Single-strand Conformation Polymorphism Analysis Research Articles

Regional bias of tumor suppressor gene mutations of STARD8 and WNK2 in colon cancers

StAR-related lipid transfer domain protein 8 (STARD8), encoding a Rho-GTPase-activating protein, and WNK2, encoding a serine/threonine kinase are candidate tumor suppressor genes (TSGs) in human cancers. Inactivation of these genes that would promote cancer pathogenesis is largely unknown in colon cancer (CC). Our study addressed to address whether STARD8 and WNK2 genes are mutated in CC. STARD8 and WNK2 genes possess mononucleotide repeats in their exons, which could be the targets for frameshift mutations in cancers with high microsatellite instability (MSI-H). By single-strand conformation polymorphism (SSCP) analysis, we analyzed the repeated sequences in 140 CCs (95 CCs with MSI-H and 45 CCs with stable MSI (MSS)). By DNA sequencing, we found that five MSI-H CCs (5/95: 5.3%) harbored the frameshift mutations, whereas MSS CCs (0/45) did not. In addition, we detected regional heterogeneous frameshift mutations of these genes in four (25%) of 16 MSI-H CCs. In immunohistochemistry for WNK2, WNK2 expression in the MSI-H CCs was significantly lower than that in the MSS CCs. Our results for the mutation and expression indicate that STARD8 and WNK2 genes are altered at various levels (frameshift mutation, expression, and regional heterogeneity) in MSI-H CCs, which might play a role in the pathogenesis by inactivating their TSG functions.

Ovine KRTAP36-2: A New Keratin-Associated Protein Gene Related to Variation in Wool Yield.

Keratin-associated proteins (KAPs) are structural components of wool fibres. High-glycine/tyrosine (HGT)-KAPs are a subset of the KAP family, and their abundance in fibres varies. In this study, we report the discovery of an ovine HGT-KAP gene to which we assigned the name KRTAP36-2. Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analyses revealed four variants of this gene in a screening population of 170 sheep from a variety of breeds. The DNA sequencing of the variants revealed four single-nucleotide polymorphisms (SNPs) and a dinucleotide deletion. Three of these SNPs were in the coding region, and one of these was non-synonymous and potentially led to the amino acid substitution p.Cys27Gly near the middle of the protein. The remaining SNP was located near the putative TATA box, and the di-nucleotide deletion was near the putative transcription initiation site. The effect of this variation in KRTAP36-2 was investigated in 274 Southdown × Merino lambs that were the progeny of five sires. Variation was only found to be associated with wool yield, that is, the proportion of the greasy fleece that remained as clean fleece upon scouring (expressed as a percentage). This may have some value in increasing wool production.

Association of ovine follistatin gene polymorphisms with body measurements, fat-tail traits and morphometric of head in Iranian Mehraban sheep

The mature body size of sheep is a polygenic trait that is used as a main selection criterion for increasing meat production through genetic selection. The purpose of this study was to characterize polymorphisms of the follistatin gene (FST locus), which is known to regulate muscle hypertrophy by inhibiting both myostatin and activin. Using single-strand conformation polymorphism analysis (SSCP) and Sanger sequencing, we examined polymorphisms of the ovine FST gene (OAR_v.3.1; Chr 16, NC_019473.1) in Iranian Mehraban sheep (n = 100, seven-month-old male lambs) with records of body measurements, fat-tails, and head morphometry. Among the nine SSCP patterns A to I, we identified five single nucleotide polymorphisms (SNPs), including g.25632659 G>C, g.25632701 G>A, g.25633069 A>G, g.25633069 A>T, and g.25634085 G>C. The only amino acid-altering variant was g.25632659 G>C in exon 3 (p.216Thr > Ser). According to a subsequent statistical analysis, SSCP patterns A (g.25632659 G/C, g.25632701 G/G), B (g.25632659 C/C, g.25632701 G/A), C (g.25632659 C/C, g.25632701 A/A), D (homozygous reference; g.25633069 A/A), E (heterozygous; g.25633069 A/G) and F (homozygous variant; g.25633069 T/T) were significantly (P < 0.05) connected with body measurements. Furthermore, patterns A and B had significantly (P ≤ 0.05) longer forehead lengths than the lambs with the pattern C. Pattern I (homozygous variant; g.25634085 C/C) was significantly associated with an increased mouth width, and decreased eye length compared to patterns H (heterozygous; g.25634085 G/C) and G (homozygous reference; g.25634085 G/G). The findings of this study provide primary evidence of linking between the polymorphisms of the FST gene and body size in a small panel of Iranian Mehraban sheep population.

Microbiomes of soil and roots of two palm species infected with ‘Candidatus Phytoplasma palmae’ in two different ecosystems: Single strand conformation polymorphism analysis

Microbiomes of soil and roots of two palm species infected with ‘Candidatus Phytoplasma palmae’ in two different ecosystems: Single strand conformation polymorphism analysis

Nucleotide Sequence Assessment of Four ORFs of Citrus Tristeza Virus: Evidence of Recombination

Citrus Tristeza Virus (CTV), usually occurs in nature as a mixture of genotypes. Six naturally infected citrus (Citrus sinensis) trees grafted on sour orange rootstock were collected from three citrus growing governorates in Egypt (Sharqia, Qalyubia and Garbia). In this study, RT-PCR, Single-Strand Conformation Polymorphism (SSCP) and nucleotide sequence analysis were used for four independent CTV genomic regions (p65, p18, p20, and p23) to detect and assess the sequence and genetic variabilities among CTV Egyptian isolates. RTPCR products (650 bp) for the CTV p23 gene obtained from the selected isolates were used for the SSCP analysis and DNA sequencing. SSCP patterns of p23 gene for individual isolates yielded different complex haplotype patterns. Nucleotide sequence analysis of p23 region amplified from six isolates under study revealed that p23 shared high nucleotide identity 98.7% with T36 isolate from USA, Florida. Phylogenetic analysis of p23 gene indicated a close evolutionary relationship between all examined isolates and Qaha isolate (T36 isolate group), suggesting that they may have originated from closely related ancestors. Nucleotide sequence analysis of the three genes located on CTV 3′-coterminal overhang, p18, p20 and p65, amplified from isolate A3, Sharqia governorate, revealed that the p18, p65, and p20 genes were related to the T3-KB isolate from South Africa with 99%–100% sequence homology. Phylogenetic relationship analysis for p65, p18 and p20 ORFs clustered the current A3 isolate with T3 genotype group. The recombination analysis identified three of six isolates from Sharqia, and Garbia as potential recombinant for p23 gene. The isolates T36 and T3 were identified as major donors for recombination events in isolate A3. Our results concluded that p23 ORF likely to be as a hotspot region for recombination and originated through recombination event. The current study indicated that recombination is an important factor for the origin of CTV strains in Egypt.

SEQUENCE VARIATIONS AND HAPLOTYPIC DIVERSITY IN THE MYOSTATIN GENE OF NIGERIAN CATTLE BREEDS

The myostatin gene (MSTN) encodes myostatin protein (MSTN), whose function involves regulating muscle growth and development. Using Polymerase Chain Reaction coupled with Single Strand Conformational Polymorphism analysis with nucleotide sequencing, a total of 161 animals from four different breeds and cross breeds of Nigerian cattle were genotyped at the introns 1 and 2 region, as well as exons 1, 2 and 3. Sixteen SNPs were identified, two (c.374- 796C&gt;T and c.374-762C&gt;T) of which are novel and are reported here. The identification of new as well as previously reported genetic variations in MSTN within this species provides more insight into the polymorphic nature of this gene. Thus, providing an important resource forfurther studies around genomic selection and breeding.

Helicobacter pylori virulent genes and host interleukin 1 are associated with demographic factors for an increased risk of gastritis

AimHelicobacter pylori is a predominant pathogen for the human race and causes gastrointestinal diseases as well as can be an increased risk for cancer. This study examined the bacterial and host genes involved in gastritis and the environmental influences to understand the disease outcome. MethodsPolymerase chain reaction assay was used to analyze the variations in virulence-associated genes of H. pylori in gastritis patients. Human interleukin 1 beta gene polymorphisms and IL-1 receptor antagonist gene (IL-1RN intron 2) variable number tandem repeats were evaluated by PCR and single-strand conformation polymorphism analysis, respectively. Various demographic factors were analyzed and all the statistical tests were two-sided. ResultsInfection with cagA, cagA – Asian lineage positive strains of H. pylori was associated with an increased risk for gastritis. IL-1B-511*T carriers homozygous for the short allele of IL-1RN (IL-1RN*2/*2) had an increased risk for gastritis for the cagA carrier. The odd ratio (OR – 0.17; 95% CI – 0.03 to 0.08; P = 0.04) was significant in certain bacterial (cagA) and host (IL-1RN*2) high-risk genotypes in the gastritis patients. ConclusionThe pathogen and the host genetics in relation to lifestyle habits provide a tool for defining the high-risk individuals and strategize H. pylori eradication programs.

Genotyping and Identification of Antigen B Gene Polymorphism of Echinococcus granulosus in Edirne, Thrace, and the First Report of Genotype G2 (Tasmanian Sheep Strain) in Turkey

Echinococcus granulosus is the causative agent of cystic echinococcosis in humans and livestock. It is common worldwide. Cystic echinococcosis is still an important public health problem in Turkey, which is an endemic region. To genotype Echinococcus granulosus isolates and investigate antigen B gene polymorphism in Thrace, Turkey. A cross-sectional study. Seventy-five hydatid cyst materials obtained between June 2020 and May 2021 were included in the study. Hydatid cyst materials were collected from 12 humans from various hospitals in Edirne and 63 from slaughterhouse animals during the same period. Cyst materials were localized in 8 livers and 4 lungs in humans, 23 livers and 17 lungs in cattle, and 13 livers and 10 lungs in sheep. In the first step, the 12S ribosomal RNA gene was amplified by polymerase chain reaction for all samples and run on an agarose gel. Band patterns were used for strain typing. Then, the selected samples that represented each of the band patterns obtained by single-strand conformation polymorphism analysis were sequenced for AgB1, AgB2, mt-CO1, and mt-ND1 genes. Three different genotypes in Edirne, Thrace, Turkey, were observed for Echinococcus granulosus: G1 (domestic sheep strain), G2 (Tasmanian sheep strain), and G3 (buffalo strain). G1 was the dominant genotype in Edirne, and G3 was the second most common. Additionally, polymorphism in AgB1 and AgB2 gene regions was found. This study is the first to report on Echinococcus granulosus G2 (Tasmania sheep strain) in Turkey and G3 (buffalo strain) and antigen B polymorphism in Thrace. The study results will contribute to the prevention and control programs for cystic echinococcosis in Turkey and worldwide.

Detection of Theileria orientalis Genotypes from Cattle in Kyrgyzstan.

The ikeda and chitose genotypes of Theileria orientalis, which for many years were thought to be benign, cause a disease that results in significant economic losses in the cattle industry. This study was carried out in order to determine the genotypes of T. orientalis in cattle in Kyrgyzstan, and 149 archived DNA samples known to be T. orientalis were analyzed by the PCR amplification of the major piroplasm surface protein (MPSP) gene region. Single-Strand Conformation Polymorphism (SSCP) analysis was performed to uncover the nucleotide changes in the archived DNA samples, and 15 samples showing different band profiles were subjected to sequence analysis. As a result of the sequence analysis, it was seen that the samples belonged to the buffeli and chitose A genotypes. In order to identify mixed genotypes, PCR was performed using primers specific for these genotypes, and buffeli (type 3), chitose (type 1) and buffeli+chitose were found to be positive in 26.2%, 2% and 71.8% of samples, respectively. As a result of this study, we showed the presence of buffeli (type 3) and chitose (type 1) genotypes of T. orientalis in cattle in Kyrgyzstan. Comprehensive epidemiological studies are needed to understand the clinical infections caused by the pathogenic chitose A and to determine the geographical distribution and different genotypes of T. orientalis.

A novel gene, Le-Dd10, is involved in fruiting body formation of Lentinula edodes

The cDNA library prepared from Lentinula edodes, Hokken 600 (H600), primordia was screened using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd1 ~ 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 an 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.

Sequence Variation in Caprine KRTAP6-2 Affects Cashmere Fiber Diameter.

Simple SummaryKeratin-associated proteins (KAPs) are a structural component of wool fibers. Despite the gene encoding the high-glycine/tyrosine KAP6-2 (called KRTAP6-2) having been described in sheep, it has not been identified goats. This study describes the identification of caprine KRTAP6-2, reports variation in the gene, and describes its association with cashmere fiber diameter in Longdong cashmere goats.Keratin-associated proteins (KAPs) are a structural component of cashmere fibers and in part determine fiber attributes. The gene encoding the high-glycine/tyrosine KAP6-2 (called KRTAP6-2) has been described in sheep, but it has not been identified goats. In this study, a 252-bp open reading frame with similarity to ovine KRTAP6-2 was found on goat chromosome 1, with its upstream and downstream flanking sequences are closely related with ovine KRTAP6-2 but are clearly distinct from other ovine KRTAP6-n sequences. Polymerase chain reaction amplification followed by single strand conformation polymorphism analysis of this region revealed five distinct banding patterns representing five different sequences (A to E) in 230 Longdong cashmere goats. Eleven diallelic single nucleotide polymorphisms (SNPs), a three-nucleotide sequence variation, and a 12-bp insertion/deletion were found among these five sequences, with most SNPs being either outside the coding region or synonymous. The presence of variant D was found to be associated with decreased mean fiber diameter (MFD; present: 13.26 ± 0.07 µm; absent: 13.55 ± 0.04 µm; p < 0.001), suggesting that variation in KRTAP6-2 may affect fiber diameter and have value as a molecular marker for improving the cashmere fiber diameter trait.

Prevalence of Virus Infections and GLRaV-3 Genetic Diversity in Selected Clones of Croatian Indigenous Grapevine Cultivar Plavac Mali.

The cultivar Plavac Mali (Vitis vinifera L.), the most important indigenous red grapevine cultivar in Croatia, was tested for the presence of 16 grapevine viruses. Thirty-five samples from the collection vineyard were tested for the presence of grapevine leafroll-associated viruses-1, -2, and -3 (GLRaV-1, GLRaV-2 and GLRaV-3, respectively), grapevine fanleaf virus (GFLV), arabis mosaic virus (ArMV), grapevine virus-A (GVA), -B (GVB), -G (GVG), -H (GVH), -I (GVI), -J (GVJ), grapevine fleck virus (GFkV), grapevine rupestris stem pitting associated virus (GRSPaV), and grapevine pinot gris virus (GPGV) by reverse transcription–polymerase chain reaction (RT-PCR). Furthermore, standard PCR was conducted for grapevine badnavirus 1 (GBV-1) and grapevine red blotch virus (GRBV). Mixed infections were most common and GLRaV-3, the most abundant virus found in 85.71% of the vines tested, was further molecularly characterised. Different genomic variants of the heat shock protein homologue (HSP70h) were separated by cloning, detected by single-strand conformation polymorphism (SSCP) analysis, sequenced, and phylogenetically analysed. The presence of phylogenetic groups I and II was only confirmed. This study demonstrates the high virus infection rate of Plavac Mali vines and the heterogeneity of GLRaV-3 present nowadays in a collection vineyard.

Detection of Polymorphism in Exon-3 Region of Leptin Gene in Bikaneri Camel

The study was undertaken on 40 Camels (Camelus dromedarius) of Bikaneri breed to identify the single nucleotide polymorphism (SNP) in leptin gene. Nucleotide sequence of Lama glama was used to design primers to amplify selected region of leptin gene in Bikaneri camel. A 540bp fragment was amplified which covered Partial intron-2, exon-3 and partial 3’ UTR region of leptin gene in Bikaneri camel. The genetic variation in partial ntron-2, exon-3 and partial 3’ UTR (540bp) region of leptin gene was studied utilising single strand conformational polymorphism (SSCP) technique followed by nucleotide sequencing. SSCP analysis revealed monomorphic pattern as only AA genotype in studied region of leptin gene was observed. For verification and validation of observed results of SSCP, amplified PCR products of 6 animals were randomly selected for sequence analysis to ascertain the sequence pattern and existence of point mutations in the selected region. All the 6 animals had same sequence pattern. Monomorphic pattern showed the conserved nature of leptin gene in dromedary camel.

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 fiber diameter-related traits, but none of these homologues have been identified in goats. In this study, we reported the identification of the sheep KRTAP6-5 homologue on goat chromosome 1 and polymerase chain reaction (PCR)-single strand conformation polymorphism analysis in 300 Longdong cashmere goats revealed the existence of 12 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 23 genotypes, which was the most polymorphism gene in KRTAPs that have been studied. Among these sequences, 15 nucleotide substitutions and a 24-bp insertion/detection were identified. Variation in goat KRTAP6-5 was associated with variation in mean-fiber diameter, suggesting that KRTAP6-5 is worthy of further study in the context of variation in cashmere traits.

Systematic Analyses of Clinically Relevant Single Nucleotide Polymorphisms (SNPs) of NR3C1 in Patients from the Italian Registry of Diamond Blackfan Anemia

The effects of GR on mRNA translation have mostly been studied in myocytes where its activation induces REDD1 that, by suppressing mTOR, reduces 4E-BP1(S65) and S6KA1(S235/236) inhibiting initiation of mRNA translation and protein biosynthesis (Wang JBC 2006;281:39128). It is then counterintuitive that GR agonists (glucocorticoids, GC) are effective in 60% of patients with DBA, a inherited form of pure RBC aplasia induced by mutations that impair protein biosynthesis by reducing the initial steps of mRNA translation (mRNA binding to the ribosomal subunit 40S and ribosme assembly). To address this paradox, we analyzed the phosphoproteomic profiling of erythroid cells generated in vitro either with or without the GR agonist dexamethasone by adult blood (AB) and cord blood (CB) (http://capmm.gmu.edu/data) for proteins affecting mRNA translation. By contrast with muscle cells, Dex significantly (p=0.049) increased S6KA1(S235/236) (fold change (FC)=4), eIF4G(S1108)(FC=4.16) and mTOR(S2448)(FC=1.75) in erythroid cells from CB. However, Dex significantly decreased RSK3(T356/S360)(FC=0.45) and MSK1(S360)(FC=0.52) and increased DEPTOR(FC=1.51) in AB. These results indicate that GR exerts, respectively, positive and negative regulatory functions on mRNA translation in fetal and adult erythroid cells.We previously reported that the single nucleotide polymorphism (SNP) rs6198 in NR3C1, the gene encoding GR, is present at frequency greater than normal in patients with DBA (Varricchio Blood 2011;118:473). This observation and the results described above led us to investigate whether additional clinically relevant NR3C1 SNPs are associated with disease manifestation in DBA. By polymerase chain reaction single strand conformation polymorphism analyses, we determined the frequency of 8 SNPs in 101 DBA patients, 67 from the Italian Registry and 34 from other European registries. Healthy volunteers (n=34) were analyzed as control. The SNPs investigated are: rs10482605 that decreases GRα and predicts GC resistance in major depression; rs10482616, rs7701443, rs6189/rs6190, rs860457, rs6198, that have unknown biological effects but predict GC resistance in Crohn's Disease; rs6196, that enhances expression of the dominant negative GR-β isoform predisposing to autoimmune diseases, myeloproliferative neoplasms and DBA, while reducing risks of diabetes in Cushing's Syndrome and rs33388/rs33389 that increase expression of GRγ which binds GC 10-times less efficiently than GRα and has not been associated with human diseases as yet. Cases versus control analyses suggests that the frequency of rs860457 (Table 1) and rs33388 (p=0.0689 by categorical analyses, not shown) is increased in this cohort of DBA patients. The reduced significance level of this observation may be due to the low numbers (34) of healthy controls used as comparison.Demographic, causal mutations and disease manifestation (GR responsiveness and age of anemia onset) are known for patients from the Italian Registry only. The patients were 55.6% females and 44.4% males and their average age at diagnosis was 9.14±24.56 months. The causal mutations were RPL5 (n=12), RPL11 (n=8); RPS26 (n=12), RPS19 (n=28), RPS17 (n=1) and unknown (n=6). Treatment outcome is known for 58 of the 67 patients: 14 responded, 41 did not respond while three patients underwent spontaneous remission and were excluded from the analyses. Age of disease manifestation is known for 57 patients: 27 (29.82) displayed the disease before and 40 (70.18%) after 2 months. Gender and causal mutations were neither associated with response nor with age of disease manifestation. None of the SNPs met the threshold in the response vs non-responsive groups (Table 1). However, two SNPs (rs6196, p=0.0509 and rs33386, p=0.1249) met the threshold criteria in the age of disease manifestation analyses (Table 1). Of note rs6196 predicts GC resistance in Crohn's disease and rs33386 favors expression of GRγ. No significant association was observed when SNPs that met the threshold were analyzed in a multivariable model.These results indicate that GR promotes protein biosynthesis in fetal but not in adult erythroid cells. The frequency of rs860457 which impairs GR functions is increased in DBA patients and rs6196 and rs33388 may be correlated with age to disease manifestation. These observations require further analyzes with larger cohorts of controls and/or patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Retinoblastoma Related Gene RB2/P130 Are Rarely Mutated in Burkitt's Lymphoma from Brazil.

Human RB2/p130 is a member of the retinoblastoma (Rb) family which also includes the retinoblastoma tumor suppressor gene RB/p105 (RB1) and the retinoblastoma related gene, p107. RB1 and other members of Rb family, p107 and p130 perform overlapping, but not fully redundant functions in cell cycle regulation and cell growth inhibition. Moreover, all three Rb family members display suppressive activities, which result in cell cycle G1 phase blocking. It has been suggested that alterations in genes controlling cell cycle probably contribute to Burkitt's lymphoma pathogenesis, in association to the 8:14 translocation that leads to c-myc protein up regulation. Mutations in tumor suppressor gene RB2/p130 were detected in cell lines and primary tumors of patients with endemic Burkitt's lymphoma (eBL). In addition, RB2/p130 mutational inactivation has been reported as a common feature in lung and nasopharyngeal carcinomas. RB2/p130 gene mutation screening was performed in 26 BL tumor pediatric samples and in mononuclear cells from 2 healthy blood donors by single strand conformation polymorphism (PCR-SSCP) analysis. Exons 19, 20, 21 and 22 were amplified separately and denatured PCR products were heated at 95°C for 5 min, chilled on ice, and immediately electrophoresed in a native 10% polyacrilamide gel, with or without the addition of glycerol at 80 volts for 20–26 hours, and silver stained. In this series, 26 children were diagnosed as BL according to histopathologic and clinical criteria. The median age was 4.5 years (range: 2 to 9 years). There were 7 females and 19 males. According to the St Jude system 19% of patients had stage I/II and 81% stage III/IV disease. Burkitt's lymphoma HIV-associated was diagnosed only in 2 patients. All four exons (19–22) were analyzed by PCR-SSCP. No changes were found upon SSCP analysis. Our results suggest that mutations in exons 19–22 of the RB2/p130 are unlikely to be involved directly in the pathogenesis of Brazilian BL. In conclusion, the characterization of tumor cells continues to emphasize the important role of Rb gene in tumor suppression but the evidence linking p107 or p130 inactivation to tumor development remains limited and controversial in Burkitt's lymphomas and other malignant tumors.

Variation in bovine leptin gene affects milk fatty acid composition in New Zealand Holstein Friesian × Jersey dairy cows.

Leptin is a protein hormone secreted from white adipose tissue. It regulates food/feed intake, body weight, immune function and reproduction. In our investigation, the polymerase chain reaction (PCR) amplification coupled with single-strand conformational polymorphism (SSCP) analysis was used to reveal variation in bovine leptin gene (LEP) in New Zealand (NZ) Holstein Friesian Jersey (HF J) dairy cows. Subsequent sequence analysis of a 430 bp amplicon spanning the entirety of exon 3 and part of the intron 2 region revealed three variant sequences (, and ) containing a total of five nucleotide substitutions, all of which have been reported previously. Using general linear mixed-effect model analyses, the presence of variant (the most common variant) was associated with a decreased level of C15:1, C18:1 trans-11, C18:1 all trans, C18:2 trans-9, cis-12, C22:0 and C24:0 levels but increased levels of C12:1 and C13:0 iso (). Variant was associated with reduced levels of C6:0, C8:0, C11:0, C13:0 and C20:0 but increased C17:0 iso and C24:0 levels (). Variant was associated with decreased C17:0 iso levels but increased C20:0 () levels. In a genotype model, the genotype was associated with increased levels of C22:0 and C24:0 but decreased C8:0, C10:0, C11:0, C13:0, C15:0 and grouped medium-chain fatty acid (MCFA) levels (). Genotype was found to be associated with decreased levels of C10:0, C11:0, C13:0 and grouped MCFA (). This is the first report of findings of this kind in NZ HF J cows, and they suggest that variation in exon 3 of bovine leptin gene could be explored as a means of decreasing the concentration of saturated fatty acids in milk.

Genetic Differentiation of the Blue Swimming Crab Portunus pelagicus Along the Coastal Thai Waters Revealed by SSCP Analysis of Cytochrome c Oxidase Subunit I

The basic information on genetic diversity and population structure is essential for the construction of appropriate management schemes leading to sustainable fisheries of the blue swimming crab (Portunus pelagicus). Here, genetic heterogeneity of P. pelagicus (N=174) was examined by single-strand conformational polymorphism (SSCP) analysis of mitochondrial cytochrome c oxidase subunit I (PpCOI270). Seven SSCP genotypes were found across all investigated samples. The average genetic distance between pairs of geographic samples was 0.0014-0.7247. Significant geographic heterogeneity (P&lt;0.05) and restricted levels of female gene flow between paired samples (0.03-1.60 individuals per generation) were observed except between Chanthaburi - Prachuap Kriri Khan and Ranong - Krabi (P&gt;0.05; 6.54 and 16.17 individuals per generation) located in the same coastal regions. Therefore, the gene pool of P. pelagicus in Thai waters was genetically differentiated to different stocks even though it is biologically regarded as a potential dispersal species. Five geographic samples of P. pelagicus in Thai waters could be differentiated to three genetic stocks; Chanthaburi and Suratthani (stock A), Prachuap Khiri Khan (stock B) and Ranong and Krabi (stock C).

Polymorphisms in hormone-sensitive lipase and leptin receptor genes and their association with growth traits in Barki lambs.

Background and Aim:Marker-assisted selection has many advantages over conventional selection in animal breeding. The candidate gene approach has been applied to identify genetic markers associated with economically important traits in livestock. This study was established to investigate variation in the hormone-sensitive lipase (HSL) and leptin receptor (LEPR) genes, and their association with growth traits in Barki lambs.Materials and Methods:Records for birth weight (BW), pre-weaning average daily gain (ADG1), weaning weight (WW), post-weaning average daily gain (ADG2), and marketing weight (MW) were obtained from 247 Barki lambs. Polymerase chain reaction–single-stranded conformational polymorphism analyses were used to detect variation in exon 9 of HSL and exon 19 of LEPR. General linear models were used to test for associations between the variation in ovine HSL and LEPR, and growth traits.Results:The SSCP banding patterns for HSL showed three variants (H1, H2, and H3), which contained two nucleotide-sequence differences (c.1865C>T and c.2038T>C). Two SSCP banding patterns (L1 and L2) were observed for LEPR and these contained two nucleotide-sequence differences (c.2800G>A and c.2978C>G). The HSL genotype showed no effect on the studied traits. The LEPR genotype was proven to have significant effects (p<0.05) on ADG2 and MW. The presence of the L1 variant was associated (p<0.01) with decreased ADG2 and MW.Conclusion:The finding of an association between LEPR gene variation and growth rate after weaning in Barki lambs warrants efforts to improve this trait.

Genetic variants of the BMP2 and GDF9 genes and their associations with reproductive performance traits in Barki ewes

• Two BMP2 variants and three GDF9 variants were identified in Barki ewes. • BMP2 variants were associated with TNLW and TWLW; however GDF9 variants were associated with twining rate, TNLB and TWLB. • Variation in ovine BMP2 and GDF9 affects reproductive performance traits of Barki ewes. The aim of this study was to investigate variation in the bone morphogenetic protein 2 ( BMP2 ) and growth differentiation factor 9 ( GDF9 ) genes, and ascertain whether it was associated with reproductive traits in Barki ewes. Records for conception rate of ewe, lambing number of ewe, lambing rate of ewe, twining rate of ewe, total number of lambs born per ewe (TNLB), total weight of lambs born per ewe (TWLB), total number of lambs weaned per ewe (TNLW), total weight of lambs weaned per ewe (TWLW) and ewe rearing ability were obtained for 296 Barki ewes. Polymerase chain reaction–single strand conformational polymorphism (PCR–SSCP) analyses were used to detect variation in exon 2 of BMP2 and exon 1 of GDF9 . The SSCP banding patterns for BMP2 revealed two variants ( A1 and A2 ), which contained one nucleotide sequence difference (c.962A > T). Three SSCP banding patterns ( C1 , C2 and C3 ) were observed for GDF9 and these contained two nucleotide-sequence differences (c.25C > T and c.260 G > A). Association analysis between the variation in BMP2 and GDF9 and reproductive performance traits revealed that BMP2 genotype was associated ( P < 0.05) with TNLW and TWLW, while GDF9 genotype was associated with twining rate of ewe ( P < 0.001), TNLB ( P < 0.001) and TWLB ( P < 0.05). This suggests that selection for the BMP2 and GDF9 genotypes might increase reproductive performance traits in Barki ewes.