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

We have analyzed the changes occurring in the chloroplast DNA (cpDNA) of taxa belonging to the Phaseolus vulgaris complex to help clarify relationships among species of this complex. Two restriction maps for 11 restriction enzymes comprising the whole chloroplast genome from a wild P. vulgaris and a wild P. coccineus accession were constructed. These maps allowed us to compare a total of 330 restriction sites between the two genomes in order to identify polymorphisms, assess the type of mutations detected, and identify regions of high variability. A region, located in the large single-copy region near the borders with the inverted repeats, accounted for a large portion of the variation. Most of the mutations detected were due to restriction sites gains or losses. Variable and conserved regions were then evaluated in 30 accessions belonging to taxa of the P. vulgaris complex. Phylogenetic analyses were made using parsimony methods. Conclusions obtained from such analyses were the following: (1) there was high cpDNA variability within P. coccineus but not in P. vulgaris. (2) P. coccineus subsp. glabellus showed a very distinct cpDNA type that strongly suggests that it actually belongs to a different but as yet undetermined section of the genus. Our cpDNA observations are supported by distinctive morphological traits and reproductive biology of this taxon. (3) In P. coccineus subsp. darwinianus (also classified as P. polyanthus), the cpDNA lineage was in disagreement with data obtained from nuclear markers and suggested a reticulated origin by hybridization between P. coccineus as the male parent and an ancestral P. polyanthus type, closely allied to P. vulgaris, as the seed parent. This initial cross was presumably followed by repeated backcrossing to P. coccineus. Our cpDNA studies illustrate the importance of molecular markers in elucidating phylogenetic relationships. They also indicate that accurate phylogenies will require analyses of both nuclear and cytoplasmic genomes. [ABSTRACT FROM AUTHOR]

Sorghum bicolor (L.) Moench is a significant grass crop globally, known for its genetic diversity. High quality genome sequences are needed to capture the diversity. We constructed high-quality, chromosome-level genome assemblies for two vital sorghum inbred lines, Tx2783 and RTx436. Through advanced single-molecule techniques, long-read sequencing and optical maps, we improved average sequence continuity 19-fold and 11-fold higher compared to existing Btx623 v3.0 reference genome and obtained 19 and 18 scaffolds (N50 of 25.6 and 14.4) for Tx2783 and RTx436, respectively. Our gene annotation efforts resulted in 29 612 protein-coding genes for the Tx2783 genome and 29 265 protein-coding genes for the RTx436 genome. Comparative analyses with 26 plant genomes which included 18 sorghum genomes and 8 outgroup species identified around 31 210 protein-coding gene families, with about 13 956 specific to sorghum. Using representative models from gene trees across the 18 sorghum genomes, a total of 72 579 pan-genes were identified, with 14% core, 60% softcore and 26% shell genes. We identified 99 genes in Tx2783 and 107 genes in RTx436 that showed functional enrichment specifically in binding and metabolic processes, as revealed by the GO enrichment Pearson Chi-Square test. We detected 36 potential large inversions in the comparison between the BTx623 Bionano map and the BTx623 v3.1 reference sequence. Strikingly, these inversions were notably absent when comparing Tx2783 or RTx436 with the BTx623 Bionano map. These inversion were mostly in the pericentromeric region which is known to have low complexity regions and harder to assemble and suggests the presence of potential artifacts in the public BTx623 reference assembly. Furthermore, in comparison to Tx2783, RTx436 exhibited 324 883 additional Single Nucleotide Polymorphisms (SNPs) and 16 506 more Insertions/Deletions (INDELs) when using BTx623 as the reference genome. We also characterized approximately 348 nucleotide-binding leucine-rich repeat (NLR) disease resistance genes in the two genomes. These high-quality genomes serve as valuable resources for discovering agronomic traits and structural variation studies., (Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics 2024.)

Weeds are attractive models for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Currently, a lack of genomic information precludes research to elucidate the genetic basis of rapid adaptation for important traits like herbicide resistance and stress tolerance and the effect of evolutionary mechanisms on wild populations. The International Weed Genomics Consortium is a collaborative group of scientists focused on developing genomic resources to impact research into sustainable, effective weed control methods and to provide insights about stress tolerance and adaptation to assist crop breeding., (© 2024. The Author(s).)

Gray leaf spot (GLS) caused by Cercospora zeina or C. zeae-maydis is a major maize disease throughout the world. Although more than 100 QTLs resistant against GLS have been identified, very few of them have been cloned. Here, we identified a major resistance QTL against GLS, qRglsSB, explaining 58.42% phenotypic variation in SB12×SA101 BC 1 F 1 population. By fine-mapping, it was narrowed down into a 928 kb region. By using transgenic lines, mutants and complementation lines, it was confirmed that the ZmWAK02 gene, encoding an RD wall-associated kinase, is the responsible gene in qRglsSB resistant against GLS. The introgression of the ZmWAK02 gene into hybrid lines significantly improves their grain yield in the presence of GLS pressure and does not reduce their grain yield in the absence of GLS. In summary, we cloned a gene, ZmWAK02, conferring large effect of GLS resistance and confirmed its great value in maize breeding., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Background: Understanding genome organization and evolution is important for species involved in transmission of human diseases, such as mosquitoes. Anophelinae and Culicinae subfamilies of mosquitoes show striking differences in genome sizes, sex chromosome arrangements, behavior, and ability to transmit pathogens. However, the genomic basis of these differences is not fully understood., Methods: In this study, we used a combination of advanced genome technologies such as Oxford Nanopore Technology sequencing, Hi-C scaffolding, Bionano, and cytogenetic mapping to develop an improved chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus., Results: We then used this assembly to annotate odorant receptors, odorant binding proteins, and transposable elements. A genomic region containing male-specific sequences on chromosome 1 and a polymorphic inversion on chromosome 3 were identified in the Cx. quinquefasciatus genome. In addition, the genome of Cx. quinquefasciatus was compared with the genomes of other mosquitoes such as malaria vectors An. coluzzi and An. albimanus, and the vector of arboviruses Ae. aegypti. Our work confirms significant expansion of the two chemosensory gene families in Cx. quinquefasciatus, as well as a significant increase and relocation of the transposable elements in both Cx. quinquefasciatus and Ae. aegypti relative to the Anophelines. Phylogenetic analysis clarifies the divergence time between the mosquito species. Our study provides new insights into chromosomal evolution in mosquitoes and finds that the X chromosome of Anophelinae and the sex-determining chromosome 1 of Culicinae have a significantly higher rate of evolution than autosomes., Conclusion: The improved Cx. quinquefasciatus genome assembly uncovered new details of mosquito genome evolution and has the potential to speed up the development of novel vector control strategies., (© 2024. The Author(s).)

Complete, gapless telomere-to-telomere chromosome assemblies are a prerequisite for comprehensively investigating the architecture of complex regions, like centromeres or telomeres and removing uncertainties in the order, spacing, and orientation of genes. Using complementary genomics technologies and assembly algorithms, we developed highly contiguous, nearly gapless, genome assemblies for two economically important soybean [Glycine max (L.) Merr] cultivars (Williams 82 and Lee). The centromeres were distinctly annotated on all the chromosomes of both assemblies. We further found that the canonical telomeric repeats were present at the telomeres of all chromosomes of both Williams 82 and Lee genomes. A total of 10 chromosomes in Williams 82 and eight in Lee were entirely reconstructed in single contigs without any gap. Using the combination of ab initio prediction, protein homology, and transcriptome evidence, we identified 58,287 and 56,725 protein-coding genes in Williams 82 and Lee, respectively. The genome assemblies and annotations will serve as a valuable resource for studying soybean genomics and genetics and accelerating soybean improvement., (© 2023 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

The house fly, Musca domestica, is a pest of livestock, transmits pathogens of human diseases, and is a model organism in multiple biological research areas. The first house fly genome assembly was published in 2014 and has been of tremendous use to the community of house fly biologists, but that genome is discontiguous and incomplete by contemporary standards. To improve the house fly reference genome, we sequenced, assembled, and annotated the house fly genome using improved techniques and technologies that were not available at the time of the original genome sequencing project. The new genome assembly is substantially more contiguous and complete than the previous genome. The new genome assembly has a scaffold N50 of 12.46 Mb, which is a 50-fold improvement over the previous assembly. In addition, the new genome assembly is within 1% of the estimated genome size based on flow cytometry, whereas the previous assembly was missing nearly one-third of the predicted genome sequence. The improved genome assembly has much more contiguous scaffolds containing large gene families. To provide an example of the benefit of the new genome, we used it to investigate tandemly arrayed immune gene families. The new contiguous assembly of these loci provides a clearer picture of the regulation of the expression of immune genes, and it leads to new insights into the selection pressures that shape their evolution., (© 2023 Wiley Periodicals LLC.)

High-quality reference genome assemblies, representative of global heterotic patterns, offer an ideal platform to accurately characterize and utilize genetic variation in the primary gene pool of hybrid crops. Here we report three platinum grade de-novo, near gap-free, chromosome-level reference genome assemblies from the active breeding germplasm in pearl millet with a high degree of contiguity, completeness, and accuracy. An improved Tift genome (Tift23D 2 B 1 -P1-P5) assembly has a contig N50 ~ 7,000-fold (126 Mb) compared to the previous version and better alignment in centromeric regions. Comparative genome analyses of these three lines clearly demonstrate a high level of collinearity and multiple structural variations, including inversions greater than 1 Mb. Differential genes in improved Tift genome are enriched for serine O-acetyltransferase and glycerol-3-phosphate metabolic process which play an important role in improving the nutritional quality of seed protein and disease resistance in plants, respectively. Multiple marker-trait associations are identified for a range of agronomic traits, including grain yield through genome-wide association study. Improved genome assemblies and marker resources developed in this study provide a comprehensive framework/platform for future applications such as marker-assisted selection of mono/oligogenic traits as well as whole-genome prediction and haplotype-based breeding of complex traits., (© 2023. Springer Nature Limited.)

Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today., (© 2023. The Author(s).)

Plant pathogens cause significant crop loss worldwide, and new resistance genes deployed to combat diseases can be overcome quickly. Understanding the existing resistance gene diversity within the germplasm of major crops, such as maize, is crucial for the development of new disease-resistant varieties. We analysed the nucleotide-binding leucine-rich repeat receptors (NLRs) of 26 recently sequenced diverse founder lines from the maize nested association mapping (NAM) population and compared them to the R gene complement present in a wild relative of maize, Zea luxurians. We found that NLRs in both species contain a large diversity of atypical integrated domains, including many domains that have not previously been found in the NLRs of other species. Additionally, the single Z. luxurians genome was found to have greater integrated atypical domain diversity than all 26 NAM founder lines combined, indicating that this species may represent a rich source of novel resistance genes. NLRs were also found to have very high sequence diversity and presence-absence variation among the NAM founder lines, with a large NLR cluster on Chr10 representing a diversity hotspot. Additionally, NLRs were shown to be mobile within maize genomes, with several putative interchromosomal translocations identified., (© 2023 Corteva Agriscience. Molecular Plant Pathology published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology.)

Northern corn leaf blight, caused by the fungal pathogen Exserohilum turcicum, is a major disease of maize. The first major locus conferring resistance to E. turcicum race 0, Ht1, was identified over 50 years ago, but the underlying gene has remained unknown. We employed a map-based cloning strategy to identify the Ht1 causal gene, which was found to be a coiled-coil nucleotide-binding, leucine-rich repeat (NLR) gene, which we named PH4GP-Ht1. Transgenic testing confirmed that introducing the native PH4GP-Ht1 sequence to a susceptible maize variety resulted in resistance to E. turcicum race 0. A survey of the maize nested association mapping genomes revealed that susceptible Ht1 alleles had very low to no expression of the gene. Overexpression of the susceptible B73 allele, however, did not result in resistant plants, indicating that sequence variations may underlie the difference between resistant and susceptible phenotypes. Modelling of the PH4GP-Ht1 protein indicated that it has structural homology to the Arabidopsis NLR resistance gene ZAR1, and probably forms a similar homopentamer structure following activation. RNA sequencing data from an infection time course revealed that 1 week after inoculation there was a threefold reduction in fungal biomass in the PH4GP-Ht1 transgenic plants compared to wild-type plants. Furthermore, PH4GP-Ht1 transgenics had significantly more inoculation-responsive differentially expressed genes than wild-type plants, with enrichment seen in genes associated with both defence and photosynthesis. These results demonstrate that the NLR PH4GP-Ht1 is the causal gene underlying Ht1, which represents a different mode of action compared to the previously reported wall-associated kinase northern corn leaf blight resistance gene Htn1/Ht2/Ht3., (© 2022 PIONEER HI-BRED INTERNATIONAL, INC. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Poa pratensis, commonly known as Kentucky bluegrass, is a popular cool-season grass species used as turf in lawns and recreation areas globally. Despite its substantial economic value, a reference genome had not previously been assembled due to the genome's relatively large size and biological complexity that includes apomixis, polyploidy, and interspecific hybridization. We report here a fortuitous de novo assembly and annotation of a P. pratensis genome. Instead of sequencing the genome of a C4 grass, we accidentally sampled and sequenced tissue from a weedy P. pratensis whose stolon was intertwined with that of the C4 grass. The draft assembly consists of 6.09 Gbp with an N50 scaffold length of 65.1 Mbp, and a total of 118 scaffolds, generated using PacBio long reads and Bionano optical map technology. We annotated 256K gene models and found 58% of the genome to be composed of transposable elements. To demonstrate the applicability of the reference genome, we evaluated population structure and estimated genetic diversity in P. pratensis collected from three North American prairies, two in Manitoba, Canada and one in Colorado, USA. Our results support previous studies that found high genetic diversity and population structure within the species. The reference genome and annotation will be an important resource for turfgrass breeding and study of bluegrasses., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Understanding and exploiting genetic diversity is a key factor for the productive and stable production of rice. Here, we utilize 73 high-quality genomes that encompass the subpopulation structure of Asian rice (Oryza sativa), plus the genomes of two wild relatives (O. rufipogon and O. punctata), to build a pan-genome inversion index of 1769 non-redundant inversions that span an average of ~29% of the O. sativa cv. Nipponbare reference genome sequence. Using this index, we estimate an inversion rate of ~700 inversions per million years in Asian rice, which is 16 to 50 times higher than previously estimated for plants. Detailed analyses of these inversions show evidence of their effects on gene expression, recombination rate, and linkage disequilibrium. Our study uncovers the prevalence and scale of large inversions (≥100 bp) across the pan-genome of Asian rice and hints at their largely unexplored role in functional biology and crop performance., (© 2023. The Author(s).)

High-quality genome assemblies are characterized by high-sequence contiguity, completeness, and a low error rate, thus providing the basis for a wide array of studies focusing on natural species ecology, conservation, evolution, and population genomics. To provide this valuable resource for conservation projects and comparative genomics studies on gyrfalcon (Falco rusticolus), we sequenced and assembled the genome of this species using third-generation sequencing strategies and optical maps. Here, we describe a highly contiguous and complete genome assembly comprising 20 scaffolds and 13 contigs with a total size of 1.193 Gbp, including 8,064 complete Benchmarking Universal Single-Copy Orthologs (BUSCOs) of the total 8,338 BUSCO groups present in the library aves_odb10. Of these BUSCO genes, 96.7% were complete, 96.1% were present as a single copy, and 0.6% were duplicated. Furthermore, 0.8% of BUSCO genes were fragmented and 2.5% (210) were missing. A de novo search for transposable elements (TEs) identified 5,716 TEs that masked 7.61% of the F. rusticolus genome assembly when combined with publicly available TE collections. Long interspersed nuclear elements, in particular, the element Chicken-repeat 1 (CR1), were the most abundant TEs in the F. rusticolus genome. A de novo first-pass gene annotation was performed using 293,349 PacBio Iso-Seq transcripts and 496,195 transcripts derived from the assembly of 42,429,525 Illumina PE RNA-seq reads. In all, 19,602 putative genes, of which 59.31% were functionally characterized and associated with Gene Ontology terms, were annotated. A comparison of the gyrfalcon genome assembly with the publicly available assemblies of the domestic chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and hummingbird (Calypte anna) revealed several genome rearrangements. In particular, nine putative chromosome fusions were identified in the gyrfalcon genome assembly compared with those in the G. gallus genome assembly. This genome assembly, its annotation for TEs and genes, and the comparative analyses presented, complement and strength the base of high-quality genome assemblies and associated resources available for comparative studies focusing on the evolution, ecology, and conservation of Aves., Competing Interests: Conflicts of interest None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Genetics Society of America.)

Although long-read sequencing can often enable chromosome-level reconstruction of genomes, it is still unclear how one can routinely obtain gapless assemblies. In the model plant Arabidopsis thaliana, other than the reference accession Col-0, all other accessions de novo assembled with long-reads until now have used PacBio continuous long reads (CLR). Although these assemblies sometimes achieved chromosome-arm level contigs, they inevitably broke near the centromeres, excluding megabases of DNA from analysis in pan-genome projects. Since PacBio high-fidelity (HiFi) reads circumvent the high error rate of CLR technologies, albeit at the expense of read length, we compared a CLR assembly of accession Eyach15-2 to HiFi assemblies of the same sample. The use of five different assemblers starting from subsampled data allowed us to evaluate the impact of coverage and read length. We found that centromeres and rDNA clusters are responsible for 71% of contig breaks in the CLR scaffolds, while relatively short stretches of GA/TC repeats are at the core of >85% of the unfilled gaps in our best HiFi assemblies. Since the HiFi technology consistently enabled us to reconstruct gapless centromeres and 5S rDNA clusters, we demonstrate the value of the approach by comparing these previously inaccessible regions of the genome between the Eyach15-2 accession and the reference accession Col-0., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Fruit development has been central in the evolution and domestication of flowering plants. In common bean (Phaseolus vulgaris), the principal global grain legume staple, two main production categories are distinguished by fibre deposition in pods: dry beans, with fibrous, stringy pods; and stringless snap/green beans, with reduced fibre deposition, which frequently revert to the ancestral stringy state. Here, we identify genetic and developmental patterns associated with pod fibre deposition. Transcriptional, anatomical, epigenetic and genetic regulation of pod strings were explored through RNA-seq, RT-qPCR, fluorescence microscopy, bisulfite sequencing and whole-genome sequencing. Overexpression of the INDEHISCENT ('PvIND') orthologue was observed in stringless types compared with isogenic stringy lines, associated with overspecification of weak dehiscence-zone cells throughout the pod vascular sheath. No differences in DNA methylation were correlated with this phenotype. Nonstringy varieties showed a tandemly direct duplicated PvIND and a Ty1-copia retrotransposon inserted between the two repeats. These sequence features are lost during pod reversion and are predictive of pod phenotype in diverse materials, supporting their role in PvIND overexpression and reversible string phenotype. Our results give insight into reversible gain-of-function mutations and possible genetic solutions to the reversion problem, of considerable economic value for green bean production., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Severe price spikes of the major grain commodities and rapid expansion of cultivated area in the past two decades are symptoms of a severely stressed global food supply. Scientific discovery and improved agricultural productivity are needed and are enabled by unencumbered access to, and use of, genetic sequence data. In the same way the world witnessed rapid development of vaccines for COVID-19, genetic sequence data afford enormous opportunities to improve crop production. In addition to an enabling regulatory environment that allowed for the sharing of genetic sequence data, robust funding fostered the rapid development of coronavirus diagnostics and COVID-19 vaccines. A similar level of commitment, collaboration, and cooperation is needed for agriculture., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The views and opinions expressed in this paper are those of the authors and not necessarily the views and opinions of the United States Agency for International Development., (© 2022 The Authors.)

Southern corn rust (SCR), caused by the fungal pathogen Puccinia polysora, is a major threat to maize production worldwide. Efficient breeding and deployment of resistant hybrids are key to achieving durable control of SCR. Here, we report the molecular cloning and characterization of RppC, which encodes an NLR-type immune receptor and is responsible for a major SCR resistance quantitative trait locus. Furthermore, we identified the corresponding avirulence effector, AvrRppC, which is secreted by P. polysora and triggers RppC-mediated resistance. Allelic variation of AvrRppC directly determines the effectiveness of RppC-mediated resistance, indicating that monitoring of AvrRppC variants in the field can guide the rational deployment of RppC-containing hybrids in maize production. Currently, RppC is the most frequently deployed SCR resistance gene in China, and a better understanding of its mode of action is critical for extending its durability., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

Phytophthora root and stem rot caused by P. sojae is a destructive soybean soil-borne disease found worldwide. Discovery of genes conferring broad-spectrum resistance to the pathogen is a need to prevent the outbreak of the disease. Here, we show that soybean Rps11 is a 27.7-kb nucleotide-binding site-leucine-rich repeat (NBS-LRR or NLR) gene conferring broad-spectrum resistance to the pathogen. Rps11 is located in a genomic region harboring a cluster of large NLR genes of a single origin in soybean, and is derived from rounds of unequal recombination. Such events result in promoter fusion and LRR expansion that may contribute to the broad resistance spectrum. The NLR gene cluster exhibits drastic structural diversification among phylogenetically representative varieties, including gene copy number variation ranging from five to 23 copies, and absence of allelic copies of Rps11 in any of the non-Rps11-donor varieties examined, exemplifying innovative evolution of NLR genes and NLR gene clusters., (© 2021. The Author(s).)

We report de novo genome assemblies, transcriptomes, annotations, and methylomes for the 26 inbreds that serve as the founders for the maize nested association mapping population. The number of pan-genes in these diverse genomes exceeds 103,000, with approximately a third found across all genotypes. The results demonstrate that the ancient tetraploid character of maize continues to degrade by fractionation to the present day. Excellent contiguity over repeat arrays and complete annotation of centromeres revealed additional variation in major cytological landmarks. We show that combining structural variation with single-nucleotide polymorphisms can improve the power of quantitative mapping studies. We also document variation at the level of DNA methylation and demonstrate that unmethylated regions are enriched for cis-regulatory elements that contribute to phenotypic variation., (Copyright © 2021, American Association for the Advancement of Science.)

Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine ( Vitis vinifera L. ssp. vinifera ). In contrast, all extant wild Vitis species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species V. cinerea and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the Vitis genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

CRISPR-Cas is a powerful double-strand-break technology with wide-ranging applications from gene discovery to commercial product development. Thus far, this tool has been almost exclusively used for gene knockouts and deletions, with a few examples of gene edits and targeted gene insertions. Here, we demonstrate the application of CRISPR-Cas9 technology to mediate targeted 75.5-Mb pericentric inversion in chromosome 2 in one of the elite maize inbred lines from Corteva Agriscience. This inversion unlocks a large chromosomal region containing substantial genetic variance for recombination, thus providing opportunities for the development of new maize varieties with improved phenotypes.

Chromosome level assemblies are accumulating in various taxonomic groups including mosquitoes. However, even in the few reference-quality mosquito assemblies, a significant portion of the heterochromatic regions including telomeres remain unresolved. Here we produce a de novo assembly of the New World malaria mosquito, Anopheles albimanus by integrating Oxford Nanopore sequencing, Illumina, Hi-C and optical mapping. This 172.6 Mbps female assembly, which we call AalbS3, is obtained by scaffolding polished large contigs (contig N50 = 13.7 Mbps) into three chromosomes. All chromosome arms end with telomeric repeats, which is the first in mosquito assemblies and represents a significant step toward the completion of a genome assembly. These telomeres consist of tandem repeats of a novel 30-32 bp Telomeric Repeat Unit (TRU) and are confirmed by analyzing the termini of long reads and through both chromosomal in situ hybridization and a Bal 31 sensitivity assay. The AalbS3 assembly included previously uncharacterized centromeric and rDNA clusters and more than doubled the content of transposable elements and other repetitive sequences. This telomere-to-telomere assembly, although still containing gaps, represents a significant step toward resolving biologically important but previously hidden genomic components. The comparison of different scaffolding methods will also inform future efforts to obtain reference-quality genomes for other mosquito species., (Copyright © 2020 Compton et al.)

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.

Improvements in long-read data and scaffolding technologies have enabled rapid generation of reference-quality assemblies for complex genomes. Still, an assessment of critical sequence depth and read length is important for allocating limited resources. To this end, we have generated eight assemblies for the complex genome of the maize inbred line NC358 using PacBio datasets ranging from 20 to 75 × genomic depth and with N50 subread lengths of 11-21 kb. Assemblies with ≤30 × depth and N50 subread length of 11 kb are highly fragmented, with even low-copy genic regions showing degradation at 20 × depth. Distinct sequence-quality thresholds are observed for complete assembly of genes, transposable elements, and highly repetitive genomic features such as telomeres, heterochromatic knobs, and centromeres. In addition, we show high-quality optical maps can dramatically improve contiguity in even our most fragmented base assembly. This study provides a useful resource allocation reference to the community as long-read technologies continue to mature.

As the human population grows from 7.8 billion to 10 billion over the next 30 years, breeders must do everything possible to create crops that are highly productive and nutritious, while simultaneously having less of an environmental footprint. Rice will play a critical role in meeting this demand and thus, knowledge of the full repertoire of genetic diversity that exists in germplasm banks across the globe is required. To meet this demand, we describe the generation, validation and preliminary analyses of transposable element and long-range structural variation content of 12 near-gap-free reference genome sequences (RefSeqs) from representatives of 12 of 15 subpopulations of cultivated Asian rice. When combined with 4 existing RefSeqs, that represent the 3 remaining rice subpopulations and the largest admixed population, this collection of 16 Platinum Standard RefSeqs (PSRefSeq) can be used as a template to map resequencing data to detect virtually all standing natural variation that exists in the pan-genome of cultivated Asian rice.

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.

RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

The MinION is a portable single-molecule DNA sequencing instrument that was released by Oxford Nanopore Technologies in 2014, producing long sequencing reads by measuring changes in ionic flow when single-stranded DNA molecules translocate through the pores. While MinION long reads have an error rate substantially higher than the ones produced by short-read sequencing technologies, they can generate de novo assemblies of microbial genomes, after an initial correction step that includes alignment of Illumina sequencing data or detection of overlaps between Oxford Nanopore reads to improve accuracy. In this study, MinION reads were generated from the multi-chromosome genome of Agrobacterium tumefaciens strain LBA4404. Errors in the consensus two-directional (sense and antisense) "2D" sequences were first characterized by way of comparison with an internal reference assembly. Both Illumina-based correction and self-correction were performed and the resulting corrected reads assembled into high-quality hybrid and non-hybrid assemblies. Corrected read datasets and assemblies were subsequently compared. The results shown here indicate that both hybrid and non-hybrid methods can be used to assemble Oxford Nanopore reads into informative multi-chromosome assemblies, each with slightly different outcomes in terms of contiguity and accuracy.

The present study was undertaken to provide the foundation for development of genome-scale resources for the fathead minnow (Pimephales promelas), an important model organism widely used in both aquatic toxicology research and regulatory testing. The authors report on the first sequencing and 2 draft assemblies for the reference genome of this species. Approximately 120× sequence coverage was achieved via Illumina sequencing of a combination of paired-end, mate-pair, and fosmid libraries. Evaluation and comparison of these assemblies demonstrate that they are of sufficient quality to be useful for genome-enabled studies, with 418 of 458 (91%) conserved eukaryotic genes mapping to at least 1 of the assemblies. In addition to its immediate utility, the present work provides a strong foundation on which to build further refinements of a reference genome for the fathead minnow., (© 2015 SETAC.)

Heterodera glycines, the soybean cyst nematode, is the number one pathogen of soybean (Glycine max). This nematode infects soybean roots and forms an elaborate feeding site in the vascular cylinder. H. glycines produces an arsenal of effector proteins in the secretory esophageal gland cells. More than 60 H. glycines candidate effectors were identified in previous gland-cell-mining projects. However, it is likely that additional candidate effectors remained unidentified. With the goal of identifying remaining H. glycines candidate effectors, we constructed and sequenced a large gland cell cDNA library resulting in 11,814 expressed sequence tags. After bioinformatic filtering for candidate effectors using a number of criteria, in situ hybridizations were performed in H. glycines whole-mount specimens to identify candidate effectors whose mRNA exclusively accumulated in the esophageal gland cells, which is a hallmark of many nematode effectors. This approach resulted in the identification of 18 new H. glycines esophageal gland-cell-specific candidate effectors. Of these candidate effectors, 11 sequences were pioneers without similarities to known proteins while 7 sequences had similarities to functionally annotated proteins in databases. These putative homologies provided the bases for the development of hypotheses about potential functions in the parasitism process.

Plant height has long been an important agronomic trait in maize breeding. Many plant height QTLs have been reported, but few of these have been cloned. In this study, a major plant height QTL, qph1, was mapped to a 1.6kb interval in Brachytic2 (Br2) coding sequence on maize chromosome 1. A naturally occurring rare SNP in qph1, which resulted in an amino acid substitution, was validated as the causative mutation. QPH1 protein is located in the plasma membrane and polar auxin transport is impaired in the short near-isogenic line RIL88(qph1). Allelism testing showed that the SNP variant in qph1 reduces longitudinal cell number and decreases plant height by 20% in RIL88(qph1) compared to RIL88(QPH1), and is milder than known br2 mutant alleles. The effect of qph1 on plant height is significant and has no or a slight influence on yield in four F2 backgrounds and in six pairs of single-cross hybrids. Moreover, qph1 could reduce plant height when heterozygous, allowing it to be easily employed in maize breeding. Thus, a less-severe allele of a known dwarf mutant explains part of the quantitative variation for plant height and has great potential in maize improvement., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Phyllotaxy describes the geometric arrangement of leaves and is important for plant productivity. Auxin is well known to regulate phyllotactic patterns via PIN1-dependent auxin polar transport, and studies of maize (Zea mays) aberrant phyllotaxy1 (abph1) mutants suggest the importance of auxin and cytokinin signaling for control of phyllotaxy. However, whether additional regulators control these patterns is poorly understood. Here, we report a new dominant maize mutant, Aberrant phyllotaxy2 (Abph2), in which the shoot meristems are enlarged and the phyllotactic pattern switches from alternate to decussate. Map-based cloning revealed that the Abph2 mutation was caused by transposition of a glutaredoxin gene, MALE STERILE CONVERTED ANTHER1 (MSCA1), which gained an altered expression pattern in Abph2 mutant embryos. msca1 loss-of-function mutants have reduced meristem size and revealed a novel function of glutaredoxins in meristem growth. In addition, MSCA1 interacts with a TGA transcription factor, FASCIATED EAR4, suggesting a novel regulatory module for regulating shoot meristem size., (© 2015 American Society of Plant Biologists. All rights reserved.)

tassel-less1 (tls1) is a classical maize (Zea mays) inflorescence mutant. Homozygous mutant plants have no tassels or very small tassels, and ear development is also impaired. Using a positional cloning approach, ZmNIP3;1 (a NOD26-like intrinsic protein) was identified as the candidate gene for tls1. The ZmNIP3;1 gene is completely deleted in the tls1 mutant genome. Two Mutator-insertional TUSC alleles of ZmNIP3;1 exhibited tls1-like phenotypes, and allelism tests confirmed that the tls1 gene encodes ZmNIP3;1. Transgenic plants with an RNA interference (RNAi) construct to down-regulate ZmNIP3;1 also showed tls1-like phenotypes, further demonstrating that TLS1 is ZmNIP3;1. Sequence analysis suggests that ZmNIP3;1 is a boron channel protein. Foliar application of boron could rescue the tls1 phenotypes and restore the normal tassel and ear development. Gene expression analysis indicated that in comparison with that of the wild type or tls1 plants treated with boron, the transition from the vegetative to reproductive phase or the development of the floral meristem is impaired in the shoot apical meristem of the tls1 mutant plants. It is concluded that the tls1 mutant phenotypes are caused by impaired boron transport, and boron is essential for inflorescence development in maize., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)