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Populus, a deciduous tree species of major economic and ecological value, grows across the range in which trees are distributed in the Northern Hemisphere. Patterns of DNA variation are often used to identify the evolutionary forces shaping the genotypes of distinctive species lineages. Sodium/hydrogen (Na+/H+) antiporter genes have been shown to play a central role in plant salt tolerance. Here, we analyzed DNA nucleotide polymorphisms in the Na+/H+ antiporter (NHX and SOS1) gene families across 30 different Populus species using several methods of phylogenetic analysis and functional verification. NHX and SOS1 gene families in the genus Populus have expanded from the state in their common ancestors by duplication events, and their distinct lineages have been retained. Signals of positive selection at certain amino acid sites in different members of the Na/H antiporter gene families show that the dynamics that drive the evolution of each gene vary. SOS1 has undergone duplication in Populus euphratica and been subjected to adaptive evolution in section Turanga; the paralog of PeSOS1 (PeSOS1.2) can complement the Escherichia coli mutant EP432; and the expression pattern of PeSOS1.2 is different from that of PeSOS1, a fact which may have been beneficial for P. euphratica, conferring a fitness advantage in saline habitats. The divergent evolution of the individual members of the NHX and SOS1 gene families is likely to have been influenced by the varied ecological and environmental niches occupied by the different poplar species, giving rise to evolutionary footprints that reflect the specific functions and subcellular localizations of the proteins encoded by these genes.

Despite the high economic and ecological importance of forests, our knowledge of the genomic evolution of trees under salt stress remains very limited. Here we report the genome sequence of the desert poplar, Populus euphratica, which exhibits high tolerance to salt stress. Its genome is very similar and collinear to that of the closely related mesophytic congener, P. trichocarpa. However, we find that several gene families likely to be involved in tolerance to salt stress contain significantly more gene copies within the P. euphratica lineage. Furthermore, genes showing evidence of positive selection are significantly enriched in functional categories related to salt stress. Some of these genes, and others within the same categories, are significantly upregulated under salt stress relative to their expression in another salt-sensitive poplar. Our results provide an important background for understanding tree adaptation to salt stress and facilitating the genetic improvement of cultivated poplars for saline soils.

Populus euphratica is well-adapted to extreme desert environments and is an important model species for studying the effects of abiotic stresses on trees. Here we present the first deep transcriptomic analysis of this species. To maximize representation of conditional transcripts, mRNA was obtained from living tissues of desert-grown trees and two types of callus (salt-stressed and unstressed). De novo assembly generated 86,777 Unigenes using Solexa sequence data. These sequences covered 92% of previously reported P. euphratica expressed sequence tags (ESTs) and 90% of the TIGR poplar ESTs, and a total of 58,499 high-quality unique sequences were annotated by BLAST similarity searches against public databases. We found that 27% of the total Unigenes were differentially expressed (up- or down-regulated) in response to salt stress in P. euphratica callus. These differentially expressed genes are mainly involved in transport, transcription, cellular communication and metabolism. In addition, we found that numerous putative genes involved in ABA regulation and biosynthesis were also differentially regulated. This study represents the deepest transcriptomic and gene-annotation analysis of P. euphratica to date. The genetic knowledge acquired should be very useful for future studies of the molecular adaptation of this tree species to abiotic stress and facilitate genetic manipulation of other poplar species.

Background: The fixed genetic differences between ecologically divergent species were found to change greatly depending on the markers examined. With such species it is difficult to differentiate between shared ancestral polymorphisms and past introgressions between the diverging species. In order to disentangle these possibilities and provide a further case for DNA barcoding of plants, we examine genetic differentiation between two ecologically divergent poplar species, Populus euphratica Oliver and P. pruinosa Schrenk using three different types of genetic marker., Methodology/principal Findings: We genotyped 290 individuals from 29 allopatric and sympatric populations, using chloroplast (cp) DNA, nuclear (nr) ITS sequences and eight simple sequence repeat (SSR) loci. Three major cpDNA haplotypes were widely shared between the two species and between-species cpDNA differentiation (F(CT)) was very low, even lower than among single species populations. The average SSR F(CT) values were higher. Bayesian clustering analysis of all loci allowed a clear delineation of the two species. Gene flow, determined by examining all SSR loci, was obvious but only slightly asymmetrical. However, the two species were almost fixed for two different nrITS genotypes that had the highest F(CT), although a few introgressed individuals were detected both in allopatric and sympatric populations., Conclusions: The two species shared numerous ancestral polymorphisms at cpDNA and a few SSR loci. Both ITS and a combination of nuclear SSR data could be used to differentiate between the two species. Introgressions and gene flow were obvious between the two species either during or after their divergence. Our findings underscore the complex genetic differentiations between ecologically diverged species and highlight the importance of nuclear DNA (especially ITS) differentiation for delimiting closely related plant species.

Populus euphratica is a salt-tolerant tree species growing in semi-arid saline areas. A Na(+)/H(+) antiporter gene was successfully isolated from this species through RACE cloning, and named PeSOS1. The isolated cDNA was 3665 bp long and contained a 3438 bp open reading frame that was predicted to encode a 127-kDa protein with 12 hypothetical transmembrane domains in the N-terminal part and a long hydrophilic cytoplasmic tail in the C-terminal part. The amino acid sequence of this PeSOS1 gene showed 64% identity with the previously isolated SOS1 gene from the glycophyte Arabidopsis thaliana. The level of protein expressed by PeSOS1 in the leaves of P. euphratica was significantly up-regulated in the presence of high (200 mM) concentrations of NaCl, while the mRNA level in the leaves remained relatively constant. Immunoanalysis suggested that the protein encoded by PeSOS1 is localized in the plasma membrane. Expression of PeSOS1 partially suppressed the salt sensitive phenotypes of the EP432 bacterial strain, which lacks the activity of the two Na(+)/H(+) antiporters EcNhaA and EcNhaB. These results suggest that PeSOS1 may play an essential role in the salt tolerance of P. euphratica and may be useful for improving salt tolerance in other tree species.