Search

Maize (Zea mays) is the third major cereal crop in the Indian subcontinent, but the crop yields per hectare of Indian maize cultivars are less than half of the global average due to the impurity of seed lots supplied to farmers. In this study, we discovered high-quality Single Nucleotide Polymorphic markers (SNPs) in two widely cultivated maize hybrids and their parental inbreds. Paired-end double digest restriction site-associated DNA sequencing was used to discover SNPs and a total of 30,764,454 reads with a read length of 151 bp per sample were generated. Genotyping of SNPs for maize hybrids 'MAH 14-5' and 'Hema' revealed a total of 47,812 and 15,815 Genetic Purity Analysis markers, respectively, of which 44,388 and 12,391 were unique with 3,424 being common to both hybrids. Identified SNPs were used to develop primers for Kompetitive Allele-Specific PCR genotyping assays to determine the genetic purity of 10 seed lots and the results were found to correlate with Grow-out-Tests. Thus, the SNPs discovered in this study proved reliable to test the genetic purity of commercial seed lots. Advances in plant molecular breeding tools especially ddRADseq for SNP discovery offer new opportunities to genotype existing cultivars and accelerate the production of genetically pure seeds.

— Glossopetalon inhabits arid regions in the American west and northern Mexico on limestone substrates. The genus comprises four species: G. clokeyi, G. pungens, G. texense, and G. spinescens. Three of the species are narrow endemics. The fourth, G. spinescens, is a widespread species with six recognized varieties. All six varieties are intricately branched shrubs that have been difficult to identify due to a lack of clearly delineating morphological characters. Characters typically used to differentiate the varieties of G. spinescens, such as stem coloration, leaf blade size, and presence of stipules, are highly variable within and among populations. A custom protocol of double digest restriction-site associated DNA sequencing (ddRAD) was used to resolve the phylogeny of Glossopetalon and address if population genetic data analyses (such as STRUCTURE, SVDquartets, and phylogenetic networks) support the recognition of six varieties of G. spinescens. Glossopetalon was fully supported as monophyletic and G. pungens was resolved sister to the remaining taxa in the genus. The varieties of G. spinescens were resolved as two distinct lineages corresponding to their biogeography, one to the northwest (lineage 1) and one to southeast (lineage 2) of the species range. Glossopetalon clokeyi was resolved at the base of lineage 1 and G. texense was embedded within lineage 2 sister to G. spinescens var. spinescens. Taxonomic changes include the recognition of G. texense and G. clokeyi as varieties of G. spinescens and description of a unique population from northern Arizona as a new variety, G. spinescens var. goodwinii.

To guarantee food safetyand sustainability, it is necessary to verify meat authenticity. This study focused on the development of single nucleotide polymorphism-based polymerase chain reaction-restriction fragment length polymorphism (SNP-based PCR-RFLP) and forensically informative nucleotide sequence (FINS) methodologies based on PCR amplification of the mitochondrial 12S rRNA gene for discrimination of six red meat species, that is, cattle, buffalo, goat, sheep, camel, and donkey. FINS allowed the unambiguous identification of all species analyzed. In addition, six SNPs, where a restriction site for TasI could be localized using a preliminary in silico analysis, gave a unique RFLP pattern for each species. The results revealed a low level of species substitution (8%) in the tested meat samples. In particular, one buffalo and goat samples have been substituted with cow and sheep, respectively. Finally, the developed techniques herein showed high potentials to be routinely used as reliable and fast tools to avoid meat species substitutions. PRACTICAL APPLICATION: This research deals with genetic techniques to trace meats. This kind of research helps the concerned agencies to build capacity to safeguard consumer sentiments as well as providing better market access and better food price and quality for the consumer.

Grape white rot (Coniothyrium diplodiella) is a major fungal disease affecting grape yield and quality. Quantitative trait locus (QTL) analysis is an important method for studying important horticultural traits of grapevine. This study was conducted to construct a high-density map and conduct QTL mapping for grapevine white rot resistance. A mapping population with 177 genotypes was developed from interspecific hybridization of a white rot-resistant cultivar (Vitis vinifera × V. labrusca ‘Zhuosexiang’) and white rot-susceptible cultivar (V. vinifera ‘Victoria’). Single-nucleotide polymorphism (SNP) markers were developed by restriction site-associated DNA sequencing. The female, male, and integrated maps contained 2,501, 4,110, and 6,249 SNP markers with average genetic distances of adjacent markers of 1.25, 0.77, and 0.50 cM, respectively. QTL mapping was conducted based on white rot resistance identification of 177 individuals in July and August of 2017 and 2018. Notably, one stable QTL related to white rot resistance was detected and located on linkage group LG14. The phenotypic variance ranged from 12.93 to 13.43%. An SNP marker (chr14_3929380), which cosegregated with white rot resistance, was discovered and shows potential for use in marker-assisted selection to generate new grapevine cultivars with resistance to white rot.

Minimal absent words (MAWs) are minimal-length oligomers absent from a genome or proteome. Although some artificially synthesized MAWs have deleterious effects, there is still a lack of a strategy for the classification of non-occurring sequences as potentially malicious or benign. In this work, by using Markovian models with multiple-testing correction, we reveal significant absent oligomers, which are statistically expected to exist. This suggests that their absence is due to negative selection. We survey genomes and proteomes covering the diversity of life and find thousands of significant absent sequences. Common significant MAWs are often mono- or dinucleotide tracts, or palindromic. Significant viral MAWs are often restriction sites and may indicate unknown restriction motifs. Surprisingly, significant mammal genome MAWs are often present, but rare, in other mammals, suggesting that they are suppressed but not completely forbidden. Significant human MAWs are frequently present in prokaryotes, suggesting immune function, but rarely present in human viruses, indicating viral mimicry of the host. More than one-fourth of human proteins are one substitution away from containing a significant MAW, with the majority of replacements being predicted harmful. We provide a web-based, interactive database of significant MAWs across genomes and proteomes.

Sorghum (Sorghum bicolor (L.) Moench) is one of the world’s leading cereal crops in agricultural production, which has a special importance in the arid regions. However, unlike other cereals, sorghum grain has a lower nutritional value, which is caused, inter alia, by the resistance of its seed storage proteins (kafirins) to protease digestion. One of the effective approaches to improve the nutritional value of sorghum grain is to obtain mutants with partially or completely suppressed synthesis or altered amino acid composition of kafirins. The employment of genome editing may allow to solve this problem by introducing mutations into the nucleotide sequences of the α- and γ-kafirin genes. In this study, genomic target motifs (23 bp sequences) were selected for the introduction of mutations into the α- and γ-KAFIRIN genes of sorghum. The design of the gRNAs was conducted using the online tools CRISPROR and CHOPCHOP. Two most suitable targets were chosen for α-KAFIRIN (k1C5) and two for γ-KAFIRIN (gKAF1) genes. The insertion of respective sequences in the generic vector pSH121 was performed at the BsaI (Eco31I) sites. Validation of the cloning procedure was performed by DNA sequencing. Subcloning of the resulting constructs was performed using the SfiI restriction sites into the compatible binary vector B479p7oUZm-LH. The correct assembly of binary vectors was confirmed by restriction analysis using the MluI and SfiI cleavage sites. The four vectors created (1C - 4C) were transferred by electroporation into the Agrobacterium tumefaciens strain AGL0. Currently, this vector series is used for stable transformation of sorghum using immature embryo explants.

At present, low-pass whole-genome sequencing (WGS) is frequently used in clinical research and in the screening of copy number variations (CNVs). However, there are still some challenges in the detection of triploids. Restriction site-associated DNA sequencing (RAD-Seq) technology is a reduced-representation genome sequencing technology developed based on next-generation sequencing. Here, we verified whether RAD-Seq could be employed to detect CNVs and triploids. In this study, genomic DNA of 11 samples was extracted employing a routine method and used to build libraries. Five cell lines of known karyotypes and 6 triploid abortion tissue samples were included for RAD-Seq testing. The triploid samples were confirmed by STR analysis and also tested by low-pass WGS. The accuracy and efficiency of detecting CNVs and triploids by RAD-Seq were then assessed, compared with low-pass WGS. In our results, RAD-Seq detected 11 out of 11 (100%) chromosomal abnormalities, including 4 deletions and 1 aneuploidy in the purchased cell lines and all triploid samples. By contrast, these triploids were missed by low-pass WGS. Furthermore, RAD-Seq showed a higher resolution and more accurate allele frequency in the detection of triploids than low-pass WGS. Our study shows that, compared with low-pass WGS, RAD-Seq has relatively higher accuracy in CNV detection at a similar cost and is capable of identifying triploids. Therefore, the application of this technique in medical genetics has a significant potential value.

In Japan, mitragynine, 7-hydroxymitragynine and Mitragyna speciosa Korth. (M. speciosa, "Kratom") were controlled as Designated Substances under the Pharmaceutical and Medical Device Act from March 2016. In this study, the origins of 16 Kratom products obtained from the illegal drug market in Japan were investigated by DNA analyses and LC-MS analyses. When the PCR-restriction fragment length polymorphism (RFLP) was performed using the restriction enzyme XmaI (as reported by Sukrong et al. to be able to distinguish M. speciosa), the same DNA fragment patterns were obtained from all 16 products. On the other hand, as a result of the identification of the plant species of each product by nucleotide sequence analyses, the sequences of M. speciosa were detected in only 14 products. Despite the facts that mitragynine and 7-hydroxymitragynine were detected also in the other two products by the LC-MS analyses, M. speciosa DNAs were not amplified from these products by the PCR. Moreover, the DNA amplicons of the other psychotropic plant (Mesembryanthemum sp., e.g. "Kanna") were detected. This plant PCR amplicon has the restriction site for the XmaI at the same position of the M. speciosa PCR amplicon and it is difficult to distinguish "Kratom" and "Kanna" by the conventional PCR-RFLP. When the restriction enzyme XhoI was used simultaneously with the Xmal, the specific DNA fragment was only observed from the M. speciosa amplicon and it was possible to distinguish both species using this improved PCR-RFLP method. This method is useful to identify the origin of Kratom products distributed in the illegal drug market.

G-quadrupex is now known to play crucial roles in various biological reactions. However, direct evidence for its presence in cells has been limited, due to the lack of versatile and non-biased methodology. We use Rif1 binding sites on the fission yeast genome, which has been shown to adopt G4 structures, as a model to prove that Rif1 BS indeed adopt G4 structure in cells. We take advantage of the presence of a single-stranded loop in the G4 structure. Rif1BS is unique in that they contain unusually long loop sequences, and we replace them with a 18 bp I-SceI restriction site. We show in vitro that I-SceI in the loop is not cleaved when G4 is formed on duplex Rif1BS DNA, but is cleaved when G4 is not formed due to a mutation in the G-tracts. This is observed both heat-induced and transcription-induced G4 structure, and gives proof of evidence for this procedure. We apply this strategy for detection of a G4 structure at the same Rif1BS in fission yeast cells. We present evidence that in vivo cleavage of I-SceI can be a measure for the presence of G4 at the target sequence in cells as well. The method described here gives a platform strategy for genome-wide analyses of cellular G4 and their dynamic formation and disruption.

Background Various polymerase chain reaction (PCR)-based methods have been applied for the development of genome walking (GW) technique. These methods which could be based on the application of restriction enzymes or primers have various efficiencies to identify the unknown nucleotide sequences. The present study was conducted to design a new innovative double-strand adaptor using MAP30 gene sequence of Momordica charantia plant as a model to improve genome walking with convenient PCR. Results The adaptor was designed using multiple restriction sites of Hind III, BamH I, EcoR I, and Bgl II enzymes with no restriction site in a known sequence of the MAP30 gene. In addition, no modification was required to add phosphate, amine, or other groups to the adaptor, since restriction enzyme digestion of double-strand adaptor provided the 5′ phosphate group. Here, preparation of the phosphate group in the genomic DNA of the plant digestion with restriction enzymes was performed followed by ligation with digested adaptor containing 5′ phosphate group. Conclusion PCR was done to amplify the unknown sequence using MAP30 gene-specific primer and adaptor primer. Results confirmed the ability of the technique for successful identification of the sequence. Consequently, a newly designed adaptor in the developed technique reduced the time and cost of the method compared to the conventional genome walking; also, cloning and culturing of bacterial steps could be eliminated.

MotivationThe application of genome-wide chromosome conformation capture (3C) methods to prokaryotes provided insights into the spatial organization of their genomes and identified patterns conserved across the tree of life, such as chromatin compartments and contact domains. Prokaryotic genomes vary in GC content and the density of restriction sites along the chromosome, suggesting that these properties should be considered when planning experiments and choosing appropriate software for data processing. Diverse algorithms are available for the analysis of eukaryotic chromatin contact maps, but their potential application to prokaryotic data has not yet been evaluated.ResultsHere, we present a comparative analysis of domain calling algorithms using available single-microbe experimental data. We evaluated the algorithms’ intra-dataset reproducibility, concordance with other tools and sensitivity to coverage and resolution of contact maps. Using RNA-seq as an example, we showed how orthogonal biological data can be utilized to validate the reliability and significance of annotated domains. We also suggest that in silico simulations of contact maps can be used to choose optimal restriction enzymes and estimate theoretical map resolutions before the experiment. Our results provide guidelines for researchers investigating microbes and microbial communities using high-throughput 3C assays such as Hi-C and 3C-seq.Availability and implementationThe code of the analysis is available at https://github.com/magnitov/prokaryotic_cids.Supplementary informationSupplementary data are available at Bioinformatics online.

Toxoplasma gondii is a widespread protozoan parasite that infects one third of the global human population. Very little information is known about the impact of T. gondii on patients with heart disease. The aim of the present study was to determine the association between T. gondii exposure and patients suffering from myocardial infarction. The infection rate of anti-Toxoplasma IgG antibodies in 86 patients with myocardial infarction (troponin‑T positive) and 86 age and gender-matched controls (troponin‑T negative) was examined using enzyme-linked immunoassays. The DNA extraction was performed on separated buffy coats of serologically positive blood samples (32 samples with high titer of anti-Toxoplasma IgG). The GRA6 gene of T. gondii was amplified using PCR. The existence of polymorphic restriction sites for endonuclease MseI was used with the PCR-RFLP method and the bases of GRA6 gene were sequenced to determine the type of strains (I, II and III). A positive anti-Toxoplasma IgG level was found in 61.6% of the myocardial infarction samples and in 24.4% of the healthy controls (P- value

Background Cold hardiness is an important agronomic trait and can significantly affect grape production and quality. Until now, there are no reports focusing on cold hardiness quantitative trait loci (QTL) mapping. In this study, grapevine interspecific hybridisation was carried out with the maternal parent ‘Cabernet sauvignon’ and paternal parent ‘Zuoyouhong’. A total of 181 hybrid offspring and their parents were used as samples for restriction-site associated DNA sequencing (RAD). Grapevine cane phloem and xylem cold hardiness of the experimental material was detected using the low-temperature exotherm method in 2016, 2017 and 2018. QTL mapping was then conducted based on the integrated map. Results We constructed a high-density genetic linkage map with 16,076, 11,643, and 25,917 single-nucleotide polymorphism (SNP) markers anchored in the maternal, paternal, and integrated maps, respectively. The average genetic distances of adjacent markers in the maps were 0.65 cM, 0.77 cM, and 0.41 cM, respectively. Colinearity analysis was conducted by comparison with the grape reference genome and showed good performance. Six QTLs were identified based on the phenotypic data of 3 years and they were mapped on linkage group (LG) 2, LG3, and LG15. Based on QTL results, candidate genes which may be involved in grapevine cold hardiness were selected. Conclusions High-density linkage maps can facilitate grapevine fine QTL mapping, genome comparison, and sequence assembly. The cold hardiness QTL mapping and candidate gene discovery performed in this study provide an important reference for molecular-assisted selection in grapevine cold hardiness breeding.

DNA has become a promising candidate as a future data storage medium; this makes DNA steganography indispensable in DNA data security. PCR primers are conventional secret keys in DNA steganography. Brute force testing of different primers will be extremely time consuming, and practically unaffordable when high‐throughput sequencing is used. However, the encrypted information can be sequenced and read once the primers are intercepted. A new steganography approach is needed to make the DNA‐encoded information safer, if not unhackable. Mixing information‐carrying DNA with a partially degenerated DNA library containing single or multiple restriction sites, we have built an additional protective layer that can be removed by desired restriction enzymes as secondary secret keys. As PCR is inevitable for reading DNA‐encrypted information, heating will cause reshuffling and generate endonuclease‐resistant mismatched duplexes, especially for DNA with high sequence diversity. Consequently, with the incorporation of randomness, DNA steganography possesses both quantum key distribution (QKD)‐like function for detecting PCR by an interceptor and a self‐destructive property. It is noteworthy that the background noise generated through the protective layer is independent from any sequencing technology including Sanger and high‐throughput sequencing. With a DNA ink incorporating the steganography, we have shown that the authenticity of a piece of writing can be confirmed only by authorized persons with knowledge of all embedded keys., Cryptic clues: We show DNA steganography using basic molecular biology techniques with a restriction enzyme as a second secret key: Alice encrypts a message and sends it to Bob. Bob needs a combination of keys 1 and 2 to sequence the DNA and retrieve the information; he can also use qPCR to detect any interception by Eve.

The phenotypic identification of sex in Sebastes rockfish is difficult and often impractical from a management perspective, and the genetic basis of sex determination in the genus is currently uncertain. We tested a previously developed sex identification polymerase chain reaction restriction fragment length polymorphism (PCR–RFLP) assay in eight species of Sebastes rockfish. Results indicated that the association of this restriction site with sex is species-dependent.

In non-model organisms, evolutionary questions are frequently addressed using reduced representation sequencing techniques due to their low cost, ease of use, and because they do not require genomic resources such as a reference genome. However, evidence is accumulating that such techniques may be affected by specific biases, questioning the accuracy of obtained genotypes, and as a consequence, their usefulness in evolutionary studies. Here, we introduce three strategies to estimate genotyping error rates from such data: through the comparison to high quality genotypes obtained with a different technique, from individual replicates, or from a population sample when assuming Hardy-Weinberg equilibrium. Applying these strategies to data obtained with Restriction site Associated DNA sequencing (RAD-seq), arguably the most popular reduced representation sequencing technique, revealed per-allele genotyping error rates that were much higher than sequencing error rates, particularly at heterozygous sites that were wrongly inferred as homozygous. As we exemplify through the inference of genome-wide and local ancestry of well characterized hybrids of two Eurasian poplar (Populus) species, such high error rates may lead to wrong biological conclusions. By properly accounting for these error rates in downstream analyses, either by incorporating genotyping errors directly or by recalibrating genotype likelihoods, we were nevertheless able to use the RAD-seq data to support biologically meaningful and robust inferences of ancestry among Populus hybrids. Based on these findings, we strongly recommend carefully assessing genotyping error rates in reduced representation sequencing experiments, and to properly account for these in downstream analyses, for instance using the tools presented here.

From simple 2-dimensional (2D) structures to highly complex 3-dimensional (3D) structures, DNA is widely used as a building block material. Despite this advancement, mass production of DNA nanostructures was a challenge due to the high cost that accompanies the preparation of diverse DNA strands. Rolling circle amplification (RCA) is one of the cost-effective methods for the mass production of repetitive DNA structures without using an expensive laboratory thermal cycler. Here, we report a DNA hydrogel containing various single-stranded DNA nanostructures (Y, X, Tetrahedron) in a one-pot system, prepared by RCA, and their selective separation with various restriction enzymes. Each DNA nanostructure in the DNA hydrogel contained distinctive restriction sites that were responsive to different kinds of restriction enzymes, achieving selective separation of Y-, X- and Tetrahedron-shaped DNA structures. This study has the potential to contribute towards DNA-based biomedical applications by employing mass production and precisely controlled release of DNA nanostructures.

Fusarium graminearum is a toxigenic plant pathogen that causes Fusarium head blight (FHB) disease on cereal crops. It has recently shown to have cross-pathogenicity on noncereals (i.e., Fusarium root rot [FRR] on soybean) in Canada and elsewhere. Specific detection and differentiation of this potent toxigenic, trichothecene-producing pathogen among other closely related species is extremely important for disease control and mycotoxin monitoring. Here, we designed a PCR restriction fragment length polymorphism protocol based on the DNA sequence of the translational elongation factor 1α (TEF1α) gene. A unique restriction site to the enzyme HpaII is only found in F. graminearum sensu stricto strains among different Fusarium strains in the F. graminearum species complex (FGSC) and other Fusarium spp. associated with FHB in cereals and FRR in soybean. Partial amplification of the TEF1α gene with newly designed primers mh1/mh2 generated a 459-bp PCR fragment. Restriction digestion of the generated fragments with the HpaII enzyme generated a unique restriction pattern that can rapidly and accurately differentiate F. graminearum sensu stricto among all other Fusarium spp. A primer pair (FgssF/FgssR) specific to F. graminearum sensu stricto also was designed and can distinguish F. graminearum sensu stricto from all other Fusarium spp. in the FGSC and other closely related Fusarium spp. involved in FHB and FRR. This finding will be very useful for the specific detection of F. graminearum sensu stricto for diagnostic purposes as well as for the accurate detection of this pathogen in breeding and other research purposes.

Pathogen causes severe loss of crop plants worldwide, the study presents isolation, identification and molecular marker development of six fungal pathogens from the different varieties of the mango from Rajkot and nearby areas. These include two strains of Colletotrichum gloeosporioides (SUF24) and C. gloeosporioides (SUF29), Fusarium fujikuroi, Phaeosphaeria nodorum, Botryosphaeria dothidea, and Pseudofusicoccum ardesiacum. They were cultured and their DNA was isolated. PCR amplification was performed using ISSR primers. Amplification was checked by agarose gel electrophoresis and band pattern was scored. PCR amplification showed monomorphic and polymorphic bands with the studied primers. ISSR primer 880 showed monomorphic band in all studied fungi. Each monomorphic band was cloned, sequenced and selected for further bioinformatics analysis. From the obtained sequences, multiple sequence analysis, sequence similarity matrix, frequency distribution plot, pairwise identity using heat map, restriction site analysis, polymorphism and haplotype identification was performed. It was observed that the monomorphic band of these fungi have nearly similar base pairs length but the nucleotide patterns were found to be different. Even two strains of Colletotrichum gloeosporioides showed distinct patterns. Each one of the sequences showed unique nucleotide sequence composition and RE cutting sites, thus monomorphic band can be converted to the polymorphic band pattern. This approach is used to design new DNA markers for the fungi studied and its applicability to discriminate fungi even at a strain level is discussed.

A total of five protoclones were successfully cultured on PDA medium out of regenerated twenty two colonies of Termitomyces protoplast and further studied. Liquid MYG grown mycelial tissue is used for protoplast isolation by enzymatic digestion in a mixture containing Lysing enzyme 2% and Cellulase R10 2% in 0.6 M mannitol. The incubation conditions like temperature, shaking and time were standardized at 24oC with shaking 60 rpm for 10 hours, respectively for healthy protoplasts liberation. The purified protoplasts showed 31.60±9.31% regeneration on specific medium and 77.12±2.72% viability by FDA test. Four ISSR primers were used in this study with a total of 27 reproducible bands with mean value 6.75 and they showed similar banding pattern in all with zero percent polymorphism ranged from 280 bp - 2700 bp. The amplified rRNA-ITS gene showed ~ 600 bp size in gel and found a single restriction site for enzyme HaeIII in all the protoclones and parent with similar fragment size in all.

Guangxi indigenous chicken breeds play a very important role in promoting the high-quality development of the broiler industry in China. However, studies on genomic information of Guangxi indigenous chicken to date remain poorly explored. To decipher the population genetic structure and differentially selected regions (DSRs) in Guangxi indigenous chickens, we dug into numerous SNPs from seven Guangxi native chickens (GX) by employing the restriction site associated with DNA sequencing (RAD-seq) technology. Another three breeds, Cobb, White Leghorn, and Chahua (CH) chicken, were used as a control. After quality control, a total of 185,117 autosomal SNPs were kept for further analysis. The results showed a significant difference in population structure, and the control breeds were distinctly separate from the Guangxi native breeds, which was also strongly supported by the phylogenetic tree. Distribution of FST indicated that there were three SNPs with big genetic differentiation (FST value all reach to 0. 9427) in GX vs. CH group, which were located on chr1-96,859,720,chr4-86,139,601, and chr12-8,128,322, respectively. Besides, we identified 717 DSRs associated with 882 genes in GX vs. Cobb group, 769 DSRs with 476 genes in GX vs. Leghorn group, and 556 DSRs with 779 genes in GX vs. CH group. GO enrichment showed that there were two significant terms, namely GPI-linked ephrin receptor activity and BMP receptor binding, which were enriched in GX vs. Leghorn group. In conclusion, this study suggests that Guangxi native chickens have a great differentiation with Cobb and Leghorn. Our findings would be beneficial to fully evaluate the genomic information on Guangxi native chicken and facilitate the application of these resources in chicken breeding.

The present study was carried out in 112 camels belonging to Bikaneri, Jaisalmeri, Kachchhi and Mewari breeds of Indian dromedary to detect point mutation in ĸ- casein encoding gene. Amplification of 488 bp fragment of ĸ-Casein gene spanning from -137 (5' flanking region) to +351 bp of ĸ-CN gene was carried out and genotyped for the g.1029T>C SNP using the restriction enzyme AluI in PCR-RFLP analysis. Three restriction patterns were resolved on 3.5% agarose gels. The pattern, comprising of 203 bp, 158 bp and 127 bp bands, was resolved successfully for the TT samples. The g.1029T>C transition created an additional restriction site for the enzyme AluI leading to the digestion of 158 bp band into two fragments of 120 bp and 38 bp resulting in the 5 band pattern of 203 bp, 158 bp, 127 bp, 120 bp and 38 bp for CT genotype and 4 band pattern of 203 bp, 127 bp, 120 bp and 38 bp for CC genotype. The genotype frequency, pooled over breed, was 0.045, 0.384 and 0.571 for the CC, CT and TT genotypes, respectively. The frequency of major allele T was observed to be 0.763 and that of C was observed to be 0.237. The existence of CT genotype in sizable number documents the dynamic nature of the locus g.1029T>C SNP, in Indian dromedary breeds. Almost comparable polymorphism was observed in both the sexes. The 3 genotypes, viz. CC, CT, TT, were almost equally distributed among the four Indian breeds (x2=3.4529; P = 0.750224). The frequency of C allele was lowest in Bikaneri and highest in the Mewari breed. Though the frequency of C allele (Cytosine) in Indian dromedary is relatively low (0.237), still a rapid directional selection might be attempted in favour of the C allele, which is responsible for the creation of an extra putative site for the Hepatocyte Nuclear Factor - 1 (HNF-1) transcription factor. The HNF-1 is reported to be involved in regulation of a number of genes associated with innate immunity, lipid and glucose transport, metabolism etc.

Insertion of silent mutations allowing for restriction site modification aids in the screening of successful mutants during site directed mutagenesis. Introducing a new restriction site requires the analysis of degenerate sequences within mutant primer. As the total number of degenerate codons increases, the analysis becomes increasingly laborious, time-consuming and prone to errors. Towards this, a software named as ‘REMP’ (for REstriction site in Mutant Primer) was developed and described here. From the input sequence, REMP instantaneously generates degenerate sequences having restriction sites that are 6–8 base pairs in length. The output sequences are arranged based on the number of bases changed compared to the input sequence. REMP software can be installed and run as a stand-alone program on different operating systems. Any user of REMP can edit the list of restriction sites to be considered by the software, without a need for writing a computer code or knowing a program language.

Preselection digests are a common strategy to reduce the background in the ligation step of molecular cloning. However, choosing fitting restriction enzymes by hand is not trivial and may lead to errors, potentially costing a lot of time and work. We therefore created preselector.uni-jena.de (https://preselector.uni-jena.de/), a free online tool to find such restriction enzymes. The tool uses regular expressions to find recognition sites of restriction enzymes in the DNA sequences provided by the user. This new tool compares the sets of restriction sites and reports the enzymes that cut one sequence but not the other sequences to the user. These enzymes are then the ones suitable for a preselection digest. Thus, preselector.uni-jena.de is a fast, reliable, and free-to-use tool to help researchers designing preselection digestion strategies for their cloning., Graphical Abstract Graphical AbstractFind your preselection enzymes with preselector.uni-jena.de.

SUMMARY Spatial organization of molecules and cells in complex tissue microenvironments provides essential organizational cues in health and disease. A significant need exists for improved visualization of these spatial relationships. Here, we describe a multiplex immunofluorescence imaging method, termed SeqStain, that uses fluorescent-DNA-labeled antibodies for immunofluorescent staining and nuclease treatment for de-staining that allows selective enzymatic removal of the fluorescent signal. SeqStain can be used with primary antibodies, secondary antibodies, and antibody fragments to efficiently analyze complex cells and tissues. Additionally, incorporation of specific endonuclease restriction sites in antibody labels allows for selective removal of fluorescent signals while retaining other signals that can serve as marks for subsequent analyses. The application of SeqStain on human kidney tissue provided a spatialomic profile of the organization of >25 markers in the kidney, highlighting it as a versatile, easy-to-use, and gentle new technique for spatialomic analyses of complex microenvironments., Graphical Abstract, In brief Multiplex imaging provides important insights into spatial organization of molecules and cells in complex tissues. In this issue of Cell Reports Methods, Rajagopalan et al. describe a method using sequential steps of staining with fluorescent-DNA-labeled antibodies and de-staining with nucleases to profile multiple molecules and cells in single tissue sections.

BackgroundMultiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher orderArabidopsis thalianamutants. Low entry, reliable assembly of CRISPR/Cas9 vectors and efficient mutagenesis is necessary to enable a maximum of researchers to break through the genetic redundancy within plant multi-gene families and allow for a plethora of gene function studies that have been previously unachievable. It will also allow routine de novo generation of mutations in ever more complex genetic backgrounds that make introgression of pre-existing alleles highly cumbersome.ResultsTo facilitate rapid and efficient use of CRISPR/Cas9 forArabidopsisresearch, we developed a CRISPR/Cas9-based toolbox for generating mutations at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either a FastRed, FastGreen or FastCyan fluorescent seed selection cassette. FastRed and FastGreen binary vectors can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors. Importantly, in the second generation after transformation,Cas9free plants are identified simply by screening the non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) fromA. thalianaor the constitutive promoter fromPetroselinum crispum(PcUBi4-2). Available enzymes are “classical”Cas9codon-optimized forA. thalianaand a recently reported, intron-containing version ofCas9codon-optimized forZea mays,zCas9i. We observed the highest efficiency in producing knockout phenotypes by using intron-containingzCas9idriven under egg-cell specificpEC1.2promoter. Finally, we introduced convenient restriction sites flanking promoter,Cas9and fluorescent selection cassette in some of the T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system.ConclusionA rapid, simple and flexible CISPR/Cas9cloning system was established that allows assembly of multi-guide RNA constructs in a robust and reproducible fashion, by avoiding generation of very big constructs. The system enables a flexible, fast and efficient screening of single or higher orderA. thalianamutants.

Polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR) is a technique used to identify single nucleotide polymorphisms (SNPs) based on the recognition of restriction sites by restriction enzymes. RFLP-PCR is an easy-to-perform and inexpensive tool for initial analysis of SNPs potentially associated with some monogenic diseases, as well as in genotyping, genetic mapping, lineage screening, forensics and ancient DNA analysis. The RFLP-PCR method employs four steps: (1) isolation of genetic material and PCR; (2) restriction digestion of amplicons; (3) electrophoresis of digested fragments; and (4) visualisation. Despite its obsolescence and the presence of high-throughput DNA analysis techniques, it is still applied in the analysis of SNPs associated with disease entities and in the analysis of genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RFLP-PCR is a low-cost and low-throughput research method allowing for the analysis of SNPs in the absence of specialised equipment, and it is useful when there is a limited budget.

Optical Mapping is a method of DNA sequencing, that can be used to detect large structural variations in genomes. To create these optical maps, a restriction enzyme is mixed with DNA where the enzyme binds to DNA creating labels called restriction sites. These restriction sites and can be captured by fluorescent microscope with a camera. One of the tools that can capture these images is Bionano Genomics Saphyr mapping instrument. Their system produces high-resolution images of DNA molecules with restriction sites and their software detects them. Molecules in these images are visualized by gray lines with restrictions sites appearing brighter. Some of these molecules have very low brightness and with static noise around them, they are almost indistinguishable from the background. This work proposes GPU accelerated method for molecule detection. A neural network was used for molecule segmentation from the image and the dataset for the network was created from the results of Bionano Genomics tools. This detection method can be used as a substitute for restriction map detection in the Bionano Genomics processing pipeline or as a tool that can highlight the regions of interest in the raw optical maps images.

Summary Chromatin immunoprecipitation sequencing (ChIP-seq) is an efficient technique to identify the binding sites of transcription factors (TFs) in both eukaryotes and prokaryotes. However, its application in bacteria is very heterogeneous. In this protocol, we optimized the methods of ChIP-seq that can be widely applied to plant pathogens. We used homologous recombination to construct pK18mobsacB-Psph plasmid instead of restriction site ligation and replaced transconjugation with electroporation transformation in Pseudomonas syringae deletion mutant construction, which is more efficient and faster than previous methods. For complete details on the use and execution of this protocol, please refer to Shao et al. (2021)., Graphical abstract, Highlights • This approach can be used to construct TF-overexpressed Pseudomonas syringae strain • Protocols for ChIP-seq library construction of Pseudomonas syringae • A simplified procedure to construct a deletion mutant of Pseudomonas syringae, Chromatin immunoprecipitation sequencing (ChIP-seq) is an efficient technique to identify the binding sites of transcription factors (TFs) in both eukaryotes and prokaryotes. However, its application in bacteria is very heterogeneous. In this protocol, we optimized the methods of ChIP-seq that can be widely applied to plant pathogens. We used homologous recombination to construct pK18mobsacB-Psph plasmid instead of restriction site ligation and replaced transconjugation with electroporation transformation in Pseudomonas syringae deletion mutant construction, which is more efficient and faster than previous methods.

Reduced representation sequencing (RRS) is a widely used method to assay the diversity of genetic loci across the genome of an organism. The dominant class of RRS approaches assay loci associated with restriction sites within the genome (restriction site associated DNA sequencing, or RADseq). RADseq is frequently applied to non-model organisms since it enables population genetic studies without relying on well-characterized reference genomes. However, RADseq requires the use of many bioinformatic filters to ensure the quality of genotyping calls. These filters can have direct impacts on population genetic inference, and therefore require careful consideration. One widely used filtering approach is the removal of loci which do not conform to expectations of Hardy-Weinberg equilibrium (HWE). Despite being widely used, we show that this filtering approach is rarely described in sufficient detail to enable replication. Furthermore, through analyses of in silico and empirical datasets we show that some of the most widely used HWE filtering approaches dramatically impact inference of population structure. In particular, the removal of loci exhibiting departures from HWE after pooling across samples significantly reduces the degree of inferred population structure within a dataset (despite this approach being widely used). Based on these results, we provide recommendations for best practice regarding the implementation of HWE filtering for RADseq datasets.

Mauremys reevesii and M. sinensis are economically important freshwater turtle species widely distributed in China. However, due to heavy overexploitation, their wild populations are near extinction and most living resources are no intensively cultured in breeding farms. To set up an effective management and conservation strategy for both species, we carried out extensive restriction site-associated DNA sequencing (RAD-seq) to develop robust single-nucelotide polymorphism (SNP) markers. A total of 144 novel high-quality SNP markers were isolated and characterized from four populations in China, including Anhui P.R., Guangdong P.R., Hubei P.R. and Taiwan P.R., five of each population. The values of observed and expected heterozygosity ranged respectively from 0.0000 to 0.7895 and from 0.0950 to 0.5000. The inbreeding coefficient varied from − 0.6087 to 1.0000, and 79 SNPs significantly deviated from Hardy–Weinberg equilibrium (P

Epizootic monitoring in recent years suggests that the highly pathogenic avian influenza A virus (H1N1) and (H7N9) actively circulate in the Eurasian countries. By 2016 - 2019 1.6 thousand outbreaks were recorded. For 2016 - 2019, 1.6 thousand cases of outbreaks were recorded. Of these, there are 872 cases in Europe. The monitoring of infected birds, both migratory and poultry, in places of cross-contact in Ukraine is relevant for preventing outbreaks of epizooties. The aim of the study. To develop an express method for the identification and determination of bird flu virus A H1N1 and H7N9 strains, based on a polymerase chain reaction with analysis of restriction fragment length polymorphism (PCR-RFLP) of the virus RNA. Results and discussion. The in silico analysis of the HA, NA, and NP gene amplicons allowed in silico to calculate the primers to the variable loci of the investigated genes, to calculate the reaction conditions, to determine restriction sites for the restriction enzyme to obtain theoretical PCR electrophoregrams. An express method for the detection and identification of influenza A H1N1 and H7N9 virus by three genes (HA, NA, and NP) of H1N1 and H7N9 RNA in polymerase chain reaction, combined with RFLP analysis, was developed. The method of rapid diagnostics is able to detect avian influenza virus A H1N1 and H7N9 and differentiate it from samples of other pathogens of viral infections of birds and animals. It was established, that the PCR-RFLP rapid diagnostic method is able to detect influenza A virus RNA of H1N1 and H7N9 strains with high sensitivity (100 % sensitivity). Conclusions. The developed method of PCR-based rapid identification, combined with RFLP analysis, makes it possible to significantly simplify the method of identification due to specific amplification of an RNA region having a polymorphic restriction site. Testing of this locus is possible by pre-PCR and restriction of the amplified fragment. The method of express - diagnosis of PLR-RFLP has been established for detecting RNA virus influenza A of high pathogenic H1N1 and H7N9 strains with high indicators of sensitivity (100 % sensitivity)

The existing molecular cloning methods are often limited by the availability of suitable restriction sites. It is still a challenge for simultaneous cloning of multiple fragments into different sites of a single vector. Here we developed a novel method named improved overlap extension PCR (IOEP) for restriction enzyme independent cloning of large fragments. The addition of primers enables IOEP to exponentially amplify the overlap extension product, thus greatly improves the amplification efficiency of large fragments. Moreover, coupled with the benefit of T4 DNA polymerase to improve cloning efficiency, our method can be used to simultaneously insert, delete and replace multiple DNA fragments at different sites.

European hazelnut (Corylus avellana L.) is an economically important edible nut producing species, which ranked sixth in world tree nut production in 2016. European hazelnut production in the United States is primarily limited to the Willamette Valley of Oregon, and currently nonexistent in the eastern United States because of the presence of a devastating endemic disease, eastern filbert blight (EFB) caused by Anisogramma anomala (Peck) E. Muller. The primary commercial means of control of EFB to date is through the development and planting of genetically resistant european hazelnut cultivars, with an R-gene introduced from the obsolete, late-shedding pollinizer ‘Gasaway’. Although the ‘Gasaway’ resistance source provides protection against EFB in the Pacific northwestern United States (PNW), recent reports have shown that it is not effective in parts of the eastern United States. This may be in part because the identification and selection of ‘Gasaway’ and ‘Gasaway’-derived cultivars occurred in an environment (PNW) with limited genetic diversity of A. anomala. The objectives of the current research were to develop a genetic linkage map using double digestion restriction site associated DNA sequencing (ddRADseq) and identify quantitative trait loci (QTL) markers associated with EFB resistance from the resistant selection Rutgers H3R07P25 from southern Russia. A mapping population composed of 119 seedling trees was evaluated in a geographic location (New Jersey) where the EFB fungus is endemic, exhibits high disease pressure, and has a high level of genetic diversity. The completed genetic linkage map included a total of 2217 markers and spanned a total genetic distance of 1383.4 cM, with an average marker spacing of 0.65 cM. A single QTL region associated with EFB resistance from H3R07P25 was located on european hazelnut linkage group (LG) 2 and was responsible for 72.8% of the phenotypic variation observed in the study. Based on its LG placement, origin, and disease response in the field, this resistance source is different from the ‘Gasaway’ source located on LG6. The current results, in combination with results from previous research, indicate that the H3R07P25 source is likely exhibiting resistance to a broader range of naturally occurring A. anomala isolates. As such, H3R07P25 will be important for the development of new european hazelnut germplasm that combines EFB resistance from multiple sources in a gene pyramiding approach. Identification of EFB resistance in high disease pressure environments representing a diversity of A. anomala populations is likely a requirement for identifying plants expressing durable EFB resistance, which is a precursor to the development of a commercially viable european hazelnut industry in the eastern United States.

The laboratory exercise described here aims to provide a relevant context for learning basic DNA techniques in an introductory animal science course at tertiary level. In two 4-hr laboratory sessions, students assess the suitability of bulls for inclusion in a gene-assisted selection program for A2 β-casein by genotyping commercial bull sperm. Sperm cells are lysed to extract the genomic DNA, and PCR with primers for the β-casein gene is performed. Using the principle of amplification-created restriction sites, restriction digestion with TaqI can be used to distinguish between the A1 allele and the A2 allele of the gene. Cut PCR amplicons are separated by gel electrophoresis to evaluate the genotype of each bull. Students then write a diagnostic report with accompanying letter to their fictional client, explaining the DNA test, and interpreting the results. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):708-711, 2019.

Background. Linseed solin varieties were created for nutrition, but the effect of oil fatty acid (FA) composition on other characters is not clear. Materials and methods. Using 6 inbreeding generations from 26 heterogeneous flax accessions were generated 19 high (HL), 7 medium (ML) and 14 low linolenic (LL) lines. For each lines contents of 5 basic FA: palmitic, stearic, oleic (OLE), linoleic (LIO) and linolenic (LIN); the ratio LIO/LIN, oil iodine number, vegetative period (VP) phases and plants size were evaluated. Development of CAPS marker for LuFAD3A gene was performed using idtdna.com. Sequencing of LIN genes sites was done in the Centre MCT SPBGU and Eurogen. Results. ANOVA showed significant differences HL, ML and LL groups for PAL, OLE, LIO, LIN, LIO/LIN, IOD. Considerable decrease of LIN, causes asymmetric changes in FA ratio and correlations between them and other traits. Factor analysis revealed the influence of two factors. The first one divided lines according to their LIN level and characters associated with it, the second one according to the VP and OLE. LIN synthesis is controlled by two complementary genes LuFAD3A and LuFAD3B. Sequencing of LuFAD3A gene 1 exon of 6 lines revealed a mutation (G255 A255), resulting in formation of stop codon. Developed developed CAPS-marker confirmed the homozygosity of hybrids between LL (gc-391) and HL lines (gc-65, 109, 121). Descendants of hybrid between gc-109 and gc-391 ripened 8-10 days earlier than gc-391. CAPS markers of LuFAD3B gene revealed differences between HL, ML, LL lines. Sequencing of this gene first exon and the beginning of the second one in 3 lines (1HL, 2LL) showed that this method reveals a mutation in the second restriction site, located in the 2 exon (C6 T6), and causing the replacement Hys Tyr. Conclusion. Lines from GC have wide variability of FA and other agronomic characters, combination of which will expand the cultivation of solin.

This study characterized Cucumber mosaic virus (CMV) infecting pepper in Southwestern Nigeria with the aim of providing up-to-date information on the taxonomic subgroup(s) of the parading strains in the study area. Fifty pepper leaf samples with one or more symptoms of mottling, mosaic, necrosis, leaf distortion and stunting were collected from eight commercial farms across Southwestern Nigeria and screened for CMV using antigen coated plate enzyme linked immunosorbent assay (ACP-ELISA). From the seropositive samples, four representative samples were selected, labelled PSWN1-4 before subjected to reverse transcription polymerase chain reaction (RT-PCR) and nucleic acid sequencing using a pair of primers specific for the amplification of RNA 3 genomic fragment of CMV. These were followed by multiple nucleotide sequence alignment and phylogenetic estimations of the isolates alongside selected CMV strains that were reported in previous studies using the molecular evolutionary genetics analysis (MEGA) software. The result of the ACP-ELISA test revealed that 14 (28%) of the samples collected were positive for CMV infection. The resulting nucleotide sequences from RT-PCR revealed 98% nucleotide homologies with several corresponding sequences of CMV strains from the Genbank. Further analysis of the PSWN nucleotide sequences through multiple nucleotide sequence alignment revealed the absence of the EcoR1 restriction site found only within the examined genomic portion of RNA 3 of subgroup II strains. These features defined the detected isolates as subgroup I strains. Phylogenetic analysis and estimations of genetic distances, conclusively distinguished the pepper-infecting CMV isolates from Southwestern Nigeria as members of subgroups IA and IB, which are the most virulent subgroups. Key words: Cucumber mosaic virus (CMV), pepper, Southwestern Nigeria.