Chloroplast DNA indicates a single origin of the allotetraploid Arabidopsis suecica

DNA sequencing was performed on up to 12 chloroplast DNA regions [giving a total of 4288 base pairs (bp) in length] from the allopolyploid Arabidopsis suecica (48 accessions) and its two parental species, A. thaliana (25 accessions) and A. arenosa (seven accessions). Arabidopsis suecica was identical to A. thaliana at all 93 sites where A. thaliana and A. arenosa differed, thus showing that A. thaliana is the maternal parent of A. suecica. Under the assumption that A. thaliana and A. arenosa separated 5 million years ago, we estimated a substitution rate of 2.9 x 10(-9) per site per year in noncoding single copy sequence. Within A. thaliana we found 12 substitution (single bp) and eight insertion/deletion (indel) polymorphisms, separating the 25 accessions into 15 haplotypes. Eight of the A. thaliana accessions from central Sweden formed one cluster, which was separated from a cluster consisting of central European and extreme southern Swedish accessions. This latter cluster also included the A. suecica accessions, which were all identical except for one 5 bp indel. We interpret this low level of variation as a strong indication that A. suecica effectively has a single origin, which we dated at 20 000 years ago or more.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Genetic differentiation in Fomitopsis pinicola (Schwarts: Fr.) Karst studied by means of arbitrary primed-PCR

Generic variation within and among one Finnish and three Swedish populations of Fomitopsis pinicola (Schwarts: Fr.) Karst. were studied by amplifying DNA from hap-loid isolates originating from single spore cultures using two arbitrary primers. Analysis offspring from single fruit bodies revealed only three pairs of codominant alleles among 42 variable genetic markers, the remaining 38 segregated independently. Genetic similarity was measured in terms of Euclidean distance. Individuals in the Finnish population tended to form a distinct cluster in the principal component analysis. Variation within and among populations/regions was partitioned by Analysis of Molecular Variance — AMOVA. Within population variation accounted for 91.6% of the total genetic variation. The remaining 7.68% was accounted for by variation between the Finnish population and each of the three Swedish ones. Variation among the Swedish populations accounted for only 0.72% of the total variation. Wright's F^st was 0.17 for all four populations and 0.13 for the three Swedish populations. These relatively low values indicate that there is gene flow among all populations or that they are derived from a common ancestral population. The observed pattern of genetic variation is probably the result of effective spore dispersal and the continuous distribution of this common early successional species.     

Microsatellite genetic variation in small and isolated populations of Magnolia sieboldii ssp. japonica

Magnolia sieboldii ssp. japonica, distributed mainly in western Japan, is restricted to high elevation areas (1000-2000 m above sea level) and usually forms small isolated populations. Four microsatellite loci were assayed for 19 populations from six regions spanning the range of distribution, and the levels and distribution of genetic variation were estimated. All four loci were variable, with a total of 39 alleles, but the overall level of microsatellite genetic variation was low, especially compared with a related species, M. obovata. Genetic structure in M. sieboldii was characterised by low intrapopulational genetic variation (A = 3.74 and H(o) = 0.366 on average) and high genetic differentiation even among regional populations. Highly significant isolation-by-distance (IBD) models at the short distance were detected. Genetic drift and limited gene flow was considered to be important in determining the genetic structure within regions. Total genetic differentiation was remarkably high (F(ST) = 0.488 and R(ST) = 0.538), suggesting genetic barriers among regions. Neighbour-joining dendrograms relating the 19 populations, and further analysis on the IBD models, revealed that a stepwise mutation model was more suited than an infinite allele model to explain the genetic differentiation among regions. It is suggested that mutation at microsatellite loci might be influential in generating the genetic differentiation among regions. These results showed the potential of hypervariable microsatellite loci to evaluate the effects of genetic drift and population isolation within regions, and to detect genetic distinctiveness, in spite of the loss of overall genetic variation in M. sieboldii.     

Multilocus analysis of variation and speciation in the closely related species Arabidopsis halleri and A. lyrata

Nucleotide variation in eight effectively unlinked genes was surveyed in species-wide samples of the closely related outbreeding species Arabidopsis halleri and A. lyrata ssp. petraea and in three of these genes in A. lyrata ssp. lyrata and A. thaliana. Significant genetic differentiation was observed more frequently in A. l. petraea than in A. halleri. Average estimates of nucleotide variation were highest in A. l. petraea and lowest in A. l. lyrata, reflecting differences among species in effective population size. The low level of variation in A. l. lyrata is concordant with a bottleneck effect associated with its origin. The A. halleri/A. l. petraea speciation process was studied, considering the orthologous sequences of an outgroup species (A. thaliana). The high number of ancestral mutations relative to exclusive polymorphisms detected in A. halleri and A. l. petraea, the significant results of the multilocus Fay and Wu H tests, and haplotype sharing between the species indicate introgression subsequent to speciation. Average among-population variation in A. halleri and A. l. petraea was approximately 1.5- and 3-fold higher than that in the inbreeder A. thaliana. The detected reduction of variation in A. thaliana is less than that expected from differences in mating system alone, and therefore from selective processes related to differences in the effective recombination rate, but could be explained by differences in population structure.     

Extremely low genetic variability and highly structured local populations of Arabidopsis thaliana at higher latitudes

The genetic diversity and population structure of Arabidopsis thaliana populations from Norway were studied and compared to a worldwide sample of A. thaliana to investigate the demographic history and elucidate possible colonization routes of populations at the northernmost species limit. We genotyped 282 individuals from 31 local populations using 149 single nucleotide polymorphism markers. A high level of population subdivision (F(ST) = 0.85 ± 0.007) was found indicating that A. thaliana is highly structured at the regional level. Significant relationships between genetic and geographical distances were found, suggesting an isolation by distance mode of evolution. Genetic diversity was much lower, and the level of linkage disequilibrium was higher in populations from the north (65-68°N) compared to populations from the south (59-62°N); this is consistent with a northward expansion pattern. A neighbour-joining tree showed that populations from northern Norway form a separate cluster, while the remaining populations are distributed over a few minor clusters. Minimal gene flow seems to have occurred between populations in different regions, especially between the geographically distant northern and southern populations. Our data suggest that northern populations represent a homogenous group that may have been established from a few founders during northward expansions, while populations in the central part of Norway constitute an admixed group established by founders of different origins, most probably as a result of human-mediated gene flow. Moreover, Norwegian populations appeared to be homogenous and isolated compared to a worldwide sample of A. thaliana, but they are still grouped with Swedish populations, which may indicate common colonization histories.     

A combined approach using ISSR and ITS analysis for the characterization of Artemisia halodendron from Horqin sandy land,northern China

Artemisia halodendron is the most dominant and constructive species among the Horqin sandy land. We evaluated its genetic variation and phylogenetic analysis within and among its populations sampled from six different habitats gradient from the Horqin sandy land in the northeast of China by using inter-simple sequence repeat polymorphism (ISSR) and the nuclear ribosomal internal transcribed spacer (ITS) molecular markers. The results showed that eight ISSR primers generated 84 bands, of which 48 (57.14%) were polymorphic. The highest genetic diversity was observed in the inter-dune lowland population, whereas the lowest diversity was found in the mobile dune population. The AMOVA analysis revealed a relatively high level of genetic variation within its populations. The alignment of 124 ITS sequences showed 401 variable sites and the GC content ranged from 54.02% to 57.32%. The ITS tree revealed the existence of three major clades, and that the semi-mobile dune population was more closely related to the inter-dune lowland population.     

Patterns of genetic variation in Gymnadenia conopsea, the fragrant orchid

Gymnadenia conopsea (L.) R. Br., or the fragrant orchid, is one of many plant species negatively influenced by new practices in agriculture and forestry during the last decades. This study describes the level of microsatellite variation within and among 10 Swedish populations of this species. It was not possible to detect strong effects of small population size or fragmentation. In general, the species had high genetic variation within and low genetic divergence among populations, although the correlation between population size and number of alleles was close to significance at the 95% level. Also, a significant isolation by distance effect was observed, indicating the presence of modest restrictions in gene dispersal between the investigated populations.     

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.     

Sensitivity of 70-mer oligonucleotides and cDNAs for microarray analysis of gene expression in Arabidopsis and its related species

Synthetic oligonucleotides (oligos) represent an attractive alternative to cDNA amplicons for spotted microarray analysis in a number of model organisms, including Arabidopsis, C. elegans, Drosophila, human, mouse and yeast. However, little is known about the relative effectiveness of 60-70-mer oligos and cDNAs for detecting gene expression changes. Using 192 pairs of Arabidopsis thaliana cDNAs and corresponding 70-mer oligos, we performed three sets of dye-swap experiments and used analysis of variance (anova) to compare sources of variation and sensitivities for detecting gene expression changes in A. thaliana, A. arenosa and Brassica oleracea. Our major findings were: (1) variation among different RNA preparations from the same tissue was small, but large variation among dye-labellings and slides indicates the need to replicate these factors; (2) sources of variation were similar for experiments with all three species, suggesting these feature types are effective for analysing gene expression in related species; (3) oligo and cDNA features had similar sensitivities for detecting expression changes and they identified a common subset of significant genes, but results from quantitative RT-PCR did not support the use of one over the other. These findings indicate that spotted oligos are at least as effective as cDNAs for microarray analyses of gene expression. We are using oligos designed from approximately 26,000 annotated genes of A. thaliana to study gene expression changes in Arabidopsis and Brassica polyploids.