Whole-genome sequencing of allotetraploid bermudagrass reveals the origin of Cynodon and candidate genes for salt tolerance.

Bermudagrass (Cynodon dactylon) is a globally distributed, extensively used warm-season turf and forage grass with high tolerance to salinity and drought stress in alkaline environments. However, the origin of the species and genetic mechanisms for salinity tolerance in the species are basically unknown. Accordingly, we set out to study evolution divergence events in the Cynodon genome and to identify genes for salinity tolerance. We developed a 604.0 Mb chromosome-level polyploid genome sequence for bermudagrass 'A12359' (n = 18). The C. dactylon genome comprises 2 complete sets of homoeologous chromosomes, each with approximately 30 000 genes, and most genes are conserved as syntenic pairs. Phylogenetic study showed that the initial Cynodon species diverged from Oropetium thomaeum approximately 19.7-25.4 million years ago (Mya), the A and B subgenomes of C. dactylon diverged approximately 6.3-9.1 Mya, and the bermudagrass polyploidization event occurred 1.5 Mya on the African continent. Moreover, we identified 82 candidate genes associated with seven agronomic traits using a genome-wide association study, and three single-nucleotide polymorphisms were strongly associated with three salt resistance genes: RAP2-2, CNG channels, and F14D7.1. These genes may be associated with enhanced bermudagrass salt tolerance. These bermudagrass genomic resources, when integrated, may provide fundamental insights into evolution of diploid and tetraploid genomes and enhance the efficacy of comparative genomics in studying salt tolerance in Cynodon.

Oryza glumaepatula: A wild relative to improve drought tolerance in cultivated rice.

Developing drought-resistant rice (Oryza sativa, L.) is essential for improving field productivity, especially in rain-fed areas affected by climate change. Wild relatives of rice are potential sources for drought-resistant traits. Therefore, we compared root growth and drought response among 22 wild Oryza species, from which Oryza glumaepatula was selected as a promising source for further exploration. A geographically diverse panel of 69 O. glumaepatula accessions was then screened for drought stress-related traits, and 6 of these accessions showed lower shoot dry weight (SDW) reduction, greater percentage of deep roots, and lower stomatal density (STO) under drought than the drought tolerant O. sativa variety, Sahbhagi dhan. Based on whole-genome resequencing of all 69 O. glumaepatula accessions and variant calling to a high-quality O. glumaepatula reference genome, we detected multiple genomic loci colocating for SDW, root dry weight at 30 to 45 cm depth, and STO in consecutive drought trials. Geo-referencing indicated that the potential drought donors originated in flood-prone locations, corroborating previous hypotheses about the coexistence of flood and drought tolerance within individual Oryza genomes. These findings present potential donor accessions, traits, and genomic loci from an AA genome wild relative of rice that, together with the recently developed reference genome, may be useful for further introgression of drought tolerance into the O. sativa backgrounds.

Pan-genome inversion index reveals evolutionary insights into the subpopulation structure of Asian rice.

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.

Long-read genome sequencing of bread wheat facilitates disease resistance gene cloning.

The cloning of agronomically important genes from large, complex crop genomes remains challenging. Here we generate a 14.7 gigabase chromosome-scale assembly of the South African bread wheat (Triticum aestivum) cultivar Kariega by combining high-fidelity long reads, optical mapping and chromosome conformation capture. The resulting assembly is an order of magnitude more contiguous than previous wheat assemblies. Kariega shows durable resistance to the devastating fungal stripe rust disease1. We identified the race-specific disease resistance gene Yr27, which encodes an intracellular immune receptor, to be a major contributor to this resistance. Yr27 is allelic to the leaf rust resistance gene Lr13; the Yr27 and Lr13 proteins show 97% sequence identity2,3. Our results demonstrate the feasibility of generating chromosome-scale wheat assemblies to clone genes, and exemplify that highly similar alleles of a single-copy gene can confer resistance to different pathogens, which might provide a basis for engineering Yr27 alleles with multiple recognition specificities in the future.

De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes.

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.

Genetic dissection of natural variation in oilseed traits of camelina by whole-genome resequencing and QTL mapping.

Camelina [Camelina sativa (L.) Crantz] is an oilseed crop in the Brassicaceae family that is currently being developed as a source of bioenergy and healthy fatty acids. To facilitate modern breeding efforts through marker-assisted selection and biotechnology, we evaluated genetic variation among a worldwide collection of 222 camelina accessions. We performed whole-genome resequencing to obtain single nucleotide polymorphism (SNP) markers and to analyze genomic diversity. We also conducted phenotypic field evaluations in two consecutive seasons for variations in key agronomic traits related to oilseed production such as seed size, oil content (OC), fatty acid composition, and flowering time. We determined the population structure of the camelina accessions using 161,301 SNPs. Further, we identified quantitative trait loci (QTL) and candidate genes controlling the above field-evaluated traits by genome-wide association studies (GWAS) complemented with linkage mapping using a recombinant inbred line (RIL) population. Characterization of the natural variation at the genome and phenotypic levels provides valuable resources to camelina genetic studies and crop improvement. The QTL and candidate genes should assist in breeding of advanced camelina varieties that can be integrated into the cropping systems for the production of high yield of oils of desired fatty acid composition.

A chromosome-scale assembly of allotetraploid Brassica juncea (AABB) elucidates comparative architecture of the A and B genomes.

Brassica juncea (AABB), commonly referred to as mustard, is a natural allopolyploid of two diploid species-B. rapa (AA) and B. nigra (BB). We report a highly contiguous genome assembly of an oleiferous type of B. juncea variety Varuna, an archetypical Indian gene pool line of mustard, with ~100× PacBio single-molecule real-time (SMRT) long reads providing contigs with an N50 value of >5 Mb. Contigs were corrected for the misassemblies and scaffolded with BioNano optical mapping. We also assembled a draft genome of B. nigra (BB) variety Sangam using Illumina short-read sequencing and Oxford Nanopore long reads and used it to validate the assembly of the B genome of B. juncea. Two different linkage maps of B. juncea, containing a large number of genotyping-by-sequencing markers, were developed and used to anchor scaffolds/contigs to the 18 linkage groups of the species. The resulting chromosome-scale assembly of B. juncea Varuna is a significant improvement over the previous draft assembly of B. juncea Tumida, a vegetable type of mustard. The assembled genome was characterized for transposons, centromeric repeats, gene content and gene block associations. In comparison to the A genome, the B genome contains a significantly higher content of LTR/Gypsy retrotransposons, distinct centromeric repeats and a large number of B. nigra specific gene clusters that break the gene collinearity between the A and the B genomes. The B. juncea Varuna assembly will be of major value to the breeding work on oleiferous types of mustard that are grown extensively in south Asia and elsewhere.

Highly accurate long-read HiFi sequencing data for five complex genomes.

The PacBio® HiFi sequencing method yields highly accurate long-read sequencing datasets with read lengths averaging 10-25 kb and accuracies greater than 99.5%. These accurate long reads can be used to improve results for complex applications such as single nucleotide and structural variant detection, genome assembly, assembly of difficult polyploid or highly repetitive genomes, and assembly of metagenomes. Currently, there is a need for sample data sets to both evaluate the benefits of these long accurate reads as well as for development of bioinformatic tools including genome assemblers, variant callers, and haplotyping algorithms. We present deep coverage HiFi datasets for five complex samples including the two inbred model genomes Mus musculus and Zea mays, as well as two complex genomes, octoploid Fragaria × ananassa and the diploid anuran Rana muscosa. Additionally, we release sequence data from a mock metagenome community. The datasets reported here can be used without restriction to develop new algorithms and explore complex genome structure and evolution. Data were generated on the PacBio Sequel II System.

Gapless assembly of maize chromosomes using long-read technologies.

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.

Evolution and Diversity of the Wild Rice Oryza officinalis Complex, across Continents, Genome Types, and Ploidy Levels.

The Oryza officinalis complex is the largest species group in Oryza, with more than nine species from four continents, and is a tertiary gene pool that can be exploited in breeding programs for the improvement of cultivated rice. Most diploid and tetraploid members of this group have a C genome. Using a new reference C genome for the diploid species O. officinalis, and draft genomes for two other C genome diploid species Oryza eichingeri and Oryza rhizomatis, we examine the influence of transposable elements on genome structure and provide a detailed phylogeny and evolutionary history of the Oryza C genomes. The O. officinalis genome is 1.6 times larger than the A genome of cultivated Oryza sativa, mostly due to proliferation of Gypsy type long-terminal repeat transposable elements, but overall syntenic relationships are maintained with other Oryza genomes (A, B, and F). Draft genome assemblies of the two other C genome diploid species, Oryza eichingeri and Oryza rhizomatis, and short-read resequencing of a series of other C genome species and accessions reveal that after the divergence of the C genome progenitor, there was still a substantial degree of variation within the C genome species through proliferation and loss of both DNA and long-terminal repeat transposable elements. We provide a detailed phylogeny and evolutionary history of the Oryza C genomes and a genomic resource for the exploitation of the Oryza tertiary gene pool.

A willow sex chromosome reveals convergent evolution of complex palindromic repeats.

BACKGROUND: Sex chromosomes have arisen independently in a wide variety of species, yet they share common characteristics, including the presence of suppressed recombination surrounding sex determination loci. Mammalian sex chromosomes contain multiple palindromic repeats across the non-recombining region that show sequence conservation through gene conversion and contain genes that are crucial for sexual reproduction. In plants, it is not clear if palindromic repeats play a role in maintaining sequence conservation in the absence of homologous recombination. RESULTS: Here we present the first evidence of large palindromic structures in a plant sex chromosome, based on a highly contiguous assembly of the W chromosome of the dioecious shrub Salix purpurea. The W chromosome has an expanded number of genes due to transpositions from autosomes. It also contains two consecutive palindromes that span a region of 200 kb, with conspicuous 20-kb stretches of highly conserved sequences among the four arms that show evidence of gene conversion. Four genes in the palindrome are homologous to genes in the sex determination regions of the closely related genus Populus, which is located on a different chromosome. These genes show distinct, floral-biased expression patterns compared to paralogous copies on autosomes. CONCLUSION: The presence of palindromes in sex chromosomes of mammals and plants highlights the intrinsic importance of these features in adaptive evolution in the absence of recombination. Convergent evolution is driving both the independent establishment of sex chromosomes as well as their fine-scale sequence structure.

Genome sequence of the model rice variety KitaakeX.

BACKGROUND: The availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to transform and propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species. RESULTS: Here, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic variations between KitaakeX and Nipponbare (ssp. japonica), and 2,785,991 variations between KitaakeX and Zhenshan97 (ssp. indica). We also compared Kitaake resequencing reads to the KitaakeX assembly and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate rice functional genomics. CONCLUSIONS: The high quality, de novo assembly of the KitaakeX genome will serve as a useful reference genome for rice and will accelerate functional genomics studies of rice and other species.

Genome-wide association mapping of date palm fruit traits.

Date palms (Phoenix dactylifera) are an important fruit crop of arid regions of the Middle East and North Africa. Despite its importance, few genomic resources exist for date palms, hampering evolutionary genomic studies of this perennial species. Here we report an improved long-read genome assembly for P. dactylifera that is 772.3 Mb in length, with contig N50 of 897.2 Kb, and use this to perform genome-wide association studies (GWAS) of the sex determining region and 21 fruit traits. We find a fruit color GWAS at the R2R3-MYB transcription factor VIRESCENS gene and identify functional alleles that include a retrotransposon insertion and start codon mutation. We also find a GWAS peak for sugar composition spanning deletion polymorphisms in multiple linked invertase genes. MYB transcription factors and invertase are implicated in fruit color and sugar composition in other crops, demonstrating the importance of parallel evolution in the evolutionary diversification of domesticated species.

The Cardamine hirsuta genome offers insight into the evolution of morphological diversity.

Finding causal relationships between genotypic and phenotypic variation is a key focus of evolutionary biology, human genetics and plant breeding. To identify genome-wide patterns underlying trait diversity, we assembled a high-quality reference genome of Cardamine hirsuta, a close relative of the model plant Arabidopsis thaliana. We combined comparative genome and transcriptome analyses with the experimental tools available in C. hirsuta to investigate gene function and phenotypic diversification. Our findings highlight the prevalent role of transcription factors and tandem gene duplications in morphological evolution. We identified a specific role for the transcriptional regulators PLETHORA5/7 in shaping leaf diversity and link tandem gene duplication with differential gene expression in the explosive seed pod of C. hirsuta. Our work highlights the value of comparative approaches in genetically tractable species to understand the genetic basis for evolutionary change.

Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63.

Asian cultivated rice consists of two subspecies: Oryza sativa subsp. indica and O. sativa subsp. japonica Despite the fact that indica rice accounts for over 70% of total rice production worldwide and is genetically much more diverse, a high-quality reference genome for indica rice has yet to be published. We conducted map-based sequencing of two indica rice lines, Zhenshan 97 (ZS97) and Minghui 63 (MH63), which represent the two major varietal groups of the indica subspecies and are the parents of an elite Chinese hybrid. The genome sequences were assembled into 237 (ZS97) and 181 (MH63) contigs, with an accuracy >99.99%, and covered 90.6% and 93.2% of their estimated genome sizes. Comparative analyses of these two indica genomes uncovered surprising structural differences, especially with respect to inversions, translocations, presence/absence variations, and segmental duplications. Approximately 42% of nontransposable element related genes were identical between the two genomes. Transcriptome analysis of three tissues showed that 1,059-2,217 more genes were expressed in the hybrid than in the parents and that the expressed genes in the hybrid were much more diverse due to their divergence between the parental genomes. The public availability of two high-quality reference genomes for the indica subspecies of rice will have large-ranging implications for plant biology and crop genetic improvement.

Global genomic diversity of Oryza sativa varieties revealed by comparative physical mapping.

Bacterial artificial chromosome (BAC) physical maps embedding a large number of BAC end sequences (BESs) were generated for Oryza sativa ssp. indica varieties Minghui 63 (MH63) and Zhenshan 97 (ZS97) and were compared with the genome sequences of O. sativa spp. japonica cv. Nipponbare and O. sativa ssp. indica cv. 93-11. The comparisons exhibited substantial diversities in terms of large structural variations and small substitutions and indels. Genome-wide BAC-sized and contig-sized structural variations were detected, and the shared variations were analyzed. In the expansion regions of the Nipponbare reference sequence, in comparison to the MH63 and ZS97 physical maps, as well as to the previously constructed 93-11 physical map, the amounts and types of the repeat contents, and the outputs of gene ontology analysis, were significantly different from those of the whole genome. Using the physical maps of four wild Oryza species from OMAP (http://www.omap.org) as a control, we detected many conserved and divergent regions related to the evolution process of O. sativa. Between the BESs of MH63 and ZS97 and the two reference sequences, a total of 1532 polymorphic simple sequence repeats (SSRs), 71,383 SNPs, 1767 multiple nucleotide polymorphisms, 6340 insertions, and 9137 deletions were identified. This study provides independent whole-genome resources for intra- and intersubspecies comparisons and functional genomics studies in O. sativa. Both the comparative physical maps and the GBrowse, which integrated the QTL and molecular markers from GRAMENE (http://www.gramene.org) with our physical maps and analysis results, are open to the public through our Web site (http://gresource.hzau.edu.cn/resource/resource.html).

Comparative BAC-based physical mapping of Oryza sativa ssp. indica var. 93-11 and evaluation of the two rice reference sequence assemblies.

Reference sequences are sequences that are used for public consultation, and therefore must be of high quality. Using the whole-genome shotgun/next-generation sequencing approach, many genome sequences of complex higher plants have been generated in recent years, and are generally considered reference sequences. However, none of these sequences has been experimentally evaluated at the whole-genome sequence assembly level. Rice has a relatively simple plant genome, and the genome sequences for its two sub-species obtained using different sequencing approaches were published approximately 10 years ago. This provides a unique system for a case study to evaluate the qualities and utilities of published plant genome sequences. We constructed a robust BAC physical map embedding a large number of BAC end sequences forrice variety 93-11. Through BAC end sequence alignments and tri-assembly comparisons of the 93-11 physical map and the two reference sequences, we found that the Nipponbare reference sequence generated using the clone-by-clone approach has a high quality but still contains small artifact inversions and missing sequences. In contrast, the 93-11 reference sequence generated using the whole-genome shotgun approach contains many large and varied assembly errors, such as inversions, duplications and translocations, as well as missing sequences. The 93-11 physical map provides an invaluable resource for evaluation and improvements toward completion of both Nipponbare and 93-11 reference sequences.

Genomic resources for gene discovery, functional genome annotation, and evolutionary studies of maize and its close relatives.

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.

Oil palm genome sequence reveals divergence of interfertile species in Old and New worlds.

Oil palm is the most productive oil-bearing crop. Although it is planted on only 5% of the total world vegetable oil acreage, palm oil accounts for 33% of vegetable oil and 45% of edible oil worldwide, but increased cultivation competes with dwindling rainforest reserves. We report the 1.8-gigabase (Gb) genome sequence of the African oil palm Elaeis guineensis, the predominant source of worldwide oil production. A total of 1.535 Gb of assembled sequence and transcriptome data from 30 tissue types were used to predict at least 34,802 genes, including oil biosynthesis genes and homologues of WRINKLED1 (WRI1), and other transcriptional regulators, which are highly expressed in the kernel. We also report the draft sequence of the South American oil palm Elaeis oleifera, which has the same number of chromosomes (2n = 32) and produces fertile interspecific hybrids with E. guineensis but seems to have diverged in the New World. Segmental duplications of chromosome arms define the palaeotetraploid origin of palm trees. The oil palm sequence enables the discovery of genes for important traits as well as somaclonal epigenetic alterations that restrict the use of clones in commercial plantings, and should therefore help to achieve sustainability for biofuels and edible oils, reducing the rainforest footprint of this tropical plantation crop.