Molecular evolution of glutathione peroxidase (<Emphasis Type="Italic">Gpx</Emphasis>) family genes in desert poplar (<Emphasis Type="Italic">Populus euphratica</Emphasis> Oliv.)

Populus euphratica Oliv. is a poplar species that is distributed mainly in deserts, making it an interesting model in which to investigate molecular mechanisms underlying different stress responses. Here, we used molecular population genetic methods to detect potential selection in candidate genes belonging to the P. euphratica glutathione (GSH) peroxidase (Gpx) family, which are associated with an enzymatic mechanism that combats oxidative damage caused by reactive oxygen species (ROS) in plant cells; earlier studies have shown that Gpx proteins play important roles in coping with increased ROS levels during biotic and abiotic stresses in plants. We analyzed the nucleotide diversity and divergence patterns of five loci encoding Gpx genes, and 16 reference loci used as controls, in order to detect departures from the neutral expectation. Gpx1 has an excess of mid-frequency alleles; high intraspecific nucleotide diversity, distributed in the upper tail of the simulated neutral model; and extensive LD, showing strong evidence of balancing selection/local adaptation. The Gpx3.2 gene exhibits very low nucleotide diversity and divergence, suggesting that it has evolved under strong purifying selection. We failed to detect any evidence for natural selection at the other loci (Gpx2, Gpx4, and Gpx5) compared with the reference loci. The results show that nucleotide diversity and/or divergence differ greatly between members of the Gpx gene family, resulting from differential selective pressure acting on genes, and that adaptive evolution influenced the distribution of P. euphratica in desert regions.     

Intraspecific Variation in Mitogenomes of Five <Emphasis Type="Italic">Crassostrea</Emphasis> Species Provides Insight into Oyster Diversification and Speciation

A large number of Crassostrea oysters are found in Asia-Pacific. While analyses of interspecific variation have helped to establish historical relationships among these species, studies on intraspecific variation are necessary to understand their recent evolutionary history and current forces driving population biology. We resequenced 18 and analyzed 31 mitogenomes of five Crassostrea species from China: Crassostrea gigas, Crassostrea angulata, Crassostrea sikamea, Crassostrea ariakensis, and Crassostrea hongkongensis. Our analysis finds abundant insertions, deletions, and single-nucleotide polymorphisms in all species. Intraspecific variation varies greatly among species with polymorphic sites ranging from 54 to 293 and nucleotide diversity ranging from 0.00106 to 0.00683. In all measurements, C. hongkongensis that has the narrowest geographic distribution exhibits the least sequence diversity; C. ariakensis that has the widest distribution shows the highest diversity, and species with intermediate distribution show intermediate levels of diversity. Low sequence diversity in C. hongkongensis may reflect recent bottlenecks that are probably exacerbated by human transplantation. High diversity in C. ariakensis is likely due to divergence of northern and southern China populations that have been separated without gene flow. The significant differences in mitogenome diversity suggest that the five sister species of Crassostrea have experienced different evolutionary forces since their divergence. The recent divergence of two C. ariakensis populations and the C. gigas/angulata species complex provides evidence for continued diversification and speciation of Crassostrea species along China’s coast, which are shaped by unknown mechanisms in a north–south divide.     

Transposition mechanism,molecular characterization and evolution of IS<Emphasis Type="Italic">6110</Emphasis>, the specific evolutionary marker of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> complex

The mycobacterial insertion sequence IS6110 proved crucial in deciphering tuberculosis (TB) transmission dynamics. This sequence was also shown to play an important role in the pathogenicity (transmission ability and/or virulence) of Mycobacterium tuberculosis, the main causative agent of TB in humans. In this study, we explored the usefulness of IS6110 and its potential as a phylogenetic/typing marker. We also analyzed the genetic polymorphism and evolutionary trends (selective pressure) of its transposase-encoding open reading frames (ORFs), A and B, using the maximum likelihood method. Both ORFs evolved chronologically through random single nucleotide polymorphisms. They were subjected to strict purifying selection more tight on orfA, with no evidence of significant recombination events. OrfA proved to have a crucial role in regulating the transpositional process. Several analyses showed that IS6110 acquisition antedated the emergence of the Mycobacterium tuberculosis complex. This original copy of IS6110 element was functionally optimal. In conclusion, this study not only demonstrated the usefulness of IS6110 in terms of phylogenetic and typing purposes and its transpositional mechanism, but also informed the scientific community on its evolutionary history.     

Single-nucleotide polymorphism,linkage disequilibrium and geographic structure in the malaria parasite <Emphasis Type="Italic">Plasmodium vivax</Emphasis>: prospects for genome-wide association studies

Background

The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized.

Results

We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance.

Conclusion

These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.See commentary: http://www.biomedcentral.com/1741-7007/8/90

     

Modern State of Populations of Endemic <Emphasis Type="Italic">Oxytropis</Emphasis> Species from Baikal Siberia and Their Phylogenetic Relationships Based on Chloroplast DNA Markers

The genetic diversity and population structure of the endemic species of Baikal Siberia Oxytropis triphylla, O. bargusinensis, and O. interposita were studied for the first time on the basis of the nucleotide polymorphism of intergenic spacers psbA–trnH, trnL–trnF, and trnS–trnG of chloroplast DNA. All populations of these species were characterized by a high haplotype (0.762–0.924) and relatively low nucleotide (0.0011–0.0022) diversity. Analysis of the distribution of variability in O. triphylla and O. bargusinensis showed that there was no significant genetic differentiation between populations of each species; the gene flow was 4.43 and 8.91, respectively. The high level of genetic diversity in the studied populations indicates a relatively stable state of these populations. A study of the phylogenetic relationships of closely related species confirms the concept of the origin of O. bargusinensis and O. tompudae as a result of intersectional hybridization of the species of the sections Orobia and Verticillares.     

Genetic diversity of MHC class II <Emphasis Type="Italic">DRB</Emphasis> alleles in the continental and Japanese populations of the sable <Emphasis Type="Italic">Martes zibellina</Emphasis> (Mustelidae,Carnivora, Mammalia)

The sable (Martes zibellina) is a medium-sized mustelid inhabiting forest environments in Siberia, northern China, the Korean Peninsula, and Hokkaido Island, Japan. To further understand the molecular evolution of the major histocompatibility complex (MHC), we sequenced part of exon 2 in MHC class II DRB genes, including codons encoding the antigen binding site, from 33 individuals from continental Eurasia and Japan. We identified 16 MHC class II DRB alleles (Mazi-DRBs), some of which were geographically restricted and others broadly distributed, and eight putative pseudogenes. A single-breakpoint recombination analysis detected a recombination site in the middle of exon 2. A mixed effects model of evolution analysis identified five amino acid sites presumably under positive selection. These sites were all located in the region 3′ to the recombination site, suggesting that positive selection and recombination could be committed to the diversity of the M. zibellina DRB gene. In a Bayesian phylogenetic tree, all Mazi-DRBs and the presumed pseudogenes grouped within a Mustelidae clade. The Mazi-DRBs showed trans-species polymorphism, with some alleles most closely related to alleles from other mustelid species. This result suggests that the sable DRBs have evolved under long-lasting balancing selection.     

A preliminary integrated genetic map distinguishes every chromosome pair and locates essential genes related to abiotic adaptation of <Emphasis Type="Italic">Crassostrea angulata</Emphasis>/<Emphasis Type="Italic">gigas</Emphasis>

Background

The re-sequencing of C. angulata has revealed many polymorphisms in candidate genes related to adaptation to abiotic stress that are not present in C. gigas; these genes, therefore, are probably related to the ability of this oyster to retain high concentrations of toxic heavy metals. There is, in addition, an unresolved controversy as to whether or not C. angulata and C. gigas are the same species or subspecies. Both oysters have 20 metacentric chromosomes of similar size that are morphologically indistinguishable. From a genomic perspective, as a result of the great variation and selection for heterozygotes in C. gigas, the assembly of its draft genome was difficult: it is fragmented in more than seven thousand scaffolds.

Results

In this work sixty BAC sequences of C. gigas downloaded from NCBI were assembled in BAC-contigs and assigned to BACs that were used as probes for mFISH in C. angulata and C. gigas. In addition, probes of H3, H4 histone, 18S and 5S rDNA genes were also used. Hence we obtained markers identifying 8 out the 10 chromosomes constituting the karyotype. Chromosomes 1 and 9 can be distinguished morphologically. The bioinformatic analysis carried out with the BAC-contigs annotated 88 genes. As a result, genes associated with abiotic adaptation, such as metallothioneins, have been positioned in the genome. The gene ontology analysis has also shown many molecular functions related to metal ion binding, a phenomenon associated with detoxification processes that are characteristic in oysters. Hence the provisional integrated map obtained in this study is a useful complementary tool for the study of oyster genomes.

Conclusions

In this study 8 out of 10 chromosome pairs of Crassostrea angulata/gigas were identified using BAC clones as probes. As a result all chromosomes can now be distinguished. Moreover, FISH showed that H3 and H4 co-localized in two pairs of chromosomes different that those previously escribed. 88 genes were annotated in the BAC-contigs most of them related with Molecular Functions of protein binding, related to the resistance of the species to abiotic stress. An integrated genetic map anchored to the genome has been obtained in which the BAC-contigs structure were not concordant with the gene structure of the C. gigas scaffolds displayed in the Genomicus database.

     

Genome-wide single nucleotide polymorphisms (SNPs) for a model invasive ascidian <Emphasis Type="Italic">Botryllus schlosseri</Emphasis>

Invasive species cause huge damages to ecology, environment and economy globally. The comprehensive understanding of invasion mechanisms, particularly genetic bases of micro-evolutionary processes responsible for invasion success, is essential for reducing potential damages caused by invasive species. The golden star tunicate, Botryllus schlosseri, has become a model species in invasion biology, mainly owing to its high invasiveness nature and small well-sequenced genome. However, the genome-wide genetic markers have not been well developed in this highly invasive species, thus limiting the comprehensive understanding of genetic mechanisms of invasion success. Using restriction site-associated DNA (RAD) tag sequencing, here we developed a high-quality resource of 14,119 out of 158,821 SNPs for B. schlosseri. These SNPs were relatively evenly distributed at each chromosome. SNP annotations showed that the majority of SNPs (63.20%) were located at intergenic regions, and 21.51% and 14.58% were located at introns and exons, respectively. In addition, the potential use of the developed SNPs for population genomics studies was primarily assessed, such as the estimate of observed heterozygosity (H _O ), expected heterozygosity (H _E ), nucleotide diversity (π), Wright’s inbreeding coefficient (F _IS ) and effective population size (Ne). Our developed SNP resource would provide future studies the genome-wide genetic markers for genetic and genomic investigations, such as genetic bases of micro-evolutionary processes responsible for invasion success.     

Genetic characterization of <Emphasis Type="Italic">Wolbachia</Emphasis> from Great Salt Lake brine flies

Wolbachia are intracellular prokaryotic endosymbionts associated with a wide distribution of arthropod and nematode hosts. Their association ranges from parasitism to mutualism, and there is growing evidence that Wolbachia can have dramatic effects on host reproduction, physiology, and immunity. Although all Wolbachia are currently considered as single species, W. pipientis, phylogenetic studies reveal about a dozen monophyletic groups, each designated as a supergroup. This study uses 16S rRNA gene sequences to examine the genetic diversity of Wolbachia present in three species of Great Salt Lake brine flies, Cirrula hians, Ephydra gracilis, and Mosillus bidentatus. The brine fly Wolbachia sequences are highly similar, with an average nucleotide sequence divergence among the three species of 0.00174. The brine fly Wolbachia form a monophyletic group that is affiliated with a subset of supergroup B, indicating that this supergroup may be more diverse than previously thought. These findings expand the phylogenetic diversity of Wolbachia and extend their host range to taxa adapted to a hypersaline environment.     

Candidate gene sequencing and validation of SNP markers linked to carotenoid content in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava is a widely grown staple in Sub-Saharan Africa and consumed as a cheap source of calories, but the crop is deficient in micronutrients including pro-vitamin A carotenoids. This challenge is currently being addressed through biofortification breeding that relies on phenotypic selection. Gene-based markers linked to pro-vitamin A content variation are expected to increase the rate of genetic gain for this critical trait. We sequenced four candidate carotenoid genes from 167 cassava accessions representing the diversity of elite breeder lines from IITA. Total carotenoid content was determined using spectrophotometer and total β-carotene was quantified by high-performance liquid chromatography. Storage root yellowness due to carotenoid pigmentation was assessed. We carried out candidate gene association analysis that accounts for population structure and kinship using genome-wide single nucleotide polymorphisms (SNPs) generated through genotyping-by-sequencing. Significant SNPs were used to design competitive allele-specific PCR assays and validated on the larger population for potential use in marker-assisted selection breeding. Candidate gene sequencing of the genes β-carotene hydroxylase (crtRB), phytoene synthase (PSY2), lycopene epsilon cyclase (lcyE), and lycopene beta cyclase (lcyB) yielded a total of 37 SNPs. Total carotenoid content, total β-carotene, and color parameters were significantly associated with markers in the PSY2 gene. The SNPs from lcyE were significantly associated with color while those of lcyB and crtRB were not significantly associated with carotenoids or color parameters. These validated and breeder-friendly markers have potential to enhance the efficiency of selection for high β-carotene cassava, thus accelerating genetic gain.     

Genetic diversity and population structure of <Emphasis Type="Italic">Taxus cuspidata</Emphasis> Sieb. et Zucc. ex Endl. (Taxaceae) in Russia according to data of the nucleotide polymorphism of intergenic spacers of the chloroplast genome

The genetic diversity and population structure of Taxus cuspidata Sieb. et Zucc. ex Endl. on the Russian part of its range was studied for the first time on the basis of the nucleotide polymorphism of intergenic spacers psbA–trnH, trnL–trnF, and trnS–trnfM of chloroplast DNA. A high level of gene (h = 0.807) and nucleotide (π = 0.0227) diversity was revealed. The data of AMOVA showed that the interpopulation component accounted for 12% of genetic variability (F _ST = 0.12044, P = 0.0000). We revealed 15 haplotypes, four of which were shown to be unique. The presence of common haplotypes in the majority of populations, the absence of phylogenetic structure, and low values or even the absence of nucleotide divergence show that the T. cuspidata populations studied are fragments of the once common ancestor population. Geographical isolation, which resulted from climatic changes in the Pleistocene–Holocene, as well as from human activities, did not produce a significant effect on the genetic structure of the species.     

Evidence of intralocus recombination at the <Emphasis Type="Italic">Glu-3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

Recombination at the Glu-3 loci was identified, and strong genetic linkage was observed only between the amplicons representing i-type and s-type genes located, respectively, at the Glu-A3 and Glu-B3 loci.

Abstract

The low-molecular weight glutenin subunits (LMW-GSs) are one of the major components of wheat seed storage proteins and play a critical role in the determination of wheat end-use quality. The genes encoding this class of proteins are located at the orthologous Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3). Due to the complexity of these chromosomal regions and the high sequence similarity between different LMW-GS genes, their organization and recombination characteristics are still incompletely understood. This study examined intralocus recombination at the Glu-3 loci in two recombinant inbred line (RIL) and one doubled haploid (DH) population, all segregating for the Glu-A3, Glu-B3, and Glu-D3 loci. The analysis was conducted using a gene marker system that consists of the amplification of the complete set of the LMW-GS genes and their visualization by capillary electrophoresis. Recombinant marker haplotypes were detected in all three populations with different recombination rates depending on the locus and the population. No recombination was observed between the amplicons representing i-type and s-type LMW-GS genes located, respectively, at the Glu-A3 and Glu-B3 loci, indicating tight linkage between these genes. Results of this study contribute to better understanding the genetic linkage and recombination between different LMW-GS genes, the structure of the Glu-3 loci, and the development of more specific molecular markers that better represent the genetic diversity of these loci. In this way, a more precise analysis of the contribution of various LMW-GSs to end-use quality of wheat may be achieved.

     

Daily activity rhythm of desert omni-seasonal Tenebrionid beetles, <Emphasis Type="Italic">Trigonoscelis gigas</Emphasis> and <Emphasis Type="Italic">T. sublaevicollis</Emphasis> (Coleoptera,Tenebrionidae) in constant darkness and under alternating 1-hour pulses of light and darkness

In the Karakum Desert (Turkmenistan) the beetles Trigonoscelis gigas are only active in the morning and evening while T. sublaevicollis are strictly nocturnal, regardless of the season and weather. The daily activity rhythm of T. gigas and T. sublaevicollis was studied in the laboratory according to the following pattern: 5 days under a light-darkness cycle of 15: 9 h (LD 15: 9), then 10 days in constant darkness (DD), and then 10 more days under alternating 1-h pulses of light and darkness (LD 1: 1). The temperature was 25°C in all the modes. At LD 15: 9, beetles of both species maintained a 24-h period and a natural pattern of the activity rhythm. In DD, the circadian rhythm ran with a period of 23.5 ± 0.3 h (n = 40) in T. gigas and 23.6 ± 0.4 h (n = 40) in T. sublaevicollis. At DD, the morning and evening activity peaks of T. gigas merged to form a rhythm with only one peak. Under LD 1: 1, both T. gigas and T. sublaevicollis recovered a 24-h period of the rhythm, while the rhythm of T. gigas regained the two-peak structure. Our research confirmed the assumption of Tshernyshev (1980) about the 24-h period of the free-running endogenous rhythm and the distorting effect of constant conditions on this rhythm.     

Adaptive evolution of osmoregulatory-related genes provides insight into salinity adaptation in Chinese mitten crab, <Emphasis Type="Italic">Eriocheir sinensis</Emphasis>

Osmoregulation is an important mechanism by which euryhaline crustaceans regulate osmotic and ionic concentrations. The Chinese mitten crab (Eriocheir sinensis) is a strong osmoregulating animal model among crustacean species, as it can maintain its hemolymph composition and survives well in either seawater or freshwater. Osmoregulation by E. sinensis during physiological adaptation has been studied extensively. However, the genetic basis of osmoregulation in E. sinensis for acclimating to changing salinities remains unclear. The current study investigated five genes involved in E. sinensis osmoregulation and compared them with a representative marine crab Portunus trituberculatus to test whether adaptive evolution has occurred changing salinity conditions. The results showed that carbonic anhydrase (CA), cytochrome P450 4C (CYP4C), glutamate dehydrogenase (GDH), and the Na⁺/H⁺ exchanger (NHE) have undergone positive selection (i.e., directional selection) in E. sinensis. Thus, the positive selection in CA and NHE suggests that E. sinensis has enhanced capacity for maintaining systemic acid-base balance and ion regulation. GDH and CYP4C also demonstrated positive selection in E. sinensis, suggesting that E. sinensis might have acquired an enhanced capacity to metabolize glutamate and synthesize ecdysteroids in response to a change in osmotic concentration. The present study provides new insight into the molecular genetic basis of salinity adaption in E. sinensis.     

Comparison of molecular genetic utilities of TD,AFLP, and MSAP among the accessions of <Emphasis Type="Italic">japonica,indica</Emphasis>, and <Emphasis Type="Italic">Tongil</Emphasis> of <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Rice is one of the most important food crops in the world. Genetic diversity is essential for cultivar improvement programs. We compared genetic diversity derived from insertion–deletion (in–del) or base substitutions by amplified fragment length polymorphism (AFLP), from transposon transposition mutations by transposon display (TD), and from cytosine methylation by methylation-sensitive amplified polymorphism (MSAP) in japonica, indica, and Tongil type varieties of Oryza sativa L. Polymorphic profiles from the three marker systems allowed us to clearly distinguish the three types of varieties. The indica type varieties showed the highest genetic diversity followed by the Tongil and japonica type varieties. Of the three marker systems, TD produced the highest marker indices, and AFLP and MSAP produced similar marker indices. Pair-wise comparisons of the three marker systems showed that the correlation between the two genetic markers systems (AFLP and TD, r = 0.959) was higher than the correlations between the genetic and epigenetic marker systems (AFLP and MSAP, r = 0.52; TD and MSAP, r = 0.505). Both genetic marker systems had similar levels of gene differentiation (G _ST ) and gene flow (N _m ), which differed in the epigenetic marker system. Although the G _ST of the epigenetic marker system was lower than the genetic marker systems, the N _m of the epigenetic marker system was higher than in the genetic marker systems, indicating that epigenetic variations have a greater influence than genetic variations among the O. sativa L. types.     

Estimates of Heritability for Growth and Shell Color Traits and Their Genetic Correlations in the Black Shell Strain of Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The Pacific oyster Crassostrea gigas has been introduced widely and massively and became an economically important aquaculture species on a global scale. We estimated heritabilities of growth and shell color traits and their genetic correlations in black shell strain of C. gigas. Analyses were performed on 22 full-sib families in a nested mating design including 410 individuals at harvest (24 months of age). The parentage assignment was inferred based on four panels of multiplex PCR markers including 10 microsatellite loci and 94.9% of the offspring were unambiguously assigned to single parent pairs. The Spearman correlation test (r = ? 0.992, P < 0.001) demonstrated the high consistency of the shell pigmentation (SP) and L* and their same efficacy in shell color measurements. The narrow-sense heritability estimated under the animal model analysis was 0.18 ± 0.12 for shell height, 0.25 ± 0.16 for shell length, 0.10 ± 0.09 for shell width, 0.42 ± 0.20 for total weight, 0.32 ± 0.18 for shell weight, and 0.68 ± 0.16 for L*, 0.69 ± 0.16 for shell pigmentation, respectively. The considerable additive genetic variation in growth and shell color traits will make it feasible to produce genetic improvements for these traits in selective breeding program. High genetic and phenotypic correlations were found among growth traits and among shell color traits. To optimize a selection strategy for both fast growth and pure dark shell strain of C. gigas, it is proposed to take both total weight and black shell as joint objective traits in selective breeding program. Our study offers an important reference in the process of selective breeding in black shell color stain of C. gigas and will facilitate to develop favorable breeding strategies of genetic improvements for this economically important strain.     

Assessment of genetic diversity among four orchids based on ddRAD sequencing data for conservation purposes

Genetic diversity was assessed in the four orchid species using NGS based ddRAD sequencing data. The assembled nucleotide sequences (fastq) were deposited in the SRA archive of NCBI Database with accession number (SRP063543 for Dendrobium, SRP065790 for Geodorum, SRP072201 for Cymbidium and SRP072378 for Rhynchostylis). Total base pair read was 1.1 Mbp in case of Dendrobium sp., 553.3 Kbp for Geodorum sp., 1.6 Gbp for Cymbidium, and 1.4 Gbp for Rhynchostylis. Average GC% was 43.9 in Geodorum, 43.7% in Dendrobium, 41.2% in Cymbidium and 42.3% in Rhynchostylis. Four partial gene sequences were used in DnaSP5 program for nucleotide diversity and phylogenetic relationship determination (Ycf2 gene of Dendrobium, matK gene of Geodorum, psbD gene of Cymbidium and Ycf2 gene of Ryhnchostylis). Nucleotide diversity (per site) Pi (π) was 0.10560 in Dendrobium, 0.03586 in Geodorum, 0.01364 in Cymbidium and 0.011344 in Rhynchostylis. Neutrality test statistics showed the negative value in all the four orchid species (Tajima’s D value ?2.17959 in Dendrobium, ?2.01655 in Geodorum, ?2.12362 in Rhynchostylis and ?1.54222 in Cymbidium) indicating the purifying selection. Result for these gene sequences (matK and Ycf2 and psbD) indicate that they were not evolved neutrally, but signifying that selection might have played a role in evolution of these genes in these four groups of orchids. Phylogenetic relationship was analyzed by reconstructing dendrogram based on the matK, psbD and Ycf2 gene sequences using maximum likelihood method in MEGA6 program.