A Novel Gene, Le-Dd 10, Is Involved in Fruiting Body Formation of Lentinula Edodes

The cDNA library prepared from Lentinula edodes, Hokken 600 (H600), primordia was screened by using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd 1~21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes. The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.

Identification of Polymorphism within Exon 8 of Bovine HSP90AA1 Gene using PCR-SSCP Technique

Heat shock proteins (HSPs) are a type of molecular chaperones that aid in the recovery of stressed cells and serve as a major system for intracellular self-defense. A study was conducted during the year 2018–19 at College of Veterinary Science Rajendranagar, Hyderabad, Telengana State, India to find polymorphisms in exon 8 of the bovine HSP90AA1 gene in Sahiwal (n=50) and crossbred (n=50) cows. Blood samples were collected from the experimental animals and genomic DNA was isolated. Physiological parameters like body temperature and respiration rate for each animal were taken during the experimental period and the heat tolerance coefficient was calculated. The data on production and reproduction traits were obtained from the history sheets of the animals. To detect the polymorphism, a 539 bp fragment of the HSP90AA1 gene covering exon 8 was subjected to the Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) technique.The PCR-SSCP of exon 8 of HSP90AA1 gene yielded two genotypic patterns AA and AB corresponding to two allelic variants with frequencies of 0.85, 0.15, 0.81 and 0.19 in Sahiwal and crossbred cows, respectively. The PCR-SSCP patterns obtained were correlated with the physiological, productive, and reproductive traits in both Sahiwal and crossbred cows. The association analysis of SSCP patterns of the exon 8 of HSP90AA1 gene revealed non-significant effect in Sahiwal cows, although the AB genotype had a significantly longer service period in crossbred cows.

​Genetic Polymorphism within Exon 3 of HSP90AA1 Gene and its Association Studies in Sahiwal and Crossbred Cows

Background: Heat shock proteins (HSPs) are molecular chaperones that play a critical role in recovering cells from stress and form a primary system for intra cellular self defense. They are highly conserved and play a crucial role in cellular thermo tolerance and heat stress response. Though there are many HSP genes, thermo tolerance is mainly correlated with HSP70 and HSP90 genes in Livestock species. Polymorphisms in these genes have shown an association with heat tolerance, milk production, fertility and disease susceptibility in livestock. They can be used as genetic markers for the selection of animals with better climate resilience, immune response and superior performance. Methods: The present study was carried out in Sahiwal (n=50) and Crossbred cows (n=50) with the objective to identify polymorphisms in HSP90AA1 gene. A 450 bp fragment of bovine HSP90AA1 gene covering exon3 was subjected to Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) technique to identify the polymorphism. PCR-SSCP patterns were correlated with the physiological, productive and reproductive traits in Sahiwal and crossbred cows using the univariate GLM model of SPSS 25. Result: The PCR-SSCP of exon 3 of HSP90AA1 gene yielded two conformational patterns AA and AB corresponding to two allelic variants A and B in both Sahiwal and crossbred cows. The allele frequencies of A and B were 0.78 and 0.22 and 0.84 and 0.16 in Sahiwal and crossbred cows, respectively. The association analysis of SSCP patterns revealed that genotype AA had higher lactation length in Sahiwal cows and higher total lactation milk yield and peak yield in crossbred cows.

Effects of Genetic Polymorphisms of Cathepsin A on Metabolism of Tenofovir Alafenamide

Cathepsin A (CatA) is important as a drug-metabolizing enzyme responsible for the activation of prodrugs, such as the anti-human immunodeficiency virus drug Tenofovir Alafenamide (TAF). The present study was undertaken to clarify the presence of polymorphisms of the CatA gene in healthy Japanese subjects and the influence of gene polymorphism on the expression level of CatA protein and the drug-metabolizing activity. Single-strand conformation polymorphism method was used to analyze genetic polymorphisms in healthy Japanese subjects. Nine genetic polymorphisms were identified in the CatA gene. The polymorphism (85_87CTG>-) in exon 2 was a mutation causing a deletion of leucine, resulting in the change of the leucine 9-repeat (Leu9) to 8-repeat (Leu8) in the signal peptide region of CatA protein. The effect of Leu8 on the expression level of CatA protein was evaluated in Flp-In-293 cells with a stably expressed CatA, resulting in the expression of CatA protein being significantly elevated in variant 2 with Leu8 compared with Leu9. Higher concentrations of tenofovir alanine (TFV-Ala), a metabolite of TAF, were observed in the Leu8-expressing cells than in the Leu9-expressing cells using LC/MS/MS. Our findings suggest that the drug metabolic activity of CatA is altered by the genetic polymorphism.

Ovine Toll-like Receptor 9 (TLR9) Gene Variation and Its Association with Flystrike Susceptibility

Toll-like receptors (TLRs) are a family of proteins that play a role in innate immune responses by recognising pathogen-associated molecular patterns derived from various microbes. Of these receptors, TLR9 recognises bacterial and viral DNA containing unmethylated cytosine-phosphate-guanine (CpG) motifs, and variation in TLR9 has been associated with resistance to various infectious diseases. Flystrike is a problem affecting the sheep industry globally and the immune response of the sheep has been suggested as one factor that influences the response to the disease. In this study, variation in ovine TLR9 from 178 sheep with flystrike and 134 sheep without flystrike was investigated using a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) approach. These sheep were collected from both commercial and stud farms throughout New Zealand and they were of 13 different breeds, cross-breds and composites. Four alleles of TLR9 were detected, including three previously identified alleles (*01, *02 and *03) and a new allele (*04). In total six single nucleotide polymorphisms (SNPs) were found. Of the three common alleles in the sheep studied, the presence of *03 was found to be associated with a reduced likelihood of flystrike being present (OR = 0.499, p = 0.024). This suggests that variation in ovine TLR9 may affect a sheep’s response to flystrike, and thus the gene may have value as a genetic marker for improving resistance to the disease.

Association of SSCP Polymorphisms of HSP70 Gene with Physiological, Production and Reproduction Performance in Sahiwal and Crossbred Cows

Background: Cellular tolerance to heat stress is mediated by heat shock proteins (HSPs). The HSPs act as molecular chaperones and are transcribed in response to stress. Among different families of these proteins, HSP70 is considered to be related to the development of temperature tolerance. Unraveling polymorphism in heat shock protein genes could be a step towards the identification of genetic markers for selecting heat-tolerant cattle. Methods: The present study was carried out in Sahiwal (n=50) and Crossbred cows (n=50) with the objective to identify polymorphisms in HSP70 gene. Two fragments (295 and 220 bp) of HSP70 gene were subjected to Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) technique. Statistical analysis was performed to study the association of each SSCP genotype on physiological, production and reproduction traits in Sahiwal and crossbred cows using the univariate GLM model of SPSS 25. Result: The PCR-SSCP of 295 bp fragment of HSP70 gene revealed two genotypes AA and AB in Sahiwal cows and two genotypes AA and AC in crossbred cows. The association analysis revealed that genotype AA had higher peak milk yield in Sahiwal cows while the same genotype had higher total lactation milk yield, lower service period and calving interval in crossbred cows. The 220 bp fragment was found to be monomorphic in both Sahiwal and crossbred cows.

Variation in the Ovine Glycogen Synthase Kinase 3 Beta-Interaction Protein Gene (GSKIP) Affects Carcass and Growth Traits in Romney Sheep

The glycogen synthase kinase 3 beta (GSK3β)-interacting protein (encoded by the gene GSKIP) is a small A-kinase anchoring protein, which complexes with GSK3βand protein kinase A (PKA) and acts synergistically with cAMP/PKA signaling to inhibit GSK3β activity. The protein plays a role in regulating glycogen metabolism, protein synthesis, the cell cycle, and in regulating gene expression. In this study, PCR-single strand conformation polymorphism (PCR-SSCP) analyses were used to screen for variation in exon 1 and exon 2 of GSKIP in 840 New Zealand (NZ) Romney sheep. Two SSCP banding patterns representing two different nucleotide variants (A and B) were detected in an exon 1 region, whereas in an exon 2 region only one pattern was detected. Variants A and B of exon 1 had one non-synonymous nucleotide difference c.37A/G (p.Met13Val). The birthweight of sheep of genotype AA (5.9 ± 0.06 kg) was different (p = 0.023) to sheep of genotype AB (5.7 ± 0.06 kg) and BB (5.7 ± 0.06 kg). The hot carcass weight (HCW) of sheep of genotype AA (17.2 ± 0.22 kg) was different (p = 0.012) to sheep of genotype AB (17.6 ± 0.22 kg) and BB (18.0 ± 0.29 kg), and the fat depth at the 12th rib (V-GR) of sheep of genotype AA (7.7 ± 0.31 mm) was different (p = 0.016) to sheep of genotype AB (8.3 ± 0.30 mm) and BB (8.5 ± 0.39 mm). The results suggest that the c.37A/G substitution in ovine GSKIP may affect sheep growth and carcass traits.

Arbuscular Mycorrhizal Fungi from Argentinean Highland Puna Soils Unveiled by Propagule Multiplication

Low arbuscular-mycorrhizal (AM) sporulation in arid field soils limits our knowledge of indigenous species when diversity studies are based only on spore morphology. Our aim was to use different approaches (i.e., spore morphological approach and PCR–SSCP (single-strand-conformation-polymorphism) analysis after trap plant multiplication strategies to improve the knowledge of the current richness of glomalean AM fungi (Glomerales; Glomeromycota) from the Argentine Puna. Indigenous propagules from two pristine sites at 3870 and 3370 m of elevation were multiplied using different host plants; propagation periods (2–6 months), and subculture cycles (1; 2; or 3) from 5 to 13 months. The propagule multiplication experiment allowed the detection of different glomoid taxa of Funneliformis spp. and Rhizoglomus spp., which were considered cryptic species since they had never been found in Puna soils before. On the other hand; almost all the generalist species previously described were recovered from cultures; except for Glomus ambisporum. Both plant host selection and culture times are critical for Glomerales multiplication. The SSCP analysis complemented the morphological approach and showed a high variability of Glomus at each site; revealing the presence of Funneliformis mosseae. This study demonstrates that AMF trap culture (TC) is a useful strategy for improving the analysis of AM fungal diversity/richness in the Argentinean highlands.

Single-Strand Conformation Polymorphism Fingerprint Method for Dictyostelids

Dictyostelid social amoebae are a highly diverse group of eukaryotic soil microbes that are valuable resources for biological research. Genetic diversity study of these organisms solely relies on molecular phylogenetics of the SSU rDNA gene, which is not ideal for large-scale genetic diversity study. Here, we designed a set of PCR–single-strand conformation polymorphism (SSCP) primers and optimized the SSCP fingerprint method for the screening of dictyostelids. The optimized SSCP condition required gel purification of the SSCP amplicons followed by electrophoresis using a 9% polyacrylamide gel under 4°C. We also tested the optimized SSCP procedure with 73 Thai isolates of dictyostelid that had the SSU rDNA gene sequences published. The SSCP fingerprint patterns were related to the genus-level taxonomy of dictyostelids, but the fingerprint dendrogram did not reflect the deep phylogeny. This method is rapid, cost-effective, and suitable for large-scale sample screening as compared with the phylogenetic analysis of the SSU rDNA gene sequences.

Short Communication: A highly polymorphic caprine keratin-associated protein gene identified and its effect on cashmere traits

Five keratin-associated protein 6 genes (KRTAP6) have been identified in sheep and variation in some KRTAP6 has been associated with wool fibre diameter-related traits, but none of these homologues has been identified in goats. In this study, we reported the identification of the sheep KRTAP6-5 homologue on goat chromosome 1 and PCR-single strand conformation polymorphism analysis in 300 Longdong cashmere goats revealed the existence of twelve variant sequences. Both coding region and 3’UTR of the putative caprine KRTAP6-5 displayed a biggest sequence similarity to ovine KRTAP6-5 gene. This suggested that the gene represents caprine KRTAP6-5 sequences, and these sequences composed twenty three genotypes which was the most polymorphism gene in KRTAPs that have been studied. Among these sequences, fifteen nucleotide substitutions and a 24-bp insertion/detection were identified. Variation in goat KRTAP6-5 was associated with variation in mean fibre diameter, suggesting that KRTAP6-5 is worthy of further study in the context of variation in cashmere traits.