cpDNA Polymorphism Research Articles

Development of a SNaPshot assay for the genotyping of organellar SNPs in four closely related pines

Mitochondrial (mtDNA) and chloroplast (cpDNA) polymorphisms are valuable resources to study past demographic changes, phylogenetics and evolution, especially in forest tree species, where these genomes are haploid and uniparentally transferred. The organellar markers were usually scored separately using direct sequencing or PCR-based approaches, which can be time-consuming and expensive, especially in large-scale population genetics research. In this study, we developed an efficient and cost-effective SNaPshot assay for genotyping preselected ­mtDNA and cpDNA polymorphism in four closely related pine species including Scots pine (Pinus sylvestris L.) and three taxa from the Pinus mugo complex. We validated the method by genotyping the samples derived from 12 populations of the species from their wide geographical distribution range in Europe. The results proved high accuracy of the method with a genotyping success rate of 99.7%. The set of assayed markers shows significant genetic variation. By using multiplex SNaPshot assay, we provided an efficient and sensitive molecular tool for intra- and interspecific genetic analyses. The presented protocol is useful for fast and relatively cheap SNP genotyping of organelle genome of closely related pine species. The assayed SNPs allow studying the species discrimination and detailed investigations of their population history and structure. Given its numerous benefits and efficient genotyping rate, the SNaPshot method appears to be a valuable and practical resource for studying the genetic makeup of forest tree species. Particularly, it proves to be advantageous for population genetics.

Glacial Expansion or Interglacial Expansion? Contrasting Demographic Models of Four Cold-Adapted Fir Species in North America and East Asia

Understanding and forecasting species’ response to climate change is a critical need for future conservation and management. Two expansion hypotheses, the glacial expansion versus the interglacial expansion, have been proposed to interpret how cold-adapted organisms in the northern hemisphere respond to Quaternary climatic fluctuations. To test these two hypotheses, we originally used two pairs of high-low elevation firs from North America (Abies lasiocarpa and Abies balsamea) and East Asia (Abies chensiensis and Abies nephrolepis). Abies lasiocarpa and Abies chensiensis are widely distributed in high-elevation regions of western North America and central China. Abies balsamea and Abies nephrolepis occur in central North America and northeast China, with much lower elevations. These fir species are typical cold-adapted species and sensitive to climate fluctuations. Here, we integrated the mtDNA and cpDNA polymorphisms involving 44 populations and 585 individuals. Based on phylogeographic analyses, recent historical range expansions were indicated in two high-elevation firs (Abies lasiocarpa and Abies chensiensis) during the last glaciation (43.8–28.4 or 21.9–14.2 kya, 53.1–34.5 or 26.6–17.2 kya). Such glacial expansions in high-elevation firs were further confirmed by the evidence of species distribution modelling, geographic-driven genetic patterns, palynological records, and current distribution patterns. Unlike the north American firs, the SDM models indicated unremarkable expansion or contraction in East Asia firs for its much more stable conditions during different historical periods. Taken together, our findings highly supported that high-elevation firs experienced glacial expansion during the Quaternary climate change in East Asia and North America, as interglacial expansion within low-elevation firs. Under this situation, the critically endangered fir species distributed in high elevation would have no enough higher elevational space to migrate. Facing the increasing global warming, thus we proposed ex-situ conservation of defining conservation units as the most meaningful strategy.

Comparative study on the chloroplast genomes of five Larix species from the Qinghai-Tibet Plateau and the screening of candidate DNA markers

Five Larix species (L. griffithii, L. speciose, L. himalaica, L. kongboensis, and L. potaninii var. australis), have survived on the Qinghai-Tibet Plateau (QTP) under specific climate conditions for decades. The lack of genomic information seriously hinders research on the evolution, conservation and ecology of these Larix resources. In this study, complete chloroplast (cp) genomes of the 5 species were assembled and compared based on next generation sequencing technology combined with polymerase chain reaction validation. The results show that the 5 cp genomes are relatively conservative in size, gene content and arrangement, and border variation. Phylogenetic analysis showed that the species are closely related as well as to seven other species of the same genus. In addition, the 5 cp genomes contained few simple sequence repeats and relatively low nucleotide variability; thus, 12 candidate polymorphic cp DNA markers will be helpful for further research on relevant population genetics. These results will provide valuable genetic information for the conservation, evolution and ecology of these species and their relatives.

TrnL-trnfF cpDNA polymorphism in some representatives of the genus Betula

We present preliminary results of the trnL-trnF cpDNA region analysis for some representatives of the g. Betula, out of which B. procurva can be considered the most interesting. The disjunctive range of this species is confined to the mountain system in southeast Central Asia (Pamir-Alai). The occurrence of the birch is isolated from the main range in the mountainous part of the Urals, in the boreal forests zone and in the Trans-Ural forest-steppe. Due to high interspecific hybridization and population variability of the g. Betula, doubts were raised about the correct identification of the representatives of B. procurva. The studied genetic variability and population structure of B. procurva, B. nana, B. pubescens, B. turkestanica, B. tianshanica and B. pendula species indicate active introgression and hybridization processes. Polymorphism in all groups is significantly reduced, increasing slightly when comparing the most distant groups. The matching of the B. procurva cpDNA haplotypes (GBS) with B. pendula, B. nana, and B. pubescens shows that this trnL-trnF cpDNA site cannot be used for molecular identification of birch species by barcoding as a single marker, but this marker use is possible for determination of certain B. procurva population. Based on the studied cpDNA region (trnL-F) we found a clear geographic subdivision in B. procurva populations of the Trans-Urals and Central Asia,.

Using reduced representation libraries sequencing methods to identify cpDNA polymorphisms in European beech (Fagus sylvatica L)

Reduced representation genomic libraries (RRLs) are increasingly used to answer diverse questions in evolutionary biology, which remained unresolved otherwise. In the case of European beech (Fagus sylvatica L.), former applications of nuclear and chloroplast genetic markers indicated that most of the beech populations of Central, Eastern, and Northern Europe have a very homogeneous genetic structure. Thus, determining differentiation among populations of F. sylvatica at regional scale in Central Europe has remained a challenge for evolutionary biologists, mainly due to lack of variable genetic markers. In this study, we used the RAD-seq and GBS approaches to identify novel cpDNA polymorphisms that could be helpful to identify population structure of European beech at geographical scale. Our datasets allowed us to indicate a finer population structure and stronger patterns of spatial genetic structure than previous studies. It is expected that the applications of cpDNA-based phylogenetic approaches using whole-genome sequencing are able to provide even more detailed picture of species phylogeography than ever before.

Whole-Genome Comparison Reveals Heterogeneous Divergence and Mutation Hotspots in Chloroplast Genome of Eucommia ulmoides Oliver.

Eucommia ulmoides (E. ulmoides), the sole species of Eucommiaceae with high importance of medicinal and industrial values, is a Tertiary relic plant that is endemic to China. However, the population genetics study of E. ulmoides lags far behind largely due to the scarcity of genomic data. In this study, one complete chloroplast (cp) genome of E. ulmoides was generated via the genome skimming approach and compared to another available E. ulmoides cp genome comprehensively at the genome scale. We found that the structure of the cp genome in E. ulmoides was highly consistent with genome size variation which might result from DNA repeat variations in the two E. ulmoides cp genomes. Heterogeneous sequence divergence patterns were revealed in different regions of the E. ulmoides cp genomes, with most (59 out of 75) of the detected SNPs (single nucleotide polymorphisms) located in the gene regions, whereas most (50 out of 80) of the indels (insertions/deletions) were distributed in the intergenic spacers. In addition, we also found that all the 40 putative coding-region-located SNPs were synonymous mutations. A total of 71 polymorphic cpDNA fragments were further identified, among which 20 loci were selected as potential molecular markers for subsequent population genetics studies of E. ulmoides. Moreover, eight polymorphic cpSSR loci were also developed. The sister relationship between E. ulmoides and Aucuba japonica in Garryales was also confirmed based on the cp phylogenomic analyses. Overall, this study will shed new light on the conservation genomics of this endangered plant in the future.

Phylogeography and population genetics of black alder (Alnus glutinosa (L.) Gaertn.) in Ireland: putting it in a European context

Black alder (Alnus glutinosa (L.) Gaertn.) is a widespread tree of European forests with a high potential for wood and biomass production. This study investigated the genetic origin and diversity in remnants of putative native alder forest stands in Ireland and compared it with other European populations to determine phylogeographic patterns. The efficiency of capture of the genetic diversity in a breeding population was also assessed. Data were obtained from chloroplast DNA (cpDNA) polymorphisms and from nuclear microsatellites. In silico PCR-RFLP was used to locate cpDNA sequence polymorphisms in order to develop flanking primers for high-resolution melting (HRM) analysis. The cpDNA polymorphism analysis detected two main haplotypes in Ireland. The same two are also found in Wales and England while only one is found in Scotland. The results suggest that Irish populations of alder have originated from at least two European glacial refugia. Black alder in Ireland exhibited no or very weak population differentiation using nuclear microsatellite markers, which is consistent for a wind-dispersed, outcrossing species. Based on the nuclear microsatellite data, a decline in population sizes is estimated to have occurred around a timeframe that coincides with a period of large-scale deforestation. The breeding population contained two haplotypes and showed a high level of genetic capture (86 % of microsatellite alleles) when compared with all samples combined. This study fills a gap in previous knowledge, provides an additional marker and an additional method for future studies. The characterisation of a breeding population provides valuable baseline data for a national alder tree improvement programme.

Towards a better understanding of the Taraxacum evolution (Compositae–Cichorieae) on the basis of nrDNA of sexually reproducing species

The genus Taraxacum is characterized by prevailing complex multiple hybridity, frequent polyploidy and widespread agamospermous reproduction, which makes the phylogenetic analysis difficult. On the basis of the previous analysis of the variation of nrDNA in Taraxacum taxa with different ploidy levels and modes of reproduction, to mitigate consequences of the reticulate complexity of the genus, a phylogenetic study of 52 samples of sexually reproducing dandelions of 26 sections (and another 13 agamospermous representatives of other sections known to include sexuals) was carried out. Both sexual and agamospermous samples were analysed using maximum parsimony and neighbour network. Exclusively sexual dandelions were analysed using the same approaches. In spite of the general agreement among various types of analyses, there is a limited overall congruence between results of nrDNA analyses and the established taxonomic system of the genus Taraxacum. The analyses shed light on the relationships among the most primitive groups. A stable clade is formed by representatives of the sections Primigenia, Orientalia, Sonchidium, Piesis and T. cylleneum. Another case of stable relationships is that of the members of the sect. Dioszegia. Relationships between the sects. Erythrosperma and Erythrocarpa were supported, and the relatedness of the members of sect. Australasica was confirmed. Rather unexpectedly, the agamospermous samples of the sect. Oligantha (T. minutilobum) are shown to be closely related with the sect. Macrocornuta. The latter section is generally considered to be close to sect. Ceratoidea (T. koksaghyz) on morphological grounds but this presumption is not corroborated by the results of nrDNA analyses. Analyses of 72 samples of sexual dandelions were also performed using the trnL–trnF region of the chloroplast DNA. The maximum parsimony analysis of this region reveals intraspecific variation in a number of ancestral diploid sexual species, all present in the two main branches of the cladogram. This phenomenon is attributed to the ancient gene flow and possibly to the persistence of ancestral cpDNA polymorphism. The strict consensus cpDNA tree information content and interpretability is quite low. The maximum parsimony analysis of combined nrDNA and cpDNA data sets was also performed with expectably low interpretability of the results.

Varietal tracing of virgin olive oils based on plastid DNA variation profiling.

Olive oil traceability remains a challenge nowadays. DNA analysis is the preferred approach to an effective varietal identification, without any environmental influence. Specifically, olive organelle genomics is the most promising approach for setting up a suitable set of markers as they would not interfere with the pollinator variety DNA traces. Unfortunately, plastid DNA (cpDNA) variation of the cultivated olive has been reported to be low. This feature could be a limitation for the use of cpDNA polymorphisms in forensic analyses or oil traceability, but rare cpDNA haplotypes may be useful as they can help to efficiently discriminate some varieties. Recently, the sequencing of olive plastid genomes has allowed the generation of novel markers. In this study, the performance of cpDNA markers on olive oil matrices, and their applicability on commercial Protected Designation of Origin (PDO) oils were assessed. By using a combination of nine plastid loci (including multi-state microsatellites and short indels), it is possible to fingerprint six haplotypes (in 17 Spanish olive varieties), which can discriminate high-value commercialized cultivars with PDO. In particular, a rare haplotype was detected in genotypes used to produce a regional high-value commercial oil. We conclude that plastid haplotypes can help oil traceability in commercial PDO oils and set up an experimental methodology suitable for organelle polymorphism detection in the complex olive oil matrices.

Genetic differentiation and delimitation of Pugionium dolabratum and Pugionium cornutum (Brassicaceae)

Species delimitation has been a major research focus in evolutionary biology. However, the genetic delimitation of recently diverged species varies depending on the markers examined. In this study, we aimed to examine genetic differentiation and delimitations between only two species of Pugionium Gaertn (Brassicaceae)—Pugionium cornutum (L.) Gaertn and Pugionium dolabratum Maxim—that occur in the desert habitats of central Asia and have parapatric distributions. We genotyped 169 individuals from 25 populations, using two chloroplast (cp) DNA fragments (trnV-trnM and trnS-trnfM), seven simple repeated sequence (SSR) loci and the nuclear ribosomal internal transcribed spacer (ITS). Four cp haplotypes were identified, three of which commonly occur in the two species, suggesting incomplete species-specific lineage sorting. Between-species cpDNA differentiation (F CT) was low, even lower than among populations of the same species. However, we found higher than average SSR F CT values, and both Bayesian clustering of SSR variables and maximum-likelihood genetic analyses divided all sampled individuals into two groups, agreeing well with morphological separation, although gene flow between species was obvious according to the SSR loci data. However, two types of ITS sequences were highly consistent with the morphological delimitation of the two species in all sampled individuals. These results together suggest that these two species shared numerous ancestral cpDNA polymorphisms and point to the importance of nuclear DNA (ITS or genetic accumulation at multiple loci) in delimiting recently diverged species.

Diversity gradients and phylogeographic patterns in Santiria trimera (Burseraceae), a widespread African tree typical of mature rainforests

New insights into the history of the African rainforest can be gathered from the phylogeographic structures of their constituent species, but few studies have been performed in this ecosystem. We studied the phylogeographic structure of Santiria trimera, a primate- and bird-dispersed, dioecious tree typical of mature African rainforests. We sequenced three chloroplast DNA (cpDNA) regions (trnL-F, rbcL, and rpl36-infA-rps8) in 377 individuals from 42 populations. Sequence chromatograms regularly displayed double peaks of unequal heights. Cloning of PCR products and sequencing of outgroup taxa led to assigning the taller peak in ambiguous sequence positions to cpDNA. A total of 14 polymorphic cpDNA sites and 12 haplotypes were detected. Populations from three distinct biogeographic regions, namely, Upper Guinea, Lower Guinea, and the volcanic island of São Tomé, did not share any haplotype, indicating allopatric divergence. In Lower Guinea, Gabonese forests had high diversity mainly from the sympatry of two genetically divergent morphotypes, whereas forests of eastern Cameroon were less diversified. The two haplotypes of the morphotype without stilt roots were distributed north and south of the Ogooué River, suggesting refuges on both sides of the river bed. The divergence between Upper and Lower Guinean rainforests is explained by the discontinuity of forest between those regions throughout most of the Quaternary. The distribution of rare endemic haplotypes concurred with proposed Pleistocene rainforest refuges in west and southwest Cameroon. Overall, phylogeographic structure is consistent with the biogeographic hypotheses largely based on patterns of species diversity.

Genomic profiling of plastid DNA variation in the Mediterranean olive tree

BackgroundCharacterisation of plastid genome (or cpDNA) polymorphisms is commonly used for phylogeographic, population genetic and forensic analyses in plants, but detecting cpDNA variation is sometimes challenging, limiting the applications of such an approach. In the present study, we screened cpDNA polymorphism in the olive tree (Olea europaea L.) by sequencing the complete plastid genome of trees with a distinct cpDNA lineage. Our objective was to develop new markers for a rapid genomic profiling (by Multiplex PCRs) of cpDNA haplotypes in the Mediterranean olive tree.ResultsEight complete cpDNA genomes of Olea were sequenced de novo. The nucleotide divergence between olive cpDNA lineages was low and not exceeding 0.07%. Based on these sequences, markers were developed for studying two single nucleotide substitutions and length polymorphism of 62 regions (with variable microsatellite motifs or other indels). They were then used to genotype the cpDNA variation in cultivated and wild Mediterranean olive trees (315 individuals). Forty polymorphic loci were detected on this sample, allowing the distinction of 22 haplotypes belonging to the three Mediterranean cpDNA lineages known as E1, E2 and E3. The discriminating power of cpDNA variation was particularly low for the cultivated olive tree with one predominating haplotype, but more diversity was detected in wild populations.ConclusionsWe propose a method for a rapid characterisation of the Mediterranean olive germplasm. The low variation in the cultivated olive tree indicated that the utility of cpDNA variation for forensic analyses is limited to rare haplotypes. In contrast, the high cpDNA variation in wild populations demonstrated that our markers may be useful for phylogeographic and populations genetic studies in O. europaea.

Heterogeneity and Spatial Autocorrelation for Chloroplast Haplotypes in Three Old Growth Populations of Northern Red Oak

AbstractIn eastern North America, evidence for cryptic northern refugia could contribute to resolving Reid’s Paradox, the disparity between the rate of oak recolonization indicated by pollen deposition and the rate indicated by contemporary seed dispersal studies. Severe anthropogenic disturbance of oak-dominated forests throughout eastern North America followed by regeneration from isolated patches and deliberate planting in some regions could obscure the signal of discontinuity expected from small, cryptic refugia. In this study of northern red oak, Quercus rubra L., the dominant representative of Quercus section Lobatae in the eastern United States, we address the question of appropriate sample size for accurate detection of the biogeographical distribution of chloroplast haplotype diversity in Q. rubra. We examined chloroplast DNA (cpDNA) variation in all Q. rubra over 17 cm in diameter (310 trees) in three forest fragments with documented histories of minimal disturbance for the last 100-190 years. cpDNA polymorphisms in three intergenic regions revealed different haplotype frequencies between the two local fragments located within 1 km of each other and complete discontinuity for the predominant haplotype between these two sites and a site 207 km distant. Haplotypes displayed positive spatial autocorrelation over 10-40 meter distances. Sample sizes of 10 or fewer taken at 50 meter intervals along a linear transect yielded poor estimates of haplotype frequencies and did not accurately detect haplotype richness.

Chloroplast DNA variations in Mediterranean olive

SummaryOver ten years, several European teams have characterised polymorphism in the olive chloroplast genome (cpDNA). A particularly low level of cpDNA polymorphism was detected; but, in a recent study based on 14 Mediterranean trees, an intergenic (trnD-trnT) spacer was reported to be sufficiently variable to distinguish at least six haplotypes in cultivated olive. Compared to previous studies, the level of polymorphism described (nine substitutions and seven short indels on a segment of approx. 1 kbp) was particularly high. In order to enlarge upon the sample analysed, new trnD-trnT sequences were generated from 18 olive trees originating from the Mediterranean Basin, Macaronesia, S. Algeria, Kenya, Yemen, Reunion Island, Hawaii, Iran, and China. Only two base substitutions were detected in this enlarged sample, one of which discriminated between Olea europaea subsp. cuspidata and other olive sub-species. Length variation at a (poly-T) microsatellite motif was also found. The relatively low variation revealed between distant populations of olive suggests that sequencing problems might be the origin of the high polymorphism previously reported in the trnD-trnT spacer. Furthermore, length polymorphism at the poly-T motif was investigated in 55 Mediterranean olive trees which had previously been analysed for other cpDNA polymorphisms. Two alleles were found in Mediterranean olive, but they did not permit detection of new cpDNA haplotypes. It is proposed that proper analyses of cpDNA polymorphism in Mediterranean olive should use a combination of seven markers in order to detect its correct phylogeographic structure.

Chloroplast DNA Diversity in Prunus and Its Implication on Genetic Relationships

Chloroplast DNA (cpDNA) in 84 Prunus L. accessions (interspecific hybrids and Prunus species) were analyzed to confirm the maternal origin of the interspecific hybrids of Prunus and to establish genetic relationships among Prunus species. The polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method, which included amplification of cpDNA regions with three universal primer pairs (K1K2, HK, DT) and subsequent digestion with three restriction enzymes (AluI, HinfI, TaqI), revealed 33 haplotypes for the 84 accessions studied. Fourteen from these cpDNA haplotypes were shared by two or more accessions, and 19 were unique. Accessions sharing the same haplotype have maternal relationships among them, which allowed identity confirmation of maternal progenitors of Prunus interspecific hybrids in these cases. Unweighted pair group method average (UPGMA) and minimum-length spanning tree (MST) analyses were performed based on shared common fragments and the number of mutational differences among the 33 haplotypes, respectively. The cpDNA polymorphisms detected made possible the analysis of genetic relationships among the studied Prunus accessions. Most of the recovered relationships are in agreement with current taxonomic hypotheses and artificial crosses.

Relationships of Anagallis foemina and A. arvensis (Myrsinaceae): New insights inferred from DNA sequence data

We investigated the relationship between Anagallis arvensis and A. foemina using nuclear and plastid molecular data. Information from the nuclear rDNA internal transcribed spacer (ITS) region and four different chloroplast loci; ndhF, trnL-F, rpl16, and rps16 was analysed using both parsimony and Bayesian inference. Anagallis foemina was found to be most closely related to the perennial A. monelli, and not to A. arvensis. The existence of two different cpDNA haplotypes was revealed; one shared by Anagallis foemina, A. monelli, A. platyphylla, and one A. arvensis individual, while all other investigated A. arvensis individuals shared the second haplotype. Ancestral cpDNA polymorphism within Anagallis arvensis or hybridization are possible explanations, however, information in ITS data is too scarce to falsify any of these hypotheses.

CHLOROPLAST SHARING IN THE TASMANIAN EUCALYPTS

Abstract The biogeographic pattern of chloroplast DNA (cpDNA) haplotypes in Eucalyptus on the island of Tasmania is consistent with reticulate evolution, involving at least 12 Tasmanian species from the subgenus Symphyomyrtus. Intraspecific cpDNA polymorphism in 14 of 17 species is coupled with extensive sharing of identical haplotypes across populations of different species in the same geographic area. Haplotype diversity is lowest in central regions of Tasmania formerly occupied by alpine vegetation during glacial intervals and in northern regions that were periodically linked to continental Australia by land bridges. The observed distribution of several cpDNA haplotypes unique to Tasmania coincides with modeled locations of glacial refugia in coastal areas of Tasmania and shows the power of cpDNA in unraveling the complex history of past distributions of Eucalyptus. The results suggest that the model of evolution of the eucalypts should be reassessed to allow for the anastomosing effects of interspecific hybridization and introgression.

Inter-generic mitochondrial genome recombination in somatic hybrids between Citrus and Poncirus

SummaryThe genetic composition of somatic hybrids between ‘Page’ tangelo (Citrus reticulata × C. paradisi) and trifoliate orange (Poncirus trifoliata) were analysed in fifteen stochastically selected, fusion-derived plants. Flow cytometry revealed that all plants were tetraploids, and nuclear simple sequence repeats (SSR) showed that all plants possessed SSR loci from both fusion parents. Organellar genome compositions were examined using cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphism (RFLP) analyses. Mitochondrial genome (mtDNA) analyses indicated that all 15 hybrids had inherited regions of mtDNA common to both parents, and some specific bands from both fusion parents were also present in the hybrids, indicating the occurrence of mtDNA recombination in these somatic hybrids. No chloroplast genome (cpDNA) polymorphisms were detected by RFLP or CAPS analyses; however, random segregation of cpDNA was revealed by chloroplast simple sequence repeat (cpSSR) analyses. To evaluate genetic variation in mtDNA among the hybrids and both fusion parents, a dendrogram based on the RFLP data for 15 polymorphic mtDNA markers was generated by the Unweighted Pair Group Method with Arithmetic mean. The results showed that the 15 hybrids, and both parents, were located in three distinct clusters, and no variation was detected among these hybrids. The hybrids showed closer genetic similarity coefficients to the suspension cell parent than to the mesophyll cell parent, which demonstrated biased transmission of mtDNA. This is the first report of mtDNA recombination between Citrus and Poncirus by somatic fusion.

Cadmium hyperaccumulation and genetic differentiation of Thlaspi caerulescens populations

Although the knowledge on heavy metal hyperaccumulation mechanisms is increasing, the genetic basis of cadmium (Cd) hyperaccumulation remains to be elucidated. Thlaspi caerulescens is an attractive model since Cd accumulation polymorphism observed in this species suggests genetic differences between populations with low versus high Cd hyperaccumulation capacities. In our study, a methodology is proposed to analyse at a regional scale the genetic differentiation of T. caerulescens natural populations in relation to Cd hyperaccumulation capacity while controlling for different environmental, soil, plant parameters and geographic origins of populations. Twenty-two populations were characterised with AFLP markers and cpDNA polymorphism. Over all loci, a partial Mantel test showed no significant genetic structure with regard to the Cd hyperaccumulation capacity. Nevertheless, when comparing the marker variation to a neutral model, seven AFLP fragments (9% of markers) were identified as presenting particularly high genetic differentiation between populations with low and high Cd hyperaccumulation capacity. Using simulations, the number of outlier loci was showed to be significantly higher than expected at random. These loci presented a genetic structure linked to Cd hyperaccumulation capacity independently of the geography, environment, soil parameters and Zn, Pb, Fe and Cu concentrations in plants. Using a canonical correspondence analysis, we identified three of them as particularly related to the Cd hyperaccumulation capacity. This study demonstrates that populations with low and high hyperaccumulation capacities can be significantly distinguished based on molecular data. Further investigations with candidate genes and mapped markers may allow identification and characterization of genomic regions linked to factors involved in Cd hyperaccumulation.

Chloroplast microsatellites in barley: the reduction of variability spectrum in cultivated forms

The chloroplast genomes of 67 accessions of Hordeum vulgare L were surveyed for simple sequence repeat polymorphism. Seven SSR loci were investigated, trnL/trnF and psbI-trnS intergenic regions, as well as two regions 3' trnS genes were polymorphic. The rare variants of these loci were revealed and five rare accessions were found. Our results show an extremely low level of cpDNA polymorphism of cultivated barley.