The molecular basis of mutations at the Waxy locus from Amaranthus caudatus L.: evolution of the waxy phenotype in three species of grain amaranth

Polymorphisms at the Waxy locus of Amaranthus caudatus L. collected from a wide range of regions were used to investigate genetic diversity and mutation sites. A comparison of the Waxy locus revealed a very high level of sequence conservation. This result clearly showed low environmental and evolutionary variability in the Waxy gene. We also performed screening to confirm the mutation sites in the coding sequences of all accessions. The results indicate that one insertion in the coding region of Waxy genes was responsible for the change in perisperm starch leading to the waxy phenotype in all accessions of this species, and thus that a single mutation event altered the regulation of the Waxy gene during the domestication of this crop. In addition, phylogenetic analysis showed that waxy phenotypes within each of three species, A. caudatus, A. cruentus and A. hypochondriacus, originated separately or differentiated from nonwaxy phenotypes of each species through a single mutational event (i.e., a frame shift or base substitution). We also compared obvious structural features of the coding sequence of waxy and nonwaxy phenotypes with those of low-amylose phenotypes in A. caudatus. The Waxy coding sequences of low-amylose phenotypes do not show polymorphisms and are identical with those of waxy phenotypes. This could mean that there is another gene that encodes a key enzyme responsible for amylose synthesis as the elementary quantity in tissues other than perisperm in A. caudatus.     

Genomic and phenotypic evidence for an incomplete domestication of South American grain amaranth (Amaranthus caudatus)

The domestication syndrome comprises phenotypic changes that differentiate crops from their wild ancestors. We compared the genomic variation and phenotypic differentiation of the two putative domestication traits seed size and seed colour of the grain amaranth Amaranthus caudatus, which is an ancient crop of South America, and its two close wild relatives and putative ancestors A. hybridus and A. quitensis. Genotyping 119 accessions of the three species from the Andean region using genotyping by sequencing (GBS) resulted in 9485 SNPs that revealed a strong genetic differentiation of cultivated A. caudatus from its two relatives. A. quitensis and A. hybridus accessions did not cluster by their species assignment but formed mixed groups according to their geographic origin in Ecuador and Peru, respectively. A. caudatus had a higher genetic diversity than its close relatives and shared a high proportion of polymorphisms with their wild relatives consistent with the absence of a strong bottleneck or a high level of recent gene flow. Genome sizes and seed sizes were not significantly different between A. caudatus and its relatives, although a genetically distinct group of A. caudatus from Bolivia had significantly larger seeds. We conclude that despite a long history of human cultivation and selection for white grain colour, A. caudatus shows a weak genomic and phenotypic domestication syndrome and proposes that it is an incompletely domesticated crop species either because of weak selection or high levels of gene flow from its sympatric close undomesticated relatives that counteracted the fixation of key domestication traits.     

Identification and Cloning of Peptide Synthetase Genes of Thermostable Bacilli Using the Polymerase Chain Reaction

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genesdhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.     

Effects of light and kinetin on amaranthin synthesis induced by cAMP

The effects of cyclic 3′,5′-adenosine monophosphate (cAMP) on amaranthin synthesis in the dark, or in the presence of kinetin or light were investigated in isolated cotyledons of Amaranthus tricolor and A. caudatus. The results suggest that sites or modes of action of cAMP and kinetin are not separated and differ from those of light and that the nucleotide cannot be considered a messenger involved in amaranthin formation stimulated by kinetin or by light.     

Nucleotide sequence of psbC,the gene encoding the CP-43 chlorophyll a-binding protein of Photosystem II,in the cyanobacterium Synechocystis 6803

The nucleotide sequence for the Photosystem II gene psbC has been determined for the cyanobacterium Synechocystis 6803. The gene overlaps the last 50 bases of the psbD gene, and both genes are transcribed in the same direction, but read in different frames. This arrangement is identical to that found in all chloroplast genomes for which psbC has been sequenced. The Synechocystis nucleotide sequence is 70% homologous to the tobacco gene and the predicted amino acid sequence shows 85% homology. A possible alternative translation start site for psbC has been conserved between seven plant sequences and the cyanobacterial sequence. The hydropathy plot for the cyanobacterial protein is very similar to plots determined for six plant species. Pairs of histidines that may play a role in binding chlorophyll are conserved between the cyanobacterial and plant amino acid sequences.     

Identification of Single Nucleotide Polymorphisms and Analysis of Linkage Disequilibrium in Different Bamboo Species Using the Candidate Gene Approach

Bamboos are one of the most beautiful and useful plants on Earth. The genetic background and population structure of bamboos are well known, which helps accelerate the process of artificial domestication of bamboo. Partial sequences of six genes involved in nitrogen use efficiency in 32 different bamboo species were analyzed for occurrence of single nucleotide polymorphisms (SNPs). The nucleotide diversity θw and total nucleotide polymorphisms π_T of the sequenced DNA regions was 0.05137 and 0.03332, respectively. Both πnonsyn /πsyn and Ka/Ks values were <1. The nucleotide sequences of these six genes were inferred to be relatively conserved, and the haplotype diversity was relatively high. The results of evolutionary neutrality tests showed that the six genes were in line with neutral evolution, and that the NRT2.1 and AMT2.1 gene sequences may have experienced negative selection. An inter-SNP recombination event at the NRT2.1 gene in the all pooled sample, of all 32 bamboo species was the lowest at 0.0645, whereas the AMT gene recombination events were all >0.1. Estimation and analysis of linkage disequilibrium of five genes revealed that with the increase in nucleotide sequence length, the degree of SNP linkage disequilibrium decreased rapidly. We inferred the population genetic structure of 32 bamboo species based on the SNP loci of six genes with frequencies >18%. 32 bamboo species were divided into five categories, which indicated that the combined population of all bamboo species had obvious multivariate characteristics and was heterogeneous; red (Group 1) and green (Group 2) were the main groups.     

The Flow of Antimicrobial Peptide Genes Through a Genetic Barrier Between <Emphasis Type="Italic">Mytilus edulis</Emphasis> and <Emphasis Type="Italic">M. galloprovincialis</Emphasis>

We studied the population genetics of two antimicrobial peptide (AMP) loci, called Mytilin B and Mytilus galloprovincialis defensin 2 (MGD2), in the secondary contact mosaic hybrid zone between Mytilus edulis and M. galloprovincialis. The isolation period between the two species was estimated to be ~1 million years (range, 0.5 million to 2 million years) long. During this period, coevolution between microbes and the immune system has likely occurred. The secondary contact, which would date back to ~25,000 (0–200,000) years, recently allowed these coadaptations to be rearranged through hybridization. Distinctive polymorphisms were uncovered in coding sequences of the two AMP loci such as insertion/deletion of codons or bisubstituted codons. Very low levels of differentiation were observed between populations of the two species at both loci, while other nuclear loci often showed marked structure among the same samples. The absence of population differentiation proved to be the consequence of secondary introgression of highly divergent alleles. While only a few recombinants were observed at the Mytilin B locus, the MGD2 locus showed a high intragenic recombination rate, which increased in the exon coding for the mature peptide. In addition, standard neutrality tests revealed significant deviations from the mutation-drift equilibrium at both loci. These results suggest that either balancing or directional selection is likely to play a role in the evolution of the two AMPs and introgression would be adaptive. However, evidence accumulated at the Mytilin B locus allows neither for identification of the direction of selection nor for any conclusions on whether selection acted directly on the antimicrobial peptide itself. At the MGD2 locus, a spatial variation of polymorphism patterns along the sequence suggests that selection was direct, although the precise nature of the selection (directional vs. balancing) remains unclear. This study concurs with previous reports of an effect of slight selection on AMP genes evolution in other invertebrates, although selection does not necessarily act on the mature peptides. E. Boon and M. F. Faure contributed equally to this work.     

The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum, a facultative CAM plant

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 by to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 by upstream of the ATG initiation codon to 9 by downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.     

A putative antimicrobial peptide from Hymenoptera in the megaplasmid pSCL4 of Streptomyces clavuligerus ATCC 27064 reveals a singular case of horizontal gene transfer with potential applications

Streptomyces clavuligerus is a Gram‐positive bacterium that is a high producer of secondary metabolites with industrial applications. The production of antibiotics such as clavulanic acid or cephamycin has been extensively studied in this species; nevertheless, other aspects, such as evolution or ecology, have received less attention. Furthermore, genes that arise from ancient events of lateral transfer have been demonstrated to be implicated in important functions of host species. This approximation discovered relevant genes that genomic analyses overlooked. Thus, we studied the impact of horizontal gene transfer in the S. clavuligerus genome. To perform this task, we applied whole‐genome analysis to identify a laterally transferred sequence from different domains. The most relevant result was a putative antimicrobial peptide (AMP) with a clear origin in the Hymenoptera order of insects. Next, we determined that two copies of these genes were present in the megaplasmid pSCL4 but absent in the S. clavuligerus ATCC 27064 chromosome. Additionally, we found that these sequences were exclusive to the ATCC 27064 strain (and so were not present in any other bacteria) and we also verified the expression of the genes using RNAseq data. Next, we used several AMP predictors to validate the original annotation extracted from Hymenoptera sequences and explored the possibility that these proteins had post‐translational modifications using peptidase cleavage prediction. We suggest that Hymenoptera AMP‐like proteins of S. clavuligerus ATCC 27064 may be useful for both species adaptation and as an antimicrobial molecule with industrial applications.     

Trichoplaxin — A new membrane-active antimicrobial peptide from placozoan cDNA

A method based on the use of signal peptide sequences from antimicrobial peptide (AMP) precursors was used to mine a placozoa expressed sequence tag database and identified a potential antimicrobial peptide from Trichoplax adhaerens. This peptide, with predicted sequence FFGRLKSVWSAVKHGWKAAKSR is the first AMP from a placozoan species, and was named trichoplaxin. It was chemically synthesized and its structural properties, biological activities and membrane selectivity were investigated. It adopts an α-helical structure in contact with membrane-like environments and is active against both Gram-negative and Gram-positive bacterial species (including MRSA), as well as yeasts from the Candida genus. The cytotoxic activity, as assessed by the haemolytic activity against rat erythrocytes, U937 cell permeabilization to propidium iodide and MCF7 cell mitochondrial activity, is significantly lower than the antimicrobial activity. In tests with membrane models, trichoplaxin shows high affinity for anionic prokaryote-like membranes with good fit in kinetic studies. Conversely, there is a low affinity for neutral eukaryote-like membranes and absence of a dose dependent response. With high selectivity for bacterial cells and no homologous sequence in the UniProt, trichoplaxin is a new potential lead compound for development of broad-spectrum antibacterial drugs.     

Antimicrobial peptides fromMirabilis jalapa andAmaranthus caudatus: expression, processing, localization and biological activity in transgenic tobacco

The cDNAs encoding the seed antimicrobial peptides (AMPs) fromMirabilis jalapa (Mj-AMP2) andAmaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolleet al., Plant Mol Biol 28:713–721 (1995) and 22:1187–1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195–1206 (1991)]; an Ac-AMP2wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing eitherwild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. Thein vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with eitherBotrytis einerea orAlternaria longipes.     

Looking into flowering time in almond (Prunus dulcis (Mill) D. A. Webb): the candidate gene approach

Blooming time is one of the most important agronomic traits in almond. Biochemical and molecular events underlying flowering regulation must be understood before methods to stimulate late flowering can be developed. Attempts to elucidate the genetic control of this process have led to the identification of a major gene (Lb) and quantitative trait loci (QTLs) linked to observed phenotypic differences, but although this gene and these QTLs have been placed on the Prunus reference genetic map, their sequences and specific functions remain unknown. The aim of our investigation was to associate these loci with known genes using a candidate gene approach. Two almond cDNAs and eight Prunus expressed sequence tags were selected as candidate genes (CGs) since their sequences were highly identical to those of flowering regulatory genes characterized in other species. The CGs were amplified from both parental lines of the mapping population using specific primers. Sequence comparison revealed DNA polymorphisms between the parental lines, mainly of the single nucleotide type. Polymorphisms were used to develop co-dominant cleaved amplified polymorphic sequence markers or length polymorphisms based on insertion/deletion events for mapping the candidate genes on the Prunus reference map. Ten candidate genes were assigned to six linkage groups in the Prunus genome. The positions of two of these were compatible with the regions where two QTLs for blooming time were detected. One additional candidate was localized close to the position of the Evergrowing gene, which determines a non-deciduous behaviour in peach.     

A Fragment of the rpoC2 Chloroplast RNA Polymerase Gene in Perennial and Annual Rye

A 1397-bp fragment corresponding to the rpoC2 chloroplast RNA polymerase gene was obtained by direct rye DNA amplification. Two rye species, Secale montanum Guss. and S. cereale L., did not practically differ in the structure of this DNA fragment (the nucleotide sequences were 99% identical). The corresponding nucleotide sequences in rye and wheat (Triticum aestivum L., Genbank accession no. AB027572) were 97–98% similar. The extent of the homology of various stretches of the rpoC2 rye gene with the corresponding sequences in maize and rice was 81–95%, whereas the deduced amino acid sequences of rpoC2 in rye, wheat, maize, and rice were considerably identical (96–97% of homology). The rye fragment of the rpoC2 gene differed from the corresponding sequences in three other grass species primarily by a short (49 bp) insert into the region of numerous short repeats corresponding to nucleotides 15750/15751, 28728/28729, and 27472/27473 in wheat, maize, and rice, respectively.     

Hipposin,a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)

A novel 51-residue antimicrobial peptide (AMP) from the skin mucus of Atlantic halibut (Hippoglossus hippoglossus L.) was isolated using acid extraction, and cationic exchange and reversed phase chromatography. The complete amino acid sequence of the AMP, termed hipposin, was determined by automated Edman degradation and mass spectrometry to be SGRGKTGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAHRVGAGAPVYL. The N-terminal amino group was acetylated. The theoretical mass of hipposin was calculated to be 5458.4 Da, which was in good agreement with the mass of 5459 Da determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Hipposin was shown to be derived from histone H2A by PCR amplifying the encoding sequences from Atlantic halibut genomic DNA. The peptide showed sequence similarity with the 39-mer AMP buforin I of Asian toad and the 19-mer AMP parasin I of catfish. Fifty of the fifty-one residues in hipposin were identical to the N-terminal region of histone H2A from rainbow trout. Hipposin showed strong antimicrobial activity against several Gram-positive and Gram-negative bacteria and activity could be detected down to hipposin concentrations of 0.3 microM (1.6 microg/ml). Hipposin without N-terminal acetylation was prepared by solid-phase peptide synthesis and shown to have the same antimicrobial activity as the natural acetylated peptide. Thus, hipposin is a new broad-spectrum histone-derived AMP found in the skin mucus of Atlantic halibut.     

Transformed Potato Plants Carrying the Gene of the Antifungal Peptide of Amaranthus caudatus

Potato plants (Solanum tuberosumL., var. Desire) were transformed with a pH22Kneo vector harboring the gene ac2, which encodes the fungicidal peptide (defensin) from the seed of amaranth (Amaranthus caudatusL.). The transformation involved cocultivation on a solid MS medium of potato stem explants (excised from aseptically grown plants) andAgrobacterium tumefaciens. The factors affecting in vitroregeneration of the explants and the transformation efficiency were optimized. Regenerated potato plants carrying the amaranth defensin gene were selected by two traits, growth and ability to form roots on a kanamycin-supplemented MS medium. The transgenic state was confirmed by PCR analysis of ac2in tissues of the kanamycin-resistant plants. The transgenic organisms thus obtained differed from the original plants in their patterns of Ambiol-induced growth and proton translocation across the plasma membrane of the tuber cells.     

Sequencing primers and SNPs for rapidly evolving reproductive loci in endangered ibex and their kin (Bovidae,Capra spp.)

Rapidly evolving genes (e.g. candidate selected loci) are of increasing interest to molecular ecologists and conservation geneticists. Here, we report primers for five regions from three independent nuclear reproductive genes that reliably generate polymorphic sequences across the widespread wild goats of the Capra ibex species group and likely many other species of bovids. From three to nine single‐nucleotide polymorphisms (SNPs) were identified in each gene region among C. ibex subspecies. Average numbers of SNPs per 1000 bp across all five gene regions was 15.0, with a high of 25.3 in the ZP3 exons 3 and 4 sequence and a low of 6.1 in the TNP1 sequence.     

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti)

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB088371–AB088374, AB089199, AB154393–AB154399, and AB162144.     

Molecular diversity of Amansieae (Ceramiales,Rhodophyta) from the Hawaiian Islands: a multi‐marker assessment reveals high diversity within Amansia glomerata

Sixty‐one Hawaiian algal specimens corresponding to members of the tribe Amansieae (Amansia and Osmundaria) were compared through DNA sequence analysis. Short DNA barcode‐like sequences of mitochondrial cytochrome c oxidase subunit I (COI) and universal plastid amplicon (UPA) markers were obtained for as many of the specimens as possible, and a subset of specimens was also used for amplification and sequencing of the nuclear small‐subunit rRNA (SSU) gene for phylogenetic inference in a broader taxonomic context. Statistical parsimony analysis of the COI and UPA markers for A. glomerata produced relationships among the samples that were largely congruent with each other, although the UPA marker was more conserved. The COI marker yielded three lineages, and nucleotide divergences for these three lineages were intermediate to those typically reported for intraspecific and interspecific comparisons, suggesting that they represent either incipient species or a complex of closely related species. The COI and UPA sequences demonstrated little to no divergence for Osmundaria obtusiloba and the taxon referred to as Amansia fimbrifolia. In contrast, specimens identified as A. daemelii were identical in sequence to lineage 3 sequences of A. glomerata, and it is recommended that this taxon no longer be included in species lists for the Hawaiian flora. Phylogenetic reconstruction based on the SSU gene was largely unresolved, indicating that this marker may be of limited utility for this purpose in this group of algae, but a small amount of nucleotide variation was found for samples of A. glomerata.     

Tetrahymena australis (Protozoa,Ciliophora): A Well‐Known But “Non‐Existing” Taxon – Consideration of Its Identification,Definition and Systematic Position

A cryptic species of the Tetrahymena pyriformis complex, Tetrahymena australis, has been known for a long time but never properly diagnosed based on taxonomic methods. The species name is thus invalid according to the International Code of Zoological Nomenclature. Recently, a population isolated from a freshwater lake in Wuhan, China was investigated using live observations, silver staining methods and gene sequence data. This organism can be separated from other described species of the T. pyriformis complex by its relatively small body size, the number of somatic kineties and differences in sequences of two genes, namely the small subunit ribosomal RNA (SSU rRNA) and the mitochondrial cytochrome c oxidase subunit I (cox1). We compared the SSU rRNA gene sequences of all available Tetrahymena species to reveal the nucleotide differences within this genus. The sequence of the Wuhan population is identical to two sequences of a previously isolated strain of T. australis (ATCC #30831). Phylogenetic analyses indicate that these three sequences (X56167, M98015, KT334373) cluster with Tetrahymena shanghaiensis (EF070256) in a polytomy. However, sequence divergence of the cox1 gene between the Wuhan population and another strain of T. australis (ATCC #30271) is 1.4%, suggesting that these may represent different subspecies.