Specific DNA Markers Research Articles

Impact of genetics on third molar agenesis

The purpose of this investigation was to determine the impact of heritability on third molar agenesis in twins. The study sample consisted of 284 same sex twins (172 monozygotic and 112 dizygotic), whose mean ages were 19.7 ± 4.3 and 18.9 ± 4.8 years, respectively. The monozygotic group consisted of 36.3% males and 63.7% females, while the dizygotic group consisted of 50.1% males and 49.9% females. The zygosity of the twins was established using 15 specific DNA markers. The prevalence of third molar agenesis in monozygotic twins was 19.6%, which was higher than in the dizygotic twins group (15.50%) (p = 0.004). In both groups, third molar agenesis was more frequent in the maxilla than in the mandible (p = 0.000). Agenesis of the maxillary third molars was mostly affected by additive genetic factors (62–63%), with the common environment and the specific environment accounting for up to 25% and 13%, respectively. In contrast, agenesis of the lower third molars was associated with a higher additive genetic determination (81–83%), with the specific environment accounting for 17% to 19%. The study’s conclusion is that the formation of the third molars follicle is strongly controlled by additive genetic factors.

Multiplex PCR identification and culture-independent quantification of Bacillus licheniformis by qPCR using specific DNA markers

Probiotics benefits in fish farming have been usually inferred appraising the effects observed on the host and not through the direct assessment of probiotic dynamics in the host gut microbiota. To overcome this gap, quantitative PCR (qPCR) can be a powerful approach to study the bacterial dynamics in fish gut microbiota. The presented work proposes four B. licheniformis-specific DNA markers and details a qPCR method to track putative probiotics B. licheniformis on fish gut. The four B. licheniformis-specific DNA markers - BL5B (hypothetical protein BL00303), BL8A (serA2), BL13C (rfaB) and BL18A (ligD) - were selected and validated by PCR and multiplex-PCR with 20 B. licheniformis isolates and a broad range of non-target bacteria. To assess the dynamics of B. licheniformis in the digesta of farmed fish, a qPCR was validated using markers BL8A and BL18A and calibration curves obtained for both markers with digesta samples spiked with B. licheniformis cells showed a high correlation (R2 > 0.99) over 6 log units (CFU/reaction), and a limit of detection (LOD) as low as 247 CFUs/reaction. Furthermore, the consistent qPCR repeatability and reproducibility underline the specificity and reliability of the qPCR proposed. Ultimately, the possibility to monitor the dynamics of B. licheniformis probiotics in the gut microbiota of farmed fish might be instrumental to optimize best practices in aquaculture.

Characterization of specific DNA markers at VRN-H1 and VRN-H2 loci for growth habit of barley genotypes

Requirement of vernalization is an important factor which plays a crucial role in cereals to transit from vegetative to reproductive phase. There are three types of growth habit in barley: winter, spring and facultative types; in which spring type does not require vernalization but winter and facultative genotypes require full and partial vernalization, respectively. Combination of two loci, Vrn-h1 and Vrn-h2, regulates vernalization in barley genotypes. Specific DNA markers have been identified for growth habit regulator genes in barley. In this study, we examined 24 barley genotypes using specific primers for detecting Vrn-h1and Vrn-h2 loci. Results showed that among all differently suggested primer combinations, a few markers were precisely correlated with seasonal growth habit in barley. The specific markers of 600, 600 and 200 bps were verified for ZCCT-Ha, ZCCT-Hb and ZCCT-Hc loci, respectively. Our field growth habit test showed that cultivar Bahman as a winter growth habit, where all the others genotypes exhibited spring growth habit. By using specific primers for Vrn-h1, only Bahman cultivar produced 616 bp and 830 bp fragments and spring genotypes showed 574 bp or 616 bp alleles without any amplification for 830 bp fragments. Therefore, presence of 616 bp and 830 bp alleles together in each genotype can be considered as an informative marker for winter growth habit in barley. These informative markers can be used easily in barley breeding programmes for detection of growth habit types in the seedling stage.

Seedling Resistance to Stem Rust (<i>Puccinia graminis</i> f.sp.<i>tritici</i>) and Molecular Marker Analysis of Resistance Genes in Some Wheat Cultivars

Stem rust caused by Puccinia graminis Pers.f.sp.<i>tritici</i> Eriks.and E.Henn.(Pgt) is one of the most destructive diseases of wheat which causing considerable yield losses in wheat growing areas worldwide. It has become a renewed threat to global wheat production after the emergence and spread of race TTKSK (also known as Ug99) and related races from Africa. Races of the pathogen in the “Ug99 lineages” are of international concern due to their virulence for widely used stem rust resistance genes and their spread throughout Africa. Disease resistant cultivars provide one of the best means for controlling stem rust. Bale zone, located on the Southeast part of Ethiopia, is one of the main wheat growing regions, playing a pivotal role in the wheat stem rust epidemic in Ethiopia. This study investigated levels of resistance in key wheat cultivars (lines) grown in Bale zone using seedling resistance evaluation and marker aided selection. Twenty wheat cultivars were evaluated for their response to stem rust infection at seedling stage under green house condition. Wheat cultivars were challenged with four stem rust races <i>viz</i> TTKSK, TRTTF, TTTTF and JRQCQ. A high level of phenotypic variation was observed in response to these races in the test entries, allowing for selection in these germplasm as a pre-breeding work. Out of the tested cultivars, three wheat cultivars exhibited low infection types (0–2) response to all the four races and hence selected as a source of resistance to stem rust. In addition, the existence of <i>Sr2, Sr22, Sr24, Sr25, Sr26, Sr35</i> and Sr36 genes in wheat cultivars were assessed using specific DNA markers. Using molecular markers, resistance gene Sr2 was identified in 2 cultivars and <i>Sr24</i> in five cultivars. However, no <i>Sr25, Sr26, Sr35</i> and <i>Sr36</i> were identified in any cultivars tested using DNA markers. The results of both seedling evaluation and marker based resistance gene identification will enable to breed wheat varieties with durable resistance to stem rust disease.

Identification of a male-specific DNA marker in the large yellow croaker (Larimichthys crocea)

Understanding sex determination mechanisms is the first and most paramount step in the development of sex control breeding biotechnologies for fish. The large yellow croaker (Larimichthys crocea) is an economically vital fish in China and East Asia, which exhibits a sex-related growth dimorphism where females grow significantly faster than males. However, the lack of a rapid and accurate sex specific marker for the large yellow croaker has hampered the application of sex control in the breeding process, as well as sex-determination investigations of this species. In this study, a male-specific 15bp deletion in the third intron of the Dmrt1 gene was identified by comparative analysis of genomic re-sequencing data from male and female samples. Two pairs of primers (MS and MFS) were carefully designed; thus, the male-specific marker could be genotyped by PCR amplification and agarose gel electrophoresis. More importantly, this male-specific marker could also be applied to precisely identify the genetic sex of multiple fish. This male specific DNA marker will have potential applications in the elucidation of the sex determination mechanism and will also accelerate the development of sex control breeding in the large yellow croaker. Moreover, our current data further validated an significant genetic evidence that an XY-male determination mechanism is utilized in the large yellow croaker.

Microfluidic electrochemical multiplex detection of bladder cancer DNA markers

Bladder cancer (BC) is one of the most difficult cancers to diagnose, and is reportedly the most expensive cancer to treat and monitor due to the lack of accurate chemical sensors; only limited methods are available, which usually are restricted to cytology and cystoscopy. Thus a system is urgently needed to detect biomarkers that give an unambiguous diagnosis on BC, as it currently is among the top five cancers occurring. Here we present a multi-sensor microchip array which efficiently detects three specific bladder cancer DNA markers simultaneously with a detection limit of 250fM, which is well below the amount of DNA markers found in urine samples. The detection is based on the electrochemical response of a free-base porphyrin marker which is embedded in a molecular beacon; this system can easily be adapted to detect other DNA markers and developed for field applications, including point-of-care diagnostics, and gives an unambiguous readout within 20min.

Overexpression of the Bx7 high molecular weight glutenin subunit on the Glu-B1 locus in a Korean wheat landrace

The Glu-B1al (Bx7OE + By8) allele is important for bread-making quality. The allele was found in a Korean wheat landrace using specific DNA markers. Molecular analyses were conducted to identify the overexpressed Bx7 (Bx7OE) subunit of the allele. The Korean wheat landrace (accession ID: IT166460) showed a similar protein expression level of Bx7 subunit, i.e., overexpression of Bx7 subunit towards cv. Glenlea, Canadian Western Red Spring wheat, which harbors Bx7OE subunit of Glu-B1al as detected on SDS–PAGE (sodium dodecyl sulfate poly-acrylamide gel electrophoresis). In addition, 2-DE (two-dimensional electrophoresis) analysis revealed similar protein expression patterns of the Bx7 subunit regions of IT166460 and Glenlea. The proportion of Bx7 to total HMW-GSs (high molecular weight glutenin subunits) in IT166460 (56.17 ± 0.22%) was higher than that of Chinese Spring (34.75 ± 1.03%) and even that of Glenlea (46.25 ± 1.76%) as assessed by RP-HPLC (reverse-phase high-performance liquid chromatography). Overexpression of Bx7 subunit was caused by gene duplication and indels of the promoter region of the Bx7 gene. IT166460 attained the 43 bp indel of the promoter region, as did Glenlea, i.e., the amplicon size of IT166460 was the same as that of Glenlea. In addition, the nucleotides present in the duplicated gene in IT166460 were the same as those in Glenlea. Bx7OE subunit is critical for dough strength. However, most wheat accessions harboring the subunit are distributed in America. Furthermore, most Korean wheats have little genetic variation in glutenin composition and are associated with inferior bread quality. Hence, IT166460 could be used to improve bread-making quality in the Korean wheat breeding program.

Multiple DNA Markers for Identification of Xanthomonas arboricola pv. juglandis Isolates and its Direct Detection in Plant Samples.

Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of walnut (Juglans regia L.) bacterial blight (WBB), and has been associated to other walnut emerging diseases, namely brown apical necrosis (BAN) and vertical oozing canker (VOC), altogether severely affecting the walnut production worldwide. Despite the research efforts carried out to disclose Xaj genetic diversity, reliable molecular methods for rapid identification of Xaj isolates and culture-independent detection of Xaj in infected plant samples are still missing. In this work, we propose nine novel specific DNA markers (XAJ1 to XAJ9) selected by dedicated in silico approaches to identify Xaj isolates and detect these bacteria in infected plant material. To confirm the efficacy and specificity of these markers, dot blot hybridization was carried out across a large set of xanthomonads. This analysis, which confirmed the pathovar specificity of these markers, allowed to identify four broad-range markers (XAJ1, XAJ4, XAJ6, and XAJ8) and five narrow-range markers (XAJ2, XAJ3, XAJ5, XAJ7, and XAJ9), originating 12 hybridization patterns (HP1 to HP12). No evident relatedness was observed between these hybridization patterns and the geographic origin from which the isolates were obtained. Interestingly, four isolates that clustered together according the gyrB phylogenetic analysis (CPBF 1507, 1508, 1514, and 1522) presented the same hybridization pattern (HP11), suggesting that these nine markers might be informative to rapidly discriminate and identify different Xaj lineages. Taking into account that a culture-independent detection of Xaj in plant material has never been described, a multiplex PCR was optimized using markers XAJ1, XAJ6, and XAJ8. This triplex PCR, besides confirming the dot blot data for each of the 52 Xaj, was able to detect Xaj in field infected walnut leaves and fruits. Altogether, these nine Xaj-specific markers allow conciliating the specificity of DNA-detection assays with typing resolution, contributing to rapid detection and identification of potential emergent and acutely virulent Xaj genotypes, infer their distribution, disclose the presence of this phytopathogen on potential alternative host species and improve phytosanitary control.

Dostignuća u oplemenjivanju suncokreta na otpornost prema volovodu

Parasitic plant broomrape (Orobanche cumana Wallr.) is a major problem in sunflower (Helianthus annuus L.) production. Yield losses can be up to 100% when susceptible genotypes are grown. Eight known races of broomrape in sunflower have been identified and designated by letters from A to H. Five races have been identified (A, B, C, D and E) in the past, while more virulent races have recently appeared in the countries around the Black Sea and Spain. The race E has been predominant in Serbia for many years, although in some places a new race has appeared. Broomrape can be confined using chemical control, but growing resistant genotypes appears to be the most efficient and reliable way of control. The sources of resistance have mainly been found in the wild species of the genus Helianthus. Sunflower resistance to broomrape is controlled by Or genes. The system of resistance control depends on the race of broomrape and sources of resistance material. Over the last five decades, significant results have been achieved in sunflower breeding for the resistance to broomrape. Five dominant genes (Or1-Or5), which provide resistance to five races of broomrape (A-E), have been identified, establishing a set of five differential lines. The set was later supplemented with lines resistant to race F. Intensive work has been done at the Institute of Field and Vegetable Crops (IFVC) on: (1) finding the source of resistance towards races which overcome races E and F, in the wild species and cultivated sunflower, (2) determining the gene effects and mode of inheritance of this trait; as well as (3) research on the molecular level, focused on finding markers closely linked to Or genes. Genotypes were tested in naturally infested fields in Serbia, where race E is fully dominant; Spain, where race F is predominant; and in Romania and Turkey, where very virulent races (over race F) of broomrape are present. The results have indicated that several genotypes were selected as potentially resistant or highly tolerant to virulent races of broomrape present in these locations. Intensive research on the molecular level was focused on the development of specific DNA markers, in order to facilitate and accelerate the introduction of resistance into cultivated sunflower. By comparing the molecular profile of resistant and susceptible genotype, a polymorphism was observed on LG3 of the SSR map. The identification of closely related molecular markers will facilitate the introgression of the gene into different sunflower lines. Another way to control broomrape is the cultivation of sunflower hybrids tolerant to imidazolinone herbicides. This measure solves the problem of broomrape regardless of its racial composition.

Characterization of a wheat mutant missing low-molecular-weight glutenin subunits encoded by the B-genome

DH20, a new wheat mutant missing low-molecular weight glutenin subunits encoded by the Glu-B3 locus, was discovered among double haploid lines obtained from a cross between the Korean wheat cultivars Keumkang and Olgeuru. Absence of the Glu-B3 LMW-GS proteins was determined by one-dimensional gel electrophoresis (SDS-PAGE) and confirmed by two-dimensional gel electrophoresis (2-DGE) and tandem mass spectrometry (MS/MS). The deletion of Glu-B3 genes was also demonstrated using allele specific DNA markers. Basic agronomic traits, protein content, dough mixing properties and bread loaf volume of DH20 and parental wheat cultivars were evaluated in field-grown wheat over a two year period. This mutant is a valuable resource for understanding the function of individual LMW-GS alleles or genes at the Glu-B3 locus using either breeding or biotechnology approaches.

Seedling Resistance to Stem Rust and Molecular Marker Analysis of Resistance Genes in Wheat Cultivars of Yunnan, China.

Stem rust is one of the most potentially harmful wheat diseases, but has been effectively controlled in China since 1970s. However, the interest in breeding wheat with durable resistance to stem rust has been renewed with the emergence of Ug99 (TTKSK) virulent to the widely used resistance gene Sr31, and by which the wheat stem rust was controlled for 40 years in wheat production area worldwide. Yunnan Province, located on the Southwest border of China, is one of the main wheat growing regions, playing a pivotal role in the wheat stem rust epidemic in China. This study investigated the levels of resistance in key wheat cultivars (lines) of Yunnan Province. In addition, the existence of Sr25, Sr26, Sr28, Sr31, Sr32, and Sr38 genes in 119 wheat cultivars was assessed using specific DNA markers. The results indicated that 77 (64.7%) tested wheat varieties showed different levels of resistance to all the tested races of Puccinia graminis f. sp. tritici. Using molecular markers, we identified the resistance gene Sr31 in 43 samples; Sr38 in 10 samples; Sr28 in 12 samples, and one sample which was resistant against Ug99 (avirulent to Sr32). No Sr25 or Sr26 (effective against Ug99) was identified in any cultivars tested. Furthermore, 5 out of 119 cultivars tested carried both Sr31 and Sr38 and eight contained both Sr31 and Sr28. The results enable the development of appropriate strategies to breed varieties resistant to stem rust.

Colorectal Cancer Screening: FIT and Colonoscopy Dominate Multitarget DNA

The Cologuard test — a combination of fecal immunochemical test (FIT) with specific DNA markers — was recently endorsed by the U.S. Preventive Services

Genotyping bacterial and fungal pathogens using sequence variation in the gene for the CCA-adding enzyme.

BackgroundTo allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology.ResultsBased on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced.ConclusionsThese results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0670-2) contains supplementary material, which is available to authorized users.

Genetic Variation for Thermotolerance in Lettuce Seed Germination Is Associated with Temperature-Sensitive Regulation of ETHYLENE RESPONSE FACTOR1 (ERF1).

Seeds of most lettuce (Lactuca sativa) cultivars are susceptible to thermoinhibition, or failure to germinate at temperatures above approximately 28°C, creating problems for crop establishment in the field. Identifying genes controlling thermoinhibition would enable the development of cultivars lacking this trait and, therefore, being less sensitive to high temperatures during planting. Seeds of a primitive accession (PI251246) of lettuce exhibited high-temperature germination capacity up to 33°C. Screening a recombinant inbred line population developed from PI215246 and cv Salinas identified a major quantitative trait locus (Htg9.1) from PI251246 associated with the high-temperature germination phenotype. Further genetic analyses discovered a tight linkage of the Htg9.1 phenotype with a specific DNA marker (NM4182) located on a single genomic sequence scaffold. Expression analyses of the 44 genes encoded in this genomic region revealed that only a homolog of Arabidopsis (Arabidopsis thaliana) ETHYLENE RESPONSE FACTOR1 (termed LsERF1) was differentially expressed between PI251246 and cv Salinas seeds imbibed at high temperature (30°C). LsERF1 belongs to a large family of transcription factors associated with the ethylene-signaling pathway. Physiological assays of ethylene synthesis, response, and action in parental and near-isogenic Htg9.1 genotypes strongly implicate LsERF1 as the gene responsible for the Htg9.1 phenotype, consistent with the established role for ethylene in germination thermotolerance of Compositae seeds. Expression analyses of genes associated with the abscisic acid and gibberellin biosynthetic pathways and results of biosynthetic inhibitor and hormone response experiments also support the hypothesis that differential regulation of LsERF1 expression in PI251246 seeds elevates their upper temperature limit for germination through interactions among pathways regulated by these hormones. Our results support a model in which LsERF1 acts through the promotion of gibberellin biosynthesis to counter the inhibitory effects of abscisic acid and, therefore, promote germination at high temperatures.

Integration of molecular approaches in a program for the establishment of a reserve gene pool of the Russian endemic Siberian crane (Grus leucogeranus Pallas)

The paper provides an overview of molecular-genetic approaches to gene pool conservation, population-genetic monitoring of captive breeding, and reintroduction of the Russian endemic and world rare species, the Siberian crane (Grus leucogeranus Pallas). The following issues are discussed: the taxonomic position of the Siberian crane in the Gruidae family (Aves); estimation of the levels of genetic diversity, differentiation, and relatedness in the captive population; individual genetic passportization and identification by microsatellite loci; DNA analysis of the paternity of birds obtained as a result of artificial insemination; problems of inbreeding and their prevention in captivity; genetic identification of interspecific hybrids; and molecular sex determination by specific DNA-markers. Key directions for further research are suggested: broadening the scale of genetic monitoring in the captive population and analysis of genetic diversity of the Siberian crane in the wild.

The use of specific DNA markers for the identification of alleles of the FAD3 genes in rape (Brassica napus L.)

A search was conducted for the alleles responsible for the quality of food-grade rapeseed oil in a collection of 21 samples of spring and winter oilseed rape of Belarusian and Russian breeding. We also developed A- and C-gene-specific DNA markers to assess the genomic polymorphisms of rape for FAD3 genes and selected plants with a low content of linolenic acid for use in the selection process. The development of a method for identifying FAD3 alleles, which control the level of linolenic acid in rapeseed oil, as well as of the design for new dCAPS markers, enabled the identification of plants homozygous for individual FAD3A and/or FAD3C genes in the F2-generation. These plants are currently involved in the selection process of new varieties with a reduced content of linolenic acid in rapeseed oil.

Cytoplasmic genome types of European potatoes and their effects on complex agronomic traits.

BackgroundVarious wild species germplasm has been used in European potato breeding since the first introduction of potato (Solanum tuberosum L.) to Europe. As the plant cytoplasmic genome including chloroplast and mitochondrial genomes is transmitted only through the maternal parent, cytoplasmic markers are useful tools in breeding programs to determine cytoplasmic genome types and to trace maternal ancestors. The potato cytoplasmic genome can be distinguished into six distinct types (M, P, A, W, T, and D). Male sterility was found in genotypes with S. demissum-derived D-type cytoplasm and S. stoloniferum-derived W/γ-type cytoplasm. These wild species were frequently used to incorporate superior pathogen resistance genes. As a result, the percentage of these two types is increasing unintentionally in the European germplasm pool. Other than cytoplasmic male sterility, little is known about effects of the cytoplasmic genome on complex agronomic traits in potato.ResultThe cytoplasm types of 1,217 European potato cultivars and breeding clones were determined with type specific DNA markers. Most frequent were T- (59.4 %), D- (27.4 %), and W- (12.2 %) type cytoplasm, while A- (0.7 %) and M-type cytoplasm (0.3 %) was rare and P-type cytoplasm was absent. When comparing varieties with breeding clones, the former showed a relatively higher frequency of T-type and lower frequency of D- and W-type cytoplasm. Correlation analysis of cytoplasm types and agronomic data showed that W/γ-type cytoplasm was correlated with increased tuber starch content and later plant maturity. Correlation with quantitative resistance to late blight was observed for D-type and M-type cytoplasm. Both cytoplasm types had a positive effect on resistance.ConclusionThis study revealed and quantified the cytoplasmic diversity in the European potato germplasm pool. Knowledge of cytoplasm type is important for maintaining genetic diversity and managing the male sterility problem in breeding programs. This is the first comprehensive study to show correlations of distinct cytoplasmic genomes with complex agronomic traits in potato. Correlations particularly with tuber starch content and resistance to late blight provided new knowledge on cytoplasmic effects on these important traits, which can be exploited for genetic improvement of potato.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0545-y) contains supplementary material, which is available to authorized users.

Extensive sequence variation in rice blast resistance gene Pi54 makes it broad spectrum in nature.

Rice blast resistant gene, Pi54 cloned from rice line, Tetep, is effective against diverse isolates of Magnaporthe oryzae. In this study, we prospected the allelic variants of the dominant blast resistance gene from a set of 92 rice lines to determine the nucleotide diversity, pattern of its molecular evolution, phylogenetic relationships and evolutionary dynamics, and to develop allele specific markers. High quality sequences were generated for homologs of Pi54 gene. Using comparative sequence analysis, InDels of variable sizes in all the alleles were observed. Profiling of the selected sites of SNP (Single Nucleotide Polymorphism) and amino acids (N sites ≥ 10) exhibited constant frequency distribution of mutational and substitutional sites between the resistance and susceptible rice lines, respectively. A total of 50 new haplotypes based on the nucleotide polymorphism was also identified. A unique haplotype (H_3) was found to be linked to all the resistant alleles isolated from indica rice lines. Unique leucine zipper and tyrosine sulfation sites were identified in the predicted Pi54 proteins. Selection signals were observed in entire coding sequence of resistance alleles, as compared to LRR domains for susceptible alleles. This is a maiden report of extensive variability of Pi54 alleles in different landraces and cultivated varieties, possibly, attributing broad-spectrum resistance to Magnaporthe oryzae. The sequence variation in two consensus region: 163 and 144 bp were used for the development of allele specific DNA markers. Validated markers can be used for the selection and identification of better allele(s) and their introgression in commercial rice cultivars employing marker assisted selection.

A quantitative hybridization approach using 17 DNA markers for identification and clustering analysis of Ralstonia solanacearum

Ralstonia solanacearum (Rs) is a quarantine phytopathogenic bacterium accountable for heavy economic losses worldwide. Monitoring and eradication programmes required for this pathogen are dependent on the availability of time‐ and cost‐efficient detection and typing methods. However, members of the Rs species complex are characterized by a high phenotypic and genetic diversity, which requires improved diagnostics methods. The currently available full genome sequences of several Rs strains allow for the selection of novel specific DNA markers using comparative genomics tools. In this work, 17 novel markers were selected based on Rs‐specific protein domains and thoroughly validated for specificity and stability, both in silico and using ‘wet lab’ assays. Polymerase chain reaction‐ and hybridization‐based validation assays revealed that the DNA regions selected as markers were unevenly distributed amongst the tested strains, with nine markers present throughout the species complex. The distribution of the remaining eight markers was highly variable between the different analysed strains and enabled the attainment of strain‐specific dot blot hybridization patterns, particularly informative for typing. The average probability value of each strain being positive for each of the 17 markers was calculated by an algorithm and used to obtain a dendrogram representing hierarchical clustering analysis of Rs, according to the similarity of their hybridization patterns. This method should prove to be a robust and straightforward procedure for genotyping members of the Rs species complex. Furthermore, this quantitative hybridization approach will allow the construction of informative databases to determine new Rs genotypes and infer epidemiological patterns.

English

A total of 150 blood and urine samples each were collected from human immunodeficiency virus (HIV) positive patients who visited selected hospitals in Ebonyi State. The subjects were made up of 57 males and 93 female patients. The blood samples were screened for the presence of four human malaria parasites using parasitological examination of blood stained films and polymerase chain reaction (PCR). Out of the 150 urine samples from the HIV positive individuals, 88 urine specimens were identified to harbor Plasmodium species. 75 (50%) urine specimens were identified to harbor Plasmodium falciparum while 10 (6.67%) and 3 (2%) were recorded against Plasmodium malariae and dual infection of P. falciparum and P. malariae, respectively. The result of the comparison of the specimens used showed the same result. None of the isolates that were negative by PCR test using DNA primers (template) from blood gave positive results by urine samples. Furthermore, when the primers (rOVA1 and rOVA2 ; rV1V1 and rV1V2) specific for Plasmodium ovale and Plasmodium vivax, respectively were used, none of the two species mentioned were detected in both urine and blood samples, signifying that these species may be absent in our environment. Our study demonstrated highly, the presence of P. falciparum and P. malariae, especially when specific Plasmodium species DNA markers were used for the analysis. Key words: Urine, Plasmodium species, human immunodeficiency virus (HIV), polymerase chain reaction (PCR), microscopy.