Restriction Enzymes AluI Research Articles

Detection of Phytoplasma belonging to the 16SrXXI group in Scott pine and mountain pine

One of the reasons for the deterioration of coniferous stands condition in a number of European countries is associated with their infection with phytoplasmas (obligate intracellular pathogens) — bacteria lacking a cell wall. The aim of the work is to identify the presence of phytoplasma infection in samples of Scots pine (Pinus sylvestris L.) and mountain pine (Pinus mugo Turra) collected in the Moscow and Samara regions, which had characteristic symptoms of the disease, and to determine the taxonomic affiliation of the phytoplasm. Phytoplasma was detected using direct and nested PCR with primer pairs P1/16S-Sr and R16F2n/R16R2 respectively. Phytoplasma DNA was found in six of the seven pine specimens, including an asymptomatic one. Analysis of restriction fragment length’s polymorphism after digestion of DNA amplicons with restriction endonucleases AluI, MseI, HhaI, HpaII, HaeIII, RsaI, and TaqI indicated the similarity of Russian strains of Scotch pine and mountain pine phytoplasmas to Lithuanian strains of PineLRN and PineBLD of mountain pine phytoplasma (GenBank Accession Number MK089821 and MK089819, respectively) identified as ‘Candidatus Phytoplasma pini’ (subgroup 16SrXXI-A). A phytoplasma related to this species has been registered on the territory of the Russian Federation for the first time.

A rapid quarantine approach for the pathogenic and invasive Phytophthora species associated with imported fruits in China.

Phytophthora spp. represent a pivotal genus of plant pathogens with a global distribution, exerting significant deleterious effects on food safety and forestry ecosystems. Numerous pathogenic and invasive Phytophthora species, introduced through imported fruits, have been frequently detected at Chinese ports. With the rise in global trade activities, the plant quarantine of imported fruits is becoming increasingly important but challenging. Fast, simple, and labor-saving techniques are necessary and anticipated. A polymerase chain reaction restriction fragment length polymorphism capillary electrophoresis (PCR-RFLP-CE) technology-based quarantine approach was developed for 16 Phytophthora species associated with the imported fruits in China. The Ypt1 gene, exhibiting abundant interspecific variations, was selected as the marker gene for PCR. The restriction endonuclease AluI was proven to be capable and compatible in simultaneously separating different Phytophthora species during CE. By combining with a fast and efficient DNA extraction kit, the developed PCR-RFLP-CE technique was successfully applied to identify Phytophthora species in artificially infested fruits. We provide a quick, practical, and high-throughput detection approach for hazardous and invasive Phytophthora species associated with imported fruits in China. This strategy can give good convenience and technological support for carrying out massive quarantine activities at Chinese ports. © 2024 Society of Chemical Industry.

Genetic diversity of the PvMSP-3α gene in Plasmodium vivax isolates circulating in the National Capital Region (NCR) of India

Malaria is still a public health problem in tropical countries like India; major malaria parasite species are Plasmodium falciparum and P. vivax. Of which, P. vivax is responsible for ∼40% of the malaria burden at least in the Indian scenario. Unfortunately, there is limited data on the population structure and genetic diversity of P. vivax parasites in India. In this study, we investigated the genetic diversity of P. vivax strains in the South-west district, Delhi and, Nuh district, Haryana [National Capital Region (NCR)], using a polymorphic marker- P. vivax merozoite surface protein-3α (PvMSP-3α) gene. Dried blood spots from microscopically confirmed P. vivax patients were used for investigation of the PvMSP-3α gene. PCR-RFLP was performed on the PvMSP-3α gene to investigate the genotypes and allelic variability with HhaI and AluI restriction enzymes. In total, 40 successfully PCR amplified PvMSP-3α gene segments were subjected to RFLP analysis. Amplified products showed three different base pair size variations viz. genotype A in 31(77.5%), genotype B in 4(10%) and genotype C in 5(12.5%) P. vivax specimens. RFLP with HhaI and AluI revealed 17 (H1-H17) and 25 (A1-A25) allelic variants, respectively. Interestingly, two similar sub-allelic variants, ie. H8 (with HhaI), and A4 (with AluI) clustered within the rural area of Nuh district, Haryana in two samples. With this study, we propose to commission such type of genetic diversity analysis of P. vivax to investigate the circulating genotypes of the parasites from distinct geographical locations across India, that can have significant implications in understanding the population structures of P. vivax.

Ace-1 Target Site Status and Metabolic Detoxification Associated with Bendiocarb Resistance in the Field Populations of Main Malaria Vector, Anopheles stephensi in Iran.

Anopheles stephensi is the main vector of malaria in Iran. This study aimed to determine the susceptibility of An. stephensi from the south of Iran to bendiocarb and to investigate biochemical and molecular resistance mechanisms in this species. Wild An. stephensi were collected from Hormozgan Province and reared to the adult stage. The susceptibility test was conducted according to the WHO protocols using bendiocarb impregnated papers supplied by WHO. Also, field An. Stephensi specimens were collected from south of Kerman and Sistan and Baluchistan Provinces. To determine the G119S mutation in the acetylcholinesterase (Ace1) gene, PCR-RFLP using AluI restriction enzyme and PCR direct-sequencing were performed for the three field populations and compared with the available GenBank data. Also, biochemical assays were performed to measure alpha and beta esterases, insensitive acetylcholinesterase, and oxidases in the strains. The bioassay tests showed that the An. stephensi field strain was resistant to bendiocarb (mortality rate 89%). Ace1 gene analysis revealed no G119S in the three field populations. Blast search of sequences revealed 98-99% identity with the Ace1 gene from Pakistan and India respectively. Also, the results of biochemical tests revealed the high activity of non-sensitive acetylcholinesterase, alpha and beta-esterase in the resistant strain compared to the susceptible strain. No G119S was detected in this study additionally the enhanced enzyme activity of esterases and acetylcholinesterase suggesting that resistance was metabolic. The use of alternative malaria control methods and the implementation of resistance management strategies are suggested in the study area.

Polymorphism of the myostatin gene exon 1 using PCR-RFLP technique in five beef cattle in Indonesia

This study aims to analyze the polymorphism of the myostatin (MSTN) gene in exon 1 at SNPc.111G>C (rs523392653) and SNPc.267G>A (rs383271508) on various beef cattle in Indonesia. A total of 136 DNA samples were analyzed in this study, consisting of 33 heads of Madura, 31 heads of PO, 36 heads of Bali, 18 heads of Simmental, and 18 heads of Limousin cattle. The DNA sample was collected from the Animal Molecular Genetic Laboratory, Faculty of Animal Science, IPB University. Along 608 bp of DNA sequence was amplified using PCR with forward primer was 5′-CAA GTT GTC TCT CAG ACT GG-3′ and reverse primer was 5′-CTC CTC CTT ACA TAC AAG CC-3′ and annealing temperature was 59°C. The amplified DNA was then digested with HaeIII and AluI restriction enzymes using the RFLP method to determine polymorphism. Genotype frequencies, allele frequencies, PIC, and heterozygosity values were calculated with the PopGen32 program. The HaeIII enzyme produces two genotypes, GG and CG, while the AluI enzyme produces three: GG, AG, and AA. SNPc.111G>C polymorphism was found in Madura and PO cattle, while SNPc.267G>A polymorphism was found in Madura, PO, and Bali cattle. Both Simmental and Limousin cattle were monomorphic.

ПОРОДНІ ОСОБЛИВОСТІ АЛЕЛЬНОГО ПРОФІЛЮ ГЕНІВ PIT1 ТА GH УКРАЇНСЬКОЇ АБОРИГЕННОЇ ЛЕБЕДИНСЬКОЇ ПОРОДИ КОРІВ

The preservation of genetic diversity is a paramount concern in addressing global challenges today. The foundation of biological diversity hinges on its genetic components, and the loss of species and genetic variability poses a genuine threat to the biosphere. This is because the sustainability of natural ecosystems and agroecosystems depends directly on their genetic adaptability to changing environmental conditions.
 In cattle, genes associated with milk yield play a crucial role, including genes related to milk proteins and hormones. Among these, the pituitary-specific transcription factor (PIT1) and growth hormone (GH) genes are of particular significance. In this study, we investigated the polymorphism of these genes in the Lebedyn breed of cattle.
 A total of 2 gene polymorphisms, PIT1 and GH, were examined through molecular and genetic research conducted at the genetics laboratory of the Institute of Animal Breeding and Genetics named after M. V. Zubets, part of the National Academy of Agrarian Science of Ukraine. The study utilized 32 DNA samples extracted from the venous blood of Lebedyn breed cows using the 'DNA Sorb-B' kit from AmpliSens. Genotyping was carried out through polymerase chain reaction (PCR-RFLP) analysis of restriction fragment length polymorphism, employing the “Tertsyk” amplifier for PCR. The resulting DNA restriction analysis products were assessed via electrophoresis in a 2–3% agarose gel containing ethidium bromide, with DNA fragment lengths determined using the GeneRuler TM 50 bp DNA Ladder and Thermo Scientific™ GeneRuler 1 kb Plus DNA Ladder markers.
 For the PIT1 gene analysis, a 451 bp amplified fragment was treated with the enzyme HinfI, yielding the following allele frequencies: A (0.38) and B (0.62). The heterozygous AB genotype was the most prevalent (0.45), while homozygotes for the A allele constituted 0.15 of the population. In the case of the GH gene fragment (223 bp), hydrolysis was performed using the restriction enzyme AluI. Analysis of allele frequency distribution indicated a high frequency of the L allele (0.85), with no animals exhibiting the homozygous GHVV genotype, and the GHLL genotype was present in 0.7 of the population.
 These findings reveal unique features of the allelic spectrum of the PIT1 (V-0.62) and GH (L-0.85) genes specific to the Ukrainian indigenous Lebedyn breed of cows. Selectionally significant genotypes include PIT1AA (15%) and GHLL (70%), while heterozygote carriers, which contribute to genetic diversity within the population, account for 45% and 30%, respectively.
 This study’s results hold particular value due to the alarming decline in the population of local indigenous breeds and the imminent threat to the preservation of agricultural genetic resources.

Identification polymorphism of LHR and FSHR genes in Indonesian Holstein dairy cattle associated with productive and reproductive traits

Abstract. Setyorini YW, Kurnianto E, Sutopo, Sutiyono. 2023. Identification polymorphism of LHR and FSHR genes in Indonesian Holstein dairy cattle associated with productive and reproductive traits. Biodiversitas 24: 2898-2905. Luteinizing and follicle-stimulating hormones and their receptors play an important role in the reproductive system’s hormonal activity and physiological function. This study aimed to identify the genotype variation of the Luteinizing Hormone Receptor (LHR) gene exon 11 and the Follicle-stimulating Hormone Receptor (FSHR) gene exon 10 and to elucidate the associating between polymorphism with the milk and reproductive traits in dairy cattle. A total of 100 samples of deoxyribonucleic acid (DNA) from Indonesian Holstein dairy cattle were used in this study. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) was performed for genotyping of the Single Nucleotide Polymorphism (SNP) of the LHR and FSHR genes with the HhaI and AluI restriction enzymes, respectively. The collected phenotype data were milk production, quality, and reproductive trait. In addition, the general linear model procedure in the SAS program was carried out to investigate the association of genotypes with productive and reproductive traits. The results showed that the LHR gene in the population of this study was monomorphic, while the FSHR gene was polymorphic with three genotypes, namely CC, CG, and GG. The sequences of the FSHR gene indicated the presence of a mutation at nucleotide number 2037 that substitutes cytosine for guanine. Cows with the CC genotype showed better parameter service per conception (P<0.05) than the other genotypes but did not correlate with other reproductive parameters and milk traits. Therefore, it was concluded that SNP g.2037C>G polymorphism at FSHR gene exon 10 was identified in the Indonesian Holstein Dairy cattle population and associated with service per conception parameters but not with milk trait.

Discrimination of Green Coffee (Coffea arabica and Coffea canephora) of Different Geographical Origin Based on Antioxidant Activity, High-Throughput Metabolomics, and DNA RFLP Fingerprinting.

The genus Coffea is known for the two species C. arabica (CA) and C. canephora (CC), which are used to prepare the beverage coffee. Proper identification of green beans of coffee varieties is based on phenotypic and phytochemical/molecular characteristics. In this work, a combination of chemical (UV/Vis, HPLC-DAD-MS/MS, GC-MS, and GC-FID) and molecular (PCR-RFLP) fingerprinting was used to discriminate commercial green coffee accessions from different geographical origin. The highest content of polyphenols and flavonoids was always found in CC accessions, whereas CA showed lower values. ABTS and FRAP assays showed a significant correlation between phenolic content and antioxidant activity in most CC accessions. We identified 32 different compounds, including 28 flavonoids and four N-containing compounds. The highest contents of caffeine and melatonin were detected in CC accessions, whereas the highest levels of quercetin and kaempferol derivatives were found in CA accessions. Fatty acids of CC accessions were characterized by low levels of linoleic and cis octadecenoic acid and high amounts of elaidic acid and myristic acid. Discrimination of species according to their geographical origin was achieved using high-throughput data analysis, combining all measured parameters. Lastly, PCR-RFLP analysis was instrumental for the identification of recognition markers for the majority of accessions. Using the restriction enzyme AluI on the trnL-trnF region, we clearly discriminated C. canephora from C. arabica, whereas the cleavage performed by the restriction enzymes MseI and XholI on the 5S-rRNA-NTS region produced specific discrimination patterns useful for the correct identification of the different coffee accessions. This work extends our previous studies and provides new information on the complete flavonoid profile, combining high-throughput data with DNA fingerprinting to assess the geographical discrimination of green coffee.

Endophytic Bacteria Showing Antioxidant Property from Periwinkle [Catharanthus roseus (L.) G. Don

The experiment was conducted in the year, April–December, 2020 at the Department of Microbiology, C. P. College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, Gujarat, India to isolate and characterize promising antioxidant producing endophytic bacteria from periwinkle plant. Twenty four endophytic bacterial cultures derived from various parts of Catharanthus roseus (L.) G. Don were tested for total antioxidant capacity (TAC) and total phenolics content (TPC). The isolate R1 showed highest TAC (615.46 µg AAE mg-1 extract) followed by the isolate R2 (308.59 µg AAE mg-1 extract). Correlation coefficient observed between TAC and TPC was 0.7591, which shows that phenolic compounds were greatly responsible for antioxidant capacity of the bacterial isolates. 15 isolates showing higher TAC were studied for morphological and molecular characteristics. All the 15 isolates were rod shaped and were monobacillus. Nine isolates were Gram positive whereas 6 were Gram negative. 16S rDNA amplification using universal primers 27F and 27R produced a band of 1.5 kb. Restriction digestion of PCR products of all the isolates with tetracutter restriction enzymes AluI, TaqI, Hae III produced polymorphic diagnostic fingerprints. The dendrogram based on ARDRA profiling grouped the 15 bacteria into two groups (cluster A and cluster B) at a Jaccard’s similarity co-efficient of 0.83. Cluster A contained eleven bacterial isolates whereas cluster B had only four. The isolates R1 and R2 may serve as an excellent source of antioxidants and may be exploited for commercial production of antioxidants.

Characterization of Plant Growth Promoting Endophytic Bacteria from Castor Plants [Ricinus communis (L.)

The present study was carried out in June–Janauary (2021–2022) at the Laboratory of the Department of Microbiology, Chimanbhai Patel College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University (SDAU), Sardarkrushinagar, Gujarat, India. 15 promising endophytic bacterial isolates from castor plants showing higher dry matter accumulation in seedlings of wheat were subjected to detailed characterization for their molecular and plant growth promoting abilities. The bacterial isolate Erc7 performed best for plant growth promoting activities viz., ammonia and IAA production, phosphate solubilization, nitrogen fixation, HCN production and antagonistic activity against Fusarium oxysporum f. sp. ricini in-vitro which cause wilt in castor plant. Pot trials involving wheat seed inoculation (rabi 2021–22) with bacterial isolates showed that the treatment with endophytic bacterial isolate Erc7 showed maximum shoot and root length (47.65 cm, 10.50 cm), shoot fresh and dry weight (824.90 mg, 308.83 mg), root fresh and dry weight (122.37 mg, 36.20 mg), spike length (09.76 cm) and number of seeds in central tiller plant-1 (15.33). PCR amplification of 16S rRNA genes and ARDRA profiling using restriction enzymes AluI, TaqI and HpaII grouped the 15 isolates in 2 clusters (A and B) at a Jaccard’s similarity co-efficient of 0.54. The best PGPR isolate Erc7 was gene sequenced for 16S rRNA gene. The sequence was submitted to the NCBI Genbank (Accession number ON514555). Upon BlastN It showed maximum similarity to Bacillus cereus. The isolates Erc7 showed presence of diplobacillus endospores which is a typical characteristic of Bacillus cereus.

Thiết lập quy trình PCR-RFLP xác định kiểu gen vi rút BK phân lập từ bệnh nhân ghép thận

The monitoring and treatment of patients after kidney transplant is important in maintaining transplant kidney function, including BK polyomavirus surveillance. BKV is a common opportunistic pathogen in kidney transplant patients and can cause BKV-associated nephropathy, which represents a major problem for these patients, causing graft dysfunction and graft loss. BKV is classified into four genotypes, I, II, III and IV. Here, thirty urine samples from kidney transplant patients were collected and quantified BKV-DNA loads by real-time PCR, viral loads were 105-1010 copies/ml. We successfully amplified and purified the specific VP1-BKV region with 580 bp in length using primers BK_(S+AS). Additionally, we also identified BKV genotypes by PCR-RFLP using the restriction enzymes RsaI and AluI. In total 20 out of 30 cases (66.7%) were genotype I and 10 cases (33.3%) were genotype IV in collected urine samples. Comparing the obtained PCR-RFLP results with direct sequencing analysis achieved complete concordance (30/30, 100%). We successfully developed a cost-effective and accurate BKV genotyping assay using PCR-RFLP, that contributes to the monitoring of patients after kidney transplantation.

Association of Quantitative Characteristics with Growth Hormone Gene (GH Gene) in Kerinci Duck Using PCR-RFLP Method

This study aims to determine the association of quantitative characteristics with growth hormone gene (GH gene) in kerinci duck using PCR-RFLP method. Samples total used was 96 Kerinci ducks consisting of 43 males and 53 females and 96 blood samples. DNA was extracted using the protocol Genomic DNA Purification Kit from Promega and then amplified by PCR using a pair of primers5'-CAA GGA ACA GAG GGT TTC CA-3' and Revers : 3'-GGG AGA TAG GGC AAA CAT CA-5', with a length of product 855 bp. The amplification product was cut using restriction enzyme AluI with the AG↓CT cutting site. Growth hormone/AluI fragments of Kerinci duck were electrophoresed using 1.5% agarose gel and visualized using doc gel. Data collected includes body weight, weight gain, body measurements, and blood of Kerinci ducks. The differences in body weight, body weight gain, and body measurements, as well as differences in body weight between genotypes were tested by T-test. The determinants of the size and shape of Kerinci duck were analyzed using PCA. This study showed that the body weight, weight gain, and body sizes of male Kerinci ducks were significantly different (P<0.05) than female Kerinci ducks. The Kerinci duck GH|AluI gene is polymorphic with three genotypes, i.e, +/+ of (49%), +/- of (39.6%), and -/- of (11.5%), and two alleles, namely (+) by 69% and (–) by 31%. Conclusion: body weight, weight gain, and body sizes of male Kerinci ducks were higher than female. The size identifier of male and female Kerinci ducks were sternum, shank lengh, and shank circumference, while shape identifier was wing length. GH Genes|AluI in Kerinci duck is polymorphic. GH gene |AluI of Kerinci ducks was associated with body weight, weight gain, and body measurements, and the best is the genotype (+/+).

Molecular characterization and association of lactoferrin gene to subclinical mastitis in goats (Capra hircus)

The study characterized the lactoferrin (Lf) mRNA gene in different goat breeds in the Philippines and determined its association with subclinical mastitis (SCM). The study involved collection of milk at second week of lactation (n=75) and blood samples (n=5) to obtain extracted RNA and using cDNA to amplify Lf gene through polymerase chain reaction. The nucleotide and amino acid sequences were determined and used as reference in the evaluation of phylogenetic relationship. Amplified products were utilized for RFLP analysis before determining the association of the gene with SCM. Results of the study demonstrated that Lf gene in goats registered a molecular weight of 2135. Nucleotide and amino acid sequence of Lf gene revealed high similarity (99%) in Saanen, Anglo-Nubian and Philippine native goats with that of Capra hircus (U53857) Lf gene submitted to GenBank. Phylogenetic studies showed that Lf gene of Anglo-Nubian, Saanen and Native goats clade together with Lf gene of C. hircus (U53857). Three genotypes in goats were documented using the restriction enzymes AluI and HaeIII. Based on the Statistical analysis, association (comp 5.65, p = 0.0308) has been established between the Lf genes of goats with genotype BB to SCM using HaeIII restriction enzyme.

First report of a ‘Candidatus Phytoplasma australasia’ (16SrII‐D) strain associated with witches’ broom and little leaf symptoms of Martynia annua in India

Martynia annua (Martyniaceae) is a small, herbaceous annual plant distributed across India. It is used for the treatment of epilepsy, tuberculosis, sore throats and wounds (Kaushik et al., 2021). In August 2021, symptoms of witches’ broom and little leaf were observed in M. annua in the Anantapur forest area, Andhra Pradesh. India. Leaves were collected from two diseased and two symptomless plants (Figures 1 and 2). Total DNA was extracted from 100 mg of leaf midribs using a modified CTAB DNA extraction method (Murray & Thompson, 1980). DNA extracts were analysed by direct and nested PCR with universal phytoplasma 16S rRNA primers, P1/P7 (Deng & Hiruki, 1991) and R16F2n/R16R2 (Gundersen & Lee, 1996), respectively. Amplicons of the expected size (c. 1.8 kb after direct PCR and c. 1.3 kb after nested PCR) were obtained from diseased samples but not symptomless plants. Nested PCR R16F2n/R16R2 products were directly sequenced (Eurofins Genomics India Pvt., Ltd., India) and the consensus sequence was deposited in GenBank (Accession No. OK135941). A BLASTn analysis revealed that the M. annua phytoplasma 16Sr DNA sequence shared 100% sequence identity with those of 16SrII group phytoplasmas, including ‘Candidatus Phytoplasma australasia’ (MN565885, MN565886) and ‘Ca. Phytoplasma aurantifolia’ (MK217101, MK421430). Phylogenetic analysis (Tamura et al., 2007) based on the R16F2n/R2 sequence and performed with the neighbour-joining method (MEGA software version 7.1) revealed that the Anantapur 16SrII phytoplasma had the closest relationship with 16SrII, subgroup D isolates, particularly with the strain ‘Ca. P. australasia’ (Y10096) (Figure 3). Virtual RFLP profiles using restriction enzyme AluI and the R16F2nR2 sequence were generated with iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi). The RFLP patterns of the Anantapur phytoplasma 16S rDNA F2nR2 sequence with AluI enzyme agreed with the phylogenetic grouping. The Anantapur phytoplasma was confirmed as a member of the 16Sr group II, subgroup D with a similarity coefficient of 1.00, compared with virtual RFLP profiles of the ‘Ca. P. australasia’ strain (Y10097). This is the first report of a ‘Ca. Phytoplasma australasia’ strain (16SrII-D) associated with witches’ broom and little leaf symptoms of M. annua in India. Since the 16SrII group has a wide host range and occurrence in India, and particularly the subgroup 16SrII-D in the south region, the present finding has a significant impact for future epidemiological studies. The authors would like to thank the support and encouragement from the Associate Director of the Research, Regional Agricultural Research Station, Tirupati.

Polymorphism Study of POU1F1 Gene in Mandya and NARI-Suwarna Sheep

Background: The mutations in POU1F1 gene result in deficiency of GH, PRL and TSH-â expression and thus have an indirect effect on development and reproduction. The present study was conducted to determine and compare the polymorphism of POU1F1 gene in Mandya and NARI-Suwarna Sheep. Methods: Blood samples were collected from 50 each of Mandya and NARI-Suwarna sheep and genomic DNA was isolated following Miller’s high salt method. PCR-RFLP analysis was performed for exon 3 and exon 4 regions of POU1F1 gene, whereas, PCR-SSCP analysis was performed for exon 6 region of POU1F1 gene. Result: The PCR products of sizes 365 bp, 508 bp and 501 bp for exon 3, exon 4 and exon 6 regions of POU1F1 gene, respectively were successfully amplified. PCR-RFLP analysis of exon 3 and exon 4 regions of POU1F1 gene with AluI and EcoRI restriction enzymes, respectively revealed monomorphism in both Mandya and NARI-Suwarna sheep. The PCR-SSCP analysis of exon 6 region of POU1F1 gene revealed two patterns, P1 and P2 with frequencies of 98 and 2 per cent, respectively in Mandya sheep and 90 and 10 per cent, respectively in NARI-Suwarna sheep. The alignment of P1 and P2 pattern sequences revealed two SNPs (109 G greater than C and 112 T greater than A) in Mandya sheep, whereas 3 SNPs (218 T greater than G, 225 G greater than A and 264 T greater than A) were revealed in NARI-Suwarna sheep. Polymorphism was successfully established at exon 6 region of POU1F1 gene.

Genetic polymorphism of DGAT1 gene in Mandya and Hassan Sheep of Karnataka

A study was carried out to identify and compare the genetic polymorphism in ovine DGAT1 (Diacylglycerol acyltransferase 1) gene of Mandya and Hassan sheep using PCR-RFLP (polymerase chain reaction – restriction fragment length polymorphism) technique. A total of 100 unrelated animals, 50 each of Mandya and Hassan sheep from farmers’ flocks in the respective breeding tracts were utilized. A 309 bp fragment of ovine DGAT1 gene sequence spanning exons 16–17 and the intervening intron was amplified by PCR employing previously published primers. The RFLP analysis using AluI restriction enzyme, polymorphic pattern revealed two alleles viz., allele C (309 bp fragment) and allele T (272 and 37bp fragments) and three genotypes, CC, CT and TT. The frequencies of CC, CT and TT genotypes were 0.84, 0.16 and 0.00 in Mandya and 0.86, 0.12 and 0.02 in Hassan sheep, respectively. The allelic frequencies for C and T alleles in Mandya and Hassan sheep were 0.92 and 0.08, respectively. Allele C and genotype CC were predominant in both the sheep populations. The sequence analysis revealed two SNPs viz., T->A (transversion) and C->T (transition) at positions 103 and 274 bp, respectively in T allele sequence of ovine DGAT1 gene. BLASTn sequence analysis showed 99% homology with predicted ovine DGAT1 gene (EU178818.1) sequence. Thus, genetic polymorphism was established at exon 1617 locus of ovine DGAT1 gene by PCR-RFLP and two SNPs were identified.

Association of GHR|AluI gene polymorphism with body weight parameters in Grati-PO cattle

The growth hormone receptor (GHR) gene is member of the class I cytokine receptor family that important in growth regulation including in cattle. This study aimed to analyze the association between GHR|AluI gene with body weight, parameters in Grati-PO cattle population. A total of 304 blood samples were collected from the experimental station at the Beef Cattle Research Station, which were also equipped with 304 phenotypic data. DNA was extracted using a zymo extraction kit. Detection of of the polymorphism of GHR gene using PCR-RFLP method with AluI restriction enzymes. polymorphism indicators include genotype frequency, allele frequency, Hardy-Weinberg Equilibrium, observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC) and number of effective alleles (Ne). Association analysis was carried out using a linear mixed model. The performance of birth weight, weaning weight, yearling weight and 18 months weight were 25.4 + 0.2 kg; 113.8 + 2.3 kg; 159.9 + 2.7 kg and 213.7 + 3.6 kg, respectively. The result of association analysis was significant (P<0.05) on weaning weight, yearling weight and 18 months weight. It could be concluded that the GHR gene has potential to be a candidate genetic marker for growth traits in Grati-PO cattle.

Microsatellite diversity and restriction enzyme-based polymorphisms of MHC loci in native Turkish goats

Playing a key role in immunity and autoimmunity, Major Histocompatibility Complex (MHC) contains microsatellite regions and polymorphisms associated with resistance to several diseases and thermo-physiologic characteristics in farm animals. The aims of this study were ı) to estimate genetic diversity and phylogenetic relationships via 5 different MHC-linked microsatellite markers (BF1, BM1818, BM1258, SMHCC1 and DYMS1) and ıı) to assess restriction ezyme-based (PstI, TaqI, BsaHI and AluI) polymorphisms of MHC Class II-DRB gene in native Turkish goats. Based on MHC-linked microsatellite markers, number of alleles ranged from 8.20 (NRD) to 8.80 (HAI and KBK) across studied goat populations. Observed heterozygosity was between 0.68 (NRD) and 0.80 (KBK), whereas expected heterozygosity ranged from 0.74 (NRD) to 0.82 (KBK) in native Turkish goats. Inbreeding coefficients were 0.04, 0.13, -0.01 and 0.09 for HAI, HNM, KBK and NRD populations, respectively. A 284 bp length PCR products belonging to MHC Class II-DRB gene region were digested separately with PstI, TaqI, BsaHI and AluI restriction endonucleases to assess polymorphism status together with Hardy-Weinberg equilibrium in studied goat populations. P allele frequency ranged from 0.73 (KBK) to 0.95 (NRD), while p allele frequency was between 0.05 (NRD) and 0.27 (KBK) in PstI polymorphism. The highest and lowest frequency of T allele were detected in HNM (0.80) and KBK (0.49), respectively, whereas frequency of t allele was between 0.20 (HNM) and 0.51 (KBK) in TaqI polymorphism. G and A allele frequency were between 0.16 (HNM) - 0.39 (HAI) and 0.84 (HNM) – 0.61 (HAI), respectively in BsaHI polymorphism, while unexpected genotype patterns were observed in AluI polymorphism. In this study, high genetic diversity and low inbreeding were detected in native Turkish goats according to MHC-linked microsatellite markers. Additionally, presence of genotypes previously reported to be associated with tuberculosis resistance and heat tolerance were observed in native Turkish goat populations. Nevertheless, association analysis between these genotypes and tuberculosis resistance and heat tolerance should be confirmed in native Turkish goats.

Detection of GHR|AluI gene polymorphism and its association with body weight of Madura cattle in Indonesian Beef Cattle Research Station

Madura cattle is a one of the native cattle that are genetically tolerant of tropical climates. Madura cattle are one of the beef cattle breeds in Indonesia. Madura cattle can be a superior breed, but improving growth trait in Madura cattle needs to be conducted. The early step that was needed was for detecting the genes which were involved in growth traits. The purpose of this research was for detecting GHR gene polymorphisms and its association with body weight of Madura cattle population in Indonesian Beef Cattle Research Station. This study used 110 DNA samples of Madura cattle that was collected from the experimental barn at Indonesian Beef Cattle Research Station. DNA samples were extracted by DNA extraction kit. Genotype of GHR gene was detected by PCR-RFLP method using AluI restriction enzymes. In this study, association genotype and body weight was analysed by univariate GLM method. The birth weight, weaning weight, yearling weight and 18 months weight of Madura cattle were 16.8±0.3 kg; 82.4±2.3 kg; 124.6±3.7 kg and 166.7±5.0 kg, respectively. The GHR genes of Madura cattle had 3 genotypes AA, AG and GG and genotype frequencies of GHR gene were 0.273 (AA), 0.373 (AG), 0.355 (GG), respectively. Whereas the allele frequencies of A was 0.459 and the allele frequencies of G was 0.541. Based on Chi-square (X2) analysis showed that the population sample was not disequilibrium. The result of association analysis was significant (P <0.05) on weaning weight, yearling weight and 18 month weight. It was concluded that the GHR gene has potential as a genetic marker for growth traits and can be used as MAS in Madura cattle in Indonesian Beef Cattle Research Station.

Allele and genotype variation of follicle stimulating hormone receptor gene of Indonesian Friesian Holstein in Boyolali District, Central Java

Follicle Stimulating Hormone Receptor (FSHR) is a gene that has function to regulate reproductive performance by controlling oogenesis in females and also spermatogenesis in male. Since dairy cattle operation lies on the reproductive trait efficiency, therefore it’s became a logical reason to use FSHR genes as part of selection criteria. This study aimed to determine allele and genotype variation of FSHR gene in local Indonesian Friesian Holstein (IFH) dairy cattle in Boyolali District, Central Java. In total 20 IFH cattle we sampled for DNA source. Allele and genotype variation was determined by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) using AluI restriction enzyme. The methods started with DNA extraction, PCR, and DNA digestion, followed by allele and genotype frequencies calculation. Two alleles were observed in this study, C and G with respective frequencies 0.75 and 0.25. According to that, we found three genotype combinations that are CC, CG, and GG, the frequencies were 0.56, 0.38, and 0.06, respectively. To use that result as a part of selection criteria in dairy cattle, especially in females, further association study with reproductive trait needs to be done.