Diversity of Micromonospora strains isolated from nitrogen fixing nodules and rhizosphere of Pisum sativum analyzed by multilocus sequence analysis

It was recently reported that Micromonospora inhabits the intracellular tissues of nitrogen fixing nodules of the wild legume Lupinus angustifolius. To determine if Micromonospora populations are also present in nitrogen fixing nodules of cultivated legumes such as Pisum sativum, we carried out the isolation of this actinobacterium from P. sativum plants collected in two man-managed fields in the region of Castilla and León (Spain). In this work, we describe the isolation of 93 Micromonospora strains recovered from nitrogen fixing nodules and the rhizosphere of P. sativum. The genomic diversity of the strains was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Forty-six isolates and 34 reference strains were further analyzed using a multilocus sequence analysis scheme developed to address the phylogeny of the genus Micromonospora and to evaluate the species distribution in the two studied habitats. The MLSA results were evaluated by DNA-DNA hybridization to determine their usefulness for the delineation of Micromonospora at the species level. In most cases, DDH values below 70% were obtained with strains that shared a sequence similarity of 98.5% or less. Thus, MLSA studies clearly supported the established taxonomy of the genus Micromonospora and indicated that genomic species could be delineated as groups of strains that share > 98.5% sequence similarity based on the 5 genes selected. The species diversity of the strains isolated from both the rhizosphere and nodules was very high and in many cases the new strains could not be related to any of the currently described species.     

Conditional allelic replacement applied to genes encoding the histone variant H3.3 in the mouse

Post‐translational modifications to residues in core histones convey epigenetic information. Their function can be evaluated in amino acid substitution mutants, although to date this method has not been used in mice. To this end, we have evaluated gene targeting vectors designed for Cre recombinase‐mediated conditional allelic replacement at the two unlinked genes encoding the histone variant H3.3. The conditional alleles consist of an uninterrupted wild‐type H3.3 coding sequence upstream of a desired alternative or proxy coding sequence. The arrangement of two loxP sites allows Cre‐mediated replacement of the wild‐type coding sequence with the proxy. To demonstrate proof of principle, at each locus we replaced the wild‐type coding sequence with a fluorescent reporter. This produced null alleles that will be useful to analyse the effects of H3.3 deficiency in development. Each targeting vector can readily be retrofitted with a proxy coding sequence encoding a modified H3.3 protein. Such vectors will allow for the conditional substitution of specific residues in order to dissect the roles of H3.3 post‐translational modifications in development and disease. genesis, 51:142?146, 2013. © 2013 Wiley Periodicals, Inc.     

Genetic diversity and population structure in the tomato-like nightshades Solanum lycopersicoides and S. sitiens

Background and Aims

Two closely related, wild tomato-like nightshade species, Solanum lycopersicoides and Solanum sitiens, inhabit a small area within the Atacama Desert region of Peru and Chile. Each species possesses unique traits, including abiotic and biotic stress tolerances, and can be hybridized with cultivated tomato. Conservation and utilization of these tomato relatives would benefit from an understanding of genetic diversity and relationships within and between populations.

Methods

Levels of genetic diversity and population genetic structure were investigated by genotyping representative accessions of each species with a set of simple sequence repeat (SSR) and allozyme markers.

Key Results

As expected for self-incompatible species, populations of S. lycopersicoides and S. sitiens were relatively diverse, but contained less diversity than the wild tomato Solanum chilense, a related allogamous species native to this region. Populations of S. lycopersicoides were slightly more diverse than populations of S. sitiens according to SSRs, but the opposite trend was found with allozymes. A higher coefficient of inbreeding was noted in S. sitiens. A pattern of isolation by distance was evident in both species, consistent with the highly fragmented nature of the populations in situ. The populations of each taxon showed strong geographical structure, with evidence for three major groups, corresponding to the northern, central and southern elements of their respective distributions.

Conclusions

This information should be useful for optimizing regeneration strategies, for sampling of the populations for genes of interest, and for guiding future in situ conservation efforts.     

Phylogeny and molecular evolution of the Acc1 gene within the StH genome species in Triticeae (Poaceae)

To estimate the phylogeny and molecular evolution of a single-copy gene encoding plastid acetyl-CoA carboxylase (Acc1) within the StH genome species, two Acc1 homoeologous sequences were isolated from nearly all the sampled StH genome species and were analyzed with those from 35 diploid taxa representing 19 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1) the StH genome species from the same areas or neighboring geographic regions are closely related to each other; (2) the Acc1 gene sequences of the StH genome species from North America and Eurasia are evolutionarily distinct; (3) Dasypyrum has contributed to the nuclear genome of Elymus repens and Elymus mutabilis; (4) the StH genome polyploids have higher levels of sequence diversity in the H genome homoeolog than the St genome homoeolog; and (5) the Acc1 sequence may evolve faster in the polyploid species than in the diploids. Our result provides some insight on evolutionary dynamics of duplicate Acc1 gene, the polyploidy speciation and phylogeny of the StH genome species.     

Genetic diversity and structure of the zombi pea (<Emphasis Type="Italic">Vigna vexillata</Emphasis> (L.) A. Rich) gene pool based on SSR marker analysis

Zombi pea (Vigna vexillata (L.) A. Rich) is an underutilized legume species and a useful gene source for resistance to biotic and abiotic stresses, although there is little understanding on its genetic diversity and structure. In this study, 422 (408 wild and 14 cultivated) accessions of zombi pea from diverse origins (201 from Africa, 126 from America, 85 from Australia, 5 from Asia and 5 from unknown origin) were analyzed with 20 simple sequence repeat (SSR) markers to determine its genetic diversity and genetic structure. The SSR markers detected 273 alleles in total with a mean of 13.6 alleles per locus. Polymorphism information content values of the markers varied from 0.58 to 0.90 with an average of 0.76. Overall gene diversity was 0.715. Gene diversity and average allelic richness was highest in Africa (0.749 and 8.08, respectively) and lowest in America (0.435 and 4.10, respectively). Nei’s genetic distance analysis revealed that the highest distance was between wild Australia and cultivated Africa (0.559), followed by wild West Africa and wild Australia (0.415). STRUCTURE, neighbor-joining (NJ), and principal coordinate analyses consistently showed that these zombi pea accessions were clustered into three major groups, viz. America, Africa and Asia, and Australia. NJ tree also suggested that American and Australian accessions are originated from East African zombi peas, and that the cultivated accessions from Africa and Asia were genetically distinct, while those from America were clustered with some cultivated accessions from Africa. These results suggest that Africa is the center of origin and diversity of zombi pea, and that domestication of this pea took place more than once in different regions.     

The complete mitochondrial genome of Microtus fortis calamorum (Arvicolinae, Rodentia) and its phylogenetic analysis

Microtus fortis is a special resource of rodent in China. It is a promising experimental animal model for the study on the mechanism of Schistosome japonicum resistance. The first complete mitochondrial genome sequence for Microtus fortis calamorum, a subspecies of M. fortis (Arvicolinae, Rodentia), was reported in this study. The mitochondrial genome sequence of M. f. calamorum (Genbank: JF261175) showed a typical vertebrate pattern with 13 protein coding genes, 2 ribosomal RNAs, 22 transfer RNAs and one major noncoding region (CR region).The extended termination associated sequences (ETAS-1 and ETAS-2) and conserved sequence block 1 (CSB-1) were found in the CR region. The putative origin of replication for the light strand (O(L)) of M. f. calamorum was 35bp long and showed high conservation in stem and adjacent sequences, but the difference existed in the loop region among three species of genus Microtus. In order to investigate the phylogenetic position of M. f. calamorum, the phylogenetic trees (Maximum likelihood and Bayesian methods) were constructed based on 12 protein-coding genes (except for ND6 gene) on H strand from 16 rodent species. M. f. calamorum was classified into genus Microtus, Arvcicolinae for the highly phylogenetic relationship with Microtus kikuchii (Taiwan vole). Further phylogenetic analysis results based on the cytochrome b gene ranged from M. f. calamorum to one of the subspecies of M. fortis, which formed a sister group of Microtus middendorfii in the genus Microtus.     

Patterns of plastid and nuclear variation among apomictic polyploids of Hieracium: evolutionary processes and taxonomic implications

Background and Aims

Apomictic species (with asexual seed production) make up for 20–50 % of all taxonomically recognized species in northern Europe, but the phylogenetic relationships of apomictic species and the mode of evolution and speciation remain largely unknown and their taxonomy is consequently disputed.

Methods

In the present study, plastid psbD-trnT sequences (349 accessions) and 12 nuclear microsatellite loci (478 accessions) were used to create an overview of the molecular variation in (mainly) northern European members of the most species-rich of all plant genera, Hieracium s.s. The results are discussed and interpreted in the context of morphological and cytological data on the same species.

Key Results and Conclusions

The complete psbD-trnT alignment was 1243 bp and 50 polymorphisms defined 40 haplotypes. All haplotypes found in the sections of the genus distributed in the northern European lowlands fell into one of two main groups, group H and group V, mutually separated by seven or eight polymorphisms. All accessions belonging to H. sects. Foliosa, Hieracioides (viz. H. umbellatum) and Tridentata and all but one accession of triploid species of H. sects. Oradea and Vulgata showed haplotypes of group V. Haplotypes of group H were found in all accessions of H. sects. Bifida and Hieracium and in all tetraploid representatives of H. sects. Oreadea and Vulgata. Additional haplotypes were found in accessions of the genus Pilosella and in southern European and Alpine sections of Hieracium. In contrast, the distribution of individual haplotypes in the two major groups appeared uncorrelated with morphology and current taxonomy, but polymorphisms within species were only rarely encountered. In total, 160 microsatellite alleles were identified. Levels of variation were generally high with only nine pairs of accessions being identical at all loci (in all cases representing accessions of the same species). In the neighbor-joining analysis based on the microsatellite data, accessions of the same species generally clustered together and some smaller groups of species congruent with morphology and/or current taxonomy were recovered but, except for H. sect. Oreadea, most larger groups were not correlated with morphology. Although the plastid DNA sequences show too little variation and the nuclear microsatellites are too variable to resolve relationships successfully among species or to fully understand processes of evolution, it is concluded that both species and sections as defined by morphology are largely congruent with the molecular data, that gene flow between the sections is rare or non-existent and that the tetraploid species may constitute the key to understanding evolution and speciation in this genus.     

Transcriptional control of hydrogen production during mixed carbon fermentation by hydrogenases 4 (hyf) and 3 (hyc) in Escherichia coli

Escherichia coli molecular hydrogen (H(2)) production was studied during mixed carbon (glucose and glycerol) fermentation at pH 6.5. Wild type cells in the assays supplemented with glucose produced H(2) at ~2 fold lower level than cells grown on glucose only. When compared to the wild type, H(2) production in the assays added with glucose was decreased by ~2 fold in fhlA, hyfG and double fhlA hyfG mutants and by ~1.5 fold in hyaB, hybC, and double hyaB hybC mutants. However, in the assays with glycerol, no measurable H(2) production was detected. Taken together, these results suggest that during mixed carbon fermentation, H(2) could be produced with low efficiency via Hyd-3 and Hyd-4. This is a novel finding for Hyd-4 activity at pH 6.5. The insignificant decrease of H(2) production in the strains with defects in Hyd-1 and Hyd-2 was probably due to an interaction between the Hyd enzymes and their organization in the bacterial membrane. In the glucose assays, H(2) production in the wild type cells was inhibited ~2 fold by 0.3mM N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F(0)F(1)-ATPase. This inhibition was the same for fhlA and hyfG fhlA mutants but not hyaB, hybC, hyfG or hyaB hybC mutants. The results indicate that the FhlA protein coded by the fhlA gene might interact with the F(0)F(1)-ATPase. We propose that this interaction is mediated by mixed carbon fermentation.     

Mitogenomic analysis for coelacanths (Latimeria chalumnae) caught in Tanzania

In recent years, a large number of individuals of the species Latimeria chalumnae, one of the living fossil coelacanths, have been landed off the coast of Tanzania. Although L. chalumnae specimens have also been landed at other localities in the western Indian Ocean, so far, viable populations of this species have been identified only at two localities, Comoros and South Africa. Therefore, the recent active catch off Tanzania suggests a new habitat for L. chalumnae. To examine the genetic background of the Tanzanian fish, we analyzed complete mtDNA sequences of two Tanzanian individuals (Kigombe-9 and Songo Mnara-1) collected from the north and south coasts of Tanzania. Using the recently reported criteria for six haplotypes established in a population genetic study for coelacanths living in the western Indian Ocean [Schartl, M., Hornung, U., Hissman, K., Schauer, J., Fricke, H., 2005. Relatedness among east African coelacanths. Nature 435, 901.], we characterized Songo Mnara-1 as haplotype 1 and Kigombe-9 as haplotype 5. We suggest that the Songo Mnara specimen is a member of the Comoran group, but was swept away by the South Equatorial current. The individual from Kigombe may be a member of an undiscovered population that exists near the boundary between Tanzania and Kenya. Further analysis using more than 19 individuals recently captured off the north coast of Tanzania will reveal whether a new population exists there. Our sequence data suggest additional variable sites in the mtDNA sequence that may define the population structure of coelacanths in the western Indian Ocean and also raise the possibility that the previously published Comoran coelacanth mtDNA sequence contains several critical errors including base changes and indels.     

Multiple origins of the determinate growth habit in domesticated common bean (Phaseolus vulgaris)

Background and Aims

The actual number of domestications of a crop is one of the key questions in domestication studies. Answers to this question have generally been based on relationships between wild progenitors and domesticated descendants determined with anonymous molecular markers. In this study, this question was investigated by determining the number of instances a domestication phenotype had been selected in a crop species. One of the traits that appeared during domestication of common bean (Phaseolus vulgaris) is determinacy, in which stems end with a terminal inflorescence. It has been shown earlier that a homologue of the arabidopsis TFL1 gene – PvTFL1y – controls determinacy in a naturally occurring variation of common bean.

Methods

Sequence variation was analysed for PvTFL1y in a sample of 46 wild and domesticated accessions that included determinate and indeterminate accessions.

Key Results

Indeterminate types – wild and domesticated – showed only synonymous nucleotide substitutions. Determinate types – observed only among domesticated accessions – showed, in addition to synonymous substitutions, non-synonymous substitutions, indels, a putative intron-splicing failure, a retrotransposon insertion and a deletion of the entire locus. The retrotransposon insertion was observed in 70 % of determinate cultivars, in the Americas and elsewhere. Other determinate mutants had a more restricted distribution in the Americas only, either in the Andean or in the Mesoamerican gene pool of common bean.

Conclusions

Although each of the determinacy haplotypes probably does not represent distinct domestication events, they are consistent with the multiple (seven) domestication pattern in the genus Phaseolus. The predominance of determinacy in the Andean gene pool may reflect domestication of common bean prior to maize introduction in the Andes.     

Experimental growing of wild pea in Israel and its bearing on Near Eastern plant domestication

Background and Aims

The wild progenitors of the Near Eastern legumes have low germination rates mediated by hardseededness. Hence it was argued that cultivation of these wild legumes would probably result in no yield gain. Based on the meagre natural yield of wild lentil and its poor germination, it was suggested that wild Near Eastern grain legumes were unlikely to have been adopted for cultivation unless freely germinating types were available for the incipient farmers. Unlike wild cereals, data from experimental cultivation of wild legumes are lacking.

Methods

Replicated nurseries of wild pea (Pisum elatius, P. humile and P. fulvum) were sown during 2007–2010 in the Mediterranean district of Israel. To assess the effect of hardseededness on the yield potential, seeds of the wild species were either subjected to scarification (to ensure germination) or left intact, and compared with domesticated controls.

Key Results

Sowing intact wild pea seeds mostly resulted in net yield loss due to poor establishment caused by wild-type low germination rates, while ensuring crop establishment by scarification resulted in net, although modest, yield gain, despite considerable losses due to pod dehiscence. Harvest efficiency of the wild pea plots was significantly higher (2–5 kg seeds h⁻¹) compared with foraging efficiency in wild pea populations (ranging from a few grams to 0·6 kg h⁻¹).

Conclusions

Germination and yield data from ‘cultivation’ of wild pea suggest that Near Eastern legumes are unlikely to have been domesticated via a protracted process. Put differently, the agronomic implications of the hardseededness of wild legumes are incompatible with a millennia-long scenario of unconscious selection processes leading to ‘full’ domestication. This is because net yield loss in cultivation attempts is most likely to have resulted in abandonment of the respective species within a short time frame, rather than perpetual unprofitable cultivation for several centuries or millennia.     

Genetic investigation of the origination of allopolyploid with virtually synthesized lines: application to the C subgenome of Brassica napus

Although there are a number of different allopolyploids in the plant kingdom, the exact ancestral parents of some allopolyploids have not been well characterized. We propose a strategy in which virtual allopolyploid lines derived from different types of parental species are used to investigate the progenitors of an allopolyploid. The genotypes of the parental lines and the natural allopolyploid were established using a set of DNA molecular markers. The genotypes of the virtual lines were then derived from those of the parental lines, and compared extensively with that of the natural allopolyploid. We applied this strategy to investigate the progenitors of the C subgenome of Brassica napus (rapeseed, AACC). A total of 39 accessions from 10 wild and 7 cultivated types of the B. oleracea cytodeme (CC), and 4 accessions of B. rapa (AA) were used to construct 156 virtual rapeseed lines. Genetic structure was compared among natural rapeseed, virtual rapeseed lines, and their parental lines by principal component analysis and analysis of ancestry. Our data showed that the C subgenome of natural rapeseed was related closely to the genome of cultivated B. oleracea and its related wild types, such as B. incana, B. bourgeaui, B. montana, B. oleracea ssp. oleracea and B. cretica. This finding indicated that these types or their progeny might be ancestral donors of the C subgenome of rapeseed. The successful application of the strategy of virtual allopolyploidy in rapeseed demonstrates that it can possibly be used to identify the progenitors of an allopolyploid species.     

The phylogeny of Ephemeroptera in Pterygota revealed by the mitochondrial genome of Siphluriscus chinensis (Hexapoda: Insecta)

The mayfly species Siphluriscus chinensis (Siphluriscidae) has valuable structures useful for phylogeny reconstruction, given its putative basal position within the Ephemeroptera. Here its nearly complete mitochondrial genome is sequenced. We built phylogenetic trees through multiple analytical strategies with some other insect mitogenomes. Structurally, the obtained mitochondrial genome of S. chinensis is 16,616 bp in length, ¹ containing 37 genes and an extra trnK-like (trnK2 (AAA)) gene. The 12 PCGs start with typical ATN codons, except the nad1 gene which starts with an unnormalized TTG. Like other known mayfly mitogenomes, the strand bias has negative AT-skew and negative GC-skew. Phylogenetically, our topologies suggest that Odonata is the basally diverged clade in Pterygota; Ephemeroptera is the sister group of the Neoptera; and S. chinensis is indeed the most basal mayfly branch.     

Development and Application of Microsatellites in Candidate Genes Related to Wood Properties in the Chinese White Poplar (Populus tomentosa Carr.)

Gene-derived simple sequence repeats (genic SSRs), also known as functional markers, are often preferred over random genomic markers because they represent variation in gene coding and/or regulatory regions. We characterized 544 genic SSR loci derived from 138 candidate genes involved in wood formation, distributed throughout the genome of Populus tomentosa, a key ecological and cultivated wood production species. Of these SSRs, three-quarters were located in the promoter or intron regions, and dinucleotide (59.7%) and trinucleotide repeat motifs (26.5%) predominated. By screening 15 wild P. tomentosa ecotypes, we identified 188 polymorphic genic SSRs with 861 alleles, 2–7 alleles for each marker. Transferability analysis of 30 random genic SSRs, testing whether these SSRs work in 26 genotypes of five genus Populus sections (outgroup, Salix matsudana), showed that 72% of the SSRs could be amplified in Turanga and 100% could be amplified in Leuce. Based on genotyping of these 26 genotypes, a neighbour-joining analysis showed the expected six phylogenetic groupings. In silico analysis of SSR variation in 220 sequences that are homologous between P. tomentosa and Populus trichocarpa suggested that genic SSR variations between relatives were predominantly affected by repeat motif variations or flanking sequence mutations. Inheritance tests and single-marker associations demonstrated the power of genic SSRs in family-based linkage mapping and candidate gene-based association studies, as well as marker-assisted selection and comparative genomic studies of P. tomentosa and related species.     

Population genetic structure and classification of cultivated and wild pea (Pisum sp.) based on morphological traits and SSR markers

Pea (Pisum sativum L.) is an important legume crop that is widely grown worldwide for human consumption and livestock feed. Despite extensive studies, the population genetic structure and classification of cultivated and wild pea (Pisum sp.) remain controversial. To characterize patterns of genetic and morphological variation and investigate the classification of Pisum, we conducted comprehensive population genetic analyses for 323 accessions from cultivated and wild pea, representing three species of Pisum and utilizing 34 morphological traits and 87 polymorphic simple sequence repeat markers. First, we identified three distinct genetic groups among all samples. Group I was primarily composed of Pisum fulvum, Pisum abyssinicum, and some wild P. sativum accessions, whereas Groups II and III consisted of the two genetic groups under P. sativum that represented different geographic distributions of cultivated pea. Analyses of morphological variation revealed significant differences among the three species. Second, among pea germplasms representing eight taxa of Pisum, P. fulvum and P. abyssinicum possessed unique genetic backgrounds and morphological characteristics, corroborating their independent species status. The intraspecific subdivisions of P. sativum described by some authors were not supported in this study, with the exception of several genotypes of P. sativum subsp. elatius that clustered with P. fulvum and P. abyssinicum. Finally, we confirmed that the Chinese pea germplasm was genetically distinct and could be divided into two genetic groups, each of which included both spring-sowing and autumn-sowing ecotypes. These results provide a robust foundation for understanding pea domestication and the utilization of wild genetic resources of pea.     

Phylogenetic relationships of the Cobitoidea (Teleostei: Cypriniformes) inferred from mitochondrial and nuclear genes with analyses of gene evolution

The superfamily Cobitoidea of the order Cypriniformes is a diverse group of fishes, inhabiting freshwater ecosystems across Eurasia and North Africa. The phylogenetic relationships of this well-corroborated natural group and diverse clade are critical to not only informing scientific communities of the phylogeny of the order Cypriniformes, the world's largest freshwater fish order, but are key to every area of comparative biology examining the evolution of traits, functional structures, and breeding behaviors to their biogeographic histories, speciation, anagenetic divergence, and divergence time estimates. In the present study, two mitochondrial gene sequences (COI, ND4+5) and four single-copy nuclear gene segments (RH1, RAG1, EGR2B, IRBP) were used to infer the phylogenetic relationships of the Cobitoidea as reconstructed from maximum likelihood (ML) and partitioned Bayesian Analysis (BA). Analyses of the combined mitochondrial/nuclear gene datasets revealed five strongly supported monophyletic Cobitoidea families and their sister-group relationships: Botiidae+(Vaillantellidae+(Cobitidae+(Nemacheilidae+Balitoridae))). These recovered relationships are in agreement with previous systematic studies on the order Cypriniformes and/or those focusing on the superfamily Cobitoidea. Using these relationships, our analyses revealed pattern lineage- or ecological-group-specific evolution of these genes for the Cobitoidea. These observations and results corroborate the hypothesis that these group-specific-ancestral ecological characters have contributed in the diversification and/or adaptations within these groups. Positive selections were detected in RH1 of nemacheilids and in RAG1 of nemacheilids and genus Vaillantella, which indicated that evolution of RH1 (related to eye's optic sense) and RAG1 (related to immunity) genes appeared to be important for the diversification of these groups. The balitorid lineage (those species inhabiting fast-flowing riverine habitats) had, as compared with other cobitoid lineages, significantly different dN/dS, dN and dS values for ND4 and IRBP genes. These significant differences are usually indicative of weaker selection pressure, and lineage-specific evolution on genes along the balitorid lineage. Furthermore, within Cobitoidea, excluding balitorids, species living in subtropics had significantly higher dN/dS values in RAG1 and IRBP genes than those living in temperate and tropical zones. Among tropical cobitoids, genes COI, ND5, EGR2B, IRBP and RH1, had a significantly higher mean dS value than those species in subtropical and temperate groups. These findings suggest that the evolution of these genes could also be ecological-group-specific and may have played an important role in the adaptive evolution and diversification of these groups. Thus, we hypothesize that the genes included in the present study were actively involved in lineage- and/or ecological-group-specific evolutionary processes of the highly diverse Cobitoidea. These two evolutionary patterns, both subject to further testing, are hypothesized as integral in the diversification with this major clade of the world's most diverse group of freshwater fishes.