (genomic and fluorescent in situ hybridization [GISH and rDNA-FISH]) to characterize both the sequence variation and genomic organization of 45S (herein ITS1-5.8S-ITS2 region) and 5S (5S gene + nontranscribed spacer) ribosomal DNA (rDNA) families in the allohexaploid grass Thinopyrum intermedium. Both rDNA families are organized within several rDNA loci within all three subgenomes of the allohexaploid species. Both families have undergone different patterns of evolution. The 45S rDNA family has evolved in a concerted manner: internal transcribed spacer (ITS) sequences residing within the arrays of two subgenomes out of three got homogenized toward one major ribotype, whereas the third subgenome contained a minor proportion of distinct unhomogenized copies. Homogenization mechanisms such as unequal crossover and/or gene conversion were coupled with the loss of certain 45S rDNA loci. Unlike in the 45S family, the data suggest that neither interlocus homogenization among homeologous chromosomes nor locus loss occurred in 5S rDNA. Consistently with other Triticeae, the 5S rDNA family in intermediate wheatgrass comprised two distinct array types-the long- and short-spacer unit classes. Within the long and short units, we distinguished five and three different types, respectively, likely representing homeologous unit classes donated by putative parental species. Although the major ITS ribotype corresponds in our phylogenetic analysis to the E-genome species, the minor ribotype corresponds to Dasypyrum. 5S sequences suggested the contributions from Pseudoroegneria, Dasypyrum, and Aegilops. The contribution from Aegilops to the intermediate wheatgrass' genome is a new finding with implications in wheat improvement. We discuss rDNA evolution and potential origin of intermediate wheatgrass.

A taxonomist's view on genomic authentication.

A brief history of taxonomy, for the most part plant oriented, is provided, which demonstrates the use of morphology early on, through the stages when different technologies became available at different times until the present use of genomic tools. Genomic authentication facilitates with greater precision than ever before the identification of an organism or part thereof. In this chapter I made an attempt to stress that, in general, but more so for genomic authentication, the use of the variation inherent in taxa down to the lowest level of the hierarchy of classification needs to be used to achieve a high degree of correct authentication.

The evolution pattern of rDNA ITS in Avena and phylogenetic relationship of the Avena species (Poaceae: Aveneae).

Ribosomal ITS sequences are commonly used for phylogenetic reconstruction because they are included in rDNA repeats, and these repeats often undergo rapid concerted evolution within and between arrays. Therefore, the rDNA ITS copies appear to be virtually identical and can sometimes be treated as a single gene. In this paper we examined ITS polymorphism within and among 13 diploid (A and C genomes), seven tetraploid (AB, AC and CC genomes) and four hexaploid (ACD genome) to infer the extent and direction of concerted evolution, and to reveal the phylogenetic and genome relationship among species of Avena. A total of 170 clones of the ITS1-5.8S-ITS2 fragment were sequenced to carry out haplotype and phylogenetic analysis. In addition, 111 Avena ITS sequences retrieved from GenBank were combined with 170 clones to construct a phylogeny and a network. We demonstrate the major divergence between the A and C genomes whereas the distinction among the A and B/D genomes was generally not possible. High affinity among the A(d) genome species A. damascena and the ACD genome species A. fatua was found, whereas the rest of the ACD genome hexaploids and the AACC tetraploids were highly affiliated with the A(l) genome diploid A. longiglumis. One of the AACC species A. murphyi showed the closest relationship with most of the hexaploid species. Both C(v) and C(p) genome species have been proposed as paternal donors of the C-genome carrying polyploids. Incomplete concerted evolution is responsible for the observed differences among different clones of a single Avena individual. The elimination of C-genome rRNA sequences and the resulting evolutionary inference of hexaploid species are discussed.

Phylogenetic inferences in Avena based on analysis of FL intron2 sequences.

The development and application of molecular methods in oats has been relatively slow compared with other crops. Results from the previous analyses have left many questions concerning species evolutionary relationships unanswered, especially regarding the origins of the B and D genomes, which are only known to be present in polyploid oat species. To investigate the species and genome relationships in genus Avena, among 13 diploid (A and C genomes), we used the second intron of the nuclear gene FLORICAULA/LEAFY (FL int2) in seven tetraploid (AB and AC genomes), and five hexaploid (ACD genome) species. The Avena FL int2 is rather long, and high levels of variation in length and sequence composition were found. Evidence for more than one copy of the FL int2 sequence was obtained for both the A and C genome groups, and the degree of divergence of the A genome copies was greater than that observed within the C genome sequences. Phylogenetic analysis of the FL int2 sequences resulted in topologies that contained four major groups; these groups reemphasize the major genomic divergence between the A and C genomes, and the close relationship among the A, B, and D genomes. However, the D genome in hexaploids more likely originated from a C genome diploid rather than the generally believed A genome, and the C genome diploid A. clauda may have played an important role in the origination of both the C and D genome in polyploids.

Codependence of repetitive sequence classes in genomes: phylogenetic analysis of 5S rDNA families in Hordeum (Triticeae: Poaceae).

To complete our study of the genus Hordeum and to elaborate a phylogeny of species based upon 5S rDNA sequences, we have cloned and sequenced PCR amplicons from seven American polyploid species to generate 164 new 5S rRNA gene sequences. These sequences were analysed along with the more than 2000 5S rDNA sequences previously generated from the majority of species in Hordeum to provide a comprehensive picture of the distribution (presence or absence) of 5S rDNA unit classes (orthologous groups) in this genus as well as insights into the phylogeny of Hordeum. Testing of substitution models for each unit class based upon the consensus sequences of all the taxa as well as for each unit class within the genus found that the general best fit was TPM3uf+G, from which a maximum-likelihood tree was calculated. A novel application of cophylogenetic analysis, where relationships among unit classes were treated as host-parasite interactions, depicted some significant pair links under tests of randomness indicative of nonrandom codivergence among several unit classes within the same taxon. The previous classification of four genomic groups is reflected in combinations of unit classes, and it is proposed that current taxa developed from ancient diploidized paleopolyploids and that some were subjected to gene loss, i.e., unit class loss. Finally, separate phylogenetic analyses performed for the tetraploid and hexaploid species were used to derive a working model describing the phylogeny of the polyploid taxa from their putative diploid ancestry.

Molecular confirmation of the genomic constitution of Douglasdeweya (Triticeae: Poaceae): demonstration of the utility of the 5S rDNA sequence as a tool for haplome identification.

A new genus Douglasdeweya containing the two species, Douglasdeweya deweyi and D. wangii was published in 2005 by Yen et al. based upon the results of cytogenetical and morphological findings. The genome constitution of Douglasdeweya-PPStSt-allowed its segregation from the genus Pseudoroegneria which contains the StSt or StStStSt genomes. Our previous work had demonstrated the utility of using 5S rDNA units, especially the non-transcribed spacer sequence variation, for the resolution of genomes (haplomes) previously established by cytology. Here, we show that sequence analysis of the 5S DNA units from these species strongly supports the proposed species relationships of Yen et al. (Can J Bot 83:413-419, 2005), i.e., the PP genome from Agropyron and the StSt genome from Pseudoroegneria. Analysis of the 5S rDNA units constitutes a powerful tool for genomic research especially in the Triticeae.

Molecular diversity of the 5S rRNA gene and genomic relationships in the genus Avena (Poaceae: Aveneae).

The molecular diversity of the rDNA sequences (5S rDNA units) in 71 accessions from 26 taxa of Avena was evaluated. The analyses, based on 553 sequenced clones, indicated that there were 6 unit classes, named according to the haplomes (genomes) they putatively represent, namely the long A1, long B1, long M1, short C1, short D1, and short M1 unit classes. The long and short M1 unit classes were found in the tetraploid A. macrostachya, the only perennial species. The long M1 unit class was closely related to the short C1 unit class, while the short M1 unit class was closely related to the long A1 and long B1 unit classes. However, the short D1 unit class was more divergent from the other unit classes. There was only one unit class per haplome in Avena, whereas haplomes in the Triticeae often have two. Most of the sequences captured belonged to the long A1 unit class. Sequences identified as the long B1 unit class were found in the tetraploids A. abyssinica and A. vaviloviana and the diploids A. atlantica and A. longiglumis. The short C1 unit class was found in the diploid species carrying the C genome, i.e., A. clauda, A. eriantha, and A. ventricosa, and also in the diploid A. longiglumis, the tetraploids A. insularis and A. maroccana, and all the hexaploid species. The short D1 unit class was found in all the hexaploid species and two clones of A. clauda. It is noteworthy that in previous studies the B genome was found only in tetraploid species and the D genome only in hexaploid species. Unexpectedly, we found that various diploid Avena species contained the B1 and D1 units. The long B1 unit class was found in 3 accessions of the diploid A. atlantica (CN25849, CN25864, and CN25887) collected in Morocco and in 2 accessions of A. longiglumis (CIav9087 and CIav9089) collected in Algeria and Libya, respectively, whereas only 1 clone of A. clauda (CN21378) had the short D1 unit. Thus there might be a clue as to where to search for diploids carrying the B and D genomes. Avena longiglumis was found to be the most diverse species, possibly harboring the A, B, and C haplomes. The long M1 and short M1 are the unit classes typical of A. macrostachya. These results could explain the roles of A. clauda, A. longiglumis, and A. atlantica in the evolution of the genus Avena. Furthermore, one clone of the tetraploid A. murphyi was found to have sequences belonging to the short D1 unit class, which could indicate that A. murphyi might have been the progenitor of hexaploid oats and not, as postulated earlier, A. insularis. The evolution of Avena did not follow the molecular clock. The path inferred is that the C genome is more ancient than the A and B genomes and closer to the genome of A. macrostachya, the only existing perennial, which is presumed to be the most ancestral species in the genus.

Molecular evolution of dimeric alpha-amylase inhibitor genes in wild emmer wheat and its ecological association.

BACKGROUND: alpha-Amylase inhibitors are attractive candidates for the control of seed weevils, as these insects are highly dependent on starch as an energy source. In this study, we aimed to reveal the structure and diversity of dimeric alpha-amylase inhibitor genes in wild emmer wheat from Israel and to elucidate the relationship between the emmer wheat genes and ecological factors using single nucleotide polymorphism (SNP) markers. Another objective of this study was to find out whether there were any correlations between SNPs in functional protein-coding genes and the environment. RESULTS: The influence of ecological factors on the genetic structure of dimeric alpha-amylase inhibitor genes was evaluated by specific SNP markers. A total of 244 dimeric alpha-amylase inhibitor genes were obtained from 13 accessions in 10 populations. Seventy-five polymorphic positions and 74 haplotypes were defined by sequence analysis. Sixteen out of the 75 SNP markers were designed to detect SNP variations in wild emmer wheat accessions from different populations in Israel. The proportion of polymorphic loci P (5%), the expected heterozygosity He, and Shannon's information index in the 16 populations were 0.887, 0.404, and 0.589, respectively. The populations of wild emmer wheat showed great diversity in gene loci both between and within populations. Based on the SNP marker data, the genetic distance of pair-wise comparisons of the 16 populations displayed a sharp genetic differentiation over long geographic distances. The values of P, He, and Shannon's information index were negatively correlated with three climatic moisture factors, whereas the same values were positively correlated by Spearman rank correlation coefficients' analysis with some of the other ecological factors. CONCLUSION: The populations of wild emmer wheat showed a wide range of diversity in dimeric alpha-amylase inhibitors, both between and within populations. We suggested that SNP markers are useful for the estimation of genetic diversity of functional genes in wild emmer wheat. These results show significant correlations between SNPs in the alpha-amylase inhibitor genes and ecological factors affecting diversity. Ecological factors, singly or in combination, explained a significant proportion of the variations in the SNPs, and the SNPs could be classified into several categories as ecogeographical predictors. It was suggested that the SNPs in the alpha-amylase inhibitor genes have been subjected to natural selection, and ecological factors had an important evolutionary influence on gene differentiation at specific loci.

The 5S DNA sequences in Hordeum bogdanii and in the H. brevisubulatum complex, and the evolution and the geographic dispersal of the diploid Hordeum species (Triticeae: Poaceae).

The molecular diversity of 5S rDNA from the closely related Asiatic diploid species, Hordeum bogdanii and the H. brevisubulatum complex has been catalogued and analysed. As in previous studies in Hordeum, we found that the sequences are constrained in such an manner that unit classes can be defined. The long H1 unit class, known to occur in all Eurasian species, was frequently found in these 2 taxa. In addition, we identified a new unit class, called the short H3 to reflect the H genome found in these 2 taxa. Although the 2 taxa are very close morphologically, the variation in the long H1 DNA units is constrained to such a great degree that, in many cases, the accessions in a unit class from a single species are clustered. In H. bogdanii, the majority of the sequences are grouped in this manner, whereas in the H. brevisubulatum complex, the tendency to be constrained is lower in some but not all subspecies. These results support keeping H. brevisubulatum ssp. violaceum and ssp. iranicum as 1 species with the long H1 and short H1 unit classes, while retaining ssp. nevskianum and ssp. turkestanicum in the H. brevisubulatum complex. We have summarized our work on the presence/absence of the 10 unit classes found in all diploid species of Hordeum. A phylogenetic analysis, based strictly on the presence/absence of unit classes, indicated clearly that all the South American diploids and all the North American diploids possess long H2 and long Y2 unit classes and, except for H. californicum and H. pusillum, which contain long H1 in addition to the long H2 and long Y2 classes, are devoid of the long H1 unit class. This suggests that the gene gain/loss process from a common ancestor has been concomitant with intercontinental dispersal between the Old and the New Worlds.

Ancient differentiation of the H and I haplomes in diploid Hordeum species based on 5S rDNA.

5S rDNA clones from 12 South American diploid Hordeum species containing the HH genome and 3 Eurasian diploid Hordeum species containing the II genome, including the cultivated barley Hordeum vulgare, were sequenced and their sequence diversity was analyzed. The 374 sequenced clones were assigned to "unit classes", which were further assigned to haplomes. Each haplome contained 2 unit classes. The naming of the unit classes reflected the haplomes, viz. both the long H1 and short I1 unit classes were identified with II genome diploids, and both the long H2 and long Y2 unit classes were recognized in South American HH genome diploids. Based upon an alignment of all sequences or alignments of representative sequences, we tested several evolutionary models, and then subjected the parameters of the models to a series of maximum likelihood (ML) analyses and various tests, including the molecular clock, and to a Bayesian evolutionary inference analysis using Markov chain Monte Carlo (MCMC). The best fitting model of nucleotide substitution was the HKY+G (Hasegawa, Kishino, Yano 1985 model with the Gamma distribution rates of nucleotide substitutions). Results from both ML and MCMC imply that the long H1 and short I unit classes found in the II genome diploids diverged from each other at the same rate as the long H2 and long Y2 unit classes found in the HH genome diploids. The divergence among the unit classes, estimated to be circa 7 million years, suggests that the genus Hordeum may be a paleopolyploid.

The utility of the nontranscribed spacer of 5S rDNA units grouped into unit classes assigned to haplomes - a test on cultivated wheat and wheat progenitors.

Data is presented on the evolutionary dynamics of non-transcribed spacers (NTSs) of 5S rRNA genes in some diploid and polyploid Triticum and Aegilops species. FISH experiments with probes representing different unit classes revealed presence and (or) absence of these sequences in genomes or separate chromosomes of the species. Among the three diploid species only Aegilops speltoides has all of the different unit classes in ribosomal clusters as detected by the probes. Triticum urartu does not have the long D1 signals and Aegilops tauschii does not have the long A1 signals. Both polyploids possess all types of sequences, but because of genome rearrangements after polyploidization there is significant repatterning of single different rDNA unit classes in chromosomal positions when compared with those in diploid progenitors. Additional refined work is needed to ascertain if the sequences in the polyploids are mixed or are located in mini clusters in close proximity to each other. Mantel tests for association between the presence of the FISH signals of the A, B, and D genomes together and separately with the unit class data of the material, i.e., the probes used in FISH, indicated that all signals were associated with their respective probe material, but that there was no association of the unit classes found and the signals to each haplome. All combinations of the partial Mantel tests, e.g., between the A and B haplomes while controlling the effect of the all probes signals, with correlations ranging from 0.48 to 0.79 were all significant. Principal coordinate analysis showed that the signals of most unit class specific probes were more or less equally distant except for the long (S1 and short G1 signals, which were not different, and that the short A1 signals were closely related to the former two, whereas the signals of the long G1 were even less related.

Sequence assessment of comigrating AFLPTM bands in Echinacea -- implications for comparative biological studies.

The extent of sequence identity among clones derived from monomorphic and polymorphic AFLPTM polymorphism bands was quantified. A total of 79 fragments from a monomorphic band of 273 bp and 48 fragments from a polymorphic band of 159 bp, isolated from individuals belonging to different populations, varieties, and species of Echinacea, were cloned and sequenced. The monomorphic fragments exhibited above 90% sequence identity among clones within samples. Sequence identity within variety ranged from 82.78% to 94.87% and within species from 75.82% to 98.9% and was 57.97% in the genus. The polymorphic fragments exhibited much less sequence identity. In some instances, even two clones from the same fragment were different in their size and sequence. Within sample, clone sequence identity ranged from 100% to 51.57%, within variety from 33.33% to 100% in one variety, and from 23.66% to 45% within species and was as low as 1.25% within the genus. In addition, sequences of the same size were aligned to verify the nature of their sequence dissimilarity/similarity. Within each size group, identical sequences were found across species and varieties. In general, comigrating bands cannot be considered homologous. Thus, the use of AFLPTM band data for comparative studies is appropriate only if the results emanating from such analyses are considered as approximations and are interpreted as phenotypic but not genotypic.

Phytochemistry of wild populations of Panax quinquefolius L. (North American ginseng).

A survey of the phytochemistry of Panax quinquefolius L. (North American ginseng) collected from wild populations in Ontario, Quebec, Maine, Vermont, and Wisconsin was undertaken. Reverse-phase HPLC was used to determine the natural variation of levels of ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd and their total in leaf, stem, and root of authentic wild-grown material. The totals in roots varied from 1 to 16%, with the greatest number of individual samples having 4-5% total ginsenosides. The lack of ginsenoside Rf in roots of authentic wild populations confirmed its status as a phytochemical marker differentiating American and Asian ginseng. Ten geographically isolated wild populations were collected, and several showed significant variation in the levels of major ginsenosides. There was no statistical difference in mean ginsenoside content between wild and cultivated P. quinquefolius roots at 4 years of age, suggesting there is no phytochemical justification for wild crafting. Baseline data on total ginsenoside levels for authentic wild P. quinquefolius reported here provide reference levels for quality assurance programs.

Phytochemical variation in echinacea from roots and flowerheads of wild and cultivated populations.

Quantitative phytochemical variation was determined from roots and inflorescences of native plant populations in the genus Echinacea. Specimens were collected in situ throughout the natural range of each putative taxon and transplanted to greenhouse cultivation. Ethanolic extracts from individual plants were separated by reversed-phase HPLC to quantify the alkamides, polyenes/ynes, and phenolics, and then grouped by age and taxonomically, according to a recent morphometric taxonomic revision of the genus. Canonical discriminant analysis revealed that cichoric acid, the diene alkamides 1-3 and 7, and ketoalkene 24 were the best taxonomic markers. Mean content for each of 26 phytochemicals revealed useful agronomic information, such as those varieties and organs with the highest accumulations, as well as the optimal age and growth conditions for each variety. The highest amounts of cichoric acid were measured from the older, wild inflorescences of E. pallida var.sanguinea, whereas the highest quantities of the alkamides 1-3 and 7 were present in roots of wild and transplanted E. purpurea. Baseline phytochemical data and chromatographic profiles for all types of wild Echinacea may be used for protection of wild stands, germplasm identification, and crop improvement.