Association between expression levels and growth trait-related SNPs located in promoters of the <Emphasis Type="Italic">MC4R</Emphasis> and <Emphasis Type="Italic">MSTN</Emphasis> genes in <Emphasis Type="Italic">Spinibarbus hollandi</Emphasis>

Melanocortin 4 receptor: (MC4R) and Myostatin (MSTN) are two important growth trait-related genes in animals. In this study, we showed that two SNPs, MC4R-719A>G and MSTN-519C>T, found in the promoters of the MC4R and MSTN genes, respectively, are both associated with growth traits in Spinibarbus hollandi. Furthermore, we observed that there were significant associations between the expression levels of the MC4R and MSTN genes and these two growth trait-related SNPs. The expression level of MC4R gene in brain was lower in GG genotype fish with extremely high growth performance than that in AA genotype fish with extremely low growth performance. Expression level of the MSTN gene in muscle was lower in TT genotype fish with extremely high growth performance than that in CC and CT genotype fish with lower growth performance. The results indicated that these SNPs located in the promoters of MC4R and MSTN are associated with growth-related traits through modification of gene expression levels. The MSTN and MC4R SNPs may have useful application in effective marker-assisted selection aimed to increase output in S. hollandi.     

Genome-wide association study of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">Paratuberculosis</Emphasis> infection in Chinese Holstein

Background

Paratuberculosis is a contagious, chronic and enteric disease in ruminants, which is caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection, resulting in enormous economic losses worldwide. There is currently no effective cure for MAP infection or a vaccine, it is thus important to explore the genetic variants that contribute to host susceptibility to infection by MAP, which may provide a better understanding of the mechanisms of paratuberculosis and benefit animal genetic improvement. Herein we performed a genome-wide association study (GWAS) to identify genomic regions and candidate genes associated with susceptibility to MAP infection in dairy cattle.

Results

Using Illumina Bovine 50?K (54,609 SNPs) and GeneSeek HD (138,893 SNPs) chips, two analytical approaches were performed, GRAMMAR-GC and ROADTRIPS in 937 Chinese Holstein cows, among which individuals genotyped by the 50?K chip were imputed to HD SNPs with Beagle software. Consequently, 15 and 11 significant SNPs (P?<?5?×?10^??5) were identified with GRAMMAR-GC and ROADTDRIPS, respectively. A total of 10 functional genes were in proximity to (i.e., within 1?Mb) these SNPs, including IL4, IL5, IL13, IRF1, MyD88, PACSIN1, DEF6, TDP2, ZAP70 and CSF2. Functional enrichment analysis showed that these genes were involved in immune related pathways, such as interleukin, T cell receptor signaling pathways and inflammatory bowel disease (IBD), implying their potential associations with susceptibility to MAP infection. In addition, by examining the publicly available cattle QTLdb, a previous QTL for MAP was found to be overlapped with one of regions detected currently at 32.5?Mb on BTA23, where the TDP2 gene was anchored.

Conclusions

In conclusion, we identified 26 SNPs located on 15 chromosomes in the Chinese Holstein population using two GWAS strategies with high density SNPs. Integrated analysis of GWAS, biological functions and the reported QTL information helps to detect positional candidate genes and the identification of regions associated with susceptibility to MAP traits in dairy cattle.

     

Haplotype-specific <Emphasis Type="Italic">MAPT</Emphasis> exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders

Background

Genome wide association studies have identified microtubule associated protein tau (MAPT) H1 haplotype single nucleotide polymorphisms (SNPs) as leading common risk variants for Parkinson’s disease, progressive supranuclear palsy and corticobasal degeneration. The MAPT risk variants fall within a large 1.8 Mb region of high linkage disequilibrium, making it difficult to discern the functionally important risk variants. Here, we leverage the strong haplotype-specific expression of MAPT exon 3 to investigate the functionality of SNPs that fall within this H1 haplotype region of linkage disequilibrium.

Methods

In this study, we dissect the molecular mechanisms by which haplotype-specific SNPs confer allele-specific effects on the alternative splicing of MAPT exon 3. Firstly, we use haplotype-hybrid whole-locus genomic MAPT vectors studies to identify functional SNPs. Next, we characterise the RNA-protein interactions at two loci by mass spectrometry. Lastly, we knockdown candidate splice factors to determine their effect on MAPT exon 3 using a novel allele-specific qPCR assay.

Results

Using whole-locus genomic DNA expression vectors to express MAPT haplotype variants, we demonstrate that rs17651213 regulates exon 3 inclusion in a haplotype-specific manner. We further investigated the functionality of this region using RNA-electrophoretic mobility shift assays to show differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213 and rs1800547 and subsequently identified candidate trans-acting splicing factors interacting with these functional SNPs sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrate a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion.

Conclusions

We identified common splice factors hnRNP F and hnRNP Q regulating the haplotype-specific splicing of MAPT exon 3 through intronic variants rs1800547 and rs17651213. This work demonstrates an integrated approach to characterise the functionality of risk variants in large regions of linkage disequilibrium.

     

Molecular characterization of <Emphasis Type="Italic">HOXC8</Emphasis> gene and methylation status analysis of its exon 1 associated with the length of cashmere fiber in Liaoning cashmere goat

Homeobox protein Hox-C8 (HOXC8) is a member of Hox family. It is expressed in the dermal papilla of the skin and is thought to be associated with the hair inductive capacity of dermal papilla cells. In the present study, we isolated and characterized a full-length open reading frame of HOXC8 cDNA from the skin tissue of Liaoning cashmere goat, as well as, established a phylogenetic relationship of goat HOXC8 with that of other species. Also, we investigated the effect of methylation status of HOXC8 exon 1 at anagen secondary hair follicle on the cashmere fiber traits in Liaoning cashmere goat. The sequence analysis indicated that the obtained cDNA was 1134-bp in length containing a complete ORF of 729-bp. It encoded a peptide of 242 amino acid residues in length. The structural analysis indicated that goat HOXC8 contained a typical homeobox domain. The phylogenetic analysis revealed that Capra hircus HOXC8 had a closer genetic relationship with that of Ovis aries, followed by Bos Taurus and Bubalus bubalis. The methylation analysis suggested that the methylation degree of HOXC8 exon 1 in anagen secondary hair follicle might be involved in regulating the growth of cashmere fiber in Liaoning cashmere goat. Our results provide new evidence for understanding the molecular structural and evolutionary characteristics of HOXC8 in Liaoning cashmere goat, as well as, for further insight into the role of methylation degree of HOXC8 exon 1 regulates the growth of cashmere fiber in goat.     

Epigenetic and genetic variation in <Emphasis Type="Italic">GATA5</Emphasis> is associated with gastric disease risk

Gastric cancer incidence varies considerably among populations, even those with comparable rates of Helicobacter pylori infection. To test the hypothesis that genetic variation plays a role in gastric disease, we assessed the relationship between genotypes and gastric histopathology in a Colombian study population, using a genotyping array of immune-related single nucleotide polymorphisms (SNPs). Two synonymous SNPs (rs6061243 and rs6587239) were associated with progression of premalignant gastric lesions in a dominant-effects model after correction for multiple comparisons (p = 2.63E?07 and p = 7.97E?07, respectively); effect sizes were β = ?0.863 and β = ?0.815, respectively, where β is an estimate of effect on histopathology scores, which ranged from 1 (normal) to 5 (dysplasia). In our replication cohort, a second Colombian population, both SNPs were associated with histopathology when additively modeled (β = ?0.256, 95 % CI = ?0.47, ?0.039; and β = ?0.239, 95 % CI = ?0.45, ?0.024), and rs6587239 was significantly associated in a dominant-effects model (β = ?0.330, 95 % CI = ?0.66, 0.00). Because promoter methylation of GATA5 has previously been associated with gastric cancer, we also tested for the association of methylation status with more advanced histopathology scores in our samples and found a significant relationship (p = 0.001). A multivariate regression model revealed that the effects of both the promoter methylation and the exonic SNPs in GATA5 were independent. A SNP-by-methylation interaction term was also significant. This interaction between GATA5 variants and GATA5 promoter methylation indicates that the association of either factor with gastric disease progression is modified by the other.     

Narrow-sense heritability and <Emphasis Type="Italic">P</Emphasis><Subscript>ST</Subscript> estimates of DNA methylation in three <Emphasis Type="Italic">Populus nigra</Emphasis> L. populations under contrasting water availability

In a context of climate change and forest decline, a better understanding of the sources of tree flexibility involved in phenotypic plasticity and adaptation is needed. These last years, the role of epigenetics in the response to environmental variations has been established in several model plants at the genotype level but little is known at the level of natural populations grown in pedoclimatic sites. Here, we focused on three French natural populations of black poplar, a key pioneer tree from watersheds, planted in common garden and subjected to controlled variations of water availability. We estimated common genetic parameters such as narrow-sense heritability (h²), phenotypic differentiation index (P_ST), and the overall genetic differentiation index (F_ST) from genome-wide SNPs to evaluate the extent of epigenetic variations. Indeed, global DNA methylation levels from individuals exposed to drought or irrigated in a common garden were used. We found that the three populations were not distinguished by their levels of DNA methylation. However, a moderate drought was associated to a significant decrease in DNA methylation in the populations. Narrow-sense heritability and P_ST estimates of DNA methylation were similar to those found for biomass productivity. Heritability and P_ST were higher when trees were subjected to drought than in control condition. Negative genetic correlations between global DNA methylation and height or biomass were detected in drought condition only. Altogether, our data highlight that global DNA methylation acts as a genetic marker of natural population differentiation under drought stress in a pedoclimatic context.     

Genetic polymorphisms in the 5'-flanking region of the melanocortin 1 receptor (<Emphasis Type="Italic">MC1R</Emphasis>) gene in foxes

The one of the key pigment genes, the melanocortin 1 receptor (MC1R) gene, plays a fundamental role in the determination of coat color in a variety of mammals. However, so far there has been no report regarding the genetic variants of the MC1R promoter region and the potential association of its mutations with coat color in foxes. This work aimed to characterize 5'-flanking region of the MC1R gene and its mutations associated with coat color variations in foxes. A total of 76 individuals including 64 red foxes (Vulpes vulpes), representing 11 color morphs, and 12 arctic foxes (Vulpes lagopus), representing 2 color morphs were studied. To explore the potential cause of coat color variation in foxes, an 1105 bp region located upstream of the MC1R gene coding region was sequenced in 76 foxes. In the present study, a 1267 bp 5'-flanking region of fox MC1R gene was obtained using a PCR-mediated chromosome-walking technique and a 1105 bp segment was sequenced. A total of 8 novel SNPs and an insertion/deletion of 4 nucleotides were detected. The results of mutations analysis indicated that SNPs g.-52G>A, g.-266A>G, g.-297T>C, g.-300G>A and the insertion/deletion spaning positions g.-382~-379 were important in distinguishing V. vulpes and V. lagopus. This work, for the first time, described and confirmed the different variants existed in the 5'-flanking region of MC1R gene between red foxes and arctic foxes. These findings may be extremely helpful for further exploring the alternative splicings or promoter activity of MC1R gene for different coat-colored foxes.     

Steroid Compounds from Far Eastern Starfishes <Emphasis Type="Italic">Henricia aspera</Emphasis> and <Emphasis Type="Italic">H. tumida</Emphasis>

Six new natural compounds were isolated from two Far Eastern starfish species, Henricia aspera and H. tumida, collected in the Sea of Okhotsk. Two new glycosylated steroid polyols were obtained from H. aspera: asperoside A and asperoside B, which were shown to be (20R,24R, 25S)-3-O-(2,3-di-O-methyl-β -D-xylopyranosyl)-24-methyl-5α-cholest-4-ene-3β, 6β,8,15α,16β,26-hexaol and (20R, 24R,25S,22E)-3-O-(2,4-di-O-methyl-β-D-xylopyranosyl)-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,26-hexaol, respectively. Two other glycosylated polyols, tumidoside A, with the structure elucidated as (20R, 22E)-3-O-(2,4-di-O-methyl-β -D-xylopyranosyl)-26,27-dinor-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,25-hexaol, and tumidoside B, whose structure was elucidated as (20R,24S)-3-O-(2,3-di-O-methyl-β-D-xylopyranosyl)-5α-cholestan-3β,4β,6β,8,15α,24-hexaol, were isolated from the two starfish species. (20R, 24S)-5α-Cholestan-3β,6β,15α,24-tetraol and (20R, 24S)-5α-cholestan-3β,6β,8,15α,24-pentaol were identified only in H. tumida. The known monoglycosides henricioside H1 and laeviuscolosides H and G were also identified in both species.     

Plant DNA methyltransferase genes: Multiplicity,expression, methylation patterns

Expression and methylation patterns of genes encoding DNA methyltransferases and their functionally related proteins were studied in organs of Arabidopsis thaliana plants. Genes coding for the major maintenance-type DNA methyltransferases, MET1 and CMT3, and the major de novo-type DNA methyltransferase, DRM2, are actively expressed in all organs. Similar constitutively active expression was observed for genes encoding their functionally related proteins, a histone H3K9 methyltransferase KYP and a catalytically non-active protein DRM3. Expression of the MET1 and CMT3 genes is significantly lower in developing endosperm compared with embryo. Vice versa, expression of the MET2a, MET2b, MET3, and CMT2 genes in endosperm is much more active compared with embryo. A special maintenance DNA methylation system seems to operate in endosperm. The DNMT2 and N6AMT genes encoding putative methyltransferases are constitutively expressed at low levels. CMT1 and DRM1 genes are expressed rather weakly in all investigated organs. Most of the studied genes have methylation patterns conforming to the “body-methylated gene” prototype. A peculiar feature of the MET family genes is methylation at all three possible site types (CG, CHG, and CHH). The most weakly expressed among genes of their respective families, CMT1 and DRM1, are practically unmethylated. The MET3 and N6AMT genes have unusual methylation patterns, promoter region, and most of the gene body devoid of any methylation, and the 3'-end proximal part of the gene body is highly methylated.     

Polymorphic Imprinting of <Emphasis Type="Italic">SLC38A4</Emphasis> Gene in Bovine Placenta

Imprinted genes are characterized by monoallelic expression that is dependent on parental origin. Comparative analysis of imprinted genes between species is a powerful tool for understanding the biological significance of genomic imprinting. The slc38a4 gene encodes a neutral amino acid transporter and is identified as imprinted in mice. In this study, the imprinting status of SLC38A4 was assessed in bovine adult tissues and placenta using a polymorphism-based approach. Results indicate that SLC38A4 is not imprinted in eight adult bovine tissues including heart, liver, spleen, lung, kidney, muscle, fat, and brain. It was interesting to note that SLC38A4 showed polymorphic status in five heterogeneous placentas, with three exhibiting paternal monoallelic expression and two exhibiting biallelic expression. Monoallelic expression of imprinted genes is generally associated with allele-specific differentially methylation regions (DMRs) of CpG islands (CGIs)-encompassed promoter; therefore, the DNA methylation statuses of three CGIs in the SLC38A4 promoter and exon 1 region were tested in three placentas (two exhibiting paternal monoallelic and one showing biallelic expression of SLC38A4) and their corresponding paternal sperms. Unexpectedly, extreme hypomethylation (<?3%) of the DNA was observed in all the three detected placentas and their corresponding paternal sperms. The absence of DMR in bovine SLC38A4 promoter region implied that DNA methylation of these three CGIs does not directly or indirectly affect the polymorphic imprinting of SLC38A4 in bovine placenta. This suggested other epigenetic features other than DNA methylation are needed in regulating the imprinting of bovine SLC38A4, which is different from that of mouse with respect to a DMR existence at the mouse’s slc38a4 promoter region. Although further work is needed, this first characterization of polymorphic imprinting status of SLC38A4 in cattle placenta provides valuable information on investigating the genomic imprinting phenomenon itself.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

Analysis of the association of allelic variants of apolypoprotein E and interleukin 1 beta genes with multiple sclerosis in ethnic Tatars

Multiple sclerosis (MS) is a multifactorial disease of the central nervous system. The apolipoprotein E (APOE) and interleukin 1 β (IL1B) genes are considered to be candidate genes of MS. The aim of the study was to examine the hypothesis of the importance of APOE and IL1B gene polymorphisms in MS development in ethnic Tatars. DNA samples isolated by phenol-chloroform extraction from peripheral blood of 383 ethnic Tatars (120 MS patients and 263 healthy donors) were studied. 112C/R and 158R/C APOE gene polymorphisms as well as ?511T/C IL1B gene polymorphism were analyzed by polymerase chain reaction (PCR) followed by PCR product digestion by endonuclease. Odds ratio (OR) values were used for evaluation of the relative risk of alleles and(or) genotype combinations. It has been shown that APOE*2/*3 genotype is associated with low risk of the disease development (OR = 0.20) in women. A combined effect of APOE and IL1B allelic variants has been discovered indicating the increased risk of the disease development in the carriers of APOE*4 and IL1B*T/*T alleles (OR = 4.76).     

Expression and DNA methylation pattern of reproduction-related genes in partially fertile triploid Pacific oysters <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

Partial or complete sterility is an obvious feature in triploid Pacific oyster (Crassostrea gigas) which contributes to improving rearing performances. Despite the significance of sterility, the molecular mechanism behind it remains elusive and related research was limited. This study focused on six reproduction-related genes and compared their different behavior in gene expression and DNA methylation pattern between triploid and diploid oysters in order to provide more molecular information. The gonadal development of triploid oyster was examined by histology before molecular analysis. Gametogenesis disturbance was observed in triploid oysters at different development stages (stage II and III) with more serious impairment in females. QPCR showed significant gene expression difference between diploid and triploid in two genes: putative Vg and cgER. Gene expression of putative Vg was delayed in triploids while for cgER triploid oyster showed higher expression and the difference was significant at stage III. DNA methylation pattern of these two genes were further investigated by bisulfite sequencing. Between diploid and triploid oysters, no difference was observed in total methylation level but some individual loci showed different patterns: significantly high methylation rate of loci 2284 in cgER was observed in triploid oyster which has a higher expression of this gene. This study indicated that putative Vg and cgER might play a role in partial sterile in triploid C. gigas. Gene expression could be regulated by the methylation pattern at specific individual locus, which deserves equivalent attention as well as total DNA methylation level.