Restriction Enzymes HaeIII Research Articles

Molecular diversity and multifarious plant growth promoting attributes of Bacilli associated with wheat (Triticum aestivum L.) rhizosphere from six diverse agro-ecological zones of India.

The diversity of culturable Bacilli was investigated in six wheat cultivating agro-ecological zones of India viz: northern hills, north western plains, north eastern plains, central, peninsular, and southern hills. These agro-ecological regions are based on the climatic conditions such as pH, salinity, drought, and temperature. A total of 395 Bacilli were isolated by heat enrichment and different growth media. Amplified ribosomal DNA restriction analysis using three restriction enzymes AluI, MspI, and HaeIII led to the clustering of these isolates into 19-27 clusters in the different zones at >70% similarity index, adding up to 137 groups. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 55 distinct Bacilli that could be grouped in five families, Bacillaceae (68%), Paenibacillaceae (15%), Planococcaceae (8%), Staphylococcaceae (7%), and Bacillales incertae sedis (2%), which included eight genera namely Bacillus, Exiguobacterium, Lysinibacillus, Paenibacillus, Planococcus, Planomicrobium, Sporosarcina, and Staphylococcus. All 395 isolated Bacilli were screened for their plant growth promoting attributes, which included direct-plant growth promoting (solubilization of phosphorus, potassium, and zinc; production of phytohormones; 1-aminocyclopropane-1-carboxylate deaminase activity and nitrogen fixation), and indirect-plant growth promotion (antagonistic, production of lytic enzymes, siderophore, hydrogen cyanide, and ammonia). To our knowledge, this is the first report for the presence of Bacillus endophyticus, Paenibacillus xylanexedens, Planococcus citreus, Planomicrobium okeanokoites, Sporosarcina sp., and Staphylococcus succinus in wheat rhizosphere and exhibit multifunctional PGP attributes. These niche-specific and multifarious PGP Bacilli may serve as inoculants for crops growing in respective climatic conditions.

Giardia duodenalis in Damascus, Syria: Identification of Giardia genotypes in a sample of human fecal isolates using polymerase chain reaction and restriction fragment length polymorphism analyzing method

Giardia duodenalis is a common gastrointestinal parasite that infects humans and many other mammals. It is most prevalent in many developing and industrialized countries. G. duodenalis is considered to be a complex species. While no morphological distinction among different assemblages exist, it can be genetically differentiated into eight major assemblages: A to H. The aim of this study was to determine the genetic heterogeneity of G. duodenalis in human isolates (a study conducted for the first time in Syria). 40 fecal samples were collected from three different hospitals during the hot summer season of 2014. Extraction of genomic DNA from all Giardia positive samples (based on a microscopic examination) was performed using QIAamp DNA Stool Mini Kit. β-giardin gene was used to differentiate between different Giardia assemblages. The 514bp fragment was amplified using the Polymerase Chain Reaction method, followed by digestion in HaeIII restriction enzyme. Our result showed that genotype A was more frequent than genotype B, 27/40 (67.5%); 4/40 (10%) respectively. A mixed genotype of A+B was only detected in 9 isolates (22.5%). This is the first molecular study performed on G. duodenalis isolates in Syria in order to discriminate among the different genotypes. Further expanded studies using more genes are needed to detect and identify the Giardia parasite at the level of assemblage and sub-assemblage.

Molecular Identification of Fungal Isolates from Textile Wastewater by ITS-RFLP Method

Eight strains isolated from textile post-finishing wastewaters were subjected to molecular identification by ITS-RFLP (Internal Transcriber Spacer-Restriction Fragment Length Polymorphism) method, using ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) DNA amplification primers for ITS region of the nuclear small-subunit rRNA gene. Restriction fragments analysis was conducted post PCR amplification, and cleaved with HaeIII, HinfI, HhaI, MseI and SduI restriction enzymes. Differences in the lengths of the obtained amplicons were detected after gel electrophoresis, and restriction analyses of the PCR fragments led to identification of six out of eight isolates as belonging to fungi T. parceramosum/ T. reesei/ T. longi, Polyporus squamosus, Fusarium oxysporum and Trichoderma atroviride groups. The PCR ITS-RFLP molecular identification technique can be used successfully for routine screening of fungal isolates from various environments.

Weissella jogaejeotgali sp. nov., isolated from jogae jeotgal, a traditional Korean fermented seafood.

Strain FOL01T was isolated from traditionally fermented Korean jogae jeotgal (fermented clams). Phylogenetic sequence analysis of the 16S rRNA gene from FOL01T revealed that it is closely related to Weissella thailandensis FS61-1T and Weissella paramesenteroides ATCC 33313T with 99.39 % and 98.50 % 16S rRNA gene sequence similarities, respectively. API and VITEK analyses showed that strain FOL01T could be separated from its nearest phylogenetic relatives with respect to carbohydrate fermentation and antibiotic resistance. Subsequent amplified rRNA gene restriction analysis of 16S rRNA genes and HaeIII-restriction enzyme profiling of genomic DNAs revealed different band patterns. In addition, DNA-DNA hybridization of genomic DNAs showed 63.9 % relatedness. Analysis of the composition of cellular fatty acids confirmed that strain FOL01T differs from its close relatives and supports the proposal to assign this organism to a novel species of the genus Weissella. Based on these results, strain FOL01T could be classified as a novel species of the genus Weissella, for which the name Weissella jogaejeotgali sp. nov. is proposed. The type strain is FOL01T ( = KCCM 43128T = JCM 30589T).

Identification of blood meals in field captured sand flies by a PCR-RFLP approach based on cytochrome b gene

Leishmaniasis is a vector-borne disease transmitted by phlebotomine sand flies. Information about blood meal preferences in sand flies is essential to understand the epidemiology of the disease to adopt control measures. In previous studies, a polymerase chain reaction (PCR) of 359bp fragment of the conserved gene cytochrome b (cyt b) and further sequencing were applied in the study of blood meal sources in sand flies collected in the area of a leishmaniasis outbreak in southwest Madrid, Spain, providing significant information about blood meal preferences in the focus.In this work, a PCR-restriction fragment length polymorphism (RFLP) targeting a fragment of 359bp of vertebrate cyt b gene was developed. Restriction endonucleases HaeIII and HinfI generated specific patterns consistent with the blood meal sources found in sand flies. The protocol has been validated with twenty six engorged females collected in the field with CDC traps. Blood meals from nine vertebrates were identified based on PCR-cyt b and sequencing—human, dog, cat, horse, hare, rabbit, sheep, goat and chicken – and mixed blood meals (sheep/human; sheep/goat) – and successfully distinguished by PCR-RFLP. Therefore, this approach is an efficient and reliable alternative method to be applied in entomological surveys.

Performance Assessment of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Method for Rapid Detection of Susceptibility to Ethambutol and Molecular Prediction of Extensively Drug-resistant Tuberculosis in Clinical Isolates of Mycobacterium tuberculosis.

The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was employed for rapid detection of ethambutol (EMB) resistant clinical isolates of Mycobacterium tuberculosis. From 182 clinical isolates of M tuberculosis collected from different regions, 103 strains were entered in the investigation. DNA was extracted by Chelex 100 method and PCR was performed using specific primers for embB gene. Polymerase chain reaction products were digested with HaeIII and NlaII restriction endonucleases and the patterns of restriction fragments were analysed. Some randomly selected samples were sequenced. Out of 103 studied strains, 52 were resistant to EMB. The cases of secondary tuberculosis were 53 (51.50 ± 1.77%), and primary cases 50 (48.50 ± 1.77%; p > 0.05). From 63 extensively drug-resistant (XDR), pre-XDR and multidrug-resistant (MDR) isolates, 27 (87%), 18 (81.8%) and 7 (70%) strains were resistant to EMB, respectively. Results of PCR-RFLP method showed that from 27R EMB XDR isolates, 13 (sensitivity 48% with CI: 0.307, 0.66 and specificity 100%), from 18R EMB pre-XDR strains, 4 (sensitivity 22% with CI: 0.09, 0.45 and specificity 100%) and of 7R EMB MDR, 2 (sensitivity 28% with CI: 0.082, 0.64 and specificity 100%) had mutation in ATG-Met codon 306. Results of sequencing were concordant with RFLP method. Overall, sensitivity of the molecular method was 36.5% (CI: 0.09, 0.45) and specificity 100%. None of the 40 pansusceptible strains was embB306 mutants. Extensively drug-resistant strains had a higher proportion of embB306 mutants (43%) than pre-XDR and MDR isolates (odds ratio 6.78; p < 0.001). Fast detection of susceptibility to EMB drug is possible by PCR-RFLP. The embB306 locus is a candidate marker for rapid prediction of high resistance consisting of MDR and XDR forms to anti-tuberculosis drugs using this method.

First report of a 16SrVI group related phytoplasma associated with cucumber phyllody in a greenhouse in Iran

Greenhouse cucumber (Cucumis sativus) is one of the most economically important vegetable crops in Iran. In September 2014, an outbreak of phyllody disease was observed in up to 82% of greenhouse cucumber in cultivars Keyhan and Negin in Nasr Abad (Taft, Yazd province). Symptoms were characterised by flower virescence, phyllody and sterility (Fig.1). Total DNA was extracted from 0.2 g of fresh phyllody-affected flowers and from symptomless cucumber plants (Zhang et al., 5). DNA samples were tested for phytoplasma by direct and nested PCR using the phytoplasma primers P1/P7 (Deng & Hiruki, 2; Schneider et al., 4) followed by R16F2n/R16R2 (Gundersen & Lee, 3). Primer pair P1/P7 amplifies an 1800 bp fragment of the phytoplasma ribosomal operon which includes the 16SrRNA gene, the 16S-23S intergenic spacer region (SR) and a portion of the 5' region of the 23S rRNA gene. R16F2n/R16R2 primer pair amplifies about 1250 bp of the phytoplasma 16S rRNA gene. PCR amplicons of ~1.8 and ~1.25 kbp were obtained from symptom-bearing, but not from symptomless greenhouse cucumber plants. Seven R16F2nR16R2 amplicons from infected areas in Nasr Abad were directly sequenced. The R16F2n/R16R2 sequences shared 100% sequence identity. A consensus sequence corresponding to the Saleh Abad (Nasr Abad, Taft, Yazd province) greenhouse cucumber phyllody (SGCP) was deposited in GenBank (Accession No. KR633068). BLAST search showed that the SGCP phytoplasma had the highest sequence identity with those of the 16SrVI (‘Candidatus Phytoplasma trifolii’) phytoplasma group. Phylogenetic analysis (MEGA software version 6.0) confirmed that the SGCP phytoplasma clustered within the 16SrVI phytoplasma clade (Fig. 2). R16F2n/R16R2 SGCP phytoplasma sequence computer-simulated restriction analysis (Fig.3) with restriction endonucleases AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI using iPhyclassifier (Zhao et. al., 6) revealed that virtual RFLP patterns of the SGCP phytoplasma differ from those previously recorded 16SrVI subgroups (A, B, C, D, E, F, H, I). Although the SGCP phytoplasma RFLP profiles were more similar to those of subgroup A (AY390261), it showed a similarity coefficient of 0.93; which suggests that the SGCP phytoplasma may represent a new subgroup within the 16SrVI phytoplasma group. This is the first report of the association of a 16SrVI phytoplasma with cucumber phyllody disease in a greenhouse in Iran. A 16SrII phytoplasma (‘Ca. P. aurantifolia’) was associated with cucumber phyllody in a greenhouse of the Fars province (Dehghan et al., 1). Therefore the SGCP phytoplasma may represent a new epidemiological constraint for the development of the phyllody disease in greenhouses in Iran.

Exploring Polymorphism in 3’ UTR Region of STAT1 Gene in different Buffalo Breeds

Study was aimed to screen the adult female of different buffalo breeds maintained at organized herd of ICAR-NDRI, Karnal (Haryana) and from field (Punjab, Himachal Pradesh and Chhattisgarh state) for the presence or absence of important genetic variations in 3’UTR region of Signal Transducer and Activator of Transcription 1 (STAT1) gene, known to be associated with mammary gland development and with milk fat% and milk yield in cows. Animals from three breeds of riverine buffaloes Murrah, Gojri and Chhattisgarhi were studied using PCR-RFLP technique with HaeIII restriction enzyme. It revealed monomorphic patterns in the 3’UTR region STAT1 gene studied in all the three breeds of riverine buffaloes, indicating absence of genetic variation at this loci and suggesting that the loci is fixed for buffaloes in contract to exotic cattle breeds.

Polymorphism and association of growth hormone gene with growth traits in Sirohi and Barbari breeds of goat.

Aim:The aim was to study the polymorphism of exon 2 and exon 3 of growth hormone (GH) gene, to test the polymorphic variants for Hardy–Weinberg equilibrium and to investigate association of these polymorphisms with chest girth and paunch girth in Sirohi and Barbari breeds of goat.Materials and Methods:A total of 80 kids involving forty each of Sirohi and Barbari breeds of goat were included in the study. A good quality genomic DNA isolated from the whole blood using standard protocol were used for polymerase chain reaction (PCR) amplification and products obtained on restriction digestion of amplicon with enzyme HaeIII were separated on 2% agarose gel, and documented in a gel doc system. The chest girth and paunch girth of kids at birth and weekly intervals up to 4 weeks of age and subsequently at 2 months, 3 months and 6 months of age were recorded. Allele frequency and genotype distribution of polymorphism were tested for Hardy–Weinberg equilibrium by program me Genepop package. Association between different genetic variants on chest girth and paunch girth were analyzed by least squares analysis employing suitable statistical model.Results:The PCR product of genomic DNA isolated from kids of Sirohi and Barbari breeds of goat on digestion with the restriction enzyme HaeIII revealed two genotypic variants viz., AB and BB. None of the two breeds was in Hardy–Weinberg equilibrium for these variants. The least squares analysis of variance revealed non-significant effect of GH genotype and breed × genotype interaction on chest girth and paunch girth from birth to 180 days of age. The effect of breed was highly significant (p<0.01) at all ages.Conclusion:The present study showed that both the breeds were polymorphic at the exon 2 and exon 3 loci of GH gene under study with respect to HaeIII restriction endonuclease. None of the breeds was in Hardy–Weinberg equilibrium for this region of GH gene. In the present study, no significant association between GH genotype and chest girth and paunch girth could be established but comparatively higher chest girth and paunch girth were observed for AB genotype across the breeds.

Molecular Study of Some Candidate Genes Affecting Milk Production Traits in Holstein Cattle

Most of the economically important traits in dairy cattle are quantitative in nature, which means that they are affected by environmental factors and by large number of gene. Selection of superior animals has been made more effective through studies of major milk protein genes that are known to affect both milk yield and composition. The present study was carried out to detect polymorphism in kappa casein (CSN3) and beta-lactoglobulin (LGB) genes in Holstein cattle under Egyptian condition through DNA sequencing and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Fifty Animals were divided into high and low milk producing according to their breeding value. PCR amplification of exon III o CSN3 and LGB was performed followed by restriction fragment length polymorphism (PCR-RFLP) using Hind-III and HinfI for CSN3 and HaeIII restriction endonuclease for LGB. Nucleotide polymorphisms between high and low producing cows were detected by DNA sequencing. The restriction enzymes digestion failed to produce restriction patterns and revealed no polymorphism in all studied animals. Comparison of nucleotide sequences between high and low producing cows revealed lack of polymorphism in CSN3 and four nucleotide changes in LGB gene; C179 T, C225T, T246C, and C294G. The further study using other specific restriction endonuclease was required to detect polymorphisms of CSN3 and LGB in Egyptian Holstein cattle. SNPs discovered in this study can be used as molecular genetic markers for marker assisted selection (MAS) to increase and accelerate the rate of genetic improvement of milk production traits.

Application of PCR-RFLP technique to species identification and phylogenetic analysis of Streptomyces associated with potato scab in Brazil based on partial atpD gene sequences

In the present study the atpD gene was evaluated as an alternative molecular marker for differentiation of Streptomyces species causing potato scab and to establish the phylogenetic relationships within this group. PCR-RFLP experiments using HaeIII and CfoI restriction enzymes allowed differentiation of most Streptomyces species, except the genomospecies S. reticuliscabiei and S. turgidiscabies which showed identical profiles between them. Phylogenetic relationships within Streptomyces spp. were also examined by comparing partial atpD gene sequences (858 nt) and the strains were separated in three main groups: Group I, supported by bootstrap values of 79%, comprised of S. scabiei, S. europaeiscabiei, S. stelliscabiei, S. reticuliscabiei, S. turgidiscabies and S. acidiscabies; Group II, supported by bootstrap values of 100%, comprised of S. sampsonii; and Group III supported by bootstrap values of 100%, comprised of S. caviscabies, S. setonii, S. luridiscabiei and S. puniciscabiei. The atpD gene sequence similarity observed among the species in this study ranged from 89.2% to 100%. Twenty-nine Streptomyces isolates obtained from potato growing areas in Brazil were analyzed by PCR-RFLP of atpD gene technique and identified as S. scabiei (15 strains), S. caviscabies/ S. setonii (12) and S. sampsonii (2). Using this tool S. europaeiscabiei strains were also detected from import material from the Netherlands. The presence of genes associated with the S. turgidiscabies pathogenicity island (PAI) was also determined and potato tuber slice assays were performed to confirm the pathogenicity of the strains. The data obtained herein indicated that the PCR-RFLP of atpD gene technique can be used for diagnosis and identification of phytopathogenic Streptomyces strains, representing a rapid and inexpensive tool. Moreover, this gene can be useful for phylogenetic analysis of Streptomyces spp. associated with potato scab.

Genotypic and phenotypic study of Lactobacillus species isolated from traditional yogurt and cheese in southwest Iran

Lactic acid bacteria (LAB) have the property of producing lactic acid from sugars by a process called fermentation. These bacteria are broadly used in the production of fermented food products, such as yogurt, cheeses, sauerkraut and sausage. Among them Lactobacillus species are used industrially for the production of yogurt and cheese. The purpose of present study was to identify the genotypic and phenotypic of Lactobacillus species in the traditional cheeses and yogurt produced in the Chaharmahal Va Bakhtiari province located in southwest Iran using molecular technique. 18 Dalameh cheeses and 15 traditional yogurt specimens were collected from various groceries and markets in the Chaharmahal Va Bakhtiari province. Genomic DNA was extracted from yogurt and cheese samples, as well as reference strains and PCR reaction was down for amplification of 16S rDNA gene. PCR products were digested by TaqI and HaeIII restriction enzymes and fragments were analyzed in 2% agarose gel electrophoresis. The results of present study showed that Lactobacillus delbrueckii (Lb. delbrueckii), Lb. acidophilus, Lb. brevis and Lb. lactis strains from traditional cheese and yogurt is important and more frequent in dairy industry. These findings could be used as starter cultures in manufacturing of cheeses and yogurt to approach better and safety dairy products with high quality.

Culturable diversity and functional annotation of psychrotrophic bacteria from cold desert of Leh Ladakh (India)

To study culturable bacterial diversity under subzero temperature conditions and their possible functional annotation, soil and water samples from Leh Ladakh region were analysed. Ten different nutrient combinations were used to isolate the maximum possible culturable morphotypes. A total of 325 bacterial isolates were characterized employing 16S rDNA-Amplified Ribosomal DNA Restriction Analysis with three restriction endonucleases AluI, MspI and HaeIII, which led to formation of 23-40 groups for the different sites at 75% similarity index, adding up to 175 groups. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 175 bacteria, grouped in four phyla, Firmicutes (54%), Proteobacteria (28%), Actinobacteria (16%) and Bacteroidetes (3%), and included 29 different genera with 57 distinct species. Overall 39% of the total morphotypes belonged to the Bacillus and Bacillus derived genera (BBDG) followed by Pseudomonas (14%), Arthrobacter (9%), Exiguobacterium (8%), Alishewanella (4%), Brachybacterium, Providencia, Planococcus (3%), Janthinobacterium, Sphingobacterium, Kocuria (2%) and Aurantimonas, Citricoccus, Cellulosimicrobium, Brevundimonas, Desemzia, Flavobacterium, Klebsiella, Paracoccus, Psychrobacter, Sporosarcina, Staphylococcus, Sinobaca, Stenotrophomonas, Sanguibacter, Vibrio (1%). The representative isolates from each cluster were screened for their plant growth promoting characteristics at low temperature (5-15°C). Variations were observed among strains for production of ammonia, hydrogen cyanide, indole-3-acetic acid and siderophore, solubilisation of phosphate, 1-aminocyclopropane-1-carboxylate deaminase activity and biocontrol activity against Rhizoctonia solani and Macrophomina phaseolina. Cold adapted microbes may have application as inoculants and biocontrol agents in crops growing at high altitudes under cold climate condition.

Polymerase chain reaction - restriction fragment length polymorphism analysis for the differentiation of mycobacterial species in bronchial washings.

To identify mycobacterial species in bronchoscopy specimens with a simple assay based on polymerase chain reaction and restriction enzyme digestion. Sputum smear negative, bronchoscopy specimens (n=202) were collected from patients attending the Central Chest Clinic and the Teaching Hospital Kandy, Sri Lanka. DNA, extracted from the mycobacterial cultures (n=43) were amplified using known mycobacterial specific Sp1 and Sp2 primers. Resulting products were digested with HaeIII and CfoI restriction enzymes and DNA sequencing was performed for the selected isolates. Among the culture positive patients, PCR was able to distinguish 12 rapid growers (~280-320 bp), 15 slow (~200-220 bp) and 10 patients having both rapid and slow and one having two rapid growing mycobacteria. DNA Sequence analysis revealed the presence of M. intracellulare (n=3), M. phocaicum (n=7), M. tuberculosis complex (n=13), Nocardia (n=2), M. smegmatis (n=1) and Mycobacterium sp (n=12). The identified organisms got digested upon exposure to HaeIII restriction enzyme whereas when exposed to CfoI, only M. phocaicum yielded 80 bp and 230 bp DNA fragments while others remained undigested. Consequently, six patients were confirmed to have M. tuberculosis complex, seven had both M. tuberculosis and non-tuberculosis bacteria (NTM) in their bronchoscopy specimens while 21 had NTM. Optimised PCR-RFLP assay was able to differentiate M. tuberculosis complex bacteria from nontuberculosis mycobacteria and Nocardia. Molecular analysis confirmed the presence of NTM in bronchoscopy specimens and according to the study a significant proportion of patients (13% to 14%) of the study population were found to have NTM in their bronchial washings.

Single nucleotide polymorphism in Egyptian cattle insulin-like growth factor binding protein-3 gene

Insulin-like growth factor binding protein-3 (IGFBP-3) gene is a structural gene respon- sible for the multiple influences of insulin-like growth factors (IGFs) system. It is considered as a candidate gene for growth and production traits. In the present study, we aimed to determine the genetic polymorphism of Egyptian cattle IGFBP-3 gene. The amplified fragment of cattle IGFBP-3 gene at 651-bp was digested with three different endonucleases; HaeIII, MspI and TaqI. The digestion of the PCR products with MspI and TaqI endonucleases revealed similar restriction patterns in all tested animals. Digestion of the PCR product with HaeIII restriction enzyme revealed three different genotypes in Egyptian cattle due to the presence of two alleles; allele A with 7 digested fragments resulting from the presence of 6 restriction sites and allele C with 8 digested fragments resulting from the presence of 7 restriction sites; six sites as allele A in the addition of another restriction site at posi- tion 298^299 as a result of SNP (A fi C) in C allele at position 299. The restriction patterns of IGFBP-3/HaeIII showed that forty-six examined animals are genotyped as AA, CC and AC with frequencies of 21.74%, 21.74% and 56.52%, respectively. It is concluded that the IGFBP-3/HaeIII polymorphism may be utilized as a good marker for genetic differentiation between cattle animals for different body functions such as growth, metab- olism, reproduction, immunity and energy balance. The nucleotide sequences of Egyptian cattle IGFBP-3 A and C alleles were submitted to GenBank with the accession numbers KF899893 and KF899894, respectively. a 2014 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.

Diversity and phylogenetic profiling of niche-specific Bacilli from extreme environments of India

The diversity of culturable, aerobic and heterotrophic Bacillus and Bacillus-derived genera (BBDG) was investigated in various extreme environments (including thermal springs, cold deserts, mangroves, salt lakes, arid regions, salt pans and acidic soils) of India. Heat treatment followed by enrichment in different media led to a total of 893 bacterial isolates. Amplified ribosomal DNA restriction analysis (ARDRA) using three restriction enzymes AluI, MspI and HaeIII led to the clustering of these isolates into 12–74 groups for the different sites at 75 % similarity index, adding up to 559 groups. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 392 bacilli, grouped in two families, Bacillaceae (89.03 %) and Paenibacillaceae (10.97 %), and included 13 different genera with 75 distinct species. It was found that among the thirteen genera, nine (Bacillus, Halobacillus, Lysinibacillus, Oceanobacillus, Pontibacillus, Salinibacillus, Sediminibacillus, Thalassobacillus and Virgibacillus) belonged to Bacillaceae and four (Ammoniphilus, Aneurinibacillus, Brevibacillus and Paenibacillus) belonged to Paenibacillaceae. Novel isolates tolerant to low and high pH and temperature, salt and low moisture were identified. The major outcome of the present investigation was the identification of niche-specific species and also the ubiquitous presence of selected species of BBDG, which illustrate the diversity and pervasive nature of BBDG in extreme environments.

Genetic polymorphism of the β-lactoglobuline gene in Friesian Holstein cows

Milk protein in dairy cattle consists of casein and whey. Whey in milk of dairy cattle is about 20% with the main component is β-lactoglobulin (7-12%). Polymorphism of the β-lactoglobulin gene affects protein and whey production in milk. Selection at a molecular base to improve whey and protein in milk requires information on genetic diversity of the β-lactoglobulin gene, besides other protein genes. This study was aimed to identify genetic polymorphism of the β-lactoglobulin gene in 88 heads of Friesian Holstein (FH) cows at Cikole Dairy Cattle Breeding and Improvement Station (Cikole DCBIS), Lembang. Genotyping was done using Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) method, and a HaeIII restriction enzyme. Genotype frequency, allele frequency, and heterozygosity value were calculated by Nei method. Hardy-Weinberg equilibrium was calculated by chi-square test. Result showed that the β-lactoglobulin gene in the observed HF cattle was polymorphic, in which there were two alleles (A and B) and three genotypes (AA, AB and BB). Frequencies of the A and B alleles were 0.40 and 0.60 respectively; while those of the AA, AB, and BB genotypes were succesively 0.10; 0.60 and 0.30. Heterozygosity value was obtained for 0.483. The β-lactoglobulin gene was in Hardy - Weinberg equilibrium (χ2cal < χ2table). It is concluded that polymorphic of the β-lactoglobulin gene can be used as an initial information for molecular selection on milk protein composition in FH cows. Key Words : HF Cattle, β-lactoglobuline Gene, Genetic Polymorphism, PCR-RFLP

First Report of Elm Yellows Subgroup 16SrV-B Phytoplasma as the Cause of Rose Balsam Phyllody in China.

Rose balsam (Impatiens balsamina L.) is an ornamental species frequently cultivated in China and the red flower is often used as nail polish in rural regions. The phytoplasmas previously reported with rose balsam phyllody in China have been classified as aster yellows group (16SrI) (1). In August 2012, some rose balsams were observed with typical phytoplasma symptoms in Handan City, Hebei Province, China, with an incidence of about 70% in the fields. The flowers turned green and petals fascicled. The new leaves wrinkled and deformed and internodes shortened. Infected plants were stunted, matured prematurely, and failed to produce seeds. To confirm phytoplasma infection, 100 mg of plant tissue (leaves, petals) was collected from five symptomatic and four asymptomatic plants and total DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method (2). The 16S rDNA gene was amplified by nested PCR using primer pair P1/P7 followed by R16F2n/R16R2 (3). No amplicons were generated with DNA from asymptomatic samples, but amplicons of approximately 1.2 kb were obtained with DNA from five symptomatic samples. The amplified products were purified with aTIANgel midi purification kit (Tiangen, Beijing) and sequenced at the Sangon Biotech facility (Shanghai, China). The sequences of the amplicons were 100% identical and deposited in NCBI GenBank (Accession No. KC993832). The 16S rDNA gene sequence from this phytoplasma was 99% similar to Jujube witches broom phytoplasma (JQ675716), Puna chicory flat stem phytoplasma (JN582266), Plum yellows phytoplasma (FJ459914), and other elm yellows group phytoplasmas by BLAST search of the NCBI database. Restriction fragment length polymorphism (RFLP) analyses were carried out by digesting the 1.2-kb R16F2n/R16R2 nested PCR product with restriction enzymes AluI, RsaI, HhaI, HpaI, Eco RI, TaqI, HaeIII, HinfI, and KpnI (Takara, Dalian). The 16S rDNA RFLP patterns matched that of Jujube witches broom phytoplasma (JWB, subgroup 16SrV-B) (4). Nucleotide sequences of rose balsam phyllody were analyzed by iPhyClassifier software, which revealed that it had maximum similarity to the reference pattern of 16Sr group V, subgroup B (AB052876). All samples were detected with transmission electron microscopy. The results showed phytoplasma-like cells in phloem sieve element of symptomatic plants, while no phytoplasma-like cells were observed in healthy phloem tissues. The phytoplasma cells ranged from 230 to 470 nm in diameter and were ellipsoidal or orbicular with visible membranes. Combining the RFLP pattern and sequence analysis by iPhyClassifier, we classified the phytoplasma causing rose balsam phyllody into subgroup 16SrV-B. To our knowledge, this is the first report of 16SrV-B group phytoplasmas infecting rose balsam in China.

Molecular Characterization of Phytoplasmas Related to Apple Proliferation and Aster Yellows Groups Associated with Pear Decline Disease in Iran

AbstractPear trees showing pear decline disease symptoms were observed in pear orchards in the centre and north of Iran. Detection of phytoplasmas using universal primer pair P1A/P7A followed by primer pair R16F2n/R16R2 in nested PCR confirmed association of phytoplasmas with diseased pear trees. However, PCR using group‐specific primer pairs R16(X)F1/R16(X)R1 and rp(I)F1A/rp(I)R1A showed that Iranian pear phytoplasmas are related to apple proliferation and aster yellows groups. Moreover, PCR results using primer pair ESFYf/ESFYr specific to 16SrX‐B subgroup indicated that ‘Ca. Phytoplasma prunorum’ is associated with pear decline disease in the north of Iran. RFLP analyses using HaeIII, HhaI, HinfI, HpaII and RsaI restriction enzymes confirmed the PCR results. Partial 16S rRNA, imp, rp and secY genes sequence analyses approved that ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma asteris’ cause pear decline disease in the centre of Iran, whereas ‘Ca. Phytoplasma prunorum’ causes disease in the north of Iran. This is the first report of the association of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma prunorum’ with pear decline disease worldwide.

Detection of Recessive Mutations (BLAD and CVM) in Holstein-Friesian Cattle Population in Republic of Macedonia

Abstract BLAD and CVM are Holstein-specific recessive mutations which have had significant economic impact on dairy cattle breeding worldwide. The aim of this study was to optimize a diagnostic test for detection of these two disorders and to obtain a preliminary picture about theire existence in Macedonian HF population. Blood samples were obtained from 84 HF cows from several different farms in Macedonia. Genomic DNA was extracted using DNeasy blood and tissue kit (Qiagen) which was also used for DNA extraction from deeply frozen sperm doses from 6 different HF bulls. Genotyping was performed by PCR-RFLP analysis with TaqI and HaeIII restriction endonucleases for BLAD and with PstI for CVM. The correctness of the genotyping was evaluated by direct sequencing of the amplified products. Among 90 HF cattle tested two cows were identified as carriers of the BLAD allele and one was carrier of the CVM allele. No bulls were found to be carriers of either of the two disorders. This study demonstrates that carriers of BLAD and CVM are present in Macedonia, although at low frequency. PCR followed by RFLP is efficient and inexpensive method for detection of the carriers and should be used as laboratory test in eradication programs. In order to establish a more accurate picture of the prevalence of these disorders, it is necessary to screen a larger number of animals. It is indispensable to test each bull before entering the A.I. program to prevent accidental transmission to a large number of offspring.