A unique recent origin of the allotetraploid species Arabidopsis suecica: Evidence from nuclear DNA markers

A coalescent-based method was used to investigate the origins of the allotetraploid Arabidopsis suecica, using 52 nuclear microsatellite loci typed in eight individuals of A. suecica and 14 individuals of its maternal parent Arabidopsis thaliana, and four short fragments of genomic DNA sequenced in a sample of four individuals of A. suecica and in both its parental species A. thaliana and Arabidopsis arenosa. All loci were variable in A. thaliana but only 24 of the 52 microsatellite loci and none of the four sequence fragments were variable in A. suecica. We explore a number of possible evolutionary scenarios for A. suecica and conclude that it is likely that A. suecica has a recent, unique origin between 12,000 and 300,000 years ago. The time estimates depend strongly on what is assumed about population growth and rates of mutation. When combined with what is known about the history of glaciations, our results suggest that A. suecica originated south of its present distribution in Sweden and Finland and then migrated north, perhaps in the wake of the retreating ice.     

Genetic variation in Arabidopsis suecica and its parental species A. arenosa and A. thaliana

Random amplified polymorphic DNA (RAPD) markers were used to estimate the level of genetic variation in Swedish accessions of the allopolyploid Arabidopsis suecica and its parental species A. thaliana and A. arenosa. The results showed clear differences among the three species with respect to the level of variation. A. arenosa was highly variable, A. thaliana showed a moderate level of variation whereas A. suecica was much less variable than the two other species. An extended analysis covering 19 Swedish populations of A. suecica corroborated the low level of variation in this species, yet 16 unique phenotypes were observed. No isolation by distance was observed. When the genetic variation was partitioned among and within populations of A. suecica, the results showed that the majority of the variation (81%) occurred among populations. This result is interpreted as a strong indication that A. suecica is autogamous in nature.     

The evolutionary history of the common chloroplast genome of Arabidopsis thaliana and A. suecica

The evolutionary history of the common chloroplast (cp) genome of the allotetraploid Arabidopsis suecica and its maternal parent A. thaliana was investigated by sequencing 50 fragments of cpDNA, resulting in 98 polymorphic sites. The variation in the A. suecica sample was small, in contrast to that of the A. thaliana sample. The time to the most recent common ancestor (T(MRCA)) of the A. suecica cp genome alone was estimated to be about one 37th of the T(MRCA) of both the A. thaliana and A. suecica cp genomes. This corresponds to A. suecica having a MRCA between 10 000 and 50 000 years ago, suggesting that the entire species originated during, or before, this period of time, although the estimates are sensitive to assumptions made about population size and mutation rate. The data was also consistent with the hypothesis of A. suecica being of single origin. Isolation-by-distance and population structure in A. thaliana depended upon the geographical scale analysed; isolation-by-distance was found to be weak on the global scale but locally pronounced. Within the genealogical cp tree of A. thaliana, there were indications that the root of the A. suecica species is located among accessions of A. thaliana that come primarily from central Europe. Selective neutrality of the cp genome could not be rejected, despite the fact that it contains several completely linked protein-coding genes.     

Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations

Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss-of-function alleles impractical. We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes. Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A. thaliana and A. arenosa. We demonstrate that A. suecica can be genetically transformed using the floral dip method for Agrobacterium-mediated transformation. Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs. Expressing a double-stranded (ds) RNA corresponding to the A. thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A. thaliana- and A. arenosa-derived centromere repeats. These results indicate that a single RNAi-inducing transgene can dominantly repress multiple orthologs.     

Contrasting patterns of transposable-element insertion polymorphism and nucleotide diversity in autotetraploid and allotetraploid Arabidopsis species

It has been hypothesized that polyploidy permits the proliferation of transposable elements, due to both the masking of deleterious recessive mutations and the breakdown of host silencing mechanisms. We investigated the patterns of insertion polymorphism of an Ac-like transposable element and nucleotide diversity at 18 gene fragments in the allotetraploid Arabidopsis suecica and the autotetraploid A. arenosa. All identified insertions were fixed in A. suecica, and many were clearly inherited from the parental species A. thaliana or A. arenosa. These results are inconsistent with a rapid increase in transposition associated with hybrid breakdown but support the evidence from nucleotide polymorphism patterns of a recent single origin of this species leading to genomewide fixations of transposable elements. In contrast, most insertions were segregating at very low frequencies in A. arenosa samples, showing a significant departure from neutrality in favor of purifying selection, even when we account for population subdivision inferred from sequence variation. Patterns of nucleotide variation at reference genes are consistent with the TE results, showing evidence for higher effective population sizes in A. arenosa than in related diploid taxa but a near complete population bottleneck associated with the origins of A. suecica.     

Nucleotide Variation in the NCED3 Region of Arabidopsis thaliana and its Association Study with Abscisic Acid Content under Drought Stress

Drought tolerance is a comprehensive quantitative trait that is being understood further at the molecular genetic level. Abscisic acid (ABA) is the main drought-induced hormone that regulates the expression of many genes related to drought responses. 9-cis-epoxycarotenoid dioxygenase (NCED3) is thought to be a key enzyme in ABA biosynthesis. In this paper, we measured the ABA content increase under drought stress, and sequenced and compared the sequence of AtNCED3 among 22 Arabidopsis thaliana accessions. The results showed that the fold of ABA content increase under drought stress was highly variable among these accessions. High density single nucleotide polymorphism (SNP) and insertion/deletion (indel) were found in the AtNCED3 region, on average one SNP per 87.4 bp and one indel per 502 bp. Nucleotide diversity was significantly lower in the coding region than that in non-coding regions. The results of an association study with ANOVA analysis suggested that the 274th site (P←→S) and the 327th site (P←→R) amino acid variations might be the cause of ABA content increase of 163av accession under drought stress.     

Genomic in situ hybridization in plants with small genomes is feasible and elucidates the chromosomal parentage in interspecific Arabidopsis hybrids.

Genomic in situ hybridization (GISH) is a useful tool to analyse natural polyploids, hybrid plants, and their backcross progenies as to their origin, genomic composition, and intergenomic rearrangements. However, in angiosperms with very small genomes (<0.6 pg/1 C), often only heterochromatic regions were found to be labeled. We have modified the GISH technique to label entire mitotic and meiotic chromosomes of Arabidopsis thaliana (2n = 10) and closely related species with very small genomes by using high concentrations of DNA (7.5-15 microg per probe per slide) or 5 microg of probe and long hybridization times (>60 h). According to our GISH data, Cardaminopsis carpatica (2n = 16) is most likely the diploid ancestor of the autotetraploid Arabidopsis arenosa (2n = 32). Furthermore, within the allotetraploid species Arabidopsis suecica (2n = 26), it was possible to elucidate the origin of chromosomes contributed by the parental species A. thaliana and A. arenosa for a specimen with 2n = 26 or a deviating chromosome number.     

Stochastic and epigenetic changes of gene expression in Arabidopsis polyploids

Polyploidization is an abrupt speciation mechanism for eukaryotes and is especially common in plants. However, little is known about patterns and mechanisms of gene regulation during early stages of polyploid formation. Here we analyzed differential expression patterns of the progenitors' genes among successive selfing generations and independent lineages. The synthetic Arabidopsis allotetraploid lines were produced by a genetic cross between A. thaliana and A. arenosa autotetraploids. We found that some progenitors' genes are differentially expressed in early generations, whereas other genes are silenced in late generations or among different siblings within a selfing generation, suggesting that the silencing of progenitors' genes is rapidly and/or stochastically established. Moreover, a subset of genes is affected in autotetraploid and multiple independent allotetraploid lines and in A. suecica, a natural allotetraploid derived from A. thaliana and A. arenosa, indicating locus-specific susceptibility to ploidy-dependent gene regulation. The role of DNA methylation in silencing progenitors' genes is tested in DNA-hypomethylation transgenic lines of A. suecica using RNA interference (RNAi). Two silenced genes are reactivated in both ddm1- and met1-RNAi lines, consistent with the demethylation of centromeric repeats and gene-specific regions in the genome. A rapid and stochastic process of differential gene expression is reinforced by epigenetic regulation during polyploid formation and evolution.     

A gene duplication/loss event in the ribulose-1,5-bisphosphate-carboxylase/oxygenase (rubisco) small subunit gene family among accessions of Arabidopsis thaliana

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), the most abundant protein in nature, catalyzes the assimilation of CO(2) (worldwide about 10(11) t each year) by carboxylation of ribulose-1,5-bisphosphate. It is a hexadecamer consisting of eight large and eight small subunits. Although the Rubisco large subunit (rbcL) is encoded by a single gene on the multicopy chloroplast genome, the Rubisco small subunits (rbcS) are encoded by a family of nuclear genes. In Arabidopsis thaliana, the rbcS gene family comprises four members, that is, rbcS-1a, rbcS-1b, rbcS-2b, and rbcS-3b. We sequenced all Rubisco genes in 26 worldwide distributed A. thaliana accessions. In three of these accessions, we detected a gene duplication/loss event, where rbcS-1b was lost and substituted by a duplicate of rbcS-2b (called rbcS-2b*). By screening 74 additional accessions using a specific polymerase chain reaction assay, we detected five additional accessions with this duplication/loss event. In summary, we found the gene duplication/loss in 8 of 100 A. thaliana accessions, namely, Bch, Bu, Bur, Cvi, Fei, Lm, Sha, and Sorbo. We sequenced an about 1-kb promoter region for all Rubisco genes as well. This analysis revealed that the gene duplication/loss event was associated with promoter alterations (two insertions of 450 and 850 bp, one deletion of 730 bp) in rbcS-2b and a promoter deletion (2.3 kb) in rbcS-2b* in all eight affected accessions. The substitution of rbcS-1b by a duplicate of rbcS-2b (i.e., rbcS-2b*) might be caused by gene conversion. All four Rubisco genes evolve under purifying selection, as expected for central genes of the highly conserved photosystem of green plants. We inferred a single positive selected site, a tyrosine to aspartic acid substitution at position 72 in rbcS-1b. Exactly the same substitution compromises carboxylase activity in the cyanobacterium Anacystis nidulans. In A. thaliana, this substitution is associated with an inferred recombination. Functional implications of the substitution remain to be evaluated.     

The pattern of insertion/deletion polymorphism in Arabidopsis thaliana

Little is known about variation of nucleotide insertion/deletions (indels) within species. In Arabidopsis thaliana, we investigated indel polymorphism patterns between two genome sequences and among 96 accessions at 1215 loci. Our study identified patterns in the variation of indel density, size, GC content and distribution, and a correlation between indels and substitutions. We found that the GC content in indel sequences was lower than that in non-indel sequences and that indels typically occur in regions with lower GC content. Patterns of indel frequency distribution among populations were more consistent with neutral expectation than substitution patterns. We also found that the local level of substitutions is positively correlated with indel density and negatively correlated with their distance to the closed indel, suggesting that indels play an important role in nucleotide variation.     

Further insights into the phylogeny of Arabidopsis (Brassicaceae) from nuclear Atmyb2 flanking sequence

Arabidopsis thaliana is the preeminent plant model organism. However, significant advances in evolution and ecology are being made by expanding the scope of research beyond this single species into the broader genus Arabidopsis. Surprisingly, few studies have rigorously investigated phylogenetic relationships between the nine Arabidopsis species, and this study evaluates both these and hypotheses related to two instances of intra-generic hybridization. DNA sequences from the 5' flanking region of the nuclear Atmyb2 gene from 12 of the 14 Arabidopsis taxa were used to reconstruct the generic phylogeny. The strict consensus tree was highly concordant with previous studies, identifying lineages corresponding to widespread species but exhibiting a large basal polytomy. Our data indicates that the paternal parent of the allopolyploid A. suecica is A. neglecta rather than A. arenosa s.l., although the need for a detailed phylogeographical study of these three species is noted. Finally, our data provided additional phylogenetic evidence of hybridization between Arabidopsis lyrata s.l. and A. halleri s.l. Taken together, the well-defined lineages within the genus and the potential for hybridization between them highlight Arabidopsis as a promising group for comparative and experimental studies of hybridization.     

Mitotic instability in resynthesized and natural polyploids of the genus Arabidopsis (Brassicaceae)

Allopolyploids contain complete sets of chromosomes from two or more different progenitor species. Because allopolyploid hybridization can lead to speciation, allopolyploidy is an important mechanism in evolution. Meiotic instability in early-generation allopolyploids contributes to high lethality, but less is known about mitotic fidelity in allopolyploids. We compared mitotic stability in resynthesized Arabidopsis suecica-like neoallopolyploids with that in 13 natural lines of A. suecica (2n = 4x = 26). We used fluorescent in situ hybridization to distinguish the chromosomal contribution of each progenitor, A. thaliana (2n = 2x =10) and A. arenosa (2n = 4x = 32). Surprisingly, cells of the paternal parent A. arenosa had substantial aneuploidy, while cells of the maternal parent A. thaliana were more stable. Both natural and resynthesized allopolyploids had low to intermediate levels of aneuploidy. Our data suggest that polyploidy in Arabidopsis is correlated with aneuploidy, but varies in frequency by species. The chromosomal composition in aneuploid cells within individuals was variable, suggesting somatic mosaicisms of cell lineages, rather than the formation of distinct, stable cytotypes. Our results suggest that somatic aneuploidy can be tolerated in Arabidopsis polyploids, but there is no evidence that this type of aneuploidy leads to stable novel cytotypes.     

Complete structure of the chloroplast genome of Arabidopsis thaliana.

The complete nucleotide sequence of the chloroplast genome of Arabidopsis thaliana has been determined. The genome as a circular DNA composed of 154,478 bp containing a pair of inverted repeats of 26,264 bp, which are separated by small and large single copy regions of 17,780 bp and 84,170 bp, respectively. A total of 87 potential protein-coding genes including 8 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acid species were assigned to the genome on the basis of similarity to the chloroplast genes previously reported for other species. The translated amino acid sequences from respective potential protein-coding genes showed 63.9% to 100% sequence similarity to those of the corresponding genes in the chloroplast genome of Nicotiana tabacum, indicating the occurrence of significant diversity in the chloroplast genes between two dicot plants. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

Genetic diversity in Arabidopsis thaliana L. Heynh. investigated by cleaved amplified polymorphic sequence (CAPS) and inter-simple sequence repeat (ISSR) markers

In this study, we investigated genetic diversity among 37 accessions in Arabidopsis thaliana from Eurasia, North Africa and North America using morphological traits and two polymerase chain reaction (PCR)-based marker systems: cleaved amplified polymorphic sequences (CAPS) and inter-simple sequence repeats (ISSR). Cluster analysis based on genetic similarities calculated from CAPS data grouped the accessions roughly according to their geographical origin: one large group contained accessions from Western, Northern and Southern Europe as well as North Africa, a second group consisted of Eastern European and Asian continental accessions. North American accessions were interspersed into these groups. Contrary to the CAPS analysis, the dendrogram obtained from the ISSR data did not reflect the geographical origin of the accessions, and the calculated genetic distances did not match the CAPS results. This could be attributable to an uneven genomic distribution of ISSR markers as substantiated by a database search for ISSR binding sites in A. thaliana genomic DNA sequence files, or to the ISSR's different mode of evolution. We recommend CAPS markers for diversity analysis in A. thaliana because a careful selection of markers can ascertain an even representation of the entire genome.     

基于短序列测序数据的四倍体拟南芥转录组研究

为了促进对四倍体拟南芥(A.suecica)的研究,阐明多倍体植物在染色体加倍过程中遗传物质的变化,从而在分子层面上解释多倍体植物的环境适应和进化机制,描述了一套基于第二代测序技术的转录组短序列组装和生物信息学分析方法.通过对23 000 000条来至于Illumina测序平台的序列数据进行SOAPdenovo组装,以...     

Genetic isolation by distance in Arabidopsis thaliana: biogeography and postglacial colonization of Europe

Arabidopsis thaliana provides a useful model system for functional, evolutionary and ecological studies in plant biology. We have analysed natural genetic variation in A. thaliana in order to infer its biogeographical and historical distribution across Eurasia. We analysed 79 amplified fragment length polymorphism (AFLP) markers in 142 accessions from the species' native range, and found highly significant genetic isolation by distance among A. thaliana accessions from Eurasia and southern Europe. These spatial patterns of genetic variation suggest that A. thaliana colonized central and northern Europe from Asia and from Mediterranean Pleistocene refugia, a trend which has been identified in other species. Statistically significant levels of multilocus linkage disequilibrium suggest intermediate levels of disequilibrium among subsets of loci, and analysis of genetic relationships among accessions reveal a star or bush-like dendrogram with low bootstrap support. Taken together, it appears that there has been sufficient historical recombination in the A. thaliana genome such that accessions do not conform to a tree-like, bifurcating pattern of evolution - there is no 'ecotype phylogeny.' Nonetheless, significant isolation by distance provides a framework upon which studies of natural variation in A. thaliana may be designed and interpreted.     

Hybrid incompatibility in Arabidopsis is determined by a multiple-locus genetic network

The cross between Arabidopsis thaliana and the closely related species Arabidopsis arenosa results in postzygotic hybrid incompatibility, manifested as seed death. Ecotypes of A. thaliana were tested for their ability to produce live seed when crossed to A. arenosa. The identified genetic variation was used to map quantitative trait loci (QTLs) encoded by the A. thaliana genome that affect the frequency of postzygotic lethality and the phenotypes of surviving seeds. Seven QTLs affecting the A. thaliana component of this hybrid incompatibility were identified by crossing a Columbia × C24 recombinant inbred line population to diploid A. arenosa pollen donors. Additional epistatic loci were identified based on their pairwise interaction with one or several of these QTLs. Epistatic interactions were detected for all seven QTLs. The two largest additive QTLs were subjected to fine-mapping, indicating the action of at least two genes in each. The topology of this network reveals a large set of minor-effect loci from the maternal genome controlling hybrid growth and viability at different developmental stages. Our study establishes a framework that will enable the identification and characterization of genes and pathways in A. thaliana responsible for hybrid lethality in the A. thaliana × A. arenosa interspecific cross.     

Postembryonic establishment of megabase-scale gene silencing in nucleolar dominance

Nucleolar dominance is an epigenetic phenomenon in plant and animal genetic hybrids that describes the expression of 45S ribosomal RNA genes (rRNA genes) inherited from only one progenitor due to the silencing of the other progenitor's rRNA genes. rRNA genes are tandemly arrayed at nucleolus organizer regions (NORs) that span millions of basepairs, thus gene silencing in nucleolar dominance occurs on a scale second only to X-chromosome inactivation in female mammals. In Arabidopsis suecica, the allotetraploid hybrid of A. thaliana and A. arenosa, the A. thaliana -derived rRNA genes are subjected to nucleolar dominance and are silenced via repressive chromatin modifications. However, the developmental stage at which nucleolar dominance is established in A. suecica is currently unknown. We show that nucleolar dominance is not apparent in seedling cotyledons formed during embryogenesis but becomes progressively established during early postembryonic development in tissues derived from both the shoot and root apical meristems. The progressive silencing of A. thaliana rRNA genes correlates with the transition of A. thaliana NORs from a decondensed euchromatic state associated with histone H3 that is trimethylated on lysine 4 (H3K4me3) to a highly condensed heterochromatic state in which the NORs are associated with H3K9me2 and 5-methylcytosine-enriched chromocenters. In RNAi-lines in which the histone deacetylases HDA6 and HDT1 are knocked down, the developmentally regulated condensation and inactivation of A. thaliana NORs is disrupted. Collectively, these data demonstrate that HDA6 and HDT1 function in the postembryonic establishment of nucleolar dominance, a process which recurs in each generation.