Single-copy Loci Research Articles

Back together: Over 1000 single-copy nuclear loci and reproductive features support the holoendoparasitic Apodanthaceae and Rafflesiaceae as sister lineages in the order Malpighiales

The systematics of the holoendoparasitic flowering plant families Apodanthaceae and Rafflesiaceae has been discussed for over two centuries. The morphological reduction of roots, shoots and leaves in all members of both families, resulting in a cryptic mycelium-like vegetative body, has been interpreted either as a key common feature, or as a result of convergent evolution due to full dependence upon their hosts. Historically, the two families have been placed together due to similar morphological features, but recent analyses based on few mitochondrial and ribosomal gene markers placed them in the distantly related orders Cucurbitales and Malpighiales. Here we reevaluate the affinities of the Apodanthaceae and the Rafflesiaceae using a phylogenomic approach. We present (1) a historical account on their affinities over the last 200 years; (2) phylogenetic analyses reinstating their sister group relationship as part of the order Malpighiales, based on over 1000 single-copy nuclear protein-coding loci; and (3) a comprehensive list of putative morphoanatomical and developmental synapomorphies in light of the phylogenomic results, with emphasis on shared reproductive traits regardless of dramatic differences in floral size.

Forensic characteristics of 38 Y-STR loci in the Hui population from Shaanxi Province, Northwest China

In this study, we genotyped 939 unrelated healthy individuals to investigate the genetic polymorphisms of 38 Y chromosome short tandem repeats(Y-STRs) loci included in the Yfiler™ Platinum kit in the Hui population of Shaanxi Province. Comprehensive population comparisons were performed using analysis of molecular variance(AMOVA), multidimensional scaling(MDS), and molecular evolutionary genetics analysis(MEGA) to explore population relationships. The allele frequencies of the single-copy loci ranged from 0.001 to 0.940 (DYS645), while those of the multi-copy loci ranged from 0.001 to 0.138 (DYS527). The highest genetic diversity (GD) value was observed for DYS385 (0.9723), and the lowest for DYS645 (0.1138). A total of 849 distinct haplotypes were identified among the 939 Hui individuals, with 808 (95.2 %) being unique. Haplotype diversity (HD), haplotype match probability, and discriminatory power (DC) in the Shaanxi Hui population were 0.9996, 0.0015, and 0.9041, respectively. Interpopulation comparisons based on Rst genetic distance values, phylogenetic trees, and MDS indicated that the Shaanxi Hui population has a close genetic relationship with the Ningxia Hui, Qinghai Hui, Xinjiang Hui, and Yunnan Hui populations. The population genetic stratification largely coincides with geographic distribution and language families. Our study revealed that the 38 Y-STR loci exhibit high genetic polymorphisms in the Hui population from Shaanxi Province.

Uncertainty in the number of contributor estimation methods applied to a Y-STR profile

Maximum allele count (MAC) and total allele count (TAC) methods are widely used for estimating the number of contributors (NoC) of autosomal short tandem repeat (STR) profile in many forensic laboratories. In this study, we applied NoC estimation methods to mixed Y-STR profiles and evaluated its uncertainty and performance. For the MAC method, as recent Y-STR typing kits involve single- and multi-copy loci, we defined “MAC-single” for use across only single-copy loci and “MAC-multi” for use across only multi-copy loci. We generated a dataset containing 120,000 Y-STR profiles for a one to six-person mixture in silico based on previously reported haplotype frequencies of 27 Y-STR loci in Yfiler Plus for the U.S. population (reported by NIST) and the Henan Han population. The dataset was randomly split into a training set and a test set. The training set was used to construct a TAC distribution (TAC curve), whereas the test set was used to calculate the performance metrics (accuracy, precision, recall, and F1-score). In addition, the effect of the upper limit of NoC considered for estimation on overall accuracy was evaluated. The overall accuracies of MAC-single, MAC-multi, and TAC methods when the upper limit of NoC was set to six-person were 0.7920, 0.4329, and 0.7877 for the U.S. population and 0.8207, 0.4609, and 0.8385 for the Henan Han population. Our results suggest that the MAC-single and TAC methods can estimate the NoC for mixed Y-STR profiles with high levels of accuracy.

Suppression by RNA Polymerase I Inhibitors Varies Greatly Between Distinct RNA Polymerase I Transcribed Genes in Malaria Parasites.

Transcription of ribosomal RNA (rRNA) by RNA Polymerase I (Pol I) is the rate-limiting step in ribosome biogenesis and a major determinant of cellular growth rates. Unlike virtually every other eukaryote, which express identical rRNA from large tandem arrays of dozens to hundreds of identical rRNA genes in every cell, the genome of the human malaria parasite Plasmodium falciparum contains only a handful single-copy 47S rRNA loci that differ substantially from one another in length, sequence and expression in different cell-types. We found that growth of malaria parasite was acutely sensitive to the Pol I inhibitors 9-hydroxyellipticine and BMH-21 and demonstrate that they greatly reduce the transcription of 47S rRNAs as well as transcription of other non-coding RNA genes. Surprisingly, we found that the various types of Pol I-transcribed genes differed by more than two orders of magnitude in their susceptibility to these inhibitors and explore the implications of these findings for regulation of rRNA in P. falciparum .

A locus-dependent mixed inheritance in the segmental allohexaploid sweetpotato (Ipomoea batatas [L.] Lam).

Two interrelated aspects of the sweetpotato genome, its polyploid origin and inheritance type, remain uncertain. We recently proposed a segmental allohexaploid sweetpotato and thus sought to clarify its inheritance type by direct analyses of homoeolog segregations at selected single-copy loci. For such analyses, we developed a digital quantitative PCR genotyping method using one nondiscriminatory and three discriminatory probes for each selected locus to discriminate and quantify three homoeolog-differentiating variation types (homoeolog-types) in genomic DNA samples for genotype fitting and constructed a F2 population for segregation analyses. We confirmed inter-subgenomic distinctions of three identified homoeolog-types at each of five selected loci by their interspecific differentiations among 14 species in Ipomoea section batatas and genotyped the loci in 549 F2 lines, selected F1 progenies, and their founding parents. Segregation and genotype analyses revealed a locus-dependent mixed inheritance (disomic, polysomic, and intermediate types) of the homoeolog-types at 4 loci in the F2 population, displaying estimated disomic-inheritance frequencies of 0, 2.72%, 14.52%, and 36.92%, and probably in the F1 population too. There were also low-frequency non-hexaploid F1 and F2 genotypes that were probably derived from double-reduction recombination or partially unreduced gametes, and F2 genotypes of apparent aneuploids/dysploids with neopolyploid-like frequencies. Additional analyses of homoeolog-type genotypes at the 5 loci in 46 lines from various regions revealed locus-dependent selection biases, favoring genotypes having more of one homoeolog-type, i.e. more of di- or homogenized homoeolog-type composition, and one-direction ploidy trending among apparent aneuploids/dysploids. These inheritance features pointed to an evolving segmental allohexaploid sweetpotato impacted by selection biases.

Development of a Target Enrichment Probe Set for Conifer (REMcon).

Conifers are an ecologically and economically important seed plant group that can provide significant insights into the evolution of land plants. Molecular phylogenetics has developed as an important approach in evolutionary studies, although there have been relatively few studies of conifers that employ large-scale data sourced from multiple nuclear genes. Target enrichment sequencing (target capture, exon capture, or Hyb-Seq) has developed as a key approach in modern phylogenomic studies. However, until now, there has been no bait set that specifically targets the entire conifer clade. REMcon is a target sequence capture probe set intended for family- and species-level phylogenetic studies of conifers that target c. 100 single-copy nuclear loci. We tested the REMcon probe set using 69 species, including 44 conifer genera across six families and four other gymnosperm taxa, to evaluate the efficiency of target capture to efficiently generate comparable DNA sequence data across conifers. The recovery of target loci was high, with, on average, 94% of the targeted regions recovered across samples with high read coverage. A phylogenetic analysis of these data produced a well-supported topology that is consistent with the current understanding of relationships among conifers. The REMcon bait set will be useful in generating relatively large-scale nuclear data sets consistently for any conifer lineage.

The complex polyploid genome architecture of sugarcane

Sugarcane, the world’s most harvested crop by tonnage, has shaped global history, trade and geopolitics, and is currently responsible for 80% of sugar production worldwide1. While traditional sugarcane breeding methods have effectively generated cultivars adapted to new environments and pathogens, sugar yield improvements have recently plateaued2. The cessation of yield gains may be due to limited genetic diversity within breeding populations, long breeding cycles and the complexity of its genome, the latter preventing breeders from taking advantage of the recent explosion of whole-genome sequencing that has benefited many other crops. Thus, modern sugarcane hybrids are the last remaining major crop without a reference-quality genome. Here we take a major step towards advancing sugarcane biotechnology by generating a polyploid reference genome for R570, a typical modern cultivar derived from interspecific hybridization between the domesticated species (Saccharum officinarum) and the wild species (Saccharum spontaneum). In contrast to the existing single haplotype (‘monoploid’) representation of R570, our 8.7 billion base assembly contains a complete representation of unique DNA sequences across the approximately 12 chromosome copies in this polyploid genome. Using this highly contiguous genome assembly, we filled a previously unsized gap within an R570 physical genetic map to describe the likely causal genes underlying the single-copy Bru1 brown rust resistance locus. This polyploid genome assembly with fine-grain descriptions of genome architecture and molecular targets for biotechnology will help accelerate molecular and transgenic breeding and adaptation of sugarcane to future environmental conditions.

Transgenic Dendra2::tau expression allows in vivo monitoring of tau proteostasis in Caenorhabditis elegans.

Protein homeostasis is perturbed in aging-related neurodegenerative diseases called tauopathies, which are pathologically characterized by aggregation of the microtubule-associated protein tau (encoded by the human MAPT gene). Transgenic Caenorhabditis elegans serve as a powerful model organism to study tauopathy disease mechanisms, but moderating transgenic expression level has proven problematic. To study neuronal tau proteostasis, we generated a suite of transgenic strains expressing low, medium or high levels of Dendra2::tau fusion proteins by comparing integrated multicopy transgene arrays with single-copy safe-harbor locus strains generated by recombinase-mediated cassette exchange. Multicopy Dendra2::tau strains exhibited expression level-dependent neuronal dysfunction that was modifiable by known genetic suppressors or an enhancer of tauopathy. Single-copy Dendra2::tau strains lacked distinguishable phenotypes on their own but enabled detection of enhancer-driven neuronal dysfunction. We used multicopy Dendra2::tau strains in optical pulse-chase experiments measuring tau turnover in vivo and found that Dendra2::tau turned over faster than the relatively stable Dendra2. Furthermore, Dendra2::tau turnover was dependent on the protein expression level and independent of co-expression with human TDP-43 (officially known as TARDBP), an aggregating protein interacting with pathological tau. We present Dendra2::tau transgenic C. elegans as a novel tool for investigating molecular mechanisms of tau proteostasis.

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae.

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

A target enrichment probe set for resolving phylogenetic relationships in the coffee family, Rubiaceae.

Rubiaceae is among the most species-rich plant families, as well as one of the most morphologically and geographically diverse. Currently available phylogenies have mostly relied on few genomic and plastid loci, as opposed to large-scale genomic data. Target enrichment provides the ability to generate sequence data for hundreds to thousands of phylogenetically informative, single-copy loci, which often leads to improved phylogenetic resolution at both shallow and deep taxonomic scales; however, a publicly accessible Rubiaceae-specific probe set that allows for comparable phylogenetic inference across clades is lacking. Here, we use publicly accessible genomic resources to identify putatively single-copy nuclear loci for target enrichment in two Rubiaceae groups: tribe Hillieae (Cinchonoideae) and tribal complex Palicoureeae+Psychotrieae (Rubioideae). We sequenced 2270 exonic regions corresponding to 1059 loci in our target clades and generated in silico target enrichment sequences for other Rubiaceae taxa using our designed probe set. To test the utility of our probe set for phylogenetic inference across Rubiaceae, we performed a coalescent-aware phylogenetic analysis using a subset of 27 Rubiaceae taxa from 10 different tribes and three subfamilies, and one outgroup in Apocynaceae. We recovered an average of 75% and 84% of targeted exons and loci, respectively, per Rubiaceae sample. Probes designed using genomic resources from a particular subfamily were most efficient at targeting sequences from taxa in that subfamily. The number of paralogs recovered during assembly varied for each clade. Phylogenetic inference of Rubiaceae with our target regions resolves relationships at various scales. Relationships are largely consistent with previous studies of relationships in the family with high support (≥0.98 local posterior probability) at nearly all nodes and evidence of gene tree discordance. Our probe set, which we call Rubiaceae2270x, was effective for targeting loci in species across and even outside of Rubiaceae. This probe set will facilitate phylogenomic studies in Rubiaceae and advance systematics and macroevolutionary studies in the family.

Calceolariaceae809: A bait set for targeted sequencing of nuclear loci.

The genus Calceolaria (Calceolariaceae) is emblematic of the Andes, is hypothesized to have originated as a recent, rapid radiation, and has important taxonomic needs. Additionally, the genus is a model for the study of specialized pollination systems, as its flowers are nectarless and many offer floral oils as a pollination reward collected by specialist bees. Despite their evolutionary and ecological significance, obtaining a resolved phylogeny for the group has proved difficult. To address this challenge, we present a new bait set for targeted sequencing of nuclear loci in Calceolariaceae and close relatives. We developed a bioinformatic workflow to use incomplete, low-coverage genomes of 10 Calceolaria species to identify single-copy loci suitable for phylogenetic studies and design baits for targeted sequencing. Our approach resulted in the identification of 809 single-copy loci (733 noncoding and 76 coding regions) and the development of 39,937 baits, which we validated in silico (10 specimens) and in vitro (29 Calceolariaceae and six outgroups). In both cases, the data allowed us to recover robust phylogenetic estimates. Our results demonstrate the appropriateness of the bait set for sequencing recent and historic specimens of Calceolariaceae and close relatives, and open new doors for further investigation of the evolutionary history of this hyperdiverse genus.

Sex-determining region complements traditionally used in phylogenetic studies nuclear and chloroplast sequences in investigation of Aigeiros Duby and Tacamahaca Spach poplars (genus Populus L., Salicaceae).

Members of the genus Populus L. play an important role in the formation of forests in the northern hemisphere and are used in urban landscaping and timber production. Populus species of closely related sections show extensive hybridization. Therefore, the systematics of the genus is rather complicated, especially for poplars of hybrid origin. We aimed to assess the efficiency of application of the sex-determining region (SDR) in addition to the nuclear and chloroplast genome loci traditionally used in phylogenetic studies of poplars to investigate relationships in sections Aigeiros Duby and Tacamahaca Spach. Targeted deep sequencing of NTS 5S rDNA, ITS, DSH 2, DSH 5, DSH 8, DSH 12, DSH 29, 6, 15, 16, X18, trnG-psbK-psbI, rps2-rpoC2, rpoC2-rpoC1, as well as SDR and ARR17 gene was performed for 379 poplars. The SDR and ARR17 gene together with traditionally used multicopy and single-copy loci of nuclear and chloroplast DNA allowed us to obtain a clustering that is most consistent with poplar systematics based on morphological data and to shed light on several controversial hypotheses about the origin of the studied taxa (for example, the inexpediency of separating P. koreana, P. maximowiczii, and P. suaveolens into different species). We present a scheme of relationships between species and hybrids of sections Aigeiros and Tacamahaca based on molecular genetic, morphological, and geographical data. The geographical proximity of species and, therefore, the possibility of hybridization between them appear to be more important than the affiliation of species to the same section. We speculate that sections Aigeiros and Tacamahaca are distinguished primarily on an ecological principle (plain and mountain poplars) rather than on a genetic basis. Joint analysis of sequencing data for the SDR and chloroplast genome loci allowed us to determine the ancestors of P. × petrovskoe - P. laurifolia (female tree) × P. × canadensis (male tree), and P. × rasumovskoe - P. nigra (female tree) × P. suaveolens (male tree). Thus, the efficiency of using the SDR for the study of poplars of sections Aigeiros and Tacamahaca and the prospects of its use for the investigation of species of the genus Populus were shown.

LoCoLotive: In silico mining for low-copy nuclear loci based on target capture probe sets and arbitrary reference genomes.

Universal target enrichment probe kits are used to circumvent the individual identification of loci suitable for phylogenetic studies in a given taxon. Under certain circumstances, however, target capture can be inefficient and costly, and lower numbers of marker loci may be sufficient. We therefore propose a computational pipeline that enables the easy identification of a subset of promising candidate loci for a taxon of interest. Target sequences used for probe design are filtered based on an assembled reference genome, resulting in presumably intron-containing single-copy loci as present in the reference taxon. The applicability of the proposed approach is demonstrated based on two probe kits (universal and family-specific) in combination with several publicly available reference genomes. Guided by commercial probe kits, LoCoLotive enables fast and cost-efficient marker mining. Its accuracy mainly depends on the quality of the reference genome and its relatedness to the taxa under study.

Reverse-ChIP Techniques for Identifying Locus-Specific Proteomes: A Key Tool in Unlocking the Cancer Regulome.

A phenotypic hallmark of cancer is aberrant transcriptional regulation. Transcriptional regulation is controlled by a complicated array of molecular factors, including the presence of transcription factors, the deposition of histone post-translational modifications, and long-range DNA interactions. Determining the molecular identity and function of these various factors is necessary to understand specific aspects of cancer biology and reveal potential therapeutic targets. Regulation of the genome by specific factors is typically studied using chromatin immunoprecipitation followed by sequencing (ChIP-Seq) that identifies genome-wide binding interactions through the use of factor-specific antibodies. A long-standing goal in many laboratories has been the development of a 'reverse-ChIP' approach to identify unknown binding partners at loci of interest. A variety of strategies have been employed to enable the selective biochemical purification of sequence-defined chromatin regions, including single-copy loci, and the subsequent analytical detection of associated proteins. This review covers mass spectrometry techniques that enable quantitative proteomics before providing a survey of approaches toward the development of strategies for the purification of sequence-specific chromatin as a 'reverse-ChIP' technique. A fully realized reverse-ChIP technique holds great potential for identifying cancer-specific targets and the development of personalized therapeutic regimens.

Molecular phylogenetics of the fresh water sleepers Odontobutis (Gobiiformes: Odontobutidae) and its implications on biogeography of freshwater ichthyofauna of East Asia

The genus Odontobutis is a group of freshwater fishes endemic to East Asia. Phylogenetic relationships among the Odontobutis species have never been fully tested due to incomplete taxon sampling and that molecular data have not been collected in many Odontobutis species. In the present study, we sampled 51 specimens from all known eight Odontobutis species with two outgroups (Perccottus glenii and Neodontobutis hainanensis). We collected sequence data of 4434 single-copy nuclear coding loci using gene capture and Illumina sequencing. A robust phylogeny of the Odontobutis with many individuals for each species was built, supporting the current taxonomy that all extant Odontobutis species are valid. The two species from Japan (O. hikimius + O. obscurus) formed an independent clade sister to the “continental odontobutids”, whereas the species from southern China (O. sinensis + O. haifengensis) separated from the rest species of the genus. Surprisingly species from the lower reaches of the Yangtze River (O. potamophilus) was more closely related to species from the Korean Peninsula and northeastern China than to the middle reaches of the Yangtze River, such that their relationship was ((O. sinensis + O. haifengensis)(O. platycephala + (O. yaluensis + (O. potamophilus + O. interruptus)))). Divergence time among the Odontobutis was estimated using 100 most clock-like loci and three fossil calibration points. The crown group of the Odontobutis was estimated at 9.0 Ma during the late Miocene (5.6–12.7 Ma, 95% HPDs). Ancestral range of the genus was reconstructed using Reconstruct Ancestral States in Phylogenies (RASP) and BioGeoBEARS. The result suggested that the common ancestor of modern Odontobutis probably was distributed in Japan, southern China or the Korean Peninsula. A series of geographical events in East Asia since the late Miocene, such as the opening of the Japan/East Sea, rapid uplift of the Tibetan Plateau and climate change in the northern region of the Yellow River might account for diversification and current distribution pattern of the Odontobutis.

Nano-Strainer: A workflow for the identification of single-copy nuclear loci for plant systematic studies, using target capture kits and Oxford Nanopore long reads.

In modern plant systematics, target enrichment enables simultaneous analysis of hundreds of genes. However, when dealing with reticulate or polyploidization histories, few markers may suffice, but often are required to be single-copy, a condition that is not necessarily met with commercial capture kits. Also, large genome sizes can render target capture ineffective, so that amplicon sequencing would be preferable; however, knowledge about suitable loci is often missing. Here, we present a comprehensive workflow for the identification of putative single-copy nuclear markers in a genus of interest, by mining a small dataset from target capture using a few representative taxa. The proposed pipeline assesses sequence variability contained in the data from targeted loci and assigns reads to their respective genes, via a combined BLAST/clustering procedure. Cluster consensus sequences are then examined based on four pre-defined criteria presumably indicative for absence of paralogy. This is done by calculating four specialized indices; loci are ranked according to their performance in these indices, and top-scoring loci are considered putatively single- or low copy. The approach can be applied to any probe set. As it relies on long reads, the present contribution also provides template workflows for processing Nanopore-based target capture data. Obtained markers are further tested and then entered into amplicon sequencing. For the detection of possibly remaining paralogy in these data, which might occur in groups with rampant paralogy, we also employ the long-read assembly tool canu. In diploid representatives of the young Compositae genus Leucanthemum, characterized by high levels of polyploidy, our approach resulted in successful amplification of 13 loci. Modifications to remove traces of paralogy were made in seven of these. A species tree from the markers correctly reproduced main relationships in the genus, however, at low resolution. The presented workflow has the potential to valuably support phylogenetic research, for example in polyploid plant groups.

First Description of the Nuclear and Mitochondrial Genomes and Associated Host Preference of Trichopoda pennipes, a Parasitoid of Nezara viridula.

Trichopoda pennipes is a tachinid parasitoid of several significant heteropteran agricultural pests, including the southern green stink bug, Nezara viridula, and leaf-footed bug, Leptoglossus phyllopus. To be used successfully as a biological control agent, the fly must selectively parasitize the target host species. Differences in the host preference of T. pennipes were assessed by assembling the nuclear and mitochondrial genomes of 38 flies reared from field-collected N. viridula and L. phyllopus. High-quality de novo draft genomes of T. pennipes were assembled using long-read sequencing. The assembly totaled 672 MB distributed among 561 contigs, having an N50 of 11.9 MB and a GC of 31.7%, with the longest contig at 28 MB. The genome was assessed for completeness using BUSCO in the Insecta dataset, resulting in a score of 99.4%, and 97.4% of the genes were single copy-loci. The mitochondrial genomes of the 38 T. pennipes flies were sequenced and compared to identify possible host-determined sibling species. The assembled circular genomes ranged from 15,345 bp to 16,390 bp and encode 22 tRNAs, two rRNAs, and 13 protein-coding genes (PCGs). There were no differences in the architecture of these genomes. Phylogenetic analyses using sequence information from 13 PCGs and the two rRNAs individually or as a combined dataset resolved the parasitoids into two distinct lineages: T. pennipes that parasitized both N. viridula and L. phyllopus, and others that parasitized only L. phyllopus.

The phylogeny and divergence times of leaf-mining flies (Diptera: Agromyzidae) from anchored phylogenomics

Leaf-mining flies (Diptera: Agromyzidae) are a diverse clade of phytophagous Diptera known largely for their economic impact as leaf- or stem-miners on vegetable and ornamental plants. Higher-level phylogenetic relationships of Agromyzidae have remained uncertain because of challenges in sampling of both taxa and characters for morphology and PCR-based Sanger-era molecular systematics. Here, we used hundreds of orthologous single-copy nuclear loci obtained from anchored hybrid enrichment (AHE) to reconstruct phylogenetic relationships among the major lineages of leaf-mining flies. The resulting phylogenetic trees are highly congruent and well-supported, except for a few deep nodes, when using different molecular data types and phylogenetic methods. Based on divergence time dating using a relaxed clock model-based analysis, leaf-mining flies are shown to have diversified in multiple lineages since the early Paleocene, approximately 65 million years ago. Our study not only reveals a revised classification system of leaf-mining flies, but also provides a new phylogenetic framework to understand their macroevolution.

Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control.

The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to known origin interactors, we find the microtubule-binding Ask1/DASH complex as an origin-regulating factor. Strikingly, tethering of Ask1 to individual origin sites advances replication timing (RT) of the targeted chromosomal domain. Targeted degradation of Ask1 globally changes RT of a subset of origins, which can be reproduced by inhibiting microtubule dynamics. Thus, our findings mechanistically connect RT and chromosomal organization via Ask1/DASH with the microtubule cytoskeleton.

CasSABER for Programmable In Situ Visualization of Low and Nonrepetitive Gene Loci.

Site-specific imaging of target genes using CRISPR probes is essential for understanding the molecular mechanisms of gene function and engineering tools to modulate its downstream pathways. Herein, we develop CRISPR/Cas9-mediated signal amplification by exchange reaction (CasSABER) for programmable in situ imaging of low and nonrepetitive regions of the target gene in the cell nucleus. The presynthesized primer-exchange reaction (PER) probe is able to hybridize multiple fluorophore-bearing imager strands to specifically light up dCas9/sgRNA target-bound gene loci, enabling in situ imaging of fixed cellular gene loci with high specificity and signal-to-noise ratio. In combination with a multiround branching strategy, we successfully detected nonrepetitive gene regions using a single sgRNA. As an intensity-codable and orthogonal probe system, CasSABER enables the adjustable amplification of local signals in fixed cells, resulting in the simultaneous visualization of multicopy and single-copy gene loci with similar fluorescence intensity. Owing to avoiding the complexity of controlling in situ mutistep enzymatic reactions, CasSABER shows good reliability, sensitivity, and ease of implementation, providing a rapid and cost-effective molecular toolkit for studying multigene interaction in fundamental research and gene diagnosis.