Dot Blot Hybridization Research Articles

Characterization of m6A in mouse ovary and testis

Characterization of m6A in mouse ovary and testis

Genotype Analysis of Pregnant Women with α- and β- Thalassemia in Fuzhou Area of Fujian Province in China

To analyze the genotype of pregnant women with α- and β- thalassemia in Fuzhou area of Fujian province in China. Blood routine examination and hemoglobin electrophoresis were performed for pregnant women, and positive samples were examined by gap polymerase chain reaction and reverse dot blot hybridization. 412 cases were diagnosed as α-thalassemia (63.9%); 201 cases were diagnosed as β-thalassemia (31.2%); 32 cases were diagnosed as α and β-composite thalassemia. There were 12 genotypes in α-thalassemia, whose major genotypes were --SEA/αα, α3.7/αα, -α4.2/αα and αQSα/αα, with carrying rate of 64.32%, 20.14%, 7.77% and 1.94%, respectively. There were 10 genotypes in β- thalassemia, whose major genotypes were CD41-42/N, CD17/N, IVS-II-654/N and -28/N, with carrying rate of 30.84%, 27.86%, 15.92% and 10.45%, respectively. There were 9 genotypes in α and β-composite thalassemia, whose major genotypes were --SEA/αα composited CD41-42/N, -α3.7/αα composited CD41-42/N, --SEA/αα composited CD17/N, with carrying rate of 18.75%, 15.62%, 15.62% respectively. The major genotypes of pregnant women with α- and β- thalassemia in Fuzhou area of Fujian province in China are --SEA/αα, α3.7/αα, CD41-42/N and CD17/N. Thalassemia screening and prenatal gene diagnosis should be strengthened in Fuzhou area of Fujian province in China.

Development of DNA controls for detection of β-thalassemia mutations commonly found in Asian.

Several DNA-based approaches including a reverse dot-blot hybridization (RDB) have been established for detection of β-thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe β-thalassemia. However, one of major concerns of these techniques is a risk of misdiagnosis due to a lack of DNA controls. Here, we constructed positive DNA controls for β-thalassemia genotyping in order to ensure that all steps in the analysis are performed properly. Four recombinant β-globin plasmids, including a normal sequence and three different mutant panels covering 10 common β-thalassemia mutations in Asia, were constructed by a conventional cloning method followed by sequential rounds of site-directed mutagenesis. These positive DNA controls were further validated by RDB analysis. We demonstrated the applicability of established positive DNA controls for β-thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant β-globin plasmids into a single positive control, which showed positive signals for both normal and mutant probes of all tested mutations. Therefore, only two positive DNA controls, normal and combined mutant β-globin plasmids, are required for detecting 10 common β-thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis. The β-globin DNA controls established here provide efficient alternatives to a conventional DNA source from peripheral blood, which is more difficult to obtain. They also provide a platform for future development of β-globin plasmid controls with other mutations, which can also be suitable for other DNA-based approaches.

Antiviral Action of Native and Methylated Lactoferrin and β-Lactoglobulin against Potato Virus Y (PVY) Infected into Potato Plants Grown in an Open Field.

Potato plants are liable to PVY infection without efficient control. Therefore, they were cultivated under greenhouse and open field conditions, artificially infected with PVY and then treated after 15 days of infection with native lactoferrin (LF) and native β-lactoglobulin (BL) and their esterified forms, MLF (methylated lactoferrin) and BLM (methylated β-lactoglobulin) to test the efficiency of this approach. Viral replication was inhibited by the applied substances, particularly the methylated forms, in a concentration-dependent manner, where the concentration of 500 μg·mL−1 was sufficient for plant protection against the PVY infection. An open field experiment showed that one single application of the antiviral substance was enough for maximum inhibitory action against PVY. The modified milk proteins induced higher inhibitory action on PVY virus replication in the plants, compared to their native forms, which was reflected by potato growth and yield. Using the dot blot hybridization and RT-PCR techniques to detect PVY in the experimental plants showed the supremacy of native and esterified LF in inhibiting the targeted virus. The generally observed scanning electronic microscopy (SEM) structural deformations and irregular appearance in PVY particles when treated with MLF and BLM revealed their direct action. BLM, MLF and LF are efficient antiviral agents against PVY. They can not only abolish the observed PVY-induced reduction in potato growth and tuber yield, but also further increase them to higher levels than negative control.

Sequence Evolution, Abundance, and Chromosomal Distribution of Ty1-copia Retrotransposons in the Saccharum spontaneum Genome

Saccharum spontaneum is a wild germplasm resource of the genus Saccharum that has many valuable traits. Ty1-copia retrotransposons constitute a large proportion of plant genomes and affect genome sequence organization and evolution. This study aims to analyze the sequence heterogeneity, phylogenetic diversity, copy number, and chromosomal dispersion patterns of Ty1-copia retrotransposons in S. spontaneum. A total of 44 Ty1-copia reverse transcriptase subclones isolated from S. spontaneum showed a range of heterogeneity, and all sequences were A-T rich, averaging approximately 54.59%. Phylogenetic analysis divided the 44 reverse transcriptase sequences into 5 distinct lineages (Retrofit/Ale, Sire/Maximus, Bianca, Tork/TAR, and Ty1-copia like). Dot-blot hybridization revealed that Ty1-copia retrotransposons consisted of a significant component of approximately 38,900 copies and 16,300 copies per genome in the accessions YN82-114 (2n = 10x = 80) and AP85-441 (2n = 4x = 32), respectively. The results of a local blast analysis showed that there are 15,069 Ty1-copia retrotransposon copies in the genome of AP85-441, of which the Retrofit/Ale lineage had the highest copy number, followed by the Tork/TAR, Sire/Maximus, and Bianca lineages. Furthermore, both FISH and the local blast analysis with AP85-441 genomic data demonstrated that the Ty1-copia retrotransposons were unevenly distributed throughout the chromosomes. Taken together, this study provides insights into the role of Ty1-copia retrotransposons in the evolution and organization of the S. spontaneum genome.

Rare β-Globin Gene Mutations including a de novo Mutation of Hemoglobin Hammersmith in Southern Thailand

Objective: The aim of this study was to characterize unknown β-globin gene mutations in individuals who attended Songklanagarind Hospital for thalassemia screening and genetic counseling.Material and Methods: β-thalassemia mutations in individuals with hemoglobin (Hb) A2 levels >3.5% originating from various provinces in southern Thailand were characterized by reverse dot blot hybridization (RDB) and multiplex gappolymerase chain reaction using a panel of 30 allele-specific probes for point mutations and 6 sets of specific primers for large deletions. Mutations which could not be identified by these two methods were further analyzed by direct deoxyribonucleic acid (DNA) sequencing.Results: Nineteen subjects found to have uncharacterized β-globin gene mutations were analyzed by direct DNA sequencing. Nine different rare mutations were identified, four of which have not been to date described in Thailand: -30 (T>C), codon 5 (-CT), Hb Monroe (codon 30, G>C) and Hb Hammersmith (codon 42, T>C). An Hb Hammersmith mutation detected in one subject appeared to be a spontaneous mutation, unrelated to family history. The other five mutations have been reported previously within Thailand, but here they were identified in the southern part of Thailand for the first time: -31 (A>G), codon 15 (-T), codon 35 (C>A), codon 95 (+A) and Hb Dhonburi (codon 126, T>G). The presence of the mutations was confirmed by RDB.Conclusion: In addition to the already reported β-globin gene mutations, 9 other different types of mutations were identified. This information should be useful for planning genetic counseling and prenatal diagnosis programs for prevention and control of thalassemia diseases.

Cloning and expression of duck C4BPα and verification of its interaction with Riemerella anatipestifer

To study the interaction between C4b-binding protein (C4BP) and Riemerella anatipestifer (RA), we cloned duck C4BPα, conducted prokaryotic expression and prepared the polyclonal antibody by immunizing mice. Then indirect immunofluorescence assay and dot blotting hybridization assay were used to verify the interaction between C4BP and RA. The full length of duck C4BPα nucleotide sequence was 1 230 bp, with the highest similarity to chicken C4BPα (82.1%). Phylogenetic tree analysis showed that duck C4BPα and chicken C4BPα were on the same phylogenetic tree branch and the genetic evolution relationship between them was the closest. C4BPα was efficiently expressed in Escherichia coli BL21 (DE3). The recombinant proteins existed in intracellular soluble form. The titer of polyclonal antibody was more than 1:10 000 and polyclonal antibodies could specifically recognize the recombinant proteins. The results of indirect immunofluorescence assay and dot blot hybridization assay showed that RA could interact with duck C4BP. The results provide a basis to further reveal the pathogenesis of RA.

Accuracy of a reverse dot blot hybridization assay for simultaneous detection of the resistance of four anti-tuberculosis drugs in Mycobacterium tuberculosis isolated from China

BackgroundDrug resistant tuberculosis poses a great challenge for tuberculosis control worldwide. Timely determination of drug resistance and effective individual treatment are essential for blocking the transmission of drug resistant Mycobacterium tuberculosis. We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization (RDBH) assay to simultaneously detect the resistance of four anti-tuberculosis drugs in M. tuberculosis isolated in China.MethodsIn this study, we applied a RDBH assay to simultaneously detect the resistance of rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) in 320 clinical M. tuberculosis isolates and compared the results to that from phenotypic drug susceptibility testing (DST) and sequencing. The RDBH assay was designed to test up to 42 samples at a time. Pearson’s chi-square test was used to compute the statistical measures of the RDBH assay using the phenotypic DST or sequencing as the gold standard method, and Kappa identity test was used to determine the consistency between the RDBH assay and the phenotypic DST or sequencing.ResultsThe results showed that the concordances between phenotypic DST and RDBH assay were 95% for RIF, 92.8% for INH, 84.7% for SM, 77.2% for EMB and the concordances between sequencing and RDBH assay were 97.8% for RIF, 98.8% for INH, 99.1% for SM, 93.4% for EMB. Compared to the phenotypic DST results, the sensitivity and specificity of the RDBH assay for resistance detection were 92.4 and 98.5% for RIF, 90.3 and 97.3% for INH, 77.4 and 91.5% for SM, 61.4 and 85.7% for EMB, respectively; compared to sequencing, the sensitivity and specificity of the RDBH assay were 97.7 and 97.9% for RIF, 97.9 and 100.0% for INH, 97.8 and 100.0% for SM, 82.6 and 99.1% for EMB, respectively. The turnaround time of the RDBH assay was 7 h for testing 42 samples.ConclusionsOur data suggested that the RDBH assay could serve as a rapid and efficient method for testing the resistance of M. tuberculosis against RIF, INH, SM and EMB, enabling early administration of appropriate treatment regimens to the affected drug resistant tuberculosis patients.

Occurrence and molecular characterization of apple scar skin viroid in Malus pumila ‘Saiwaihong’ (small apple tree) from North China

Viroids are naked non-coding RNAs that can infect plants and cause severe disease. Apple scar skin viroid (ASSVd) is the causal agent of apple scar skin disease, which can infect apple trees and cause dappling and scar skin symptoms. In this study, we investigated the incidence of apple scar skin disease in apple-grafted small apple tree (Malus pumila ‘Saiwaihong’) in North China. Of 1322 tested trees, 90 exhibited obvious dapple symptoms in fruit skin (disease incidence of 6.8%). Dot-blot hybridization analysis proved that the peel from 25 diseased small apple tree were all positive for ASSVd, confirming that disease was associated with ASSVd infection. RT-PCR, cloning and sequencing were performed, and 12 complete sequences of ASSVd variants from Malus pumila ‘Saiwaihong’ were obtained. Sequence comparison showed that the 12 sequence variants shared 90.6–99.7% sequence similarity, phylogenetic analysis, performed based on the 12 ASSVd Malus pumila ‘Saiwaihong’ variants and other ASSVd variants reported from different hosts, did not show any clustering according to the respective host species. This is the first report of ASSVd infection in small apple tree (Malus pumila ‘Saiwaihong’).

Genetic epidemiology of beta-thalassemia in the Maldives: 23years of a beta-thalassemia screening program.

The Maldives is an archipelago of 407,660 people according to population census of 2014, made up of 20 atolls, which has one of the highest prevalence of β-thalassemia worldwide. However, there is a dearth of studies related to β-thalassemia in the Maldives; therefore, in this study, we aimed to investigate the genetic epidemiology of β-thalassemia in Maldives. Blood samples were collected from 110,504 participants (1992-2015). Hemoglobin and RBC indices were measured on automated hematology analyzers. The quantitation of hemoglobin, HbA2, Hb F, and other abnormal Hb variants were assessed by HPLC. Molecular analysis was performed for the most common mutations in Southeast Asia for only 874 individuals either heterozygous or homozygous for these mutations using reverse dot blot hybridization. We screened 110,504 individuals for β-thalassemia between 1992 and 2015, which is~30% of the entire population. The β-thalassemia carrier frequency was estimated to be 16.2%. Molecular diagnosis of 874 β-thalassemia carriers/major was performed for the most common seven mutations in Southeast Asia; of these, 139 patients were diagnosed as β-thalassemia major. This analysis showed that the most common mutations were IVS1+5G>C, (678; 77.6%), followed by the CD 30 (136; 15.6%). The least frequent mutation was FS8/9, (1, 0.001%), followed by IVS1+1G>T and CD15 (2; 0.2%). The frequency of β-thalassemia varies significantly among the 20 different atolls in Maldives. This study is expected to improve genetic counseling, creating awareness, enhance premarital screening, and customize the prevention and treatment strategies based on the needs of each atoll.

Application of PCR reverse dot blot in non-syndromic deafness gene detection

Objective:To detect 20 common deafness gene mutations in non- syndromic deafness patients in China using PCR- RDB, and analyze and summarize the mutation data to explore the clinical value of this method. Method:The PCR- RDB and Sanger sequencing were used to detect 20 common mutations of four deafness genes（GJB2, GJB3, SLC26A4 and mtDNA） in 500 patients with non- syndromic hearing loss . The Sanger sequencing was used to compare the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the deafness mutation detected by PCR- RDB. Result:A total of 500 samples were detected. 147 wild- type samples, 81 homozygous mutant samples, 240 heterozygous mutant samples, 32 composite heterozygous mutant samples were detected using the PCR- RDB within the range of 20 gene mutations, which were identical to the Sanger sequencing results. GJB2 c.235delC and SLC26A4 c.919- 2 A>G are the most common hotspot mutations in this study, followed by mtDNA m. 1555 A>G. Compared with the Sanger sequencing method, the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the real- time fluorescence PCR melting curve method were 100%, and the Kappa value was one. Conclusion:PCR reverse dot-blot hybridization is a simple, rapid, sensitive and specific method for detecting 20 mutations of 4 common deafness genes in Chinese population, it is expected to be used in clinical detection of deafness genes in the future.

A Dual-Amplification Electrochemical Aptasensor for Profenofos Detection

The present study reported a dual-amplification electrochemical aptasensor for sensitive detection of profenofos (PFF) in vegetables. A screen-printed carbon electrode (SPCE) modified with graphitized multi-walled carbon nanotubes (MWCNTGr) and Au nanoshell was used as a test platform, which ensured a rapid detection process and showed a favorable electrochemical performance. MWCNTGr and Au nanoshell enhanced the electrical conductivity and the surface area, thus the detection signal was amplified. The affinity between PFF and its aptamer (Apt) was verified firstly by dot blot hybridization (DBH), and the result was exciting. Furthermore, the effects of the aptamers modified respectively with -NH2 and -SH on the current signal were compared with each other by cyclic voltammetry (CV), and results showed that the aptamers modified with -NH2 made the current signal change more obvious. Based on all above, a high-efficiency electrochemical aptasensor was fabricated with a wide linear range from 0.1–1 × 105 ng · ml−1 and a detection limit of 0.052 ng · ml−1 under the optimized conditions. This aptasensor had great specificity, stability and reproducibility. Hence, the developed aptasensor was successfully used to detect PFF in vegetables. The proposed method also has a potential for the detection of other organophosphorus pesticide (OPs).

Human papillomavirus genotype distribution in genital warts among women in Harare-Zimbabwe

This study aimed to determine the prevalence of HPV genotypes in genital warts among women in Harare, Zimbabwe. Women aged 18–45 years attending gynaecology and genitourinary clinics with a clinical diagnosis of genital-warts were recruited. HPV-DNA was extracted from tissue biopsies. HPV-DNA testing and typing was done by Southern Dot Blot Hybridisation. A hundred samples from 100 women were analysed. Median age of participants was 30.3 years (range 18–45 years). Seventy-eight percent of participants were HIV infected. HPV prevalence was 98%. Low risk genotypes predominated at 86% prevalence. The most prevalent genotypes were 11 (47%), 6 (42%) and 16 (14%). This is the first study on HPV genotype distribution among women with genital warts in Zimbabwe. The high prevalence of HR-HPV 16 in clinically benign lesions shows that warts should have histological analysis to exclude pre-malignancy and malignancy.Impact statementWhat is already known on this subject? Genital warts (GWs), also known as condylomata acuminata (EAC), are a clinical manifestation of persistent infection with ‘low risk’ or non-oncogenic HPV genotypes. HPV 6 and 11 are examples of low risk genotypes, and both are associated with 90% of GWs. Data on HPV genotypes causing genital warts in the population under study are scarce.What do the results of this study add? A high prevalence (98%) of HPV DNA in genital warts, confirms that the biopsied lesions were HPV related. Over and above the high prevalence of low risk HPV 11 (47%) and HPV 6 (42%), the women had 14% prevalence of HPV 16, an oncogenic genotype, in genital warts. Seventy-eight percent of the participants were HIV infected. The HIV infected women had a 33.3% prevalence of HR-HPV as compared to the 15.8% prevalence in the HIV uninfected women.What are the implications of these findings for clinical practice and/or further research? The population under study will benefit more if an HPV vaccine that includes anti-HPV 6 and 11 is used. The high prevalence of the HR-HPV in apparently benign lesions shows that warts should have histological analysis to exclude vulvar cancer and vulvar intraepithelial neoplasia. All women presenting with genital warts should be offered an HIV test.

Molecular analysis of α-thalassemia and β-thalassemia in Quanzhou region Southeast China

AimsThalassemia is one of the most prevalent inherited disorders in south China. However, there still has no comprehensive research on molecular characterisation of α-thalassemia and β-thalassemia in the Quanzhou region...

First Report of Tomato Chlorosis Virus Infecting Tectona grandis Associated With Infestation of Bemisia tabaci Mediterranean in Central Brazil

The commercial production of teak (Tectona grandis L. f., family Lamiaceae) has been increasing in Brazil due to the adaptation of this timber species to the predominant environmental conditions. Symptoms of interveinal yellowing in older leaves similar to that induced by species of the genus Crinivirus (family Closteroviridae) were observed (≈90% incidence) in 100-day-old teak seedlings in one orchard in Brasilia–DF in 2018. Moreover, a severe whitefly infestation was observed in association with these symptomatic plants. Fifteen leaf samples collected at random from distinct symptomatic plants were tested for the presence of tomato chlorosis virus (ToCV) and tomato infectious chlorosis virus (TICV), two criniviruses reported infecting a wide range of crops in Brazil (Fonseca et al. 2016). Leaf samples from asymptomatic teak plants (n = 10) were also collected and employed as negative controls. Total RNA extraction was performed using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) from all samples of symptomatic and asymptomatic plants. RT-PCR assays were conducted using the universal primer pair HS-11/HS-12 (which amplifies a 587-bp RNA-2 segment encompassing the HSP70h protein gene), followed by a nested-PCR performed with the primer pair ToCV- (ToC-5/ToC-6) and TICV-specific primers (TIC-3/TIC-4) (Dovas et al. 2002). All 15 symptomatic teak samples were positive only for ToCV (≈463-bp amplicons). PCR product from one randomly selected sample was directly sequenced. The obtained sequence (GenBank MH688047) displayed 99.7% identity with the equivalent sequence of a tomato-infecting ToCV isolate reported in Brazil (EU868927). Five symptomatic samples were also positive in dot-blot hybridization assays using a coat protein-derived RNA probe (436-nt fragment of RNA-2; primers MA-380/MA-381) (Fortes et al. 2012) labeled with digoxigenin (DIG)-11-UTP and with the chemiluminescent substrate CDP Star kit (Roche Diagnostics). Total DNA extraction and PCR assays were also conducted using pooled (three pools of n = 10 each) whitefly samples collected on symptomatic teak plants. A segment of the mitochondrial cytochrome oxidase I gene (mtCOI) was amplified using the specific primers C1-J-2195-FW and L2-N-3014- RV (Simon et al. 1994), yielding ≈860 bp amplicons. Direct sequencing of three amplicons obtained from the whitefly samples (MH688048) indicated high identity (99%) to B. tabaci Mediterranean (MED) species (= Q biotype). ToCV-transmission assays were performed using B. tabaci MED adults. Thirty viruliferous whiteflies derived from healthy whiteflies fed on ToCV-infected teak plants were confined in separated cages with healthy teak (n = 6) and tomato cv. Santa Clara (n = 6) seedlings. Aviruliferous whiteflies were also confined to healthy teak (n = 6) and tomato (n = 6) plants (negative controls). After 72 h, the whiteflies were eliminated by insecticide spraying. Thirty days after the feeding access period, only teak and tomato plants exposed to viruliferous B. tabaci MED exhibited crinivirus-like symptoms and were positive to ToCV in RT-PCR assays. To our knowledge, this is the first report of T. grandis as a host of ToCV as well as the first report of virus infection in this woody plant in Brazil. The introduction of B. tabaci MED into southern Brazil was recently reported (de Moraes et al. 2017). Our results also confirm the presence of B. tabaci MED in central Brazil and its association with ToCV infection of T. grandis seedlings. Although expressing overall mild symptoms, the potential negative effects of ToCV in teak plants need to be investigated in more detail.

Changes in methylation status of the whole genome and GCH1 gene in patients with herpes zoster neuralgia

Objective To analyze the correlation between herpes zoster neuralgia and the methylation status of the whole genome and GCH1 gene. Methods From June to October in 2017, patients with confirmed herpes zoster and obvious neuralgia were selected in Department of Dermatology, The Affiliated Hospital of Xuzhou Medical University, who achieved complete remission (no effect was observed on normal sleep) of neuralgia after antiviral and neurotrophic treatment. Finally, 36 patients and 36 healthy controls were enrolled into this study. Peripheral blood samples were obtained from the healthy controls and patients before and after the treatment. Dot-blot hybridization assay was performed to determine the methylation status of the whole genome, methylated-DNA IP kit was used to enrich the methylation sites of the GCH1 gene, and real-time quantitative PCR was conducted to detect changes in methylation status of the GCH1 gene. Statistical analysis was carried out with GraphPad Prism v7.00 software by using paired t test for the comparison of methylation status before and after the treatment, and two-sample t test for the comparison between the patient group and control group. Results The relative methylation level of the whole genome was 135.94 ± 2.52 in the patients before treatment, significantly lower than that in the patients after treatment (144.76 ± 3.48, t=2.056, P 0.05) . Compared with the patients after treatment (0.89 ± 0.13) and healthy control group (0.97 ± 0.07) , the methylation status of the GCH1 gene significantly decreased in the patients before treatment (0.65 ± 0.17; t=3.977, 4.648 respectively, P 0.05) . Conclusion The methylation status of the whole genome and GCH1 gene markedly decreased in the patients with herpes zoster neuralgia. Key words: Neuralgia, postherpetic; Methylation; Epigenomics; GCH1 gene

Isolation and characterisation of Ty1-copia retrotransposons from Pongamia pinnata

This manuscript mainly deals about the diversity and transcriptional activity of Ty1-copia in Pongamia genome. Pongamia pinnata, a leguminous tree, is popularly known for its biodiesel and medicinal application. However, the structure and the composition of Pongamia nuclear genome are largely unknown. Ty1-copia elements occupy a significant fraction of the plant genome and are responsible for sequence organisation. Here, we have isolated the reverse transcriptase (RT) and RNase H domain of Ty1-copia from Pongamia. In total, 28 Ty1-copia RT sequences were isolated with high levels of sequence heterogeneity. Besides this, the Ty1-copia like elements were also identified in the mitochondrial genome and in the transcriptome library. The analysis revealed that transcriptional activity of Ty1-copia element exists in leaf, root, and seed of salt-stressed and control plants. Dot blot hybridisation reveals that Ty1-copia like retrotransposons occupy around 8.5% of the haploid genome of Pongamia and the phylogenetic study categorised this class of retrotransposons into seven different lineages. These results are useful in understanding the diversity of Ty1-copia lineages and their transcriptional activity present in different tissues of Pongamia.

Genotyping of Patients with α and β Thalassemia in Fujian Province Area in China

To investigate the gene-carrying rate and gene mutation types of α- and β-thalassemia in population of Fujian area and to analyze the differences in hemoglobin A2 (HbA2), mean cell volume (MCV) and mean cell hemoglobin (MCH) between different types of thalassemia, so as to provide the reference basis for screening and classification, genetic diagnosis and counseling about thalassemia. Total 1474 samples from different areas of Fujian province were detected for α- and β-thalassemsia genotypes by gap single PCR (GS-PCR) combined with reverse dot blot hybridization (RDB). The detection of peripheral RBC, hemoglobin and primary screening of thalassemia in each set of sample were carried out before test. Among the detected 1474 samples, 704 (47.76%) were diagnosed as α-thalassemia, out of them 416 (28.22%) were diagnosed as α-thalassemia, 267(18.11%) as β-thalassemia, 21 (1.43%) as αβ-thalassemia. The α-thalassemia further was divide into 3 types: silent (5.09%), minor (22.18%) and HbH disease (0.95%), and their MCV, MCH and HbA2 levels were detected. The detection results showed obvious decrease trend with significant difference (P＜0.05). The gene mutation types of thalassemia display obvious heterogenity in Fujian area. The gene type in α-thalassemia mostly is --SEA/αα, the gene type in β-thalassemia mostly is IVS-Ⅱ-654. Moreover, the sings of anemia in Hb H disease of α-thalassemia are mostly serious, which obviously are different from other types of α-thalassemia.

PB2405 PHENOTYPES AND GENOTYPES OF HEMOGLOBINOPATHIES IN AZERBAIJAN

Background:Azerbaijan is located in Transcaucasia, which is a high prevalent region of hemoglobinopathies. Besides β‐thalassemia, the local population is also known for the presence of α‐thalassemia and some hemoglobin variants. Several previous studies reported the incidences of α‐thalassemia and hemoglobin variants such as HbS, HbE and HbD, as well as incidence rate and mutation spectrum of β‐thalassemia in the local population. Nevertheless, these were mainly conducted in small study groups and they were limited on β‐globin genotyping, which necessitated a large‐scale screening and genotyping to reveal phenotypes and mutational background of all common hemoglobinopathies in Azerbaijan.Aims:The aim of our study was to screen a large sample of population in order to reveal the prevalence of thalassemias and variant hemoglobins as well as their mutational background in Azerbaijan.Methods:The sample group included 436,144 individuals (population = 9,982,952) applying for marriage certificate, which were screened via Thalassemia Prevention Program of Azerbaijan. Premarital screening of thalassemia is mandatory since 2015, covering almost all areas of the country. Total hemoglobin (Hb) levels, red blood cell indices and Hb pattern analysis were evaluated in all samples. Genotyping of selected samples were performed via reverse dot blot hybridization and sequencing techniques.Results:The screening of more than 4% of the population revealed following carrier frequencies of hemoglobinopathies: α‐thalassemia – 10.1%, β‐thalassemia – 3.71%, HbS – 0.082%, HbC – 0.382%, HbE – 0.087% and HbD Punjab – 0.068%. The frequency of α‐thalassemia was calculated via Hardy–Weinberg equation, based on the count of individuals with HbH. Genotyping of the carriers revealed 33 β‐thalassemia and 9 α‐thalassemia mutations. The most common β‐thalassemia mutations were frameshifts and mRNA processing mutations accounting for β0 phenotype: Codon 8 [‐AA] – 34.7%, IVS‐II‐1 [G > A] – 16.7%, IVS‐I‐110 [G > A] – 9.7%, Codon 8/9 [+G] – 4.8%, and Codon 36/37 [‐T] – 4.3% (N = 1252). The most prevalent α‐thalassemia mutations, on the other hand, were deletions accounting for milder phenotype of HbH disease: 20.5 kb DEL – 43.8% and 3.7 kb DEL – 35.4% (N = 144).Summary/Conclusion:This is the largest study to date evaluating phenotypic and genotypic diversity of hemoglobinopathies in Azerbaijan, and we hereby provided the first report on carrier frequencies of hemoglobin variants in the country. The screening and genotyping results revealed that hemoglobinopathies are highly heterogenous in Azerbaijan. β‐Thalassemia, although accounts for a lower frequency comparing to α‐thalassemia, is associated with severe phenotypes, including the possibility of coinheritance with clinically significant variants as HbS and HbE.

RNAi-Based Biocontrol of Wheat Nematodes Using Natural Poly-Component Biostimulants.

With the growing global demands on sustainable food production, one of the biggest challenges to agriculture is associated with crop losses due to parasitic nematodes. While chemical pesticides have been quite successful in crop protection and mitigation of damage from parasites, their potential harm to humans and environment, as well as the emergence of nematode resistance, have necessitated the development of viable alternatives to chemical pesticides. One of the most promising and targeted approaches to biocontrol of parasitic nematodes in crops is that of RNA interference (RNAi). In this study we explore the possibility of using biostimulants obtained from metabolites of soil streptomycetes to protect wheat (Triticum aestivum L.) against the cereal cyst nematode Heterodera avenae by means of inducing RNAi in wheat plants. Theoretical models of uptake of organic compounds by plants, and within-plant RNAi dynamics, have provided us with useful insights regarding the choice of routes for delivery of RNAi-inducing biostimulants into plants. We then conducted in planta experiments with several streptomycete-derived biostimulants, which have demonstrated the efficiency of these biostimulants at improving plant growth and development, as well as in providing resistance against the cereal cyst nematode. Using dot blot hybridization we demonstrate that biostimulants trigger a significant increase of the production in plant cells of si/miRNA complementary with plant and nematode mRNA. Wheat germ cell-free experiments show that these si/miRNAs are indeed very effective at silencing the translation of nematode mRNA having complementary sequences, thus reducing the level of nematode infestation and improving plant resistance to nematodes. Thus, we conclude that natural biostimulants produced from metabolites of soil streptomycetes provide an effective tool for biocontrol of wheat nematode.