Exome resequencing reveals signatures of demographic and adaptive processes across the genome and range of black cottonwood (Populus trichocarpa)

Extant variation in temperate and boreal plant species has been influenced by both demographic histories associated with Pleistocene glacial cycles and adaptation to local climate. We used sequence capture to investigate the role of these neutral and adaptive processes in shaping diversity in black cottonwood (Populus trichocarpa). Nucleotide diversity and Tajima's D were lowest at replacement sites and highest at intergenic sites, while LD showed the opposite pattern. With samples grouped into three populations arrayed latitudinally, effective population size was highest in the north, followed by south and centre, and LD was highest in the south followed by the north and centre, suggesting a possible northern glacial refuge. F_ST outlier analysis revealed that promoter, 5′‐UTR and intronic sites were enriched for outliers compared with coding regions, while no outliers were found among intergenic sites. Codon usage bias was evident, and genes with synonymous outliers had 30% higher average expression compared with genes containing replacement outliers. These results suggest divergent selection related to regulation of gene expression is important to local adaptation in P. trichocarpa. Finally, within‐population selective sweeps were much more pronounced in the central population than in putative northern and southern refugia, which may reflect the different demographic histories of the populations and concomitant effects on signatures of genetic hitchhiking from standing variation.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Molecular Evolution of CXCR1, a G Protein-Coupled Receptor Involved in Signal Transduction of Neutrophils

Human neutrophils are a type of white blood cell, which forms an early line of defense against bacterial infections. Neutrophils are highly responsive to the chemokine, interleukin-8 (IL-8) due to the abundant distribution of CXCR1, one of the IL-8 receptors on the neutrophil cell surface. As a member of the GPCR family, CXCR1 plays a crucial role in the IL-8 signal transduction pathway in neutrophils. We sequenced the complete coding region of the CXCR1 gene in worldwide human populations and five representative nonhuman primate species. Our results indicate accelerated protein evolution in the human lineage, which was likely caused by Darwinian positive selection. The sliding window analysis and the codon-based neutrality test identified signatures of positive selection at the N-terminal ligand/receptor recognition domain of human CXCR1. [Reviewing Editor: Dr. Manyuan Long] The GenBank accession numbers of sequences reported herein are AY916760–AY916773.     

Rates and relations of mitochondrial genome evolution across the Echinoidea,with special focus on the superfamily Odontophora

In order to better characterize the placement of genus Tripneustes, as a representative of the Toxopneustidae family within the broader sea urchin mitochondrial (MT) phylogeny, the complete MT genome of Tripneustes gratilla was generated and compared with all published echinoid MT genomes currently available on NCBI GenBank. The MT genome phylogeny supports the existence of the superfamily Odontophora (consisting of the families Strongylocentrotidae, Echinometridae, and Toxopneustidae). A relaxed molecular‐clock time calibration suggests a split between the three key Odontophore MT lineages occurred during the late Eocene/Oligocene. Major global oceanographic changes have been inferred during this time frame, potentially driving species diversification through environmental selection pressures. To test for signatures of selection acting on the mitochondria, the historical rate of gene evolution of individual MT genes was assessed through a branch‐site comparison of nonsynonymous to synonymous substitution ratios (ω). Models of positive selection and neutral evolution, as compared via a likelihood ratio test, show no evidence of strong historical positive selection on mitochondrial genes at the genesis of the Odontophora. However, while pairwise ω comparison revealed signatures of strong negative selection, relatively elevated ω values were observed within the Strongylocentrotus genus.     

Isolation of DNA from the Uncultured “Candidatus Liberobacter” Species Associated with Citrus Huanglongbing by RAPD

“Candidatus Liberobacter,” the uncultured bacterium associated with citrus Huanglongbing (HLB) disease, is an α-Proteobacteria, and two species, “Candidatus L. africanum” and “Candidatus L. asiaticum,” have been characterized by sequence analysis of the 16S rDNA and β operon (rplKAJL-rpoBC) genes. These genes were isolated by PCR and random cloning of DNA from infected plants. However, this strategy is laborious and allowed selection of only three Liberobacter DNA fragments. In this paper, we described isolation of additional genes using Random Amplified Polymorphic DNA (RAPD). In total, 102 random 10-mer primers were used in PCR reactions on healthy and Liberobacter-infected plant DNA. Eight DNA bands amplified from infected plant DNA were cloned and analyzed. Six of them were found to be part of the Liberobacter genome by sequence and hybridization experiments. On these DNA fragments, four genes were identified: nusG, pgm, omp, and a hypothetical protein gene. These results indicate that RAPD can be used to clone DNA of uncultured organisms. Received: 14 September 1998 / Accepted: 6 October 1998     

Nucleotide diversity patterns of three divergent soybean populations: evidences for population‐dependent linkage disequilibrium and taxonomic status of Glycine gracilis

The level of linkage disequilibrium (LD) is a major factor to determine DNA polymorphism pattern of a population and to construct high‐resolution maps useful in localizing and gene cloning of complicated traits. Here, we investigated LD level of three soybean populations with different genetic backgrounds and taxonomic status of G. gracilis by comparing the DNA polymorphism patterns of four high‐diversity single‐copy nuclear genes. A total of 152, 22, and 77 accessions of G. soja, G. gracilis, and G. max were observed. The results indicated that G. max retained only 75.3 (π) and 39% (θ) of the nucleotide polymorphism found in G. soja. Four gene loci evolved according to neutrality in both G. max and G. gracilis populations, and three gene loci evolved according to neutrality in G. soja population by Tajima's and Fu and Li's test. However, one gene locus deviated from neutrality by Fu and Li's test in the G. soja population. Further, medial level of LD (average r² = 0.2426) was found in intragene in G. max and G. gracilis populations, but unexpected low level of LD (r² ≤ 0.0539) was found in G. soja population. Significant genetic differentiation was detected between G. max and G. soja populations and also between G. max and G. gracilis populations; however, nonsignificant genetic differentiation was found between G. gracilis and G. soja populations. The results suggest that LD level depends on genetic background of soybean population, and implicit that G. gracilis should be regarded as the variant of G. soja, not as an independent species.     

Genetic analysis of disease resistance to all strains of BaYMV in a Chinese barley landrace, Mokusekko 3

 A Chinese landrace of barley, Mokusekko 3, is unique in being completely resistant against all strains of barley yellow mosaic virus (BaYMV). The present investigation revealed that the resistance of Mokusekko 3 is governed by two recessive genes. As one of the resistance genes was known to be tightly linked with alleles at the Est complex locus, consisting of the Est1, Est2 and Est4 loci for esterase isozymes, each of the resistance genes could be separated by means of marker-assisted selection using an isozyme allelic combination as a marker. One of the resistance genes, ym1, is linked to K (hooded lemma) and gl3 (glossy leaf 3) with recombination values of 25.3% and 9.7% respectively, and these three genes are located in the order K-gl3-ym1 on chromosome 4. Another newly designated resistance gene, ym5, is linked to alleles at the Est complex locus and cu2 (curly growth 2), with recombination values of 1.9% and 19.5% respectively, in the order cu2-Est-ym5 from proximal to distal on the long arm of chromosome 3. The complete resistance of Mokusekko 3 is caused by combining two resistance genes, ym1 and ym5. However, almost all the “resistant” cultivars derived from crosses with Mokusekko 3 are susceptible to the recently detected strain BaYMV-III in Japan, since they contain only one resistance gene, ym5. Marker-assisted selection to combine resistance genes into a cultivar is discussed for the breeding of stabilizing resistance to BaYMV. Received: 23 September 1996 / Accepted: 8 November 1996     

Patterns of nucleotide sequence variation in <Emphasis Type="Italic">ICAM1</Emphasis> and <Emphasis Type="Italic">TNF</Emphasis> genes in twelve ethnic groups of India: roles of demographic history and natural selection

We have studied DNA sequence variation in and around the genes ICAM1 and TNF, which play functional and correlated roles in inflammatory processes and immune cell responses, in 12 diverse ethnic groups of India, with a view to investigating the relative roles of demographic history and natural selection in shaping the observed patterns of variation. The total numbers of single nucleotide polymorphisms (SNPs) detected at the ICAM1 and TNF loci were 29 and 12, respectively. Haplotype and allele frequencies differed significantly across populations. The site frequency spectra at these loci were significantly different from those expected under neutrality, and showed an excess of intermediate-frequency variants consistent with balancing selection. However, as expected under balancing selection, there was no significant reduction of F _ST values compared to neutral autosomal loci. Mismatch distributions were consistent with population expansion for both loci. On the other hand, the phylogenetic network among haplotypes for the TNF locus was similar to expectations under population expansion, while that for the ICAM1 was as expected under balancing selection. Nucleotide diversity at the ICAM1 locus was an order of magnitude lower in the promoter region, compared to the introns or exons, but no such difference was noted for the TNF gene. Thus, we conclude that the pattern of nucleotide variation in these genes has been modulated by both demographic history and selection. This is not surprising in view of the known allelic associations of several polymorphisms in these genes with various diseases, both infectious and noninfectious.     

Revisiting protein heterozygosity in plants��nucleotide diversity in allozyme coding genes of conifer Pinus sylvestris

Allozyme variation has been and continues to be a major source of information on the level of genetic variation among plant species. Deciphering the molecular basis of electrophoretic variation is essential for understanding the forces affecting the protein level variation. In this study, the relationship between allozyme heterozygosity and nucleotide diversity in plants is investigated among and within species. Allozyme and nucleotide diversity in 27 plant species was reviewed. At the multilocus level, the two methods are congruent: a clear correlation between the two measures of genetic diversity among plant species was observed, strengthening the view that effective population size is the major determinant of genome-wide diversity. Nucleotide diversity at six allozyme coding genes (6pgdB, aco, gdh, gotC, mdhA, and mdhB) in conifer Pinus sylvestris was investigated jointly with electrophoretic data. Single non-synonymous charge-changing mutations were found together with electrophoretic alleles that consequently were mutationally unique. Synonymous site nucleotide diversity (point estimate of θ _W—0.009 per bp) and silent site divergence from Pinus pinaster at allozyme coding loci were at comparable levels with other loci in the species. Linkage disequilibrium was extensive compared to earlier estimates from P. sylvestris and other trees, spanning several kilobases. Allozyme coding genes had an excess of closely related haplotypes whose frequency has recently increased possibly as a result of partial selective sweeps or balancing selection, but complex demographic effects cannot be excluded.     

Molecular phylogenetic studies of Lachnum and its allies based on the Japanese material

Molecular phylogenetic studies were carried out based on ITS-5.8S rDNA, the D1–D2 region of the large subunit rRNA gene, RPB2, and combined data of D1–D2 and RPB2 as well as these three genes on 36 species among 7 genera for Lachnum and allied genera in the family Hyaloscyphaceae. In the combined data of all three regions, seven strongly supported clades were obtained. The same clades were also recognized in most of the trees based on each gene, and the combined data of D1–D2 and RPB2, although some of them were not strongly supported. Four clades represented Albotricha, Brunnipila, Incrucipulum, and Lachnellula, respectively, whereas Lachnum was distributed to the remaining three clades. The molecular phylogenies strongly supported a group of species with granulate hairs, and we suggest the concept of Lachnaceae should be restricted to these species. Based on the molecular phylogenetic analysis, three new combinations—Incrucipulum longispineum, I. radiatum, and Lachnellula pulverulentum from Lachnum—are proposed.     

Characterization of expressed class II MHC sequences in the banner-tailed kangaroo rat (<Emphasis Type="Italic">Dipodomys spectabilis</Emphasis>) reveals multiple <Emphasis Type="Italic">DRB</Emphasis> loci

Genes of the major histocompatibility complex (MHC) are exceptionally polymorphic due to the combined effects of natural and sexual selection. Most research in wild populations has focused on the second exon of a single class II locus (DRB), but complete gene sequences can provide an illuminating backdrop for studies of intragenic selection, recombination, and organization. To this end, we characterized class II loci in the banner-tailed kangaroo rat (Dipodomys spectabilis). Seven DRB-like sequences (provisionally named MhcDisp-DRB*01 through *07) were isolated from spleen cDNA and most likely comprise ≥5 loci; this multiformity is quite unlike the situation in muroid rodents such as Mus, Rattus, and Peromyscus. In silico translation revealed the presence of important structural residues for glycosylation sites, salt bonds, and CD4+ T-cell recognition. Amino-acid distances varied widely among the seven sequences (2–34%). Nuclear DNA sequences from the Disp-DRB*07 locus (∼10 kb) revealed a conventional exon/intron structure as well as a number of microsatellites and short interspersed nuclear elements (B4, Alu, and IDL-Geo subfamilies). Rates of nucleotide substitution at Disp-DRB*07 are similar in both exons and introns (π = 0.015 and 0.012, respectively), which suggests relaxed selection and may indicate that this locus is an expressed pseudogene. Finally, we performed BLASTn searches against Dipodomys ordii genomic sequences (unassembled reads) and find 90–97% nucleotide similarity between the two kangaroo rat species. Collectively, these data suggest that class II diversity in heteromyid rodents is based on polylocism and departs from the muroid architecture. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers EU817477–EU817485.     

Genetic diversity analysis of abiotic stress response gene <Emphasis Type="Italic">TaSnRK2.7</Emphasis>-<Emphasis Type="Italic">A</Emphasis> in common wheat

Sucrose non-fermenting1-related protein kinase 2 (SnRK2) plays a key role in plant stress signaling transduction pathways. In this study, one copy of TaSnRK2.7, a SnRK2 member of common wheat, was isolated and characterized for nucleotide diversity among 45 wheat accessions with different stress-response features. Most of the accessions were elite wheat cultivars, which had been subject to population bottlenecks and intensive selection during breeding. Nucleotide and haplotype diversity across the entire TaSnRK2.7-A region was 0.00076 and 0.590, respectively, and diversity in non-coding regions was higher than that in coding regions. Sliding-window analysis showed variable levels of nucleotide variation along the entire TaSnRK2.7-A region; the sixth intron and ninth exon represented variation-enriched regions. As predicted, neutrality tests revealed that population bottlenecks or purifying selection had acted on the TaSnRK2.7-A gene, a relatively conserved gene. Furthermore, strong linkage disequilibrium between SNP loci extends across the entire TaSnRK2.7-A region. These findings demonstrate that the TaSnRK2.7-A genomic region has evolved under extensive selection pressure during crop breeding.     

Molecular population genetics of the<Emphasis Type="Italic">β-esterase</Emphasis> gene cluster of<Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We have investigated nucleotide polymorphism at theβ-esterase gene cluster including theEst-6 gene andψEst-6 putative pseudogene in four samples ofDrosophila melanogaster derived from natural populations of southern Africa (Zimbabwe), Europe (Spain), North America (USA: California), and South America (Venezuela). A complex haplotype structure is revealed in bothEst-6 andψEst-6. Total nucleotide diversity is twice inψEst-6 as inEst-6; diversity is higher in the African sample than in the non-African ones. Strong linkage disequilibrium occurs within theβ-esterase gene cluster in non-African samples, but not in the African one. Intragenic gene conversion events are detected withinEst-6 and, to a much greater extent, withinyEst-6; intergenic gene conversion events are rare. Tests of neutrality with recombination are significant for theβ-esterase gene cluster in the non-African samples but not significant in the African one. We suggest that the demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the pattern of nucleotide polymorphism in theb-esterase gene cluster. However there are some ’footprints’ of directional and balancing selection shaping specific distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection betweenEst-6 andψEst-6 may play an important role in the evolution of theβ-esterase gene cluster preserving the putative pseudogene from degenerative destruction and reflecting possible functional interaction between the functional gene and the putative pseudogene.Est-6 andyEst-6 may represent an indivisible intergenic complex (‘intergene’) in which each single component (Est-6 orψEst-6) cannot separately carry out the full functional role.     

Patterns of genetic variation within a captive population of Amur tigerPanthera tigris altaica

Eight founders and thirty-one descendants were sampled as the Founder group and the Offspring group respectively from a captive population of Amur tigerPanthera tigris altaica Temminck, 1844 for population genetic analysis with RAPD and ISSR markers. Integrated with demographic data during the initial recovery stage, results showed: (1) increasing the population size (N) and the effective population size (N _e) greatly retard lose of genetic variation induced mainly by genetic drift and selection; (2) recombination and admixture could cause the Offspring group (5.711%) and the Founder group (10.383%) to hold different linkage disequilibrium (LD); (3) further Ohta’s variance analysis indicated genetic drift (87.3%) and epistatic selection (12.7%) maintained LD in population, whereas GENEDROP analysis supported epistatic selection largely derived from artificial selection of managers; (4) both Tajima’s test and Fu’s test confirmed the statistic neutrality of genetic markers used, moreover the positive value of Tajima’sD (0.090) together with the result that π (25.286) was bigger than ϑ (24.898) revealed the Founder group was admixture population, while the negative Tajima’sD value (−0.053) together with the result that π (23.679) was less than ϑ (23.912) disclosed the Offspring group experienced selective sweep.     

Signatures of selection in mammalian clock genes with coding trinucleotide repeats: Implications for studying the genomics of high‐pace adaptation

Climate change is predicted to affect the reproductive ecology of wildlife; however, we have yet to understand if and how species can adapt to the rapid pace of change. Clock genes are functional genes likely critical for adaptation to shifting seasonal conditions through shifts in timing cues. Many of these genes contain coding trinucleotide repeats, which offer the potential for higher rates of change than single nucleotide polymorphisms (SNPs) at coding sites, and, thus, may translate to faster rates of adaptation in changing environments. We characterized repeats in 22 clock genes across all annotated mammal species and evaluated the potential for selection on repeat motifs in three clock genes (NR1D1, CLOCK, and PER1) in three congeneric species pairs with different latitudinal range limits: Canada lynx and bobcat (Lynx canadensis and L. rufus), northern and southern flying squirrels (Glaucomys sabrinus and G. volans), and white‐footed and deer mouse (Peromyscus leucopus and P. maniculatus). Signatures of positive selection were found in both the interspecific comparison of Canada lynx and bobcat, and intraspecific analyses in Canada lynx. Northern and southern flying squirrels showed differing frequencies at common CLOCK alleles and a signature of balancing selection. Regional excess homozygosity was found in the deer mouse at PER1 suggesting disruptive selection, and further analyses suggested balancing selection in the white‐footed mouse. These preliminary signatures of selection and the presence of trinucleotide repeats within many clock genes warrant further consideration of the importance of candidate gene motifs for adaptation to climate change.     

Validation and application of reference genes for quantitative gene expression analyses in various tissues of <Emphasis Type="Italic">Bupleurum chinense</Emphasis>

It is crucial to select stable references in gene expression analyses using quantitative real-time PCR (qRT-PCR). In this work, seven frequently used reference genes, 18S, Actin, EF1α, α-tubulin, β-tubulin, Cyclophilin and Cytoplasmic ribosomal protein L2 (L2), from Bupleurum chinense DC. were evaluated as the internal control in five tissues, roots, stems, leaves, flowers and fruits, before tissue specific gene expression assays. The results showed that β-tubulin was the most stable and reliable reference gene among the seven candidate genes in the measured tissues. The expression levels of four genes involved in saikosaponins (the pharmacological active compounds of B. chinense) biosynthesis, HMGR, IPPI, FPS and β-AS, were assayed with β-tubulin as the internal control in the five tissues. All the four genes were expressed in the five tissues with different profiles and HMGR in the order of roots > flowers, stems and leaves > fruits, IPPI of stems > leaves and fruits > roots and flowers, FPS of flowers > fruits > stems and roots > leaves and β-AS of roots > flowers, stems and fruits > leaves. The genes of FPS and β-AS were expressed predominantly in flowers and roots, respectively. This study may provide a suitable internal control for quantitative gene expression assays in various tissues and give insight into the tissue expression profiles of four saikosaponins biosynthesis-involved genes of medicinal B. chinense.