Avirulence gene and insertion element-based RFLP as well as RAPD markers reveal high levels of genomic polymorphism in the rice pathogen Xanthomonas oryzae pv. oryzae

Genetic polymorphism within the genomes of bacterial pathogens determines their evolutionary potential during long-term interaction with their hosts. To investigate the level of genetic variation in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of rice bacterial blight disease, three DNA marker systems, including (i) restriction fragment length polymorphism (RFLP) of the avrBs3/PthA family genes (avrXa27), (ii) RFLP of insertion (IS) elements and (iii) random amplified polymorphic DNA (RAPD) markers, were used to detect polymorphism among 32 Xoo strains that differed in their virulence patterns. All these strains contained multiple avrXa27 homologs that were variable in copy number and genomic location. RFLP of six IS elements revealed that these mobile sequences were abundant in Xoo genomes, with 150 of the total of 165 discernable markers being variable. Thirty-eight decamer primers of RAPD amplified a total of 691 bands, with 100% of them being variable. In addition, analysis of molecular variance (AMOVA) of data from RFLP analysis of IS elements and from RAPD analysis showed that most of the genetic variation residues were within Xoo populations, rather than between populations. Although all three DNA marker systems supported that substantial variation was maintained in Xoo genomes, Mantel tests did not identify significant correlation between the similarity coefficients calculated from them. The results of the present study indicated that Xoo genomes contain a high level of genetic polymorphism, which greatly facilitates the evolution of this important pathogen during interaction with its host rice plant.     

Comparative genomics in the grass family: molecular characterization of grass genome structure and evolution

The genomes of grasses are very different in terms of size, ploidy level and chromosome number. Despite these significant differences, it was found by comparative mapping that the linear order (colinearity) of genetic markers and genes is very well conserved between different grass genomes. The potential of such conservation has been exploited in several directions, e.g. in defining rice as a model genome for grasses and in designing better strategies for positional cloning in large genomes. Recently, the development of large insert libraries in species such as maize, rice, barley and diploid wheat has allowed the study of large stretches of DNA sequence and has provided insight into gene organization in grasses. It was found that genes are not distributed randomly along the chromosomes and that there are clusters of high gene density in species with large genomes. Comparative analysis performed at the DNA sequence level has demonstrated that colinearity between the grass genomes is retained at the molecular level (microcolinearity) in most cases. However, detailed analysis has also revealed a number of exceptions to microcolinearity, which have given insight into mechanisms that are involved in grass-genome evolution. In some cases, the use of rice as a model to support gene isolation from other grass genomes will be complicated by local rearrangements. In this Botanical Briefing, we present recent progress and future prospects of comparative genomics in grasses.     

Inverse correlation of population similarity and introduction date for invasive ascidians

The genomes of many marine invertebrates, including the purple sea urchin and the solitary ascidians Ciona intestinalis and Ciona savignyi, show exceptionally high levels of heterozygosity, implying that these populations are highly polymorphic. Analysis of the C. savignyi genome found little evidence to support an elevated mutation rate, but rather points to a large population size contributing to the polymorphism level. In the present study, the relative genetic polymorphism levels in sampled populations of ten different ascidian species were determined using a similarity index generated by AFLP analysis. The goal was to determine the range of polymorphism within the populations of different species, and to uncover factors that may contribute to the high level of polymorphism. We observe that, surprisingly, the levels of polymorphism within these species show a negative correlation with the reported age of invasive populations, and that closely related species show substantially different levels of genetic polymorphism. These findings show exceptions to the assumptions that invasive species start with a low level of genetic polymorphism that increases over time and that closely related species have similar levels of genetic polymorphism.     

Genetic Variation Within and Among Populations of Rhodiola alsia (Crassulaceae) Native to the Tibetan Plateau as Detected by ISSR Markers

Genetic variation of 10 Rhodiola alsia (Crassulaceae) populations from the Qinghai–Tibet Plateau of China was investigated using intersimple sequence repeat (ISSR) markers. R. alsia is an endemic species of the Qinghai–Tibet Plateau. Of the 100 primers screened, 13 were highly polymorphic. Using these primers, 140 discernible DNA fragments were generated with 112 (80%) being polymorphic, indicating pronounced genetic variation at the species level. Also there were high levels of polymorphism at the population level with the percentage of polymorphic bands (PPB) ranging from 63.4 to 88.6%. Analysis of molecular variance (AMOVA) showed that the genetic variation was mainly found among populations (70.3%) and variance within populations was 29.7%. The main factors responsible for the high level of differentiation among populations are probably the isolation from other populations and clonal propagation of this species. Occasional sexual reproduction might occur in order to maintain high levels of variation within populations. Environmental conditions could also influence population genetic structure as they occur in severe habitats. The strong genetic differentiation among populations in our study indicates that the conservation of genetic variability in R. alsia requires maintenance of as many populations as possible.     

Extremely high molecular diversity within the East Asian nematode Caenorhabditis sp. 5

Most relatives of the self-fertilizing hermaphroditic nematode model organism Caenorhabditis elegans reproduce via obligate outbreeding between males and females, which also represents the ancestral mode of reproduction within the genus. However, little is known about the scope of genetic diversity and differentiation within such gonochoristic species, especially those found outside of temperate Europe and North America. It is critical to understand the evolutionary processes operating in these species to provide a framework for deciphering the evolution of hermaphroditism and a baseline for the application of outcrossing Caenorhabditis to problems in evolutionary genetics. Here, we investigate for the first time molecular sequence variation for Caenorhabditis sp. 5, a species found commonly in eastern Asia. We identify enormous levels of standing genetic variation that approach the levels observed in the marine broadcast-spawning sea squirt, Ciona savignyi. Although we document significant isolation by distance, we demonstrate that the high polymorphism within C. sp. 5 is not because of strong differentiation among populations or to the presence of cryptic species. These findings illustrate that molecular population genetic approaches to studying obligately outbreeding species of Caenorhabditis will prove powerful in identifying and characterizing functionally and evolutionarily important features of the genome.     

Diversification of Sulawesi macaque monkeys: decoupled evolution of mitochondrial and autosomal DNA

Abstract. In macaque monkeys, females are philopatric and males are obligate dispersers. This social system is expected to differently affect evolution of genetic elements depending on their mode of inheritance. Because of this, the geographic structure of molecular variation may differ considerably in mitochondrial DNA (mtDNA) and in autosomal DNA (aDNA) in the same individuals, even though these genomes are partially co-inherited. On the Indonesian island of Sulawesi, macaque monkeys underwent an explosive diversification as a result of range fragmentation. Today, barriers to dispersal have receded and fertile hybrid individuals can be found at contact zones between parapatric species. In this study, we examine the impact of range fragmentation on Sulawesi macaque mtDNA and aDNA by comparing evolution, phylogeography, and population subdivision of each genome. Our results suggest that mtDNA is paraphyletic in some species, and that mtDNA phylogeography is largely consistent with a pattern of isolation by distance. Autosomal DNA, however, is suggestive of fragmentation, in that interspecific differentiation across most contact zones is significant but intraspecific differentiation between contact zones is not. Furthermore, in mtDNA, most molecular variation is partitioned between populations within species but in aDNA most variation is partitioned within populations. That mtDNA has a different geographic structure than aDNA (and morphology) in these primates is a probable consequence of (1) a high level of ancestral polymorphism in mtDNA, (2) differences between patterns of ancestral dispersal of matrilines and contemporary dispersal of males, and (3) the fact that female philopatry impedes gene flow of macaque mtDNA.     

Making the most of mitochondrial genomes--markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea)

An increasing number of complete sequences of mitochondrial (mt) genomes provides the opportunity to optimise the choice of molecular markers for phylogenetic and ecological studies. This is particularly the case where mt genomes from closely related taxa have been sequenced; e.g., within Schistosoma. These blood flukes include species that are the causative agents of schistosomiasis, where there has been a need to optimise markers for species and strain recognition. For many phylogenetic and population genetic studies, the choice of nucleotide sequences depends primarily on suitable PCR primers. Complete mt genomes allow individual gene or other mt markers to be assessed relative to one another for potential information content, prior to broad-scale sampling. We assess the phylogenetic utility of individual genes and identify regions that contain the greatest interspecific variation for molecular ecological and diagnostic markers. We show that variable characters are not randomly distributed along the genome and there is a positive correlation between polymorphism and divergence. The mt genomes of African and Asian schistosomes were compared with the available intraspecific dataset of Schistosoma mansoni through sliding window analyses, in order to assess whether the observed polymorphism was at a level predicted from interspecific comparisons. We found a positive correlation except for the two genes (cox1 and nad1) adjoining the putative control region in S. mansoni. The genes nad1, nad4, nad5, cox1 and cox3 resolved phylogenies that were consistent with a benchmark phylogeny and in general, longer genes performed better in phylogenetic reconstruction. Considering the information content of entire mt genome sequences, partial cox1 would not be the ideal marker for either species identification (barcoding) or population studies with Schistosoma species. Instead, we suggest the use of cox3 and nad5 for both phylogenetic and population studies. Five primer pairs designed against Schistosoma mekongi and Schistosoma malayensis were tested successfully against Schistosoma japonicum. In combination, these fragments encompass 20-27% of the variation amongst the genomes (average total length approximately 14,000bp), thus providing an efficient means of encapsulating the greatest amount of variation within the shortest sequence. Comparative mitogenomics provides the basis of a rational approach to molecular marker selection and optimisation.     

Analysis of the evolutionary forces shaping mitochondrial genomes of a Neotropical malaria vector complex

Many vectors of human malaria belong to complexes of morphologically indistinguishable cryptic species. Here we report the analysis of the newly sequenced complete mitochondrial DNA molecules from six recognized or putative species of one such group, the Neotropical Anopheles albitarsis complex. The molecular evolution of these genomes had been driven by purifying selection, particularly strongly acting on the RNA genes. Directional mutation pressure associated with the strand-asynchronous asymmetric mtDNA replication mechanism may have shaped a pronounced DNA strand asymmetry in the nucleotide composition in these and other Anopheles species. The distribution of sequence polymorphism, coupled with the conflicting phylogenetic trees inferred from the mitochondrial DNA and from the published white gene fragment sequences, indicates that the evolution of the complex may have involved ancient mtDNA introgression. Six protein coding genes (nad5, nad4, cox3, atp6, cox1 and nad2) have high levels of sequence divergence and are likely informative for population genetics studies. Finally, the extent of the mitochondrial DNA variation within the complex supports the notion that the complex consists of a larger number of species than until recently believed.     

Quantifying the Elevation of Mitochondrial DNA Evolutionary Substitution Rates Over Nuclear Rates in the Intertidal Copepod Tigriopus californicus

Mitochondrial DNA (mtDNA) genomes generally evolve rapidly in animals, but considerable variation in the rates of evolution of mtDNA occurs among taxa. Higher levels of mutation will tend to increase the amount of polymorphism, which should also scale with population size, but there are mixed signals from previous studies on the evolutionary outcomes of the interactions of these processes. The copepod Tigriopus californicus provides an interesting model in which to study the evolution of mtDNA because it has high levels of divergence among populations and there is the suggestion that this divergence could be involved in reproductive isolation. This species also appears to have an elevated mtDNA substitution rate, but previous studies did not provide an accurate measurement. This article examines the rate of mtDNA substitution versus nuclear substitution in T. californicus and finds that the mtDNA rate for synonymous sites averages 55-fold higher, a level that exceeds the rates found in most other invertebrates. Levels of polymorphism are also examined in both mtDNA and nuclear genes, and it is shown that the effective population size of mtDNA genes is much lower than that of nuclear genes. In addition, no correlation between polymorphism in mtDNA and nuclear genes is found across populations, which suggest factors other than demography may shape polymorphism in this species. The results from this study suggest that mtDNA is evolving at a very rapid rate in this copepod species, and this could increase the likelihood that mtDNA evolution is involved in the generation of reproductive isolation.     

THE QUANTITATIVE AND MOLECULAR GENETIC ARCHITECTURE OF A SUBDIVIDED SPECIES

In an effort to elucidate the evolutionary mechanisms that determine the genetic architecture of a species, we have analyzed 17 populations of the microcrustacean Daphnia pulex for levels of genetic variation at the level of life-history characters and molecular markers in the nuclear and mitochondrial genomes. This species is highly subdivided, with approximately 30% of the variation for nuclear molecular markers and 50% of the variation for mitochondrial markers being distributed among populations. The average level of genetic subdivision for quantitative traits is essentially the same as that for nuclear markers, which superficially suggests that the life-history characters are diverging at the neutral rate. However, the existence of a strong correlation between the levels of population subdivision and broadsense heritabilities of individual traits argues against this interpretation, suggesting instead that the among-population divergence of some quantitative traits (most notably body size) is being driven by local adaptation to different environments. The fact that the mean phenotypes of the individual populations are also strongly correlated with local levels of homozygosity indicates that variation in local inbreeding plays a role in population differentiation. Rather than being a passive consequence of local founder effects, levels of homozygosity may be selected for directly for their effects on the phenotype (adaptive inbreeding depression). There is no relationship between the levels of variation within populations for molecular markers and quantitative characters, and this is explained by the fact that the average standing genetic variation for life-history characters in this species is equivalent to only 33 generations of variation generated by mutation.     

反转录转座子标记及在作物遗传育种中的应用

反转录转座子通过RNA中间体进行反转录而转座,广泛分布于各种植物基因组中,拷贝数多,异质性高,在种内和种间表现出较高的序列差异性和丰富的插入多态性。针对这些特点,开发出了几种基于反转录转座子的分子标记,如SSAP、RIVPI、RAP、REMAP和RBIP等。由于反转录转座子标记能揭示出丰富的多态性,因而在遗传多样性和系谱研究、遗传连锁图谱构建及性状基因定位等方面得到了应用。随着分离技术的不断改进,获取序列信息更加容易,反转录转座子作为分子标记用于作物遗传育种将具有广阔前景。     

Evolutionary rate variation at multiple levels of biological organization in plant mitochondrial DNA

We examined patterns of mitochondrial polymorphism and divergence in the angiosperm genus Silene and found substantial variation in evolutionary rates among species and among lineages within species. Moreover, we found corresponding differences in the amount of polymorphism within species. We argue that, along with our earlier findings of rate variation among genes, these patterns of rate heterogeneity at multiple phylogenetic scales are most likely explained by differences in underlying mutation rates. In contrast, no rate variation was detected in nuclear or chloroplast loci. We conclude that mutation rate heterogeneity is a characteristic of plant mitochondrial sequence evolution at multiple biological scales and may be a crucial determinant of how much polymorphism is maintained within species. These dramatic patterns of variation raise intriguing questions about the mechanisms driving and maintaining mutation rate heterogeneity in plant mitochondrial genomes. Additionally, they should alter our interpretation of many common phylogenetic and population genetic analyses.     

Cytonuclear incompatibility contributes to the early stages of speciation

Genetic incompatibility is a hallmark of speciation. Cytonuclear incompatibilities are proposed to be among the first genetic barriers to arise during speciation. Accordingly, reproductive isolation (RI) within species should be heavily influenced by interactions between the organelle and nuclear genomes. However, there are few clear examples of cytonuclear incompatibility within a species. Here, we show substantial postzygotic RI in first‐generation hybrids between differentiated populations of an herbaceous plant (up to 92% reduction in fitness). RI was primarily due to germination and survival, with moderate RI for pollen viability. RI for survival was asymmetric and caused by cytonuclear incompatibility, with the strength of incompatibility linearly related to chloroplast genetic distance. This cytonuclear incompatibility may be the result of a rapidly evolving plastid genome. Substantial asymmetric RI was also found for germination, but was not associated with cytonuclear incompatibility, indicating endosperm or maternal‐zygote incompatibilities. These results demonstrate that cytonuclear incompatibility contributes to RI within species, suggesting that initial rates of speciation could be influenced by rates of organelle evolution. However, other genetic incompatibilities are equally important, indicating that even at early stages, speciation can be a complex process involving multiple genes and incompatibilities.     

Levels of genetic polymorphism: marker loci versus quantitative traits.

Species are the units used to measure ecological diversity and alleles are the units of genetic diversity. Genetic variation within and among species has been documented most extensively using allozyme electrophoresis. This reveals wide differences in genetic variability within, and genetic distances among, species, demonstrating that species are not equivalent units of diversity. The extent to which the pattern observed for allozymes can be used to infer patterns of genetic variation in quantitative traits depends on the forces generating and maintaining variability. Allozyme variation is probably not strictly neutral but, nevertheless, heterozygosity is expected to be influenced by population size and genetic distance will be affected by time since divergence. The same is true for quantitative traits influenced by many genes and under weak stabilizing selection. However, the limited data available suggest that allozyme variability is a poor predictor of genetic variation in quantitative traits within populations. It is a better predictor of general phenotypic divergence and of postzygotic isolation between populations or species, but is only weakly correlated with prezygotic isolation. Studies of grasshopper and planthopper mating signal variation and assortative mating illustrate how these characters evolve independently of general genetic and morphological variation. The role of such traits in prezygotic isolation, and hence speciation, means that they will contribute significantly to the diversity of levels of genetic variation within and among species.     

Population genetics of the cytoplasm and the units of selection on mitochondrial DNA in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Biological variation exists across a nested set of hierarchical levels from nucleotides within genes to populations within species to lineages within the tree of life. How selection acts across this hierarchy is a long-standing question in evolutionary biology. Recent studies have suggested that genome size is influenced largely by the balance of selection, mutation and drift in lineages with different population sizes. Here we use population cage and maternal transmission experiments to identify the relative strength of selection at an individual and cytoplasmic level. No significant trends were observed in the frequency of large (L) and small (S) mtDNAs across 14 generations in population cages. In all replicate cages, new length variants were observed in heteroplasmic states indicating that spontaneous length mutations occurred in these experimental populations. Heteroplasmic flies carrying L genomes were more frequent than those carrying S genomes suggesting an asymmetric mutation dynamic from larger to smaller mtDNAs. Mother-offspring transmission of heteroplasmy showed that the L mtDNA increased in frequency within flies both between and within generations despite sampling drift of the same intensity as occurred in population cages. These results suggest that selection for mtDNA size is stronger at the cytoplasmic than at the organismal level. The fixation of novel mtDNAs within and between species requires a transient intracellular heteroplasmic stage. The balance of population genetic forces at the cytoplasmic and individual levels governs the units of selection on mtDNA, and has implications for evolutionary inference as well as for the effects of mtDNA mutations on fitness, disease and aging.     

Sequence divergence of rice microsatellites in Oryza and other plant species

To determine the basis of genetic variation at microsatellite loci, eleven primer pairs, developed to amplify microsatellite markers in rice, were evaluated for their ability to amplify a PCR product and for both electromorphic and sequence-based polymorphism of the resulting products in 12 plant samples, including representatives from six different species within the genus Oryza and one genotype each from Zea (maize), Triticum (wheat) and Arabidopsis. PCR amplification was reliable in the four O. sativa samples as well as in the closely related Oryza relatives with AA genomes, while only 73% (8/11) of primers amplified in the BB/CC and CC genomes of Oryza, and 27% (3/11) amplified in the other genera. Three out of seven DNA fragments that were amplified from all genera were determined to be orthologous to their rice counterparts. A total of 115 amplicons were detected using polyacrylamide gel electrophoresis and these clustered into 74 distinct electromorphs. Sequencing of 108 amplicons revealed size homoplasy, exposing 13 new sequence-based variants. Allelic diversity within a species was predominantly due to changes in the number of repeats in the microsatellite region, but the frequency of insertions/deletions (indels) and base substitutions increased as the genetic distance between samples increased. This study suggests that electromorph size polymorphism is an adequate measure of genetic difference in studies involving closely-related individuals, but that when phylogenetic or evolutionary inferences are being made over longer time scales, evaluation of SSR variation at the sequence level is essential.     

Phylogeography and monophyly of the swordtail fish species Xiphophorus birchmanni (Cyprinodontiformes, Poeciliidae)

We used sequences of the mitochondria control region to assess the distribution of genetic variation within and among populations of the poeciliid fish species Xiphophorus birchmanni . We collected 122 X. birchmanni samples from 11 sites in three drainage systems comprising the distribution of the species. We found low levels of polymorphism among aligned sequences and low levels of genetic variation within populations but high levels of genetic differentiation among populations. Haplotypes are exclusive to three river drainages (Los Hules, Calabozo and San Pedro). Mantel tests revealed correlations between geographical (both straight-line and river distances) and genetic distance, consistent with an isolation by distance scenario, while nested clade analysis suggested allopatric fragmentation between haplotypes from two of the major drainages, and isolation by distance with restricted gene flow within those drainages. Finally, monophyly of X. birchmanni is strongly supported while the previous hypothesis of the evolutionary origin of this species from X. malinche is not.     

Gynodioecy in structured populations: understanding fine-scale sex ratio variation in Beta vulgaris ssp. maritima

Natural selection, random processes and gene flow are known to generate sex ratio variations among sexually polymorphic plant populations. In gynodioecious species, in which hermaphrodites and females coexist, the relative effect of these processes on the maintenance of sex polymorphism is still up for debate. The aim of this study was to document sex ratio and cytonuclear genetic variation at a very local scale in wind-pollinated gynodioecious Beta vulgaris ssp. maritima and attempt to elucidate which processes explained the observed variation. The study sites were characterized by geographically distinct patches of individuals and appeared to be dynamic entities, with recurrent establishment of distinct haplotypes through independent founder events. Along with substantial variation in sex ratio and unexpectedly low gene flow within study sites, our results showed a high genetic differentiation among a mosaic of genetically distinct demes, with isolation by distance or abrupt genetic discontinuities taking place within a few tens of metres. Overall, random founder events with restricted gene flow could be primary determinants of sex structure, by promoting the clumping of sex-determining genes. Such high levels of sex structure provide a landscape for differential selection acting on sex-determining genes, which could modify the conditions of maintenance of gynodioecy in structured populations.     

Do density‐driven mating system differences explain reproductive incompatibilities between populations of a placental fish?

Matrotrophy, the provisioning of embryos between fertilization and birth, creates the potential for conflict between mothers and embryos over the level of maternal investment. This conflict is predicted to drive the evolution of reproductive isolation between populations with different mating systems. In this study, we examine whether density‐driven mating system differences explain the patterns of asymmetric reproductive isolation observed in previous studies involving four populations of the matrotrophic least killifish, Heterandria formosa. Minimum sire number reconstructions suggested that two populations characterized by low densities had lower levels of concurrent multiple paternity than two populations characterized by high densities. However, low levels of genetic variation in the low‐density populations greatly reduced our probability of detecting multiple mating in them. Once we took the lower level of genetic variation into account in our estimations, high levels of multiple paternity appeared the rule in all four populations. In the population where we had the greatest power of detecting multiple mating, we found that multiple paternity in H. formosa typically involves multiple sires contributing to offspring within the same brood instead of different fathers contributing to distinct, simultaneously provisioned broods. Paternity was often skewed towards one sire. Our results suggest that differences between H. formosa populations in the levels of multiple paternity are not sufficient to explain the reproductive isolation seen in previous studies. We suggest that other influences on maternal–foetal conflict may contribute to the pattern of reproductive isolation observed previously. Alternatively, the asymmetric reproductive isolation seen in previous studies might reflect the disruption of maternal–foetal coadaptation.