Biochemical Genetics

Analysis of Genetic Diversity and Development of a SCAR Marker for Green Tea ( Camellia sinensis ) Cultivars in Zhejiang Province: The Most Famous Green Tea-Producing Area in China Abstract Camellia sinensis (L.) O. Kuntze is one of the most important non-alcoholic beverage crops in Asian and African countries. In recent years, many green tea cultivars have been released and played an important role in improving the production and quality of tea trees. The objectives of this study were to assess the genetic diversity of the eighteen main green tea cultivars in Zhejiang Province—the most famous green tea-producing area of China—using start codon-targeted (SCoT) markers and to develop a specific sequence-characterized amplified region (SCAR) marker for application in cultivar diagnosis. Thirty-one SCoT primers produced 264 loci, 226 of which were polymorphic. The genetic similarity coefficients among these green tea cultivars ranged from 0.587 to 0.814, indicating that a high level of genetic diversity was present. Both a UPGMA dendrogram and a PCoA plot grouped the tea cultivars into three groups. The partitioning of groups in the UPGMA and PCoA was similar, and much of the clustering was highly consistent with the classification of tea cultivars according to their genetic backgrounds. A unique SCoT band, SCoT4-1649, specific to the tea cultivar ‘Yingshuang,’ was transformed into a SCAR marker. This SCAR marker is highly useful for the identification and germplasm conservation of green tea cultivars.

Population Genetics of Calotropis gigantea , a Medicinal and Fiber Resource Plant, as Inferred from Microsatellite Marker Variation in two Native Countries Abstract Calotropis gigantea is well known for its aesthetic, medicinal, pharmacological, fodder, fuel, and fiber production potential. Unfortunately, this plant species is still undomesticated, and the genetic information available for crop improvement is limited. For this study, we sampled 21 natural populations of C. gigantea from two key areas of its natural distribution range (Bangladesh and China) and genotyped 379 individuals using nine nuclear microsatellite markers. Population genetic diversity was higher in Bangladesh than that observed in Chinese populations. Overall, a moderate level of genetic diversity was found (Na = 3.73, HE = 0.466), with most of the genetic variation detected within populations (65.49%) and substantial genetic differentiation (FST = 0.345) between the study regions. We observed a significant correlation between genetic and geographic distances (r  =  0.287, P  =  0.001). The Bayesian clustering, UPGMA tree, and PCoA analyses yielded three distinct genetic pools, but the number of migrants per generation was high (NM = 0.52–2.78) among them. Our analyses also revealed that some populations may have experienced recent demographic bottlenecks. Our study provides a baseline for exploitation of the genetic resources of C. gigantea in domestication and breeding programs as well as some insights into the germplasm conservation of this valuable plant.

Genetic Polymorphisms of CCDC26 rs891835, rs6470745, and rs55705857 in Glioma Risk: A Systematic Review and Meta-analysis Abstract A genetic component is accepted in the etiology of the glioma. Evidence from candidate genes studies and GWAS reveal that CCDC26 gene could increase the risk of glioma. We performed a systematic review and up-to-date meta-analysis to explore if polymorphisms of CCDC26 gene (rs891835, rs6470745, and rs55705857) may be a susceptibility factor in developing glioma. An online search in PubMed, Web of Science, and SCOPUS up to September 2018 was performed. The pooled odds ratios were evaluated by fixed effects model and random effects model. Analyses of the overall sample and ethnic sub-groups were performed. In all the analyses, the allelic, additive, dominant, and recessive models were used. We found an association between all polymorphisms evaluated and an increased risk for glioma in the overall population in all the models studied. In sub-group analysis, we found that rs891835 and rs6470745 increased the risk of glioma in Europeans and Caucasians. On the other hand, the rs891835 polymorphism did not reveal any statistical association in Chinese population. Taken into consideration the limitations of this study, the present findings suggest a possible participation of rs891835, rs6470745, and rs55705857 as risk factors to develop glioma. Furthermore, it is possible that the involvement of CCDC26 variants depends on ethnicity. However, we recommend to perform further studies to have conclusive outcomes.

A Novel CNGA1 Gene Mutation (c.G622A) of Autosomal Recessive Retinitis Pigmentosa Leads to the CNGA1 Protein Reduction on Membrane Abstract CNGA1 encodes a membrane protein on rod photoreceptor related to phototransduction. The present study was to identify a novel mutation of CNGA1 associated with autosomal recessive retinitis pigmentosa by using next generation sequencing of a Chinese family. Next generation sequencing and Sanger sequencing has identified a compound heterozygous mutation in CNGA1 gene, c0.472 del C (reported) and c0.829G>A (novel mutation, same as c0.622G>A according to NM_000087.3) of the proband. SIFT and Polyphen-2 predicted the CNGA1 G622A site to be possibly deleterious. Evolutionary conservation analysis of amino acid residues showed this aspartic acid is highly conserved between species, and protein structure prediction by I-TASSER server indicated that the D208N mutation induced a large disappear of interactions between S2 and S4. Flag-tagged CNGA1 and mutant G622A cDNA were generated and inserted into pCIG-eGFP vectors. Transfection of human embryonic kidney 293T cells was performed with lipofectamine. Interestingly, western blot and immunofluorescence results indicated that the expression of mutant CNGA1 (D208N) decreased significantly, especially on the membrane of transfected HEK293T cells. The novel variant c0.622G>A (p. D208N) in this study enriched the CNGA1 mutation spectrum. Besides, this mutant was predicted “possibly damaging” due to bioinformatics analysis and validated by laboratorial experiments. Our study suggests that this mutation lead to the CNGA1 protein reduction from the cell membrane.

Omega-3 PUFA Alters the Expression Level but Not the Methylation Pattern of the WIF1 Gene Promoter in a Pancreatic Cancer Cell Line (MIA PaCa-2) Abstract Pancreatic cancer is the fourth leading cause of death in both males and females, with a 5-year relative survival rate of 8%. The Wnt signaling pathway has a significant role in the pathogenesis of many tumors, including those of pancreatic cancer. Hypermethylation of the Wnt inhibitory Factor-1 (WIF1) gene promoter have been detected in different types of cancer. In contrast, the anticancer effects of long-chain omega-3 PUFA (ALA) have been reported. Regarding its anticancer effects, in this study, we investigated the effects of various concentrations of omega-3 PUFA on expression level and promoter methylation of the WIF1 gene in MIA PaCa-2 cells in 24, 48, and 72 h after treatment. MIA PaCa-2 cells were treated with different concentrations of omega-3 PUFA (25, 50, 100, 250, 500, and 1000 μM). Cell viability assay was carried out followed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and methylation-specific PCR (MSP). This investigation suggested that dietary consumption of omega-3 PUFAs (250–1000 μM) has a significant effect on the proliferation and WIF1 gene expression of the MIA PaCa-2 cancer cell line but no effect on the promoter methylation of this gene. Changes in promoter methylation were not observed in any of the treatments.

Down-Regulation of SIRT1 Expression by mir-23b Contributes to Lipid Accumulation in HepG2 Cells Abstract Non-alcoholic fatty liver disease is one of the main causes of chronic liver disease and therefore is currently considered a major public health problem. Sirtuin 1 (SIRT1) is an NAD-dependent deacetylase enzyme that contributes in the regulation of metabolic processes and protects against lipid accumulation in hepatocytes. Its expression is potentially regulated by microRNAs which attach to the 3′ untranslated region (3′-UTR) of their target mRNA. HepG2 cells were incubated by glucose to induce lipid accumulation and were subsequently transfected with mir-23b mimic and inhibitor. Real-time PCR was used for measuring the expression of mir-23b and SIRT1 mRNA. Cell survival assay and intracellular triglyceride measurement were performed using colorimetric methods. Determination of SIRT1 protein level and activity were done by western blot and fluorometric analysis, respectively. The interaction of miR-23b with 3′-UTR of SIRT1 mRNA was confirmed by dual luciferase. miR-23b mimic inhibited gene and protein expression of SIRT1, while the inhibitor of miR-23b significantly elevated the expression levels of SIRT1 mRNA and protein. The results showed that the 3′-UTR of SIRT1 mRNA is a direct target for miR-23b. The intracellular triglyceride level was increased following the inhibition of SIRT1 in transfected HepG2 cell by miR-23b mimic. Cell viability was decreased in response to miR-23b upregulation compared to control cells. miR-23b reduces the expression and activity of SIRT1 and therefore may be a causative factor in the enhancement of lipid accumulation in HepG2 cells.

Genome-Wide Identification and Characterization of DIR Genes in Medicago truncatula Abstract Dirigent proteins (DIRs) are critically involved in the formation of lignans, a diverse and widely distributed class of secondary plant metabolites exhibiting interesting pharmacological activities and implicated in natural plant defense. However, no detailed information is available about DIR gene family in Medicago truncatula. In this study, a total of 45 DIR genes were identified in M. truncatula. DIR proteins have variability in sequence. Most MtDIR genes have no intron. All MtDIR proteins contain single dirigent domain. A large number of MtDIR genes were expanded via gene duplication, and 37 MtDIR genes were duplicated in tandem. Digital expression data showed that 40% MtDIR genes had a higher expression level in the root. Analysis of RNA-seq and microarray data indicated that more than 30% MtDIR genes were responsive to biotic and/or abiotic treatments. This study will facilitate further studies on DIR family and provide useful clues for functional validation of DIR genes in higher plants.

Variant Ionotropic Receptors are Expressed in the Antennae of Anopheles sinensis (Diptera: Culicidae) Abstract Mosquitoes transmit many harmful diseases that seriously threaten public health. The mosquito’s olfactory system is of great significance for host selection. Inotropic receptors (IRs) and olfactory receptors (ORs) have been demonstrated to be capable of odorant molecular recognition. Analyzing the molecular principles of mosquito olfaction facilitates the development of prevention and therapy techniques. Advances in the understanding of IRs have been seriously inadequate compared to those of ORs. Here, we provide evidence that 35 Anopheles sinensis IR (AsIR) genes are expressed, 7 of which are in the antennae and 2 have expression levels that are upregulated with a blood meal. A homologous analysis of the sequences showed that AsIRs are a subfamily of ionotropic glutamate receptors (iGLURs). This is the first that time IRs have been identified in Anopheles sinensis in vitro. The ultrastructure of the antennae supports the theory that diverse sensilla are distributed in the antennae. The results here may facilitate the revelation of the regulation mechanism in AsIRs, which could mitigate the transmission of diseases by mosquitoes.

Biological Effects of Single-Nucleotide Polymorphisms in the Drosophila melanogaster Malic Enzyme Locus Abstract A pair of amino acid polymorphisms within the Drosophila melanogaster Malic enzyme (Men) locus presents an interesting case of genetic variation that appears to be under selection. The two alleles at each site are biochemically distinct, but their biological effects are unknown. One polymorphic site is near the active site and the other is buried within the protein. Strikingly, in twelve different populations, the first polymorphism is always found at approximately a 50:50 allelic frequency, whereas the second polymorphism is always found at approximately 90:10. The consistency of the frequencies between populations suggests that the polymorphisms are under selection and it is possible that balancing selection is at play. We used 16 lines of flies to create the nine genotypes needed to quantify both effects of the polymorphic sites and possible genetic background effects, which we found to be widespread. The alleles at each site differ, but in different biochemical characteristics. The first site significantly influences MEN Km and Vmax, whereas the second site affects the Km and the Vmax/Km ratio (relative activity). Interestingly, the rarest allele is the most biochemically distinct. We also assayed three more distal phenotypes, triglyceride concentration, carbohydrate concentration, and longevity. In all cases, the phenotypes of the heterozygous genotypes are intermediate between those of the respective homozygotes suggesting that if balancing selection is maintaining the observed allele frequencies it is not through non-linear combinations of the biochemical phenotypes.

Morphological Characterization and Gene Expression Patterns for Melanin Pigmentation in Rex Rabbit Abstract Animal melanin has an important role in the formation of animal fur and skin, which is determined by its quantities, character, and distribution. To identify the effect of melanin on the formation of multi-colored Rex rabbits (Black, Chinchilla, Beaver, Protein cyan, Protein yellow, White), the structure of hair follicles and melanin content in multi-colored Rex rabbit skins were observed by Hematoxylin and Eosin (H&E) staining and melanin staining, respectively. The melanin granules were primarily found in the epidermis and hair follicle roots. The melanin content of skin was measured by extracting melanin from skin tissue. The results demonstrated that the melanin content was the highest in the skin of black Rex rabbit. Additionally, we measured the mRNA and protein expression levels of melanin-related key genes (MITF and TYR) in the skin of different hair color by quantitative real-time PCR and Wes assay, respectively. The results revealed that the mRNA expression levels in the skin of black Rex rabbit was highly expressed when as compared with other Rex rabbit skin (P < 0.01), and they were the lowest in the skin of white Rex rabbit. Finally, correlation analysis was conducted between melanin content and the expression levels of mRNA and protein. The results indicated a significant correlation between melanin content and the mRNA expression of MITF (P < 0.05), but it was not correlated with the mRNA expression of TYR (P > 0.05). In summary, melanin deposition has important economic value, and the coat color of fur-bearing animals is partly determined by the melanin-related genes.