Nucleotide Sequence Variation in Long-Term Tissue Cultures of Chinese Ginseng (Panax ginseng C. A. Mey.)

Plants have the salient biological property of totipotency, i.e., the capacity to regenerate a whole plant from virtually any kind of fully differentiated somatic cells after a process of dedifferentiation. This property has been well-documented by successful plant regeneration from tissue cultures of diverse plant species. However, the accumulation of somaclonal variation, especially karyotype alteration, during the tissue culture process compromises cell totipotency. In this respect, Chinese ginseng (Panax ginseng C. A. Mey.) is an exception in that it shows little decline in cell totipotency accompanied by remarkable chromosomal stability even after prolonged tissue cultures. However, it remains unclear whether chromosomal level stability necessarily couples with molecular genetic stability at the nucleotide sequence level, given that the two types of stabilities are generated by largely distinct mechanisms. Here, we addressed this issue by genome-wide comparisons at the single-base resolution of long-term tissue culture-regenerated P. ginseng plants. We identified abundant single nucleotide polymorphisms (SNPs) that have accumulated in cultured ginseng callus and are retained in the process of plant regeneration. These SNPs did not occur at random but showed differences among chromosomes and biased regional aggregation along a given chromosome. In addition, our results demonstrate that, compared with the overall genes, genes related to processes of cell totipotency and chromosomal stability possess lower mutation rates at both coding and flanking regions. In addition, collectively, the mutated genes exhibited higher expression levels than non-mutated genes and are significantly enriched in fundamental biological processes, including cellular component organization, development, and reproduction. These attributes suggest that the precipitated molecular level genetic variations during the process of regeneration in P. ginseng are likely under selection to fortify normal development. As such, they likely did not undermine chromosomal stability and totipotency of the long-term ginseng cultures.

Molecular Signatures of Reticulate Evolution within the Complex of European Pine Taxa

Speciation mechanisms, including the role of interspecific gene flow and introgression in the emergence of new species, are the major focus of evolutionary studies. Inference of taxonomic relationship between closely related species may be challenged by past hybridization events, but at the same time, it may provide new knowledge about mechanisms responsible for the maintenance of species integrity despite interspecific gene flow. Here, using nucleotide sequence variation and utilizing a coalescent modeling framework, we tested the role of hybridization and introgression in the evolutionary history of closely related pine taxa from the Pinus mugo complex and P. sylvestris. We compared the patterns of polymorphism and divergence between taxa and found a great overlap of neutral variation within the P. mugo complex. Our phylogeny reconstruction indicated multiple instances of reticulation events in the past, suggesting an important role of interspecific gene flow in the species divergence. The best-fitting model revealed P. mugo and P. uncinata as sister species with basal P. uliginosa and asymmetric migration between all investigated species after their divergence. The magnitude of interspecies gene flow differed greatly, and it was consistently stronger from representatives of P. mugo complex to P. sylvestris than in the opposite direction. The results indicate the prominent role of reticulation evolution in those forest trees and provide a genetic framework to study species integrity maintained by selection and local adaptation.

Variation in PLIN2 and its association with milk traits and milk fat composition in dairy cows

The current study investigated associations between variation in the bovine perilipin-2 gene (PLIN2) and milk traits (milk fat content, milk protein content, milk yield and milk fatty acid (FA) component levels) in 409 New Zealand pasture-grazed Holstein-Friesian × Jersey-cross (HF × J-cross or Kiwicross™) cows. Five nucleotide sequence variants were found in three regions of the gene, including c.17C>T in exon 2, c.53A>G in exon 3, c.595+23G>A and c.595+104_595+108del in intron 5, and c.*302T>C in the 3′-untranslated region. The c.*302T>C substitution produces two nucleotide sequence variants (A5 and B5), and this variation was associated with variation in milk protein content and milkfat composition for C10:0, C11:0, C12:0, C13:0 and C16:0 FA and medium-chain fatty acid (MCFA) and long-chain fatty acid (LCFA) groups. After correcting for the effect of variation in the diacylglycerol acyl-CoA acyltransferase 1 gene (DGAT1) that results in the amino acid substitution p.K232A, variation in the FA binding protein 4 gene (FABP4) and variation in the stearoyl-CoA desaturase (Δ-9-desaturase) gene (SCD) that results in the amino acids substitution p.A293V, significant differences between A5A5 and B5B5 cows were found for C10:0, C11:0, C12:0, C13:0, C16:0, and the MCFA, LCFA, total saturated FA and C10:1 index groups. This suggests that nucleotide sequence variation in PLIN2 may be affecting milk FA component levels.

Genetic Variation in the Domain II, 3′ Untranslated Region of Human and Mosquito Derived Dengue Virus Strains in Sri Lanka

Genetic variations in dengue virus (DENV) play a distinct role in epidemic emergence. The DENV 3′ UTR has become a recent interest in research. The objective of the study was to examine the genetic variation in the domain II, 3′ UTR region of human and mosquito-derived DENV. DENV-infected human sera were orally infected to laboratory reared Aedes aegypti mosquitoes. The domain II, 3′ UTR of each human- and mosquito-derived sample was amplified. The nucleotide sequence variation, phylogenetic and secondary structure analysis was carried out incorporating respective regions of so far recorded Sri Lankan and the reference genotype strains of the DENV3 and DENV1 serotypes. The human- and mosquito-derived domain II, 3′ UTR were identical in nucleotide sequences within the serotypes isolated, indicating the conserved nature of the region during host switch. The sequence analysis revealed distinct variations in study isolates compared to so far recorded Sri Lankan isolates. However, despite single nucleotide variations, the maintenance of structural integrity was evident in related strains within the serotypes in the secondary structure analysis. The phylogenetic analysis revealed distinct clade segregation of the study sequences from so far reported Sri Lankan isolates and illustrated the phylogenetic relations of the study sequences to the available global isolates of respective serotypes.

Identification of epigenetic variation associated with synchronous pod maturity in mungbean (Vigna radiata L.)

Cytosine methylation in genomic DNA affects gene expression, potentially causing phenotypic variation. Mungbean, an agronomically and nutritionally important legume species, is characterized by nonsynchronous pod maturity, resulting in multiple harvest which costs extra time and labor. To elucidate the epigenetic influences on synchronous pod maturity (SPM) in mungbean, we determined the genome-wide DNA methylation profiles of eight mungbean recombinant inbred lines (RILs) and their parental genotypes, and compared DNA methylation profiles between high SPM and low SPM RILs, thus revealing differentially methylated regions (DMRs). A total of 3, 18, and 28 pure DMRs, defined as regions showing no significant correlation between nucleotide sequence variation and methylation level, were identified in CpG, CHG, and CHH contexts, respectively. These DMRs were proximal to 20 genes. Among the 544 single nucleotide polymorphisms identified near the 20 genes, only one caused critical change in gene expression by early termination. Analysis of these genome-wide DNA methylation profiles suggests that epigenetic changes can influence the expression of proximal genes, regardless of nucleotide sequence variation, and that SPM is mediated through gibberellin-mediated hormone signaling pathways. These results provide insights into how epialleles contribute to phenotypic variation and improve SPM in mungbean cultivars.

Associations between the Bovine Myostatin Gene and Milk Fatty Acid Composition in New Zealand Holstein-Friesian × Jersey-Cross Cows

The myostatin gene (MSTN), which encodes the protein myostatin, is pleiotropic, and its expression has been associated with both increased and decreased adipogenesis and increased skeletal muscle mass in animals. In this study, the polymerase chain reaction, coupled with single strand conformation polymorphism analysis, was utilized to reveal nucleotide sequence variation in bovine MSTN in 410 New Zealand (NZ) Holstein-Friesian × Jersey (HF × J)-cross cows. These cows ranged from 3 to 9 years of age and over the time studied, produced an average 22.53 ± 2.18 L of milk per day, with an average milk fat content of 4.94 ± 0.17% and average milk protein content of 4.03 ± 0.10%. Analysis of a 406-bp amplicon from the intron 1 region, revealed five nucleotide sequence variants (A–E) that contained seven nucleotide substitutions. Using general linear mixed-effect model analyses the AD genotype was associated with reduced C10:0, C12:0, and C12:1 levels when compared to levels in cows with the AA genotype. These associations in NZ HF × J cross cows are novel, and they suggest that this variation in bovine MSTN could be explored for increasing the amount of milk unsaturated fatty acid and decreasing the amount of saturated fatty acid.

Genetic diversity and taxonomic issues in Gastridium P.Beauv (Poaceae) inferred from plastid and nuclear DNA sequence analysis

Incomplete taxonomic knowledge may seriously hamper biodiversity conservation efforts that are crucial in a context of global change. Gastridium P.Beauv. is a Mediterranean-Paleotropical member of the Poaceae family, inhabiting ephemeral grass habitats, whose species number and diversity are still imperfectly known. In order to progress towards a comprehensive taxonomic treatment of this genus, we examined patterns of DNA diversity in the four taxa (Gastridium lainzii, G. phleoides, G. scabrum, and G. ventricosum) that have been recently advanced by different authors, based on new morpho-ecological descriptors. We explored nucleotide sequence variation at two plastid (trnH-psbA, trnL-F) and one nuclear (ITS) DNA markers in 44 total individuals. Diversity data were treated with multiple statistical and phylogenetic tools, and integrated with available GenBank sequences of Gastridium and other closely related genera. Despite the limited variability detected, evidence of within-taxon genetic cohesion and estimates of molecular divergence comparable with those of species in the same subtribal lineage (Agrostidinae) were recovered. The identified plastid genealogies appeared congruent with a subdivision of the genus into (at least) three distinct entities, and coherent with collected morphological descriptors. Phylogenetic reconstructions with ITS were less corresponding to taxa identities, likely due to reticulation and polyploidization. Once placed in a broader taxonomic context, the investigated dataset produced plastid and nuclear tree topologies consistent with previous assessments, highlighting the overall little resolution of species and genera within Agrostidinae. In the plastid tree, a sister relationship between Gastridium and Triplachne was weakly supported. In the ITS tree, relationships among these genera were unresolved. The hypothesis of closely related but separately evolving lineages within Gastridium is discussed, suggesting a re-evaluation of its current assessment in taxonomic authorities to enhance our knowledge of the grass family, and assist future biodiversity surveys of key Mediterranean grassland ecosystems.

Kinetics of Genetic Variation of the Mycoplasma genitalium MG192 Gene in Experimentally Infected Chimpanzees

Mycoplasma genitalium, a human pathogen associated with sexually transmitted diseases, is capable of causing chronic infections, though mechanisms for persistence remain unclear. Previous studies have found that variation of the MgPa operon occurs by recombination of repetitive chromosomal sequences (known as MgPars) into the MG191 and MG192 genes carried on this operon, which may lead to antigenic variation and immune evasion. In this study, we determined the kinetics of MG192 sequence variation during the course of experimental infection using archived specimens from two chimpanzees infected withM. genitaliumstrain G37. The highly variable region of MG192 was amplified by PCR fromM. genitaliumisolates obtained at various time points postinfection (p.i.). Sequence analysis revealed that MG192 sequence variation began at 5 weeks p.i. With the progression of infection, sequence changes accumulated throughout the MG192 variable region. The presence of MG192 variants at specific time points was confirmed by variant-specific PCR assays and sequence analysis of single-colony clonedM. genitaliumorganisms. MG192 nucleotide sequence variation correlated with estimated recombination events, predicted amino acid changes, and time of seroconversion, a finding consistent with immune selection of MG192 variants. In addition, we provided evidence that MG192 sequence variation occurred during the process ofM. genitaliumsingle-colony cloning. Such spontaneous variation suggests that some MG192 variation is independent of immune selection but may form the basis for subsequent immune selection.