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September 22, 2019  |  

Variation in human chromosome 21 ribosomal RNA genes characterized by TAR cloning and long-read sequencing.

Despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. We recovered ribosomal DNA segments from a single human chromosome 21 using transformation-associated recombination (TAR) cloning in yeast. Accurate long-read sequencing of 13 isolates covering ~0.82 Mb of the chromosome 21 rDNA complement revealed substantial variation among tandem repeat rDNA copies, several palindromic structures and potential errors in the previous reference sequence. These clones revealed 101 variant positions in the 45S transcription unit and 235 in the intergenic spacer sequence. Approximately 60% of the 45S variants were confirmed in independent whole-genome or RNA-seq data, with 47 of these further observed in mature 18S/28S rRNA sequences. TAR cloning and long-read sequencing enabled the accurate reconstruction of multiple rDNA units and a new, high-quality 44 838 bp rDNA reference sequence, which we have annotated with variants detected from chromosome 21 of a single individual. The large number of variants observed reveal heterogeneity in human rDNA, opening up the possibility of corresponding variations in ribosome dynamics.

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September 22, 2019  |  

A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits.

Rose is the world’s most important ornamental plant, with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Roses are outbred and can have various ploidy levels. Our objectives were to develop a high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short reads, and anchoring to a high-density genetic map, and to study the genome structure and genetic basis of major ornamental traits. We produced a doubled haploid rose line (‘HapOB’) from Rosa chinensis ‘Old Blush’ and generated a rose genome assembly anchored to seven pseudo-chromosomes (512?Mb with N50 of 3.4?Mb and 564 contigs). The length of 512?Mb represents 90.1-96.1% of the estimated haploid genome size of rose. Of the assembly, 95% is contained in only 196 contigs. The anchoring was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features, including the pericentromeric regions, through annotation of transposable element families and positioned centromeric repeats using fluorescent in situ hybridization. The rose genome displays extensive synteny with the Fragaria vesca genome, and we delineated only two major rearrangements. Genetic diversity was analysed using resequencing data of seven diploid and one tetraploid Rosa species selected from various sections of the genus. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and the number of flower petals. A rose APETALA2/TOE homologue is proposed to be the major regulator of petal number in rose. This reference sequence is an important resource for studying polyploidization, meiosis and developmental processes, as we demonstrated for flower and prickle development. It will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.

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September 22, 2019  |  

Whole genome and transcriptome maps of the entirely black native Korean chicken breed Yeonsan Ogye.

Yeonsan Ogye (YO), an indigenous Korean chicken breed (Gallus gallus domesticus), has entirely black external features and internal organs. In this study, the draft genome of YO was assembled using a hybrid de novo assembly method that takes advantage of high-depth Illumina short reads (376.6X) and low-depth Pacific Biosciences (PacBio) long reads (9.7X).The contig and scaffold NG50s of the hybrid de novo assembly were 362.3 Kbp and 16.8 Mbp, respectively. The completeness (97.6%) of the draft genome (Ogye_1.1) was evaluated with single-copy orthologous genes using Benchmarking Universal Single-Copy Orthologs and found to be comparable to the current chicken reference genome (galGal5; 97.4%; contigs were assembled with high-depth PacBio long reads (50X) and scaffolded with short reads) and superior to other avian genomes (92%-93%; assembled with short read-only or hybrid methods). Compared to galGal4 and galGal5, the draft genome included 551 structural variations including the fibromelanosis (FM) locus duplication, related to hyperpigmentation. To comprehensively reconstruct transcriptome maps, RNA sequencing and reduced representation bisulfite sequencing data were analyzed from 20 tissues, including 4 black tissues (skin, shank, comb, and fascia). The maps included 15,766 protein-coding and 6,900 long noncoding RNA genes, many of which were tissue-specifically expressed and displayed tissue-specific DNA methylation patterns in the promoter regions.We expect that the resulting genome sequence and transcriptome maps will be valuable resources for studying domestic chicken breeds, including black-skinned chickens, as well as for understanding genomic differences between breeds and the evolution of hyperpigmented chickens and functional elements related to hyperpigmentation.

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September 22, 2019  |  

A mosaic monoploid reference sequence for the highly complex genome of sugarcane.

Sugarcane (Saccharum spp.) is a major crop for sugar and bioenergy production. Its highly polyploid, aneuploid, heterozygous, and interspecific genome poses major challenges for producing a reference sequence. We exploited colinearity with sorghum to produce a BAC-based monoploid genome sequence of sugarcane. A minimum tiling path of 4660 sugarcane BAC that best covers the gene-rich part of the sorghum genome was selected based on whole-genome profiling, sequenced, and assembled in a 382-Mb single tiling path of a high-quality sequence. A total of 25,316 protein-coding gene models are predicted, 17% of which display no colinearity with their sorghum orthologs. We show that the two species, S. officinarum and S. spontaneum, involved in modern cultivars differ by their transposable elements and by a few large chromosomal rearrangements, explaining their distinct genome size and distinct basic chromosome numbers while also suggesting that polyploidization arose in both lineages after their divergence.

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September 22, 2019  |  

GC content elevates mutation and recombination rates in the yeast Saccharomyces cerevisiae.

The chromosomes of many eukaryotes have regions of high GC content interspersed with regions of low GC content. In the yeast Saccharomyces cerevisiae, high-GC regions are often associated with high levels of meiotic recombination. In this study, we constructed URA3 genes that differ substantially in their base composition [URA3-AT (31% GC), URA3-WT (43% GC), and URA3-GC (63% GC)] but encode proteins with the same amino acid sequence. The strain with URA3-GC had an approximately sevenfold elevated rate of ura3 mutations compared with the strains with URA3-WT or URA3-AT About half of these mutations were single-base substitutions and were dependent on the error-prone DNA polymerase ?. About 30% were deletions or duplications between short (5-10 base) direct repeats resulting from DNA polymerase slippage. The URA3-GC gene also had elevated rates of meiotic and mitotic recombination relative to the URA3-AT or URA3-WT genes. Thus, base composition has a substantial effect on the basic parameters of genome stability and evolution. Copyright © 2018 the Author(s). Published by PNAS.

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September 22, 2019  |  

Using XCAVATOR and EXCAVATOR2 to Identify CNVs from WGS, WES, and TS Data.

Copy Number Variants (CNVs) are structural rearrangements contributing to phenotypic variation but also associated with many disease states. In recent years, the identification of CNVs from high-throughput sequencing experiments has become a common practice for both research and clinical purposes. Several computational methods have been developed so far. In this unit, we describe and give instructions on how to run two read count-based tools, XCAVATOR and EXCAVATOR2, which are tailored for the detection of both germline and somatic CNVs from different sequencing experiments (whole-genome, whole-exome, and targeted) in various disease contexts and population genetic studies. © 2018 by John Wiley & Sons, Inc.© 2018 John Wiley & Sons, Inc.

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September 22, 2019  |  

A chromosome scale assembly of the model desiccation tolerant grass Oropetium thomaeum

Oropetium thomaeum is an emerging model for desiccation tolerance and genome size evolution in grasses. A high-quality draft genome of Oropetium was recently sequenced, but the lack of a chromosome scale assembly has hindered comparative analyses and downstream functional genomics. Here, we reassembled Oropetium, and anchored the genome into ten chromosomes using Hi-C based chromatin interactions. A combination of high-resolution RNAseq data and homology-based gene prediction identified thousands of new, conserved gene models that were absent from the V1 assembly. This includes thousands of new genes with high expression across a desiccation timecourse. The sorghum and Oropetium genomes have a surprising degree of chromosome-level collinearity, and several chromosome pairs have near perfect synteny. Other chromosomes are collinear in the gene rich chromosome arms but have experienced pericentric translocations. Together, these resources will be useful for the grass comparative genomic community and further establish Oropetium as a model resurrection plant.

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September 22, 2019  |  

Human copy number variants are enriched in regions of low mappability.

Copy number variants (CNVs) are known to affect a large portion of the human genome and have been implicated in many diseases. Although whole-genome sequencing (WGS) can help identify CNVs, most analytical methods suffer from limited sensitivity and specificity, especially in regions of low mappability. To address this, we use PopSV, a CNV caller that relies on multiple samples to control for technical variation. We demonstrate that our calls are stable across different types of repeat-rich regions and validate the accuracy of our predictions using orthogonal approaches. Applying PopSV to 640 human genomes, we find that low-mappability regions are approximately 5 times more likely to harbor germline CNVs, in stark contrast to the nearly uniform distribution observed for somatic CNVs in 95 cancer genomes. In addition to known enrichments in segmental duplication and near centromeres and telomeres, we also report that CNVs are enriched in specific types of satellite and in some of the most recent families of transposable elements. Finally, using this comprehensive approach, we identify 3455 regions with recurrent CNVs that were missing from existing catalogs. In particular, we identify 347 genes with a novel exonic CNV in low-mappability regions, including 29 genes previously associated with disease.

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September 22, 2019  |  

Creating a functional single-chromosome yeast.

Eukaryotic genomes are generally organized in multiple chromosomes. Here we have created a functional single-chromosome yeast from a Saccharomyces cerevisiae haploid cell containing sixteen linear chromosomes, by successive end-to-end chromosome fusions and centromere deletions. The fusion of sixteen native linear chromosomes into a single chromosome results in marked changes to the global three-dimensional structure of the chromosome due to the loss of all centromere-associated inter-chromosomal interactions, most telomere-associated inter-chromosomal interactions and 67.4% of intra-chromosomal interactions. However, the single-chromosome and wild-type yeast cells have nearly identical transcriptome and similar phenome profiles. The giant single chromosome can support cell life, although this strain shows reduced growth across environments, competitiveness, gamete production and viability. This synthetic biology study demonstrates an approach to exploration of eukaryote evolution with respect to chromosome structure and function.

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September 22, 2019  |  

Optical and physical mapping with local finishing enables megabase-scale resolution of agronomically important regions in the wheat genome.

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.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.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.

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September 22, 2019  |  

A gene-rich fraction analysis of the Passiflora edulis genome reveals highly conserved microsyntenic regions with two related Malpighiales species.

Passiflora edulis is the most widely cultivated species of passionflowers, cropped mainly for industrialized juice production and fresh fruit consumption. Despite its commercial importance, little is known about the genome structure of P. edulis. To fill in this gap in our knowledge, a genomic library was built, and now completely sequenced over 100 large-inserts. Sequencing data were assembled from long sequence reads, and structural sequence annotation resulted in the prediction of about 1,900 genes, providing data for subsequent functional analysis. The richness of repetitive elements was also evaluated. Microsyntenic regions of P. edulis common to Populus trichocarpa and Manihot esculenta, two related Malpighiales species with available fully sequenced genomes were examined. Overall, gene order was well conserved, with some disruptions of collinearity identified as rearrangements, such as inversion and translocation events. The microsynteny level observed between the P. edulis sequences and the compared genomes is surprising, given the long divergence time that separates them from the common ancestor. P. edulis gene-rich segments are more compact than those of the other two species, even though its genome is much larger. This study provides a first accurate gene set for P. edulis, opening the way for new studies on the evolutionary issues in Malpighiales genomes.

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September 22, 2019  |  

Opposite polarity monospore genome de novo sequencing and comparative analysis reveal the possible heterothallic life cycle of Morchella importuna.

Morchella is a popular edible fungus worldwide due to its rich nutrition and unique flavor. Many research efforts were made on the domestication and cultivation of Morchella all over the world. In recent years, the cultivation of Morchella was successfully commercialized in China. However, the biology is not well understood, which restricts the further development of the morel fungus cultivation industry. In this paper, we performed de novo sequencing and assembly of the genomes of two monospores with a different mating type (M04M24 and M04M26) isolated from the commercially cultivated strain M04. Gene annotation and comparative genome analysis were performed to study differences in CAZyme (Carbohydrate-active enzyme) enzyme content, transcription factors, duplicated sequences, structure of mating type sites, and differences at the gene and functional levels between the two monospore strains of M. importuna. Results showed that the de novo assembled haploid M04M24 and M04M26 genomes were 48.98 and 51.07 Mb, respectively. A complete fine physical map of M. importuna was obtained from genome coverage and gene completeness evaluation. A total of 10,852 and 10,902 common genes and 667 and 868 endemic genes were identified from the two monospore strains, respectively. The Gene Ontology (GO) and KAAS (KEGG Automatic Annotation Serve) enrichment analyses showed that the endemic genes performed different functions. The two monospore strains had 99.22% collinearity with each other, accompanied with certain position and rearrangement events. Analysis of complete mating-type loci revealed that the two monospore M. importuna strains contained an independent mating-type structure and remained conserved in sequence and location. The phylogenetic and divergence time of M. importuna was analyzed at the whole-genome level for the first time. The bifurcation time of morel and tuber was estimated to be 201.14 million years ago (Mya); the two monospore strains with a different mating type represented the evolution of different nuclei, and the single copy homologous genes between them were also different due to a genetic differentiation distance about 0.65 Mya. Compared with truffles, M. importuna had an extension of 28 clusters of orthologous genes (COGs) and a contraction of two COGs. The two different polar nuclei with different degrees of contraction and expansion suggested that they might have undergone different evolutionary processes. The different mating-type structures, together with the functional clustering and enrichment analysis results of the endemic genes of the two different polar nuclei, imply that M. importuna might be a heterothallic fungus and the interaction between the endemic genes may be necessary for its complete life history. Studies on the genome of M. importuna facilitate a better understanding of morel biology and evolution.

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September 22, 2019  |  

Improved Brassica rapa reference genome by single-molecule sequencing and chromosome conformation capture technologies.

Brassica rapa comprises several important cultivated vegetables and oil crops. Current reference genome assemblies of Brassica rapa are quite fragmented and not highly contiguous, thereby limiting extensive genetic and genomic analyses. Here, we report an improved assembly of the B. rapa genome (v3.0) using single-molecule sequencing, optical mapping, and chromosome conformation capture technologies (Hi-C). Relative to the previous reference genomes, our assembly features a contig N50 size of 1.45?Mb, representing a ~30-fold improvement. We also identified a new event that occurred in the B. rapa genome ~1.2 million years ago, when a long terminal repeat retrotransposon (LTR-RT) expanded. Further analysis refined the relationship of genome blocks and accurately located the centromeres in the B. rapa genome. The B. rapa genome v3.0 will serve as an important community resource for future genetic and genomic studies in B. rapa. This resource will facilitate breeding efforts in B. rapa, as well as comparative genomic analysis with other Brassica species.

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September 22, 2019  |  

Draft genome sequence of wild Prunus yedoensis reveals massive inter-specific hybridization between sympatric flowering cherries.

Hybridization is an important evolutionary process that results in increased plant diversity. Flowering Prunus includes popular cherry species that are appreciated worldwide for their flowers. The ornamental characteristics were acquired both naturally and through artificially hybridizing species with heterozygous genomes. Therefore, the genome of hybrid flowering Prunus presents important challenges both in plant genomics and evolutionary biology.We use long reads to sequence and analyze the highly heterozygous genome of wild Prunus yedoensis. The genome assembly covers >?93% of the gene space; annotation identified 41,294 protein-coding genes. Comparative analysis of the genome with 16 accessions of six related taxa shows that 41% of the genes were assigned into the maternal or paternal state. This indicates that wild P. yedoensis is an F1 hybrid originating from a cross between maternal P. pendula f. ascendens and paternal P. jamasakura, and it can be clearly distinguished from its confusing taxon, Yoshino cherry. A focused analysis of the S-locus haplotypes of closely related taxa distributed in a sympatric natural habitat suggests that reduced restriction of inter-specific hybridization due to strong gametophytic self-incompatibility is likely to promote complex hybridization of wild Prunus species and the development of a hybrid swarm.We report the draft genome assembly of a natural hybrid Prunus species using long-read sequencing and sequence phasing. Based on a comprehensive comparative genome analysis with related taxa, it appears that cross-species hybridization in sympatric habitats is an ongoing process that facilitates the diversification of flowering Prunus.

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September 22, 2019  |  

Asymmetric processing of DNA ends at a double-strand break leads to unconstrained dynamics and ectopic translocation.

Multiple pathways regulate the repair of double-strand breaks (DSBs) to suppress potentially dangerous ectopic recombination. Both sequence and chromatin context are thought to influence pathway choice between non-homologous end-joining (NHEJ) and homology-driven recombination. To test the effect of repetitive sequences on break processing, we have inserted TG-rich repeats on one side of an inducible DSB at the budding yeast MAT locus on chromosome III. Five clustered Rap1 sites within a break-proximal TG repeat are sufficient to block Mre11-Rad50-Xrs2 recruitment, impair resection, and favor elongation by telomerase. The two sides of the break lose end-to-end tethering and show enhanced, uncoordinated movement. Only the TG-free side is resected and shifts to the nuclear periphery. In contrast to persistent DSBs without TG repeats that are repaired by imprecise NHEJ, nearly all survivors of repeat-proximal DSBs repair the break by a homology-driven, non-reciprocal translocation from ChrIII-R to ChrVII-L. This suppression of imprecise NHEJ at TG-repeat-flanked DSBs requires the Uls1 translocase activity. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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