Primer Pair Combinations Research Articles

A full-length 18S ribosomal DNA metabarcoding approach for determining protist community diversity using Nanopore sequencing.

Protist diversity studies are frequently conducted using DNA metabarcoding methods. Currently, most studies have utilized short read sequences to assess protist diversity. One limitation of using short read sequences is the low resolution of the markers. For better taxonomic resolution longer sequences of the 18S rDNA are required because the full-length has both conserved and hypervariable regions. In this study, a new primer pair combination was used to amplify the full-length 18S rDNA and its efficacy was validated with a test community and then validated with field samples. Full-length sequences obtained with the Nanopore MinION for protist diversity from field samples were compared with Illumina MiSeq V4 and V8-V9 short reads. Sequences generated from the high-throughput sequencers are Amplicon Sequence Variants (ASVs). Metabarcoding results show high congruency among the long reads and short reads in taxonomic annotation at the major taxonomic group level; however, not all taxa could be successfully detected from sequences. Based on the criteria of ≥95% similarity and ≥1000 bp query length, 298 genera were identified by all markers in the field samples, 250 (84%) were detected by 18S, while only 226 (76%) by V4 and 213 (71%) by V8-V9. Of the total 85 dinoflagellate genera observed, 19 genera were not defined by 18S dinoflagellate ASVs compared to only three among the total 52 diatom genera. The discrepancy in this resolution is due to the lack of taxonomically available 18S reference sequences in particular for dinoflagellates. Overall, this preliminary investigation demonstrates that application of the full-length 18S rDNA approach can be successful in field studies.

Establishment and application of a rapid molecular diagnostic platform for the isothermal visual amplification of group B Streptococcus based on recombinase polymerase.

With growing concerns about Group B streptococcal (GBS) infections and their adverse effects on perinatal pregnancies, including infection, premature delivery, neonatal septicemia, and meningitis, it is urgent to promote GBS screening at all pregnancy stages. The purpose of this study is to establish a device-independent, fast, sensitive, and visual GBS detection method. Taking advantage of the characteristics of the recombinase polymerase isothermal amplification (RPA), the activity of the nfo nuclease cleavage base analog (tetrahydrofuran, THF) site, and the advantages of visual reading of the lateral flow chromatography strip (LFS), a GBS detection method was developed. This method focused on the conservative region of the Christie-Atkins-Munch-Petersen factor encoded by the cfb gene, a virulence gene specific to GBS. Two forward primers, two biotin-labeled reverse primers, and one fluorescein isothiocyanate (FITC)-labeled and C3spacer-blocked probe were designed. The study involved optimizing the primer pair and probe combination, determining the optimal reaction temperature and time, evaluating specificity, analyzing detection limits, and testing the method on 87 vaginal swabs from perinatal pregnant women. The results showed that the visual detection method of GBS-RPA-LFS, using the cfb-F1/R2/P1 primer probe, could detect GBS within 15 min at the temperature ranging from 39°C to 42°C. Furthermore, the method specifically amplified only GBS, without cross-reacting with pathogens like Lactobacillus iners, Lactobacillus crispatus, Candida albicans, Listeria monocytogenes, Yersinia enterocolitica, Klebsiella Pneumoniae, Enterobacter cloacae, Citrobacter freundii, Vibrio alginolyticus, Vibrio parahaemolyticus, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, or Trichomonas vaginalis. It could detect a minimum of 100 copies per reaction. In clinical 98 samples of vaginal swabs from pregnant women, the agreement rate between the GBS-RPA-LFS method and TaqMan real-time fluorescence quantification method was 95.92%. In conclusion, this study successfully established a combined RPA and LFS GBS in situ detection platform, with short reaction time, high sensitivity, high specificity, portability, and device independence, providing a feasible strategy for clinical GBS screening.

Novel amplified fragment length polymorphism (AFLP) markers for typing medically relevant Fusarium and allied fusarioid genera

Fusariosis is an emerging mycosis caused by diverse Fusarium and allied fusarioid genera that are characterized by spindle-shaped macroconidia. These fungi possess a broad ecological distribution, causing infections in a wide diversity of hosts, spanning the animal and plant kingdoms. The spectrum of human fusariosis encompasses superficial lesions like keratitis and onychomycosis to invasive fungal diseases. Notable genera within the medically relevant fusarioid group include Neocosmospora, Fusarium s. str., and Bisifusarium. While Neocosmospora species (formerly F. solani species complex) are primary causative agents of human fusariosis, instances involving Fusarium s. str. and Bisifusarium (formerly F. dimerum species complex) have been reported. There is an urgent need for DNA-based markers to explore the epidemiology of these emerging fusarioid pathogens using molecular methods. We took advantage of fusarioid genomes available in NCBI (n = 20) to optimize the development of novel amplified fragment length polymorphism (AFLP) markers by conducting in-depth in silico analyses to refine their applicability for studies on these pathogens’ genetic epidemiology. In-silico screening highlighted eight primer pair combinations (C1–C8) to be tested in vitro. The AFLP protocol was used for genotyping 40 medically relevant fusarioid fungi. Based on the overall scored AFLP markers (77–93 fragments), the values of polymorphism information content (PIC = 0.3474–0.3725), marker index (MI = 0.0038–0.0056), effective multiplex ratio (E = 26.3750–40.4750), resolving power (Rp = 40.1500–54.6000), discriminating power (D = 0.7978–0.8857), expected heterozygosity (H = 0.4476–0.4949), and mean heterozygosity (Havp = 0.0001) demonstrated the utility of these primer combinations for discriminating Neocosmospora, Fusarium s. str., and Bisifusarium species. Of relevance, some AFLP panels were better than others at studying genetic trends in Neocosmospora (#2 EcoRI-AT/MseI-TA, #3 EcoRI-AA/MseI-TT, and #5 EcoRI-AT/MseI-AG) or Fusarium s. str. (mainly #2 EcoRI-AT/MseI-TA and #6 EcoRI-GA/MseI-TT) and Bisifusarium (#1 EcoRI-GA/MseI-AG and #6 EcoRI-GA/MseI-TT), and these combinations will better resolve disease transmission routes. Our DNA fingerprint assay has proven effective by exhibiting rapidity, reproducibility, and high discriminatory capabilities, which represents a valuable asset in the ongoing efforts to combat fusariosis and enhance our scientific understanding of medically relevant Fusarium and allied fusarioid genera.

Effects of primers, PCR approaches and sample preservation methods on diversity studies of myxobacteria

Myxobacteria have potential application value in developing new antibiotics and environmental protection. In this study, in order to establish a more suitable method for diversity studies of myxobacteria, the effects of primers, polymerase chain reaction (PCR) approaches and sample preservation methods on the results were compared by Illumina high-throughput sequencing. The results showed that the relative abundance and operational taxonomic unit (OTU) ratio of myxobacteria amplified by the universal primers accounted for 0.91–1.85% and 2.82–4.10% of total bacteria, indicating that myxobacteria were the dominant bacteria both in population and species numbers. The relative abundance and OTU number and ratio of myxobacteria amplified by the myxobacteria semi-specific primers were significantly higher than those amplified by the universal primers, of which the primer pair W2/802R specifically amplified myxobacteria of suborder Cystobacterineae, while the primer pair W5/802R mainly amplified myxobacteria of suborder Sorangineae and also amplified more species of suborder Nannocystineae at the same time. Among three PCR approaches, the relative abundance and OTU ratio of myxobacteria amplified by the touch-down PCR were the highest. More myxobacterial OTUs were detected in most dried samples. In conclusion, the combination of the myxobacteria semi-specific primer pairs W2/802R and W5/802R, touch-down PCR, and dry preservation of samples were more conducive to diversity studies of myxobacteria.

Development of a fluorescent recombinase-aided isothermal amplification-based nucleic acid assay for detection of Leishmania

To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania. Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum. After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA. A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.

Morphological and Molecular Characterization of Quinoa Genotypes

Quinoa cultivation has expanded from South America to many countries because of its wide adaptability and nutritional value. We evaluated 32 introduced quinoa genotypes using 17 qualitative and 11 quantitative traits under Saudi Arabia conditions during the 2018–2019 season. The quinoa genotypes showed considerable variation during the vegetative and maturity stages. Plant height values varied between 60 and 18 cm, and maturity ranged from 98 to 177 days. Leaf shapes were rhomboidal or triangular, with dentate or serrate margins. Green was the standard color for leaves and panicles at the flowering stage. The leaf granule colors were white, purple, and white-red. At 150 units of the Euclidean distance, the genotypes aggregated into four major groups based on their morphological traits. Twenty-one sequence-related amplified polymorphism (SRAP) primer pair combinations generated 75 amplified fragments (alleles), with a mean of 3.57 alleles per primer pair combination. Unweighted Pair-Group Method with Arithmetic means (UPGMA) clustering analysis showed that the quinoa genotypes were grouped based on origin or according to genetic background. Genotypes from South America presented higher mean values for the average number of alleles, Shannon index (0.411), gene diversity (0.271), and polymorphic percentage (83.95). Analysis of molecular variance (AMOVA) showed that most of the genetic variation was because of differences within populations (86%). The wide variability of the genotypes studied herein is of great importance for quinoa breeders.

Development of a set of oligonucleotides for the identification of the Sakhalin sturgeon Acipenser mikadoi Hilgendorf, 1892 by PCR

Sturgeons (family Acipenseridae) are valuable commercial fish and aquaculture resources. The Acipenser genus includes about 20 species, 12 of which are recorded for the Russian territory. One of the rarest is the Sakhalin sturgeon A. mikadoi Hilgendorf, 1892. At present, its population size is significantly decreased and the species is close to extinction. Natural populations of the species have survived in the Tumnin River in Khabarovsk Region and the Viakhtu River in Sakhalin Region. Vihtu River in Sakhalin Region. Due to the small numbers of rare and endangered fish species, noninvasive approaches, including the use of DNA from the environment, appear to be the most suitable for monitoring their species and genetic diversity. The method is well established in this area and has been successfully tested for the monitoring of several species of rare and endangered sturgeons. This paper presents the results of the development of primers specific for the mitochondrial DNA of the species A. mikadoi. A matrix of 15 sequences of complete mitochondrial genomes from 3 sturgeon species whose ranges may overlap - A. mikadoi (KX276658), A. dauricus (KJ402277) and A. schrenckii (MH973728- MH973734, KX276660, KX276659, KF150287, KC905169, KC820796) - was generated. A. medirostris (NC_028405), which is genetically closest to the Sakhalin sturgeon, was also included in the analysis. A sliding window algorithm was applied to the sequence matrix. A graph showing the distribution of divergence values along the mitochondrial genome of sturgeon was plotted based on the results of the analysis Fig. 1. Based on matrices of individual genome fragments (16S rRNA, COI, ND1, and D-loop), a search for species-specific primers was performed in the DECIPHER program. A Primer-BLAST algorithm was run based on each pair of primers. Taxa Actinopterygii and Acipenseridae were used as references separately in different runs. In addition, primers were checked using local reference sequences of the complete mitochondrial genome of representatives of the genus Acipenser. The results of primer evaluation are summarized in the table Table 1. As a result, a combination of primer pairs developed on the basis of D-loop and COI fragments seems to be the most successful for error-free identification of Sakhalin sturgeon.

The development of SSR markers based on RNA-sequencing and its validation between and within Carex L. species

BackgroundCarex L. is one of the largest genera in the Cyperaceae family and an important vascular plant in the ecosystem. However, the genetic background of Carex is complex and the classification is not clear. In order to investigate the gene function annotation of Carex, RNA-sequencing analysis was performed. Simple sequence repeats (SSRs) were generated based on the Illumina data and then were utilized to investigate the genetic characteristics of the 79 Carex germplasms.ResultsIn this study, 36,403 unigenes with a total length of 41,724,615 bp were obtained and annotated based on GO, KOG, KEGG, NR databases. The results provide a theoretical basis for gene function exploration. Out of 8776 SSRs, 96 pairs of primers were randomly selected. One hundred eighty polymorphic bands were amplified with a polymorphism rate of 100% based on 42 pairs of primers with higher polymorphism levels. The average band number was 4.3 per primer, the average distance value was 0.548, and the polymorphic information content was ranged from 0.133 to 0.494. The number of observed alleles (Na), effective alleles (Ne), Nei’s (1973) gene diversity (H), and the Shannon information index (I) were 2.000, 1.376, 0.243, and 0.391, respectively. NJ clustering divided into three groups and the accessions from New Zealand showed a similar genetic attribute and clustered into one group. UPGMA and PCoA analysis also revealed the same result. The analysis of molecular variance (AMOVA) revealed a superior genetic diversity within accessions than between accessions based on geographic origin cluster and NJ cluster. What’s more, the fingerprints of 79 Carex species are established in this study. Different combinations of primer pairs can be used to identify multiple Carex at one time, which overcomes the difficulties of traditional identification methods.ConclusionsThe transcriptomic analysis shed new light on the function categories from the annotated genes and will facilitate future gene functional studies. The genetic characteristics analysis indicated that gene flow was extensive among 79 Carex species. These markers can be used to investigate the evolutionary history of Carex and related species, as well as to serve as a guide in future breeding projects.

754. Performance Characterization of a Real Time PCR Assay for Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid and Sputum

Background Pneumocystis jirovecii pneumonia (PJP) affects immunocompromised patients and contributes significantly to mortality. Outcomes depend on early treatment, making timely and accurate diagnosis critical. Typically, PJP diagnosis is through identification of trophozoite or cyst forms in bronchoalveolar lavage (BAL) fluid or sputum, a labor-intensive and insensitive process. Options for more accessible and sensitive molecular detection are limited. It is known that patients may be colonized, which can cast doubt on the clinical significance of low levels of DNA amplification in qualitative result reporting. In this study, we describe a real time (rt) PCR assay utilizing analyte specific reagent primers targeting the mtLSU gene of P. jirovecii and correlate amplification with morphological PJP identification.MethodsIUHPL Clinical Microbiology assessed sputum or BAL fluid from 109 patients with clinical concern for PJP microscopically via fungal stains (GMS, calcofluor white). Comparative rtPCR was conducted as follows. First, 2µL of residual specimen or control were mixed with an 8µL combination of rtPCR mastermix, control DNA, and primer pairs (Simplexa). No nucleic acid extraction was performed. Real time PCR was executed and analyzed on the LIAISON MDX (DiaSorin) platform. Qualitative amplification results and cycle threshold (CT) values were correlated with microscopic methods to establish performance. Chart review was performed to assess the clinical impact of this assay.Results P. jirovecii was microscopically detected in 26% (29/109) of samples, while 31.1% (34/109) exhibited amplification by rtPCR. Agreement between the two methods was 95.4%; rtPCR demonstrated 100% sensitivity and 93.8% specificity in comparison.ConclusionOur results indicate that this assay has exceptional negative predictive value (100%), and therefore may be valuable as a screening test. Considering this data alone, the positive predictive value is lower (85.3%). Further examination of the data, however, revealed that 80% (4/5) of discrepant results demonstrated CT values of >34, while the highest CT for a microscopically positive sample was 31.2. Further clinical correlation may establish a CT cutoff that will reduce false positive and potentially clinically insignificant cases.Disclosures All Authors: No reported disclosures

Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus

Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect ≤ 141 fg of RNA when cDNA used as a template. The assay detected ≤ 0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15–20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.

Genome-wide mapping using new AFLP markers to explore intraspecific variation among pathogenic Sporothrix species.

Sporotrichosis is a chronic subcutaneous mycosis caused by Sporothrix species, of which the main aetiological agents are S. brasiliensis, S. schenckii, and S. globosa. Infection occurs after a traumatic inoculation of Sporothrix propagules in mammals' skin and can follow either a classic route through traumatic inoculation by plant debris (e.g., S. schenckii and S. globosa) or an alternative route through zoonotic transmission from animals (e.g., S. brasiliensis). Epizootics followed by a zoonotic route occur in Brazil, with Rio de Janeiro as the epicenter of a recent cat-transmitted epidemic. DNA-based markers are needed to explore the epidemiology of these Sporothrix expansions using molecular methods. This paper reports the use of amplified-fragment-length polymorphisms (AFLP) to assess the degree of intraspecific variability among Sporothrix species. We used whole-genome sequences from Sporothrix species to generate 2,304 virtual AFLP fingerprints. In silico screening highlighted 6 primer pair combinations to be tested in vitro. The protocol was used to genotype 27 medically relevant Sporothrix. Based on the overall scored AFLP markers (97-137 fragments), the values of polymorphism information content (PIC = 0.2552-0.3113), marker index (MI = 0.002-0.0039), effective multiplex ratio (E = 17.8519-35.2222), resolving power (Rp = 33.6296-63.1852), discriminating power (D = 0.9291-0.9662), expected heterozygosity (H = 0.3003-0.3857), and mean heterozygosity (Havp = 0.0001) demonstrated the utility of these primer combinations for discriminating Sporothrix. AFLP markers revealed cryptic diversity in species previously thought to be the most prevalent clonal type, such as S. brasiliensis, responsible for cat-transmitted sporotrichosis, and S. globosa responsible for large sapronosis outbreaks in Asia. Three combinations (#3 EcoRI-FAM-GA/MseI-TT, #5 EcoRI-FAM-GA/MseI-AG, and #6 EcoRI-FAM-TA/MseI-AA) provide the best diversity indices and lowest error rates. These methods make it easier to track routes of disease transmission during epizooties and zoonosis, and our DNA fingerprint assay can be further transferred between laboratories to give insights into the ecology and evolution of pathogenic Sporothrix species and to inform management and mitigation strategies to tackle the advance of sporotrichosis.

Phylotype and sequevar determination and AFLP fingerprinting of Ralstonia solanacearum strains causing bacterial wilt of potato in southeastern Iran

Bacterial wilt of potato caused by Ralstonia solanacearum is a severe threat to potato cultivation in Kerman province, located in southeastern Iran and the third largest potato producer in the country. The present study intends to determine the precise phylogenetic position, sequevar, and genetic diversity of R. solanacearum strains associated with the disease. To this end, the major potato-growing regions of the province (Jiroft and Laleh Zaar) were surveyed to collect tuber samples from affected potato plants showing typical wilt symptoms. Twenty-three strains were collectively purified and subjected to phenotypic and genotypic characterization. Biochemical tests showed that all the strains belonged to R. solanacearum biovar 2A. Furthermore, phylotype-specific multiplex polymerase chain reaction results indicated that the strains belonged to phylotype II. For sequevar typing as well as the determination of the phylotype subcluster (IIA/IIB), polymerase chain reaction amplicons of the endoglucanase precursor gene of 11 representative strains were sequenced. In phylogenetic analysis, the strains were grouped with the reference strains of phylotype IIB-1 retrieved from the GenBank. Then, to assess genetic diversity, 19 selected strains were subjected to genomic fingerprinting, i.e., amplified fragment length polymorphism (AFLP) using seven primer pair combinations. The AFLP data showed heterogeneity among the strains of R. solanacearum and were grouped into four clusters. With our knowledge, this is the first report of the phylotype, and sequevar determination (i.e., IIB-1) in addition to AFLP-based genomic fingerprinting of R. solanaceraum strains causing potato wilt in southeastern Iran.

Exploring the Archaeome: Detection of Archaeal Signatures in the Human Body.

Due to their fundamentally different biology, archaea are consistently overlooked in conventional microbiome surveys. Using amplicon sequencing, we evaluated methodological set-ups to detect archaea in samples from five different body sites: respiratory tract (nasal cavity), digestive tract (mouth, appendix, and stool) and skin. With optimized protocols, the detection of archaeal ribosomal sequence variants (RSVs) was increased from one (found in currently used, so-called “universal” approach) to 81 RSVs in a representative sample set. The results from this extensive primer-evaluation led to the identification of the primer pair combination 344f-1041R/519F-806R which performed superior for the analysis of the archaeome of gastrointestinal tract, oral cavity and skin. The proposed protocol might not only prove useful for analyzing the human archaeome in more detail but could also be used for other holobiont samples.

Amplified fragment length polymorphism analysis to assess genetic diversity in rice

Amplified Fragment Length polymorphism (AFLP) was studied involving 48 diverse rice genotypes, comprising of indica, japonica, and Tongil type (indica × japonica) varieties to assess the genetic diversity. Initially 64 primer pair combinations were screened of which 5 were found to be suitable producing clear fragments (35–500 bp). In total 700 amplicons (bands) were produced with an average of 140 bands per primer pair. Maximum (207) from EcoRI AC*/MseI-CAC and minimum (75) amplicons from EcoRI TG*/MseI-CTT primer pairs were observed. Minimum (0.973) and maximum (0.990) PIC values recorded for primer pair EcoRI AC*/MseI-CAC and EcoRI AC*/MseI-CAT, respectively. Pairwise genetic similarity estimates ranged in between 0.6412 and 0.943 with a mean of 0.797. Triguna, an indica high yielding variety (HYV), and a new plant type (NPT) rice, IR 7946-46-1-3-2 having indica x japonica parentage showed minimum genetic similarity coefficient (0.641) with maximum genetic divergence, whereas ADT 41 and Sasyasree, two indica HYVs displayed maximum genetic similarity coefficient—0.953 with lowest genetic distance. All accessions could be clearly identified by using five primer pairs, offers immense scope of AFLP in assessing genetic diversity in rice. The present study also identified prospective varieties for use in selective hybridization and productive progeny selection. Most of the varieties shared distinct clusters based on varietal types, race, parentage and growing area where those are cultivated traditionally albeit with a few exceptions. The dendrogram could demarcate the varieties irrespective of race with unique traits, albeit with exceptions, which warrants further experimentation involving more number of primers in future.

Morphological characteristics and genetic diversity of Colletotrichum horii infecting persimmon tree in China

Anthracnose is the most devastating disease affecting persimmon (Diospyros kaki Thunb.) cultivars in China, and is caused by the pathogen recently identified as Colletotrichum horii. In this study, the symptoms of anthracnose in persimmon fruits and trees in China, and the morphological characteristics of C. horii, were observed and recorded. Thirty simple sequence repeat (SSR) markers for C. horii were developed from the whole genome of the closely related species C. gloeosporioides, which is available from the GenBank database. After screening 30 combinations of primer pairs, six SSR markers were selected to amplify the genomic DNA of 23 isolates, to assess the degree of polymorphism and the reproducibility of the SSR markers. A total of 164 discernible bands were obtained when visualizing the amplicons by electrophoresis, 156 of which showed polymorphism. A dendrogram was constructed with FreeTree software (ver. 0.9.1.50) and the similarity coefficients ranged from 0.58–0.98. The result indicated there was genetic diversity in the C. horii population in China. There was an obvious correlation between the cluster groups and the sites from which the samples were isolated. The results of this study will facilitate a more detailed understanding of the population structure of C. horii in China.

Agromorphologic, genetic and methylation profiling of Dioscorea and Musa species multiplied under three micropropagation systems.

Plant in vitro vegetative propagation using classical semi-solid culture medium is limited due to the low degree of automation, suboptimal nutrient availability and induced physiological stress which often reduce its efficiency. Temporary Immersion System (TIS) emerged as an innovative approach to optimize and eliminate the drawbacks associated with the conventional system of micropropagation. In this study, both Dioscorea and Musa spp. were subjected to conventional semi-solid culture media, complete immersion in shaking liquid culture media and TIS using RITA bioreactor. In vitro grown plantlets were screened for possible vegetative changes using agro-morphological descriptors while genetic and methylation differences were assessed using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP). In vitro results showed that the number of shoots produced in Musa spp. varied significantly (P≤0.001) with the type of culture system. The highest mean shoot produced was observed with TIS (28.40) and the least using semi-solid culture medium (1.13). For Dioscorea spp., there was no significant interaction between the hormone combination and the culture system. However, the lowest mean shoot value (1.55) was observed in the semi-solid culture medium. Genetic analysis via AFLP using 15 primer pair combinations revealed that the 3 culture systems maintained genetic variation for Musa and Dioscorea spp. under in vitro and field conditions. Results showed 99% and 91% of the total bands were polymorphic under in vitro and field conditions respectively for Musa and 100% polymorphism for Dioscorea under in vitro and field conditions. Methylation investigation via MSAP using 12 primer pair combinations showed 25% and 46% polymorphic methylated-sensitive loci, 100% and 78% of non-methylated loci of the total bands generated under in vitro and field conditions respectively. Unmethylated (HPA+/MSP+) levels were highest in TIS (0.0842) as compared to CI (0.0227) and SS (0.0161) while full methylation or absence of target (HPA-/MSP-) was lowest in TIS (0.5890) and highest in SS (0.7138). For Dioscorea, 52% and 53% methylated sensitive loci and 100% non-methylated loci were polymorphic under in vitro and field conditions respectively. Although in vitro plant tissue culture techniques led to methylation at some loci of both species, there were no observable changes in the phenotype of both crops under field conditions. This also confirmed that not all methylation events lead to phenotypic changes.

Elit Domuz Ayrığı (Dactylis glomerata L.) Genotiplerinde Genetik Çeşitliliğin SSR Markörleri ile Belirlenmesi

Domuz ayrığı (Dactylis glomerata L.) yaygın olarak yetiştiriciliği yapılan, ekonomik açıdan önemli çok yıllık bir buğdaygil yem bitkisidir. Bu araştırma, Ege Tarımsal Araştırma Enstitüsü yem bitkileri ıslah programı kapsamında geliştirilen elit domuz ayrığı klonlarının akrabalık derecelerini ve genetik çeşitliliğini belirlemek amacıyla yürütülmüştür. Çalışmada 32 genotip ve 24 SSR primeri kullanılmıştır. Moleküler analiz sonucunda SSR primerleri toplamda 126 allel üretmiştir. Allel sayısı 3 ila 7 arasında değişmiş ve lokus başına düşen ortalama allel sayısı 5,25 olarak bulunmuştur. Elde edilen allel büyüklükleri ise 101 bp ile 354 bp arasında değişmiş ve polimorfizm oranı her primer için %100 olarak gerçekleşmiştir. Bireyler arasında uzaklık derecesi Jaccard genetik uzaklık katsayısı kullanılarak elde edilmiş ve 0,21 ile 0,84 arasında değişmiş ve genetik çeşitlilik seviyesi yüksek bulunmuştur. Genotiplerden elde edilen 126 allelin 28‘nin nadir alleller olduğu ortaya çıkmıştır. Jaccard genetik uzaklık katsayısı kullanılarak yapılan neighbor-joining analizi sonucunda oluşturulan dendrogram 3 ana gruba ayrılmıştır. A grubu en büyük grubu oluşturmuş ve bünyesinde 15 genotip barındırmıştır. B grubu orijini aynı bölge olan 13 genotiple ikinci büyük grubu oluşturmuştur. C grubu ise en küçük grup olup orijini Türkiye‘nin kuzeyi olan genotipleri barındırmıştır. Moleküler analizler domuz ayrığı genotiplerinin önemli derecede genetik varyasyon taşıdığı ve ıslah programı için değerli kaynaklar olduğu ortaya çıkmıştır. Bunlara ek olarak, SSR tekniğinin domuz ayrığı genotiplerini moleküler olarak tanımlamada oldukça uygun ve etkili bir teknik olduğu sonucuna varılmıştır.

AFLP Fingerprinting Analysis of Citrus Cultivars and Wild Accessions from Oman Suggests the Presence of Six Distinct Cultivars

Abstract A study was conducted to evaluate genetic relatedness of 27 citrus cultivars and 6 wild citrus accessions using AFLP fingerprinting. The 27 citrus cultivars belonged to Citrus sinensis, C. aurantifolia, C. aurantium, C. paradise, C. reticulata, C. limon, C. latifolia, C. maxima, C. limettoides, C. limetta, C. medica and C. Jambhiri. The wild cultivars were obtained from Oman while the other cultivars originated from Oman and other countries. AFLP analysis using 4 primer pair combinations resolved 910 polymorphic alleles. All citrus cultivars and accessions had low genetic diversity (H = 0.0281 to 0.1300), with the percent polymorphic loci ranging from 8 to 35%. Populations of the six wild citrus accessions showed a very low level of genetic diversity (< 0.0700). Cluster analysis of the 33 cultivars and accessions showed that they share a high level of genetic similarity (81‒99%; mean = 92%). The six wild accessions clustered into two main clusters, with the analysis indicating that the six wild accessions may make up six distinct cultivars. The study provides information on the phylogeny of citrus cultivars and citrus diversity in Oman, a country through which citrus moved in the past from Asia to different African and European countries. In addition, it shows that some distinct citrus cultivars are present in this part of the world.

Biochemical and molecular characterization of cowpea landraces using seed storage proteins and SRAP marker patterns.

Seven landraces of cowpea [Vigna unguiculata (L.) Walp.] were assessed for genetic variability in total proteins, protein fractions viz. albumins, globulins, prolamins, and glutelins by SDS-polyacrylamide gel electrophoresis and DNA polymorphism using sequence-related amplified polymorphisms (SRAP) markers. The solubility-based protein fractionation data indicated that the salt soluble fraction (globulin) and water-soluble fraction (albumin) proteins were the predominant fractions in cowpea seeds comprising 45–50.3% and 31.2–35.5% of total soluble proteins, respectively. The electrophoretic pattern revealed the molecular heterogeneity among total proteins as well as different protein fractions. The molecular weights of protein bands obtained by SDS-PAGE varied between 10 to 250, 15 to 110, 15 to 150, and 15 to 130 kDa for total proteins, albumins, globulins, and glutelins, respectively. A large number of bands were found common to the various landraces, indicative of their close relationship with one another. However, a few bands distinctive to some specific landraces were also detected, indicating varietal differences. A 34 SRAP primer pair combination generated a total of 1003 amplicons (loci) showed 100% polymorphism with an average of 0.93 polymorphism information content (PIC) value. Landraces displayed an average 0.50 similarity coefficient which clustered the landraces corresponding to their growth habit in main clusters and to their geographical origin in subcultures. Molecular and biochemical analysis were correlated with a medium level (Mantel test, r = 0.56, P < 0.02). These findings revealed that seed proteins and DNA polymorphism provide valuable information regarding the variability among landraces and this information could be utilized for breeding purposes in the enhancement of protein quality and quantity in grain legumes.

Elucidation of Genetic Markers for the Essential Oils Quantity in Mentha Plants F-AFLP

Medicinal plants are an essential source of therapeutic ingredients, being also the basis of traditional or original healing systems. Many medicinal plants belong to the family Lamiaceae. The Mint is the common name of about 25 perennial species of the genus Mentha. The essential oils of the Mentha species and their extracts are well documented and possess antimicrobial, antifungal, antiviral, pesticide, and antioxidant properties. The current study aimed to collect and genotype mint species from different sources using fluorescently labeled AFLP technique to asses the genetic variability and elucidate high-resolution genetic markers related to oil content and/or oil quantity. A total of 11 mint samples were analyzed using AFLP technique; among the three primer pair combinations showed number of peaks ranged between 49 to 117, with a minimum band size in base pair of 50 bp to 661 bp. The overall mean of number of bands per individual is 99, while the total number of polymorphic loci is 254. Based on the maximum likelihood hybrid index method, two samples were found misclassified or possibly hybrids. Out of the 254 polymorphic bands, 16 were found specific to the high oil-content group [A] compared to 95 bands that were specific for the low oil-content group [B]. In group A, two bands, P1.23 and P2.28, were found fixed as present in group A while absent from group B, the bands were scored from the primer pairs 1 and 2 of 297 and 140 bp, respectively; and considered as positive markers to high-oil content. However, no negative marker was found. When the two possible hybrids were inspected, the positive marker number 1 [PP1.23] was found consistent and absents in the two samples, which increase its validity as a positive marker for high-oil content, while was not the same case for the second marker. Comparing both the whole dataset and the selected markers using AMOVA and PCoA, the elucidated markers should higher genetic differentiation among the studied groups than the whole dataset. The selected markers can be applied for marker-assistant selection breeding program and early preselection of high-oil content samples for future studies related to the oil content in Mentha sp.