The draft genome of a wild barley genotype reveals its enrichment in genes related to biotic and abiotic stresses compared to cultivated barley

Wild barley (Hordeum spontaneum) is the progenitor of cultivated barley (Hordeum vulgare) and provides a rich source of genetic variations for barley improvement. Currently, the genome sequences of wild barley and its differences with cultivated barley remain unclear. In this study, we report a high‐quality draft assembly of wild barley accession (AWCS276; henceforth named as WB1), which consists of 4.28 Gb genome and 36 395 high‐confidence protein‐coding genes. BUSCO analysis revealed that the assembly included full lengths of 95.3% of the 956 single‐copy plant genes, illustrating that the gene‐containing regions have been well assembled. By comparing with the genome of the cultivated genotype Morex, it is inferred that the WB1 genome contains more genes involved in resistance and tolerance to biotic and abiotic stresses. The presence of the numerous WB1‐specific genes indicates that, in addition to enhance allele diversity for genes already existing in the cultigen, exploiting the wild barley taxon in breeding should also allow the incorporation of novel genes. Furthermore, high levels of genetic variation in the pericentromeric regions were detected in chromosomes 3H and 5H between the wild and cultivated genotypes, which may be the results of domestication. This H. spontaneum draft genome assembly will help to accelerate wild barley research and be an invaluable resource for barley improvement and comparative genomics research.     

Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ)

Next‐generation whole‐genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence‐based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost‐efficient establishment of powerful genomic information for many species.     

High quality genome of Erigeron breviscapus provides a reference for herbal plants in Asteraceae

Erigeron breviscapus is an important medicinal plant in Compositae and the first species to realize the whole process from the decoding of the draft genome sequence to scutellarin biosynthesis in yeast. However, the previous low‐quality genome assembly has hindered the optimization of candidate genes involved in scutellarin synthesis and the development of molecular‐assisted breeding based on the genome. Here, the E. breviscapus genome was updated using PacBio RSII sequencing data and Hi‐C data, and increased in size from 1.2 Gb to 1.43 Gb, with a scaffold N50 of 156.82 Mb and contig N50 of 140.95 kb, and a total of 43,514 protein‐coding genes were obtained and oriented onto nine pseudo‐chromosomes, thus becoming the third plant species assembled to chromosome level after sunflower and lettuce in Compositae. Fourteen genes with evidence for positive selection were identified and found to be related to leaf morphology, flowering and secondary metabolism. The number of genes in some gene families involved in flavonoid biosynthesis in E. breviscapus have been significantly expanded. In particular, additional candidate genes involved in scutellarin biosynthesis, such as flavonoid‐7‐O‐glucuronosyltransferase genes (F7GATs) were identified using updated genome. In addition, three candidate genes encoding indole‐3‐pyruvate monooxygenase YUCCA2 (YUC2), serine carboxypeptidase‐like 18 (SCPL18), and F‐box protein (FBP), respectively, were identified to be probably related to leaf development and flowering by resequencing 99 individuals. These results provided a substantial genetic basis for improving agronomic and quality traits of E. breviscapus, and provided a platform for improving other draft genome assemblies to chromosome‐level.     

A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.)

Cicer arietinum L. (chickpea) is the third most important food legume crop. We have generated the draft sequence of a desi‐type chickpea genome using next‐generation sequencing platforms, bacterial artificial chromosome end sequences and a genetic map. The 520‐Mb assembly covers 70% of the predicted 740‐Mb genome length, and more than 80% of the gene space. Genome analysis predicts the presence of 27 571 genes and 210 Mb as repeat elements. The gene expression analysis performed using 274 million RNA‐Seq reads identified several tissue‐specific and stress‐responsive genes. Although segmental duplicated blocks are observed, the chickpea genome does not exhibit any indication of recent whole‐genome duplication. Nucleotide diversity analysis provides an assessment of a narrow genetic base within the chickpea cultivars. We have developed a resource for genetic markers by comparing the genome sequences of one wild and three cultivated chickpea genotypes. The draft genome sequence is expected to facilitate genetic enhancement and breeding to develop improved chickpea varieties.     

Unlocking the secondary gene‐pool of barley with next‐generation sequencing

Crop wild relatives (CWR) provide an important source of allelic diversity for any given crop plant species for counteracting the erosion of genetic diversity caused by domestication and elite breeding bottlenecks. Hordeum bulbosum L. is representing the secondary gene pool of the genus Hordeum. It has been used as a source of genetic introgressions for improving elite barley germplasm (Hordeum vulgare L.). However, genetic introgressions from H. bulbosum have yet not been broadly applied, due to a lack of suitable molecular tools for locating, characterizing, and decreasing by recombination and marker‐assisted backcrossing the size of introgressed segments. We applied next‐generation sequencing (NGS) based strategies for unlocking genetic diversity of three diploid introgression lines of cultivated barley containing chromosomal segments of its close relative H. bulbosum. Firstly, exome capture‐based (re)‐sequencing revealed large numbers of single nucleotide polymorphisms (SNPs) enabling the precise allocation of H. bulbosum introgressions. This SNP resource was further exploited by designing a custom multiplex SNP genotyping assay. Secondly, two‐enzyme‐based genotyping‐by‐sequencing (GBS) was employed to allocate the introgressed H. bulbosum segments and to genotype a mapping population. Both methods provided fast and reliable detection and mapping of the introgressed segments and enabled the identification of recombinant plants. Thus, the utilization of H. bulbosum as a resource of natural genetic diversity in barley crop improvement will be greatly facilitated by these tools in the future.     

Whole‐exome targeted sequencing of the uncharacterized pine genome

The large genome size of many species hinders the development and application of genomic tools to study them. For instance, loblolly pine (Pinus taeda L.), an ecologically and economically important conifer, has a large and yet uncharacterized genome of 21.7 Gbp. To characterize the pine genome, we performed exome capture and sequencing of 14 729 genes derived from an assembly of expressed sequence tags. Efficiency of sequence capture was evaluated and shown to be similar across samples with increasing levels of complexity, including haploid cDNA, haploid genomic DNA and diploid genomic DNA. However, this efficiency was severely reduced for probes that overlapped multiple exons, presumably because intron sequences hindered probe:exon hybridizations. Such regions could not be entirely avoided during probe design, because of the lack of a reference sequence. To improve the throughput and reduce the cost of sequence capture, a method to multiplex the analysis of up to eight samples was developed. Sequence data showed that multiplexed capture was reproducible among 24 haploid samples, and can be applied for high‐throughput analysis of targeted genes in large populations. Captured sequences were de novo assembled, resulting in 11 396 expanded and annotated gene models, significantly improving the knowledge about the pine gene space. Interspecific capture was also evaluated with over 98% of all probes designed from P. taeda that were efficient in sequence capture, were also suitable for analysis of the related species Pinus elliottii Engelm.     

Draft genome assembly and annotation of Glycyrrhiza uralensis,a medicinal legume

Chinese liquorice/licorice (Glycyrrhiza uralensis) is a leguminous plant species whose roots and rhizomes have been widely used as a herbal medicine and natural sweetener. Whole‐genome sequencing is essential for gene discovery studies and molecular breeding in liquorice. Here, we report a draft assembly of the approximately 379‐Mb whole‐genome sequence of strain 308‐19 of G. uralensis; this assembly contains 34 445 predicted protein‐coding genes. Comparative analyses suggested well‐conserved genomic components and collinearity of gene loci (synteny) between the genome of liquorice and those of other legumes such as Medicago and chickpea. We observed that three genes involved in isoflavonoid biosynthesis, namely, 2‐hydroxyisoflavanone synthase (CYP93C), 2,7,4′‐trihydroxyisoflavanone 4′‐O‐methyltransferase/isoflavone 4′‐O‐methyltransferase (HI4OMT) and isoflavone‐7‐O‐methyltransferase (7‐IOMT) formed a cluster on the scaffold of the liquorice genome and showed conserved microsynteny with Medicago and chickpea. Based on the liquorice genome annotation, we predicted genes in the P450 and UDP‐dependent glycosyltransferase (UGT) superfamilies, some of which are involved in triterpenoid saponin biosynthesis, and characterised their gene expression with the reference genome sequence. The genome sequencing and its annotations provide an essential resource for liquorice improvement through molecular breeding and the discovery of useful genes for engineering bioactive components through synthetic biology approaches.     

Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes

Hierarchical shotgun sequencing remains the method of choice for assembling high‐quality reference sequences of complex plant genomes. The efficient exploitation of current high‐throughput technologies and powerful computational facilities for large‐insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole‐genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high‐quality assemblies of a large number of clones to assemble map‐based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.     

Assembly and annotation of a draft genome sequence for Glycine latifolia,a perennial wild relative of soybean

Glycine latifolia (Benth.) Newell & Hymowitz (2n = 40), one of the 27 wild perennial relatives of soybean, possesses genetic diversity and agronomically favorable traits that are lacking in soybean. Here, we report the 939‐Mb draft genome assembly of G. latifolia (PI 559298) using exclusively linked‐reads sequenced from a single Chromium library. We organized scaffolds into 20 chromosome‐scale pseudomolecules utilizing two genetic maps and the Glycine max (L.) Merr. genome sequence. High copy numbers of putative 91‐bp centromere‐specific tandem repeats were observed in consecutive blocks within predicted pericentromeric regions on several pseudomolecules. No 92‐bp putative centromeric repeats, which are abundant in G. max, were detected in G. latifolia or Glycine tomentella. Annotation of the assembled genome and subsequent filtering yielded a high confidence gene set of 54 475 protein‐coding loci. In comparative analysis with five legume species, genes related to defense responses were significantly overrepresented in Glycine‐specific orthologous gene families. A total of 304 putative nucleotide‐binding site (NBS)‐leucine‐rich‐repeat (LRR) genes were identified in this genome assembly. Different from other legume species, we observed a scarcity of TIR‐NBS‐LRR genes in G. latifolia. The G. latifolia genome was also predicted to contain genes encoding 367 LRR‐receptor‐like kinases, a family of proteins involved in basal defense responses and responses to abiotic stress. The genome sequence and annotation of G. latifolia provides a valuable source of alternative alleles and novel genes to facilitate soybean improvement. This study also highlights the efficacy and cost‐effectiveness of the application of Chromium linked‐reads in diploid plant genome de novo assembly.     

Chromosome‐level genome assembly of the predator Propylea japonica to understand its tolerance to insecticides and high temperatures

The ladybird beetle Propylea japonica is an important natural enemy in agro‐ecological systems. Studies on the strong tolerance of P. japonica to high temperatures and insecticides, and its population and phenotype diversity have recently increased. However, abundant genome resources for obtaining insights into stress‐resistance mechanisms and genetic intra‐species diversity for P. japonica are lacking. Here, we constructed the P. japonica genome maps using Pacific Bioscience (PacBio) and Illumina sequencing technologies. The genome size was 850.90 Mb with a contig N50 of 813.13 kb. The Hi‐C sequence data were used to upgrade draft genome assemblies; 4,777 contigs were assembled to 10 chromosomes; and the final draft genome assembly was 803.93 Mb with a contig N50 of 813.98 kb and a scaffold N50 of 100.34 Mb. Approximately 495.38 Mb of repeated sequences was annotated. The 18,018 protein‐coding genes were predicted, of which 95.78% were functionally annotated, and 1,407 genes were species‐specific. The phylogenetic analysis showed that P. japonica diverged from the ancestor of Anoplophora glabripennis and Tribolium castaneum ~ 236.21 million years ago. We detected that some important gene families involved in detoxification of pesticides and tolerance to heat stress were expanded in P. japonica, especially cytochrome P450 and Hsp70 genes. Overall, the high‐quality draft genome sequence of P. japonica will provide invaluable resource for understanding the molecular mechanisms of stress resistance and will facilitate the research on population genetics, evolution and phylogeny of Coccinellidae. This genome will also provide new avenues for conserving the diversity of predator insects.     

Sequencing of 15 622 gene‐bearing BACs clarifies the gene‐dense regions of the barley genome

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole‐genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene‐containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical‐mapped gene‐bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene‐enriched BACs and are characterized by high recombination rates, there are also gene‐dense regions with suppressed recombination. We made use of published map‐anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D‐genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley–Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map‐based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene‐dense but low recombination is particularly relevant.     

The Ficus erecta genome aids Ceratocystis canker resistance breeding in common fig (F. carica)

Ficus erecta, a wild relative of the common fig (F. carica), is a donor of Ceratocystis canker resistance in fig breeding programmes. Interspecific hybridization followed by recurrent backcrossing is an effective method to transfer the resistance trait from wild to cultivated fig. However, this process is time consuming and labour intensive for trees, especially for gynodioecious plants such as fig. In this study, genome resources were developed for F. erecta to facilitate fig breeding programmes. The genome sequence of F. erecta was determined using single‐molecule real‐time sequencing technology. The resultant assembly spanned 331.6 Mb with 538 contigs and an N50 length of 1.9 Mb, from which 51 806 high‐confidence genes were predicted. Pseudomolecule sequences corresponding to the chromosomes of F. erecta were established with a genetic map based on single nucleotide polymorphisms from double‐digest restriction‐site‐associated DNA sequencing. Subsequent linkage analysis and whole‐genome resequencing identified a candidate gene for the Ceratocystis canker resistance trait. Genome‐wide genotyping analysis enabled the selection of female lines that possessed resistance and effective elimination of the donor genome from the progeny. The genome resources provided in this study will accelerate and enhance disease‐resistance breeding programmes in fig.     

De novo assembly of a wild pear (Pyrus betuleafolia) genome

China is the origin and evolutionary centre of Oriental pears. Pyrus betuleafolia is a wild species native to China and distributed in the northern region, and it is widely used as rootstock. Here, we report the de novo assembly of the genome of P. betuleafolia‐Shanxi Duli using an integrated strategy that combines PacBio sequencing, BioNano mapping and chromosome conformation capture (Hi‐C) sequencing. The genome assembly size was 532.7 Mb, with a contig N50 of 1.57 Mb. A total of 59 552 protein‐coding genes and 247.4 Mb of repetitive sequences were annotated for this genome. The expansion genes in P. betuleafolia were significantly enriched in secondary metabolism, which may account for the organism's considerable environmental adaptability. An alignment analysis of orthologous genes showed that fruit size, sugar metabolism and transport, and photosynthetic efficiency were positively selected in Oriental pear during domestication. A total of 573 nucleotide‐binding site (NBS)‐type resistance gene analogues (RGAs) were identified in the P. betuleafolia genome, 150 of which are TIR‐NBS‐LRR (TNL)‐type genes, which represented the greatest number of TNL‐type genes among the published Rosaceae genomes and explained the strong disease resistance of this wild species. The study of flavour metabolism‐related genes showed that the anthocyanidin reductase (ANR) metabolic pathway affected the astringency of pear fruit and that sorbitol transporter (SOT) transmembrane transport may be the main factor affecting the accumulation of soluble organic matter. This high‐quality P. betuleafolia genome provides a valuable resource for the utilization of wild pear in fundamental pear studies and breeding.     

Improved genome assembly provides new insights into genome evolution in a desert poplar (Populus euphratica)

Populus euphratica is well adapted to extreme desert environments and is an important model species for elucidating the mechanisms of abiotic stress resistance in trees. The current assembly of P. euphratica genome is highly fragmented with many gaps and errors, thereby impeding downstream applications. Here, we report an improved chromosome‐level reference genome of P. euphratica (v2.0) using single‐molecule sequencing and chromosome conformation capture (Hi‐C) technologies. Relative to the previous reference genome, our assembly represents a nearly 60‐fold improvement in contiguity, with a scaffold N50 size of 28.59 Mb. Using this genome, we have found that extensive expansion of Gypsy elements in P. euphratica led to its rapid increase in genome size compared to any other Salicaceae species studied to date, and potentially contributed to adaptive divergence driven by insertions near genes involved in stress tolerance. We also detected a wide range of unique structural rearrangements in P. euphratica, including 2,549 translocations, 454 inversions, 121 tandem and 14 segmental duplications. Several key genes likely to be involved in tolerance to abiotic stress were identified within these regions. This high‐quality genome represents a valuable resource for poplar breeding and genetic improvement in the future, as well as comparative genomic analysis with other Salicaceae species.