Molecular analysis of many problems in genetics would be facilitated by the ability to construct restriction site maps of long stretches of genomic DNA and to directly place genes on these maps. Pulsed-field gradient gel electrophoresis allows measurement of the size of DNA fragments up to at least 2,000 kilobase pairs (kb) long and we have used this technique here to map sites for one class of infrequently cutting restriction enzyme over a total of 1,500 kb of mouse genomic DNA. The sites for these enzymes tend to be clustered in the genome. These clusters may correspond to the short stretches of C + G-rich unmethylated DNA often associated with mammalian genes.

Upland cotton (Gossypium hirsutum L., 2n = 52, AADD) is an allotetraploid, therefore the discovery of single nucleotide polymorphism (SNP) markers is difficult. The recent emergence of genome complexity reduction technologies based on the next-generation sequencing (NGS) platform has greatly expedited SNP discovery in crops with highly repetitive and complex genomes. Here we applied restriction-site associated DNA (RAD) sequencing technology for de novo SNP discovery in allotetraploid cotton. We identified 21,109 SNPs between the two parents and used these for genotyping of 161 recombinant inbred lines (RILs). Finally, a high dense linkage map comprising 4,153 loci over 3500-cM was developed based on the previous result. Using this map quantitative trait locus (QTLs) conferring fiber strength and Verticillium Wilt (VW) resistance were mapped to a more accurate region in comparison to the 1576-cM interval determined using the simple sequence repeat (SSR) genetic map. This suggests that the newly constructed map has more power and resolution than the previous SSR map. It will pave the way for the rapid identification of the marker-assisted selection in cotton breeding and cloning of QTL of interest traits.

Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is promising for anchoring such assemblies, but traditional linkage mapping methods are hindered by the infrequency and unevenness of meiotic recombination that limit attainable map resolution. Here we develop a sequencing-based "in vitro" linkage mapping approach (called RadMap), where chromosome breakage and segregation are realized by generating hundreds of "subhaploid" fosmid/bacterial-artificial-chromosome clone pools, and by restriction site-associated DNA sequencing of these clone pools to produce an ultradense whole-genome restriction map to facilitate genome scaffolding. A bootstrap-based minimum spanning tree algorithm is developed for grouping and ordering of genome-wide markers and is implemented in a user-friendly, integrated software package (AMMO). We perform extensive analyses to validate the power and accuracy of our approach in the model plant Arabidopsis thaliana and human. We also demonstrate the utility of RadMap for enhancing the contiguity of a variety of whole-genome shotgun assemblies generated using either short Illumina reads (300 bp) or long PacBio reads (6-14 kb), with up to 15-fold improvement of N50 (∼816 kb-3.7 Mb) and high scaffolding accuracy (98.1-98.5%). RadMap outperforms BioNano and Hi-C when input assembly is highly fragmented (contig N50 = 54 kb). RadMap can capture wide-range contiguity information and provide an efficient and flexible tool for high-resolution physical mapping and scaffolding of highly fragmented assemblies.

The APOBEC3 (A3) family of cellular cytidine deaminases comprises seven members (A, B, C, D, F, G, and H) that potently inhibit retroviral replication. Human immunodeficiency virus type 1 (HIV-1) Vif is a small pleiotropic protein that specifically inactivates these enzymes, targeting them for ubiquitin-mediated proteasomal degradation. A3 Vif-interaction sites are presumed to fall into three distinct types: A3C/D/F, A3G, and A3H. To date, two types of A3G and A3C/D/F sites have been well characterized, whereas the A3H Vif-binding site remains poorly defined. Here, we explore the residues critical for the A3H-type Vif interaction. To avoid technical difficulties in performing experiments with human A3H haplotype II (hapII), which is relatively resistant to HIV-1 Vif, we employed its ortholog chimpanzee A3H (cA3H), which displays high Vif sensitivity, for a comparison of sensitivity with that of A3H hapII. The Vif susceptibility of A3H hapII-cA3H chimeras and their substitution mutants revealed a single residue at position 97 as a major determinant for the difference in their Vif sensitivities. We further surveyed critical residues by structure-guided mutagenesis using an A3H structural model and thus identified eight additional residues important for Vif sensitivity, which mapped to the α3 and α4 helices of A3H. Interestingly, this area is located on a surface adjacent to the A3G and A3C/D/F interfaces and is composed of negatively charged and hydrophobic patches. These findings suggest that HIV-1 Vif has evolved to utilize three dispersed surfaces for recognizing three types of interfaces on A3 proteins under certain structural constraints.

Recent advances in genotyping-by-sequencing have enabled genome-wide association studies in nonmodel species including those in aquaculture programs. As with other aquaculture species, rainbow trout and steelhead (Oncorhynchus mykiss) are susceptible to disease and outbreaks can lead to significant losses. Fish culturists have therefore been pursuing strategies to prevent losses to common pathogens such as Flavobacterium psychrophilum (the etiological agent for bacterial cold water disease [CWD]) and infectious hematopoietic necrosis virus (IHNV) by adjusting feed formulations, vaccine development, and selective breeding. However, discovery of genetic markers linked to disease resistance offers the potential to use marker-assisted selection to increase resistance and reduce outbreaks. For this study we sampled juvenile fish from 40 families from 2-yr classes that either survived or died after controlled exposure to either CWD or IHNV. Restriction site-associated DNA sequencing produced 4661 polymorphic single-nucleotide polymorphism loci after strict filtering. Genotypes from individual survivors and mortalities were then used to test for association between disease resistance and genotype at each locus using the program TASSEL. After we accounted for kinship and stratification of the samples, tests revealed 12 single-nucleotide polymorphism markers that were highly associated with resistance to CWD and 19 markers associated with resistance to IHNV. These markers are candidates for further investigation and are expected to be useful for marker assisted selection in future broodstock selection for various aquaculture programs.

Protection from DNA invasion is afforded by restriction-modification systems in many bacteria. The efficiency of protection depends crucially on the relative expression levels of restriction versus methytransferase genes. This regulation is provided by a controller protein, named C protein. Studies of the Bcll system in E. coli suggest that C.Bcll functions as a negative regulator for M.Bcll expression, implying that it plays a role in defense against foreign DNA during virus infection. C.Bcll binds (Kd = 14.3 nM) to a 2-fold symmetric C box DNA sequence that overlaps with the putative -35 promoter region upstream of the bcllM and bcllC genes. The C.Bcll fold comprises five alpha helices: two helices form a helix-turn-helix motif, and the remaining three helices form the extensive dimer interface. The C.Bcll-DNA model proposed suggests that DNA bending might play an important role in gene regulation, and that Glu27 and Asp31 in C.Bcll might function critically in the regulation.

Quantitative trait loci (QTL) studies provide insight into the complexity of drought tolerance mechanisms. Molecular markers used in these studies also allow for marker-assisted selection (MAS) in breeding programs, enabling transfer of genetic factors between breeding lines without complete knowledge of their exact nature. However, potential for recombination between markers and target genes limit the utility of MAS-based strategies. Candidate gene mapping offers an alternative solution to identify trait determinants underlying QTL of interest. Here, we used restriction site polymorphisms to investigate co-location of candidate genes with QTL for seedling drought stress-induced premature senescence identified previously in cowpea. Genomic DNA isolated from 113 F(2:8) RILs of drought-tolerant IT93K503-1 and drought susceptible CB46 genotypes was digested with combinations of EcoR1 and HpaII, Mse1, or Msp1 restriction enzymes and amplified with primers designed from 13 drought-responsive cDNAs. JoinMap 3.0 and MapQTL 4.0 software were used to incorporate polymorphic markers onto the AFLP map and to analyze their association with the drought response QTL. Seven markers co-located with peaks of previously identified QTL. Isolation, sequencing, and blast analysis of these markers confirmed their significant homology with drought or other abiotic stress-induced expressed sequence tags (EST) from cowpea and other plant systems. Further, homology with coding sequences for a multidrug resistance protein 3 and a photosystem I assembly protein ycf3 was revealed in two of these candidates. These results provide a platform for the identification and characterization of genetic trait determinants underlying seedling drought tolerance in cowpea.

Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.

Restriction site associated DNA sequencing (RAD-seq), a next-generation sequencing technology, has greatly facilitated genetic linkage mapping studies in outbred species. RAD-seq is capable of discovering thousands of genetic markers for linkage mapping across many individuals, and can be applied in species with or without a reference genome. Although several analytical tools are available for RAD-seq data, alternative strategies are necessary for improving the marker quality and hence the genetic mapping accuracy.

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.

Mass spectrometric analysis of microbial metabolism provides a long list of possible compounds. Restricting the identification of the possible compounds to those produced by the specific organism would benefit the identification process. Currently, identification of mass spectrometry (MS) data is commonly done using empirically derived compound databases. Unfortunately, most databases contain relatively few compounds, leaving long lists of unidentified molecules. Incorporating genome-encoded metabolism enables MS output identification that may not be included in databases. Using an organism's genome as a database restricts metabolite identification to only those compounds that the organism can produce.

Virus-specific CD4(+) T cells contribute to effective virus control through a multiplicity of mechanisms including direct effector functions as well as "help" for B cell and CD8(+) T cell responses. Here, we have used the lymphocytic choriomeningitis virus (LCMV) system to assess the minimal constraints of a dominant antiviral CD4(+) T cell response. We report that the core epitope derived from the LCMV glycoprotein (GP) is 11 amino acids in length and provides optimal recognition by epitope-specific CD4(+) T cells. Surprisingly, this epitope is also recognized by LCMV-specific CD8(+) T cells and thus constitutes a unique viral determinant with dual MHC class I- and II-restriction.

In the present study, quantitative trait loci (QTLs) analysis was performed to identify the chromosomal positions of growth and carcass-related trait QTLs using 319 F2 chickens obtained from intercrosses of an Oh-Shamo male and four White Plymouth Rock females. Body weight was measured weekly until the birds were 7 weeks old. Carcass-related traits were also measured at this timepoint. A genetic linkage map was constructed using 545 single nucleotide polymorphism (SNP) markers that were developed using a restriction-site associated DNA sequencing method. The linkage map included the 23 autosomes and the Z chromosome. Using simple interval QTL mapping, we were able to identify 10 significant and suggestive main-effect QTLs for growth and carcass-related traits present on chromosomes 1, 2, 3, 5, 8, 19, 24, and Z. These loci explained 5.60-16.52% of the phenotypic variances. The chromosomal positions of the 10 QTLs overlapped with those of previously reported QTLs, whereas the targeted traits varied. Our QTLs will aid future breeding programs in improving growth and meat yield of chickens (e.g., via marker-assisted selection), particularly in the Japanese brand chicken industry.

Mapping of quantitative trait loci (QTL) is essential for the discovery of genetic structures that related to complex quantitative traits. In this study, we identified 264,072 raw SNPs (single-nucleotide polymorphisms) by double digest restriction site associated DNA sequencing (ddRADseq), and utilized 3029 of these SNPs to construct a genetic linkage map in orange-spotted grouper (Epinephelus coioides) using a regression mapping algorithm. The genetic map contained 24 linkage groups (LGs) spanning a total genetic distance of 1231.98 cM. Twenty-seven significant growth-related QTLs were identified. Furthermore, we identified 17 genes (fez2, alg3, ece2, arvcf, sla27a4, sgk223, camk2, prrc2b, mchr1, sardh, pappa, syk, tert, wdrcp91, ftz-f1, mate1 and notch1) including three (tert, ftz-f1 and notch1) that have been reported to be involved in fish growth. To summarize, we mapped growth-related QTLs in the orange-spotted grouper. These QTLs will be useful in marker-assisted selection (MAS) efforts to improve growth-related traits in this economically important fish.

Mining activities in the Chini Lake catchment area have been extensive for several years, contributing to acid mine drainage (AMD) events with high concentrations of iron (Fe) and other heavy metals impacting the surface water. However, during the restriction period due to the COVID-19 outbreak, anthropogenic activities have been suspended, which clearly shows a good opportunity for a better environment. Therefore, we aimed to analyze the variation of AMD-associated water pollution in three main zones of the Chini Lake catchment area using Sentinel-2 data for the periods pre-movement control order (MCO), during MCO, and post-MCO from 2019 to 2021. These three zones were chosen due to their proximity to mining areas: zone 1 in the northeastern part, zone 2 in the southeastern part, and zone 3 in the southern part of the Chini Lake area. The acid mine water index (AMWI) was a specific index used to estimate acid mine water. The AMWI values from Sentinel-2 images exhibited that the mean AMWI values in all zones during the MCO period decreased by 14% compared with the pre-MCO period. The spatiotemporal analysis found that the highest polluted zones were recorded in zone 1, followed by zone 3 and zone 2. As compared with during the MCO period, the maximum percentage of increment during post-MCO in all zones was up to 25%. The loosened restriction policy has resulted in more AMD flowing into surface water and increased pollution in Chini Lake. As a whole, our outputs revealed that Sentinel-2 data had a major potential for assessing the AMD-associated pollution of water.

Understanding the genetic mechanisms that underlie innate fear behavior is essential for improving the management and performance of the poultry industry. This study aimed to map QTL associated with innate fear responses in open field (OF) and tonic immobility (TI) tests, using an F2 chicken intercross population between 2 behaviorally distinct breeds: the aggressive Japanese Oh-Shamo (OSM) and the docile White Leghorn T-line (WL-T). Genome-wide QTL analysis for the OF and TI traits was conducted using 2,109 single nucleotide polymorphism (SNP) markers obtained through restriction site-associated DNA sequencing (RAD-seq). While several suggestive QTL were identified for TI and OF traits at genome-wide 20% significance threshold levels, the analysis revealed 2 significant QTL for 2 OF traits (total distance and maximum speed) at genome-wide 5% significance threshold levels. These significant QTL were located between 12.34 and 30.49 megabase (Mb) on chromosome 1 and between 40.02 and 63.38 Mb on chromosome 2, explaining 6.75 to 7.40% of the total variances. These findings provide valuable insights for the poultry industry, particularly in refining chicken management strategies and informing targeted breeding efforts.

Temperature plays an important role in bacteria-host interactions and can be a determining factor for host switching. In this study we sought to investigate the reasons behind growth temperature restriction in the entomopathogenic enterobacterium Photorhabdus. Photorhabdus has a complex dual symbiotic and pathogenic life cycle. The genus consists of 19 species but only one subgroup, previously all classed together as Photorhabdus asymbiotica, have been shown to cause human disease. These clinical isolates necessarily need to be able to grow at 37°C, whilst the remaining species are largely restricted to growth temperatures below 34°C and are therefore unable to infect mammalian hosts. Here, we have isolated spontaneous mutant lines of Photorhabdus laumondii DJC that were able to grow up to 36-37°C. Following whole genome sequencing of 29 of these mutants we identified a single gene, encoding a protein with a RecG-like helicase domain that for the majority of isolates contained single nucleotide polymorphisms. Importantly, provision of the wild-type allele of this gene in trans restored the temperature restriction, confirming the mutations are recessive, and the dominant effect of the protein product of this gene. The gene appears to be part of a short three cistron operon, which we have termed the Temperature Restricting Locus (TRL). Transcription reporter strains revealed that this operon is induced upon the switch from 30 to 36°C, leading to replication arrest of the bacteria. TRL is absent from all of the human pathogenic species so far examined, although its presence is not uniform in different strains of the Photorhabdus luminescens subgroup. In a wider context, the presence of this gene is not limited to Photorhabdus, being found in phylogenetically diverse proteobacteria. We therefore suggest that this system may play a more fundamental role in temperature restriction in diverse species, relating to as yet cryptic aspects of their ecological niches and life cycle requirements.

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical "contig" maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning approximately 10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual "BAC-HAPPY-mapping" to convert BAC landing data into BAC linkage contigs is possible.

Restriction endonucleases are highly specific in recognizing the particular DNA sequence they act on. However, their activity is affected by sequence context, enzyme concentration and buffer composition. Changes in these factors may lead to either ineffective cleavage at the cognate restriction site or relaxed specificity allowing cleavage of degenerate 'star' sites. Additionally, uncharacterized restriction endonucleases and engineered variants present novel activities. Traditionally, restriction endonuclease activity is assayed on simple substrates such as plasmids and synthesized oligonucleotides. We present and use high-throughput Illumina sequencing-based strategies to assay the sequence specificity and flanking sequence preference of restriction endonucleases. The techniques use fragmented DNA from sequenced genomes to quantify restriction endonuclease cleavage on a complex genomic DNA substrate in a single reaction. By mapping millions of restriction site-flanking reads back to the Escherichia coli and Drosophila melanogaster genomes we were able to quantitatively characterize the cognate and star site activity of EcoRI and MfeI and demonstrate genome-wide decreases in star activity with engineered high-fidelity variants EcoRI-HF and MfeI-HF, as well as quantify the influence on MfeI cleavage conferred by flanking nucleotides. The methods presented are readily applicable to all type II restriction endonucleases that cleave both strands of double-stranded DNA.

In mammals, caloric restriction consistently results in extended lifespan. Epigenetic information encoded by DNA methylation is tightly regulated, but shows a striking drift associated with age that includes both gains and losses of DNA methylation at various sites. Here, we report that epigenetic drift is conserved across species and the rate of drift correlates with lifespan when comparing mice, rhesus monkeys, and humans. Twenty-two to 30-year-old rhesus monkeys exposed to 30% caloric restriction since 7-14 years of age showed attenuation of age-related methylation drift compared to ad libitum-fed controls such that their blood methylation age appeared 7 years younger than their chronologic age. Even more pronounced effects were seen in 2.7-3.2-year-old mice exposed to 40% caloric restriction starting at 0.3 years of age. The effects of caloric restriction on DNA methylation were detectable across different tissues and correlated with gene expression. We propose that epigenetic drift is a determinant of lifespan in mammals.Caloric restriction has been shown to increase lifespan in mammals. Here, the authors provide evidence that age-related methylation drift correlates with lifespan and that caloric restriction in mice and rhesus monkeys results in attenuation of age-related methylation drift.