PCR Products Of Size Research Articles

SSR marker analysis and DNA fingerprinting of mutant rice variety VTL 10 (Lavanya)

VTL-3 is a tall high yielding saline tolerant rice variety developed by Rice Research Station, Vyttila in 1987, which is having a tendency to lodge at maturity. Researchers hence, developed semi-tall lines of VTL-3 using induced mutagenesis. The selected semi-tall mutants were screened with markers linked to Saltol QTL and it was found that this region was not affected in the mutant. A promising line selected after comparative yield trials was later released as VTL 10, Lavanya in the year 2018. In order to differentiate the variety Lavanya from its parent VTL 3, SSR marker analysis and DNA fingerprinting was carried out. These two varieties were screened with 48 SSR markers for DNA fingerprinting and eight markers were found to be polymorphic between VTL-10 andVTL-3. The polymorphic markers include RM600, RM3362,RM279, RM7, RM85, RM149, RM144 and RM1880. The number of alleles ranged from 1 to 3 and the size of PCR products ranged from 110 to 300bp with Polymorphic Information Content (PIC) from 0 to 0.38. From the present study, it was observed that these eight markers can be used to distinguish the mutant line VTL-10 from its original parent VTL-3.

Molecular Identification of Aspergillus flavus isolated from some maize (Zea mays L.) varieties across Abuja, Nigeria

This study aims at isolating and molecularly characterizing Aspergillus flavus from maize (Zea mays L.) varieties (Samaz 15,20, 37, 52, Oba super 6, 9, 39, 98, and DK 777) obtained across Abuja, Nigeria. Maize varieties were obtained across the district areas of Abuja namely; Kuje, Kwali, Gwagalada, AMAC, Abaji, Bwari and UniAbuja Experimental field. The potato dextrose agar as a medium was used in growing the fungi from the maize varieties seeds for 7 days at 25oC and then sub-cultured to obtain an isolate into potato dextrose broth. The mycelial mass produced was processed to extract its DNA using genomic DNA extraction of Zymo, D6005 for fungi and bacteria. Molecular Identification of the Internal transcribed spacer (ITS) region for each A. flavus isolate was carried out with ITS1 and ITS4 primers. anf18S rRNA gene region was also amplified with ITS5 and ITS4 primers. 2.0% agarose gels were electrophoresed for 30 minutes in 0.5× buffer, Dye and 60 mM EDTA at 80 V. A gel documentation system was used in determining PCR product sizes by evaluating with internal Mol. Wt. Standards of Thermo Scientific GeneRuler 100 bp DNA Ladder. Fifteen (15) of the twenty (20) fungal isolates were found to be positive with PCR amplification of ITS regions and amplicon sizes of 600 base pairs (bp) for A. flavus were obtained. All the twenty isolates have the presence of 18S rRNA 300-400bp. Thirteen of the fungal growth from the fourteen maize varieties obtained across Abuja were identified as A. flavus.

Assessment of marker genotypes associated with the resistant genes in a set of rice cultivars for brown planthopper, blast disease, and bacterial leaf blight

Brown planthopper, bacterial leaf blight and rice blast disease are identified as devastating stresses of rice cultivation. Developing host plant resistance is considered an effective and environmentally friendly management strategy. In Sri Lanka, limited studies have been carried out to assess the genetic basis of disease resistance. Bph3, Pikh and Xa21 genes convey significant resistance against biotic stresses. The current study was conducted to identify whether markers linked to Bph3, Pikh and Xa21 genes can facilitate the identification of resistant varieties. A total of 34 varieties were selected for the study and their morphometric and marker allelic data were subjected to descriptive and statistical analyses. Moderate height varieties depicted high yield. Most of the varieties showed significant moderate resistance against stresses. Molecular marker analysis yielded polymorphic banding patterns for all markers. RM 589 marker linked to Bph3 dominant gene provides overlapping banding patterns for the cultivars with different resistance levels making it impossible to discriminate resistant and susceptible cultivars. RM 206 marker linked to gene pikh provided the expected PCR product size for RM 206 marker is 147 bp. The resistant varieties gave band lengths of 140 bp, 170 bp and susceptible or moderately susceptible varieties produced band lengths of 160 bp and 155 bp. pTA 248 marker linked to Xa21 locus produced bands length of 670 bp, 740 bp and 930 bp. The 930 bp band was observed only in IRBB 60 variety. IRBB60 amplified 930 bp fragment confirms the possession of the resistant allele of Xa21 in its background. Presence of 730 bp indicates the susceptible allele. None of the Sri Lankan varieties carries a resistant allele for Xa21 as they have 730 bp allele for pTA 248. The rice varieties such as Bg251, Bg455, Bg450, Bg305, At402, At308 and At354 contain high yielding traits but need to improve for the pest and disease resistance.

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte.

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes' zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve's regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (± standard deviation) of 137,904 ± 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 ± 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 ± 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P < 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.

DNA sexing of Sumatran Tiger (Panthera tigris sumatrae) based on amelogenin genes

Nowadays, forensic and wildlife research, especially the Sumatran tiger needs further research with a molecular approach. Molecular approaches are needed for forensic and wildlife research including for sex identity. This study used the amelogenin gene as a marker for identification based on previous studies of Felidae species. The sample used consisted of 10 blood samples from Sumatran tigers of known sex were collected by the Dharmasraya Sumatran Tiger Rehabilitation Center (PRHSD), one hair sample, and two bone samples whose sex was unknown were collected from Natural Resources Conservation Center West Sumatra (BKSDA). The PCR results of the amelogenin gene of the Sumatran tiger confirmed the sex of 10 samples of known sex’s Sumatran tiger (Four male samples and six female samples), and Three samples of unknown sex were identified as females. Male was characterized by the electrophoresis appearance of two bands, while in female’s only one band, with PCR product sizes of at least 190 bp for AMELY and at least 210 bp for AMELX.

Independent development and validation of a novel six-color fluorescence multiplex panel including 61 diallelic DIPs and 2 miniSTRs for forensic degradation sample.

Current forensic DNA profiles are obtained based on analyses of PCR product sizes or DNA sequence polymorphisms. Sometimes routine forensic analysis using short tandem repeat (STR) generates unsuccessful DNA testing result if the biological sample encountered is excessively degraded and low-template DNA. Herein, a new six-color fluorescence labeling system, including 59 autosomal diallelic deletion or insertion polymorphisms (DIPs), 2 miniSTRs, 2 Y-chromosome DIPs, and 1 Amelogenin gene with the amplicon sizes of less than 200bp, was self-developed. According to the validation guidelines for DNA analysis methods formulated by the Scientific Working Group on DNA Analysis Methods, the validation studies have also been carried out for the multiplex system. This novel panel possessed the features of strong stability, high sensitivity, and good specificity, which was especially suitable for the forensic degraded and mixed sample detections. The cumulative power of exclusion and cumulative matching probability of the system were 0.9999978 and 9.833E-28, respectively, in Han Chinese in Hunan, China. Moreover, this system will be an effective new tool that can be independently applied to forensic personal identification and paternity testing in the populations from the East Asia region, even from the South Asia, America, and Europe regions. The system can also contribute to population phylogenetic affinity and genetic structure analyses among different populations.

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.

Study association of bPRL (Bovine Prolactin) gene and milk production trait in Indonesian Holstein dairy cattle

This study was aimed to analyze the genetic variation of bPRL (Bovine Prolactin) gene and its association to milk production in Indonesian Friesian Holstein (IFH) dairy cattle. In total 49 DNA from whole blood were extracted and followed with PCR-RFLP to distinguish the genotype difference between individual samples. The fragment of bPRL gene was amplified using pair of primer forward 5’-GAGTCCTTATGAGCTTGATTCTT-3’ and reverse 5’-GCCTTCCAGAAGTCGTTTGTTTTC-3’, resulting in 156 bp PCR product size. DNA digestion then performed using RsaI restriction enzyme that recognizes 5’-GT↓AC-3’ sites with blunt-end termini. According to the study, two genotypes were discovered that are AA and AB, with respective frequencies of 0.84 and 0.16. Based on the genotype grouping of milk production data, AB genotype yielded 4016.63±1344 kg while AA genotype yielded 3314.32±1153 Kg of milk per lactation. However, there is no statistical (P>0,05) difference between both genotypes on the amount of milk yielded. It is concluded that bPRL gene was not associated with milk production traits in the studied Indonesian Dairy Cattle population.

Genetic Polymorphisms of Chicken Antiviral Mx1 and TVB Genes in Indigenous and Giriraja Chicken

Background: Recent developments in molecular genetics lead to addressing certain poultry diseases via breeding for disease resistance. The present study was carried to identify and compare the genetic polymorphism in Chicken Mx1 and TVB genes among the indigenous and Giriraja chicken using PCR-RFLP technique. Methods: Blood samples were collected from 50 indigenous and 50 Giriraja birds and DNA isolation was done by Phenol: Chloroform: Isoamyl alcohol method. PCR amplification of Chicken Mx1 (exon 14) and Chicken TVB (exon 3) genes was carried out followed by RFLP analysis. Result: PCR product sizes of 301 bp and 303 bp of Mx1 and TVB genes, respectively were successfully amplified. RFLP analysis of Mx1 gene with Hyp8I restriction enzyme revealed three genotypes AA, AB and BB. In indigenous birds genotypic frequencies of AA, AB and BB were 0.314, 0.493 and 0.194, respectively and gene frequencies were 0.56 and 0.44 for alleles A and B, respectively. In Giriraja birds, genotypic frequencies for AA, AB and BB were 0.27, 0.499 and 0.23, respectively and gene frequencies were 0.52 and 0.48 for alleles A and B, respectively. RFLP analysis of TVB gene with NlaIII restriction enzyme revealed two genotypes viz., AA and AB. In indigenous birds genotypic frequencies of AA and AB were 0.81 and 0.18, respectively and gene frequencies were 0.9 and 0.1 for alleles A and B, respectively. In Giriraja birds genotypic frequencies for AA and AB were 0.774 and 0.211, respectively and gene frequencies were 0.88 and 0.12 for alleles A and B, respectively.

Bacteriological and molecular characterization of Mycobacterium bovis isolates from tuberculous lesions collected among slaughtered cattle, Northwest Ethiopia

BackgroundIn Ethiopia, the distribution of bovine tuberculosis (BTB) has long been known and documented as a major problem of animal health. However, the burden of circulating M. bovis strains is poorly understood in the country. Therefore; this study aimed to identify and characterize the mycobacterial isolates responsible for BTB in Northwest Ethiopia.MethodsA cross-sectional study was conducted on tuberculous lesions that had been collected from slaughtered cattle between September 2018 to June 2019. Collected lesions were cultured and tested for tuberculous bacilli. The MPT64 assay and Genotype line probe assay (LPA) were used for identification of mycobacterial isolates, and region of deletion 4 (RD4) typing and spoligotyping were used to characterize the M. bovis strains.ResultsOf the total 1458 examined slaughtered cattle, only 62 (4.3, 95%CI; 0.0328–0.0542) had tuberculous lesions. The highest number of gross tuberculous lesions were observed from the lymph nodes of the thoracic cavity; at the mediastinal (40.3%, 25/62) and bronchial (22.6%, 14/62) lymph nodes. Of the 62 collected tuberculous lesions; 18 (29.0%) were culture positive for mycobacterium isolates, and only five isolates were confirmed for M. tuberculosis complex (MTBc) by the MPT64 assay and LPA. All the five MTBc isolates were positive for RD4 typing of M. bovis with a PCR product size of 446 bp, and no isolate was noticed to have M. tuberculosis. The detected M. bovis strains displayed five spoligotypes; with the common SB1176 and SB0133 M. bovis strains, although the two spoligotypes had not been previously reported.ConclusionThe present study shows that BTB in North Gondar, Ethiopia, is caused by M. bovis strains SB1176 and SB0033, with low frequency. Thus, the finding highlights the importance of continuous surveillance for mycobacterial strains in cattle populations.

PCR-based SNP Markers for Sex Identification in Date Palm (Phoenix dactylifera L.) cv. KL1

The date palm (Phoenix dactylifera L.) is a valuable commercial plant in the Arecaceae family. It is a sweet, edible fruit containing a lot of nutrients including carbs, sugar, protein, and potassium. Maejo 36 or KL1 is a Thai date palm with excellent fresh fruit quality that is mostly grown in Thailand's northern and northeastern areas. The main issue with date palm farming is that there is no reliable way to discriminate between male and female seedlings prior to flowering, which takes 3 - 5 years after planting. An increase in the number of female date palm plants in commercial orchards may increase in date production and profitable investment. The present research aims to develop Single Nucleotide Polymorphisms (SNP) markers for the sex identification of date palm cv. KL1. The SNP markers were designed by analysis of nucleotide sequence difference from GenBank (accession number GL746268.1). The results revealed that SNP primers; PH02F - PdaNu01 (PdaNU02 or PdaNU03) - PdaNU05 used in multiplex PCR technique can precisely distinguish the sex of date palm by generating 2 different sizes of PCR-products in male plants, and only 1 size of PCR-product in female plants. This research indicates that SNP markers with multiplex-PCR technique are a high potential method for identifying the sex of date palm cv. KL1 may be used to increase female plants for cultivation at the seedling stage, which is highly beneficial for commercial date palm production. HIGHLIGHTS Date palm is currently an economic fruits crops in Thailand The sex of date palm can be identified by phenotype in flowering stage until five years after planting The amplicon patterns in male and female date palm generated by SNP marker are applicable to predict their genders at an early seedling stage The use of SNP marker is highly beneficial for commercial date palm production GRAPHICAL ABSTRACT

Identification of polymorphism of FecX gene in indigenous sheep of Meghalaya

Fecundity gene such as FecX, govern fertility and litter size in sheep. Selection of animals with the help of molecular markers through identification of polymorphism of fecundity genes is an impactful method of genetic improvement. The present study was carried out to identify the polymorphism of FecX gene in indigenous sheep of Meghalaya. Blood samples were collected from 50 randomly selected ewes from 2 districts of Meghalaya namely, East Khasi Hills and West Khasi Hills. Genomic DNA was extracted from the samples and PCR amplification of FecX gene with specific primer set was done. PCR product size of 141 bp was obtained. Further, digestion of the PCR product with restriction enzyme HinfI through PCR-RFLP technique was conducted to identify the variants in FecX gene. Upon digestion, 2 types of fragment pattern identified, arbitrarily designated as AA and AB genotype, where AA genotype yielded one fragment (141 bp) and AB genotype yielded 2 fragments (100 and 41 bp). Presence of polymorphism can be an important indicator for selection with the help of molecular markers.

Evaluation of curcumin and CM11 peptide alone and in combination against amastigote form of Iranian strain of L. major (MRHO/IR75/ER) in vitro

Curcumin (diferuloylmethane) is the main phytochemical of Curcuma longa Linn, an extract of the rhizome turmeric. For thousands of years, turmeric among other natural products has been used as a dietary spice and as a medicinal plant in Asian countries. The present study reports the leishmanicidal activity of curcumin in different concentrations (10 μM, 20 μM, 40 μM). It is also showing the effect of CM11 peptide (8 μM) alone and in combination with curcumin (10 and 20 μM) as a leishmanicidal drug. The experiments were performed with the amastigote form of Leishmania major (MRHO/IR/75/ER) in vitro and the leishmanicidal activity was analyzed after 12 and 24 h of incubation by Giemsa and DAPI staining. Further investigation was done by using semi-quantitative PCR with new designed common primer pair derived from an 18S rRNA gene belonging to the L. major and mouse, which amplified the above-mentioned gene segments simultaneously with different PCR product size. Our findings showed that curcumin had leishmanicidal activity in a dose and time-dependent manner and its lowest effective dose was at concentrations of 40 μM afetr12 h and 10 μM after 24 h. The IC50 value of curcumin against amastigote forms of L. major was 21.12 μM and 11.77 μM after 12 and 24 h, respectively. Treatment of amastigote form with CM11 (8 μM) alone and in combination with curcumin (10 μM and 20 μM) showed less leishmanicidal activity. Interestingly, CM11 in combination with curcumin (10 μM and 20 μM) had even less leishmanicidal effect compared to curcumin alone in the same concentrations (10 μM and 20 μM). The semi-quantitative PCR analysis confirmed the data achieved by Giemsa and DAPI staining and showed that curcumin reduced the PCR product derived from amastigote form in concentration and time-dependent manner compared to the genome of the host cells.

Application of DNA Fingerprinting using the &lt;em&gt;D1S80&lt;/em&gt; Locus in Lab Classes

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences. This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard. Following this guide, students should be able to: • Harvest and extract DNA from buccal mucosa epithelial cells • Perform a PCR experiment and understand the function of various reaction components • Analyze the amplicons by agarose gel electrophoresis and interpret the results • Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes.

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences. This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard. Following this guide, students should be able to: • Harvest and extract DNA from buccal mucosa epithelial cells • Perform a PCR experiment and understand the function of various reaction components • Analyze the amplicons by agarose gel electrophoresis and interpret the results • Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences.

Non-invasive DNA extraction for molecular identification of royal Bengal tiger Panthera tigris tigris

The flagship animal species of Sundarbans, the Royal Bengal tiger (Panthera tigris tigris) is under threat of extinction. Its natural population is declining day by day. So, to avoid killing and harming the animal, the use of non-invasive samples such as scat, hair, or scent is preferred for DNA extraction and subsequent genotyping of tiger species. DNA has been extracted from scat samples of the Bengal tiger in the present study, and a fragment of the cytochrome b gene has been sequenced after PCR with species-specific primers. DNA has been extracted manually using a previously described methodology with slight modifications. The size of the PCR product and sequence of cytochrome b gene indicates that tiger DNA is successfully extracted from scat samples using tigerspecific primers. Thus, presence of tiger DNA can be detected by using this method just by the PCR product size in the gel. This is the first report of a partial sequence of mitochondrial cytochrome b gene of P. t. tigris from Bangladesh.&#x0D; Dhaka Univ. J. Biol. Sci. 30(2): 325-330, 2021 (July)

Identification of a novel haplotype of the Ms locus controlling restoration of male-fertility and its implication in origination of cytoplasmic male-sterility in onion (Allium cepa L.)

ABSTRACT Seeds of F1 hybrid cultivars of onions have been exclusively produced by utilising cytoplasmic male-sterility (CMS). A single locus, Ms, controls restoration of male-fertility of CMS onions. When a molecular marker tagging the AcPMS1 gene, a candidate gene for the Ms locus, was used to genotype diverse breeding lines, an unusual marker genotype showing an intermediate size of PCR product was identified in one breeding line (ORI854). The male-fertility phenotype of ORI854 was all male-sterile. Full-length genomic DNA sequence (16,464 bp) of this novel AcPMS1 allele from ORI854 was more closely related to a recessive AcPMS1 allele than to a dominant allele. However, there were more than 200 polymorphic sequences between this novel allele and the recessive allele, indicating that this novel AcPMS1 allele might not have recently diverged from the recessive allele. Phylogenetic analysis implied that onion male-sterility might have first appeared in a common ancestor before divergence of this novel allele and recessive allele of AcPMS1. Analysis of full-length cDNA sequences of additional 12 genes showing almost perfect linkage disequilibrium with the Ms locus indicated that no recombination might have happened among 13 genes including AcPMS1 in the novel Ms haplotype after divergence from the recessive Ms haplotype.

Genetic characterization of donkeys of Braj region of India

The grey type donkeys of Braj region of Uttar Pradesh in India were genetically characterized using ten heterologous microsatellite loci. At these loci the PCR product size ranged from 75-95 bp at locus HTG6 to 251-277 bp at locus COR18. The observed number of alleles varied from 4 (HTG15) to 10 (HTG7 and AHT4) with a mean of 7.50 ± 1.96. The observed heterozygosity ranged from 0.46 (HTG6 and NVHEQ54) to 0.79 (COR18) with a mean of 0.66 ± 0.12. The mean genetic diversity estimate (FIS) was 0.157. When these donkeys were compared on the basis of allelic frequency data at these loci to the brown type donkeys of Ladakh, Spiti and Rayalseema regions in the union territories/states of Ladakh, Himachal Pradesh and Andhra Pradesh, respectively, they clustered independently from these three donkey populations in a dendrogram based on Goldstein’s average square distances indicating their genetic distinctness.

Endometriosis Is Associated with Functional Polymorphism in the Promoter of Heme Oxygenase 1 (HMOX1) Gene.

Endometriosis is a common gynecological disorder characterized by the ectopic growth of endometrial-like tissue outside the uterine cavity. Etiopathogenesis of endometriosis is poorly understood; it is plausible, however, that the disease may be associated with oxidative stress related to local heme and iron metabolism. Therefore, the aim of the study was to reveal a possible association of endometriosis with a stress-inducible heme oxygenase 1 (HMOX1). For this purpose, 228 patients with clinically confirmed endometriosis and 415 control parous women from general Polish population were examined for functional –413A>T (rs2071746) single-nucleotide polymorphism (SNP) and (GT)n dinucleotide repeat length polymorphism in the promoter of HMOX1 gene. In addition, –413A>T SNP was assessed by the specific TaqMan® SNP Genotyping Assay, and (GT)n polymorphism was determined by PCR product size analysis. We found that endometriosis is associated with an increased frequency of −413A(GT)31,32 haplotype (OR (95%CI) = 1.27 (1.01–1.60), p = 0.0381) and −413A(GT)31,32 homozygous genotype [OR (95%CI) = 1.51 (1.06–2.17), p = 0.0238]. These data suggest that endometriosis is associated with functional polymorphism of HMOX1 gene, and this gene may play a part in the pathogenesis of this disorder.

Conventional and Molecular Identification of Mycotic Mastitis Caused by Candida in Farm Animals

Mycotic mastitis due to Candida is increasing and the identification of Candida species is crucial in choosing the ideal antifungal drugs. Identification of Candida to species level is essential as many non-albicans Candida developed drug resistance against antifungal agents. A total of 138 milk samples (24, 15, 46 and 53 from goats, sheep, buffaloes and cows, respectively) were streaked on plain plates of SDA and incubated. CHROM agar was used to differentiate Candida isolates. Mycological examination revealed the presence of yeast in 8.3, 6.7, 32.6 and 47.2% milk samples of goats, sheep, buffaloes and cows, respectively. The total isolation percent of yeast in all milk samples was 31.2%. Species identification of Candida in the present work using both CHROM agar and the bio-typing assay (API 20 C AUX) revealed the highest presence of Candida albicans (11.6%) followed C. glabrata (5.8%), C. tropicalis (5.1%) and C. krusei (2.9%). Multiplex PCR gave two different sized PCR products specific for every tested Candida isolates separately. It was concluded that phenotypic and biochemical assays are time-consuming and exposed to misdiagnosis. Whereas multiplex PCR is a genotypic-based technique that permits the identification of species that is difficult to be identified conventionally because of their abnormal morphology or biochemical features and also this assay is simple, easy and time saving so it is recommended to be implemented in clinical laboratories.