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Construction of a physical chromosome map of a species is important for positional cloning, targeted marker development, fine mapping of genes, selection of candidate genes for molecular breeding, as well as understanding the genome organization. The genomic libraries in the form of bacterial artificial chromosome (BAC) clones are also a very useful resource for physical mapping and identification and isolation of full-length genes and the related cis acting elements. Some BAC-FISH based studies reported in the past were gene based physical chromosome maps of Clarias magur (magur) to understand the genome organization of the species and to establish the relationships with other species in respect to genes' organization and evolution in the past. In the present study, we generated end sequences of the BAC clones and analyzed those end sequences within the scaffolds of the draft genome of magur to identify and map the genes bioinformatically for each clone. A total of 36 clones mostly possessing genes were identified and used in probe construction and their subsequent hybridization on the metaphase chromosomes of magur. This study successfully mapped all 36 specific clones on 16 chromosome pairs, out of 25 pairs of magur chromosomes. These clones are now recognized as chromosome-specific makers, which are an aid in individual chromosome identification and fine assembly of the genome sequence, and will ultimately help in developing anchored genes' map on the chromosomes of C. magur for understanding their organization, inheritance of important fishery traits and evolution of magur with respect to channel catfish, zebrafish and other species.

The pleiotropic drug resistance (PDR) proteins of the ATP-binding cassette (ABC) family play essential roles in physiological processes and have been characterized in many plant species. However, no comprehensive investigation of tobacco (Nicotiana tabacum), an important economic crop and a useful model plant for scientific research, has been presented. We identified 32 PDR genes in the tobacco genome and explored their domain organization, chromosomal distribution and evolution, promoter cis-elements, and expression profiles. A phylogenetic analysis revealed that tobacco has a significantly expanded number of PDR genes involved in plant defense. It also revealed that two tobacco PDR proteins may function as strigolactone transporters to regulate shoot branching, and several NtPDR genes may be involved in cadmium transport. Moreover, tissue expression profiles of NtPDR genes and their responses to several hormones and abiotic stresses were assessed using quantitative real-time PCR. Most of the NtPDR genes were regulated by jasmonate or salicylic acid, suggesting the important regulatory roles of NtPDRs in plant defense and secondary metabolism. They were also responsive to abiotic stresses, like drought and cold, and there was a strong correlation between the presence of promoter cis-elements and abiotic/biotic stress responses. These results provide useful clues for further in-depth studies on the functions of the tobacco PDR genes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Recent sequencing and software enhancements have advanced our understanding of the evolution of genomic structure and function, especially addressing novel evolutionary biology questions. Yet fragmentary turtle genome assemblies remain a challenge to fully decipher the genetic architecture of adaptive evolution. Here, we use optical mapping to improve the contiguity of the painted turtle ( Chrysemys picta ) genome assembly and use de novo fluorescent in situ hybridization (FISH) of bacterial artificial chromosome (BAC) clones, BAC-FISH, to physically map the genomes of the painted and slider turtles ( Trachemys scripta elegans ). Optical mapping increased C. picta 's N50 by ~242% compared to the previous assembly. Physical mapping permitted anchoring ~45% of the genome assembly, spanning 5544 genes (including 20 genes related to the sex determination network of turtles and vertebrates). BAC-FISH data revealed assembly errors in C. picta and T. s. elegans assemblies, highlighting the importance of molecular cytogenetic data to complement bioinformatic approaches. We also compared C. picta 's anchored scaffolds to the genomes of other chelonians, chicken, lizards, and snake. Results revealed a mostly one-to-one correspondence between chromosomes of painted and slider turtles, and high homology among large syntenic blocks shared with other turtles and sauropsids. Yet, numerous chromosomal rearrangements were also evident across chelonians, between turtles and squamates, and between avian and non-avian reptiles.

Creating gapless telomere-to-telomere assemblies of complex genomes is one of the ultimate challenges in genomics. We use two independent assemblies and an optical map-based merging pipeline to produce a maize genome (B73-Ab10) composed of 63 contigs and a contig N50 of 162 Mb. This genome includes gapless assemblies of chromosome 3 (236 Mb) and chromosome 9 (162 Mb), and 53 Mb of the Ab10 meiotic drive haplotype. The data also reveal the internal structure of seven centromeres and five heterochromatic knobs, showing that the major tandem repeat arrays (CentC, knob180, and TR-1) are discontinuous and frequently interspersed with retroelements.

Bacterial artificial chromosome (BAC) library is an important genomic resource useful in targeted marker development, positional cloning, physical mapping and a substrate for genome sequencing for better understanding the genome organization of a species. The present manuscript elucidates the improvement in protocols for economical and efficient BAC insert DNA isolation, BAC end sequencing and FISH for physical localization on the metaphase chromosome complements. BAC clones of Clarias magur, maintained in 384-well plate format in our laboratory, were used in this study. The protocols gave consistent and efficient results. We use routinely these protocols for BAC insert DNA extraction, generating end sequence data of the clone and constructing DNA probes to hybridize on the metaphase spreads of C. magur using FISH for physical their localization.

KEY MESSAGE: ddRAD-seq-based high-density genetic map comprising 2595 loci identified a major and consensus QTL with a linked marker in a 0.8-Mb physical interval for oil content in peanut. Enhancing oil content is an important breeding objective in peanut. High-resolution mapping of quantitative trait loci (QTLs) with linked markers could facilitate marker-assisted selection in breeding for target traits. In the present study, a recombined inbred line population (Xuhua 13 × Zhonghua 6) was used to construct a genetic map based on double-digest restriction-site-associated DNA sequencing (ddRAD-seq). The resulting high-density genetic map contained 2595 loci, and spanned a length of 2465.62 cM, with an average distance of 0.95 cM/locus. Seven QTLs for oil content were identified on five linkage groups, including the major and stable QTL qOCA08.1 on chromosome A08 with 10.14-27.19% phenotypic variation explained. The physical interval of qOCA08.1 was further delimited to a ~ 0.8-Mb genomic region where two genes affecting oil synthesis had been annotated. The marker SNPOCA08 was developed targeting the SNP loci associated with oil content and validated in peanut cultivars with diverse oil contents. The major and stable QTL identified in the present study could be further dissected for gene discovery. Furthermore, the tightly linked marker for oil content would be useful in marker-assisted breeding in peanut.

Chimonanthus campanulatus R.H. Chang & C.S. Ding is a good horticultural tree because of its beautiful yellow flowers and evergreen leaves. In this study, fluorescence in situ hybridization (FISH) was used to analyse mitotic metaphase chromosomes of Ch. campanulatus with 5S rDNA and (AG 3 T 3 ) 3 oligonucleotides. Twenty-two small chromosomes were observed. Weak 5S rDNA signals were observed only in proximal regions of two chromosomes, which were adjacent to the (AG 3 T 3 ) 3 proximal signals. Weak (AG 3 T 3 ) 3 signals were observed on both chromosome ends, which enabled accurate chromosome counts. A pair of satellite bodies was observed. (AG 3 T 3 ) 3 signals displayed quite high diversity, changing in intensity from weak to very strong as follows: far away from the chromosome ends (satellites), ends, subtelomeric regions, and proximal regions. Ten high-quality spreads revealed metaphase dynamics from the beginning to the end and the transition to anaphase. Chromosomes gradually grew larger and thicker into linked chromatids, which grew more significantly in width than in length. Based on the combination of 5S rDNA and (AG 3 T 3 ) 3 signal patterns, ten chromosomes were exclusively distinguished, and the remaining twelve chromosomes were divided into two distinct groups. Our physical map, which can reproduce dynamic metaphase progression and distinguish chromosomes, will powerfully guide cytogenetic research on Chimonanthus and other trees.

Background: In computational biology, the physical mapping of DNA is a key problem. We know that the double digest problem (DDP) is NP-complete. Many algorithms have been proposed for solving the DDP, although it is still far from being resolved., Results: We present DDmap, an open-source MATLAB package for solving the DDP, based on a newly designed genetic algorithm that combines six genetic operators in searching for optimal solutions. We test the performance of DDmap by using a typical DDP dataset, and we depict exact solutions to these DDP instances in an explicit manner. In addition, we propose an approximate method for solving some hard DDP scenarios via a scaling-rounding-adjusting process., Conclusions: For typical DDP test instances, DDmap finds exact solutions within approximately 1 s. Based on our simulations on 1000 random DDP instances by using DDmap, we find that the maximum length of the combining fragments has observable effects towards genetic algorithms for solving the DDP problem. In addition, a Maple source code for illustrating DDP solutions as nested pie charts is also included.

Background: The mammalian major histocompatibility complex (MHC) harbours clusters of genes associated with the immunological defence of animals against infectious pathogens. At present, no complete MHC physical map is available for any of the wild ruminant species in the world., Results: The high-density physical map is composed of two contigs of 47 overlapping bacterial artificial chromosome (BAC) clones, with an average of 115 Kb for each BAC, covering the entire addax MHC genome. The first contig has 40 overlapping BAC clones covering an approximately 2.9 Mb region of MHC class I, class III, and class IIa, and the second contig has 7 BAC clones covering an approximately 500 Kb genomic region that harbours MHC class IIb. The relative position of each BAC corresponding to the MHC sequence was determined by comparative mapping using PCR screening of the BAC library of 192,000 clones, and the order of BACs was determined by DNA fingerprinting. The overlaps of neighboring BACs were cross-verified by both BAC-end sequencing and co-amplification of identical PCR fragments within the overlapped region, with their identities further confirmed by DNA sequencing., Conclusions: We report here the successful construction of a high-quality physical map for the addax MHC region using BACs and comparative mapping. The addax MHC physical map we constructed showed one gap of approximately 18 Mb formed by an ancient autosomal inversion that divided the MHC class II into IIa and IIb. The autosomal inversion provides compelling evidence that the MHC organizations in all of the ruminant species are relatively conserved.

Background: Localized functional domains within chromosomes, known as topologically associating domains (TADs), have been recently highlighted. In Drosophila, TADs are biochemically defined by epigenetic marks, this suggesting that the 3D arrangement may be the "missing link" between epigenetics and gene activity. Recent observations (Boettiger et al. in Nature 529(7586):418-422, 2016) provide access to structural features of these domains with unprecedented resolution thanks to super-resolution experiments. In particular, they give access to the distribution of the radii of gyration for domains of different linear length and associated with different transcriptional activity states: active, inactive or repressed. Intriguingly, the observed scaling laws lack consistent interpretation in polymer physics., Results: We develop a new methodology conceived to extract the best information from such super-resolution data by exploiting the whole distribution of gyration radii, and to place these experimental results on a theoretical framework. We show that the experimental data are compatible with the finite-size behavior of a self-attracting polymer. The same generic polymer model leads to quantitative differences between active, inactive and repressed domains. Active domains behave as pure polymer coils, while inactive and repressed domains both lie at the coil-globule crossover. For the first time, the "color-specificity" of both the persistence length and the mean interaction energy are estimated, leading to important differences between epigenetic states., Conclusion: These results point toward a crucial role of criticality to enhance the system responsivity, resulting in both energy transitions and structural rearrangements. We get strong indications that epigenetically induced changes in nucleosome-nucleosome interaction can cause chromatin to shift between different activity states.

Key Message: A major grain length QTL on chromosome 5H was fine mapped from 180.5 to 1.7 Mb. Quantitative trait loci (QTLs) mapping has been used extensively in barley to detect QTLs that underlie complex traits such as grain size. In the present study, we utilised 312 double haploid lines derived from a cross between two Australian malting varieties, Vlamingh and Buloke, to dissect the genetic control of a number of grain size characteristics. Digital image analysis was used to measure grain size characteristics including length, width, thickness and plumpness which are important traits influencing barley yield and grain physical quality. Using data from four independent environments and molecular marker genotype data, we identified 23 significant QTLs for these four traits, ten of which were consensus QTLs and identified in two or more environments. A QTL region on chromosome 5H designated qGL5H that was associated with grain size was fine mapped to a 1.7 Mb interval. qGL5H was able to explain 21.6% of phenotypic variation for grain length within the population. This major QTL is an appropriate candidate for further genetic dissection.

Key Message: The tomato gray leaf spot resistance gene Sm was fine-mapped in a 185-kb region through a map-based cloning strategy and genome-wide association study; a candidate gene was proved to be involved in Sm-mediated resistance through transient gene silencing. Gray leaf spot, caused by Stemphylium spp., is a warm weather foliar disease in tomato (Solanum lycopersicum L). Resistance against gray leaf spot is conferred by a single incompletely dominant gene (Sm) located on chromosome 11. This study aimed to map and identify molecular marker tightly linked to the Sm gene for the use of marker-assisted selection in breeding. Using an F 2 population derived from a cross between the resistant line '9706' and the susceptible line 'Heinz 1706', the Sm gene was mapped to a 185-kb interval between two markers, InDel343 and InDel-FT-32 on chromosome 11, which was consistent with the result of a genome-wide association study using 289 diverse accessions. An ORF predicted in this region was proved to be involved in Sm-mediated resistance through transient gene silencing and seems to be a good candidate of the Sm locus. To clone the Sm gene, a bacterial artificial chromosome (BAC) library was screened and one BAC clone B80B15 containing the predicted ORF was identified. The analysis of sequence and structure characteristics demonstrated that the candidate gene was not a typical type resistance gene. Additionally, a co-dominant marker Sm-InDel, which produced a 122-bp or 140-bp fragment for resistant or susceptible alleles, respectively, was developed. This marker was validated in 289 germplasm and could be used in marker-assisted selection for gray leaf spot resistance.

Bread wheat (Triticum aestivum L.) is a staple food for a significant part of the world's population. The growing demand on its production can be satisfied by improving yield and resistance to biotic and abiotic stress. Knowledge of the genome sequence would aid in discovering genes and QTLs underlying these traits and provide a basis for genomics-assisted breeding. Physical maps and BAC clones associated with them have been valuable resources from which to generate a reference genome of bread wheat and to assist map-based gene cloning. As a part of a joint effort coordinated by the International Wheat Genome Sequencing Consortium, we have constructed a BAC-based physical map of bread wheat chromosome arm 7DS consisting of 895 contigs and covering 94% of its estimated length. By anchoring BAC contigs to one radiation hybrid map and three high resolution genetic maps, we assigned 73% of the assembly to a distinct genomic position. This map integration, interconnecting a total of 1713 markers with ordered and sequenced BAC clones from a minimal tiling path, provides a tool to speed up gene cloning in wheat. The process of physical map assembly included the integration of the 7DS physical map with a whole-genome physical map of Aegilops tauschii and a 7DS Bionano genome map, which together enabled efficient scaffolding of physical-map contigs, even in the non-recombining region of the genetic centromere. Moreover, this approach facilitated a comparison of bread wheat and its ancestor at BAC-contig level and revealed a reconstructed region in the 7DS pericentromere., (Copyright © 2018. Published by Elsevier B.V.)

The development of genomic resources and tools is an important step in designing novel approaches to genetic control of mosquitoes. Physical genome maps enhance the quality of the genome assemblies, improve gene annotation, and provide a better framework for comparative and population genomics studies in mosquitoes. In this chapter, we describe protocols for an important procedure in physical genome mapping-fluorescence in situ hybridization (FISH). We provide details on (1) dissection of salivary glands, ovaries, and imaginal discs for obtaining high-quality polytene or mitotic chromosome preparations; (2) DNA-labeling procedures and extraction of repetitive DNA fractions; and (3) approaches to FISH on polytene and mitotic chromosomes.

Long-read sequencing technologies have greatly facilitated assemblies of large eukaryotic genomes. In this paper, Oxford Nanopore sequences generated on a MinION sequencer are combined with Bionano Genomics Direct Label and Stain (DLS) optical maps to generate a chromosome-scale de novo assembly of the repeat-rich Sorghum bicolor Tx430 genome. The final assembly consists of 29 scaffolds, encompassing in most cases entire chromosome arms. It has a scaffold N 50 of 33.28 Mbps and covers 90% of the expected genome length. A sequence accuracy of 99.85% is obtained after aligning the assembly against Illumina Tx430 data and 99.6% of the 34,211 public gene models align to the assembly. Comparisons of Tx430 and BTx623 DLS maps against the public BTx623 v3.0.1 genome assembly suggest substantial discrepancies whose origin remains to be determined. In summary, this study demonstrates that informative assemblies of complex plant genomes can be generated by combining nanopore sequencing with DLS optical maps.

Due to recent advances in experimental and theoretical approaches, the dynamic three-dimensional organization (3D) of the nucleus has become a very active area of research in life sciences. We now understand that the linear genome is folded in ways that may modulate how genes are expressed during the basic functioning of cells. Importantly, it is now possible to build 3D models of how the genome folds within the nucleus and changes over time (4D). Because genome folding influences its function, this opens exciting new possibilities to broaden our understanding of the mechanisms that determine cell fate. However, the rapid evolution of methods and the increasing complexity of data can result in ambiguity and reproducibility challenges, which may hamper the progress of this field. Here, we describe such challenges ahead and provide guidelines to think about strategies for shared standardized validation of experimental 4D nucleome data sets and models.

Background: Numerous scaffold-level sequences for wheat are now being released and, in this context, we report on a strategy for improving the overall assembly to a level comparable to that of the human genome., Results: Using chromosome 7A of wheat as a model, sequence-finished megabase-scale sections of this chromosome were established by combining a new independent assembly using a bacterial artificial chromosome (BAC)-based physical map, BAC pool paired-end sequencing, chromosome-arm-specific mate-pair sequencing and Bionano optical mapping with the International Wheat Genome Sequencing Consortium RefSeq v1.0 sequence and its underlying raw data. The combined assembly results in 18 super-scaffolds across the chromosome. The value of finished genome regions is demonstrated for two approximately 2.5 Mb regions associated with yield and the grain quality phenotype of fructan carbohydrate grain levels. In addition, the 50 Mb centromere region analysis incorporates cytological data highlighting the importance of non-sequence data in the assembly of this complex genome region., Conclusions: Sufficient genome sequence information is shown to now be available for the wheat community to produce sequence-finished releases of each chromosome of the reference genome. The high-level completion identified that an array of seven fructosyl transferase genes underpins grain quality and that yield attributes are affected by five F-box-only-protein-ubiquitin ligase domain and four root-specific lipid transfer domain genes. The completed sequence also includes the centromere.

Background: Pathogen avirulence (Avr) genes can evolve rapidly when challenged by the widespread deployment of host genes for resistance. They can be effectively isolated by positional cloning provided a robust and well-populated genetic map is available., Results: An updated, SSR-based physical map of the rice blast pathogen Magnaporthe oryzae (Mo) has been constructed based on 116 of the 120 SSRs used to assemble the last map, along with 18 newly developed ones. A comparison between the two versions of the map has revealed an altered marker content and order within most of the Mo chromosomes. The avirulence gene AvrPi12 was mapped in a population of 219 progeny derived from a cross between the two Mo isolates CHL42 and CHL357. A bulked segregant analysis indicated that the gene was located on chromosome 6, a conclusion borne out by an analysis of the pattern of segregation shown by individual isolates. Six additional PCR-based markers were developed to improve the map resolution in the key region. AvrPi12 was finally located within the sub-telomeric region of chromosome 6, distal to the SSR locus LSM6-5., Conclusions: The improved SSR-based linkage map should be useful as a platform for gene mapping and isolation in Mo. It was used to establish the location of AvrPi12, thereby providing a starting point for its positional cloning.

A quantitative pachytene chromosome map of rice (Oryza sativa L.) was developed using imaging methods. The map depicts not only distribution patterns of chromomeres specific to pachytene chromosomes, but also the higher order information of chromosomal structures, such as heterochromatin (condensed regions), euchromatin (decondensed regions), the primary constrictions (centromeres), and the secondary constriction (nucleolar organizing regions, NOR). These features were image analyzed and quantitatively mapped onto the map by Chromosome Image Analyzing System ver. 4.0 (CHIAS IV). Correlation between H3K9me2, an epigenetic marker and formation and/or maintenance of heterochromatin, thus was, clearly visualized. Then the pachytene chromosome map was unified with the existing somatic chromosome and linkage maps by physically mapping common DNA markers among them, such as a rice A genome specific tandem repeat sequence (TrsA), 5S and 45S ribosomal RNA genes, five bacterial artificial chromosome (BAC) clones, four P1 bacteriophage artificial chromosome (PAC) clones using multicolor fluorescence in situ hybridization (FISH). Detailed comparison between the locations of the DNA probes on the pachytene chromosomes using multicolor FISH, and the linkage map enabled determination of the chromosome number and short/long arms of individual pachytene chromosomes using the chromosome number and arm assignment designated for the linkage map. As a result, the quantitative pachytene chromosome map was unified with two other major rice chromosome maps representing somatic prometaphase chromosomes and genetic linkages. In conclusion, the unification of the three rice maps serves as an indispensable basic information, not only for an in-depth comparison between genetic and chromosomal data, but also for practical breeding programs.

Chromosome conformation capture (3C) and its derivatives (e.g., 4C, 5C and Hi-C) are used to analyze the 3D organization of genomes. We recently developed targeted chromatin capture (T2C), an inexpensive method for studying the 3D organization of genomes, interactomes and structural changes associated with gene regulation, the cell cycle, and cell survival and development. Here, we present the protocol for T2C based on capture, describing all experimental steps and bio-informatic tools in full detail. T2C offers high resolution, a large dynamic interaction frequency range and a high signal-to-noise ratio. Its resolution is determined by the resulting fragment size of the chosen restriction enzyme, which can lead to sub-kilobase-pair resolution. T2C's high coverage allows the identification of the interactome of each individual DNA fragment, which makes binning of reads (often used in other methods) basically unnecessary. Notably, T2C requires low sequencing efforts. T2C also allows multiplexing of samples for the direct comparison of multiple samples. It can be used to study topologically associating domains (TADs), determining their position, shape, boundaries, and intra- and inter-domain interactions, as well as the composition of aggregated loops, interactions between nucleosomes, individual transcription factor binding sites, and promoters and enhancers. T2C can be performed by any investigator with basic skills in molecular biology techniques in ∼7-8 d. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments.

Background: Although draft genomes are available for most agronomically important plant species, the majority are incomplete, highly fragmented, and often riddled with assembly and scaffolding errors. These assembly issues hinder advances in tool development for functional genomics and systems biology., Findings: Here we utilized a robust, cost-effective approach to produce high-quality reference genomes. We report a near-complete genome of diploid woodland strawberry (Fragaria vesca) using single-molecule real-time sequencing from Pacific Biosciences (PacBio). This assembly has a contig N50 length of ∼7.9 million base pairs (Mb), representing a ∼300-fold improvement of the previous version. The vast majority (>99.8%) of the assembly was anchored to 7 pseudomolecules using 2 sets of optical maps from Bionano Genomics. We obtained ∼24.96 Mb of sequence not present in the previous version of the F. vesca genome and produced an improved annotation that includes 1496 new genes. Comparative syntenic analyses uncovered numerous, large-scale scaffolding errors present in each chromosome in the previously published version of the F. vesca genome., Conclusions: Our results highlight the need to improve existing short-read based reference genomes. Furthermore, we demonstrate how genome quality impacts commonly used analyses for addressing both fundamental and applied biological questions., (© The Authors 2017. Published by Oxford University Press.)

The study is focused on addressing the problem of building genetic maps in the presence of ∼10 3 -10 4 of markers per chromosome. We consider a spectrum of situations with intrachromosomal heterogeneity of recombination rate, different level of genotyping errors, and missing data. In the ideal scenario of the absence of errors and missing data, the majority of markers should appear as groups of cosegregating markers ("twins") representing no challenge for map construction. The central aspect of the proposed approach is to take into account the structure of the marker space, where each twin group (TG) and singleton markers are represented as points of this space. The confounding effect of genotyping errors and missing data leads to reduction of TG size, but upon a low level of these effects surviving TGs can still be used as a source of reliable skeletal markers. Increase in the level of confounding effects results in a considerable decrease in the number or even disappearance of usable TGs and, correspondingly, of skeletal markers. Here, we show that the paucity of informative markers can be compensated by detecting kernels of markers in the marker space using a clustering procedure, and demonstrate the utility of this approach for high-density genetic map construction on simulated and experimentally obtained genotyping datasets., (Copyright © 2017 by the Genetics Society of America.)

The comparative analysis of genetic and physical maps as well as of whole genome sequences had revealed that in the Drosophila genus, most structural rearrangements occurred within chromosomal elements as a result of paracentric inversions. Genome sequence comparison would seem the best method to estimate rates of chromosomal evolution, but the high-quality reference genomes required for this endeavor are still scanty. Here, we have obtained dense physical maps for Muller elements A, C, and E of Drosophila subobscura, a species with an extensively studied rich and adaptive chromosomal polymorphism. These maps are based on 462 markers: 115, 236, and 111 markers for elements A, C, and E, respectively. The availability of these dense maps will facilitate genome assembly and will thus greatly contribute to obtaining a good reference genome, which is a required step for D. subobscura to attain the model species status. The comparative analysis of these physical maps and those obtained from the D. pseudoobscura and D. melanogaster genomes allowed us to infer the number of fixed inversions and chromosomal evolutionary rates for each pairwise comparison. For all three elements, rates inferred from the more closely related species were higher than those inferred from the more distantly related species, which together with results of relative-rate tests point to an acceleration in the D. subobscura lineage at least for elements A and E.

Introduction: Preimplantation genetic diagnosis and screening (PGD/PGS) has been applied clinically for >25 years however inherent drawbacks include the necessity to tailor each case to the trait in question, and that technology to detect monogenic and chromosomal disorders respectively is fundamentally different. Areas covered: The area of preimplantation genetics has evolved over the last 25 years, adapting to changes in technology and the need for more efficient, streamlined diagnoses. Karyomapping allows the determination of inheritance from the (grand)parental haplobocks through assembly of inherited chromosomal segments. The output displays homologous chromosomes, crossovers and the genetic status of the embryos by linkage comparison, as well as chromosomal disorders. It also allows for determination of heterozygous SNP calls, avoiding the risks of allele dropout, a common problem with other PGD techniques. Manuscripts documenting the evolution of preimplantation genetics, especially those investigating technologies that would simultaneously detect monogenic and chromosomal disorders, were selected for review. Expert commentary: Karyomapping is currently available for detection of single gene disorders; ~1000 clinics worldwide offer it (via ~20 diagnostic laboratories) and ~2500 cases have been performed. Due an inability to detect post-zygotic trisomy reliably however and confounding problems of embryo mosaicism, karyomapping has yet to be applied clinically for detection of chromosome disorders.

Applications that use Bacterial Artificial Chromosome (BAC) libraries often require paired-end sequences and knowledge of the physical location of each clone in plates. To facilitate obtaining this information in high-throughput, we generated pBACode vectors: a pool of BAC cloning vectors, each with a pair of random barcodes flanking its cloning site. In a pBACode BAC library, the BAC ends and their linked barcodes can be sequenced in bulk. Barcode pairs are determined by sequencing the empty pBACode vectors, which allows BAC ends to be paired according to their barcodes. For physical clone mapping, the barcodes are used as unique markers for their linked genomic sequence. After multi-dimensional pooling of BAC clones, the barcodes are sequenced and deconvoluted to locate each clone. We generated a pBACode library of 94,464 clones for the flounder Paralichthys olivaceus and obtained paired-end sequence from 95.4% of the clones. Incorporating BAC paired-ends into the genome preassembly improved its continuity by over 10-fold. Furthermore, we were able to use the barcodes to map the physical locations of each clone in just 50 pools, with up to 11 808 clones per pool. Our physical clone mapping located 90.2% of BAC clones, enabling targeted characterization of chromosomal rearrangements., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in FIP1 , and a loxPsym site affecting promoter function of ATP2 PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping., (Copyright © 2017, American Association for the Advancement of Science.)

Leaves are the main organs in which photosynthates are produced. Leaf senescence facilitates the translocation of photosynthates and nutrients from source to sink, which is important for plant development and especially for crop yield. However, the molecular mechanism of leaf senescence is unknown. Here, we identified a mutant, yellow leaf and dwarf 1 (yld1), which exhibited decreased plant height and premature leaf senescence. Nitroblue tetrazolium and diamiobenzidine staining analyses revealed that the concentrations of reactive oxygen species were higher in yld1 leaves than in wild type leaves. The photosynthetic pigment contents were significantly decreased in yld1. The yld1 chloroplasts had collapsed and were filled with abnormal starch granules. Combining bulk segregant and MutMap gene mapping approaches, the mutation responsible for the yld1 phenotype was mapped to a 7.3 Mb centromeric region, and three non-synonymous single nucleotide polymorphisms located in three novel genes were identified in this region. The expression patterns of the three candidate genes indicated that LOC_Os06g29380 had the most potential for functional verification. Plant hormone measurements showed that salicylic acid was highly accumulated in yld1 leaves when compared with wild type leaves, and yld1 was more sensitive to salicylic acid than wild type. This work lays the foundation for understanding the molecular regulatory mechanism of leaf senescence, and may reveal new connections among the molecular pathways related to leaf senescence, starch metabolism and salicylic acid signaling., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Chromatin structure at the length scale encompassing local nucleosome-nucleosome interactions is thought to play a crucial role in regulating transcription and access to DNA. However, this secondary structure of chromatin remains poorly understood compared with the primary structure of single nucleosomes or the tertiary structure of long-range looping interactions. Here we report the first genome-wide map of chromatin conformation in human cells at the 1-3 nucleosome (50-500 bp) scale, obtained using ionizing radiation-induced spatially correlated cleavage of DNA with sequencing (RICC-seq) to identify DNA-DNA contacts that are spatially proximal. Unbiased analysis of RICC-seq signal reveals regional enrichment of DNA fragments characteristic of alternating rather than adjacent nucleosome interactions in tri-nucleosome units, particularly in H3K9me3-marked heterochromatin. We infer differences in the likelihood of nucleosome-nucleosome contacts among open chromatin, H3K27me3-marked, and H3K9me3-marked repressed chromatin regions. After calibrating RICC-seq signal to three-dimensional distances, we show that compact two-start helical fibre structures with stacked alternating nucleosomes are consistent with RICC-seq fragmentation patterns from H3K9me3-marked chromatin, while non-compact structures and solenoid structures are consistent with open chromatin. Our data support a model of chromatin architecture in intact interphase nuclei consistent with variable longitudinal compaction of two-start helical fibres.

Genetic maps are based on the recombination frequency of molecular markers which often show different positions in comparison to the corresponding physical maps. To decipher the position and order of DNA sequences genetically mapped to terminal and interstitial regions of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes was performed with 16 genomic single-copy probes derived from fingerprinted BAC contigs. Long genetic distances at subterminal regions translated into short physical distances, confirming that recombination events occur more often at distal regions of chromosome 3H. Nonoverlapping FISH signals were frequently obtained for probes with a physical distance of at least 30-60 kb. Only 8% of the analyzed chromosomes showed a symmetric order of FISH signals on both sister chromatids. Due to the dynamic packing of metaphase chromatin, the order of 2 adjacent single-copy signals along the chromosome arms outside the (peri)centromeric region can only reliably be determined if the cytological distance is approximately 3%, corresponding to 21.6 Mb., (© 2017 S. Karger AG, Basel.)

Centromeres usually consist of hundreds of kilobases of repetitive sequence which renders them difficult to assemble. As a consequence, centromeres are often missing from assembled genomes and their locations on physical chromosome maps have to be inferred from flanking sequences via fluorescence in situ hybridization (FISH). Alternatively, centromere positions can be mapped using linkage analyses in accidentally triploid individuals formed by half-tetrads (resulting from the inheritance of two chromatids from a single meiosis). The current genome assembly of the zebra finch (Taeniopygia guttata) comprises 32 chromosomes, but only for the ten largest chromosomes centromere positions have been mapped using FISH. We here map the positions of most of the remaining centromeres using half-tetrad analyses. For this purpose, we genotyped 37 zebra finches that were triploid or tetraploid due to inheritance errors (and mostly died as embryos) together with their parents at 64 microsatellite markers (at least two per chromosome). Using the information on centromere positions on the ten largest chromosomes, we were able to identify 12 cases of non-disjunction in maternal meiosis I and 10 cases of non-disjunction in maternal meiosis II. These 22 informative cases allowed us to infer centromere positions on additional 19 microchromosomes in reference to the current genome assembly. This knowledge will be valuable for studies of chromosome evolution, meiotic drive and species divergence in the avian lineage., Competing Interests: Compliance with ethical standards Ethical standards All institutional and national guidelines for the care and use of laboratory animals were followed. Conflict of interest The authors declare that they have no competing interests.

Background: Physical mapping of DNA with restriction enzymes allows for the characterization and assembly of much longer molecules than is feasible with sequencing. However, assemblies of physical map data are sensitive to input parameters, which describe noise inherent in the data collection process. One possible way to determine the parameter values that best describe a dataset is by trial and error., Results: Here we present OMWare, a tool that efficiently generated 405 de novo map assemblies of a single datasets collected from the cotton species Gossypium raimondii. The assemblies were generated using various input parameter values, and were completed more efficiently by re-using compatible intermediate results. These assemblies were assayed for contiguity, internal consistency, and accuracy., Conclusions: Resulting assemblies had variable qualities. Although highly accurate assemblies were found, contiguity and internal consistency metrics were poor predictors of accuracy.

Eukaryotic chromatin is organized in contiguous domains that differ in protein binding, histone modifications, transcriptional activity, and in their degree of compaction. Genome-wide comparisons suggest that, overall, the chromatin organization is similar in different cells within an organism. Here, we compare the structure and activity of the 61C7-61C8 interval in polytene and diploid cells of Drosophila. By in situ hybridization on polytene chromosomes combined with high-resolution microscopy, we mapped the boundaries of the 61C7-8 interband and of the 61C7 and C8 band regions, respectively. Our results demonstrate that the 61C7-8 interband is significantly larger than estimated previously. This interband extends over 20 kbp and is in the range of the flanking band domains. It contains several active genes and therefore can be considered as an open chromatin domain. Comparing the 61C7-8 structure of Drosophila S2 cells and polytene salivary gland cells by ChIP for chromatin protein binding and histone modifications, we observe a highly consistent domain structure for the proximal 13 kbp of the domain in both cell types. However, the distal 7 kbp of the open domain differs in protein binding and histone modification between both tissues. The domain contains four protein-coding genes in the proximal part and two noncoding transcripts in the distal part. The differential transcriptional activity of one of the noncoding transcripts correlates with the observed differences in the chromatin structure between both tissues. The significance of our findings for the organization and structure of open chromatin domains will be discussed.

Key Message: Tyramide FISH was used to locate relatively small genomic amplicons from molecular markers linked to Ms locus onto onion chromosome 2 near the centromere, a region of relatively low recombination. Fluorescence in situ hybridization (FISH) has not been readily exploited for physical mapping of molecular markers in plants due to the technical challenge of visualizing small single-copy probes. Signal amplification using tyramide (tyr) FISH can increase sensitivity up to 100-fold. We used tyr-FISH to physically locate molecular markers tightly linked to the nuclear male-fertility (Ms) restoration locus of onion onto mitotic metaphase, pachytene, and super-stretched pachytene chromosomes. Relatively short genomic amplicons (846-2251 bp) and a cDNA clone (666 bp) were visualized in 9-42 % of observed cells. The markers were assigned to proximal locations close to the centromere on the long arm of chromosome 2, a region of lower recombination, revealing that tightly linked markers may be physically distant from Ms. This result explains why several labs have identified molecular markers tightly linked to the Ms locus after screening relatively few DNA clones or primers and segregating progenies. Although these markers are still useful for marker-aided selection, our results indicate that map-based cloning of Ms will likely be difficult due to reduced recombination near this gene.

Sex chromosomes are particularly interesting regions of the genome for both molecular genetics and evolutionary studies; yet, for most species, we lack basic information, such as the gene order along the chromosome. Because they lack recombination, Y-linked genes cannot be mapped genetically, leaving physical mapping as the only option for establishing the extent of synteny and homology with the X chromosome. Here, we developed a novel and general method for deletion mapping of non-recombining regions by solving "the travelling salesman problem", and evaluate its accuracy using simulated datasets. Unlike the existing radiation hybrid approach, this method allows us to combine deletion mutants from different experiments and sources. We applied our method to a set of newly generated deletion mutants in the dioecious plant Silene latifolia and refined the locations of the sex-determining loci on its Y chromosome map.

We present a high-resolution map of genomic transformation-competent artificial chromosome (TAC) clones extending over all Arabidopsis thaliana (Arabidopsis) chromosomes. The Arabidopsis genomic TAC clones have been valuable genetic tools. Previously, we constructed an Arabidopsis genomic TAC library consisting of more than 10,000 TAC clones harboring large genomic DNA fragments extending over the whole Arabidopsis genome. Here, we determined 13,577 end sequences from 6987 Arabidopsis TAC clones and mapped 5937 TAC clones to precise locations, covering approximately 90% of the Arabidopsis chromosomes. We present the large-scale data set of TAC clones with high-resolution mapping information as a Java application tool, the Arabidopsis TAC Position Viewer, which provides ready-to-go transformable genomic DNA clones corresponding to certain loci on Arabidopsis chromosomes. The TAC clone resources will accelerate genomic DNA cloning, positional walking, complementation of mutants and DNA transformation for heterologous gene expression., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Background: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat., Results: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B., Conclusions: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

Soybean mosaic virus (SMV) disease is one of the most serious and broadly distributed soybean (Glycine max (L.) Merr.) diseases. Here, we combine the advantages of association and linkage analysis to identify and fine-map the soybean genes associated with resistance to SMV strain SC7. A set of 191 soybean accessions from different geographic origins and 184 recombinant inbred lines (RILs) derived from Kefeng No.1 (resistant) × Nannong 1138-2 (susceptible) were used in this study. The SC7 resistance genes were previously mapped to a 2.65 Mb region on chromosome 2 and a 380 kb region on chromosome 13. Among 19 single nucleotide polymorphisms (SNPs) detected via association analysis in the study, the SNP BARC-021625-04157 was located in the 2.65 Mb region, and the SNP BARC-041671-08065 was located near the 380 kb region; three genes harboring the SNPs were probably related to SC7 resistance. The resistance gene associated with BARC-021625-04157 was then fine-mapped to a region of approximately 158 kb on chromosome 2 using 184 RILs. Among the 15 genes within this region, one NBS-LRR type gene, one HSP40 gene and one serine carboxypeptidase-type gene might be candidate SC7 resistance genes. These results will be useful for map-based cloning and marker-assisted selection in soybean breeding programs., (© 2014 Institute of Botany, Chinese Academy of Sciences.)

Background: The substantially large bread wheat genome, organized into highly similar three sub-genomes, renders genomic research challenging. The construction of BAC-based physical maps of individual chromosomes reduces the complexity of this allohexaploid genome, enables elucidation of gene space and evolutionary relationships, provides tools for map-based cloning, and serves as a framework for reference sequencing efforts. In this study, we constructed the first comprehensive physical map of wheat chromosome arm 5DS, thereby exploring its gene space organization and evolution., Results: The physical map of 5DS was comprised of 164 contigs, of which 45 were organized into 21 supercontigs, covering 176 Mb with an N50 value of 2,173 kb. Fifty-eight of the contigs were larger than 1 Mb, with the largest contig spanning 6,649 kb. A total of 1,864 molecular markers were assigned to the map at a density of 10.5 markers/Mb, anchoring 100 of the 120 contigs (>5 clones) that constitute ~95 % of the cumulative length of the map. Ordering of 80 contigs along the deletion bins of chromosome arm 5DS revealed small-scale breaks in syntenic blocks. Analysis of the gene space of 5DS suggested an increasing gradient of genes organized in islands towards the telomere, with the highest gene density of 5.17 genes/Mb in the 0.67-0.78 deletion bin, 1.4 to 1.6 times that of all other bins., Conclusions: Here, we provide a chromosome-specific view into the organization and evolution of the D genome of bread wheat, in comparison to one of its ancestors, revealing recent genome rearrangements. The high-quality physical map constructed in this study paves the way for the assembly of a reference sequence, from which breeding efforts will greatly benefit.

Background: Southern house mosquito Culex quinquefasciatus belongs to the C. pipiens cryptic species complex, with global distribution and unclear taxonomy. Mosquitoes of the complex can transmit human and animal pathogens, such as filarial worm, West Nile virus and avian malarial Plasmodium. Physical gene mapping is crucial to understanding genome organization, function, and systematic relationships of cryptic species, and is a basis for developing new vector control strategies. However, physical mapping was not established previously for Culex due to the lack of well-structured polytene chromosomes., Methods: Inbreeding was used to diminish inversion polymorphism and asynapsis of chromosomal homologs. Identification of larvae of the same developmental stage using the shape of imaginal discs allowed achievement of uniformity in chromosomal banding pattern. This together with high-resolution phase-contrast photography enabled the development of a cytogenetic map. Fluorescent in situ hybridization was used for gene mapping., Results: A detailed cytogenetic map of C. quinquefasciatus polytene chromosomes was produced. Landmarks for chromosome recognition and cytological boundaries for two inversions were identified. Locations of 23 genes belonging to 16 genomic supercontigs, and 2 cDNA were established. Six supercontigs were oriented and one was found putatively misassembled. The cytogenetic map was linked to the previously developed genetic linkage groups by corresponding positions of 2 genetic markers and 10 supercontigs carrying genetic markers. Polytene chromosomes were numbered according to the genetic linkage groups., Conclusions: This study developed a new standard cytogenetic photomap of the polytene chromosomes for C. quinquefasciatus and was applied for the fine-scale physical mapping. It allowed us to infer chromosomal position of 1333 of annotated genes belonging to 16 genomic supercontigs and find orientation of 6 of these supercontigs; the new cytogenetic and previously developed genetic linkage maps were integrated based on 12 matches. The map will further assist in finding chromosomal position of the medically important and other genes, contributing into improvement of the genome assembly. Better assembled C. quinquefasciatus genome can serve as a reference for studying other vector species of C. pipiens complex and will help to resolve their taxonomic relationships. This, in turn, will contribute into future development of vector and disease control strategies.

Sorghum is one of the most promising bioenergy crops. Stem juice yield, together with stem sugar concentration, determines sugar yield in sweet sorghum. Bulked segregant analysis (BSA) is a gene mapping technique for identifying genomic regions containing genetic loci affecting a trait of interest that when combined with deep sequencing could effectively accelerate the gene mapping process. In this study, a dry stem sorghum landrace was characterized and the stem water controlling locus, qSW6, was fine mapped using QTL analysis and the combined BSA and deep sequencing technologies. Results showed that: (i) In sorghum variety Jiliang 2, stem water content was around 80% before flowering stage. It dropped to 75% during grain filling with little difference between different internodes. In landrace G21, stem water content keeps dropping after the flag leaf stage. The drop from 71% at flowering time progressed to 60% at grain filling time. Large differences exist between different internodes with the lowest (51%) at the 7th and 8th internodes at dough stage. (ii) A quantitative trait locus (QTL) controlling stem water content mapped on chromosome 6 between SSR markers Ch6-2 and gpsb069 explained about 34.7-56.9% of the phenotypic variation for the 5th to 10th internodes, respectively. (iii) BSA and deep sequencing analysis narrowed the associated region to 339 kb containing 38 putative genes. The results could help reveal molecular mechanisms underlying juice yield of sorghum and thus to improve total sugar yield.

RAD sequencing was performed using DH962 and Jimian5 as upland cotton mapping parents. Sequencing data for DH962 and Jimian5 were assembled into the genome sequences of ≈55.27 and ≈57.06 Mb, respectively. Analysing genome sequences of the two parents, 1,323 SSR, 3,838 insertion/deletion (InDel), and 9,366 single-nucleotide polymorphism (SNP) primer pairs were developed. All of the SSRs, 121 InDels, 441 SNPs, and other 6,747 primer pairs were screened in the two parents, and a total of 535 new polymorphic loci were identified. A genetic map including 1,013 loci was constructed using these results and 506 loci previously published for this population. Twenty-seven new QTLs for yield and fibre quality were identified, indicating that the efficiency of QTL detection was greatly improved by the increase in map density. Comparative genomics showed there to be considerable homology and collinearity between the AT and A2 genomes and between the DT and D5 genomes, although there were a few exchanges and introgressions among the chromosomes of the A2 genome. Here, the development of markers using parental RAD sequencing was effective, and a high-density intraspecific genetic map was constructed. This map can be used for molecular marker-assisted selection in cotton., (© The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

The genome assembly of southern house mosquito Cx. quinquefasciatus is represented by a high number of supercontigs with no order or orientation on the chromosomes. Although cytogenetic maps for the polytene chromosomes of this mosquito have been developed, their utilization for the genome mapping remains difficult because of the low number of high-quality spreads in chromosome preparations. Therefore, a simple and robust mitotic-chromosome-based approach for the genome mapping of Cx. quinquefasciatus still needs to be developed. In this study, we performed physical mapping of 37 genomic supercontigs using fluorescent in situ hybridization on mitotic chromosomes from imaginal discs of 4th instar larvae. The genetic linkage map nomenclature was adopted for the chromosome numbering based on the direct positioning of 58 markers that were previously genetically mapped. The smallest, largest, and intermediate chromosomes were numbered as 1, 2, and 3, respectively. For idiogram development, we analyzed and described in detail the morphology and proportions of the mitotic chromosomes. Chromosomes were subdivided into 19 divisions and 72 bands of four different intensities. These idiograms were used for mapping the genomic supercontigs/genetic markers. We also determined the presence of length polymorphism in the q arm of sex-determining chromosome 1 in Cx. quinquefasciatus related to the size of ribosomal locus. Our physical mapping and previous genetic linkage mapping resulted in the chromosomal assignment of 13% of the total genome assembly to the chromosome bands. We provided the first detailed description, nomenclature, and idiograms for the mitotic chromosomes of Cx. quinquefasciatus. Further application of the approach developed in this study will help to improve the quality of the southern house mosquito genome.

Congenital anomalies of the kidney and urogenital tract (CAKUT) occur in approximately 0.5% of live births and represent the most frequent cause of end-stage renal disease in neonates and children. The genetic basis of CAKUT is not well defined. To understand more fully the genetic basis of one type of CAKUT, unilateral renal agenesis (URA), we are studying inbred ACI rats, which spontaneously exhibit URA and associated urogenital anomalies at an incidence of approximately 10%. URA is inherited as an incompletely dominant trait with incomplete penetrance in crosses between ACI and Brown Norway (BN) rats and a single responsible genetic locus, designated Renag1, was previously mapped to rat chromosome 14 (RNO14). The goals of this study were to fine map Renag1, identify the causal genetic variant responsible for URA, confirm that the Renag1 variant is the sole determinant of URA in the ACI rat, and define the embryologic basis of URA in this rat model. Data presented herein localize Renag1 to a 379 kilobase (kb) interval that contains a single protein coding gene, Kit (v-kit Hardy-Zukerman 4 feline sarcoma viral oncogene homolog); identify an endogenous retrovirus-derived long terminal repeat located within Kit intron 1 as the probable causal variant; demonstrate aberrant development of the nephric duct in the anticipated number of ACI rat embryos; and demonstrate expression of Kit and Kit ligand (Kitlg) in the nephric duct. Congenic rats that harbor ACI alleles at Renag1 on the BN genetic background exhibit the same spectrum of urogenital anomalies as ACI rats, indicating that Renag1 is necessary and sufficient to elicit URA and associated urogenital anomalies. These data reveal the first genetic link between Kit and URA and illustrate the value of the ACI rat as a model for defining the mechanisms and cell types in which Kit functions during urogenital development.

The reshuffling of existing genetic variation during meiosis is important both during evolution and in breeding. The reassortment of genetic variants relies on the formation of crossovers (COs) between homologous chromosomes. The pattern of genome-wide CO distributions can be rapidly and precisely established by the short-read sequencing of individuals from F2 populations, which in turn are useful for quantitative trait locus (QTL) mapping. Although sequencing costs have decreased precipitously in recent years, the costs of library preparation for hundreds of individuals have remained high. To enable rapid and inexpensive CO detection and QTL mapping using low-coverage whole-genome sequencing of large mapping populations, we have developed a new method for library preparation along with Trained Individual GenomE Reconstruction, a probabilistic method for genotype and CO predictions for recombinant individuals. In an example case with hundreds of F2 individuals from two Arabidopsis thaliana accessions, we resolved most CO breakpoints to within 2 kb and reduced a major flowering time QTL to a 9-kb interval. In addition, an extended region of unusually low recombination revealed a 1.8-Mb inversion polymorphism on the long arm of chromosome 4. We observed no significant differences in the frequency and distribution of COs between F2 individuals with and without a functional copy of the DNA helicase gene RECQ4A. In summary, we present a new, cost-efficient method for large-scale, high-precision genotyping-by-sequencing., (Copyright © 2015 Rowan et al.)

Genetic linkage map is helpful for analysis on heredity of some gene families and map-based gene cloning because of its simple and elegant manifestation. One software is in need to draw a gene physical map, which shows a manner similar to the genetic linkage map, based on the relative physical distance between genes. Although some tools like GBrowse and MapViewer etc. are available to draw gene physical map, there are obvious limitations for them: (1) the data need to be decorated in advance; (2) users can't modify results. Therefore, we developed a bio-assisted mapping software--MapGene2Chrom with PC and web versions, which is based on Perl and SVG languages. The software can be used to draw the corresponding physical map quickly in SVG format based on the input data. It will become a useful tool for drawing gene physical map with the advantages of simple input data format, easily modified output and very good portability.

The single-copy gene Period was located in five grasshopper species belonging to the Acridomorpha group through permanent in situ hybridization (PISH). The mapping revealed one copy of this gene in the L1 chromosome pair in Ommexecha virens, Xyleus discoideus angulatus, Tropidacris collaris, Schistocerca pallens, and Stiphra robusta. A possible second copy was mapped on the L2 chromosome pair in S. robusta, which should be confirmed by further studies. Except for the latter case, the chromosomal position of the Period gene was highly conserved among the four families studied. The S. robusta karyotype also differs from the others both in chromosome number and morphology. The position conservation of the single-copy gene Period contrasts with the location diversification of multigene families in these species. The localization of single-copy genes by PISH can provide new insights about the genomic content and chromosomal evolution of grasshoppers and others insects.

In Carangidae, Caranx is taxonomically controversial because of slight morphological differences among species, as well as because of its relationship with the genus Carangoides. Cytogenetic data has contributed to taxonomic and phylogenetic classification for some groups of fish. In this study, we examined the chromosomes of Caranx latus, Caranx lugubris, and Carangoides bartholomaei using classical methods, including conventional staining, C-banding, silver staining for nuclear organizer regions, base-specific fluorochrome, and 18S and 5S ribosomal sequence mapping using in situ hybridization. These 3 species showed chromosome numbers of 2n = 48, simple nuclear organizer regions (pair 1), and mainly centromeric heterochomatin. However, C. latus (NF = 50) and C. bartholomaei (NF = 50) showed a structurally conserved karyotype compared with C. lugubris (NF = 54), with a larger number of 2-armed chromosomes. The richness of GC-positive heterochromatic segments and sites in 5S rDNA in specific locations compared to the other 2 species reinforce the higher evolutionary dynamism in C. lugubris. Cytogenetic aspects shared between C. latus and C. bartholomaei confirm the remarkable phylogenetic proximity between these genera.

The Genetic Investigation of Anthropometric Traits (GIANT) consortium identified 14 loci in European Ancestry (EA) individuals associated with waist-to-hip ratio (WHR) adjusted for body mass index. These loci are wide and narrowing the signals remains necessary. Twelve of 14 loci identified in GIANT EA samples retained strong associations with WHR in our joint EA/individuals of African Ancestry (AA) analysis (log-Bayes factor >6.1). Trans-ethnic analyses at five loci (TBX15-WARS2, LYPLAL1, ADAMTS9, LY86 and ITPR2-SSPN) substantially narrowed the signals to smaller sets of variants, some of which are in regions that have evidence of regulatory activity. By leveraging varying linkage disequilibrium structures across different populations, single-nucleotide polymorphisms (SNPs) with strong signals and narrower credible sets from trans-ethnic meta-analysis of central obesity provide more precise localizations of potential functional variants and suggest a possible regulatory role. Meta-analysis results for WHR were obtained from 77 167 EA participants from GIANT and 23 564 AA participants from the African Ancestry Anthropometry Genetics Consortium. For fine mapping we interrogated SNPs within ± 250 kb flanking regions of 14 previously reported index SNPs from loci discovered in EA populations by performing trans-ethnic meta-analysis of results from the EA and AA meta-analyses. We applied a Bayesian approach that leverages allelic heterogeneity across populations to combine meta-analysis results and aids in fine-mapping shared variants at these locations. We annotated variants using information from the ENCODE Consortium and Roadmap Epigenomics Project to prioritize variants for possible functionality., (Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Key Message: Fine mapping by recombinant backcross populations revealed that a preharvest sprouting QTL on 2B contained two QTLs linked in coupling with different effects on the phenotype. Wheat preharvest sprouting (PHS) occurs when grain germinates on the plant before harvest, resulting in reduced grain quality. Previous mapping of quantitative trait locus (QTL) revealed a major PHS QTL, QPhs.cnl-2B.1, located on chromosome 2B significant in 16 environments that explained from 5 to 31 % of the phenotypic variation. The objective of this project was to fine map the QPhs.cnl-2B.1 interval. Fine mapping was carried out in recombinant backcross populations (BC1F4 and BC1F5) that were developed by backcrossing selected doubled haploids to a recurrent parent and self-pollinating the BC1F4 and BC1F5 generations. In each generation, three markers in the QPhs.cnl-2B.1 interval were used to screen for recombinants. Fine mapping revealed that the QPhs.cnl-2B.1 interval contained two PHS QTLs linked in coupling. The distal PHS QTL, located between Wmc453c and Barc55, contributed 8 % of the phenotypic variation and also co-located with a major seed dormancy QTL determined by germination index. The proximal PHS QTL, between Wmc474 and CNL415-rCDPK, contributed 16 % of the variation. Several candidate genes including Mg-chelatase H subunit family protein, GTP-binding protein and calmodulin/Ca(2+)-dependent protein kinase were linked to the PHS QTL. Although many recombinant lines were identified, the lack of polymorphism for markers in the QTL interval prevented the localization of the recombination breakpoints and identification of the gene underlying the phenotype.

Background: An initial comparative genomic study of the malaria vector Anopheles gambiae and the yellow fever mosquito Aedes aegypti revealed striking differences in the genome assembly size and in the abundance of transposable elements between the two species. However, the chromosome arms homology between An. gambiae and Ae. aegypti, as well as the distribution of genes and repetitive elements in chromosomes of Ae. aegypti, remained largely unexplored because of the lack of a detailed physical genome map for the yellow fever mosquito., Results: Using a molecular landmark-guided fluorescent in situ hybridization approach, we mapped 624 Mb of the Ae. aegypti genome to mitotic chromosomes. We used this map to analyze the distribution of genes, tandem repeats and transposable elements along the chromosomes and to explore the patterns of chromosome homology and rearrangements between Ae. aegypti and An. gambiae. The study demonstrated that the q arm of the sex-determining chromosome 1 had the lowest gene content and the highest density of minisatellites. A comparative genomic analysis with An. gambiae determined that the previously proposed whole-arm synteny is not fully preserved; a number of pericentric inversions have occurred between the two species. The sex-determining chromosome 1 had a higher rate of genome rearrangements than observed in autosomes 2 and 3 of Ae. aegypti., Conclusions: The study developed a physical map of 45% of the Ae. aegypti genome and provided new insights into genomic composition and evolution of Ae. aegypti chromosomes. Our data suggest that minisatellites rather than transposable elements played a major role in rapid evolution of chromosome 1 in the Aedes lineage. The research tools and information generated by this study contribute to a more complete understanding of the genome organization and evolution in mosquitoes.