Dynamic genetic features of chromosomes revealed by comparison of soybean genetic and sequence-based physical maps

Despite the intensive soybean [Glycine max (L.) Merrill] genome studies, the high chromosome number (20) of the soybean plant relative to many other major crops has hindered the development of a high-resolution genomewide genetic map derived from a single population. Here, we report such a map, which was constructed in an F₁₅ population derived from a cross between G. max and G. soja lines using indel polymorphisms detected via a G. soja genome resequencing. By targeting novel indel markers to marker-poor regions, all marker intervals were reduced to under 6 cM on a genome scale. Comparison of the Williams 82 soybean reference genome sequence and our genetic map indicated that marker orders of 26 regions were discrepant with each other. In addition, our comparison showed seven misplaced and two absent markers in the current Williams 82 assembly and six markers placed on the scaffolds that were not incorporated into the pseudomolecules. Then, we showed that, by determining the missing sequences located at the presumed beginning points of the five major discordant segments, these observed discordant regions are mostly errors in the Williams 82 assembly. Distributions of the recombination rates along the chromosomes were similar to those of other organisms. Genotyping of indel markers and genome resequencing of the two parental lines suggested that some marker-poor chromosomal regions may represent introgression regions, which appear to be prevalent in soybean. Given the even and dense distribution of markers, our genetic map can serve as a bridge between genomics research and breeding programs.     

Chromosomal features revealed by comparison of genetic maps of Glycine max and Glycine soja

Recombination is a crucial component of evolution and breeding. New combinations of variation on chromosomes are shaped by recombination. Recombination is also involved in chromosomal rearrangements. However, recombination rates vary tremendously among chromosome segments. Genome-wide genetic maps are one of the best tools to study variation of recombination. Here, we describe high density genetic maps of Glycine max and Glycine soja constructed from four segregating populations. The maps were used to identify chromosomal rearrangements and find the highly predictable pattern of cross-overs on the broad scale in soybean. Markers on these genetic maps were used to evaluate assembly quality of the current soybean reference genome sequence. We find a strong inversion candidate larger than 3 Mb based on patterns of cross-overs. We also identify quantitative trait loci (QTL) that control number of cross-overs. This study provides fundamental insights relevant to practical strategy for breeding programs and for pan-genome researches.     

Interactions and structure of the nuclear pore complex revealed by cryo- electron microscopy

Nuclear pore complexes (NPCs) play a central role in mediating nucleocytoplasmic transport and exchange processes in eukaryotic cells. The arrangement and interactions of NPCs within amphibian nuclear envelopes have been studied using cryo-electron microscopy of unfixed and frozen hydrated specimens. The nuclear lamina in Necturus forms an orthogonal network with crossover distances which vary between 1,600 and 4,000 A and which may be related to the basic filament repeat of lamins. Furthermore, the NPCs are attached randomly within the confines of the lamin network, presumably by their nucleoplasmic rings. Image analysis of edge-on and en face projections of detergent-extracted NPCs has been combined with data on the coaxial thin rings to provide a quantitative evaluation of the triple ring model of NPC architecture proposed previously (Unwin, P. N. T., and R. Milligan. 1982. J. Cell Biol. 93:63-75). Additional details of the complex have been visualized including an intimate association of the inner spoke domains as an inner spoke ring, extensive domains within the spokes and coaxial thin rings, and interestingly, a central channel-like feature. Membrane-associated NPCs and detergent-extracted NPCs both possess peripherally located radial arms resulting in an effective diameter of approximately 1,450-1,500 A. In projection, the radial arms possess approximate mirror symmetry suggesting that they originate from both sides of the assembly. Moreover, membrane-associated NPCs are asymmetric at most radii and right-handed as viewed from the cytoplasm; detergent-extracted NPCs appear to be symmetric and have approximately 822 symmetry. Taken together, the data suggests that the framework of membrane-associated NPCs is perturbed from a symmetrical configuration, either during isolation of nuclei or by interactions with the lamina and the nuclear envelope in vivo. However, detergent extraction of nuclei appears to result in a more symmetrical alignment of components in apposing halves of the assembly.     

Reticulate evolution of the Daphnia pulex complex as revealed by nuclear markers

The study of species complexes is of particular interest to understand how evolutionary young species maintain genomic integrity. The Daphnia pulex complex has been intensively studied as it includes species that dominate freshwater environments in the Northern hemisphere and as it is the sole North American complex that shows transitions to obligate parthenogenesis. Past studies using mitochondrial markers have revealed the presence of 10 distinct lineages in the complex. This study is the first to examine genetic relationships among seven species of the complex at nuclear markers (nine microsatellite loci and one protein-coding gene). Clones belonging to the seven species of the Daphnia pulex complex were characterized at the mitochondrial NADH dehydrogenase (ND5) gene and at the Lactate dehydrogenase (LDH) locus. K-means, principal coordinate analyses and phylogenetic network analyses on the microsatellite data all separated European D. pulicaria, D. tenebrosa, North American D. pulex, D. pulicaria and their hybrids into distinct clusters. The hybrid cluster was composed of diploid and polyploid hybrids with D. pulex mitochondria and some clones with D. pulicaria mitochondria. By contrast, the phylogeny of the D. pulex complex using Rab4 was not well resolved but still showed clusters consisting mostly of D. pulex alleles and others of D. pulicaria alleles. Incomplete lineage sorting and hybridization may obscure genetic relationships at this locus. This study shows that hybridization and introgression have played an important role in the evolution of this complex.     

Bayesian inference of a complex invasion history revealed by nuclear and chloroplast genetic diversity in the colonizing plant,Silene latifolia

Species invading new ranges are subject to a series of demographic events that can strongly shape genetic diversity. Describing this demographic history is important for understanding where invasive species come from and how they spread, and is critical to testing hypotheses of postinvasion adaptation. Here, we analyse nuclear and chloroplast genetic diversity to study the invasion history of the widespread colonizing weed, Silene latifolia (Caryophyllaceae). Bayesian clustering and PCA revealed strong population structure in the native range of Europe, and although genotypes from multiple native sources were present in the introduced range of North America, the spatial distribution of genetic variance was dramatically reorganized. Using approximate Bayesian computation (ABC), we compared support for different invasion scenarios, including the number and size of independent introduction events and the amount of admixture occurring between sources of introduced genotypes. Our results supported independent introductions into eastern and western North America, with the latter forming a bridgehead for a secondary invasion into the Great Lakes region of central North America. Despite small estimated founder population sizes, the duration of the demographic bottleneck after the initial introduction appeared extremely short‐lived. This pattern of repeated colonization and rapid expansion has effectively eroded the strong population structure and cytonuclear associations present in Europe, but has retained overall high genetic diversity since invasion. Our results highlight the flexibility of the ABC approach for constructing a narrative of the demographic history of species invasions and provide baseline for future studies of evolutionary changes in introduced S. latifolia populations.     

The human nuclear pore complex as revealed by cryo-electron tomography

Nuclear pore complexes (NPCs) are the sole passage through the nuclear envelope, connecting the cytoplasm to the nucleoplasm. These gigantic molecular machines, over 100 MDa in molecular weight, allow free diffusion of small molecules and ions while mediating selective energy-dependent nucleocytoplasmic transport of large macromolecules. Here, we applied cryo-electron tomography to human fibroblast cells, reconstructing their nuclear envelopes without applying any purification steps. From these reconstructions, we extracted subtomograms containing individual NPCs and utilized in silico subtomogram averaging procedures to determine the structure of the mammalian pore complex at a resolution of ～6.6?nm. Beyond revealing the canonical features of the human NPC, our analysis identified inner lateral channels and fusing bridge-like structures, suggesting alternative routes of peripheral nuclear passage. Finally, we concluded from our structural analysis that the human NPC is structurally distinct from that of lower eukaryotes in terms of dimension and organization but resembles its amphibian (frog) counterpart.     

Architecture of the Xenopus nuclear pore complex revealed by three- dimensional cryo-electron microscopy

The nuclear pore complex spans the nuclear envelope and functions as a macromolecular transporter in the ATP-dependent process of nucleocytoplasmic transport. In this report, we present three dimensional (3D) structures for both membrane-associated and detergent- extracted Xenopus NPCs, imaged in frozen buffers by cryo-electron microscopy. A comparison of the differing configurations present in the 3D maps suggests that the spokes may possess an intrinsic conformational flexibility. When combined with recent data from a 3D map of negatively stained NPCs (Hinshaw, J. E., B. O. Carragher, and R. A. Milligan. 1992. Cell. 69:1133-1141), these observations suggest a minimal domain model for the spoke-ring complex which may account for the observed plasticity of this assembly. Moreover, lumenal domains in adjacent spokes are interconnected by radial arm dimers, forming a lumenal ring that may be responsible for anchoring the NPC within the nuclear envelope pore. Importantly, the NPC transporter is visualized as a centrally tapered cylinder that spans the entire width of the NPC, in a direction normal to the nuclear envelope. The central positioning, tripartite structure, and hollow nature of the transporter suggests that it may form a macromolecular transport channel, with a globular gating domain at each end. Finally, the packing of the transporter within the spokes creates a set of eight internal channels that may be responsible, in part, for the diffusion of ions and small molecules across the nuclear envelope.     

Morphological changes in the cochlear nuclear complex in primate phylogeny and development

The primate cochlear nuclear complex exhibits several characteristic morphological differences in the various primate families from Lorisidae through Hominidae. The most striking differences occur in the organization of the dorsal cochlear nucleus in which the laminar pattern becomes progressively obscured. Granule cells form an external granular layer as well as being intermixed within the molecular and pyramidal layers in slow lorises and squirrel and rhesus monkeys. Whereas a prominent external granular layer remains in chimpanzees, granule cells are scant in other portions of the nucleus. Human adults lack an external granular layer. A small number of granule cells occur but with inconstant distribution. Primates lack the linear array of pyramidal cells oriented perpendicularly to the epithelial surface as seen in cats. The granule cell layer exhibits similar regression in development of the human cochlear complex. The external granular layer is prominent in the fetus but rapidly decreases in size after birth. It achieves its adult form prior to 18 months. The data suggest that neuronal attrition, or programmed cell death, may be the major mechanism accounting for the alterations that occur in the human granule cell layer. Other differences in cytoarchitecture, within the great apes and humans, include decreases in the small and giant cell populations of the cochlear complex. These changes, in consort with the organizational changes and reduction of granule cells as noted above, suggest a trend towards reduced intranuclear integration at the level of the cochlear nucleus coupled with encephalization of the auditory system.     

Introgressive hybridization and the evolutionary history of the herring gull complex revealed by mitochondrial and nuclear DNA

Background  

Based on extensive mitochondrial DNA (mtDNA) sequence data, we previously showed that the model of speciation among species of herring gull (Larus argentatus) complex was not that of a ring species, but most likely due more complex speciation scenario's. We also found that two species, herring gull and glaucous gull (L. hyperboreus) displayed an unexpected biphyletic distribution of their mtDNA haplotypes. It was evident that mtDNA sequence data alone were far from sufficient to obtain a more accurate and detailed insight into the demographic processes that underlie speciation of this complex, and that extensive autosomal genetic analysis was warranted.     

Taxon delimitation and genetic similarities of the Sphagnum imbricatum complex,as revealed by enzyme electrophoresis

Abstract

Isozyme electrophoretic studies were used to assess genetic variation in the Sphagnum imbricatum complex in a sample of 1332 plants from 39 populations from western Europe, eastern North America and Japan. Mean pairwise genetic distance among populations clustered and depicted in a UPGMA phenogram correspond to the recognition of four species, viz. Sphagnum portoricense , S. affine , S. imbricatum and S. austinii. The mean pairwise genetic identity among conspecific populations were 0.976, 0.847 and 0.841 for S. austinii , S. affine and S. portoricense, respectively. The mean pairwise genetic identity among taxa was 0.525 (S. austinii–S. affine), 0.476 (S. affine –S. portoricense), 0.600 (S. affine–S. imbricatum), 0.484 (S. imbricatum–S. austinii), 0.629 (S. imbricatum –S. portoricense) and 0.285 (S. austinii–S. portoricense). Populations of S. austinii in Europe are found to be genetically eroded (Hs = 0.001 ± 0.000), (P₉₅ = 0.00), probably due to severe bottlenecks caused by a series of founder effects during postglacial migration from a limited number of glacial refugia in S.W. Europe. The mean genetic diversity of S. affine (Hs = 0.122 ± 0.020) is at the same level as up to now reported for the more variable congeneric species. Among individuals of S. affine, 0.27% displayed mixed markers, indicating that, on rare occasions, hybridization may occur between S. affine and S. austinii. Preliminary genetic analysis of S. steerei supports the recognition of this taxon.     

RNA degradation by the exosome is promoted by a nuclear polyadenylation complex

The exosome complex of 3'-5' exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein complexes in vivo. However, the purified exosome showed weak in vitro activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation complex containing a known exosome cofactor, the RNA helicase Mtr4p; a poly(A) polymerase, Trf4p; and a zinc knuckle protein, Air2p. In vitro, the Trf4p/Air2p/Mtr4p polyadenylation complex (TRAMP) showed distributive RNA polyadenylation activity. The presence of the exosome suppressed poly(A) tail addition, while TRAMP stimulated exosome degradation through structured RNA substrates. In vivo analyses showed that TRAMP is required for polyadenylation and degradation of rRNA and snoRNA precursors that are characterized exosome substrates. Poly(A) tails stimulate RNA degradation in bacteria, suggesting that this is their ancestral function. We speculate that this function was maintained in eukaryotic nuclei, while cytoplasmic mRNA poly(A) tails acquired different roles in translation.     

Development of nuclear gene-derived molecular markers linked to legume genetic maps

The systematic identification of the orthologous features of related organisms greatly facilitates comparative genomics, including research on genome evolution and comparative genetic mapping. In this study, we selected 274 unique gene sequences for the development of PCR-based genetic markers across fifteen legume genomes, representing six crop or model legume species from the phaseoloid and inverted repeat loss clades (IRLC). DNA sequence analysis demonstrated that 129 of the amplified fragments represented single copy loci across most target diploid genomes. The majority of these markers are intron-spanning (70.5%) and linked to legume genetic maps (85.3%). The markers were grouped into four main categories: (1) intron-spanning relatively conserved, (2) intron-spanning diverged, (3) exon-derived conserved, and (4) exon-derived diverged. The extent of sequence divergence within each category indicates that the corresponding markers may have utility for assessing phylogenetic relationships at different, but overlapping, taxonomic levels. We tested marker performance on genomes that had not been previously sampled, representing 95 different species that span the diversity of the Fabaceae. Phylogenetic analyses support the orthology of amplified sequences, with the notable exception of an ambiguous affiliation of Lotus relative to the IRLC and phaseoloid clades.     

Tissue-inherent fate of GPI revealed by GPI-anchored GFP transgenesis

To clarify the fate of glycosylphosphatidylinositol (GPI) in mammals, we developed GPI-anchored enhanced green fluorescent protein (EGFP-GPI) and transgenic mice carrying this fusion construct. When it was introduced to culture cells, the EGFP-GPI protein was correctly sorted to plasma membranes and microsomes depending on GPI biosynthesis. Transgenic mice carrying EGFP-GPI were found to show a broad transgene expression. Histologically, a prominent polarized localization of EGFP-GPI protein was observed in various epithelia, the nervous system and liver and secreted from some exocrine glands, as well as non-polarized presence in non-epithelial tissues, demonstrating a tissue-inherent manner of GPI sorting.     

Molecular genetic divergence of the centric diatom Cyclotella and Discostella (Bacillariophyceae) revealed by nuclear ribosomal DNA comparisons

The centric diatom Cyclotella, including the recently separated Discostella, is commonly present in freshwater and several species are important bio-indicators. Here, we describe molecular characteristics of the nuclear rDNA, spanning 18S to D1/D2 region of the 28S rDNA, of two genera Cyclotella and Discostella, particularly using Korean isolates of C. meneghiniana, Discostella sp. c.f. D. pseudostelligera. Molecular and morphological analyses showed that our isolates had nearly identical genotypes in rDNA and similar morphology as compared to presumably the same species from other geographical areas. Phylogenetic analyses of individual 18S and partial 28S rDNAs of Cyclotella sensu lato showed that all sequences were separated into two clades: one containing Cyclotella, the other Discostella including C. ocellata and C. bodanica. Statistical tests with pairwise genetic distance scores showed that the two genera were significantly different (one-factor ANOVA, p?<?0.01). In addition, divergence in the partial 28S rDNA was significantly high (p?<?0.01) as compared to 18S rDNA. This provides evidence that the two genera, Cyclotella and Discostella, belong to genetically well-separated groups. In addition, 28S rDNAs is a more suitable molecular marker for the discrimination of Cyclotella sensu lato.     

Genetic differences between two strains of Xylella fastidiosa revealed by suppression subtractive hybridization

Suppression subtractive hybridization was used to rapidly identify 18 gene differences between a citrus variegated chlorosis (CVC) strain and a Pierce's disease of grape (PD) strain of Xylella fastidiosa. The results were validated as being highly representative of actual differences by comparison of the completely sequenced genome of a CVC strain with that of a PD strain.     

Assembly of pre-mRNA splicing complex is cap dependent.

To study the influence of the ubiquitous cap structure of nuclear pre-mRNAs on the assembly of a functional splicing complex, the in vitro splicing of a truncated human metallothionein pre-mRNA was examined in the presence of the cap analogue m7GTP. Significant inhibition of splicing was observed at a concentration as low as 5 microM m7GTP. Analysis of the splicing reaction on glycerol density gradients showed two complexes sedimenting at 45S and 22S. When the reaction was carried out in presence of m7GTP a marked decrease of the material sedimenting at 45S, representing the active splicing complex, was observed. When capped pre-mRNA was replaced by uncapped pre-mRNA, complex formation was significantly reduced. These data indicate that the cap structure plays an important yet unknown role in the assembly of spliceosomes.