ALLELIC VARIATIONS OF SOS, HVNHX AND SDO GENES IN SOME BARLEY (Hordeum Vulgare L.) GENOTYPES

This study has been carried out at the Biotechnology Lab., Department of Field Crops, Faculty of Agriculture, Damascus University during the growing season 2019 - 2020, in order to detect the variations of SOS, HVNHX and SDO genes in different barley genotypes. Clear variations in the SOS, HVNHX and SDO genes, which are responsible for salinity tolerance were found among the investigated genotypes. It has been found that the variation in the amplicon size between loci per gene was very high in some cases, while there was a high degree of symmetry in other cases, and could be easily distinguished on 2% Agarose gel. The PCR results for the SOS genes (SOS1, SOS2, SOS3), HVNHX genes (HVNHX1, HVNHX2, HVNHX3) and SDO genes (Cu/Zn SODII, Cu/Zn SODI, CAT, GRI , APXIII) have shown only one morphological pattern in most of the studied genotypes, while revealed two patterns for the SOS3 gene, but the rest of genes (HVNHX1, HVNHX2, HVNHX3 Cu/Zn SODI, CAT) exhibited only one morphological pattern. The SOS3 was superior in the number of polymorphic patterns, as the number of total patterns was 14 in all the studied genotypes, but the Cu/Zn SODI showed the least number of polymorphic patterns with only 1 pattern, while the largest number (7 patterns) was detected in the genotype (H9), but the two genotypes Fourat9 and Fourat7 showed only one polymorphic pattern.

Fungal biodiversity in Arctic paleoecosystems assessed by metabarcoding of lake sedimentary ancient DNA

Fungi are crucial organisms in most ecosystems as they exert ecological key functions and are closely associated with land plants. Fungal community changes may therefore help reveal biodiversity changes in past ecosystems. Lake sediments contain DNA of organisms in the catchment area, which allows reconstructing past biodiversity by using metabarcoding of ancient sedimentary DNA. We developed a novel PCR primer combination for fungal metabarcoding targeting a short amplicon to account for length bias of amplification due to ancient DNA degradation. In-silico PCRs showed higher diversity using this primer combination than using previously established fungal metabarcoding primers. We analyzed existing data from sediment cores from four artic and one boreal lake in Siberia. These cores had been stored for 2-22 years and examined degradation effects of ancient DNA and storage time-related bias in fungal communities. Amplicon size differed between fungal divisions, however, we observed no significant effect of sample age on amplicon length and GC content, suggesting robust results. We also found no indication of post-coring fungal growth during storage distorting ancient fungal communities. Terrestrial soil fungi, including mycorrhizal fungi and saprotrophs, were predominant in all lakes, which supports the use of lake sedimentary ancient DNA for reconstructing terrestrial communities.

Incidence of Alternaria Species Associated with Watermelon Leaf Blight in Korea

Alternaria leaf blight is one of the most common diseases in watermelon worldwide. In Korea, however, the Alternaria species causing the watermelon leaf blight have not been investigated thoroughly. A total of 16 Alternaria isolates was recovered from diseased watermelon leaves with leaf blight symptoms, which were collected from 14 fields in Korea. Analysis of internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II second largest subunit (RPB2) were not competent to differentiate the Alternaria isolates. On the contrary, analysis of amplicon size of the histone H3 (HIS3) gene successfully differentiated the isolates into three Alternaria subgroups, and further sequence analysis of them identified three Alternaria spp. Alternaria tenuissima, A. gaisen, and A. alternata. Representative Alternaria isolates from three species induced dark brown leaf spot lesions on detached watermelon leaves, indicating that A. tenuissima, A. gaisen, and A. alternata are all causal agents of Alternaria leaf blight. Our results indicate that the Alternaria species associated watermelon leaf blight in Korea is more complex than reported previously. This is the first report regarding the population structure of Alternaria species causing watermelon leaf blight in Korea.

SEROLOGICAL AND MOLECULAR EVALUATION OF MIGRATORY TRICHINELLA SPIRALIS LARVAE IN BLOOD OF HUMANS IN KADUNA METROPOLIS, NIGERIA

Trichinellosis is an important food-borne zoonotic disease with public health implications and a worldwide distribution. In this study, Polymerase Chain Reaction (PCR) procedure using species specific ATP6 primers was used to detect the presence of migratory Trichinella spiralis larval mitochondrial ATP6 synthase F0 subunit (ATP6) gene, after detection of antibodies to Trichinella excretory-secretory (E/S) antigen using Enzyme-linked Immunosorbent Assay (ELISA), in blood of humans in Kaduna metropolis, Nigeria. The sera of 210 participants were tested for antibodies to Trichinella E/S antigen. Overall seroprevalence rate of 39% (82/210) was recorded using ELISA. Out of the 9 ELISA samples selected randomly, PCR detected migratory Trichinella spiralis larval ATP6 gene in 4 (44.4%) at the amplicon size of 250 base pairs using the whole blood of the participants. The 9 samples comprised 7 seropositive and 2 seronegative. The bands at lanes 1, 2, 3 and 4 were positive for ATP6 while lanes 5,6,7,8 and 9 were negative for ATP6. Lanes 4 and 5 were ELISA negative for anti-Trichinella antibodies. One in 5 of the 128 ELISA negative samples was positive for ATP6 representing a 25.6% prevalence rate by extrapolation. PCR using ATP6 gene as a genetic marker is valuable for the detection of T. spiralis migratory larvae in blood samples of humans and consequently the early diagnosis of trichinellosis in humans.

ERBB2 copy number (CN) as a quantitative biomarker for real-world (RW) outcomes to anti-HER2 therapy in advanced gastroesophageal adenocarcinoma (adv GEA).

4045 Background: HER2 ( ERBB2) overexpression or amplification (amp) are biomarkers for approved anti-HER2 therapies. ERBB2amp may be a superior predictor of anti-HER2 therapy outcome compared to IHC/ISH, and degree of CN gain may further stratify patients (pts). We investigated the distribution of ERBB2amp in adv GEA and hypothesized that increased CN was associated with better outcome to trastuzumab (T). Methods: Genomic analysis was performed using the Foundation Medicine (FM) tissue database (DB) of 313,896 pts with solid tumors including 12,749 pts with GEA and 34,629 pts with breast cancer (BC) used for comparison. ERBB2amp was defined as predicted CN ≥5 with > 80% of exons amplified. Using the nationwide US-based Flatiron Health-FM clinico-genomic DB linking de-identified EHR-derived clinical data to FM genomic data, pts with ERBB2amp adv GEA (01/2011 - 12/2020) were selected. RW progression (rwP) was obtained via technology-enabled abstraction of EHR. Multivariable Cox proportional hazard models were used for outcome comparisons. Results: ERBB2amp was detected in 15% (1,920/12,749) of GEA samples; median CN 22 (IQR 9-73), and 97% of cases had full gene amp. Median ERBB2 amplicon size was 0.27Mbp (IQR 0.13-0.95). In comparison, ERBB2amp was detected in 9.2% (3,193/34,629) of BC samples; median CN 20 (IQR 9-40), median amplicon size 0.32Mbp (IQR 0.13-1.37). In both cancers, smaller amplicons were associated with higher CN (P < 0.001), excluding amplicons < 0.1Mbp where less than 100% target amp was common. ERBB2amp was additionally seen in 2.7% of other solid tumors, and specifically in 2.3% of NSCLC and 3.1% of CRC. In the RW DB of 183 pts with ERBB2amp adv GEA, chemo + T (45%) and chemo alone (17%) were the most common first therapies after genomic report. In 101 evaluable first-line T treated pts ERBB2 CN was a significant predictor of rwP free survival (rwPFS) as a continuous variable (aHR = 0.74 [95% CI: 0.61 - 0.89], P = 0.002) and a range of cutoffs were similarly predictive. For control, in ERBB2amp pts treated with chemo ERBB2 CN was not predictive of rwPFS (aHR = 0.93, [95% CI: 0.72 – 1.20], P = 0.060). Among T treated pts, co- PIK3CA mutation was more common with lower CN (p = 0.03 by Wilcox test); no significant differences were observed for primary tumor location, age, stage at adv diagnosis, co- KRASmut, EGFRamp or F GF R1/2amp. Conclusions: ERBB2amp was detected in 15% of GEA tissue samples, with significant diversity in ERBB2 CN and amplicon focality, but with a similar CN distribution and amplicon focality seen in ERBB2amp BC. ERBB2 CN was predictive of rwPFS as a continuous variable for pts with GEA treated with T in the RW setting. Further studies exploring the clinical utility of quantitative ERBB2 CN, and extending to ctDNA, particularly in the setting of the evolving anti-HER2 landscape and combination therapies, are warranted.

Direct Detection and Identification of the Most Common Bacteria and Fungi Causing Otitis Externa by a Stepwise Multiplex PCR

BackgroundConsidering the importance of differential diagnosis of infectious otitis externa (OE), a stepwise PCR-based assay using universal and genus- or species-specific primers for the detection/identification of the most prevalent bacterial and fungal OE was developed and evaluated on the ear aspiration specimens of clinically suspected patients.Methods and MaterialsA total of 120 ear aspiration specimens with otomycosis suspicion were subjected to manual DNA extraction using phenol–chloroform extraction after tissue digestion with a lysis buffer. The multiplex PCR was initially performed using pan-fungal and bacterial homemade primers. Pseudomonas and Staphylococcus specific primers were simultaneously used in one reaction mixture to identify the bacterial genera. Furthermore, for the identification of fungal agents, Candida species-specific multiplex primers targeting the most clinically important Candida species causing OE (i.e., C. albicans, C. parapsilosis, and C. auris), as well as Aspergillus related multiplex PCR identifying the most prevalent Aspergillus species were used in two separate reaction mixtures. All the results of multiplex PCR were interpreted based on the amplicon size.ResultsThe overall multiplex PCR-based detection rate of bacterial (n = 88; 73.3%) and fungal (n = 97; 81%) OE was documented to be 100% along with and complete consistency with the results of direct examination and Giemsa staining. Double amplicon bands of bacterial and fungal pathogens were evidenced in 76 specimens (63.3%). Moreover, the positivity rate of pan-fungal PCR was higher than that of the culture result. Out of 88 pan-bacterial positive PCR specimens, 66 and 47 ones were positive for Staphylococcus and Pseudomonas, respectively. In addition, 30 samples exhibited mixed infection of both, and five specimens remained negative. Out of 97 pan-fungal positive PCR specimens, 67 and 51 ones contained Candida and Aspergillus species, respectively. It should be noted that dual amplicon bands of Candida and Aspergillus-related multiplex PCR were yielded in 30 specimens.ConclusionThe stepwise multiplex PCR assay proved to be more sensitive, more rapid, as well as less cumbersome in detection and identification of fungal and bacterial OE, compared to culture.

Genotyping of Avasirkhva, an indigenous Abkhasian grapevine variety

Molecular genotyping of native varieties of Vitis vinifera L. from different winegrowing areas is a current trend in the grapevine genetic diversity research. Abkhazia is among the world cradles of tamed grape, and its indigenous gene pool is of particular interest. Avasirkhva is a native Abkhasian grapevine variety mainly grown in the Gudauta District. Te research aimed to obtaining a genetic passport of Avasirkhva grapevine using microsatellite polymorphism data. Te study sampled grape plants from private farmsteads of the Gudauta District. Te plant phenotype corresponded to the variety’s ampelographic description. DNA was isolated from young shoot tip leaves with a CTAB-based protocol. Genotyping was performed with polymerase chain reaction (PCR) followed by capillary fragment separation. High-polymorphic SSR loci (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62, VrZAG79) recommended for grapevine varietal identification were used as markers. Te amplicon size was estimated with an ABI Prism 3130 automated genetic analyser using the GeneMapper and PeakScanner soſtware and Pinot Noir as a reference variety genotype. Four samples exhibited an identical microsatellite profile. Te microsatellite assay-based genetic passport of the Avasirkhva variety is as follows: VVS2141-145 VVMD5234-242 VVMD7239- 249, VVMD25239-249, VVMD27184-190, VVMD28234-248, VVMD32248- 262, VrZAG62200-204, VrZAG79251-257. Te obtained passport is unique with respect to the known genotypes in the Vitis International Variety Catalogue (VIVC). A Principal Coordinate Analysis of microsatellite data was used to infer the genetic relationships between Avasirkhva and the Abkhasian varieties Kachich and Azhizhkvakva genotyped in our earlier studies, as well as nine native grapevines of Georgia, the nearest viticultural area. Te Avasirkhva genetic passport can be used in grapevine genotyping studies to clarify the varietal identity.

pr2-primers: an 18S rRNA primer database for protists

Metabarcoding of microbial eukaryotes (collectively known as protists) has developed tremendously in the last decade, almost uniquely relying on the 18S rRNA gene. As microbial eukaryotes are extremely diverse, many primers and primer pairs have been developed. To cover a relevant and representative fraction of the protist community in a given study system, a wise primer choice is needed as no primer pair can target all protists equally well. As such, a smart primer choice is very difficult even for experts and there are very few on-line resources available to list existing primers. We built a database listing 179 primers and 76 primer pairs that have been used for eukaryotic 18S rRNA metabarcoding. In silico performance of primer pairs was tested against two sequence databases: PR2 for eukaryotes and a subset of Silva for prokaryotes. This allowed to determine the taxonomic specificity of primer pairs, the location of mismatches as well as amplicon size. We developed a R-based web application that allows to browse the database, visualize the taxonomic distribution of the amplified sequences with the number of mismatches, and to test any user-defined primer set (https://app.pr2-primers.org). This tool will provide the basis for guided primer choices that will help a wide range of ecologists to implement protists as part of their investigations.

Occurrence, Distribution and Molecular Characterization of Phytoplasma Infecting Chickpea (Cicer arietinum L.) from Tamil Nadu

Phytoplasma is an obligate prokaryote infecting a wide array of crops such as urdbean, sesame, brinjal and many other vegetable crops in India. Several workers have noticed the recent outbreak of phytoplasma disease in several pulse crops. A study was conducted to identify the phytoplasma infection in chickpea from Tamil Nadu. The average disease incidence due to phytoplasma was recorded with a range of 5-20% under field condition. The infected plants produced symptoms including stunting of plants, bushy appearance, reduced leaf size, chlorosis and reddening of infected leaves. The association of phytoplasma with these symptoms was confirmed by nested PCR assay using the universal primers P1/P7 and R16F2n/R16R2. The infected samples were amplified with an amplicon size of 1.2 kb and sequence analysis showed more than 99% similarity with phytoplasma belongs to Candidatus Phytoplasma aurantifolia. Phylogenetic analysis of nucleotide sequences confirmed the chickpea phyllody phytoplasma forms a single subgroup with other Indian isolates of various crops.

Characterization of Little millet (Panicum sumatrense) varieties using Morphological descriptors and SSR based DNA fingerprinting

Little millet varieties are generally distinguished by morphological descriptors which are being used for seed certification and DUS characterization [1]. But in practical terms, these key differentiation descriptors between varieties of little millet are very fewer and hence difficult to differentiate germplasm accessions. Germplasm registration in NBPGR needs DNA fingerprint to show the uniqueness of germplasm in comparison to existing varieties. DNA fingerprinting is a better option to identify unique markers to differentiate the varieties. Available genomic resources are scarce since little millet is still considered to be an orphan crop. Therefore markers from other cereal genomes such as maize, pearl millet and barnyard millet that are been utilized for DNA fingerprinting purpose with a clue of cereal synteny relationship. Twenty-one morphological descriptors studies revealed that the variety ATL 1 is different from the other varieties for more than 16 morphological characters studied. DNA fingerprinting is attempted in five genotypes of little millets such as BL6, ATL 1, TNPsu 176, Co (Samai) 4, Paiyur 2 using cereal SSR markers. Among the 25 maize SSR markers used two markers viz., phi213984 and phi295450 scored polymorphism by the amplicon size of 310bp and 600bp respectively. From the 25 Pearl millet SSR markers used only one SSR marker found polymorphic at 305bp allele size for ATL 1 and Hence, SSR based DNA fingerprinting helped to differentiate ATL1, the newly released high yielding variety from other genotypes of little millets which can be used for varietal identification purpose.