Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3–5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

Barcoded reciprocal hemizygosity analysis via sequencing illuminates the complex genetic basis of yeast thermotolerance

Decades of successes in statistical genetics have revealed the molecular underpinnings of traits as they vary across individuals of a given species. But standard methods in the field can’t be applied to divergences between reproductively isolated taxa. Genome-wide reciprocal hemizygosity mapping (RH-seq), a mutagenesis screen in an inter-species hybrid background, holds promise as a method to accelerate the progress of interspecies genetics research. Here we describe an improvement to RH-seq in which mutants harbor barcodes for cheap and straightforward sequencing after selection in a condition of interest. As a proof of concept for the new tool, we carried out genetic dissection of the difference in thermotolerance between two reproductively isolated budding yeast species. Experimental screening identified dozens of candidate loci at which variation between the species contributed to the thermotolerance trait. Hits were enriched for mitosis genes and other housekeeping factors, and among them were multiple loci with robust sequence signatures of positive selection. Together, these results shed new light on the mechanisms by which evolution solved the problems of cell survival and division at high temperature in the yeast clade, and they illustrate the power of the barcoded RH-seq approach.

Evaluation of odorant-binding protein-1 as a molecular marker for identifying biological forms and delimitating sibling species of Anopheles stephensi

BackgroundAnopheles stephensi, an invasive malaria vector, has been reported to have three biological forms identifiable based on the number of ridges present on the egg’s floats and the dimension of eggs. Recently, these forms have been designated as sibling species based on the fixed differences in the DNA sequence of the first intron of the odorant-binding protein-1 (AsteObp1). In this study, we evaluated the utility of this neutral marker in designating sibling species or identifying biological forms.MethodsField collected and laboratory-reared An. stephensi were characterized for biological forms based on the number of floats on egg-ridge. DNA sequencing of the partial AsteObp1 gene of An. stephensi individuals were performed by Sanger’s method, either directly or after cloning with a plasmid vector.ResultsAsteObp1 intron-1 in Indian An. stephensi populations are highly polymorphic with the presence of more than 12 haplotypes exhibiting nucleotide-as well as length-polymorphism (90-to-121 bp). A majority of the field samples were heterozygous (up to 89% in the field populations). The phasing of haplotypes in heterozygotes through Sanger’s sequencing was challenging due to indels (1-to-24 bp) at multiple loci. No specific haplotype or monophyletic clade of intron-1 was found associated with a specific biological form. The inbreeding coefficient for this marker was close to zero in field and laboratory populations which refute the existence of sibling species based on the AsteObp1 marker.ConclusionsAsteObp1 cannot serve as a marker for the identification of biological forms of An. stephensi. The probable existence of sibling species in An. stephensi based on the AsteObp1 intron-1 is refuted.

Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.

On the Effect of Intralocus Recombination on Triplet-Based Species Tree Estimation

We consider species tree estimation from multiple loci subject to intralocus recombination. We focus on R∗, a summary coalescent-based methods using rooted triplets. We demonstrate analytically that intralocus recombination gives rise to an inconsistency zone, in which correct inference is not assured even in the limit of infinite amount of data. In addition, we validate and characterize this inconsistency zone through a simulation study that suggests that differential rates of recombination between closely related taxa can amplify the effect of incomplete lineage sorting and contribute to inconsistency.

Development of a Panel of Genotyping-in-Thousands by Sequencing in Capsicum

Genotyping by sequencing (GBS) enables genotyping of multiple loci at low cost. However, the single nucleotide polymorphisms (SNPs) revealed by GBS tend to be randomly distributed between individuals, limiting their direct comparisons without applying the various filter options to obtain a comparable dataset of SNPs. Here, we developed a panel of a multiplex targeted sequencing method, genotyping-in-thousands by sequencing (GT-seq), to genotype SNPs in Capsicum spp. Previously developed Fluidigm® SNP markers were converted to GT-seq markers and combined with new GT-seq markers developed using SNP information obtained through GBS. We then optimized multiplex PCR conditions: we obtained the highest genotyping rate when the first PCR consisted of 25 cycles. In addition, we determined that 101 primer pairs performed best when amplifying target sequences of 79 bp. We minimized interference of multiplex PCR by primer dimer formation using the PrimerPooler program. Using our GT-seq pipeline on Illumina Miseq and Nextseq platforms, we genotyped up to 1,500 (Miseq) and 1,300 (Nextseq) samples for the optimum panel size of 100 loci. To allow the genotyping of Capsicum species, we designed 332 informative GT-seq markers from Fluidigm SNP markers and GBS-derived SNPs. This study illustrates the first application of GT-seq in crop plants. The GT-seq marker set developed here will be a useful tool for molecular breeding of peppers in the future.

Multiplex Genome-Editing Technologies for Revolutionizing Plant Biology and Crop Improvement

Multiplex genome-editing (MGE) technologies are recently developed versatile bioengineering tools for modifying two or more specific DNA loci in a genome with high precision. These genome-editing tools have greatly increased the feasibility of introducing desired changes at multiple nucleotide levels into a target genome. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) [CRISPR/Cas] system-based MGE tools allow the simultaneous generation of direct mutations precisely at multiple loci in a gene or multiple genes. MGE is enhancing the field of plant molecular biology and providing capabilities for revolutionizing modern crop-breeding methods as it was virtually impossible to edit genomes so precisely at the single base-pair level with prior genome-editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Recently, researchers have not only started using MGE tools to advance genome-editing applications in certain plant science fields but also have attempted to decipher and answer basic questions related to plant biology. In this review, we discuss the current progress that has been made toward the development and utilization of MGE tools with an emphasis on the improvements in plant biology after the discovery of CRISPR/Cas9. Furthermore, the most recent advancements involving CRISPR/Cas applications for editing multiple loci or genes are described. Finally, insights into the strengths and importance of MGE technology in advancing crop-improvement programs are presented.

Genotyping of Chlamydia Abortus Using Multiple Loci Variable Number of Tandem Repeats Analysis Technique

Background: The correlation between various factors (geographical region, clinical incidence, and host type) and the genomic heterogeneity has been shown in several bacterial strains including Chlamydia abortus. Methods: The aim of this study was to survey the predominant types of C. abortus strains isolated from ruminants in Iran by the multiple loci variable number of tandem repeats (VNTR) analysis (MLVA) method. C. abortus infection was evaluated in a total of 117 aborted fetuses by real-time PCR. The isolation was done via the inoculation of the positive samples in chicken embryo and the L929 cell line. Genotyping was carried out by MLVA typing technique. Results: Forty samples (34.2%) were detected with C. abortus infection; however, chlamydial infection in ruminants of Charmahal/Bakhtiari (3 bovines and 35 sheep) was higher than that of Khuzestan (2 sheep). All MLVA types (MT1-MT8) were detected in the collected samples from Charmahal/Bakhtiari but only 2 types (MT1 and MT3) were reported in samples from Khuzestan. The main MT type was MT1 (32% of samples). Although in this study only 9 cow samples were investigated, they possessed similar clusters to those obtained from sheep (MT1 and MT6).Variation of type in sheep samples (MT1 to MT8) was more than that of bovine samples (MT1, and MT6); this can be attributed to the fact that more samples of sheep were studied as against the bovine samples. Conclusion: Although the difference between the detected same MT types in several animal species or between 2 geographic areas is significant, comprehensive studies are still needed. In Iran, due to traditional intercourse between animals of different provinces, the spread of other types is typically highly probable.

Niveoporofomes (Basidiomycota, Fomitopsidaceae) in Tropical Africa: Niveoporofomes oboensis sp. nov. and N.widdringtoniae comb. nov., two species from Afromontane Forests

During a survey of polypores in the montane forest of the Ôbo de São Tomé National Park, in the western African, equatorial island of São Tomé, a specimen that was, a priori, related to Fomitopsis, based on the gross morphology of the basidiome and a brown rot, showed deviating features including subglobose basidiospores with a large gutta, what pointed toward Niveoporofomes. Phylogenetic inferences based on multiple loci dataset (ITS-nLSU-nSSU-tef1-rpb2) confirmed these affinities, and Niveoporofomes oboensis is described as new. The species is compared to Fomitopsis widdringtoniae, known from southeast Africa, which is characterized also by subglobose basidiospores; hence, the new combination N. widdringtoniae is proposed. The new combination Niveoporofomes globosporus (basionym Trametes globospora) is also proposed based on phylogenetic analyses. A key to the species of Fomitopsis, Niveoporofomes, Rhodofomes, and Rhodofomitopsis in Tropical Africa is presented.

QTL analysis in multiple sorghum mapping populations facilitates dissection of the genetic control of agronomic and yield-related traits in sorghum [Sorghum bicolor (Moench)]

The genetic architectures of agronomic and yield-related traits are expected to involve multiple loci that are unlikely all to segregate for alternative alleles in a single bi-parental population. Therefore, the identification of quantitative trait loci (QTL) that are expressed in diverse genetic backgrounds of multiple bi-parental populations provides evidence about both background-specific and common genetic variants. The purpose of this study was to map QTLs for agronomic and yield related traits using three connected mapping populations of different genetic backgrounds, to gain insight into the genomic landscape of these important traits in elite Ethiopian sorghum germplasm. The three bi-parental populations, each with 207 F 2:3 lines were evaluated using an alpha lattice design with two replications under two moisture stress environments. Data analysis was done separately for each population using composite interval mapping, finding a total of 105 QTLs. All the QTLs identified from individual populations were projected on a combined consensus map, comprising a total of 25 meta QTLs for seven traits. The consensus map allowed us to deduce locations of a larger number of markers than possible in any individual map, providing a reference for genetic studies in different genetic backgrounds. The meta QTLs identified in this study could be used for marker-assisted breeding programs in sorghum after validation. Only one trait reduced leaf senescence, showed a striking bias of allele distribution, indicating substantial standing variation among the lines that might be employed in improving drought tolerance of sorghum.