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Fast and Sensitive Silver Staining of DNA in Polyacrylamide Gels
Abstract
The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437–1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage φX174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).
... Silver staining method is described by Bassam et al. (1991) and it is highly efficient. It can detect nucleic acid in the pictogram range by the specific chemical reduction of silver ions. ...
... 1. Silver staining is carried out according to Bassam et al. (1991) with few modifications. 2. The gel is placed on clean surface by keeping the notched plate facing upwards 3. The gel along with the plate is placed in a suitable sized tray. ...
... The detailed protocol for isolation of DNA is as under1. Two volume of ice-cold lysis buffer is added to whole blood and mixed well by vortexing. ...
... PCR products of SSR markers are commonly separated using agarose or polyacrylamide gel electrophoresis and then visualized with silver staining or under UV light after staining with ethidium bromide. Silver staining of DNA fragments in polyacrylamide gels is more sensitive than the other staining methods 5,6 and has been widely used to detect DNA fragments such as SSR markers 7 . ...
... Like many biological laboratory techniques, silver staining of polyacrylamide gels has steadily improved since its first being reported as a fragment visualization technique in 1979 8 . The technique was initially modified for detection of DNA fragments by Bassam et al. 6 in 1991 and then improved by Sanguinetti and coworkers 9 in 1994. The method has been further optimized in the last few decades 6,7,9,10,11,12,13,14,15 . ...
... The technique was initially modified for detection of DNA fragments by Bassam et al. 6 in 1991 and then improved by Sanguinetti and coworkers 9 in 1994. The method has been further optimized in the last few decades 6,7,9,10,11,12,13,14,15 . However, most of these updated versions of the protocols still have some drawbacks such as high technical demand and long processing time for fixation and mounting 6 , that limit the application of these protocols 7,11 . ...
... The mixture was loaded on 8% polyacrylamide gel (acrylamide/bis-acrylamide 29:1, w/w) and electrophoresis was carried out using×0.5 tris-borate EDTA (45 mM tris-borate 1 -1 mM EDTA) at 150 V for 4 h. Distinct band patterns were detected by silver staining of the SSCP product (Bassam et al., 1991). Each animal showing different conformation banding pattern was assigned a specific genotype. ...
The experiment was conducted during January, 2022 to January, 2023 at the Department of Animal Genetics & Breeding, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Navsari, Gujarat, India to identify polymorphisms in HSP 70 gene. Heat Shock Proteins (HSPs) known as molecular chaperones are essential for cells’ ability to recover from stress and serve as the primary defence mechanism within cells. They are extremely conserved and essential to the response to heat stress and cellular thermotolerance. Even though there are numerous HSP genes, in livestock species, heat tolerance is primarily associated with HSP 70. This gene polymorphisms have been linked to heat tolerance, milk production, fertility and cattle susceptibility to disease. They can be utilised as genetic markers to help choose animals that are more resilient to climate change, have stronger immune systems and perform better overall. A 220 bp fragment of bovine HSP 70 gene was subjected to Polymerase Chain Reaction- Single-Strand Conformation Polymorphism (PCR-SSCP) technique to identify the polymorphism. PCR-SSCP pattern was associated with the thermotolerance traits in Kankrej cattle using the univariate GLM model of SPSS 26. HSP 70 gene (220 bp fragment) was found to be monomorphic documented on SSCP gel which revealed only one genotype (AA) in all Kankrej cattle. It is concluded that genotype and its association with thermotolerance traits were found to be non-significant. However, The HSP 70 polymorphism is expected to strongly predict cattle heat tolerance, aiding in selection for thermotolerance.
... SSR assays were performed according to standard protocols (Sayed et al., 2002). products were separated using a 6% polyacrylamide sequencing gel system (GIBCO/BRL, Life Technologies) and visualised by silver staining (Bassam et al., 1991). All the SSR markers used in mapping are described in Table S1. ...
Barley production is severely affected by drought caused by the unpredictable Mediterranean weather patterns, which include uneven rainfall and extreme temperatures. This leads to a decrease in crop yield. However, to tackle this issue, landraces and wild species are crucial sources of variation for stress adaptive traits. By incorporating these traits into improved varieties, we may see an increase in yield and stability under drought conditions. Seventy‐six quantitative traits loci (QTLs) identified traits were mapped using recombinant inbred lines (RIL) population Arta × Harmal‐2//Esp/1808‐4L, evaluated at six dry and semi‐dry areas over 3 years. The study investigated traits such as grain yield, biological yield, harvest index, kernel weight, seed per head, days to heading, kernel filling duration, growth vigour, growth habit, lodging and plant height. Numerous QTLs were discovered that are associated with various phenotypic traits related to grain yield, kernel yield, duration of filling period and days to heading. For areas with less than 250 mm/annum of rainfall, QTLs were identified on chromosome 2H for biological yield, days to heading, and kernel weight, on 1H for harvest index, and on 2H, 4H, and 5H for kernel weight. For semi‐dry areas with rainfall less than 450 mm, QTLs were found on chromosome 6H for grain yield, 2H and 5H for kernel weight, 1H and 6H for seed per head, and 2H for days to heading. Notably, these QTLs significantly explain more than 10% of phenotypic variation. The 2H chromosome was found to have the most important QTL and pleiotropic effect for yield and its components, such as kernel weight, days to heading, and biological yield. The cross Arta/Harmal was adapted, and mechanisms were developed to cope with drought stress, reflected by the significant and positive correlation of biological yield and harvest index with grain yield. Chromosomes 1H, 2H, 4H and 5H harbour more than 60% of mapped QTLs for dry areas. It is worth noting that the QTLs mentioned earlier, along with the kernel weight QTLs (QKW 1.5, QKW2.7b, QKW4.1, QKW6.7, QKW6.9), have consistently exhibited positive effects on crop yield in semi‐dry and dry areas, making them potential candidates for breeding drought‐tolerant crops. Genomic co‐localisation of the QTL for Arta/Harmal population suggested that selection for drought through linked markers can be an option for drought tolerance selection for barley in dry areas.
... For validation of the dCAPS marker reliability, DNA samples extracted from 344 individuals that were randomly selected from an F 2 population consisting of 3,000 plants and 34 inbred lines with varying degrees of white colour fruits were used for PCR amplification, where the dCAPS primers were used. After TaqI digestion, the products were detected using silver staining post 8% polyacryamide gel electrophoresis [32]. Polyacryamide gel (8%) electrophoresis was conducted using 0.5x TBE buffer at 200 V, 300 mA, around 1.5-2 h. ...
Fruit colour is a crucial factor influencing both the marketability and quality of pepper (Capsicum annuum), particularly ornamental varieties. Fruit colour is a complex multigenic trait in plant species. Previously, the Arabidopsis pseudo-response regulator 2 (APRR2) gene, one of the regulators that control fruit chlorophyll content and chloroplast development, thereby influencing fruit colour at the green immature stage in tomato (Solanum lycopersicum), cucumber (Cucumis sativus) and pepper, was reported. Functional molecular markers associated with this homologous gene in pepper could be employed to discern fruit colour at the seedling stage, thus improving the efficiency of green/white-fruited pepper breeding. In this study, a derived cleaved amplified polymorphic sequences (dCAPS) marker was developed based on the mutation site in the sequence of a CaAPRR2-like gene that was cloned from the inbred line B1-2, which exhibits milky white fruit at the green immature stage. The marker was subsequently validated in a CaAPRR2-like progeny segregation population. After digestion with the restriction endonuclease TaqI, the amplification product exhibited evident polymorphic bands, enabling it to distinguish between peppers with milky white and green fruit colours. The developed molecular marker displayed remarkable stability and repeatability, thus offering a straightforward and effective tool for enhancing fruit colour in pepper breeding programs.
... PCRs were performed using a Veriti 96-well Thermal Cycler (Applied Biosystems, Foster City, CA, USA) with the following program: initial denaturation at 95°C for 3 min; 30 cycles at 95°C for 15 s, 55°C for 15 s, 72°C for 30 s, and a final extension at 72°C for 10 min (or adjusted to give the best results). Two microliters of PCR product was run on 8% polyacylamide gels and visualized by silver staining (Bassam et al. 1991). ...
Chromosome segmental introgression lines (ILs)
are an effective way to utilize germplasm resources in crops.
To improve agronomic traits of wheat cultivar (Triticum
aestivum) Shi 4185, four sets of ILs were developed. The
donors were Chinese endemic subspecies accessions Yunnan
wheat (T. aestivum ssp. yunnanense) YN3, Tibetan semiwild
wheat (T. aestivum ssp. tibetanum) XZ-ZM19450, and
Xinjiang wheat (T. aestivum ssp. petropavlovskyi) XJ5, and
synthetic wheat HC-XM1620 derived from a cross between
T. durum acc. D67.2/P66.270 with Aegilops tauschii acc. 218.
Totals of 356, 366, 445 and 457 simple sequence repeat (SSR)
markers were polymorphic between Shi 4185 and YN3, XZZM19450,
XJ5 and HC-XM1620, respectively. In total, 991 ILs
were identified, including 300 derived from YN3, covering 95%
of the genome of Shi 4185, 218 from XZ-ZM19450 (79%), 279
from XJ5 (97%), and 194 from HC-ZX1620 (84%). The sizes and
locations of each introgression were determined from a
consensus SSR linkage map. Using the ILs, 11 putative quantitative trait loci (QTLs) were identified for plant height
(PH), spike length (SL) and grain number per spike (GNS).
Comparative analyses of 24 elite ILs with the parents revealed
that the four donor parents could be important resources to
improve wheat SL and GNS. Our work offers a case for utilizing
endemic landraces for QTL mapping and improvement of
wheat cultivars using introgression lines.
... ( var. ) (Fagaceae) [1] 15 ~ 20 m CTAB [2] DNA SSR ( ) [3] ( ) [4] 49 SSR GENEPOP [5] (http://genepop.curtin.edu.au/) SSR -FSTAT V.2.9.3 [6] SSR [7] SSR 1 7 (2) SSR RAPD [13] ISSR [9] AFLP [14] EST [15] ( ...
为探讨人工砍伐后短柄枹栎(Quercus glandulifera var.brevipetiolata Nakai)居群的遗传关系,采用SSR分子标记技术,对短柄枹栎天然林中的4个次生林居群的遗传多样性、遗传结构进行研究。结果表明,筛选的7对SSR引物在4个居群内共揭示了101个等位基因。砍伐后恢复20年(20YAH)的短柄枹栎居群的遗传多样性最高,而砍伐后恢复1年(1YAH)和10年(10YAH)的遗传多样性相对较低。从长远来看,试验地居民特殊的砍伐习惯不会造成短柄枹栎天然林遗传多样性的变化。
... Electrophoresis (100 V for 17 h) was performed in 1 TAE buffer maintained at 60°C. After that, DNA was visualized using silver stain according to Bassam et al. (1991). ...
This study investigated (1) the effects of salinity and temperature on the bacterial
community composition of developing biofilms, and (2) the responses of marine invertebrate larvae
(the polychaete Hydroides elegans and the barnacles Balanus amphitrite and B. trigonus) to these
biofilms during settlement (i.e. attachment to a surface and metamorphosis into juveniles). Biofilms
developed in a 3 × 3 array of salinity and temperature treatments resulted in different bacterial
community compositions (revealed by DGGE and T-RFLP), bacterial densities and total biomasses.
Larval settlement of B. amphitrite and B. trigonus was induced by biofilms developed at high
temperatures (23 and 30°C), but was unaffected (B. amphitrite) or inhibited (B. trigonus) by those
developed at a low temperature (16°C). The settlement response of these barnacles did not correlate
with the biomass or the bacterial density of the biofilms, but did coincide with the marked differences
in bacterial community composition between the biofilms developed at different temperatures. In
contrast, larval settlement of H. elegans differed slightly among biofilms developed in different
salinities, but not among those developed at different temperatures. This settlement response was
moderately correlated with bacterial density but had no apparent relationship with bacterial
community composition of the biofilms. Our results implied that the community composition and cell
density of bacteria in biofilms, which can vary with local environmental conditions, may allow larvae
of the 2 barnacles and H.
... The final step involved storing the samples at 4˚C. The PCR products were separated on an 8% polyacrylamide gel and visualized using silver nitrate staining, following established protocols [38]. The primer synthesis and PCR product detection were conducted by Sangon in Shanghai, a trusted service provider for these procedures in our study. ...
Iron-Heart Cunninghamia lanceolata , a wild relative of Chinese fir with valuable genetic and breeding traits, has been limited in genetic studies due to a lack of genomic resources and markers. In this study, we conducted transcriptome sequencing of Iron-Heart C . lanceolata leaves using Illumina NovaSeq 6000 and performed assembly and analysis. We obtained 45,326,576 clean reads and 115,501 unigenes. Comparative analysis in five functional databases resulted in successful annotation of 26,278 unigenes, with 6,693 unigenes annotated in all databases (5.79% of the total). UniProt and Pfam databases provided annotations for 22,673 and 18,315 unigenes, respectively. Gene Ontology analysis categorized 23,962 unigenes into three categories. KEGG database alignment annotated 10,195 unigenes, classifying them into five categories: metabolism, genetic information, biological systems, cellular processes, and environmental information processing. From the unigenes, we identified 5,645 SSRs, with dinucleotides repeats being the most common (41.47%). We observed variations in repeat numbers and base compositions, with the majority of markers ranging from 12 to 29 bp in length. We randomly selected 200 primer pairs and successfully amplified 15 pairs of polymorphic SSR primers, which effectively distinguished Chinese fir plants of different origins. This study provides insights into the genetic characteristics of Iron-Heart C . lanceolata and offers a foundation for future molecular marker development, breeding programs, genetic diversity analysis, and conservation strategies.
... Single strand conformation polymorphism (SSCP) analysis: SSCP analysis of the amplified genes (Table 2) were carried out by electrophoresis of 5 μl PCR amplicon product in 12% and 15% polyacryamide gel at 150V at 4°C for 12 h followed by silver staining of gels for visualization of banding pattern (Bassam et al. 1991). ...
The usefulness of polymerase chain reaction-single stranded confirmation polymorphism (PCR-SSCP) for determination of rifampicin and isoniazid resistance in Mycobacterium tuberculosis and M. bovis cultures from human and animal origin was investigated. Mycobacteria (81) in the study included, were 12 MDR-TB samples, 35 sputum samples, 3 lung and lymph node tissues from bovines and 11 M. tuberculosis and 18 M. bovis culture, M. tuberculosis H37Rv and M. bovis BCG strain. All the mycobacterial cultures were characterized on growth characteristics, biochemical test pattern, MTB complex specific IS6110 PCR and species specific 12.7 kb multiplex PCR. PCR-SSCP was used to determine resistance against rifiampin by targeting rpoB gene (305bp) and isoniazid by targeting katG (237bp) and inhA (261 bp). Rifampicin resistance was detected by PCR-SSCP in 1 out of 12 MDR-TB samples (8.3%), while isoniazid resistance was detected in 66.7% of MDR-TB samples using PCRSSCP of katG and 75% of MDR-TB samples using inhA SSCP analysis.
... All PCR reactions were repeated at least twice. The resulting amplification was separated by using a 4% polyacrylamide gel with 5 M of urea and visualized using a silver-staining protocol according to Bassam et al. (1991). For the SRAP analysis, fragments were scored manually and the presence/absence of fragments was used to construct a binary matrix. ...
Brachypodium distachyon has been accepted as a model grass species for genetics and molecular genomics in cereals since 2001. However, the genetic variability present in Brachypodium spp. continue being the aim of many research. Therefore, the aim of this work was to analyze the genetic variability present in different Brachypodium spp. from different countries and to find different open reading frames (ORFs) among them by using sequence-related amplified polymorphism (SRAP) markers. In our study, a total of 12 Brachypodium spp. accessions were selected from different countries. By using SRAP markers, the genetic variability was evaluated and some fragments were sequenced to obtain differential ORF information among genotypes. SRAP molecular markers revealed high variability among accessions being USA the accession that showed the most variability at the DNA level. The analysis of sequenced SRAP fragments from monomorphic and polymorphic fragments as well as those obtained from new primer combinations targeted coding regions into B. distachyon available genome. SRAP molecular marker showed not only to be an excellent tool for Brachypodium variability studies but also to provide additional information about differential amplification of open reading frames. ARTICLE HISTORY
... The amplification consisted of a denaturing step of 4 min at 94°C, followed by 35 cycles at 94°C for 1 min, 50°C for 1 min, 72°C for 1 min, and a final extension of 72°C for 10 min, followed by cooling to 4°C. The PCR products were electrophoresed on 6% nondenatured polyacrylamide gels in 1× TBE buffer, running at 120 V constant voltage for 3 h, and then silver stained (Bassam et al., 1991;Liu et al., 2007a, b). The band patterns on the gels were then photographed over white fluorescent light. ...
Egusi' melon Citrullus lanatus (Thunb.) Matsum. & Nakai is an important vegetable crop in Nigeria, grown for its edible seeds and oils. The diverse areas in which the crop is cultivated make it a rich source of genetic resources for the species. To explore its diversity, 50 accessions of 'egusi' melon were collected from different agro-ecological parts of Nigeria and were evaluated using 25 simple sequence repeat (SSR) markers. A total of 49 bands were scored, of which 42 were polymorphic, accounting for 93.60% of the polymorphic loci. The PIC value ranged from 0.36-0.80. UPGMA cluster analysis revealed five distinct groups for SSR. PCA analysis revealed the distinction of the accessions NG/OE/MAR/09/014, NG/TO/APR/09/027 and A8. Based on the results of this study, SSR markers appear to be particularly useful for the estimation of genetic similarity among diverse accessions of melon.
... Each reaction was made at least twice. Amplified fragments were separated by vertical electrophoresis in polyacrylamide gels at 54 mA for 4 h and then revealed by silver staining, according to Bassam et al. (1991). All generated fragments were analyzed. ...
Alternaria is one of the main fungal contaminants of cereal grains worldwide with the potential to produce mycotoxins hazardous to human and animal health. Many studies have been carried out to characterize Alternaria sp.-grp. using traditional morphology or polyphasic approach, but a good correlation between morphological sp.-grp., molecular, and chemotaxonomic groups has not always been achieved. For this reason, this study aimed to investigate the usefulness of a cheaper alternative tool, SRAP markers, in identifying Alternaria sp.-grps. obtained from Argentinean barley grains and to compare it with preliminary characterization using morphological traits, phylogeny, and metabolite profiles. Fifty-three Alternaria isolates from barley grains of the main producing regions of Argentina were analyzed with four combinations of SRAP markers. The UPGMA dendrogram, based on the Simple Matching similarity coefficient, revealed three distinct groups. SRAP markers allowed the separation of Alternaria from Infectoriae sections in agreement with the results of a polyphasic approach previously made. Besides, isolates of A. arborescens sp.-grp. were clustered in a separate group from isolates of A. tenuissima and A. alternata sp.-grp., which were grouped in the same cluster. SRAP markers are a recommended tool for classifying Alternaria isolates because of its simplicity, reliability, and cost-effectiveness compared to other molecular markers.
... (Bio-Rad. Inc., Hercules, CA, USA) [43]. ...
The deployment of Eucalyptus pellita trees that are resistant to Ceratocystis manginecans is essential for the commercial plantations and therefore the sustainability of forest industries in Southeast Asia that utilize this resource. Current screening procedures are time-consuming and expensive but could be expedited with the aid of marker-assisted selection and breeding. The identification of genotypes with resistance to the disease may be facilitated if microsatellite markers developed in other Eucalyptus species are transferable and can be linked to quantitative trait loci (QTL) for disease resistance. This possibility was tested in 111 full-sib progenies and their parents by genotyping with 49 microsatellite markers developed in other Eucalyptus species. Disease development was assessed after stem inoculation with C. manginecans isolate Am60C. The disease index (DI) varied from 0 to 20% of stem length. There was a continuous distribution of resistant and susceptible seedlings with 60% in the resistant category. Of the 30 acceptable markers, 17 (56%) defined two linkage groups (LG). In each LG, one QTL with a significant logarithm of odds (LODs > 13) was identified. The transferability of microsatellite markers developed in other Eucalyptus species facilitated the rapid identification of LGs and QTLs in E. pellita. To further refine the linkage map, the testing of more microsatellite markers and a larger population of progenies are required.
... Each reaction was made at least twice. Amplified fragments were separated by vertical electrophoresis in polyacrylamide gels at 54 mA for 4 h and then revealed by silver staining, according to Bassam et al. (1991). All generated fragments were analyzed. ...
... The ampli ed DNA fragments were separated by electrophoreses in a 6.0% agarose gel with a TBE buffer system. Gels were stained with Silver Nitrate (AgNO 3 ) and fragment patterns were photographed for further analyses (Bassam et al. 1991). Thus, the SSR data were scored for presence (1) or absence (0) of bands detected on the DNA bands shown on the gel electrophoresis (vertical electrophoresis tank, Cleaver Scienti c) with the power supply (CS-300V-Cleaver Scienti c). ...
Indonesia is known as the fourth biggest coffee producing countries in the world. There are over 124 species within the Coffea family, however, only arabica (C. arabica L.) and robusta (C. canephora) have played an immense economic role. The region of Aceh; especially the Gayo highlands [800- 2,200 m. above sea level (a.s.l.)] is known as the largest arabica coffee plantation across the nation, and an average production of 700 up to 800 kg
ha⁻¹, and produce almost 25% of the total arabica coffee at the national level. This is the first publication about the genetic diversity of coffee arabica (Coffea arabica L.) cultivated on the Gayo Highlands, although it was already existed almost two centuries. Based on history, the Dutch initially introduced coffee to Aceh in the early of 18th century. There were up to 52 accessions of coffee arabica and their genetic diversities were measured via their (i) morphological characteristics (n= 33 traits); (ii) their simplification bi-plot diagram via Principle Component Analyses (PCA); (iii) molecular variation via Simple Sequence Repeat (SSR) marker (n= 8). Our result showed that high morphological diversities was existed, although, low to moderate genetic diversity was confirmed among those commercial accessions based on these parameters: the PCA biplot diagram, and dendrogram, Polymorphic Information Content (PIC) that showed a range of 0.00-0.84, and 0.157-0.610, respectively. By conducting genetic diversity study intended for local germplasm conservation, a sustainable coffee production in Middle Aceh, and their economic benefits could be still maintained for a long term.
... Primers were designed with DNAMAN (Lynnon Biosoft, Quebec, Canada) and synthesized by the Tsingke Biological Technology Company. PCR reactions were performed on a thermocycler (Eppendorf, Hamburg, Germany), and products were separated in 8% polyacrylamide gels (40% acrylamide bisacrylamide) [45]. ...
Wheat sharp eyespot, a stem disease caused by the soilborne fungus Rhizoctonia cerealis van der Hoeven, has become a threat to wheat production worldwide. Exploiting resistance resources from wild relatives of wheat is a promising strategy for controlling this disease. In this study, a new wheat–Dasypyrum villosum T2DS·2V#4L translocation line in the background of Chinese Spring (CS) showed stable resistance to R. cerealis. Introgression of the T2DS·2V#4L chromosome into wheat cultivar Aikang 58 by backcrossing produced a marked increase in sharp eyespot resistance in NIL-T2DS·2V#4L in comparison with NIL-T2DS·2DL, and no detrimental effects of 2V#4L on agronomic traits were observed in the BC2F2, BC2F2:3, and BC2F2:4 generations. Flow-sorted sequencing of 2V#4L yielded 384.3 Mb of assembled sequence, and 8836 genes were predicted of which 6154 had orthologs in at least one of the 2AL, 2BL, and 2DL arms of CS, whereas 1549 genes were unique to 2V#4L. About 100,000 SNPs were detected in genes of 2V#4L and 2DL in 10 sequenced bread wheat cultivars. A Kompetitive Allele Specific Polymerase chain reaction and 30 conserved ortholog sequence markers were developed to trace the 2V#4L chromatin in wheat backgrounds. T2DS·2V#4L compensating translocation lines represent novel germplasm with sharp eyespot resistance and the markers will allow rapid detection in breeding programs.
... In cases where this cycling failed to produce amplicons, a touchdown program was used, i.e., started with 94°C for 5 min; then 10 cycles at 94°C for 30 s, 60°C for 30 s (touchdown with -0.5°C each cycle), and 72°C for 30 s; followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and ended with 72°C for 10 min. PCR products were differentiated using 6% denaturing polyacrylamide gels and stained with AgNO 3 (Bassam et al., 1991). Thirty-two of the 988 markers were selected to genotype the 112 RILs. ...
Wheat yield has been constrained by stripe rust disease globally. A wheat landrace (Qishanmai, QSM) consistently showed lower stripe rust severities in multiple year studies than susceptible check varieties including Suwon11 (SW) at the adult plant stage. To detect QTL for reducing the severity in QSM, 1218 recombinant inbred lines (RILs) were developed from SW × QSM. QTL detection was conducted firstly using 112 RILs selected for similarity in pheno-morphological characters. The 112 RILs were assessed for stripe rust severity at the 2nd leaf, 6th leaf and flag leaf stages under field and greenhouse conditions, and genotyping was done primarily with a single nucleotide polymorphism (SNP) array. On the basis of these phenotypic and genotypic data, a major QTL (QYr.cau-1DL) was detected on chromosome 1D at the 6th leaf and flag leaf stages. Further mapping was conducted by genotyping 1218 RILs using new simple sequence repeat (SSR) markers, which were developed by referring to the sequences of the wheat line Chinese Spring (IWGSC RefSeq v1.0). QYr.cau-1DL was mapped within a 0.5 cM (5.2 Mb) interval delimited by the SSR markers 1D-320.58 and 1D-325.79. These markers were applied to select for QYr.cau-1DL by screening F2 or BC4F2 plants of the wheat crosses RL6058 × QSM, Lantian10 × QSM and Yannong21 × QSM. F2:3 or BC4F2:3 families derived from the selected plants were assessed for stripe rust resistance in the fields of two locations and in a greenhouse. Wheat plants carrying the resistant marker haplotype in homozygous state for QYr.cau-1DL showed lower stripe rust severities (by 44% to 48%) than plants lacking this QTL. The trial of RL6058 (a carrier of Yr18) × QSM also indicated that QYr.cau-1DL had larger effect than Yr18 on reducing severity; they acted synergistically, yielding an elevated level of stripe rust resistance.
... Among these DNA markers, RAPD and AFLP markers have some obvious disadvantages, including less reproducibility [42,43], failing to detect heterozygotes' loci [44], etc. As for SSR markers, the main shortcoming is labor-intensive wet experiments where silver staining steps are usually necessary before recording of the DNA polymorphisms [45]. On the other hand, due to the increasingly growing throughput, the decreasing cost [46], and the ability to detect tens of thousands of genome-wide and evenly distributed SNP markers, high-throughput sequencing technology has been extensively applied in population genetics and thus provided us an unprecedented comprehensive spectrum of plant genetic diversity [47][48][49]. ...
Hollyhock (Alcea rosea (Linn). Cavan) is an herbaceous flowering plant with significant applications in urban greening, soil remediation, and traditional medicine. However, its genetic diversity and molecular characteristics at the population level have not been explored yet. Here, the phenotypic and genetic diversity of 162 hollyhock accessions from China revealed extensive variation among 11 traits and strong correlations between several quantitative traits. Whole-genome re-sequencing of 32 randomly chosen accessions identified 10,468,760 core single-nucleotide polymorphisms (SNPs) distributed evenly across the genome, except for on chromosome 21, and the average nucleotide diversity (π) was calculated to be 0.00397. Principal component analysis showed that these 32 accessions could be divided into four subpopulations, which was in agreement with the population structure analysis, and the subpopulations were strongly correlated with geographic location. A neighbor-joining dendrogram displayed similar clusters, except for accessions HuB25 and HLJ28, which formed two separate clusters. Our findings illuminate the genetic diversity in hollyhock and provide valuable information for hollyhock breeding.
... Alteration in electrophoretic mobility of any band as earlier given by [75], the purified PCR product was assayed. Silver staining was carried out as earlier described [76,77]. The labeled silver-stained gel was scanned for analysis of computer imaging and documentation. ...
Introduction:
The BORIS, 11 zinc-finger transcription factors, is a member of the cancer-testis antigen (CTA) family. It is mapped to chromosome number 20q13.2 and this region is genetically linked to the early onset of breast cancer. The current study analyzed the correlation between BORIS mutations and the expression of the protein in breast cancer cases.
Materials and methods:
A population-based study including a total of 155 breast cancer tissue samples and an equal number of normal adjacent tissues from Indian female breast cancer patients was carried out. Mutations of the BORIS gene were detected by polymerase chain reaction-single standard confirmation polymorphisms (PCR-SSCP) and automated DNA sequencing and by immunohistochemistry for BORIS protein expression were performed. The observed findings were correlated with several clinicopathological parameters to find out the clinical relevance of associations.
Results:
Of all the cases 16.12% (25/155) showed mutations in the BORIS gene. The observed mutations present on codon 329 are missense, leading to Val> Ile (G>A) change on exon 5 of the BORIS gene. A significant association was observed between mutations of the BORIS gene and some clinicopathological features like nodal status (p = 0.013), estrogen receptor (ER) expression (p = 0.008), progesterone receptor (PR) expression (p = 0.039), clinical stage (p = 0.010) and menopausal status (p = 0.023). The protein expression analysis showed 20.64% (32/155) samples showing low or no expression (+), 34.19% (53/155) with moderate expression (++), and 45.17% (70/155) showing high expression (+++) of BORIS protein. A significant association was observed between the expression of BORIS protein and clinicopathological features like clinical stage (p = 0.013), nodal status (p = 0.049), ER expression (p = 0.039), and PR expression (p = 0.027). When mutation and protein expression were correlated in combination with clinicopathological parameters a significant association was observed in the category of high (+++) level of BORIS protein expression (p = 0.017).
Conclusion:
The BORIS mutations and high protein expression occur frequently in carcinoma of the breast suggesting their association with the onset and progression of breast carcinoma. Further, the BORIS has the potential to be used as a biomarker.
... Each 10-μL PCR reaction contained 5 μLof Taq Master Mix (Vazyme, Nanjing, China), 1 μL of DNA solution, 0.3 μL of reverser primer, 0.3 μL forward primer, and 3.4 μLof ddH 2 O. PCR was conducted at 94˚C for 5 min for pre-denaturation, followed by 30 cycles at 94˚C for 30 s, with annealing at 58-62˚C for 30 s, elongation at 72˚C for 30 s, and finally an extension at 72˚C for 7 min. Polyacrylamide gel electrophoresis was performed as stated in a previous study [44] to display the PCR product. ...
Investigating the genetic diversity and population structure is important in conserving narrowly distributed plants. In this study, 90 Clematis acerifolia (C. acerifolia) plants belonging to nine populations were collected from the Taihang Mountains in Beijing, Hebei, and Henan. Twenty-nine simple sequence repeats (SSR) markers developed based on RAD-seq data were used to analyze the genetic diversity and population structure of C. acerifolia. The mean PIC value for all markers was 0.2910, indicating all SSR markers showed a moderate degree of polymorphism. The expected heterozygosity of the whole populations was 0.3483, indicating the genetic diversity of both C. acerifolia var. elobata and C. acerifolia were low. The expected heterozygosity of C. acerifolia var. elobata (He = 0.2800) was higher than that of C. acerifolia (He = 0.2614). Genetic structure analysis and principal coordinate analysis demonstrated that C. acerifolia and C. acerifolia var. elobata showed great genetic differences. Molecular variance analysis (AMOVA) demonstrated that within-population genetic variation (68.31%) was the main contributor to the variation of the C. acerifolia populations. Conclusively, C. acerifolia var. elobata had higher genetic diversity than C. acerifolia, and there are significant genetic differences between C. acerifolia and C. acerifolia var. elobata, and small genetic variations within the C. acerifolia populations. Our results provide a scientific and rational basis for the conservation of C. acerifolia and provide a reference for the conservation of other cliff plants.
... Forty SSR primer pairs from Röder et al. [35] and Tsuruta et al. [36] were used in this study. SSR-PCR and SRAP-PCR was performed in a total volume of 20 µL containing 1 µL genomic DNA, 10 µL of 2 × Mix (Yeasen Biotechnology Co., Ltd., Shanghai, China), 1 µL of 10 µmol·L −1 each PCR primer and 7 µL purified water. ...
Zoysia spp. germplasm exhibit genetic variation between and within species. A comprehension of the genetic diversity of Zoysia germplasm could enable the effective utilization of these germplasms in future breeding endeavors. Ten simple sequence repeats (SSR) primer pairs and nine sequence-related amplified polymorphism (SRAP) primer pairs were used to analyze genetic diversity and construct DNA fingerprints for 45 Chinese Zoysia germplasm collections. We detected 231 SSR polymorphic bands and 149 SRAP polymorphic bands with 97.18% and 93.43% polymorphism ratios, respectively. The genetic similarity coefficient of the 45 germplasm collections ranged from 0.623 to 0.856, with an average of 0.727. Forty-five germplasm collections were divided into six major clusters when the genetic similarity coefficient was 0.71 based on the unweighted pair group method with the arithmetic averaging (UPGMA) method. Both SSR and SRAP molecular marker systems can be used to identify all germplasm collections, the SSR primer pair (Xgwm234-5B) and SRAP primer pairs (Me3-Em1 and Me3-Em2) can effectively distinguish 45 Zoysia spp. accessions. Collectively, we utilized both SSR and SRAP molecular markers to generate DNA fingerprints in this study providing a theoretical foundation for germplasm conservation and assisting in selecting and breeding new varieties of Zoysia.
... The PCRs were conducted using the following thermal protocol: initial denaturation at 95 °C for 15 min, 10 touchdown cycles at 94 °C for 30 s, 60 °C for 30 s (− 1 °C/cycle), and 72 °C for 40 s; 30 cycles at 94 °C for 30 s, 50 °C for 50 s, and 72 °C for 40 s; and a final extension at 72 °C for 10 min. PCR products were resolved on 8% polyacrylamide gel and stained with silver nitrate following the protocol of Merril et al. (1981) modified by Bassam et al. (1991) to increase sensitivity and reduce unspecific background. The Gel Pro Analyzer package 3.9 (Gene, USA) was used to determine the fragment size of the amplified products (SSR variants). ...
Maintenance of standing genetic diversity and effective population size (Ne) in trees is essential for sustainability, adaptation, and evolution, especially for dioecious tree species under changed climatic conditions. We examined the gene pool of Taxus baccata along latitudinal gradients in the Hyrcanian forest from west to east using 15 simple sequence repeat markers (SSR). A high level of genetic diversity and a significant level of fixation index was observed, and the populations showed spatial genetic structure. The fixation index ranged from 0.027 to 0.197. In six out of eleven populations inbreeding was the significant factor influencing deviation from the Hardy–Weinberg equilibrium. There was no bottleneck effect in any of the analysed populations and the global Fst with ENA correction was 0.044. The average total number of migrants per generation ranged from 4.93 to 2.14. Under the six tested scenarios, the DIYABC analysis showed that the eastern and western parts of the Hyrcanian yew forests split about 134 generations ago but these two pools meet in the central part of these forests about 49 generations ago. Although the genetic diversity of T. baccata in the Hyrcanian forest is relatively high, it is expected that the inbreeding depression will increase over time, causing the accumulation of destructive alleles and intensifying the extinction process. This process is caused by anthropogenic activities, habitat fragmentations, the age of the trees, low regenerations, the existence of high geographical barriers, and a significant reduction in gene flow among the habitats.
... PCR products of the DRD2 gene were initially resolved on 1.5% agarose gel electrophoresis and documented using gel documentation system (Syngene, Cambridge, U.K.) to confirm the size of the fragment (233 bp). Single Strand Conformational Polymorphism (SSCP) study was done to know the SNP polymorphism (Bassam et al., 1991). Briefly, formamide dye (12 ìl) was added to the PCR product (6 ìl) in PCR tubes, mixed, centrifuged, and denatured at 95°C for 10 min. ...
Ghagus breed was characterized for broodiness, its effect on production traits and polymorphisms at 5’ regulatory region of
dopamine D2 receptor (DRD2) and prolactin genes for their association with production and broody traits. A high incidence (85.4%) of broodiness was observed and it had a significant effect on production traits. The broody hens were (P<0.001) lesser in body weight than non-broody hens and the bodyweight decreased with the increasing duration of broodiness (DB). Non-broody hens produced more (P<0.001) eggs than the broody hens. The number of clutches and pauses was (P<0.001) higher in non-broody hens as compared to broody hens. However, the average pause size was significantly (P<0.001) lesser in non-broody hens. The number of clutches and pauses reduced while pause size increased significantly as DB increased. Insertion-deletion (InDel) genetic marker at 5' region of the DRD2 gene was associated (P<0.05) with the age at first broody cycle, duration of the first broody cycle (P<0.01), egg production, and 40 weeks body weight (P<0.05) while SNP at 5' region of DRD2 gene had significant (P<0.004) influence on 40 weeks body weight. The
study concluded that broodiness had a significant influence on production traits and InDel and SNP markers at the 5’ regulatory region of DRD2 gene had significantly (P<0.004) affected the bodyweight at 40 weeks. The findings of the study could be useful for the development of breeding programs for the improvement of indigenous chickens like Ghagus.
Keywords: Broodiness, Body weight, Ghagus, Indigenous chicken, Marker
... Three microliters of each sample were then run on a polyacrylamide gel (MDEx, FMC Bioproducts, Rockland, ME, USA) under non-denaturing conditions at 4 W for 17 h on a vertical electrophoresis system. Gels were then silver stained according to Bassam et al. (1991) and dried at 60°C for 3 h before being scanned. Alleles (SSCP-band profiles) scored for each mtDNA marker were summarized in an Excel sheet and submitted for statistical analyses. ...
A total of 108 isolates of the wheat pathogen Zymoseptoria tritici were collected from four distinct locations of Tunisia and characterized for three mitochon-drial DNA sequences to provide insight into the genetic diversity and population structure of the fungus in this country. The number of different alleles averaged over all loci within locations ranged from 3.33 to 5.67, with an average of 4.42 per location. Multilocus analysis identified 27 (25 %) distinct haplotypes among the 108 assessed isolates, revealing a high mtDNA-based clonality within the population. Three of the highlighted haplotypes occurred in all locations and nine of them covered at least two locations. Significant levels of genetic diversity were found for the whole population and within each of the sampled locations, as indicated by the Nei's gene diversity (0.52), unbiased gene diversity (0.58) and allele richness (4.43) indices. Further analyzes using Bayesian and non-Bayesian statistical models, as well as AMOVA, showed a lack of mtDNA-based genetic structure (G ST = 0.06), hence supporting previous reports on Z. tritici in Tunisia performed using nuclear-DNA markers. A high level of gene flow (Nm = 8.27) corroborates the lack of structure and suggests regular cycles of sexual reproduction, leading to allelic pool homogenization via wind-born ascospores in the Tunisian population of Z. tritici.
... PCR reaction mixture contained 1.0 μL 10× PCR buffer (Mg 2+ Fu et al. (2005) and Yan et al. (2004Yan et al. ( , 2006 for Vrn, in Ellis et al. (2002) for Rht, and in Beales et al. (2007) and Mohler et al. (2004) and ended with 72 °C for 10 min. Amplicons were separated in 6% denaturing polyacrylamide gels and visualized with the silver staining method (Bassam et al. 1991). Genetic map was constructed with JoinMap 4.0 (Stam 1993), and genetic distance (in cM) was calculated based on Kosambi mapping function. ...
Key message
A novel QTL (QSt.nftec-2BL) was mapped to a 0.7 cM interval on chromosome 2B. Plants carrying QSt.nftec-2BL produced higher grain yields by up to 21.4% than otherwise in salinized fields.
Abstract
Wheat yield has been limited by soil salinity in many wheat-growing areas globally. The wheat landrace Hongmangmai (HMM) possesses salt tolerance as it produced higher grain yields than other tested wheat varieties including Early Premium (EP) under salt stresses. To detect QTL underlying this tolerance, wheat cross EP × HMM was chosen to serve as mapping population that was homozygous at Ppd (photoperiod response gene), Rht (reduced plant height gene) and Vrn (vernalization gene); thus, interference with QTL detection by these loci could be minimized. QTL mapping was conducted firstly using 102 recombinant inbred lines (RILs) that were selected from the EP × HMM population (827 RILs) for similarity in grain yield under non-saline condition. Under salt stresses, however, the 102 RILs varied significantly in grain yield. These RILs were genotyped using a 90 K SNP (single nucleotide polymorphism) array; consequently, a QTL (QSt.nftec-2BL) was detected on chromosome 2B. Then, using 827 RILs and new simple sequence repeat (SSR) markers developed according to the reference sequence IWGSC RefSeq v1.0, location of QSt.nftec-2BL was refined to a 0.7 cM (6.9 Mb) interval flanked by SSR markers 2B-557.23 and 2B-564.09. Selection for QSt.nftec-2BL was performed based on the flanking markers using two bi-parental wheat populations. Trials for validating effectiveness of the selection were conducted in salinized fields in two geographical areas and two crop seasons, demonstrating that wheat plants with the salt-tolerant allele in homozygous status at QSt.nftec-2BL produced higher grain yields by up to 21.4% than otherwise.
... The electrophoresis was performed at 4°C temperature at 110 V for 10-12 h. Gel was stained with silver nitrate staining (Bassam et al., 1991) with slight modification to visualize the banding pattern. ...
The aim of the study was to identify growth hormone gene polymorphisms and theirassociation with body weights and morphometric characters in Nellore sheep. A total of 50 Nelloresheep (12 months old) maintained at Instructional Livestock Farm Complex, Mamnoor weregenotyped at 5ꞌ regulatory region and exon 4 by using polymerase chain reaction-single strandedchain polymorphism (PCR-SSCP) technique. The PCR-SSCP of 5ꞌ regulatory region (210 bp)yielded three genotypes AA, AB and BB corresponding to two allelic variants A and B. Thegenotypic frequencies were 0.24, 0.42 and 0.34 for AA, AB and BB, respectively and the allelicfrequencies of A and B were 0.45 and 0.55, respectively. The exon-4 (191 bp) yielded twogenotypes, AA (0.60) and AB (0.40) corresponding to two allelic variants A and B. The allelicfrequencies of A and B were 0.80 and 0.20, respectively. No significant associations were foundbetween different SSCP genotypes and body weights and morphometric characters. The studyshowed that two loci were polymorphic and hence further studies are required to find outassociation of polymorphism, if any, with different economic traits in Nellore sheep
(PDF) GROWTH HORMONE GENE AND THEIR ASSOCIATION WITH BODY WEIGHTS AND MORPHOMETRIC CHARACTERS IN NELLORE SHEEP. Available from: https://www.researchgate.net/publication/369042663_GROWTH_HORMONE_GENE_AND_THEIR_ASSOCIATION_WITH_BODY_WEIGHTS_AND_MORPHOMETRIC_CHARACTERS_IN_NELLORE_SHEEP [accessed Mar 07 2023].
... DNA was extracted from blood samples taken from the male and the nestlings using a resin-based technique (Walsh et al. 1991), and amplified at three polymorphic microsatellite loci (Pdoµ3 and Pdoµ4, Neumann andWetton 1996, andPdoµ6, Griffith et al. 1999). PCR products were electrophoresed through 4% denaturing polyacrylamide gels and visualized using silver staining (Bassam et al. 1991). All five nestlings shared at least one allele with the attendant male at each of the three loci. ...
... Samples were run for 15h at 200 V in 4°C on a 12% acrylamide: bisacrylamide gel (37.5:1). Bands were appeared by 0.1 percent silver nitrate and NaOH solution (containing 0.1 percent formaldehyde) using the protocol of Bassam, Caetano-Anollés, and Gresshoff (1991). The PCR fragments from different SSCP patterns were sequenced in both directions. ...
The aim of current study was to survey genetic variability of PALM gene’s exon 3 and 4 by PCR-SSCP and DNA sequencing in Zel and Lori Bakhtiari sheep breeds. The SIFT (Sorting Intolerant from Tolerant) and PHyre2 program were used to predict the possible impact of amino acid substitutions on performance and structure of the paralemmin protein. A total of 140 animal's from 2 Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Zel sheep breeds were considered. The results showed that there are two polymorphic sites including a nonsynonymous substitution and an insertion mutation (49bp). Non-synonymous mutation deduced Thr20Ala amino acid exchange and ensuing two different structures for paralemmin protein that could be potentially affect protein structure and function during the interaction with glutamate in the cytosolic surface of plasma membrane. PALM gene, according to evolutionary path, is classified into two separate categories. In first covey, Gallus gallus and in second one, other species in several branches, so that the sequence of cow and sheep is placed in a sub-branch which forms a clade beside goat. Comparison of illustrated coding region sequences, PALM gene among different species, is of orthologous which are derived from a common ancestor.
... Los fragmentos amplificados fueron separados por electroforesis en geles de poliacrilamida desnaturalizantes de 6% con urea 7 M, empleando el sistema Sequi-Gen GT BIORAD 38 x 50 cm a 80 W, 50 ºC por 4 h. La visualización de los fragmentos se realizó con nitrato de plata según el protocolo descrito por Bassam et al. (1991) y el tamaño se determinó con un marcador de peso molecular de 25 bp (GIBCO BRL.). ...
Pelliciera rhizophorae es una especie neotropical de mangle, la única especie representante de su género, cuya distribución actual está casi restringida a la costa del Pacífico americano. Como una contribución al conocimiento de la ecogenética de P. rhizophorae se realizó un análisis de su variabilidad genética y morfológica en el Pacífico colombiano y los factores ambientales asociados en seis localidades: Virudó, Charambirá, Isla La Plata, Tumaco, Milagros y Chontal (Colombia). El análisis de diversidad genética realizado mediante marcadores moleculares AFLP, produjo 225 fragmentos amplificados con 155 (69%) polimórficos en 57 individuos de P. rhizophorae colectados en las seis zonas del Pacífico colombiano. La diversidad genética dentro de poblaciones reveló niveles de variación más bajos en Isla La Plata y Tumaco y el más alto en Chontal (Hep = 0,187). Esta especie en el Pacífico colombiano resultó estar significativamente estructurada (fst = 0,2654), con un 73,5% de variación dentro de poblaciones. La diferenciación genética no mostró correlación con la distancia geográfica entre zonas. Esto sugiere que la dinámica poblacional de la especie está asociada con procesos históricos influenciados por factores ecológicos y ambientales. El estudio de la variabilidad morfológica, reveló un óptimo desarrollo de los árboles en la localidad de Virudó, una zona constituida por manglar de ribera; en contraste, Tumaco, Milagros y Charambirá, mostraron un fenotipo disminuido, a pesar de ser también manglares de ribera. El análisis de suelo determinó que el sustrato de anclaje de la especie está compuesto en su mayoría por arenas muy finas y arcillas y, aunque la salinidad medida en agua intersticial mostró una ligera variación entre las zonas de estudio, estas características parecen ser adecuadas para la sobrevivencia de la especie.
... The accessions were selected based on a prior evaluation of genetic diversity (Azevedo et al. 2012) and represented the maximum diversity discovered in the germplasm bank. PCR was performed under the same conditions as described above, and the amplified products were loaded onto 12% native polyacrylamide gel electrophoresis for 5 hours at 500V and stained with silver nitrate (Bassan et al. 1991). Gel scoring was performed using GelAnalyzer 19.1 (www.gelanalyzer.com), ...
... For SRAP analysis, samples were subjected to the following thermal profile: the five cycles including initial denaturation of 1 min at 94˚C, annealing of 1 min at 35˚C, and extension of 1 min at 72˚C, followed by 35 cycles of 3 min at 50˚C, with a final extension of 10 min at 72˚C. PCR products were separated by electrophoresis on 12% non-denatured polyacrylamide gels and stained by AgNO3 solution [49]. Polymorphic SRAP markers were scored as binary data with presence (1) or absence (0). ...
Association analysis has been proven as a powerful tool for the genetic dissection of complex traits. This study was conducted to identify association of recovery, persistence, and summer dormancy with sequence related amplified polymorphism (SRAP) markers in 36 smooth bromegrass genotypes under two moisture conditions and find stable associations. In this study, a diverse panel of polycross-derived progenies of smooth bromegrass was phenotyped under normal and water deficit regimes for three consecutive years. Under water deficit, dry matter yield of cut 1 was approximately reduced by 36, 39, and 37% during 2013, 2014, and 2015, respectively, compared with the normal regime. For dry matter yield of cut 2, these reductions were approximately 38, 60, and 56% in the same three consecutive years relative to normal regime. Moreover, water deficit decreased the RY and PER of the genotypes by 35 and 28%, respectively. Thirty primer combinations were screened by polymerase chain reaction (PCR). From these, 541 polymorphic bands were developed and subjected to association analysis using the mixed linear model (MLM). Population structure analysis identified five main subpopulations possessing significant genetic differences. Association analysis identified 69 and 46 marker-trait associations under normal and water deficit regimes, respectively. Some of these markers were associated with more than one trait; which can be attributed to pleiotropic effects or tightly linked genes affecting several traits. In normal and water-deficit regimes, these markers could potentially be incorporated into marker-assisted selection and targeted trait introgression for the improvement of drought tolerance of smooth bromegrass.
... After completion of the selective amplification phase, 6% polyacrylamide sequencing gel was used for observation and separation of the bands. In order to observe the fragments obtained from electrophoresis, staining was performed by using silver nitrate under the hood on rotating shaker at the constant speed of 40 rpm (Bassam et al., 1991). ...
Ziziphus jujuba Mill is an important medicinal plant which is of high importance in medicinal industry due to containing mucilage, different types of vitamin, pectin, alkaloid, phenolic compounds and fatty acids. The current study was conducted to evaluate phylogenetic relations of jujube accessions using morphological, phytochemical and AFLP molecular markers. In morphological and phytochemical experiments, minimum, maximum, and average of fruit weight, were respectively 1.52, 2.99 and 2.31 g. In the same way, the minimum (1.3 g), maximum (2.65 g) and average (1.99 g) of fruit flesh weight were observed in accessions. The lowest (11.58 %) and highest (28.85 %) of mucilage quantity were related to Kalaghneshin1 and Kasva accessions. Simple correlation analysis of the traits showed that there were significant positive and negative correlations among some important traits including the positive correlation between the amount of mucilage and flesh to kernel ratio and kernel shape, and negative correlation between mucilage rate and kernel weight and diameter and fruit shape. Analysis of factors based on Varimax method defined four factors of fruit volume, kernel, fruit flesh and mucilage. Cluster analysis classified accessions into four groups, and segregated three groups with Esfahani, Qomi and Mazandarani origins. In a similar matrix molecular experiment using Jaccard coefficient, cluster analysis was performed by using UPGMA algorithm. 12 out of 15 pairs of AFLP primer showed polymorphism, with these primers producing 689 bands, out of which 44 bands presented polymorphism. Based on the results obtained from dendrogram, the studied accessions were divided into 8 discrete groups at similarity level of 0.75. AFLP marker could clearly cluster three groups of Qom, Esfahan and Khorasan. The results obtained from cluster analysis were confirmed by the principal component analysis. Two groups of Qom and Esfahan were distinctively separated in all markers; therefore these regions (central regions) can be introduced with high probability as one of the origins of jujube. In general, diversity of traits, efficiency of markers and high correlation between the makers were considerable in segregation and separation of the accessions, and can provide an appropriate foundation for breeding of this valuable medicinal plant.
... The PCR Thermo cycles were carried out with a program of 35 three-step cycles, including 94 °C for 1 min, 56 °C for 30 s, and 72 °C for 1 min. On 6% acrylamide gel in vertical electrophoresis, Bio-Rad, the DNA fragments were separated, and to reveal the bands, the silver staining method was applied 69 . The banding pattern of the amplified DNA fragments was turned into binary codes as presence (1) or absence (0), and only the consistently repeatable bands were scored. ...
Understanding how environmental factors shape patterns of genetic and phenotypic variations in a species is necessary for conservation and plant breeding. However, these factors have not yet been completely understood in tuberous orchid species used to make ‘Salep’, an important ingredient in traditional medicine and beverages in middle eastern countries and India. In many areas, increasing demand has pushed species to the brink of extinction. In this study, 198 genotypes from 18 populations of the endangered species Orchis mascula L. spanning a large-scale climatic gradient in northern Iran were used to investigate patterns of genetic diversity and plant functional traits. Populations were sampled from three land cover types (woodland, shrubland, and pastureland/grassland). Plant height, stem length, number of flowers, bulb fresh and dry weight, glucomannan, and starch concentrations showed high variation among populations and were significantly related to land cover type. In general, genetic diversity was high, particularly in those from eastern Hyrcanian; additionally, populations showed a high level of genetic differentiation (G'st = 0.35) with low gene flow (Nm = 0.46). The majority of genetic differentiation occurred within populations (49%) and land cover types (20%). The population structural analysis using the AFLP marker data in K = 4 showed a high geographical affinity for 198 O. mascula genotypes, with some genotypes having mixed ancestry. Temperature and precipitation were found to shape genetic and phenotypic variation profoundly. Significant isolation by the environment was observed, confirming the strong effect of environmental variables on phenotypic and genetic variation. Marker-trait association studies based on MLM1 and MLM2 models revealed significant associations of P-TGG + M-CTT-33 and E-AGG + M-CGT-22 markers with plant height and glucomannan content. Overall, a combination of large-scale climatic variables and land cover types significantly shaped genetic diversity and functional trait variation in O. mascula populations.
... DCode System for PCR-SSCP (Bio-Rad) coupled to a cooling bath device (Lauda E100) was optimized at 26 °C and 120 V (10 mA) for 18 h for the separation on 20 cm × 20 cm × 0,75 mm 0.6X MDE gels using TBE running buffer 0.7× 46 . The obtained ssDNA from 100 to 400 ng dsDNA in the gel was amplified and analyzed by PCR-SSCP and silver stained 48 . ...
Polychlorinated Biphenyls (PCBs) are persistence in the contaminated sites as a result of lacking PCBs-degrading microorganisms. Cultivation-independent technique called single-strand-conformation polymorphism (SSCP) based on 16SrRNA genes was chosen to characterize the diversity of bacterial communities in PCBs polluted soil samples. The bacterial communities showed an increasing diversity from the genetic profiles using SSCP technique. 51 single products were identified from the profiles using PCR reamplification and cloning. DNA sequencing of the 51 products, it showed similarities to Acidobacteria, Actinobacteria, Betaproteobateria, Gammaproteobacteria and Alphaproteobacteria, the range of similarities were 92.3 to 100%. Pure 23 isolates were identified from PCBs contaminated sites. The identified isolates belonged to genus Bacillus, Brevibacillus, Burkholderia, Pandoraea, Pseudomonas, and Rhodococcus. The new strains have the capability to use PCBs as a source of sole carbon and harbor 2,3-dihydroxybiphenyl dioxygenase (DHBDO) which could be used as molecular marker for detection PCBs-degrading bacteria in the PCBs contaminated sites. This finding may enhance the PCBs bioremediation by monitoring and characterization of the PCBs degraders using DHBDO in PCBs contaminated sites.
... The procedure for polyacrylamide gel electrophoresis (PAGE) was as previously published . The silver staining method was used to visualize the PCR products as described by Bassam, Caetano-Anollés & Gresshoff (1991). Based on the PAGE results, '1' or '0' were assigned to the presence or absence of bands, respectively, and the same method was used for frequency of virulence analysis, '1' or '0' were assigned to resistance (ITs: 0-2) or susceptibility (ITs: 3-4), respectively, in the host. ...
Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (an obligate biotrophic pathogen) is a worldwide threat to wheat production that occurs over a wide geographic area in China. For monitoring genetic variation and virulence structure of Blumeria graminis f. sp. tritici in Liaoning, Heilongjiang, and Sichuan in 2015, 31 wheat lines with known Powdery mildew resistance genes and 2 EST-SSR markers were used to characterize the virulence and genetic diversity. Results indicated that 90% of all isolates were virulent on Pm3c , Pm3e , Pm3f , Pm4a , Pm5 , Pm6 (Timgalen), Pm7 , Pm16 , Pm19 , and Pm1 + 2 + 9 and 62.6% to 89.9% of isolates were virulent on Pm3a , Pm3b , Pm3d , Pm4b , Pm6 (Coker747), Pm8 , Pm17 , Pm20 , Pm23 , Pm30 , Pm4 + 8 , Pm5 + 6 , Pm4b + mli , Pm2 + mld , Pm4 + 2X , Pm2 + 6 . The Pm13 and PmXBD genes were effective against most collected isolates from Liaoning and Heilongjiang Provinces. Only Pm21 exhibited an immune infection response to all isolates. Furthermore, closely related isolates within each region were distinguished by cluster analyses using EST-SSR representing some gene exchanges and genetic relationships between the flora in Northeast China (Liaoning, Heilongjiang) and Sichuan. Only 45% of the isolates tested show a clear correlation between EST-SSR genetic polymorphisms and the frequency of virulence gene data. However, the EST-SSR polymorphism of isolated genes did not correspond to the virulence diversity of isolates in the single-gene lineage identification of hosts.
... The amplified products were separated on 20 cm long and 1 mm thick non-denaturing 8% acrylamide gels through electrophoresis in 1× TBE buffer at a constant voltage of 100 V, 50 W, and 50 mA for 1.0-1.5 h. After electrophoresis, the gels were silver stained according to Bassam et al. [31]. ...
Aneuploids are valuable materials of genetic diversity for genetic analysis and improvement in diverse plant species, which can be propagated mainly via in vitro culture methods. However, somaclonal variation is common in tissue culture-derived plants including euploid caladium. In the present study, the genetic stability of in vitro-propagated plants from the leaf cultures of two types of caladium (Caladium × hortulanum Birdsey) aneuploids obtained previously was analyzed morphologically, cytologically, and molecularly. Out of the randomly selected 23 and 8 plants regenerated from the diploid aneuploid SVT9 (2n = 2x − 2 = 28) and the tetraploid aneuploid SVT14 (2n = 4x − 6 = 54), respectively, 5 plants from the SVT9 and 3 plants from the SVT14 exhibited morphological differences from their corresponding parent. Stomatal analysis indicated that both the SVT9-derived variants and the SVT14-originated plants showed significant differences in stomatal guard cell length and width. In addition, the variants from the SVT14 were observed to have rounder and thicker leaves with larger stomatal guard cells and significantly reduced stomatal density compared with the regenerants of the SVT9. Amongst the established plants from the SVT9, two morphological variants containing 3.14–3.58% less mean fluorescence intensity (MFI) lost one chromosome, and four variants containing 4.55–11.02% more MFI gained one or two chromosomes. As for the plants regenerated from the SVT14, one variant with significantly higher MFI gained two chromosomes and three plants having significantly lower MFI resulted in losing four chromosomes. Three, out of the twelve, simple sequence repeat (SSR) markers identified DNA band profile changes in four variants from the SVT9, whereas no polymorphism was detected among the SVT14 and its regenerants. These results indicated that a relatively high frequency of somaclonal variation occurred in the in vitro-propagated plants from caladium aneuploids, especially for the tetraploid aneuploid caladium. Newly produced aneuploid plants are highly valuable germplasm for future genetic improvement and research in caladium.
... The annealing temperature of SSR primers was set as that described by Wu et al. (2015) [32], and ISSRs were set as those described in the ISSR primer database. The PCR products of ISSRs were detected using 1.5% agarose gel, and the SSR products were separated via 8% polyacrylamide gels and stained with silver [79]. ...
Previous studies indicated that extensive genetic variations could be generated due to polyploidy, which is considered to be closely associated with the manifestation of polyploid heterosis. Our previous studies confirmed that triploid loquats demonstrated significant heterosis, other than the ploidy effect, but the underlying mechanisms are largely unknown. This study aimed to overcome the narrow genetic distance of loquats, increase the genetic variation level of triploid loquats, and systematically illuminate the heterosis mechanisms of triploid loquats derived from two cross combinations. Here, inter-simple sequence repeats (ISSRs) and simple sequence repeats (SSRs) were adopted for evaluating the genetic diversity, and transcriptome sequencing (RNA-Seq) was performed to investigate gene expression as well as pathway changes in the triploids. We found that extensive genetic variations were produced during the formation of triploid loquats. The polymorphism ratios of ISSRs and SSRs were 43.75% and 19.32%, respectively, and almost all their markers had a PIC value higher than 0.5, suggesting that both ISSRs and SSRs could work well in loquat assisted breeding. Furthermore, our results revealed that by broadening the genetic distance between the parents, genetic variations in triploids could be promoted. Additionally, RNA-Seq results suggested that numerous genes differentially expressed between the triploids and parents were screened out. Moreover, KEGG analyses revealed that "photosynthetic efficiency" and "glyco-metabolism" were significantly changed in triploid loquats compared with the parents, which was consistent with the results of physiological indicator analyses, leaf micro-structure observations, and qRT-PCR validation. Collectively, our results suggested that extensive genetic variations occurred in the triploids and that the changes in the "photosynthetic efficiency" as well as "glyco-metabolism" of triploids might have further resulted in heterosis manifestation in the triploid loquats.
... Samples were run for 15h at 200 V in 4°C on a 12% acrylamide: bisacrylamide gel (37.5:1). Bands were appeared by 0.1 percent silver nitrate and NaOH solution (containing 0.1 percent formaldehyde) using the protocol of Bassam, Caetano-Anollés, and Gresshoff (1991). The PCR fragments from different SSCP patterns were sequenced in both directions. ...
The aim of current study was to survey genetic variability of PALM gene's exon 3 and 4 by PCR-SSCP and DNA sequencing in Zel and Lori Bakhtiari sheep breeds. The SIFT (Sorting Intolerant from Tolerant) and PHyre2 program were used to predict the possible impact of amino acid substitutions on performance and structure of the paralemmin protein. A total of 140 animal's from 2 Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Zel sheep breeds were considered. The results showed that there are two polymorphic sites including a nonsynonymous substitution and an insertion mutation (49bp). Non-synonymous mutation deduced Thr20Ala amino acid exchange and ensuing two different structures for paralemmin protein that could be potentially affect protein structure and function during the interaction with glutamate in the cytosolic surface of plasma membrane. PALM gene, according to evolutionary path, is classified into two separate categories. In first covey, Gallus gallus and in second one, other species in several branches, so that the sequence of cow and sheep is placed in a sub-branch which forms a clade beside goat. Comparison of illustrated coding region sequences, PALM gene among different species, is of orthologous which are derived from a common ancestor.
Frosty pod rot (FPR) disease, caused by Moniliophthora roreri, is the most important cocoa disease in the Western Hemisphere. In Guatemala, the presence of the pathogen is attributed to a rapid and clonal dissemination throughout Central America after one or very few introductions of M. roreri from South America. We analyzed the genetic diversity of isolates from the main cocoa producing departments in Guatemala using AFLP. In total, 15 different multilocus genotypes were found among 119 isolates, indicating a low genetic diversity (percentage of polymorphic loci and Nei’s gene diversity, 12.28% and 0.321, respectively). The obtained linkage disequilibrium through the observed and standardized indexes of association (IA and r̄d) confirmed clonality and asexual reproduction in the populations of M. roreri. The discriminant analysis of principal components suggested three genetic groups; nonetheless, the minimum spanning network and fixation index (ΦST = 0.043 P = 0.09) revealed a weak population structure, mainly attributed to high human-mediated gene flow (Nm = 11.13). Given the high mutation rate of M. roreri, constant monitoring of its evolution is suggested along with quarantine practices that limit its dispersal and evaluation of cocoa clones tolerant to the new genotypes of M. roreri, thus preventing increases in losses of Guatemalan cocoa production.
The use of molecular markers is one of the most sensitive, powerful technologies for genetic purity assessment of seed lots. In this study, we aimed to develop a set of insertion and deletion (InDel) markers, through bioinformatics approaches, that may effectively distinguish three representative japonica rice ( Oryza sativa L.) varieties, Nipponbare, Taichung 65 and Zhonghua11. The published whole-genome sequences of these varieties were aligned using BWA-MEM, followed by manual inspection for InDels of more than ten base pairs in size with the tview function of SAMtools. A set of ten InDel markers were thus identified and then validated by PCR in the three japonica rice varieties and their intercross F 1 hybrids. Results showed that the InDel markers developed in this study could reliably distinguish these three japonica rice varieties. These molecular markers together with the detection method developed here can be applied to DNA-based genetic purity evaluation in rice breeding.
Background
Faba bean ( Vicia faba L) is one of the most important legumes in the world. However, there is relatively little genomic information available for this species owing to its large genome. The lack of data impedes the discovery of molecular markers and subsequent genetic research in faba bean. The objective of this study was to analyze the faba bean transcriptome, and to develop simple sequence repeat (SSR) markers to determine the genetic diversity of 226 faba bean varieties derived from different regions in China.
Methods
Faba bean varieties with different phenotype were used in transcriptome analysis. The functions of the unigenes were analyzed using various database. SSR markers were developed and the polymorphic markers were selected to conduct genetic diversity analysis.
Results
A total of 92.43 Gb of sequencing data was obtained in this study, and 133,487 unigene sequences with a total length of 178,152,541 bp were assembled. A total of 5,200 SSR markers were developed on the basis of RNA-Seq analysis. Then, 200 SSR markers were used to evaluate polymorphisms. In total, 103 (51.5%) SSR markers showed significant and repeatable bands between different faba bean varieties. Clustering analysis revealed that 226 faba bean materials were divided into five groups. Genetic diversity analysis revealed that the relationship between different faba beans in China was related, especially in the same region. These results provided a valuable data resource for annotating genes to different categories and developing SSR markers.
The polymorphism in ovine relaxin family peptide receptor 2 gene and the relevance of earlier established human and mice cryptorchidism associated SNP’s in Mandya and Hassan Sheep was studied. Genomic DNA was extracted from 60 cryptorchid and 80 normal unrelated sheep. Two sets of primers were designed to amplify exon 8 and exon 12–13 regions of ovine RXFP2 gene. SSCP revealed no polymorphism at exon 8, exon 12 and exon 13 of ovine RXFP2 indicating absence of T222P in exon 8 and D294G in exon 12 as reported in human and mice cryptorchids respectively. A novel SNP (KF527573.1, 171T>A) in intron12 of ovine RXFP2 was observed. The sheep population studied was in Hardy Weinberg equilibrium for the genotypes of the SNP. The distribution of genotypes was significantly different for Hassan and Mandya sheep breeds. However, the SNP in both the breeds studied was not associated with the cryptorchid phenotype.
Rice blast disease caused by the fungus Pyricularia oryzae is one of the most devastating diseases of rice (Oryza sativa L.). Therefore, the use of resistant rice varieties would be the most effective way to control this disease. Based on disease evaluation, upland rice varieties were classified into two groups: resistant (35%) and moderately resistant (65%). Forty upland rice varieties and two lowland rice varieties were genotyped for seven major rice blast resistance genes Pi37, Pid2, Pi9, Pi36(t), Pi5, Pik-m, and Pi54. The gene frequencies of the seven major R genes ranged from 2.38% to 100%. The 42 varieties contained one to five R genes. Two varieties had five blast resistance genes, whereas 21 varieties contained four R genes, 15 varieties contained three R genes, 3 varieties contained two R genes, and only one variety contained one R gene. Furthermore, the relationship between the presence of different R genes and disease reactions was investigated using a multiple stepwise regression model. Three markers, Pi5, Pi54MAS, and Ckm2, for three R genes (Pi5, Pi54, and Pik-m) were moderately correlated with blast disease with partial correlation coefficients of 0.35 to 0.47. These results provide new sources of resistance genes for designing future breeding program to develop leaf blast-resistant rice varieties.
Genetic diversity is essential for sustainability, adaptation and evolution of forest tree populations, especially under changed climate conditions. Maintenance of standing genetic diversity and effective population size (Ne) in relict forest species is of special interest to preserve their genetic heritage. Forest harvesting and management practices can affect genetic diversity and population structure by affecting demography and several evolutionary processes. We examined the effect of shelterwood harvesting on genetic diversity, population structure and Ne of Oriental beech (Fagus orientalis L.) populations in the relict Hyrcanian forest region by comparing pairwise unmanaged and manged populations at five locations using nuclear microsatellites. High levels of genetic diversity and significant levels of fixation index were observed, and the populations showed geographical genetic structure. There was a trend of reduced genetic diversity and Ne in the managed compared to unmanaged populations, but the differences were not significant. The unmanaged populations had almost twice the number of private alleles that of the managed populations. The unmanaged populations were genetically differentiated from their managed counterparts to varying degrees. The two unmanaged populations from Asalem and Sangedeh which had other human disturbance of livestock grazing or illegal harvesting were genetically the most differentiated from their managed counterparts as well as all other populations. FST estimates indicated similar levels of genetic differentiation among unmanaged and among managed populations. Overall, our study shows that shelterwood harvesting although does not significantly reduce genetic diversity and Ne and increase inbreeding levels in the extant managed populations, which are only one generation apart from the unmanaged populations, it does cause some genetic diversity and Ne erosion. Our study has implications and applications for sustainable management and conservation of genetic resources of Oriental beech inside and outside of the Hyrcanian forest as well as for other beech species.
Purpose
The Seasonally dry forests of South America are known as the Gran Chaco are areas vulnerable in the world, the highest percentage of protected areas is found in South America. Anthropogenic processes as clearing of native forests makes ecosystems more fragile to changes, due to agricultural frontier expansion. We purpose study as the soil fungal community has been modified due to land use changes caused by clearing and agricultural activities.
Methods
We observed the response of the soil fungal community due to anthropogenic actions through to use phenotypic and genotypic tools to detecting changes in the diversity, at three study sites under different land uses in Chaco dry forest in Argentina. Soil samples were obtained from relicts of native forests of Schinopsis spp., cleared soils that are used later for agricultural activities and soil of soybean monoculture.
Results
The results provided a signal of consequences of human activity on soil fungal communities. This was visualized by the grouping of different soils by species fungi abundance, the presence of detector species in both sampling years and in the ordering of sampling sites through analysis with traditional and molecular tools such as PCR-DGGE. Soil organic carbon and phosphorous parameters were significantly modified by the interactions of sampling sites and years.
Conclusion
The present study emphasizes the different land use change between fungal communities of native soils and soils for agricultural purposes, being replaced by others with different soil roles.
In the present study, the genetic diversity of 120 barley genotypes was investigated through 14 phenologic and agronomic traits as well as 50 SSR markers that cover the seven chromosomes of barley. In addition, the relationships between these marker loci and the studied traits were assessed via multiple regressions in two conditions. The field experiment was conducted in two planting dates, namely before the ideal time of the region planting date and the ideal planting date of the region in the experimental field of the Faculty of Agriculture and Natural Resources, Gonbad-e-Kavous University. A randomized complete block design with two replications was laid out for both planting dates. The results of cluster analysis based on SSR data classified the studied barley genotypes into six groups. For all the traits, except for spike length, more than one informative marker was detected. The results of stepwise regression analysis revealed significant associations between the traits and 60 SSR marker alleles under cold stress conditions. Under normal conditions, 63 SSR marker alleles showed a significant correlation with the studied traits. Among the 50 SSR markers, particular attention should be paid to a number of them, including the Bmag0211 marker with the highest coefficient of determination (R²) in regression analyses of grain yield, 1000-kernel weight under non-stress conditions, and grain width and days to maturity under cold stress conditions as well as HVBM5A marker associated with spike length, 1000-kernel weight, and days to flowering under cold stress conditions. These markers may be a novel finding and relatively more reliable than the other identified markers. The informative markers could be suitable for marker-assisted breeding in barley after confirming them in other experiments.
A new silver stain for electrophoretically separated polypeptides can be rapidly and easily used and can detect as little
as 0.01 nanogram of protein per square millimeter. When employed with two-dimensional electrophoresis, it should permit qualitative
and quantitative characterization of protein distributions in body fluids and tissues. It has been used to demonstrate regional
variations in cerebrospinal fluid proteins.
The study of effects of several parameters on silver staining of proteins has led to the development of a staining method which is simple and reliable, requires only few stable solutions, and can be applied to all gel types such as sodium dodecyl sulfate (SDS) containing polyacrylamide or isoelectric focusing gels. It can be used for ultrathin layers (0.1–0.2 mm) or thicker slab gels (up to 3 mm). With this method not only proteins but also polypeptides of molecular weights as low as 2500 are detectable with high sensitivity. Comparison of isoelectric focusing and SDS-containing gels and a simple spot test on thin gellayers show that the detection sensitivity depends not so much on the type of proteins but rather on their structure. The redox properties of the gel are important for the staining mechanism.
A simplified, inexpensive silver stain for proteins in polyacrylamide gels also stains DNA with a sensitivity of 1 ng (< 0.03 ng/mm2). Sensitivity of detection is superior to ethidium bromide and, for 11 double-stranded fragments of ϕX174 DNA, staining is linear with respect to concentration. Surprisingly, the slope of staining is similar for pieces of DNA larger than 300 base pairs, but is greater for fragments of smaller size. Staining of DNA with silver is a practical alternative to visualization with ethidium bromide and may serve as a tool for elucidating the basis of silver staining.
A silver staining method is described for detecting subnanogram amounts of DNA and RNA on polyacrylamide and agarose gels. The key step for increased sensitivity and linearity is an initial soaking of the gel in a 0.1% solution of cetyltrimethylammonium bromide (CTAB). Agarose-containing gels can be silver stained after treatment with formaldehyde. The methods described are comparatively rapid, extremely sensitive (≅ 150 pg of DNA) and inexpensive (0.4 g of AgNo per 25 ml of slab volume).
By using light (“photodevelopment”) to develop a metallic silver image of proteins separated on polyacrylamide gels, it is possible to visualize protein and nucleic acid patterns within 10 min after electrophoretic separation. This light catalyzed silver stain is based on establishing gel conditions in which ionic silver is reduced to silver metal in the presence of light energy at a different rate in gel regions containing biopolymers (such as proteins or nucleic acids) than the rate of reduction in regions which are devoid of such biopolymers. This photodevelopment method requires only two solutions: a solution to “fix” the proteins and a solution containing silver ions, which produces an image when exposed to light. This type of protein stain has achieved a sensitivity of 0.5 ng of protein. DNA separated on polyacrylamide may also be visualized with this stain. However, DNA generally produces a negative image. Studies of photodevelopment silver stains allow further insight into the processes which are involved in the ability of silver to form images of biopolymers separated by electrophoresis.
A new modification of silver staining is presented which utilizes two chemical properties of thiosulfate: image enhancement by pretreatment of fixed gels, and formation of soluble silver complexes which prevents unspecific background staining during image development. This procedure provides high sensitivity for proteins, RNA and DNA in the nanogram range on a colorless, transparent background. The performance of this method is documented by staining one-and two-dimensional patterns of plant leaf proteins. Moreover, we achieved, for the first time, the detection of the non-structural, tobacco mosaic virus-specific 126 kDa protein directly in the one-dimensional protein pattern of infected protoplasts by a staining procedure.
A technique is described for staining DNA in polyacrylamide gels with silver. It is rapid, requiring about 30 min for whole staining and development procedure, very simple and at least 20 times more sensitive than ethidium bromide for the staining of double-stranded DNA in polyacrylamide gels. This technique can also be applied for the staining of denatured, single-stranded DNA as well as RNA after their electrophoretic separation on polyacrylamide gels, having the same sensitivity as for double-stranded DNA fragments.
The ultrasensitive silver staining procedure developed for proteins also stains nanogram quantities of RNA and DNA in polyacrylamide gels. A gradient polyacrylamide gel system is described which separates proteins from 104 to 106 Mr, RNA from 5S to 23S and DNA from 0.4 to 21 Kb. The sensitivity of nucleic acid silver staining in this gel system considerably exceeds that of commonly used DNA and RNA dye-binding stains.
The surprising finding that amplification of genomic DNA can be directed by only one oligonucleotide primer of arbitrary sequence to produce a characteristic spectrum of short DNA products of varying complexity, was applied as a strategy to detect genetic differences between organisms. This approach, DNA amplification fingerprinting (DAF), does not depend on cloning or DNA sequence information and can generate fingerprints from DNA of viral, bacterial, fungal, plant and animal origins. Primers as short as 5 nucleotides in length can produce complex banding patterns that are resolved by polyacrylamide gel electrophoresis and silver staining. Amplification fragment length polymorphisms (AFLPs) were detected between different human individuals as well as between soybean cultivars. It is anticipated that DAF will have wide application for DNA analysis.
Separation of polymerase chain reaction (PCR) amplification of specific fragment length polymorphisms was carried out on rehydratable polyacrylamide gels on a horizontal flat slab system. A discontinuous sulfate-borate buffer system was employed on 5-8% T gels crosslinked with 3.5% C. Samples were diluted in leading sulfate ion buffer at 1/10 the ionic strength of the separating gel buffer and placed directly onto the surface of the rehydrated gels in 0.5-10 microliters volumes. The trailing ion and counterion were contained in a gel plug and placed directly onto the anodal and cathodal ends of the gel, and the electrodes placed directly onto the surface of the gel plugs. Filter paper wicks, soaked in diluted leading ion buffer, were placed along each side to lower the ionic strength of the edges, thereby increasing mobility at the edge and thus preventing smile effects. The gel-gel contact of the plug and separating gel prevent the production of a junction potential which occurs between dissimilar materials such as a paper wick and the gel. Ten- to 20-cm separations were carried out from 2-5 h, respectively, and resolution in the 20 cm system was 1.6-4 bp (base pairs) between 100 and 500 bp, 4-7 bp between 500 and 1000 bp, 12-20 bp between 1000 and 2000 bp and about 50 bp between 2000 and 3000 bp. Between 3000 and 4000 bp, resolution fell off to +/- 100 bp. Sensitivity, using a silver stain, indicated that one could readily distinguish less than 10 pg of DNA per mm width on the gels.(ABSTRACT TRUNCATED AT 250 WORDS)
A method for ultrasensitive detection of proteins on polyacrylamide gels by staining with silver, recently described by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert (Science211, 1437–1438 (1981)), was applied with slight modifications to staining nucleic acids. Silver staining of double-stranded DNA was at least 100 times as sensitive as fluorescence staining with ethidium bromide, and at least 20 times as sensitive as staining with ammoniacal silver. The limit of detection of double-stranded DNA was approximately 25–50 pg/band with a cross-sectional area of 5 mm2. The intensities of silver staining of double-stranded fragments 271 bp or longer from HaeIII endonuclease digests of φX174 RF DNA were linear over a concentration range of 0.25 to 4 ng DNA/band. RNA and single-stranded DNA species as short as 10 to 20 nucleotides were detected with high sensitivity after electrophoresis on denaturing gels containing urea, suggesting that silver staining may be applicable to the sequencing of a few micrograms of unlabeled DNA. Methods for staining DNA using ammoniacal silver were relatively insensitive for small DNA fragments.
Staining of polyacrylamide gels with methylene blue prior to silver staining increases band resolution and sensitivity. This method permits resolution of multiple bands less than 1 mm apart, and is able to detect bands containing only 100 pg of RNA.
Using a modified silver stain of Merril et al. [(1981) Science 211, 1437-1438] for staining polypeptides in sodium dodecyl sulfate-polyacrylamide gels, protein bands reproducibly stain different shades of blue, yellow, red, and gray. The procedure is highly temperature dependent, with optimal color formation at 42 degrees C. The procedure may be completed within 2 h. Color formation is due to silver ion complexes with charged amino acid side chains. The color of the silver-protein complex can be predicted if the amino acid sequence is known, although some exceptions are discussed. This provides another dimension to the characterization of proteins by gel electrophoresis.