Molecular Identification of Dendrobium Species (Orchidaceae) Based on the DNA Barcode ITS2 Region and Its Application for Phylogenetic Study

The over-collection and habitat destruction of natural Dendrobium populations for their commercial medicinal value has led to these plants being under severe threat of extinction. In addition, many Dendrobium plants are similarly shaped and easily confused during the absence of flowering stages. In the present study, we examined the application of the ITS2 region in barcoding and phylogenetic analyses of Dendrobium species (Orchidaceae). For barcoding, ITS2 regions of 43 samples in Dendrobium were amplified. In combination with sequences from GenBank, the sequences were aligned using Clustal W and genetic distances were computed using MEGA V5.1. The success rate of PCR amplification and sequencing was 100%. There was a significant divergence between the inter- and intra-specific genetic distances of ITS2 regions, while the presence of a barcoding gap was obvious. Based on the BLAST1, nearest distance and TaxonGAP methods, our results showed that the ITS2 regions could successfully identify the species of most Dendrobium samples examined; Second, we used ITS2 as a DNA marker to infer phylogenetic relationships of 64 Dendrobium species. The results showed that cluster analysis using the ITS2 region mainly supported the relationship between the species of Dendrobium established by traditional morphological methods and many previous molecular analyses. To sum up, the ITS2 region can not only be used as an efficient barcode to identify Dendrobium species, but also has the potential to contribute to the phylogenetic analysis of the genus Dendrobium.     

The Uneven Rate of the Molecular Evolution of Gene Sequences of DNA-Dependent RNA Polymerase I of the Genus Lamium L

RNA polymerase type I (plastid-encoded polymerase, PEP) is one of the key chloroplast enzymes. However, the rpo genes that encode its subunits (rpoA, rpoB, rpoC1 and rpoC2) are relatively rapidly evolving sequences. The aim of this study was to investigate the rate of the molecular evolution of rpo genes and to evaluate them as phylogenetic markers on the example of the genus Lamium L. (Lamiaceae). The analyzed genes were shown to differ in the level of variation, rate of intragenic mutations, phylogenetic informativeness, and in the impact of these mutations on the properties of encoded peptides. Destabilizing effects of the positive pressure were observed in all genes examined coding for PEP enzyme. We have demonstrated the relationship between mutations fixed by positive selection and the separation of phylogenetic lines within the genus Lamium. The study showed also that the rpo genes were reliable phylogenetic markers, useful in the reconstruction of interconnections of species belonging to the same genus. Of the four tested genes, the most promising phylogenetic marker was rpoA gene, while the least useful gene appeared to be rpoC1.     

Avoidance and Potential Remedy Solutions of Chimeras in Reconstructing the Phylogeny of Aphids Using the 16S rRNA Gene of Buchnera: A Case in Lachninae (Hemiptera)

It is known that PCR amplification of highly homologous genes from complex DNA mixtures can generate a significant proportion of chimeric sequences. The 16S rRNA gene is not only widely used in estimating the species diversity of endosymbionts in aphids but also used to explore the co-diversification of aphids and their endosymbionts. Thus, chimeric sequences may lead to the discovery of non-existent endosymbiont species and mislead Buchnera-based phylogenetic analysis that lead to false conclusions. In this study, a high probability (6.49%) of chimeric sequence occurrence was found in the amplified 16S rRNA gene sequences of endosymbionts from aphid species in the subfamily Lachninae. These chimeras are hybrid products of multiple parent sequences from the dominant species of endosymbionts in each corresponding host. It is difficult to identify the chimeric sequences of a new or unidentified species due to the high variability of their main parent, Buchnera aphidicola, and because the chimeric sequences can confuse the phylogenetic analysis of 16S rRNA gene sequences. These chimeras present a challenge to Buchnera-based phylogenetic research in aphids. Thus, our study strongly suggests that using appropriate methods to detect chimeric 16S rRNA sequences may avoid some false conclusions in endosymbiont-based aphid research.     

Enhanced Muscle Fibers of Epinephelus coioides by Myostatin Autologous Nucleic Acid Vaccine

Epinephelus coioides is a fish species with high economic value due to its delicious meat, high protein content, and rich fatty acid nutrition. It has become a high-economic fish in southern parts of China and some other Southeast Asian countries. In this study, the myostatin nucleic acid vaccine was constructed and used to immunize E. coioides. The results from body length and weight measurements indicated the myostatin nucleic acid vaccine promoted E. coioides growth performance by increasing muscle fiber size. The results from RT-qPCR analysis showed that myostatin nucleic acid vaccine upregulated the expression of myod, myog and p21 mRNA, downregulated the expression of smad3 and mrf4 mRNA. This preliminary study is the first report that explored the role of myostatin in E. coioides and showed positive effects of autologous nucleic acid vaccine on the muscle growth of E. coioides. Further experiments with increased numbers of animals and different doses are needed for its application to E. coiodes aquaculture production.     

Effects of Polymorphisms in Pepsinogen (PEP), Amylase (AMY) and Trypsin (TRY) Genes on Food Habit Domestication Traits in Mandarin Fish

Mandarin fish (Siniperca chuatsi) have a peculiar feeding habit of only accepting live fish prey and refusing dead prey and artificial diets. However, previous research has shown that some individuals accept dead prey after gradual domestication. Digestive enzymes are correlated with feeding habits in fish. In the current study, SNPs in the mandarin fish genes for pepsinogen (PEP), amylase (AMY), and trypsin (TRY) were evaluated for associations with feeding habits in domesticated mandarin fish by scanning their complete genomic sequence. In total, two SNPs were found in PEP, one was found in TRY, and none were found in AMY. The D1(CTCC) and D5(TTTT) diplotypes in the PEP gene tended to show strong effects on the feeding habits of domesticated fish (p < 0.01). The results indicate that PEP may be associated with the genetic mechanism for feeding habits in mandarin fish, and the D1(CTCC) and D5(TTTT) diplotypes in the PEP gene may be useful markers for selecting mandarin fish with appropriate feeding habits for domestication.     

Elucidating the Diversity of Aquatic Microdochium and Trichoderma Species and Their Activity against the Fish Pathogen Saprolegnia diclina

Animals and plants are increasingly threatened by emerging fungal and oomycete diseases. Amongst oomycetes, Saprolegnia species cause population declines in aquatic animals, especially fish and amphibians, resulting in significant perturbation in biodiversity, ecological balance and food security. Due to the prohibition of several chemical control agents, novel sustainable measures are required to control Saprolegnia infections in aquaculture. Previously, fungal community analysis by terminal restriction fragment length polymorphism (T-RFLP) revealed that the Ascomycota, specifically the genus Microdochium, was an abundant fungal phylum associated with salmon eggs from a commercial fish farm. Here, phylogenetic analyses showed that most fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride species. Phylogenetic and quantitative PCR analyses showed both a quantitative and qualitative difference in Trichoderma population between diseased and healthy salmon eggs, which was not the case for the Microdochium population. In vitro antagonistic activity of the fungi against Saprolegnia diclina was isolate-dependent; for most Trichoderma isolates, the typical mycoparasitic coiling around and/or formation of papilla-like structures on S. diclina hyphae were observed. These results suggest that among the fungal community associated with salmon eggs, Trichoderma species may play a role in Saprolegnia suppression in aquaculture.     

Genetic Diversity Characterization of Porcine Reproductive and Respiratory Syndrome Virus Isolates in Romania, Based on Phylogenetic Analysis

Porcine reproductive and respiratory syndrome (PRRS) is a disease produced by the (PRRS) virus, characterized by endemic evolution in the majority of countries, which remains in actuality being a permanent threat to health and economic free farms, as well as for those infected. The aim of this study was to evaluate the genetic diversity of Romanian PRRSV isolates from the four most important pig farms in Romania by comparing the nucleotide sequences obtained for ORF5 and ORF7 with a wide range of sequences from GenBank belonging to the main types of PRRSV; the type 1. Eighteen different sequences were obtained for ORF5 gene and 10 for ORF7 gene. One Romanian isolate (Rom3) was found in three of the four different investigated farms. The phylogenetic analysis revealed that the Romanian PRRSV nucleotide sequences clustered in three groups within the subtype 1 of the virus. The analysis of amino acid sequences evidenced for GP5 and N-nucleocapsid proteins confirmed that the Romanian virus belonged to type 1.     

Conservation Genetics of an Endangered Lady’s Slipper Orchid: Cypripedium japonicum in China

Knowledge about the population genetic variation of the endangered orchid, Cypripedium japonicum, is conducive to the development of conservation strategies. Here, we examined the levels and partitioning of inter-simple sequence repeat (ISSR) diversity (109 loci) in five populations of this orchid to gain insight into its genetic variation and population structure in Eastern and Central China. It harbored considerably lower levels of genetic diversity both at the population (percentage of polymorphic loci (PPL) = 11.19%, Nei’s gene diversity (H) = 0.0416 and Shannon’s information index (I) = 0.0613) and species level (PPL = 38.53%, H = 0.1273 and I = 0.1928) and a significantly higher degree of differentiation among populations (the proportion of the total variance among populations (Φ_pt) = 0.698) than those typical of ISSR-based studies in other orchid species. Furthermore, the Nei’s genetic distances between populations were independent of the corresponding geographical distances. Two main clusters are shown in an arithmetic average (UPGMA) dendrogram, which is in agreement with the results of principal coordinate analysis (PCoA) analysis and the STRUCTURE program. In addition, individuals within a population were more similar to each other than to those in other populations. Based on the genetic data and our field survey, the development of conservation management for this threatened orchid should include habitat protection, artificial gene flow and ex situ measures.     

Single Nucleotide Polymorphisms in Growth Hormone Gene and Their Association with Growth Traits in Siniperca chuatsi (Basilewsky)

Growth hormone (GH) has been considered as a candidate gene for growth traits in fish. In this study, polymorphisms of the GH gene were evaluated for associations with growth traits in 282 Siniperca chuatsi individuals. Using directly sequencing, four single nucleotide polymorphisms (SNPs) were identified in GH gene, with two mutations in intron 4 (g.4940A>C, g.4948A>T), one mutation in exon 5 (g.5045T>C) and one in intron 5 (g.5234T>G). Notably, three of them were significantly associated with growth performance, particularly for g.4940A>C which was highly correlated with all the four growth traits. In conclusion, our results demonstrated that these SNPs in GH gene could influence growth performance of S.chuatsi and could be used for marker-assisted selection (MAS) in this species.     

Genetic Diversity and Population Differentiation of Calanthe tsoongiana,a Rare and Endemic Orchid in China

Calanthe tsoongiana is a rare terrestrial orchid endemic to China, and this species has experienced severe habitat loss and fragmentation. Inter-simple sequence repeat (ISSR) markers were employed to assess the genetic diversity and differentiation of six populations of C. tsoongiana. Based on 124 discernible fragments yielded by eleven selected primers, high genetic diversity was revealed at the species level; however, genetic diversity at the population level was relatively low. High-level genetic differentiation among populations was detected based on analysis of molecular variance (AMOVA), indicating potential limited gene flow. No significant relationship was observed between genetic and geographic distances among the sampled populations. These results suggested that restricted gene flow might be due to habitat fragmentation and reduced population size as a result of human activities. Based on the findings, several conservation strategies were proposed for the preservation of this threatened species.     

Rapid Development of Microsatellite Markers for the Endangered Fish Schizothorax biddulphi (Günther) Using Next Generation Sequencing and Cross-Species Amplification

Tarim schizothoracin (Schizothorax biddulphi) is an endemic fish species native to the Tarim River system of Xinjiang and has been classified as an extremely endangered freshwater fish species in China. Here, we used a next generation sequencing platform (ion torrent PGM™) to obtain a large number of microsatellites for S. biddulphi, for the first time. A total of 40577 contigs were assembled, which contained 1379 SSRs. In these SSRs, the number of dinucleotide repeats were the most frequent (77.08%) and AC repeats were the most frequently occurring microsatellite, followed by AG, AAT and AT. Fifty loci were randomly selected for primer development; of these, 38 loci were successfully amplified and 29 loci were polymorphic across panels of 30 individuals. The H_o ranged from 0.15 to 0.83, and H_e ranged from 0.15 to 0.85, with 3.5 alleles per locus on average. Cross-species utility indicated that 20 of these markers were successfully amplified in a related, also an endangered fish species, S. irregularis. This study suggests that PGM™ sequencing is a rapid and cost-effective tool for developing microsatellite markers for non-model species and the developed microsatellite markers in this study would be useful in Schizothorax genetic analysis.     

Nuclear Microsatellite Primers for the Endangered Relict Fir,Abies pinsapo (Pinaceae) and Cross-Amplification in Related Mediterranean Species

Twelve nuclear microsatellite primers (nSSR) were developed for the endangered species Abies pinsapo Boiss. to enable the study of gene flow and genetic structure in the remaining distribution areas. Microsatellite primers were developed using next-generation sequencing (454) data from a single Abies pinsapo individual. Primers were applied to thirty individuals from the three extant localities. The number of alleles per locus ranged from one to four. Cross-amplification was tested for other Abies species from the Mediterranean Basin, and most of the loci showed higher polymorphisms in the Mediterranean species than in A. pinsapo. These microsatellite markers provide tools for conservation genetic studies in Abies pinsapo as well other Abies species from the Mediterranean Basin.     

Genetic Diversity of the Critically Endangered Thuja sutchuenensis Revealed by ISSR Markers and the Implications for Conservation

Thuja sutchuenensis Franch. is a critically endangered plant endemic to the North-East Chongqing, China. Genetic variation was studied to assess the distribution of genetic diversity within and among seven populations from the single remnant locations, using inter-simple sequence repeat (ISSR) markers. A total of 15 primers generated 310 well defined bands, with an average of 20.7 bands per primer. The seven populations revealed a relatively high level of genetic diversity in the species. The percentage of polymorphic bands, Nei’s gene diversity and Shannon’s information index at the population and species level were 76.1%, 0.155, 0.252 and 100%, 0.165, 0.295, respectively. A low level of genetic differentiation among populations (G_ST = 0.102), in line with the results of Analyses of Molecular Variance (AMOVA), and a high level of gene flow (N_m = 4.407) were observed. Both the Unweighted Pair Group Method with Arithmatic Mean (UPGMA) cluster analysis and Principal Coordinates Analysis (PCoA) supported the grouping of all seven populations into two groups. In addition, Mantel test revealed no significant correlation between genetic and geographical distances (r = 0.329, p = 0.100). The low genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered species.     

Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria

Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.     

Molecular Identification of Fusarium Species in Gibberella fujikuroi Species Complex from Rice, Sugarcane and Maize from Peninsular Malaysia

The objective of this study was to identify Fusarium species in the Gibberella fujikuroi species complex from rice, sugarcane and maize as most of the Fusarium species in the species complex are found on the three crops. Isolates used were collected from the field and obtained from culture collection. The Fusarium isolates were initially sorted based on morphology and identifications confirmed based on the DNA sequence of the translation elongation factor 1-α (TEF-1α) gene. Based on the closest match of BLAST analysis, five species were recovered, namely, F. sacchari, F. fujikuroi, F. proliferatum, F. andiyazi and F. verticillioides. This is the first report regarding F. andiyazi from rice in Malaysia and Southeast Asia. The phylogenetic tree generated by using the neighbor joining method showed that isolates from the same species were grouped in the same clade. The present study indicated that Fusarium species in the G. fujikuroi species complex are widespread in rice, sugarcane and maize in Peninsular Malaysia. The findings also suggest that the use of morphological characters for identification of Fusarium species in the G. fujikuroi species complex from the three crops will lead to incorrect species designation.     

A Molecular Genetic Linkage Map of Eucommia ulmoides and Quantitative Trait Loci (QTL) Analysis for Growth Traits

Eucommia ulmoides is an economically important tree species for both herbal medicine and organic chemical industry. Effort to breed varieties with improved yield and quality is limited by the lack of knowledge on the genetic basis of the traits. A genetic linkage map of E. ulmoides was constructed from a full-sib family using sequence-related amplified polymorphism, amplified fragment length polymorphism, inter-simple sequence repeat and simple sequence repeat markers. In total, 706 markers were mapped in 25 linkage groups covering 2133 cM. The genetic linkage map covered approximately 89% of the estimated E. ulmoides genome with an average of 3.1 cM between adjacent markers. The present genetic linkage map was used to identify quantitative trait loci (QTL) affecting growth-related traits. Eighteen QTLs were found to explain 12.4%–33.3% of the phenotypic variance. This genetic linkage map provides a tool for marker-assisted selection and for studies of genome in E. ulmoides.     

Application of Novel Polymorphic Microsatellite Loci Identified in the Korean Pacific Abalone (Haliotis diversicolor supertexta (Haliotidae)) in the Genetic Characterization of Wild and Released Populations

The small abalone, Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important species of marine shellfish in eastern Asia. Over the past few decades, this species has drastically declined in Korea. Thus, hatchery-bred seeds have been released into natural coastal areas to compensate for the reduced fishery resources. However, information on the genetic background of the small abalone is scarce. In this study, 20 polymorphic microsatellite DNA markers were identified using next-generation sequencing techniques and used to compare allelic variation between wild and released abalone populations in Korea. Using high-throughput genomic sequencing, a total of 1516 (2.26%; average length of 385 bp) reads containing simple sequence repeats were obtained from 86,011 raw reads. Among the 99 loci screened, 28 amplified successfully, and 20 were polymorphic. When comparing allelic variation between wild and released abalone populations, a total of 243 different alleles were observed, with 18.7 alleles per locus. High genetic diversity (mean heterozygosity = 0.81; mean allelic number = 15.5) was observed in both populations. A statistical analysis of the fixation index (F_ST) and analysis of molecular variance (AMOVA) indicated limited genetic differences between the two populations (F_ST = 0.002, p > 0.05). Although no significant reductions in the genetic diversity were found in the released population compared with the wild population (p > 0.05), the genetic diversity parameters revealed that the seeds released for stock abundance had a different genetic composition. These differences are likely a result of hatchery selection and inbreeding. Additionally, all the primer pair sets were effectively amplified in another congeneric species, H. diversicolor diversicolor, indicating that these primers are useful for both abalone species. These microsatellite loci may be valuable for future aquaculture and population genetic studies aimed at developing conservation and management plans for these two abalone species.     

Expression Patterns and Functional Novelty of Ribonuclease 1 in Herbivorous Megalobrama amblycephala

Ribonuclease 1 (RNase1) is an important digestive enzyme that has been used to study the molecular evolutionary and plant-feeding adaptation of mammals. However, the expression patterns and potential biological function of RNase1 in herbivorous fish is not known. Here, we identified RNase1 from five fish species and illuminated the functional diversification and expression of RNase1 in herbivorous Megalobrama amblycephala. The five identified fish RNase1 genes all have the signature motifs of the RNase A superfamily. No expression of Ma-RNase1 was detected in early developmental stages but a weak expression was detected at 120 and 144 hours post-fertilization (hpf). Ma-RNase1 was only expressed in the liver and heart of one-year-old fish but strongly expressed in the liver, spleen, gut, kidney and testis of two-year-old fish. Moreover, the immunostaining localized RNase1 production to multiple tissues of two-year-old fish. A biological functional analysis of the recombinant protein demonstrated that M. amblycephala RNase1 had a relatively strong ribonuclease activity at its optimal pH 6.1, which is consistent with the pH of its intestinal microenvironment. Collectively, these results clearly show that Ma-RNase1 protein has ribonuclease activity and the expression patterns of Ma-RNase1 are dramatically different in one year and two-year-old fish, suggesting the functional differentiation during fish growing.