Single-strand Conformation Polymorphism Patterns Research Articles

Transmission of isolates of Citrus tristeza virus by single aphids (Toxoptera citricida)

Citrus tristeza virus (CTV) can be transmitted by several aphid species in a semi-persistent mode, with Toxoptera citricida being the most efficient vector. In Brazil, mild CTV isolates are used for pre-immunization of citrus trees against severe isolates. We aimed to determine the capacity and efficiency of T. citricida in separating the viral complex into haplotypes from three well-characterized CTV isolates (PIAC, CS1, and Forte Rolândia). Single-aphid transmission assays were conducted to determine CTV transmission efficiency. The results showed that T. citricida transmitted only haplotypes from the PIAC and CS1 isolates, with efficiencies of 8 and 4%, respectively. Both isolates caused mild CTV symptoms in Brazil. However, isolate Forte Rolândia, which causes severe symptoms in citrus trees, was not transmitted by T. citricida. The detection of CTV haplotypes from PIAC and CS1 isolates in sweet orange (Citrus sinensis) plants after a single aphid transmission occurred at different time points. The first haplotype observed was from PIAC 150 days after citrus was challenged by a single aphid, followed by CS1 at day 210 after transmission. In addition, differences in the single-strand conformation polymorphism patterns between the CTV isolates and CTV haplotypes were determined, suggesting that an aphid can acquire and transmit only one CTV haplotype to citrus plants during phloem feeding. The study of the mechanism of transmission by the vector can increase our knowledge of the interactions among hosts, vectors, and pathogens, which are often neglected.

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

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

The relationship between mitochondrial ND5 gene polymorphisms and in vitro embryo production in Sanjabi sheep.

The aim of this study was to investigate mitochondrial ND5 gene polymorphisms and their relationship with in vitro maturation (IVM) and in vitro culture (IVC) of Sanjabi sheep. Blood and ovarian samples of adult ewes were obtained from a local slaughterhouse. For each ovarian sample, cumulus-oocyte complexes larger than 3 mm in diameter were aspirated from follicles, and their IVM and IVC rates were recorded. A 666-bp fragment of the ND5 gene was amplified using the polymerase chain reaction. The samples were genotyped using a modified single-stranded conformation polymorphism (SSCP) method, and an association study was conducted with IVM and IVC rates. Six different SSCP patterns, designated A, B, C, D, E and F with respective frequencies of 8, 47, 4, 4, 32 and 5%, respectively, were observed. According to the results of association analysis, there was no significant association between the ND1 gene polymorphisms and the IVM and IVC rates (P > 0.05).

A Novel Missense Single Nucleotide Polymorphism in the GREM1 Gene is Highly Associated with Higher Reproductive Traits in Awassi Sheep.

GREM1 (gremlin1) is a known inhibitor for BMP15 (bone morphogenetic protein 15) family, but its genetic diversity in sheep is unknown. The present study was conducted to analyze the polymorphism of GREM1 gene using PCR- single-strand conformation polymorphism (SSCP) and DNA sequencing methods and to assess the possible association of GREM1 gene polymorphism with reproductive traits in Awassi ewes. A total of 224 ewes, 124 producing singles and 100 producing twins, were included in the study. Two SSCP patterns were detected in two amplified loci within the exon 2. Two exonic novel single nucleotide polymorphism (SNP)s were identified, c.74T > G (the silent SNP p.Met123 =) and c.30T > A with (the missense SNP p.Ile237Phe). Statistical analyses indicated a non-significant (P > 0.05) association of p.Met123 = with the analyzed reproductive traits of fecundity, prolificacy, litter size, and twinning rate. Meanwhile, p.Ile237Phe SNP exhibited a highly significant (P < 0.01) association with the measured reproductive traits, in which ewes with TA genotype (with p.Ile237Phe SNP) exhibited higher litter size, twinning ratio, fecundity, and prolificacy than those with TT genotype (without p.Ile237Phe SNP). The deleterious impact of p.Ile237Phe SNP was observed by the means of ten different state-of-the-art in silico tools that predicted a highly damaging effect of p.Ile237Phe SNP on the structure, function, and stability of gremlin1. In conclusion, the results of our study suggest that p.Ile237Phe SNP has a remarkable negative impact on the gremlin1 structure, function, and stability. Since gremlin1 is a known inhibitor of reproductive performance, a consequent higher reproductive performance was observed in ewes with damaged gremlin1 (with p.Ile237Phe SNP) than those with non-damaged gremlin1 (without p.Ile237Phe SNP). Therefore, it can be stated that the implementation of the novel p.Ile237Phe SNP in the GREM1 gene could be a useful marker in marker-assisted selection. This manuscript is the first one to describe GREM1 gene variations in sheep.

A novel T177P missense variant in the HSPA8 gene associated with the low tolerance of Awassi sheep to heat stress.

This study was conducted to identify the association of coding variations in the HSPA8 gene with heat stress in two different breeds of sheep. All the coding regions of the HSPA8 gene of Awassi and Arabi sheep were covered by amplifying nine exons. A single-strand conformation polymorphism (SSCP) was utilized to assess the genetic variations in both breeds. The possible association of the observed genotypes with rectal temperature (RT), respiratory rate (RR), and heat tolerance coefficient (HTC) was analyzed in different seasons. While all the coding regions of both sheep were monomorphous, a remarkable heterogeneity was observed in exon 4, of which two SSCP patterns, a normal TT and a mutant TG, were detected. The TG genotype was characterized by a missense variant of T177P with frequencies of 77% in Awassi and 54% in Arabi. Cumulative in silico tools indicated extremely deleterious consequences for T177P on protein structure, function, and stability. Results indicated that sheep with the TT genotype had significantly (P< 0.05) lower RT, RR, and HTC values than sheep with the TG genotype. Therefore, a significant association of T177P with a lower tolerance of Awassi to higher temperature conditions was revealed. In conclusion, the identified T177P may have damaging effects in the HSPA8, which affects the ability of Awassi sheep to cope up with elevated temperatures compared with Arabi sheep. This manuscript describes a novel description of a highly deleterious missense variant in the HSPA8 gene that may reduce the ability of sheep to withstand high-temperature conditions.

Mutation analysis of VSX1 gene in vernal keratoconjunctivitis patients

ObjectivesVernal keratoconjunctivitis (VKC) is a multifactorial disease of conjunctiva that usually involves young adults. Heretofore, the role of several genes has been studied in VKC pathogenesis. The aim of this study was to discover new modifications in Visual System Homeobox 1 (VSX1) gene and evaluate their possible correlation with the protein activity and VKC pathogenesis in Iranian patients. MethodsFor this cross-sectional study, DNA samples of 100 sporadic patients with VKC and 100 healthy people were analyzed for detection of novel mutations in exon 3, intron 3 and exon 4 of VSX1 gene, using single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA). After DNA sequencing of samples with different SSCP and HA pattern, conservation level and pathogenicity of the detected variations were investigated using bioinformatics software and databases. ResultsSequencing results revealed a (c. 628-31 T > A) g. 25,075,355 T > A variation in intron 3 and a c.697G > A variation in the coding region of exon 4. RFLP results did not confirm the existence of c.697G > A mutation in the control group and this missense variation was recognized as a pathogenic risk factor, using SIFT software (P SIC>2). ConclusionSince the modified sequence is located within the conserved region, it can be proposed that the detected modification may have an effect on the encoded protein function. Nevertheless, more studies are required for analyzing and evaluating the exact effects of this substitution.

Molecular characterization of Echinococcus granulosus in livestock of Al-Madinah (Saudi Arabia).

Echinococcus granulosus is the causative agent of cystic echinococcosis, which has serious impacts on human and/or animal health, resulting in significant economic losses. Echinococcus granulosus comprises a number of intra-specific variants or strains at the genetic level. In Saudi Arabia, few studies were performed on genetic variations in Echinococcus species. Therefore, the present study aimed to investigate the phenotypic and genetic characterization of hydatid cysts harboured by sheep and camels in Al-Madinah Al-Munawarah. Samples of hydatid cysts were collected from local sheep (n = 25) and camels (n = 8). The morphological criteria of protoscoleces were investigated. To investigate the molecular characterization, random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR), single-stranded conformation polymorphism (SSCP) were carried out. DNA was extracted from individual fertile cysts and subjected to RAPD-PCR analysis (using five arbitrary primers) and PCR amplification of cytochrome c oxidase I (cox1) and 12S ribosomal ribonucleic acid (12S rRNA) genes. The PCR products were subjected to SSCP analysis for genetic discrimination in E. granulosus isolates. In addition, partially sequencing of the mitochondrial DNA cox1 genes was achieved for assessing the phylogenetic positions of collected isolates using some global published sequence data of cox1 genes. The rostellar hooks of camel and local sheep isolates show remarkable variability in their dimensions. Five distinct SSCP patterns were identified in the 12S rRNA gene, showing intraspecific variations in E. granulosus of camels and local sheep. Sequencing of (cox1) genes of both local sheep and camels exhibit high similarity with those of the same gene (E. granulosus sensu stricto) published in NCBI BLAST.

Polymorphism of avirulence genes in potato late blight pathogen Phytophthora infestans as characterized by SSCP analysis

Fighting late blight, economically the most important of potato diseases, is greatly hampered by rapid changes in the populations of the pathogen Phytophthora infestans: new pathotypes turn up owing to pathogen evolution and migration and overcome potato varieties that were previously considered resistant. Early recognition of the changes in P. infestans populations would greatly assist potato protection against late blight. The pathogenicity of P. infestans depends on the (a)virulence genes (Avr genes), among which the best known are the genes encoding RXLR effectors. This is the first report on polymorphism of these genes when characterized by single-strand conformation polymorphism (SSCP) analysis. Highly reproducible SSCP patterns combine electrophoretic zones corresponding to the Avr alleles. Ten Avr genes were studied in two series of P. infestans samples: single cell lines obtained from the isolates collected in the potato genetic collection of the N.I. Vavilov Institute of Plant Genetic Resources (VIR), Pushkin, St. Petersburg, and the reference lines from the Western European and USA collections. The Avr3b, Avr4, and Avr8 genes were monomorphic, while in Avr1, Avr2, Avr3a, Avr9, Avr-blb1, Avr-blb2, and Avr-vnt1, we discerned two to five SSCP patterns, with their frequencies different in two series of P. infestans lines. Sequences of Avr alleles differed in synonymous and non-synonymous single nucleotide substitutions, with 93–100% identity between variants. When Avr polymorphisms were compared with the profiles of virulence factors recognized with the Mastenbroeck-Black differentials, the acceptable agreement between two independent indices was observed only for Avr1, Avr3a and Avr4.

Arg677Trp and Arg753Gln Polymorphisms in TLR2 Genes Detected in Patients With Tuberculosis in Golestan Province, Iran

Background: Toll-like receptors (TLRs), especially TLR2, play an important role in the immune responses of patients with tuberculosis (TB). The prevalence of TB is estimated 38.15 per 100,000 population in Golestan Province, Iran, which is higher than the rates reported in other provinces. Objectives: The current study aimed at detecting Arg677Trp and Arg753Gln polymorphisms in TLR2 genes detected in patients with TB in Golestan province. Methods: A total of 130 blood samples were collected from patients with sputum smear-positive TB, and 130 blood samples were collected from healthy individuals. Two polymorphisms of TLR2 gene, i e, Arg677Trp and Arg753Gln, were investigated via polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) and sequencing methods. Results: History of corticosteroid use was more frequently reported in patients, compared to the controls, while the frequency of Bacillus calmette-guerin (BCG) vaccination was more reported in controls. PCR-SSCP analysis of the TLR2 Arg677Trp region showed an almost identical pattern for patients and controls, while 2 SSCP patterns were observed in the amplicon, related to the Arg753Gln polymorphism. DNA sequencing for the Arg677Trp polymorphism showed a duplicated pseudogene, similar to the mutant sequence (C > T), as this polymorphism cannot be considered conclusive. The Arg753Gln polymorphism was not found in the samples, although nucleotide deletion (G) was detected at position 808 among the patients. Conclusions: The Arg753Gln polymorphism was not involved in susceptibility to TB in the current study population. The presence of pseudogenes in some genes such as TLR2 necessitates careful interpretation of such studies. © 2018, Jundishapur Journal of Microbiology.

High genetic stability of potato yellow mosaic Panama virus infecting tomato in Panama

The relevant regions in Panama involved in commercial tomato production, including the Chiriqui, Veraguas, Herrera, Los Santos and Panama Oeste provinces, were surveyed for the distribution and genetic diversity of potato yellow mosaic Panama virus (PYMPV) in the growing seasons of 2011 and 2012. A total of 28 tomato plots were surveyed and 314 individual tomato plants were sampled. DNA was extracted from each plant for a subsequent rolling circle amplification (RCA) analysis, to confirm the presence of begomovirus infections. The samples displaying a positive RCA reaction were subsequently analysed by PCR with a specific primer pair to identify PYMPV. This virus was detected in samples collected in all the above-mentioned provinces. The population structure of PYMPV was assessed by single-strand conformation polymorphism (SSCP) analysis. In total, four distinct SSCP patterns were distinguished. Based on the geographical distribution and the different patterns obtained, 42 viral isolates were selected for cloning and sequencing. Sequences obtained were compared with the ancestral sequence of PYMPV reported in Panama in 1998. These sequences showed very low variability, with two lineages probably originated by a single introduction.

Sequence Characterization in 3′-Flanking Region of Bovine TNF-α: Association with Milk Production Traits and Somatic Cell Score in Holstein Cattle of Iran

Background: Tumor necrosis factor- a (TNF-α) is a cytokine that was identified as a factor with a wide range of proinflammatory activities. The expression of bovine TNF-α in mammary tissue during pregnancy seems to have a role in development of the corresponding glands.Objective: Single nucleotide polymorphisms (SNPs) were defined in 3′-flanking region of bovine TNF-α in cattle. Moreover, its association with performance traits in Holstein dairy cattle was evaluated.Materials and Methods: The 3′-flanking region of TNF-α was screened by single strand conformation polymorphism (SSCP) and DNA sequencing in Holstein cattle breed. SAS statistical software was used to analyze the relationship between different genotypes of amplified fragment with milk production traits (daily milk yield, fat and protein percentage) and somatic cell score (SCS).Results: A total of 6 distinct SSCP patterns were observed. It was further revealed to be 3 novel SNPs. Statistical analysis revealed that different haplotypes of amplified fragment in the TNF-α 3′-region had a significant effect on average daily milk production (p < 0.05), but no such correlation was established with fat percentage, protein percentage and SCS.Conclusion: The association identified in the 3′-flanking region of TNF-α may have potential to serve as candidate genetic marker for genomic selection in dairy cattle.

Genetic characterization of FSH beta-subunit gene and its association with buffalo fertility

Objective: To study genetic variation in buffalo follicle stimulating hormone beta-subunit (FSHB) gene and its association with fertility. Methods: In this experimental study, blood samples were collected by standard methods using EDTA anticoagulant and transrectal ultrasound examination was conducted on fertile (n=74) and infertile buffaloes with a history of anestrum (n=30) or repeat breeding (n=12). The genomic DNA was extracted for PCR followed by single strand conformation polymorphism analysis. DNA sequencing was performed for the determination of single nucleotide polymorphism of FSHB gene. Results: The study results showed that there was genetic polymorphism with two different single strand conformation polymorphism patterns, AA and AB. The former pattern was associated with fertility in Egyptian buffaloes. Pair wise alignment of the two patterns sequences revealed that FSHB pattern II (AB) has C nucleotide insertion as SNP at the site of 208 bp of sequenced fragment. Conclusions: FSHB is polymorphic in the Egyptian buffaloes, suggesting its practicability as a candidate marker for female fertility.

Expression of catechol O-methyltransferase during ovarian development and association between its SNP and reproduction-related parameters of the giant tiger shrimp Penaeus monodon

Catechol O-methyltransferase cDNA and gene of the giant tiger shrimp Penaeus monodon (PmCOMT) were characterized. Genomic sequence of PmCOMT spanned 1466bp in length containing 3 exons (194, 111 and 361bp) and 2 introns (143 and 147bp). The full-length cDNA of PmCOMT was 1176bp in length with an open reading frame (ORF) of 666bp corresponding to a polypeptide of 221 amino acids. PmCOMT mRNA was not differentially expressed during ovarian development in wild intact P. monodon adults (P>0.05). Eyestalk ablation resulted in up-regulation of PmCOMT in stages II (vitellogenic) and IV (mature) relative to stage I (previtellogenic) ovaries (P<0.05). In domesticated shrimp, its expression was reduced in 18-month-old adults compared with 6-month-old juveniles and 14-month-old adults (P<0.05). In situ hybridization indicated that PmCOMT mRNA was clearly localized in ooplasm of previtellogenic oocytes but not in oogonia and other oocytes stages. Recombinant PmCOMT protein and anti-rPmCOMT polyclonal antibody were successfully produced. The PmCOMT protein was localized in ooplasm of previtellogenic oocytes. It was temporally found in both ooplasm and nucleus of vitellogenic oocytes and subsequently in cortical rods (Crs) and the Cr membrane of mature oocytes. In addition, single nucleotide polymorphism (SNP) in PmCOMT of domesticated P. monodon (14-month-old shrimp, N=64) was identified by single-strand conformational polymorphism (SSCP) and PCR-direct sequencing. Two SSCP patterns (A and B) corresponding to G/Cc.305+g.84 and C/Cc.305+g.84 SNP genotypes were found. Shrimp carrying the former genotype (OW=0.92±0.37g and GSI=1.06±0.39%, N=31) had a greater average ovarian weight and GSI value than those carrying the latter genotype (OW=0.75±0.30g and GSI=0.86±0.32%, N=33) (P<0.05). Taken the information together, PmCOMT should play the functional role during ovarian development of P. monodon. Statement of relevanceReduced reproductive performance of the giant tiger shrimp Penaeus monodon in captivity prevents the potential application of domestication and selective breeding program for genetic improvement of this economically important species. Understanding molecular mechanisms controlling the development and maturation of ovaries/oocytes is essential for finding an approach to promote the reproductive maturation of P. monodon without the use of eyestalk ablation. Two kinds of O-methyltransferase (OMT) transcripts; farnesoic acid O-methyltransferase (FAMeT) and catechol O-methyltransferase (COMT) have been characterized in penaeid species. P. monodon FAMeT was differentially expressed during ovarian development and its SNPs showed association with ovarian weight of domesticated adults. Similarly, the expression profile of P. monodon COMT was examined and eyestalk ablation affected its expression level during vitellogenic and mature stages. Relationships between a SNP of P. monodon COMT and reproduction-related parameters (ovarian weight and GSI) were found.

Identification of novel SNP in caprine β-lactoglobulin gene

β-lactoglobulin (β-LG) gene is suggested as a functional candidate gene for milk yield and milk composition. β-LG polymorphism has been reported to be associated with milk yield in cows, sheep and Indian goats. This study was performed to identify SNPs in exon 7 of β-LG gene and their association with milk traits in Iranian local Mahabadi goats using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) and PCR-sequencing. Three SSCP patterns were observed with frequencies 0.678, 0.096 and 0.226, respectively. Subsequently, after sequencing each unique pattern nine novel mutations were identified. These mutations include: T InDel at nucleotide position 93 and substitutions T/C, T/G, T/C, G/T, T/G,T/C, G/A and A/T at nucleotide positions 99, 124, 126, 134, 147, 156, 176 and 177, respectively. Of these, seven mutations were same among the genotypic patterns while differences were related to T deletion and insertion (-/T) at nucleotide position 93 with frequencies 0.22 and 0.78 in the presence and absence of T allele, respectively; and substitution (A/T) at nucleotide position 177 with frequencies 0.16 and 0.84 for A and T alleles, respectively. Milk traits including milk production (gr), milk fat and protein (%) were also measured. These findings demonstrated that β-LG gene had a significant effect on milk protein percentage (P < 0.05), but had no significant effect on milk production and milk fat percentage.

9. Ovine DRB1 Polymorphism and Its Associations with Body Weight, Milk Contents and Immunological Parameters

Abstract The DRB1 gene is known as a part of the Major Histocompatibility Complex (MHC) with important effects on disease resistance and immunological response in mammals. Single strand conformational polymorphism (SSCP) analysis and direct DNA sequencing methods were employed to study the second exon of DRB1 gene polymorphism in Mehraban and Lori-Bakhtiari sheep, using 155 Mehraban and 167 Lori-Bakhtiari (totally 322) ewes. Eleven different SSCP patterns with 26 SNPs (five new mutations) were found in both breeds. Associations of different genotypes with some traits including body weight, milk contents, milk somatic cell count and some immunological parameters were also studied. The observed genotypes had significant associations (P&lt;0.05) with immunological parameters including serum total protein, α2-globulin, β-globulin, γ-globulin and total globulins and body weight at 3 months of age (weaning). The effects of the genotypes on serum albumin and α1-globulin, body weights at 9 and 12 months of age and milk lactose tended to be significant (P&lt;0.10). The results of the present study indicated that the DRB1 exon 2 is highly polymorphic and could be considered as an important fragment in marker assisted selection, especially for improvement of immunity in sheep.

Identification of processed tuna products sold by the Brazilian market

The composition of processed food is a concern of consumers, of the fishery industry and governmental agencies. The specific composition of canned tuna in three Brazilian brands was evaluated using Single-Strand Conformational Polymorphism (SSCP) of the cytochrome b gene by comparison with patterns of fresh tuna samples and tuna-related species landed at Santa Catarina ports (South Brazil). Thirteen SSCP patterns were obtained from fresh samples, and the specific variety was confirmed by analyzing their sequences. Inconsistencies were found in the fishery statistics of Auxis thazard thazard and Scomber japonicus reported from Brazilian ports, where fresh samples instead were represented mostly by Auxis rochei rochei and Scomber colias. A case of introgression was detected between A. t. thazard and Katsuwonus pelamis. The skipjack tuna K. pelamis composed seven out of eight of the processed tuna products analyzed, and two brands showed products containing a mixture of tuna (K. pelamis or Thunnus albacares) and the less commercially valuable bonito meat (A. t. thazard), which reinforces the need for the control of canned tuna in the Brazilian market.

Sequence characterization and polymorphism detection in lactoferrin gene of Deoni (Bos indicus) cattle

Lactoferrin is an iron binding glycoprotein which plays an essential role in antimicrobial defense and a potential candidate gene in dairy cattle breeding. The present study was undertaken with the objective of sequence characterization and identification of polymorphisms within exons 2, 3 and 14 and their flanking intronic regions in lactoferrin gene of Deoni (Bos indicus) cattle by single strand conformation polymorphism (SSCP). Amplicons of exons 4, 5 and 16 showed monomorphic patterns. PCR-SSCP analysis revealed a total of eight different variants in three investigated regions of the lactoferrin gene. The locus LtfE2 revealed four different variants, viz. LtfE2-A, LtfE2-B, LtfE2-C and LtfE2-D with the frequency of 0.42, 0.26, 0.22 and 0.10, respectively. Analysis of exon 3 and 14 revealed two unique SSCP patterns with the frequencies of 0.54 and 0.46, 0.86 and 0.14 respectively. Comparison of nucleotide sequences of lactoferrin gene of the Deoni cattle with taurine cattle revealed a total of 12 point mutations, 11 of which were found to be in coding region with 10 transitions. Conceptualized translation of nucleotide sequence revealed 5 amino acid changes. The SNPs identified in the coding region of lactoferrin gene may serve as potential genetic marker (s) in cattle for disease resistance.

Screening of the CFTR gene in Indian patients.

Cystic fibrosis (CF) has been observed to be far more common in India, than was previously thought. Variability in CF clinical symptoms among individuals, results in diagnostic errors. Also, CF diagnostic facilities are not available at all diagnostic centers across India. Sweat test (gold standard for CF diagnosis) has some limitations. Mutation analysis, therefore, would be useful in detecting the mutant CF alleles in Indian patients. This study, aimed at identifying common CF transmembrane conductance regulator (CFTR) mutations, to develop a molecular diagnostic test in Indian patients, and establish genotype-phenotype correlation. Mutation identification was performed by single stranded conformation polymorphism (SSCP) screening, followed by DNA sequencing of regions with an abnormal SSCP pattern. ∆F508 accounts for about 53% of CF alleles. A substantial proportion of these patients have rare and/or novel mutations. Eight novel and 12 known polymorphisms were also identified. Considering the high percentage of rare/novel mutations, along with ethnic history of Indian population, we can speculate that the remaining uncharacterized mutations might also not be prevalent mutations. The total number of CF disease-causing mutations in Indian patients is very large. Thus, DNA-based population screening will be complicated, and an indirect genetic diagnosis (screening entire gene) would be necessary to characterize all mutations.

Differential expression of X-box binding protein 1 during ovarian development and association between its SNP and growth-related parameters of the giant tiger shrimp Penaeus monodon

Abstract The full-length cDNA of X-box binding protein 1 ( PmXbp1 ) in the giant tiger shrimp, Penaeus monodon was identified. It was 1762 bp in length containing an ORF of 855 bp corresponding to 284 amino acids. The expression level of PmXbp1 in different ovarian stages of wild intact broodstock was comparable but greater than that of cultured juveniles ( P PmXbp1 in stages III (late vitellogenic) and IV (mature) ovaries of wild P. monodon broodstock ( P PmXbp1 in ovaries of domesticated 18-month-old broodstock at 24 hpi ( P PmXbp1 (185 bp) were examined in domesticated 3-month-old juveniles (average body weight and total length = 12.32 ± 5.13 g and 11.30 ± 1.58 cm, N = 162) by single-strand conformational polymorphism (SSCP) analysis. Association between SSCP patterns of PmXbp1 and growth-related parameters was found where the average body weight (15.07 ± 5.04, N = 44) of juveniles carrying pattern A (corresponding to a T/T 349 SNP) was significantly greater than that (11.37 ± 4.80, N = 116) of juveniles carrying pattern B (corresponding to a T/C 349 SNP). The relative expression levels of PmXbp1 in the hepatopancreas of juveniles carrying different SSCP/SNP genotypes were significantly different (A P PmXbp1 in shrimp exhibiting a greater growth performance (22.08 ± 2.97 g and 13.60 ± 0.56 cm; N = 11) was significantly lower than that with a lesser growth performance (5.00 ± 0.89 g and 8.60 ± 0.98 cm; N = 8) ( P PmXbp1 seems to play an important role in reproduction and growth of P. monodon . Statement of relevance In aquaculture, the primary goal of genetic selection is the growth improvement of economically important species. Molecular markers that allow for selection of juveniles and broodstock with a high breeding value for reproductive maturation and/or growth rates are useful for the shrimp industry. Information on correlation between genotypic and phenotypic variations in penaeid shrimp is rather limited. Identification of molecular markers linked with growth/reproduction or those regulating the growth/reproduction mechanisms can be applied to improve artificial selection processes of P. monodon . To assess the possible biological roles of X-box binding protein 1 in reproduction and growth of P. monodon , the PmXbp1 cDNA sequence was characterized. Expression patterns of PmXbp1 during ovarian development in wild P. monodon broodstock were examined. Effects of eyestalk ablation (wild broodstock) and serotonin induction (domesticated broodstock) on expression levels of ovarian PmXbp1 were examined. In addition, analysis of relationships between SNP detected by SSCP and DNA sequencing and expression levels of PmXbp1 , and growth traits in juvenile shrimp was examined.

Genetic diversity of Grapevine leafroll-associated virus 3 in Algeria.

Several vineyards and a Vitis germplasm collection were surveyed in Algeria for the presence Grapevine leafroll-associated virus 3 (GLRaV-3) by DAS-ELISA. The virus was found in almost half (44%) of the samples tested (212 out of 484). The genetic diversity of selected GLRaV-3 isolates was determined by characterizing fragments of the heat shock 70 protein homologue (HSP70h) and coat protein (CP) genes by single-strand conformation polymorphism (SSCP) and sequencing. Algerian GLRaV-3 isolates showed three SSCP patterns and belonged to previously described phylogenetic groups I, II and III. Analysis of phylogenetic trees obtained with partial HSP70h gene sequences suggested the existence of two new subgroups and confirmed a few stand-alone variants. To the best of our knowledge, this is the first characterization of GLRaV-3 isolates from Algeria.