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July 19, 2019

De novo assembly of haplotype-resolved genomes with trio binning.

Complex allelic variation hampers the assembly of haplotype-resolved sequences from diploid genomes. We developed trio binning, an approach that simplifies haplotype assembly by resolving allelic variation before assembly. In contrast with prior approaches, the effectiveness of our method improved with increasing heterozygosity. Trio binning uses short reads from two parental genomes to first partition long reads from an offspring into haplotype-specific sets. Each haplotype is then assembled independently, resulting in a complete diploid reconstruction. We used trio binning to recover both haplotypes of a diploid human genome and identified complex structural variants missed by alternative approaches. We sequenced an F1 cross between the cattle subspecies Bos taurus taurus and Bos taurus indicus and completely assembled both parental haplotypes with NG50 haplotig sizes of >20 Mb and 99.998% accuracy, surpassing the quality of current cattle reference genomes. We suggest that trio binning improves diploid genome assembly and will facilitate new studies of haplotype variation and inheritance.

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July 19, 2019

Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L.

Modern sugarcanes are polyploid interspecific hybrids, combining high sugar content from Saccharum officinarum with hardiness, disease resistance and ratooning of Saccharum spontaneum. Sequencing of a haploid S. spontaneum, AP85-441, facilitated the assembly of 32 pseudo-chromosomes comprising 8 homologous groups of 4 members each, bearing 35,525 genes with alleles defined. The reduction of basic chromosome number from 10 to 8 in S. spontaneum was caused by fissions of 2 ancestral chromosomes followed by translocations to 4 chromosomes. Surprisingly, 80% of nucleotide binding site-encoding genes associated with disease resistance are located in 4 rearranged chromosomes and 51% of those in rearranged regions. Resequencing of 64 S. spontaneum genomes identified balancing selection in rearranged regions, maintaining their diversity. Introgressed S. spontaneum chromosomes in modern sugarcanes are randomly distributed in AP85-441 genome, indicating random recombination among homologs in different S. spontaneum accessions. The allele-defined Saccharum genome offers new knowledge and resources to accelerate sugarcane improvement.

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July 19, 2019

The Dominant and Poorly Penetrant Phenotypes of Maize Unstable factor for orange1 Are Caused by DNA Methylation Changes at a Linked Transposon.

The maize (Zea mays) mutant Unstable factor for orange1 (Ufo1) has been implicated in the epigenetic modifications of pericarp color1 (p1), which regulates the production of the flavonoid pigments phlobaphenes. Here, we show that the ufo1 gene maps to a genetically recalcitrant region near the centromere of chromosome 10. Transcriptome analysis of Ufo1-1 mutant and wild-type plants identified a candidate gene in the mapping region using a comparative sequence-based approach. The candidate gene, GRMZM2G053177, is overexpressed by >45-fold in multiple tissues of Ufo1-1, explaining the dominance of Ufo1-1 and its phenotypes. In the mutant stock, GRMZM2G053177 has a unique transcript originating within a CACTA transposon inserted in its first intron, and it is missing the first four codons of the wild-type transcript. GRMZM2G053177 expression is regulated by the DNA methylation status of the CACTA transposon, explaining the incomplete penetrance and poor expressivity of Ufo1-1 Transgenic overexpression lines of GRMZM2G053177 (Ufo1-1) phenocopy the p1-induced pigmentation in coleoptiles, tassels, leaf sheaths, husks, pericarps, and cob glumes. Transcriptome analysis of Ufo1 versus wild-type tissues revealed changes in several pathways related to abiotic and biotic stress. Thus, this study addresses the enigma of Ufo1 identity in maize, which had gone unsolved for more than 50 years.© 2018 American Society of Plant Biologists. All rights reserved.

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July 7, 2019

Construction of a reference genetic map of Raphanus sativus based on genotyping by whole-genome resequencing.

This manuscript provides a genetic map of Raphanus sativus that has been used as a reference genetic map for an ongoing genome sequencing project. The map was constructed based on genotyping by whole-genome resequencing of mapping parents and F 2 population. Raphanus sativus is an annual vegetable crop species of the Brassicaceae family and is one of the key plants in the seed industry, especially in East Asia. Assessment of the R. sativus genome provides fundamental resources for crop improvement as well as the study of crop genome structure and evolution. With the goal of anchoring genome sequence assemblies of R. sativus cv. WK10039 whose genome has been sequenced onto the chromosomes, we developed a reference genetic map based on genotyping of two parents (maternal WK10039 and paternal WK10024) and 93 individuals of the F2 mapping population by whole-genome resequencing. To develop high-confidence genetic markers, ~83 Gb of parental lines and ~591 Gb of mapping population data were generated as Illumina 100 bp paired-end reads. High stringent sequence analysis of the reads mapped to the 344 Mb of genome sequence scaffolds identified a total of 16,282 SNPs and 150 PCR-based markers. Using a subset of the markers, a high-density genetic map was constructed from the analysis of 2,637 markers spanning 1,538 cM with 1,000 unique framework loci. The genetic markers integrated 295 Mb of genome sequences to the cytogenetically defined chromosome arms. Comparative analysis of the chromosome-anchored sequences with Arabidopsis thaliana and Brassica rapa revealed that the R. sativus genome has evident triplicated sub-genome blocks and the structure of gene space is highly similar to that of B. rapa. The genetic map developed in this study will serve as fundamental genomic resources for the study of R. sativus.

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July 7, 2019

Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions.

Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

The draft genome of Primula veris yields insights into the molecular basis of heterostyly.

The flowering plant Primula veris is a common spring blooming perennial that is widely cultivated throughout Europe. This species is an established model system in the study of the genetics, evolution, and ecology of heterostylous floral polymorphisms. Despite the long history of research focused on this and related species, the continued development of this system has been restricted due the absence of genomic and transcriptomic resources.We present here a de novo draft genome assembly of P. veris covering 301.8 Mb, or approximately 63% of the estimated 479.22 Mb genome, with an N50 contig size of 9.5 Kb, an N50 scaffold size of 164 Kb, and containing an estimated 19,507 genes. The results of a RADseq bulk segregant analysis allow for the confident identification of four genome scaffolds that are linked to the P. veris S-locus. RNAseq data from both P. veris and the closely related species P. vulgaris allow for the characterization of 113 candidate heterostyly genes that show significant floral morph-specific differential expression. One candidate gene of particular interest is a duplicated GLOBOSA homolog that may be unique to Primula (PveGLO2), and is completely silenced in L-morph flowers.The P. veris genome represents the first genome assembled from a heterostylous species, and thus provides an immensely important resource for future studies focused on the evolution and genetic dissection of heterostyly. As the first genome assembled from the Primulaceae, the P. veris genome will also facilitate the expanded application of phylogenomic methods in this diverse family and the eudicots as a whole.

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July 7, 2019

Chloroplast genome of Aconitum barbatum var. puberulum (Ranunculaceae) derived from CCS reads using the PacBio RS platform.

The chloroplast genome (cp genome) of Aconitum barbatum var. puberulum was sequenced using the third-generation sequencing platform based on the single-molecule real-time (SMRT) sequencing approach. To our knowledge, this is the first reported complete cp genome of Aconitum, and we anticipate that it will have great value for phylogenetic studies of the Ranunculaceae family. In total, 23,498 CCS reads and 20,685,462 base pairs were generated, the mean read length was 880 bp, and the longest read was 2,261 bp. Genome coverage of 100% was achieved with a mean coverage of 132× and no gaps. The accuracy of the assembled genome is 99.973%; the assembly was validated using Sanger sequencing of six selected genes from the cp genome. The complete cp genome of A. barbatum var. puberulum is 156,749 bp in length, including a large single-copy region of 87,630 bp and a small single-copy region of 16,941 bp separated by two inverted repeats of 26,089 bp. The cp genome contains 130 genes, including 84 protein-coding genes, 34 tRNA genes and eight rRNA genes. Four forward, five inverted and eight tandem repeats were identified. According to the SSR analysis, the longest poly structure is a 20-T repeat. Our results presented in this paper will facilitate the phylogenetic studies and molecular authentication on Aconitum.

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July 7, 2019

A draft genome of field pennycress (Thlaspi arvense) provides tools for the domestication of a new winter biofuel crop.

Field pennycress (Thlaspi arvense L.) is being domesticated as a new winter cover crop and biofuel species for the Midwestern United States that can be double-cropped between corn and soybeans. A genome sequence will enable the use of new technologies to make improvements in pennycress. To generate a draft genome, a hybrid sequencing approach was used to generate 47 Gb of DNA sequencing reads from both the Illumina and PacBio platforms. These reads were used to assemble 6,768 genomic scaffolds. The draft genome was annotated using the MAKER pipeline, which identified 27,390 predicted protein-coding genes, with almost all of these predicted peptides having significant sequence similarity to Arabidopsis proteins. A comprehensive analysis of pennycress gene homologues involved in glucosinolate biosynthesis, metabolism, and transport pathways revealed high sequence conservation compared with other Brassicaceae species, and helps validate the assembly of the pennycress gene space in this draft genome. Additional comparative genomic analyses indicate that the knowledge gained from years of basic Brassicaceae research will serve as a powerful tool for identifying gene targets whose manipulation can be predicted to result in improvements for pennycress. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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July 7, 2019

The genome of Dendrobium officinale illuminates the biology of the important traditional Chinese orchid herb.

Dendrobium officinale Kimura et Migo is a traditional Chinese orchid herb that has both ornamental value and a broad range of therapeutic effects. Here, we report the first de novo assembled 1.35 Gb genome sequences for D. officinale by combining the second-generation Illumina Hiseq 2000 and third-generation PacBio sequencing technologies. We found that orchids have a complete inflorescence gene set and have some specific inflorescence genes. We observed gene expansion in gene families related to fungus symbiosis and drought resistance. We analyzed biosynthesis pathways of medicinal components of D. officinale and found extensive duplication of SPS and SuSy genes, which are related to polysaccharide generation, and that the pathway of D. officinale alkaloid synthesis could be extended to generate 16-epivellosimine. The D. officinale genome assembly demonstrates a new approach to deciphering large complex genomes and, as an important orchid species and a traditional Chinese medicine, the D. officinale genome will facilitate future research on the evolution of orchid plants, as well as the study of medicinal components and potential genetic breeding of the dendrobe. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

Best practices in insect genome sequencing: What works and what doesn’t.

The last decade of decreasing DNA sequencing costs and proliferating sequencing services in core labs and companies has brought the de-novo genome sequencing and assembly of insect species within reach for many entomologists. However, sequence production alone is not enough to generate a high quality reference genome, and in many cases, poor planning can lead to extremely fragmented genome assemblies preventing high quality gene annotation and other desired analyses. Insect genomes can be problematic to assemble, due to combinations of high polymorphism, inability to breed for genome homozygocity, and small physical sizes limiting the quantity of DNA able to be isolated from a single individual. Recent advances in sequencing technology and assembly strategies are enabling a revolution for insect genome reference sequencing and assembly. Here we review historical and new genome sequencing and assembly strategies, with a particular focus on their application to arthropod genomes. We highlight both the need to design sequencing strategies for the requirements of the assembly software, and new long-read technologies that are enabling a return to traditional assembly approaches. Finally, we compare and contrast very cost effective short read draft genome strategies with the long read approaches that although entailing additional cost, bring a higher likelihood of success and the possibility of archival assembly qualities approaching that of finished genomes.

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July 7, 2019

Saccharina genomes provide novel insight into kelp biology.

Seaweeds are essential for marine ecosystems and have immense economic value. Here we present a comprehensive analysis of the draft genome of Saccharina japonica, one of the most economically important seaweeds. The 537-Mb assembled genomic sequence covered 98.5% of the estimated genome, and 18,733 protein-coding genes are predicted and annotated. Gene families related to cell wall synthesis, halogen concentration, development and defence systems were expanded. Functional diversification of the mannuronan C-5-epimerase and haloperoxidase gene families provides insight into the evolutionary adaptation of polysaccharide biosynthesis and iodine antioxidation. Additional sequencing of seven cultivars and nine wild individuals reveal that the genetic diversity within wild populations is greater than among cultivars. All of the cultivars are descendants of a wild S. japonica accession showing limited admixture with S. longissima. This study represents an important advance toward improving yields and economic traits in Saccharina and provides an invaluable resource for plant genome studies.

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July 7, 2019

Human gene-centered transcription factor networks for enhancers and disease variants.

Gene regulatory networks (GRNs) comprising interactions between transcription factors (TFs) and regulatory loci control development and physiology. Numerous disease-associated mutations have been identified, the vast majority residing in non-coding regions of the genome. As current GRN mapping methods test one TF at a time and require the use of cells harboring the mutation(s) of interest, they are not suitable to identify TFs that bind to wild-type and mutant loci. Here, we use gene-centered yeast one-hybrid (eY1H) assays to interrogate binding of 1,086 human TFs to 246 enhancers, as well as to 109 non-coding disease mutations. We detect both loss and gain of TF interactions with mutant loci that are concordant with target gene expression changes. This work establishes eY1H assays as a powerful addition to the toolkit of mapping human GRNs and for the high-throughput characterization of genomic variants that are rapidly being identified by genome-wide association studies. Copyright © 2015 Elsevier Inc. All rights reserved.

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July 7, 2019

It’s more than stamp collecting: how genome sequencing can unify biological research.

The availability of reference genome sequences, especially the human reference, has revolutionized the study of biology. However, while the genomes of some species have been fully sequenced, a wide range of biological problems still cannot be effectively studied for lack of genome sequence information. Here, I identify neglected areas of biology and describe how both targeted species sequencing and more broad taxonomic surveys of the tree of life can address important biological questions. I enumerate the significant benefits that would accrue from sequencing a broader range of taxa, as well as discuss the technical advances in sequencing and assembly methods that would allow for wide-ranging application of whole-genome analysis. Finally, I suggest that in addition to ‘big science’ survey initiatives to sequence the tree of life, a modified infrastructure-funding paradigm would better support reference genome sequence generation for research communities most in need. Copyright © 2015 Elsevier Ltd. All rights reserved.

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July 7, 2019

Complete genome sequence of endophytic nitrogen-fixing Klebsiella variicola strain DX120E.

Klebsiella variicola strain DX120E (=CGMCC 1.14935) is an endophytic nitrogen-fixing bacterium isolated from sugarcane crops grown in Guangxi, China and promotes sugarcane growth. Here we summarize the features of the strain DX120E and describe its complete genome sequence. The genome contains one circular chromosome and two plasmids, and contains 5,718,434 nucleotides with 57.1% GC content, 5,172 protein-coding genes, 25 rRNA genes, 87 tRNA genes, 7 ncRNA genes, 25 pseudo genes, and 2 CRISPR repeats.

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