DNA Fingerprinting Analysis Research Articles

Genetic Diversity Analysis and Development of DNA Fingerprints of 20 Indonesian Local Chili Pepper Varieties Based on SSR Markers

<p>Chili pepper is one of the most valuable<em> </em>horticultural crops, widely cultivated in Indonesia. Analysis of its genetic diversity is needed to develop successful breeding programs of local varieties. Simple sequence repeat (SSR), a robust molecular marker used for genetic diversity analysis in plant species, offers potential, reliable DNA fingerprinting method to assess genetic variation and varietal identification of chili pepper. Fifteen SSR markers were used in this study to analyze the genetic diversity and develop profiling identification of DNA fingerprint of local chili pepper varieties. Twenty local and two improved varieties of three chili pepper species, consisting of 3, 1, and 18 varieties of <em>Capsicum </em><em>frutescens</em>, <em>C. chinense</em>, and <em>C. annuum</em>, respectively, were assessed for their SSR polymorphism. A total of 87 alleles was obtained from the polymorphism analysis with high alleles variation (2–16 alleles) with average total allele of 5.8 and average polymorphism information content (PIC) of 0.59 (0.34–0.83). Clustering and Principle Coordinate Analyses (PCoA) classified the varieties into two groups with coefficient of similarity of 0.65 indicating their high genetic variability. Most local varieties belonged to the same cluster and separated from the two improved varieties. Based on PIC values and dendrogram with selected markers, five SSR markers, i.e. EPMS441, EPMS331, EPMS335, GPMS194, and CaSSRBio1.1, were identified as SSR marker set for DNA fingerprinting purposes. SSR marker set used in this study was successful in developing the varietal identity of local chili pepper varieties, as indicated by unique code of each variety.</p>

Examination of unidentifiable spined loach individuals found in the overlap zone of two tetraploid species within a single river in Japan

An endangered tetraploid spined loach species, Cobitis takenoi (Cypriniformes: Cobitidae; hereafter called Tango loach) is known to inhabit only a single river in Kyoto Prefecture, Japan. Since Tango loach was discovered recently, in 2010, and only described in 2016, its morphology, ecology, and genetics are not well studied. Another tetraploid spined loach species Cobitis sp. BIWAE type A (hereafter, called Ohshima loach) inhabits the same river. The two loaches are reported as morphologically distinguishable from each other. Although the habitats of the two species in the river are segregated (Ohshima loach and Tango loach inhabit the upper and lower reaches, respectively), they overlap to a small degree in the boundary area. Recently, some individuals with morphological characteristics that are intermediate between the two species were found in the overlap zone. It was suspected that hybrids between the two species were produced since breeding seasons of the two species overlapped. To investigate whether the two species produce hybrids, we performed mitochondrial and nuclear DNA analyses on the unidentifiable individuals. Eight individuals unidentifiable to the species level collected in the river between 2017 and 2018 were examined and compared with the Tango and Ohshima loach species. Using mitochondrial DNA (mtDNA) cytochrome b analysis, we found that six individuals had mtDNA types identical to Tango loach and two individuals had mtDNA types identical to Ohshima loach. Furthermore, sequencing analysis of nuclear recombination activating gene 1 (RAG-1) revealed that each species had species-specific alleles. The phylogenetic analysis indicated that alleles in Tango loach were divided into two clusters and those from Ohshima loach formed a single cluster. There were no discrepancies in the combination between mtDNA and nuclear DNA species types within each specimen. DNA fingerprinting analysis (AFLP) showed that the species-unidentifiable individuals exhibited distinctly segregated genetic groups corresponding with Tango and Ohshima loaches. In summary, no hybrids were detected from among any unidentifiable individual examined in this study. New conventional genetic method for discriminating the two sympatric loach species developed here can be effective tool for the conservation of the Tango loach since there was no strict diagnostic morphological character between them.

Evaluation of bacterial communities of Grana Padano cheese by DNA metabarcoding and DNA fingerprinting analysis

The composition of the bacterial community of Grana Padano (GP) cheese was evaluated by an amplicon-based metagenomic approach (DNA metabarcoding) and RAPD-PCR fingerprinting. One hundred eighteen cheeses, which included 118 dairies located in the production area of GP, were collected. Two hundred fifty-four OTUs were detected, of which 82 were further discriminated between dominant (32 OTUs; > 1% total reads) and subdominant (50 OTUs; between 0.1% and 1% total reads) taxa. Lactobacillus (L.) delbrueckii, Lacticaseibacillus (Lact.) rhamnosus, Lact. casei, Limosilactobacillus fermentum, Lactococcus (Lc.) raffinolactis, L. helveticus, Streptococcus thermophilus, and Lc. lactis were the major dominant taxa (‘core microbiota’). The origin of samples significantly impacted on both richness, evenness, and the relative abundance of bacterial species, with peculiar pattern distribution among the five GP production regions. A differential analysis allowed to find bacterial species significantly associated with specific region pairings. The analysis of pattern similarity among RAPD-PCR profiles highlighted the presence of a ‘core’ community banding pattern present in all the GP samples, which was strictly associated with the core microbiota highlighted by DNA metabarcoding. A trend to group samples according to the five production regions was also observed. This study widened our knowledge on the bacterial composition and ecology of Grana Padano cheese.

Elicitation of salt stress-tolerant mutants in bread wheat (Triticum aestivum L.) by using gamma radiation

BackgroundGiven the strategic importance of wheat being the staple food for the vast majority of people, it was necessary to know reasons for the contraction and decline of its area globally and consequently its lower yield. Among the most important of these problems is the problem of a high level of salinity in both soil and irrigation water coming at the forefront of environmental challenges that hinder its production process at the local and global levels. Therefore, the genetic improvement of wheat for salinity tolerance was one of the most important priorities of this investigation.ResultsThe seven wheat accessions (Sakha 8 and its six M5 derived mutants) succeeded in drawing unique cases of salinity tolerance and were excellent especially mutants 1, 2, 3, and 5. The rest genotypes were coming in the second rank for this purpose and were very good in this regard. The promising wheat genotypes which recorded high salinity tolerance in the recent investigation exhibited also high genetic stability. This fact was proved after estimating some agro-morphological and physiological traits related to salinity tolerance based on evaluating some important genetic parameters besides salinity tolerance indices under stress experiment compared to the control treatment within two seasons. Data evaluated of expected genetic advance (GA) based on 5% selection proved that all values calculated during the two seasons under both treatments appeared low for all studied traits in this regard. However, it reflects the success of breeding for salinity tolerance in wheat using mutations but in relative terms. Molecular marker analysis profile using the six ISSR primers exhibited a total of 173 markers, 12 of them were monomorphic, while that 161 bands appeared polymorphic included 56 unique bands or positive markers and 7 negative markers with 93.06 % (polymorphism).ConclusionThe original wheat variety (Sakha 8) and its six M5 derived mutants exhibited high tolerance of salinity stress in all studied traits based on all genetic parameters and salinity tolerance indices calculated for both seasons under salinity treatment compared to the normal conditions. DNA fingerprinting analysis as well for the six wheat mutants besides the local variety proved that these genotypes were recorded highly genetic differences among them.

Rapid Protocol for Characterizing Klebsiella pneumoniae Isolates by Pulsed Field Gel Electrophoresis (PFGE) in Contour Clamped Homogeneous Electric Field (CHEF) Minigels

Klebsiella pneumoniae strains may act as opportunistic pathogens infecting susceptible patients, or as hypervirulent germs infecting healthy people, or as multidrug-resistant organisms due to extended-spectrum beta-lactamase (ESBL)-production provoking infection outbreaks in hospital settings and communities. Consequently, it is a priority to encourage the development of simple, rapid and economical methods for the molecular subtyping of the outbreak strains and then to facilitate the infection control. Pulsed field gel electrophoresis (PFGE) remains a good choice for K. pneumoniae subtyping and for the laboratory confirmation of outbreaks. We report the implementation of a rapid protocol for characterizing K. pneumoniae isolates by contour clamped homogeneous electric field (CHEF) in mini gels in a total analysis time of less than 12 h. This approach is based on the preparation of K. pneumoniae DNA of PFGE-quality by a single incubation step of immobilized bacteria for 2 h with a nonenzymatic solution containing 4 M urea and nonionic detergents with further XbaI DNA fingerprint resolution in CHEF mini gels in 5.5 h. The DNA suitable for PFGE obtained by this procedure was approximately 91% of the total DNA in the plugs. This procedure enabled the reproducible subtyping of 26 isolates of K. pneumoniae and three of K. oxytoca. The XbaI DNA fingerprint analysis in CHEF mini gels was able to discriminate the seven isolates of K. pneumoniae that were epidemiologically related and the nineteen that were unrelated. The nonenzymatic DNA plugs preparation reported in this paper is a cost-effective alternative for K. pneumoniae PFGE subtyping in laboratories where the technique is already implemented.

Stress tolerant rhizobia inhabiting the root nodules of Gliricidia sepium from selected locations of Anuradhapura district, Sri Lanka

The symbiotic association between Rhizobium and leguminous species plays a significant role in sustainable agricultural systems as it contributes higher amounts of fixed nitrogen to soil through biological nitrogen fixation. Gliricidia sepium is one of the most commonly cultivated agro forestry trees in the world. This leguminous tree is widely distributed throughout Sri Lanka. A very limited number of studies are available on G. sepium–Rhizobium symbiosis in Sri Lanka. The main objective of this study is to isolate and identify the stress tolerant Rhizobium sp. in G. sepium as a preliminary approach to use rhizobial strains for cross inoculation of crop legumes in order to minimize the heavy use of chemical nitrogen fertilizers. The root nodules of G. sepium were collected from seven sites in Anuradhapura district which is located in the dry zone of Sri Lanka. A total of 34 strains were tested for their tolerance to different physiological conditions namely pH, salinity, drought and temperature. Fifteen strains showed a higher tolerance among the isolated 34 strains when grown under a wide range of physiological conditions. When these 15 stress tolerant rhizobial strains were grown under extreme physiological conditions, 12 strains could survive well as they often experience adverse environmental conditions in their natural habitat. DNA fingerprinting analysis with ERIC 1R and ERIC 2R showed that the 15 strains are genetically diverse and they belong to 9 clusters at 71% similarity level

DNA fingerprinting and genetic diversity analysis with SCoT markers of Persian walnut populations (Juglans regia L.) in Iran

Juglans regia L. is the most widely cultivated and economically important species of edible nuts. Iran is known as a center of origin of genetically rich walnut germplasm and expected to be found a large diversity within Iranian walnut populations. A detailed population genetic of local populations is useful for developing an optimal strategy for in situ conservation and can assist the breeders in crop improvement programmes. The main aim of the present study was genetic fingerprinting of 20 Persian walnut populations, including 3 wild and 17 cultivated populations by using SCoT markers. We also aimed to evaluate the discriminating power of SCoT markers in these populations. The correlation between genetic distance and geographical distance has also investigated. The results showed that SCoT marker has a good discrimination power and can differentiate the studied populations. This marker can be used to evaluate genetic diversity, identify genotypes and DNA finger printing of Persian walnut populations. The present study also revealed that there are at least two genetic groups in the studied Persian walnut populations and the geographical features of cultivated populations can be influenced grouping of them. The studied populations differed genetically and showed isolation by distance.

Microbial community analysis using high-throughput sequencing technology: a beginner's guide for microbiologists.

Microbial communities present in diverse environments from deep seas to human body niches play significant roles in the complex ecosystem and human health. Characterizing their structural and functional diversities is indispensable, and many approaches, such as microscopic observation, DNA fingerprinting, and PCR-based marker gene analysis, have been successfully applied to identify microorganisms. Since the revolutionary improvement of DNA sequencing technologies, direct and high-throughput analysis of genomic DNA from a whole environmental community without prior cultivation has become the mainstream approach, overcoming the constraints of the classical approaches. Here, we first briefly review the history of environmental DNA analysis applications with a focus on profiling the taxonomic composition and functional potentials of microbial communities. To this end, we aim to introduce the shotgun metagenomic sequencing (SMS) approach, which is used for the untargeted ("shotgun") sequencing of all ("meta") microbial genomes ("genomic") present in a sample. SMS data analyses are performed in silico using various software programs; however, in silico analysis is typically regarded as a burden on wet-lab experimental microbiologists. Therefore, in this review, we present microbiologists who are unfamiliar with in silico analyses with a basic and practical SMS data analysis protocol. This protocol covers all the bioinformatics processes of the SMS analysis in terms of data preprocessing, taxonomic profiling, functional annotation, and visualization.

Simple Sequence Repeat-Based Genetic Diversity and Analysis of Molecular Variance among on-Farm Native Potato Landraces from the Influence Zone of Camisea Gas Project, Northern Ayacucho, Peru

The Andean native potato is an important food security crop and constitutes a gene pool for potato-breeding. To establish the current state of the native potatoes diversity in the influence zone of the Camisea Gas Project, North Ayacucho, Peru, landraces (n = 144) were collected. Three sampling populations (Anco, Chungui and Ticllas) were resolved using 10 simple sequence repeats (SSRs) markers that amplified 67 polymorphic alleles. Principal component, correspondence and cluster analysis revealed a minimum set of six SSRs to achieve DNA fingerprinting and cost-effective genetic diversity analysis. Analysis of molecular variance (AMOVA), average fixation index (FST = 0.279), and genetic distance index (SB) indicated that the genetic diversity of the native potatoes is high at the intra- and inter-population levels, and each of the three sampling populations constituted closed populations. The three populations were genetically distinct and contained unique genotypes and exclusive alleles. Misidentified native potatoes (10.41%) were found.

Vulcaniibacterium gelatinicum sp. nov., a moderately thermophilic bacterium isolated from a hot spring.

The present study aimed to determine the taxonomic positions of strains designated R-5-52-3T, R-5-33-5-1-2, R-5-48-2 and R-5-51-4 isolated from hot spring water samples. Cells of these strains were Gram-stain-negative, non-motile and rod-shaped. The strains shared highest 16S rRNA gene sequence similarity with Vulcaniibacterium thermophilum KCTC 32020T (95.1%). Growth occurred at 28-55 °C, at pH 6-8 and with up to 3 % (w/v) NaCl. DNA fingerprinting, biochemical, phylogenetic and 16S rRNA gene sequence analyses suggested that R-5-52-3T, R-5-33-5-1-2, R-5-48-2 and R-5-51-4 were different strains but belonged to the same species. Hence, R-5-52-3T was chosen for further analysis and R-5-33-5-1-2, R-5-48-2 and R-5-51-4 were considered as additional strains of this species. R-5-52-3T possessed Q-8 as the only quinone and iso-C15:0, iso-C11:0, C16 : 0 and iso-C17 : 0 as major fatty acids. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified polar lipids and two unidentified phospholipids. The genomic G+C content was 71.6 mol%. Heat shock proteins (e.g. Hsp20, GroEL, DnaK and Clp ATPases) were noted in the R-5-52-3T genome, which could suggest its protection in the hot spring environment. Pan-genome analysis showed the number of singleton gene clusters among Vulcaniibacterium members varied. Average nucleotide identity (ANI) values between R-5-52-3T, Vulcaniibacterium tengchongense YIM 77520T and V. thermophilum KCTC 32020T were 80.1-85.8 %, which were below the cut-off level (95-96 %) recommended as the ANI criterion for interspecies identity. Thus, based on the above results, strain R-5-52-3T represents a novel species of the genus Vulcaniibacterium, for which the name Vulcaniibacterium gelatinicum sp. nov. is proposed. The type strain is R-5-52-3T (=KCTC 72061T=CGMCC 1.16678T).

Utilizing Start Codon Targeted (SCoT) markers for DNA fingerprinting and diversity analysis of Artemisia herba-alba plant species

Utilizing Start Codon Targeted (SCoT) markers for DNA fingerprinting and diversity analysis of Artemisia herba-alba plant species

Keragaman Isolat Khamir Osmofilik pada Madu Hutan dari Sulawesi Tengah Ditinjau Menggunakan Teknik RAPD

Honey is a sweet substance with high sugar content produced by bees from flower nectar. Osmophilic yeast is one of organisms that can grow in honey because their ability to adapt in high osmotic pressure environment. Twenty-seven osmophilic yeast isolates were obtained from 11 forest honey samples to this research. This research objective is to determine the diversity of yeast isolates from genotypic characterization. Genotypic characterization was done using DNA fingerprint analysis using the RAPD PCR technique. This research used 2 types of primers, namely CDU primer (5′-GCGATCCCCA-3 ′) and M13 primer (5′-GAGGGTGGCGGTTCT GAGGGTGGCGGTTCT-3 ′). Data in the form of electrophoregram are represented in table n (yeast strain) x t (character units) using binary scoring type. The data were analyzed using a numerical taxonomy method with similarity (OTU) and determined using Jaccard coefficient (SJ) in 70% of IS limit. The results of this research showed that 27 yeast isolates were divided into 2 clusters and 10 outliers with a correlation coefficient (r) of 0.924624

Low-energy heavy-ion beam induced high-yield mutation breeding of Thai jasmine rice (Oryza sativa L. cv. KDML 105)

Thai jasmine rice was improved by low-energy heavy-ion beam induced mutation breeding for high yields. Seeds of Thai rice KDML105 and its ion-beam-induced primary mutant BKOS6 were bombarded by mixed atomic and molecular nitrogen ions accelerated by tens kV in a home-developed high-current ion implanter. Both phenotypes and genotypes of induced mutants were investigated. In M1 generation, more than a hundred plants with the photoperiod-insensitivity potential were obtained. In the subsequent generations, tens of rice mutants with a broad spectrum of phenotypic variations dominantly supporting the high yield potential were selected and studied for the mutation stability till M5 generation. The mutants possessed not only high crop yields but also other properties improved or altered in the grains. DNA fingerprinting analysis revealed polymorphisms in the mutants distinguished from that of KDML105. The cDNA fingerprinting investigation indicated four additional fragments in the mutant profiles encoding proteins which could be involved in the high yield characteristics of the mutants.

Impact of fruit shape selection on genetic structure and diversity uncovered from genome-wide perfect SNPs genotyping in eggplant

The recently constructed eggplant genome provides an opportunity for genome-wide marker exploration to be used for variety identification and genetic analysis. SNPs with high density across the genome and genetic stability are suitable for DNA fingerprint and genetic analysis. In this study, we used the eggplant reference genome of line 63/7 and resequencing data of 45 unique lines to select 219 genome-wide perfect SNPs in the eggplant. The high-throughput SNP genotyping technique target SNP-seq was used to successfully validate the 219 SNPs across 377 eggplant varieties to establish the unique DNA fingerprint for each variety. In addition, we chose 36 SNPs as a core SNP set with the ability to differentiate 95% of 377 eggplant varieties. Model-based structure and principal component analysis suggested three major population groups and indicated a significant impact of long-term selection for fruit shape in eggplant varieties. Further genetic diversity analysis within the three population groups showed a considerably narrow genetic background in round- and oval-fruited eggplants. This could be a result of limited choices of varieties used for breeding of round- and oval-fruited eggplants in Asia, indicated by its low observed heterozygosity (Ho) value (0.152) and high inbreeding coefficient (0.442). Finally, a genome-wide association study based on the 219 SNPs identified five associated SNPs located near the SUN and OVATE homologs, which had conserved function in controlling the fruit shape. This study signals a risk of genetic erosion in the round- and oval-fruited eggplants and provides valuable information for future variety management and breeding programs.

Changes in rhizosphere bacterial communities associated with tree decline: grapevine esca syndrome case study.

An investigation was carried out on rhizosphere bacteria to determine if they may be associated with perennial crops affected by nonspecific decline, a phenomenon that is difficult to diagnose and prevent. Esca disease of grapevine was chosen for this case study because of its easy foliar symptom identification. Ribosomal DNA fingerprint analysis by polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE), quantitative PCR (qPCR), and rDNA amplicon sequencing by next-generation sequencing (NGS) were adopted to investigate the bacterial communities associated with grapevines, which were selected for the presence and absence of external foliar symptoms in 11 vineyards. According to PCR-DGGE and qPCR, bacterial communities differed in site of origin (vineyards), but not between symptomatic and asymptomatic plants, whereas qPCR gave a significantly higher presence of total bacteria and Pseudomonas spp. in asymptomatic plants. NGS confirmed no difference between symptomatic and asymptomatic plants, apart from a few minor genera (<0.5%) such as Salinibacterium, Flavobacterium, Nocardia, and Janthinobacterium, which were, in all cases, higher in asymptomatic plants and whose functional role should be the object of further investigation. The fact that total bacteria and Pseudomonas were more abundant in the rhizosphere of asymptomatic grapevines and that some bacterial genera were associated with the latter, represents a new element when investigating the multiple-origin phenomenon such as esca disease of grapevine.

Virulence and DNA fingerprinting analysis of Xanthomonas oryzae pv. oryzae identify a new pathotype in Guangxi, South China.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases affecting rice worldwide. However, little is known about the population structure of this organism in Guangxi Zhuang Autonomous Region, South China. Here, pathotypic and DNA fingerprint analyses were conducted to characterize the isolates of Xoo collected from rice leaves in five districts of the region from 2013 to 2016. Their pathogenicity was tested by leaf clipping, and the DNA fingerprints were analyzed by repetitive sequence-based polymerase chain reaction and endogenous insertion sequence element-based polymerase chain reaction assays using the repetitive extragenic palindromic sequence and enterobacterial repetitive intergenic consensus primers, respectively. Pathogenicity assays of 70 representative isolates were conducted using a series of near-isogenic lines and two new pathotypes were identified. All the pathotypes were found to be incompatible with xa5 and Xa7. One pathotype was virulent to Xa14, Xa21, and Xa23, whereas another virulent to Xa21 and Xa23, but incompatible with Xa14. A dendrogram generated for the data sets obtained from DNA fingerprinting suggested the prevalence of high genetic diversity of Xoo throughout Guangxi, and no association between the molecular haplotypes and pathotypes was identified.

Diversity and guaiacol production of Alicyclobacillus spp. from fruit juice and fruit-based beverages

Alicyclobacillus acidoterrestris is an acido-thermophilic, spore-forming bacterial species that can spoil acidic fruit juice and fruit-based beverages. The metabolism of taint compounds by this bacterial species has led to its status as a targeted microorganism in the fruit juice industry. This study aims to assess the genetic diversity of Alicyclobacillus spp. including A. acidoterrestris and its correlation to spoilage taint metabolism.Alicyclobacillus cultures, which were previously isolated from a wide range of domestic and international products including fruit juice, fruit drinks and fruit juice concentrates, were subjected to DNA fingerprint analysis by using randomly amplified polymorphic DNA (RAPD) – polymerase chain reaction. Isolates were classified on the basis of their RAPD profile and the results were used to select representative strains to undergo taint production assessment. The taint guaiacol produced by Alicyclobacillus spp. was measured by headspace gas chromatography and mass spectrometry.From produced RAPD profiles, two genotypic groups and two sub-groups were identified. The groups were independent of product types and geographical origins. A significant number of isolates were clustered in genotypic group I, including A. acidoterrestris ATCC 49025. These isolates produced significant levels of guaiacol, 8.7 mg/L on average. A smaller number of isolates was found in genotypic group II including A. acidocaldarius and they produced no guaiacol. Primer F-64 was useful to identify Alicyclobacillus at the species level, and permitted rapid identification of strains producing fruit juice taint compounds such as guaiacol.

Development and Application of EST-SSR Markers for DNA Fingerprinting and Genetic Diversity Analysis of the Main Cultivars of Black Locust (Robinia pseudoacacia L.) in China

Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree species which is used for pillar construction, honey production and soil improvement. More EST-SSR (Expressed sequence tag simple sequence repeat) markers of black locust can be used as a complement and improvement of Genomic-SSR markers for the identification of the function of gene and the construction of genetic map. Additionally, currently there is no simple method for identifying black locust cultivars. In this study, we obtained 2702 unigenes from 3095 expressed sequence tags (ESTs) from the National Center of Biotechnology Information (NCBI) database to identify simple sequence repeats (SSRs) in R. pseudoacacia samples. A total of 170 SSR loci were found to be distributed in 162 non-redundant sequences with a frequency of 6.29%. Dinucleotide repeats were the most predominant types among microsatellites (62.35%), followed by tri-nucleotide repeats (25.88%); the remaining SSRs accounted for less than 12%. The repeat motifs AG/TC (29.25%) and CT/GA (29.25%) were the most abundant among dinucleotides, and AAT/TTA (15.91%) was the most common among tri-nucleotides. A total of 62 primer pairs were designed to screen polymorphic and stable SSR loci. The resulting 25 EST-SSR markers capable of amplifying polymorphic, stable, and repeatable products. Eight newly developed EST-SSR markers and four published SSR markers were selected for DNA fingerprinting and genetic diversity analysis of the 123 main R. pseudoacacia cultivars in China. The 12 SSR loci amplified 102 alleles, with an average number of alleles per locus of 8.5 (range 4–15). The average polymorphism information content at the 12 SSR loci for the 123 cultivars was 0.670 (range 0.427–0.881). The 123 cultivars clustered into six main groups based on similarity coefficients, with most cultivars in one subgroup. Fingerprinting was performed using eight SSR markers; 110 black locust cultivars were distinguished. The results of this study increase the availability of EST-SSR markers in black locust and make it a simple method for checking the collection, the certification, and the correct attribution of clones and cultivars.

Characteristics of a novel cell line ZJU-0430 established from human gallbladder carcinoma

BackgroundGallbladder cancer is the most common malignant neoplasm of the biliary tract, responsible for 80–95% of cases. Appropriate models are required for investigating the molecular pathogenesis of gallbladder cancer.MethodsIn this study, we aimed to establish a gallbladder cancer cell line from primary tumour. Single cell RNA sequencing, Light and electron microscopy, DNA content analysis, cytogenetic analysis, short tandem repeat (STR) DNA fingerprint analysis, immunophenotypic characterization, and xeno-transplantation were utilized to characterize the novel ZJU-0430 cell line in vitro and in vivo.ResultsThe cell line showed multiple cell shapes and characteristic epithelial morphologies under the microscope, but no too much heterogeneity by scRNA-Seq, with a population doubling time (PDT) of 19.81 h, which was shorter than that for GBC-SD cells. An immunophenotypic analysis revealed that ZJU-0430 cells were positive for CD24, CD44, CD29 and CD133 expression, and partially positive for CD184, and CD326 expression, and negative for CD34, CD90, CD117, and CD338 expression, similar to the primary cancer cells. A pathological analysis confirmed the origination of cell line from gallbladder tumour. ZJU-0430 cells had higher migration, invasion and proliferation properties than GBC-SD cells in vitro, and showed in vivo tumorigenicity in nude mouse xenograft settings.ConclusionsThe results confirm the potential utility of ZJU-0430 cell line as a representative model of gallbladder cancer and suggest that it could be used in the in vitro and in vivo studies of gallbladder cancer pathogenesis and to develop new therapeutics.

DNA Fingerprinting and Diversity Analysis of Some Aus Rice Landraces

Objective: The allelic diversity and relationship among 48Aus rice landraces were determined through SSR markers. A total of 11 SSR markers were used to characterize all Aus rice genotypes. Methods/Findings: All of them were polymorphic showing different chromosome numbers. The number of alleles per locus varied from 3 alleles (RM234 and RM277) to 15 alleles (RM493). A total number of 72 alleles were detected with an average number of 8.91 alleles per locus. The polymorphic information content (PIC) values ranged from 0.19 (RM277) to 0.90 (RM493) with an average 0.70. The highest PIC value was obtained in RM493 (0.89) followed by RM526 (0.85) and The highest genetic dissimilarity (1.00) was found among the nineteen Aus genotypes combinations viz., Kalojira TAPL-68, Busmoti-sufoid, Hatisail, Sakharkhara, Khasa, Black, Ukinmodhu, Fulkari, Porbotjira, Straw THPL-554, Kalonunia, China IRRI, Mehedhan, Tulsimoni, Meny, Tilkapur, Nunia, Lalmota and Tapu. This material may be selected as potential parents for crop improvement for their distinctive characters. Whereas lowest genetic dissimilarity was found between Chini sagor-1 vs Malshira and Kalijir TAPL74 vs Kalojira (0.18) followed by three other pairs (0.28). Application: The genetic distance-based results seen in the Unweighted Pair Group Method of Arithmetic means (UPGMA) revealed six genetic groups. These landraces will be useful for the selection of diverse parents, background selection during backcross breeding programs and assist in broadening germplasm-based rice breeding programs in the near future. Keywords: DNA Fingerprinting, Genetic Diversity, Landraces, Microsatellites.