Genomic DNA Extraction Research Articles

A DNA Extraction Method for Nondestructive Testing and Evaluation of Cotton Seeds (Gossypium L.).

Kernels of cotton provide lint and linter for textiles, oil and protein for food and feed. Cotton seed is formed following fertilization between an ovule and a pollen grain. The seed coat is maternal in origin, whereas the embryo and attached cotyledonary leaves are hybrids of parental lines. The extraction of genomic DNA from an ungerminated whole, a portion or mixed seeds are prerequisite in genetic and genomic studies of cotton. As far as our knowledge, there is only one method of nondescriptive DNA extraction from ungerminated cotton seeds without affecting the seed germination capability, but it has technical difficulties and requires special equipment. Furthermore, the amount of DNA extracted using the published method is low and, therefore, it is only suitable for routine marker assisted selection studies. In this study, a DNA extraction protocol referred to as the CTAB-LiCl was developed for single whole cotton seed, a portion of cotton seed and bulked cotton seeds. This protocol uses a combination of CTAB and LiCl to lyse cells and deplete RNAs simultaneously. The CTAB-LiCl DNA extraction method was evaluated in ninety-six individuals of six different cotton cultivars along with two genetic standards of cotton, TM-1 (G. hirsutum L.), Pima 3-79 (G. barbadense L.), and several other plant species of different plant genera. Results revealed that this method produced high quality and amounts of DNA as confirmed by spectrophotometry, agarose gel, restriction enzyme digestion, polymerase chain reaction, and library production for next generation sequencing studies of whole genome bisulfite sequencing. It does not require the use of liquid nitrogen, RNase, proteinase K, or beta-mercaptoethanol and can be completed in approximately 2h. Small tissues of the chalaza ends of ungerminated cotton seeds could be used to obtain high quality and quantity of DNA ranging from 14 to 28µg without affecting the seeds' germination ability, allowing marker-assisted selection before planting and flowering.

First Report of Rhizopus stolonifer Causing Premature Soft Rot of Jackfruit in Bangladesh.

The jackfruit tree (Artocarpus heterophyllus) is native to South and South-east Asia including Bangladesh. It is a commercially important tropical tree species that produces fruit, food, fodder, and high-quality wood (Gupta et al. 2022). During surveys in February 2022, soft rot on immature fruit at approximately 70% incidence was observed in several plantations and homesteads in the Sylhet district of Bangladesh. Infected fruit had black patches surrounded by wide bands of white, powdery masses. The patches enlarged with fruit maturation, and in some cases, covered the entire fruit. Symptomatic fruit were collected, surface sterilized with 70% ethanol for 1 min, and washed 3 times with sterile distilled water. Fen air-dried, and small pieces from the margins of lesions were transferred to potato dextrose agar (PDA). The plates were incubated at 25°C in the dark. Two-day-old colonies had diffuse, gray cottony mycelia that were hyaline and aseptate under the microscope. Sporangiophores measuring 0.6-2.5mm in length and 18 to 23μm in diameter had rhizoids and stolons at their bases. Sporangia were almost spherical and were 125μm (±65μm, n=50) in diameter. Sporangiospores were ellipsoid to ovoid and measured 3.5 to 9.32μm × 2.82 to 5.86μm (x̄= 5.86×4.1μm, n=50). Based on these morphological features, the isolates were identified preliminarily as Rhizopus stolonifer (García-Estrada et al. 2019; Lin et al. 2017). To identify the pathogen molecularly, genomic DNA was extracted using the FavorPrep Fungi/Yeast Genomic DNA extraction Mini Kit (Taiwan). Polymerase chain reaction (PCR) amplification of ITS1-5.8S-ITS2 rDNA was done using primers ITS4 and ITS5 (White et al. 1990) following the procedure of Khan and Bhadauria (2019). The PCR product was then sequenced by Macrogen, Korea. A BLAST search in GenBank revealed that isolate JR02 (GenBank accession OP692731) was 100% identical to R. stolonifer (GenBank accession MT256940). In pathogenicity tests,10 healthy young fruit at a similar maturity stage as the ones found diseased were collected from a orchard where the disease was not observed. Fruit were surface sterilized with 70% ethylalcohol and washed with sterile distilled water. Wounded (using a sterilized needle) and non-wounded fruits were inoculated with 20µl of a spore suspension (1×106/ml). Sterile distilled water was used for the controls. Inoculated fruit were covered with sterile cloth, transferred to perforated plastic bags with moistened blotting paper, and incubated at 25°C in the dark. Symptoms were first observed after 2 days on wounded fruit, but no symptoms developed on controls and non-wounded fruit. Rhizopus stolonifer was re-isolated from infected fruit, thus fulfilling Koch's postulates. Rhizopus rot is a devastating disease causing premature fruit drop, reduced crop yield, and post-harvest rot of jackfruit and other fruits and vegetables (Sabtu et al. 2019). Three Rhizopus species namely R. stolonifer, R. artocarpi and R. oryzae have been reported causing fruit rot of jackfruit in the tropics including Mexico, India and Hawaii (García-Estrada et al., 2019; Babu et al., 2018; Nelson, 2005). Appropriate management strategies are needed to be developed to prevent premature rot of jackfruit. To our knowledge, this is the first report of R. stolonifer causing premature soft rot of jackfruit in Bangladesh.

Comparative Study of Genomic DNA Extraction Protocols from Whole Blood for P53 Gene Polymorphism in Persons with and without Prostate Cancer

In latest decades, genetic methods have developed into a potent tool in a number of life-attaching applications. In research looking at demographic genetic diversity, QTL detection, marker-assisted selection, and food traceability, DNA-based technologies like PCR are being employed more and more. These approaches call for extraction procedures that provide efficient nucleic acid extraction and the elimination of PCR inhibitors. The first and most important stage in molecular biology is the extraction of DNA from cells. For a molecular scientist, the high quality and integrity of the isolated DNA as well as the extraction method's ease of use and affordability are crucial factors. The present study was designed to establish a simple, fast and inexpensive method for DNA extraction from human peripheral blood (normal male n=2, age 24 years old, patient male (prostate cancer) n=2, age 65 years old) by comparing between them, and aimed to standardize a protocol of DNA extraction using five extraction protocols. The first method was the modified organic method by using sodium perchlorate instead of organic solvent (phenol, chloroform), sodium perchlorate advantage comes from its cheap price and low storage and shipping requirements, the second was the enzymatic method by using proteinase K, third method was done by using detergent, the fourth used phenol-chloroform; finally fifth one was salting out method. The result showed that the organic method gives a good DNA yield and needs relatively short time while the enzymatic method gives an excellent DNA purity which are more suitable for PCR by comparing five protocols using the spectrophotometer and Nanodrop technetium in addition to electrophoresis. Through the use of the five suggested procedures, the PCR multiplication of the P53 gene with the isolated DNA was effectively carried out. This indicates that, with the exception of the detergent approach, there were no significant inhibiting substances for Taq polymerase in the final solution.

First Report of Anthracnose Caused by Colletotrichum siamense on Hydrangea macrophylla in China.

Hydrangea macrophylla (Thunb.) Ser. (Hydrangeaceae), a shrubby perennial plant, is widely used as an ornamental flowering plant because of its showy inflorescences and colorful sepals. In October 2022, leaf spot symptom was observed on H. macrophylla in Meiling Scenic Spot, which covers an area of about 143.58 km2 in Nanchang, Jiangxi Province, China (28.78°N, 115.83°E). An investigation was carried out in a 500 m2 mountain area with 60 H. macrophylla plants in a residential garden, the incidence of disease observed was 28~35%. The symptoms were visible as nearly round dark brown spots on the leaves in the early stages of infection. At later stages, the spots gradually developed grayish white center with dark brown margins. To isolate the pathogen, seven leaves randomly selected from 30 infected leaves were cut into 4-mm2 pieces, surface disinfected with 75% ethanol for 30s followed by 5% NaClO for 1 min, rinsed in sterile water three times, placed on potato dextrose agar (PDA), and cultured at 25 °C in the dark for 7 days, and four strains with similar morphological characteristics were obtained from 7 diseased samples. Conidia were aseptate, cylindrical, hyaline, obtuse at both ends, and measured 13.31 to 17.53 × 4.43 to 7.45 µm (15.47 ± 0.83 × 5.91 ± 0.62 µm, n = 60). Morphological characteristics matched Colletotrichum siamense (Weir et al. 2012; Sharma et al. 2013). For molecular identification, two representative isolates (HJAUP CH003 and HJAUP CH004) were used for genomic DNA extraction, and the internal transcribed spacer (ITS), partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB2) and partial calmodulin (CAL) were amplified, using primer pairs ITS4/ITS5 (White et al. 1990), ACT-512F/ACT-783R, GDF1/GDR1, Bt2a/Bt2b and CL1C/CL2C (Weir et al. 2012), respectively. The sequences were deposited in GenBank (accessions nos. ITS: OQ449415, OQ449416; ACT: OQ455197, OQ455198; GAPDH: OQ455203, OQ455204; TUB2: OQ455199, OQ455200; CAL: OQ455201, OQ455202). Concatenated sequences of the five genes were used to conduct phylogenetic analyses using the maximum-likelihood method in MEGA7.0 (Sudhir et al. 2016) and Bayesian inference analysis in MrBayes 3.2 (Ronquist et al. 2012). Our two isolates cluster together with four strains of C. siamense with 93%ML/1.00BI bootstrap support. The isolates were identified as C. siamense based on the morpho-molecular approach. Pathogenicity of HJAUP CH003 was tested indoors by inoculating detached wounded leaves of six healthy H. macrophylla plants. Three healthy plants with three leaves were punctured with flamed needles and sprayed with a 1 × 106 spores/ml spores suspension, and another three healthy plants were wounded inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and PDA plugs on three leaves each. Treated plant tissue were incubated in an artificial climate box at 25°C, 90% relative humidity and a photoperiod of 12 h. After 4 days, symptoms similar to those of natural infection were observed on all wounded inoculated leaves, while no symptoms appeared on mock-inoculated leaves. The fungus isolated from inoculated leaves was identical to the original pathogen based on morphological and molecular data, confirming Koch's hypothesis. It has been reported that C. siamense can cause anthracnose on numerous plants (Rong et al. 2021; Tang et al. 2021; Farr and Rossman 2023). This is the first report of C. siamense causing anthracnose on H. macrophylla in China. The disease is of major concern to the horticultural community as it seriously affects the aesthetic value of ornamentals.

Mediation effect of JAK2 methylation on the association between sitting time and abdominal obesity in rural adults.

Sitting time may affect health by altering the methylation of certain genes. This research aimed to estimate the association of sitting time with abdominal obesity and the role of Janus kinase 2 (JAK2) methylation in the association among rural adults. A total of 1062 rural adults from the Henan Rural Cohort Study were included. Whole blood was used to extract genomic DNA. JAK2 DNA methylation level was assessed by MethylTargetTM. The logistic regression model was utilized to assess the association of sitting time with abdominal obesity, and the possible effect of JAK2 DNA methylation on the association were conducted by using mediation analyses. Average time of sitting of participants was 7.28 ± 3.37 h/d. For per 1 h increment in sitting time, the odd ratio (OR) and 95% confidence interval (CI) of abdominal obesity was 1.153 (1.095, 1.214) after controlling potential risk factors. Simultaneously, the methylation levels of Chr9: 4985407 site and Chr9: 4985238-4985455 region were negatively correlated with abdominal obesity (OR: 0.549, 95% CI: 0.394, 0.765; OR: 0.189, 95% CI: 0.056, 0.640, respectively). Moreover, Chr9: 4985407 site and Chr9: 4985238-4985455 region methylation levels mediated the association of sitting time with abdominal obesity, and the indirect effects account for 6.78% and 4.24%, respectively. Longer sitting time was positively correlated with abdominal obesity in the rural population, and methylation level of JAK2 may be an underlying mediation of the effect.

Evaluation of Easy and Economic Protocols for High Quality Genomic DNA Extraction and Sex Determination in Papaya Using SCAR Markers

Rapid and precise identification of three sex types (male, hermaphrodite and female flowers) is the key in achieving good returns in papaya (Carica papaya L.) cultivation. This study presents a simple, reliable, fast and cost-efficient DNA isolation protocol and a rapid and precise method of sex determination in papaya. Three DNA isolation protocols viz., CTAB method, Modified Dellaporta method and BioBasic DNA isolation kit were comparatively evaluated for their simplicity, economic, DNA quantity and purity. Among them, CTAB method was regarded as relatively economic, with a cost of Rs. 11.80943 per sample with higher amount of DNA (4667.4 µg/ µl). However, it was noticed that DNA isolation with the BioBasic kit was fastest method, which took only 6 hours 7 minutes. On the other hand, relatively better DNA purity was noticed with the Modified Dellaporta method (average ratio of absorbance at 260 nm and 280 nm range between 1.8-2.0) and yielded clear, intact and distinct DNA bands. Hence, Modified Dellaporta method is suggested as an ideal DNA isolation protocol for papaya. Molecular markers (such as SCAR derived from RAPD T12, W11, NAPF-2 and PKBT-4) were employed in this study to identify the sex type and they have successfully distinguished between male and hermaphrodite plants in both dioecious and gynodioecious varieties; especially, NAPF-2 has successfully distinguished male in dioecious varieties. Evaluation of different kinds of molecular markers has shown that the SCAR marker T12 can be used as reliable marker for rapid and precise identification of papaya sex type.

Analysis of CNNM2 gene variant in a child with Hypomagnesemia, seizures, and mental retardation syndrome

To explore the genetic etiology of a child with Hypomagnesemia, epilepsy and mental retardation syndrome (HSMR). A child who was admitted to the Children's Hospital of Shandong University on July 9, 2021 due to repeated convulsions for 2 months was selected as the study subject. Clinical data of the child was collected. Peripheral blood samples of the child and his pedigree members were collected for the extraction of genomic DNA. Whole exome sequencing was carried out, and candidate variant was verified by Sanger sequencing and bioinformatic analysis. The child, a 1-year-and-7-month-old male, had presented with epilepsy and global developmental delay. Serological testing revealed that he has low serum magnesium. Genetic testing showed that the child has harbored a heterozygous c.1448delT (p.Val483GlyfsTer29) variant of the CNNM2 gene, which was de novo in origin. The variant has caused substitution of the Valine at position 483 by Glycine and formation of a termination codon after 29 amino acids at downstream. As predicted by Swiss-Model online software, the variant may alter the protein structure, resulting in a truncation. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.1448delT (p.Val483GlyfsTer29) was predicted as a pathogenic variant (PVS1+PS2+PM2_Supporting+PP4). The heterozygous c.1448delT variant of the CNNM2 gene probably underlay the HSMR in this child. Above finding has enriched the phenotype-genotype spectrum of the CNNM2 gene.

An Efficient Extraction Method Allowing the Genetic Evaluation of Host DNA from Samples Collected for Virus Infection Diagnosis in Viral Transport Medium.

Introduction: During the COVID-19 pandemic, an extraordinary number of nasopharyngeal secretion samples inoculated in viral transport medium (VTM) were collected and analyzed to detect SARS-CoV-2 infection. In addition to viral detection, those samples can also be a source of host genomic material, providing excellent opportunities for biobanking and research. Objective: To describe a simple, in-house-developed DNA extraction method to obtain high yield and quality genomic DNA from VTM samples for host genetic analysis and assess its relative efficiency by comparing its yield and suitability to downstream applications to two different commercial DNA extraction kits. Methods: In this study, 13 VTM samples were processed by two commercial silica-based kits and compared with an in-House-developed protocol for host DNA extraction. An additional 452 samples were processed by the in-House method. The quantity and quality of the differentially extracted DNA samples were assessed by Qubit and spectrophotometric measurements. The suitability of extracted samples for downstream applications was tested by polymerase chain reaction (PCR) amplification followed by amplicon sequencing and allelic discrimination in real-time PCR. Results: The in-House method provided greater median DNA yield (0.81 μg), being significantly different from the PureLink® method (0.14 μg, p < 0.001), but not from the QIAamp® method (0.47 μg, p = 0.980). Overall satisfactory results in DNA concentrations and purity, in addition to cost, were observed using the in-House method, whose samples were able to produce clear amplification in PCR and sequencing reads, as well as effective allelic discrimination in real-time PCR TaqMan® assay. Conclusion: The described in-House method proved to be suitable and economically viable for genomic DNA extraction from VTM samples for biobanking purposes. These results are extremely valuable for the study of the COVID-19 pandemic and other emergent infectious diseases, allowing host genetic studies to be performed in samples initially collected for diagnosis.

Development of a rapid detection method for Karenia mikimotoi by using CRISPR-Cas12a.

Harmful algal blooms (HABs), mainly formed by dinoflagellates, have detrimental effects on marine ecosystems and public health. Therefore, detecting HABs is crucial for early warning and prevention of HABs as well as the mitigation of their adverse effects. Although various methods, such as light microscopy, electron microscopy, real-time PCR, and microarrays, have already been established for the detection of HABs, they are still cumbersome to be exploited in the field. Therefore, rapid nucleic detection methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)-lateral flow dipstick (LFD) have been developed for monitoring bloom-forming algae. However, the CRISPR/Cas-based detection of HABs has yet to be applied to this field. In this study, we developed a method for detecting Karenia mikimotoi (K. mikimotoi), a typical ichthyotoxic dinoflagellate responsible for global blooms. Our method utilized Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) to target and cleave the internal transcribed spacer (ITS) of K. mikimotoi, guided by RNA. We leveraged the target-activated non-specific single-stranded deoxyribonuclease cleavage activity of LbCas12a to generate signals that can be detected using fluorescence-read machines or LFDs. By combining RPA and LbCas12a with reporters, we significantly enhanced the sensitivity, enabling the detection of ITS-harboring plasmids at concentrations as low as 9.8 aM and genomic DNA of K. mikimotoi at levels as low as 3.6 × 10-5 ng/μl. Moreover, we simplified the genomic DNA extraction method using cellulose filter paper (CFP) by directly eluting the DNA into RPA reactions, reducing the extraction time to < 30 s. The entire process, from genomic DNA extraction to result reporting, takes less than an hour, enabling the identification of nearly a single cell. In conclusion, our method provided an easy, specific, and sensitive approach for detecting K. mikimotoi, offering the potential for efficient monitoring and management of K. mikimotoi blooms.

Automated and robust extraction of genomic DNA from various leftover blood samples

With the development of genomic technologies, the isolation of genomic DNA (gDNA) from clinical samples is increasingly required for clinical diagnostics and research studies. In this study, we explored the potential of utilizing various leftover blood samples obtained from routine clinical tests as a viable source of gDNA. Using an automated method with optimized pre-treatments, we obtained gDNA from seven types of clinical leftover blood, with average yields of gDNA ranging from 3.11 ± 0.45 to 22.45 ± 4.83 μg. Additionally, we investigated the impact of storage conditions on gDNA recovery, resulting in yields of 8.62–68.08 μg when extracting gDNA from EDTA leftover blood samples stored at 4 °C for up to 13 weeks or −80 °C for up to 78 weeks. Furthermore, we successfully obtained sequenceable gDNA from both Serum Separator Tube and EDTA Tube using a 96-well format extraction, with yields ranging from 0.61 to 71.29 μg and 3.94–215.98 μg, respectively. Our findings demonstrate the feasibility of using automated high-throughput platforms for gDNA extraction from various clinical leftover blood samples with the proper pre-treatments.

First Report of Corynespora cassiicola Causing Leaf Spot on Jasminum nudiflorum in China.

Winter jasmine (Jasminum nudiflorum Lindl.), a trailing, deciduous shrub, is widely used as an ornamental plant. Its flowers and leaves also has great medicinal value for treatment of inflammatory swelling, purulent eruptions, bruises and traumatic bleeding (Takenaka et al. 2002). In October 2022, leaf spot symptoms were observed on J. nudiflorum distributed in Meiling Scenic Spot (28.78°N, 115.83°E) and Jiangxi Agricultural University (28.75°N, 115.83°E), Nanchang, Jiangxi Province, China. In a week-long series of investigations, the incidences of disease could range up to 25%. Initially, the symptoms of the lesions were small yellow circular spots (0.5 to 1.8 mm), and gradually developing irregular spots (2.8 to 4.0 mm) with grayish white central parts, a dark brown inner ring, and outer yellow halo. To identify the pathogen, sixty symptomatic leaves from fifteen different plants were collected, of which twelve were randomly selected, cut into 4-mm2 pieces, and surface sterilized with 75% ethanol for 30s followed by 5% NaClO for 1 min, rinsed four times with sterile water, and then placed on potato dextrose agar (PDA) medium at 25 °C in the dark for 5 to 7 days. Six isolates with similar morphological characteristics were obtained. Aerial mycelium was vigorous, downy and exhibited white to grayish-green coloration. Conidia were solitary or catenate, pale brown, obclavate to cylindrical, apex obtuse, one to 11 pseudosepta, 24.9 to 125.7 × 7.9 to 12.9 μm (n = 50). Morphological characteristics matched Corynespora cassiicola (Ellis 1971). For molecular identification, two representative isolates (HJAUP C001 and HJAUP C002) were selected for genomic DNA extraction, and the ITS, TUB2 and TEF1-α gene were amplified, using the primer ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Lousie and Donaldson 1995) and EF1-728F/EF-986R (Carbone and Kohn 1999), respectively. The sequenced loci (GenBank accession nos. ITS: OP957070, OP957065; TUB2: OP981639, OP981640; TEF1-α: OP981637, OP981638) of the isolates were 100, 99 and 98% similar to the corresponding sequences of C. cassiicola strains (GenBank accession nos. OP593304, MW961419, MW961421, respectively). Phylogenetic analyses of combined ITS and TEF1-α sequences was performed using maximum-likelihood method in MEGA 7.0 (Kuma et al. 2016). The result showed that our isolates (HJAUP C001 and HJAUP C002) clustered with four strains of C. cassiicola at 99% bootstrap values in the bootstrap test (1000 replicates). Based on the morpho-molecular approach, the isolates were identified as C. cassiicola. The pathogenicity of one representative strain (HJAUP C001) was tested by inoculating the wounded leaves of six healthy J. nudiflorum plants under natural condition. Three leaves from each of three plants were punctured with flamed needles and sprayed with a conidial suspension (1 × 106 conidia/ml), and three wounded leaves from each of other three plants were inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and PDA plugs on three leaves each, respectively. Leaves from all treatments were incubated in a greenhouse at high relative humidity, 25°C, and 12-hour photoperiod. After one week, all wounded inoculated leaves appeared similar symptoms as described above, whereas the mock inoculated leaves were still healthy. Similar isolates with grayish white and vigorous aerial mycelium were reisolated from inoculated and symptomatic leaves and identified as C. cassiicola by DNA sequencing, fulfilling Koch's postulates. It has been reported that C. cassiicola can cause leaf spots on numerous plant species (Tsai et al. 2015; Lu et al. 2019; Farr and Crossman 2023). However, to our knowledge, this is the first report of C. cassiicola causing leaf spots on J. nudiflorum in China. This finding aids in protection of J. nudiflorum, a medicinal and ornamental plant with high economic value.

Root rot Caused by Setophoma terrestris on Illicium verum in Yunnan Province, China.

Star anise (Illicium verum Hook. f.), a genus of star anise in the family Magnoliaceae, is an important cash crop of "medicinal and food" origin, mainly from China. In August 2021, root rot of I. verum was first observed on more than 80% of the plants grown within a 500 hectares area in Wenshan city, Yunnan Province. At the early stage of the disease, the phloem of the root was dark yellow-brown, and the leaves turn yellow. With further disease development, the whole root became black (Fig. 1a, 1b), and the leaves gradually fall off, affecting the growth, yield and eventually caused death of the whole plant. A total of 20 root samples were collected from typical symptomatic plant roots with 20 years old in Wenshan City (23°18'12″N, 103°56'98″E) and were cut into 2 × 2 mm pieces at the junction of infected and healthy tissue. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before rinsing three times with distilled water. The sterile filter paper (5×5 cm) was used to dry the tissue, and samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/ml). Plates were incubated at 25°C in the dark in the incubator. From 9 isolates obtained in culture, 7 exhibited the morphology described by Boerema et al. (Boerema et al. 2004) for Setophoma sp. The hyphae were hyaline and septate (Fig.1c). After 14 days of culture on V8 juice agar, white round colonies are formed, but there is no groove in the middle of the colonies (Fig.1d), and transparent, oval, or cylindrical conidia were produced, 6.0-8.0 x 2.5 to 4.0 um (Fig.1e). DNA was extracted from a representative isolate BJGF-04 for molecular identification using a fungal genomic DNA extraction kit (Solarbio, Beijing, China). Polymerase chain reactions (PCRs) were performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990) and primers T1/β-Sandy-R for the β-tubulin gene (TUB) region (Yang et al. 2017) and primers NL3/ LR5 for 28S large subunit rDNA (LSU) region (Hu et al. 2021) and NS1/ NS4 for 5.8S large subunit rDNA (SSU) region (Mahesha et al. 2021). Newly generated representative sequences were deposited in GenBank: ITS sequence (ON645256), TUB sequence (ON854484), and LSU sequence (ON644445), SSU sequence (ON644451). were sequenced and blasted, showing 99 to 100% sequence homology with known S. terrestris. Pathogenicity was performed using one-year asymptomatic plants of I. verum. A conidial suspension (1 x 106 conidia/ml) collected from V8 juice cultures with 0.05% Tween buffer was poured at a volume of 10 ml/plant. Three individual seedlings were used as replicates for each treatment, and sterile water was used as the negative control. All plants were placed in an artificial climate incubator at 25°C under 90% relative humidity. After 20 days, all inoculated plants showed symptoms identical to those described above, whereas controls remained healthy. Setophoma terrestris was reisolated from the infected roots, which was confirmed by morphological and molecular identification, which completed Koch's postulates. To our knowledge, this is the first report of S. terrestris as a causal agent of root rot on I. verum in China.

Brown leaf spot of Cunninghamia lanceolata Caused by Colletotrichum kahawae in Sichuan province, China.

Chinese fir (Cunninghamia lanceolata) is an important timber species that has been widely cultivated in southern China. It is extensively applied in medicine, environmental monitoring, furniture, urban (e.g., street trees) and rural landscaping, windbreak forest, soil and water conservation. In January 2022, distinct leaf spot symptoms were observed in Chinese fir in Hongya Forestry (29°45'N, 103°11'E) Meishan City, Sichuan Province, China. Field surveys showed that the disease was widespread, with around 70% disease incidence. The typical symptoms initially appeared as yellowish-brown necrotic lesions on the margin of the leaves. Subsequently, lesions gradually expanded and developed into larger necrotic areas with red-brown irregular shape. The lesions later expanded throughout the leaf. Infected leaves turned dark brown and wilted, leading to seeding's death. Diseased leaves with typical symptoms were collected for pathogen isolation and identification. Infected tissues from ten samples were cut into small pieces of 2 × 2 mm. Infected tissues were surface disinfected with 3% sodium hypochlorite and 75% ethanol for 30s and 60s, respectively, and rinsed with sterile water 3 times. They were blotted dry with autoclaved paper towels and incubated on potato dextrose agar (PDA) with streptomycin sulfate (50 μg/mL) for 5 ~ 8 days at 25°C. and 12 h light/dark period. The diameter of the colonies reached 65.7 to 75.9 mm, with a gray to black center, and white edges while the reverse sides were gray to orange. Conidia were single-celled, colorless, straight, cylindrical, bluntly rounded at both ends, Conidia dimensions varied from, 7.3 μm to 15.7 μm in length and 3.3 μm to 6.1 μm in width (n = 100). For molecular identification, the genomic DNA of isolate SM2290708, SM229070801 and SM229070802 were extracted using a fungal genomic DNA extraction kit (Beijing Solarbio Science & Technology Co., Ltd., City, China). The internal transcribed spacers of the ribosomal RNA (ITS) [ITS1/ITS4 (White et al., 1990), calmodulin (CAL) (Weir et al., 2012), β-tubulin (TUB2) (O'Donnell et al., 1997), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al. 1992) were amplified. Sequences were deposited in GenBank (ITS: ON564877, OQ535027 and OQ535028; CAL: ON583827, OQ538101 and OQ538102; TUB2: ON583830, OQ538104 and OQ538105; and GAPDH: ON583831, OQ538108 and OQ538109). BLAST results showed that our ITS, CAL, TUB2 and GAPDH sequences were >99% identical to the corresponding sequences of Colletotrichum kahawae deposited at NCBI (GenBank JX010231, JX009642, JX010444, and JX010012). Identification was confirmed by Bayesian inference using MrBayes (Fig 2). The conidial suspension (1 × 106 conidia/ml) was used for inoculation by spraying leaves of ten 3-year-old Chinese fir plants for pathogenicity test. Fifteen leaves of each plant were inoculated. An equal number of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 28°C under a 16 h/8 h photoperiod and in 73% to 79% relative humidity. After fifteen days, the symptoms observed on the inoculated plants were similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum kahawae was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on C. lanceolata caused by C. kahawae in China. The results of this study provide basic information for diagnosis of the pathogen and developing prevention strategies to manage C. lanceolata leaf spot disease.

Molecular evidence and phylogenetic delineation of spotted fever group Rickettsia species in Amblyomma ticks from cattle in Gauteng and Limpopo Provinces, South Africa

Objective: To determine the prevalence of tick-borne pathogens with a particular focus on Rickettsia spp. in ticks collected from cattle in Gauteng and Limpopo Provinces, South Africa. Methods: A total of 200 ticks were collected from cattle within the Madala livestock, Pretoria, Gauteng Province and in Mankweng Township, Polokwane, Limpopo Province in 2019. The ticks were morphologically identified and processed individually for a total genomic DNA extraction. Specific primers targetting ompA, ompB, and the 17KDa genes were used for a molecular screening and delineation of Rickettsia from the extracted genetic materials using polymerase chain reaction (PCR) technique. PCR amplicons of positive samples were sequenced bidirectionally using the Sanger sequencing method. Sequences generated were processed and analysed using appropriate bioinformatics software. Results: The ticks were morphologically identified as Amblyomma spp. PCR profiling of the genomic DNA samples revealed the presence of the Rickettsia pathogen in 42 (21%) of the ticks collected from both Provinces. Out of the genes profiled, 14 (7%) were positive for 17KDa, 42 (21%) for ompA and 32 (16%) were positive for ompB genes respectively. The nucleotide blast of the sequenced genomes showed high similarity, as high as 100% with other reference Rickettsia (R.) africae in the GenBank. The phylogenetic analysis of the sequences further validated them as R. africae with their characteristic clustering pattern with related reference sequences. Conclusions: There is an abundance of R. africae in Amblyomma ticks collected from cattle in the study areas. This has serious public health implications as individuals who accidentally get infested with the ticks could acquire R. africae. Hence, adequate precautions in terms of sensitization of farmers about the risk and mass mobilization drive to control the vectors in the areas are highly recommended to safeguard public health.

Gastric Microbiota Gender Differences in Subjects with Healthy Stomachs and Autoimmune Atrophic Gastritis.

Gender differences and microbiota are gaining increasing attention. This study aimed to assess gender differences in gastric bacterial microbiota between subjects with healthy stomachs and those with autoimmune atrophic gastritis. This was a post hoc analysis of 52 subjects undergoing gastroscopy for dyspepsia (57.7% healthy stomach, 42.3% autoimmune atrophic gastritis). Gastric biopsies were obtained for histopathology and genomic DNA extraction. Gastric microbiota were assessed by sequencing the hypervariable regions of the 16SrRNA gene. The bacterial profile at the phylum level was reported as being in relative abundance expressed as 16SrRNA OTUs (>0.5%) and biodiversity calculated as Shannon-diversity index-H. All data were stratified for the female and male gender. Results showed that women with healthy stomachs had a higher gastric bacterial abundance and less microbial diversity compared to men. Likely due to hypochlorhydria and the non-acid intragastric environment, autoimmune atrophic gastritis seems to reset gender differences in gastric bacterial abundance and reduce biodiversity in males, showing a greater extent of dysbiosis in terms of reduced biodiversity in men. Differences between gender on taxa frequency at the phylum and genus level in healthy subjects and autoimmune atrophic gastritis were observed. The impact of these findings on the gender-specific natural history of autoimmune atrophic gastritis remains to be elucidated; in any case, gender differences should deserve attention in gastric microbiota studies.

OPTIMIZATION OF COLONY POLYMERASE CHAIN REACTION FOR THE 16SRRNA OF DIFFERENT STRAINS OF ESCHERICHIA COLI

Objective: This work aimed to enhance colony polymerase chain reaction (PCR) for the 16S rRNA of several Escherichia coli strains. Methods: The isolation of E. coli is done from the gut of the chicken and soil. Then, we optimized the condition for colony PCR for the amplification of 16s ribosomal RNA. We successfully designed primer 3 for 16s ribosomal RNA and made the dilution solution with PCR grade water that is 1:10. Moreover, finally, we made a 20 μL solution that contains the master mix of our isolated colony and forward and reverse base primer for amplification. After the conventional PCR, the amplified 16s ribosomal RNA was then run on Gel to obtain the desired bands. And finally saw the bands in the Gel Doc picture. Results: Our result shows that the technique of colony PCR is an efficient and quick method than other existing methods that are too costly, tedious, and time-consuming procedures that deter their exploitation in various experimentations and for the identification of E. coli strains. Conclusion: This study concluded that 16s ribosomal RNA can be amplified without the extraction and purification of total genomic DNA from a bacterial colony using colony PCR. Therefore, by designing rRNA primers for E. coli species, we can evaluate their various types of mutations, strain detection, and antibiotic resistance.

First Report of a Rust Disease on Bletilla striata Caused by Coleosporium bletiae in Anhui province of China.

Bletilla striata is a perennial Orchidaceae which is widely distributed in Anhui, Hubei, Guizhou, Guangxi, Sichuan, Yunnan and other provinces in China (He et al. 2017). It has the effects of convergence, haemostasis, clearing away heat and detoxification, reducing swelling and generating muscle (He et al. 2017; Xu et al. 2019). With the reduction in wild resources of B. striata, cultivation can promote the effective conservation of B. striata germplasm resources. Fusarium root rot, leaf spot disease, and other diseases have been reported on this plant in China (Wang et al. 2022, Xu et al. 2022, Zhou et al. 2022). Coleosporium bletiae on B. striata has been reported in Guizhou, Sichuan, and Hubei provinces in China, with frequent occurrence and serious disease incidences in Guizhou province (Xu et al. 2022). In April to November 2021, rust was found in Anhui Bletilla planting base (30°09' to 30°46'N; 115°45' to 116°30'E). Approximately 17% of the B. striata plants showed disease symptoms (Figure S1). Uredinia are hypophyllous, scattered, round, powdery, and are associated with chlortic spots 1-4 mm wide on the upper side of the leaves. After mid-October, the temperature gradually decreased, and reddish-brown telia appeared around uredinia in circular shape. A total of 60 samples with typical symptoms were collected. Urediospore and teliospores were obtained by sterile needles, and their sizes were observed and measured under a microscope with sterile water. Urediospores were orange‒yellow, mainly oval in shape, 27.2-36.8 µm× 17.6-22.8 µm (n=60), and uniformly covered by verrucae on the surface (Figure S2 a-b). Teliospores were clavate, with a smooth surface and golden yellow contents, and gradually narrowed from top to base, ranging in size from 85.33-109.66 μm× 15.27-20.35 μm (n=60) (Figures S2 c-e). Three samples with typical symptoms were selected; approximately 200 μg of Urediospore of the rust pathogen were colleced per sampleand placed in 1.5 mL sterile centrifuge tubes for genomic DNA extraction by the CTAB method (Xu et al. 2022). The internal transcribed spacer (ITS) region of rDNA and large subunit (LSU) RNA genes were amplified using primer pairs ITS1/ITS4 (Leclerc et al., 2000) and NL1/NL4 (Zhang et al. 2012), respectively. The obtained sequences were deposited in GenBank (OP363678 for ITS and OP363683 for LSU), which were 100% identical with 100% coverage to the ITS sequence (MN108161, locality: Hubei) and the LSU sequence (KX386038) of Coleosporium bletiae. Based on morphological characteristics and DNA sequences, the isolates were identified as C. bletiae (Figures S2 and Figure S3). To confirm pathogenicity, 2-year-old healthy plants of B. striata were sprayed with 5 mL of urediospore suspension (2.6×105/mL). Plants sprayed with sterile distilled water were used as control. The test was conducted in an artificial climate incubator at 25 ± 1°C with a 12 h light/12 h dark photoperiod (relative humidity = 90%). The experiment was repeated three times. Two weeks after inoculation, typical yellowish chlorosis spots appeared on the leaf surface and a few yellow spore piles appeared on the leaves, which was consistent with the field symptoms (Figure S4). Control plants remained healthy. To our knowledge, this is the first report of C. bletiae causing rust on B. striata in Anhui province of China. Correct identification of this disease is of great importance to develop management strategies to control the disease. There may be seedling transport and spore transmission between Hubei and Anhui.

The oxytocin receptor rs2254298 polymorphism and alcohol withdrawal symptoms: a gene-environment interaction in mood disorders.

Alcohol use disorder (AUD) is a common mental disorder characterized by repeated withdrawal episodes. Negative emotions during withdrawal are the primary factors affecting successful abstinence. Oxytocin is a critical modulator of emotions. OXTR, the oxytocin receptor, may also be a promising candidate for treating alcohol withdrawal symptoms. Previous studies indicated that people with different genotypes of OXTR rs2254298 were reported to suffer from more significant depressive or heightened anxiety symptoms when experiencing early adversity. The present study aims to explore the modulatory role of the polymorphism OXTR rs2254298 on mood disorders during alcohol withdrawal and to help researchers better understand and develop effective relapse prevention and interventions for alcohol use disorders. We recruited 265 adult Chinese Han men with AUD. Anxiety and depressive symptoms were measured using the Self-Rating Anxiety Scale and Self-Rating Depression Scale. Alcohol dependence levels were measured using Michigan Alcoholism Screening Test. Genomic DNA extraction and genotyping from participants' peripheral blood samples. First, a multiple linear regression was used to set the alcohol dependence level, OXTR.rs2254298, interaction terms as the primary predictor variable, and depression or anxiety as an outcome; age and educational years were covariates. There was a significant interaction between OXTR rs2254298 and alcohol dependence level on anxiety (B = 0.23, 95% confidence interval [CI]: 0.01-0.45) but not on depression (B = -0.06, 95% CI: -0.30 - 0.18). The significance region test showed that alcohol-dependent men who are GG homozygous were more likely to experience anxiety symptoms than subjects with the A allele (A allele: β = 0.27, p < 0.001; GG homozygote: β = 0.50, p < 0.001). Finally, re-parameterized regression analysis demonstrated that this gene-environment interaction of OXTR rs2254298 and alcohol dependence on anxiety fits the weak differential susceptibility model (R2 = 0.17, F (5,259) = 13.46, p < 0.001). This study reveals a gene-environment interactive effect between OXTR rs2254298 and alcohol withdrawal on anxiety but not depression. From the perspective of gene-environment interactions, this interaction fits the differential susceptibility model; OXTR rs2254298 GG homozygote carriers are susceptible to the environment and are likely to experience anxiety symptoms of alcohol withdrawal.

Clinical manifestations and genetic analysis of 4 patients with variants of FBN1 gene

To explore the genetic basis for four patients suspected for Marfan syndrome (MFS). Four male patients with suspected MFS and their family members who were treated at West China Second Hospital of Sichuan University from September 12, 2019 to March 27, 2021 were selected as the study subjects. Peripheral venous blood samples were collected from the patients and their parents or other pedigree members for the extraction of genomic DNA. Whole exome sequencing was carried out, and candidate variants were validated by Sanger sequencing. The pathogenicity of the variants was determined based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). Genetic testing revealed that all four patients have harbored variants of the FBN1 gene, including c.430_433del (p.His144fs) deletional variant in exon 5, c.493C>T (p.Arg165*) nonsense variant in exon 6, c.5304_5306del (p.Asp1768del) deletional variant in exon 44 and c.5165C>G (p.Ser1722Cys) missense variant in exon 42. According to the ACMG guidelines, the c.430_433del and c.493C>T were classified as pathogenic variants (PVS1+PM2_Supporting+PP4; PVS1+PS1+PS2+PM2_Supporting+PP4). c.5304_5306del and c.5165C>G were classified as likely pathogenic variants (PS2+PM2_Supporting+PM4+PP4; PS2_Moderate+PS1+PM1+PM2_Supporting). The c.430_433del and c.5304_5306del variants of the FBN1 gene identified in this study were unreported previously. Above results have enriched the variation spectrum of the FBN1 gene and provided a basis for genetic counseling and prenatal diagnosis of patients with MFS and acromicric dysplasia.

Analysis of COL1A1 and COL1A2 gene variants in two fetuses with osteogenesis imperfecta

To explore the genetic basis of two fetuses with an osteogenesis imperfecta (OI) phenotype. Two fetuses diagnosed at the Affiliated Hospital of Weifang Medical College respectively on June 11, 2021 and October 16, 2021 were selected as the study subjects. Clinical data of the fetuses were collected. Amniotic fluid samples of the fetuses and peripheral blood samples of their pedigree members were collected for the extraction of genomic DNA. Whole exome sequencing (WES) and Sanger sequencing were carried out to identify the candidate variants. Minigene splicing reporter analysis was used to validate the variant which may affect the pre-mRNA splicing. For fetus 1, ultrasonography at 17+6 weeks of gestation had revealed shortening of bilateral humerus and femurs by more than two weeks, in addition with multiple fractures and angular deformities of long bones. WES revealed that fetus 1 had harbored a heterozygous c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in exon 49 of the COL1A1 gene (NM_000088.4). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), it was classified as a pathogenic variant (PVS1+PS2+PM2_Supporting) for disrupting the downstream open reading frame resulting in premature translational termination, being de novo in origin, and lacking records in the population and disease databases.For fetus 2, ultrasonography at 23 weeks of gestation also revealed shortening of bilateral humerus and femurs by one and four weeks, respectively, in addition with bending of bilateral femurs, tibias and fibulas. Fetus 2 had harbored a heterozygous c.1557+3A>G variant in intron 26 of the COL1A2 gene (NM_000089.4). Minigene experiment showed that it has induced skipping of exon 26 from the COL1A2 mRNA transcript, resulting in an in-frame deletion (c.1504_1557del) of the COL1A2 mRNA transcript. The variant was inherited from its father and had been previously reported in a family with OI type 4. It was therefore classified as a pathogenic variant (PS3+PM1+PM2_Supporting+PP3+PP5). The c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in the COL1A1 gene and c.1557+3A>G variant in the COL1A2 gene probably underlay the disease in the two fetuses. Above findings not only have enriched the mutational spectrum of OI, but also shed light on the correlation between its genotype and phenotype and provided a basis for genetic counseling and prenatal diagnosis for the affected pedigrees.