High Repeat Content Research Articles

The genomes of all lungfish inform on genome expansion and tetrapod evolution.

The genomes of living lungfishes can inform on the molecular-developmental basis of the Devonian sarcopterygian fish-tetrapod transition. We de novo sequenced the genomes of the African (Protopterus annectens) and South American lungfishes (Lepidosiren paradoxa). The Lepidosiren genome (about 91 Gb, roughly 30 times the human genome) is the largest animal genome sequenced so far and more than twice the size of the Australian (Neoceratodus forsteri)1 and African2 lungfishes owing to enlarged intergenic regions and introns with high repeat content (about 90%). All lungfish genomes continue to expand as some transposable elements (TEs) are still active today. In particular, Lepidosiren's genome grew extremely fast during the past 100 million years (Myr), adding the equivalent of one human genome every 10 Myr. This massive genome expansion seems to be related to a reduction of PIWI-interacting RNAs and C2H2 zinc-finger and Krüppel-associated box (KRAB)-domain protein genes that suppress TE expansions. Although TE abundance facilitates chromosomal rearrangements, lungfish chromosomes still conservatively reflect the ur-tetrapod karyotype. Neoceratodus' limb-like fins still resemble those of their extinct relatives and remained phenotypically static for about 100 Myr. We show that the secondary loss of limb-like appendages in the Lepidosiren-Protopterus ancestor was probably due to loss of sonic hedgehog limb-specific enhancers.

Lake Baikal amphipods and their genomes, great and small.

Endemic amphipods (Crustacea: Amphipoda) of Lake Baikal represent an outstanding example of large species flocks occupying a wide range of ecological niches and originating from a handful of ancestor species. Their development took place at a restricted territory and is thus open for comprehensive research. Such examples provide unique opportunities for studying behavioral, anatomic, or physiological adaptations in multiple combinations of environmental conditions and thus attract considerable attention. The existing taxonomies of this group list over 350 species and subspecies, which, according to the molecular phylogenetic studies of marker genes, full transcriptomes and mitochondrial genomes, originated from at least two introductions into the lake. The studies of allozymes and marker genes have revealed a significant cryptic diversity in Baikal amphipods, as well as a large variance in genetic diversity within some morphological species. Crossing experiments conducted so far for two morphological species suggest that the differences in the mitochondrial marker (cytochrome c oxidase subunit I gene) can potentially be applied for making predictions about reproductive isolation. For about one-tenth of the Baikal amphipod species, nuclear genome sizes and chromosome numbers are known. While genome sizes vary within one order of magnitude, the karyotypes are relatively stable (2n = 52 for most species studied). Moreover, analysis of the diversity of repeated sequences in nuclear genomes showed significant between-species differences. Studies of mitochondrial genomes revealed some unusual features, such as variation in length and gene order, as well as duplications of tRNA genes, some of which also underwent remolding (change in anticodon specificity due to point mutations). The next important steps should be (i) the assembly of whole genomes for different species of Baikal amphipods, which is at the moment hampered by complicated genome structures with high repeat content, and (ii) updating species taxonomy taking into account all the data.

Remarkably High Repeat Content in the Genomes of Sparrows: The Importance of Genome Assembly Completeness for Transposable Element Discovery.

Transposable elements (TE) play critical roles in shaping genome evolution. Highly repetitive TE sequences are also a major source of assembly gaps making it difficult to fully understand the impact of these elements on host genomes. The increased capacity of long-read sequencing technologies to span highly repetitive regions promises to provide new insights into patterns of TE activity across diverse taxa. Here we report the generation of highly contiguous reference genomes using PacBio long-read and Omni-C technologies for three species of Passerellidae sparrow. We compared these assemblies to three chromosome-level sparrow assemblies and nine other sparrow assemblies generated using a variety of short- and long-read technologies. All long-read based assemblies were longer (range: 1.12 to 1.41 Gb) than short-read assemblies (0.91 to 1.08 Gb) and assembly length was strongly correlated with the amount of repeat content. Repeat content for Bell's sparrow (31.2% of genome) was the highest level ever reported within the order Passeriformes, which comprises over half of avian diversity. The highest levels of repeat content (79.2% to 93.7%) were found on the W chromosome relative to other regions of the genome. Finally, we show that proliferation of different TE classes varied even among species with similar levels of repeat content. These patterns support a dynamic model of TE expansion and contraction even in a clade where TEs were once thought to be fairly depauperate and static. Our work highlights how the resolution of difficult-to-assemble regions of the genome with new sequencing technologies promises to transform our understanding of avian genome evolution.

Telomere-to-Telomere Genome Assembly of Tibetan Medicinal Mushroom Ganoderma leucocontextum and the First Copia Centromeric Retrotransposon in Macro-Fungi Genome.

A complete telomere-to-telomere (T2T) genome has been a longstanding goal in the field of genomic research. By integrating high-coverage and precise long-read sequencing data using multiple assembly strategies, we present here the first T2T gap-free genome assembly of Ganoderma leucocontextum strain GL72, a Tibetan medicinal mushroom. The T2T genome, with a size of 46.69 Mb, consists 13 complete nuclear chromosomes and typical telomeric repeats (CCCTAA)n were detected at both ends of 13 chromosomes. The high mapping rate, uniform genome coverage, a complete BUSCOs of 99.7%, and base accuracy exceeding 99.999% indicate that this assembly represents the highest level of completeness and quality. Regions characterized by distinct structural attributes, including highest Hi-C interaction intensity, high repeat content, decreased gene density, low GC content, and minimal or no transcription levels across all chromosomes may represent potential centromeres. Sequence analysis revealed the first Copia centromeric retrotransposon in macro-fungi genome. Phylogenomic analysis identified that G. leucocontextum and G. tsugae diverged from the other Ganoderma species approximately 9.8-17.9 MYA. The prediction of secondary metabolic clusters confirmed the capability of this fungus to produce a substantial quantity of metabolites. This T2T gap-free genome will contribute to the genomic 'dark matter' elucidation and server as a great reference for genetics, genomics, and evolutionary studies of G. leucocontextum.

Chromosome-Level Genome Assembly of the Blacktail Brush Lizard, Urosaurus nigricaudus, Reveals Dosage Compensation in an Endemic Lizard.

Urosaurus nigricaudus is a phrynosomatid lizard endemic to the Baja California Peninsula in Mexico. This work presents a chromosome-level genome assembly and annotation from a male individual. We used PacBio long reads and HiRise scaffolding to generate a high-quality genomic assembly of 1.87 Gb distributed in 327 scaffolds, with an N50 of 279 Mb and an L50 of 3. Approximately 98.4% of the genome is contained in 14 scaffolds, with 6 large scaffolds (334-127 Mb) representing macrochromosomes and 8 small scaffolds (63-22 Mb) representing microchromosomes. Using standard gene modeling and transcriptomic data, we predicted 17,902 protein-coding genes on the genome. The repeat content is characterized by a large proportion of long interspersed nuclear elements that are relatively old. Synteny analysis revealed some microchromosomes with high repeat content are more prone to rearrangements but that both macro- and microchromosomes are well conserved across reptiles. We identified scaffold 14 as the X chromosome. This microchromosome presents perfect dosage compensation where the single X of males has the same expression levels as two X chromosomes in females. Finally, we estimated the effective population size for U. nigricaudus was extremely low, which may reflect a reduction in polymorphism related to it becoming a peninsular endemic.

Improved haplotype resolution of highly duplicated MHC genes in a long-read genome assembly using MiSeq amplicons.

Long-read sequencing offers a great improvement in the assembly of complex genomic regions, such as the major histocompatibility complex (MHC) region, which can contain both tandemly duplicated MHC genes (paralogs) and high repeat content. The MHC genes have expanded in passerine birds, resulting in numerous MHC paralogs, with relatively high sequence similarity, making the assembly of the MHC region challenging even with long-read sequencing. In addition, MHC genes show rather high sequence divergence between alleles, making diploid-aware assemblers incorrectly classify haplotypes from the same locus as sequences originating from different genomic regions. Consequently, the number of MHC paralogs can easily be over- or underestimated in long-read assemblies. We therefore set out to verify the MHC diversity in an original and a haplotype-purged long-read assembly of one great reed warbler Acrocephalus arundinaceus individual (the focal individual) by using Illumina MiSeq amplicon sequencing. Single exons, representing MHC class I (MHC-I) and class IIB (MHC-IIB) alleles, were sequenced in the focal individual and mapped to the annotated MHC alleles in the original long-read genome assembly. Eighty-four percent of the annotated MHC-I alleles in the original long-read genome assembly were detected using 55% of the amplicon alleles and likewise, 78% of the annotated MHC-IIB alleles were detected using 61% of the amplicon alleles, indicating an incomplete annotation of MHC genes. In the haploid genome assembly, each MHC-IIB gene should be represented by one allele. The parental origin of the MHC-IIB amplicon alleles in the focal individual was determined by sequencing MHC-IIB in its parents. Two of five larger scaffolds, containing 6-19 MHC-IIB paralogs, had a maternal and paternal origin, respectively, as well as a high nucleotide similarity, which suggests that these scaffolds had been incorrectly assigned as belonging to different loci in the genome rather than as alternate haplotypes of the same locus. Therefore, the number of MHC-IIB paralogs was overestimated in the haploid genome assembly. Based on our findings we propose amplicon sequencing as a suitable complement to long-read sequencing for independent validation of the number of paralogs in general and for haplotype inference in multigene families in particular.

Small cardamom genome: development and utilization of microsatellite markers from a draft genome sequence of Elettaria cardamomum Maton.

Small cardamom (Elettaria cardamomum Maton), the queen of spices, is the third most expensive spice in the world after saffron and vanilla, valued highly for its aroma and taste. This perennial herbaceous plant is a native of coastal parts of Southern India and displays a significant amount of morphological diversity. Its genetic potential has not been exploited due to lack of genomic resources limiting our understanding of the genome and important metabolic pathways which give it the economic advantage in the spice industry. Here, we report upon the de novo assembled, draft whole genome sequence of cardamom variety, Njallani Green Gold. We used a hybrid assembly strategy using the reads from the Oxford Nanopore, Illumina and 10x Genomics GemCode sequencing chemistries. The assembled genome length was 1.06 Gb (gigabases) which is close to the estimated genome size of cardamom. More than 75% of the genome was captured in 8000 scaffolds with a N50 of 0.15 Mb. The genome appears to have a high repeat content and 68055 gene models were predicted. The genome is close to Musa species and displays an expansion and contraction in different gene families. The draft assembly was used for in silico mining of simple sequence repeats (SSRs). A total of 2,50,571 SSRs were identified of which 2,18,270 were perfect SSRs and 32,301 were compound SSRs. Among the perfect SSRs, trinucleotides were most abundant (1,25,329) and hexanucleotide repeats appear least (2,380). From the 2,50,571 SSRs mined, 2,27,808 primer pairs were designed based on flanking sequence information. Wet lab validation was performed for 246 SSR loci and based on their amplification profiles, 60 SSR markers were used for diversity analysis of a set of 60 diverse cardamom accessions. The average number of alleles detected per locus were 14.57 with a minimum of 4 and maximum of 30 alleles. Population structure analysis revealed the presence of high degree of admixtures which could primarily be due to cross-pollination prevalent in this species. The SSR markers identified would help in the development of gene or trait-linked markers which can be subsequently used for marker-assisted breeding for crop improvement in cardamom. The information on utilization of the SSR loci for generation of markers has been developed into a public database, 'cardamomSSRdb' that is freely available for use by the cardamom community.

Sizing Up the Onychophoran Genome: Repeats, Introns, and Gene Family Expansion Contribute to Genome Gigantism in Epiperipatus broadwayi.

Genome assemblies are growing at an exponential rate and have proved indispensable for studying evolution but the effort has been biased toward vertebrates and arthropods with a particular focus on insects. Onychophora or velvet worms are an ancient group of cryptic, soil dwelling worms noted for their unique mode of prey capture, biogeographic patterns, and diversity of reproductive strategies. They constitute a poorly understood phylum of exclusively terrestrial animals that is sister group to arthropods. Due to this phylogenetic position, they are crucial in understanding the origin of the largest phylum of animals. Despite their significance, there is a paucity of genomic resources for the phylum with only one highly fragmented and incomplete genome publicly available. Initial attempts at sequencing an onychophoran genome proved difficult due to its large genome size and high repeat content. However, leveraging recent advances in long-read sequencing technology, we present here the first annotated draft genome for the phylum. With a total size of 5.6Gb, the gigantism of the Epiperipatus broadwayi genome arises from having high repeat content, intron size inflation, and extensive gene family expansion. Additionally, we report a previously unknown diversity of onychophoran hemocyanins that suggests the diversification of copper-mediated oxygen carriers occurred independently in Onychophora after its split from Arthropoda, parallel to the independent diversification of hemocyanins in each of the main arthropod lineages.

Haplotype-phased and chromosome-level genome assembly of Puccinia polysora, a giga-scale fungal pathogen causing southern corn rust.

Rust fungi are characterized by large genomes with high repeat content and have two haploid nuclei in most life stages, which makes achieving high-quality genome assemblies challenging. Here, we described a pipeline using HiFi reads and Hi-C data to assemble a gigabase-sized fungal pathogen, Puccinia polysora f.sp. zeae, to haplotype-phased and chromosome-scale. The final assembled genome is 1.71 Gbp, with ~850 Mbp and 18 chromosomes in each haplotype, being currently one of the two giga-scale fungi assembled to chromosome level. Transcript-based annotation identified 47,512 genes for the dikaryotic genome with a similar number for each haplotype. A high level of interhaplotype variation was found with 10% haplotype-specific BUSCO genes, 5.8 SNPs/kbp, and structural variation accounting for 3% of the genome size. The P.polysora genome displayed over 85% repeat contents, with genome-size expansion and copy number increasing of species-specific orthogroups. Interestingly, these features did not affect overall synteny with other Puccinia species having smaller genomes. Fine-time-point transcriptomics revealed seven clusters of coexpressed secreted proteins that are conserved between two haplotypes. The fact that candidate effectors interspersed with all genes indicated the absence of a "two-speed genome" evolution in P.polysora. Genome resequencing of 79 additional isolates revealed a clonal population structure of P.polysora in China with low geographic differentiation. Nevertheless, a minor population differentiated from the major population by having mutations on secreted proteins including AvrRppC, indicating the ongoing virulence to evade recognition by RppC, a major resistance gene in Chinese corn cultivars. The high-quality assembly provides valuable genomic resources for future studies on disease management and the evolution of P.polysora.

Genome characterization and comparative analysis among three swimming crab species

In this study we sequenced the genomes of three economically important swimming crabs Portunus trituberculatus, Charybdis japonica, and Callinectes sapidus using the next-generation sequencing approach and made a basic assembly. The genomes of the three species are characterized with high heterozygosity (>1.2%) and high repeat content (>50%). Genome comparative analysis revealed 40 long conserved fragments (>5,000 bp) among the three species, most of them are involved in cardiac-related biological process. Relative higher genome similarity was found between P. trituberculatus and C. japonica that are belong to different subfamilies, compared to that between P. trituberculatus and C. sapidus which are from the same subfamily. It is inconsistent with their phylogenetic evolutionary trees inferred from previous mitochondrial DNA coding fragments and a conserved ANK2 protein fragment from this study. We speculated that the high genome similarity between P. trituberculatus and C. japonica might be attributed to their same inhabit range in which the genome is subject to the same environment selection, and the inconsistence between genome similarity and phylogenetic relationship is caused by the different evolutionary rates of coding DNA and non-coding DNA under environment selection.

The evolution of extremely diverged plastomes in Selaginellaceae (lycophyte) is driven by repeat patterns and the underlying DNA maintenance machinery.

Two factors are proposed to account for the unusual features of organellar genomes: the disruptions of organelle-targeted DNA replication, repair, and recombination (DNA-RRR) systems in the nuclear genome and repetitive elements in organellar genomes. Little is known about how these factors affect organellar genome evolution. The deep-branching vascular plant family Selaginellaceae is known to have a deficient DNA-RRR system and convergently evolved organellar genomes. However, we found that the plastid genome (plastome) of Selaginella sinensis has extremely accelerated substitution rates, a low GC content, pervasive repeat elements, a dynamic network structure, and it lacks direct or inverted repeats. Unexpectedly, its organelle DNA-RRR system is short of a plastid-targeted Recombinase A1 (RecA1) and a mitochondrion-targeted RecA3, in line with other explored Selaginella species. The plastome contains a large collection of short- and medium-sized repeats. Given the absence of RecA1 surveillance, we propose that these repeats trigger illegitimate recombination, accelerated mutation rates, and structural instability. The correlations between repeat quantity and architectural complexity in the Selaginella plastomes support these conclusions. We, therefore, hypothesize that the interplay of the deficient DNA-RRR system and the high repeat content has led to the extraordinary divergence of the S. sinensis plastome. Our study not only sheds new light on the mechanism of plastome divergence by emphasizing the power of cytonuclear integration, but it also reconciles the longstanding contradiction on the effects of DNA-RRR system disruption on genome structure evolution.

Telomere-to-telomere human DNA replication timing profiles

The spatiotemporal organization of DNA replication produces a highly robust and reproducible replication timing profile. Sequencing-based methods for assaying replication timing genome-wide have become commonplace, but regions of high repeat content in the human genome have remained refractory to analysis. Here, we report the first nearly-gapless telomere-to-telomere replication timing profiles in human, using the T2T-CHM13 genome assembly and sequencing data for five cell lines. We find that replication timing can be successfully assayed in centromeres and large blocks of heterochromatin. Centromeric regions replicate in mid-to-late S-phase and contain replication-timing peaks at a similar density to other genomic regions, while distinct families of heterochromatic satellite DNA differ in their bias for replicating in late S-phase. The high degree of consistency in centromeric replication timing across chromosomes within each cell line prompts further investigation into the mechanisms dictating that some cell lines replicate their centromeres earlier than others, and what the consequences of this variation are.

A multiple alignment workflow shows the effect of repeat masking and parameter tuning on alignment in plants.

Alignments of multiple genomes are a cornerstone of comparative genomics, but generating these alignments remains technically challenging and often impractical. We developed the msa_pipeline workflow (https://bitbucket.org/bucklerlab/msa_pipeline) to allow practical and sensitive multiple alignment of diverged plant genomes and calculation of conservation scores with minimal user inputs. As high repeat content and genomic divergence are substantial challenges in plant genome alignment, we also explored the effect of different masking approaches and parameters of the LAST aligner using genome assemblies of 33 grass species. Compared with conventional masking with RepeatMasker, a masking approach based on k-mers (nucleotide sequences of k length) increased the alignment rate of coding sequence and noncoding functional regions by 25 and 14%, respectively. We further found that default alignment parameters generally perform well, but parameter tuning can increase the alignment rate for noncoding functional regions by over 52% compared with default LAST settings. Finally, by increasing alignment sensitivity from the default baseline, parameter tuning can increase the number of noncoding sites that can be scored for conservation by over 76%. Overall, tuning of masking and alignment parameters can generate optimized multiple alignments to drive biological discovery in plants.

The genomic architecture of the passerine MHC region: High repeat content and contrasting evolutionary histories of single copy and tandemly duplicated MHC genes

The major histocompatibility complex (MHC) is of central importance to the immune system, and an optimal MHC diversity is believed to maximize pathogen elimination. Birds show substantial variation in MHC diversity, ranging from few genes in most bird orders to very many genes in passerines. Our understanding of the evolutionary trajectories of the MHC in passerines is hampered by lack of data on genomic organization. Therefore, we assembled and annotated the MHC genomic region of the great reed warbler (Acrocephalus arundinaceus), using long-read sequencing and optical mapping. The MHC region is large (>5.5Mb), characterized by structural changes compared to hitherto investigated bird orders and shows higher repeat content than the genome average. These features were supported by analyses in three additional passerines. MHC genes in passerines are found in two different chromosomal arrangements, either as single copy MHC genes located among non-MHC genes, or as tandemly duplicated tightly linked MHC genes. Some single copy MHC genes are old and putative orthologues among species. In contrast tandemly duplicated MHC genes are monophyletic within species and have evolved by simultaneous gene duplication of several MHC genes. Structural differences in the MHC genomic region among bird orders seem substantial compared to mammals and have possibly been fuelled by clade-specific immune system adaptations. Our study provides methodological guidance in characterizing complex genomic regions, constitutes a resource for MHC research in birds, and calls for a revision of the general belief that avian MHC has a conserved gene order and small size compared to mammals.

A Genome for Bidens hawaiensis: A Member of a Hexaploid Hawaiian Plant Adaptive Radiation.

The plant genus Bidens (Asteraceae or Compositae; Coreopsidae) is a species-rich and circumglobally distributed taxon. The 19 hexaploid species endemic to the Hawaiian Islands are considered an iconic example of adaptive radiation, of which many are imperiled and of high conservation concern. Until now, no genomic resources were available for this genus, which may serve as a model system for understanding the evolutionary genomics of explosive plant diversification. Here, we present a high-quality reference genome for the Hawaiʻi Island endemic species B. hawaiensis A. Gray reconstructed from long-read, high-fidelity sequences generated on a Pacific Biosciences Sequel II System. The haplotype-aware, draft genome assembly consisted of ~6.67 Giga bases (Gb), close to the holoploid genome size estimate of 7.56 Gb (±0.44 SD) determined by flow cytometry. After removal of alternate haplotigs and contaminant filtering, the consensus haploid reference genome was comprised of 15 904 contigs containing ~3.48 Gb, with a contig N50 value of 422 594. The high interspersed repeat content of the genome, approximately 74%, along with hexaploid status, contributed to assembly fragmentation. Both the haplotype-aware and consensus haploid assemblies recovered >96% of Benchmarking Universal Single-Copy Orthologs. Yet, the removal of alternate haplotigs did not substantially reduce the proportion of duplicated benchmarking genes (~79% vs. ~68%). This reference genome will support future work on the speciation process during adaptive radiation, including resolving evolutionary relationships, determining the genomic basis of trait evolution, and supporting ongoing conservation efforts.

An Overview of the Genetics and Genomics of the Urochloa Species Most Commonly Used in Pastures.

Pastures based on perennial monocotyledonous plants are the principal source of nutrition for ruminant livestock in tropical and subtropical areas across the globe. The Urochloa genus comprises important species used in pastures, and these mainly include Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, and Urochloa ruziziensis. Despite their economic relevance, there is an absence of genomic-level information for these species, and this lack is mainly due to genomic complexity, including polyploidy, high heterozygosity, and genomes with a high repeat content, which hinders advances in molecular approaches to genetic improvement. Next-generation sequencing techniques have enabled the recent release of reference genomes, genetic linkage maps, and transcriptome sequences, and this information helps improve our understanding of the genetic architecture and molecular mechanisms involved in relevant traits, such as the apomictic reproductive mode. However, more concerted research efforts are still needed to characterize germplasm resources and identify molecular markers and genes associated with target traits. In addition, the implementation of genomic selection and gene editing is needed to reduce the breeding time and expenditure. In this review, we highlight the importance and characteristics of the four main species of Urochloa used in pastures and discuss the current findings from genetic and genomic studies and research gaps that should be addressed in future research.

Genomic analyses provide insights into spinach domestication and the genetic basis of agronomic traits

Spinach is a nutritious leafy vegetable belonging to the family Chenopodiaceae. Here we report a high-quality chromosome-scale reference genome assembly of spinach and genome resequencing of 305 cultivated and wild spinach accessions. Reconstruction of ancestral Chenopodiaceae karyotype indicates substantial genome rearrangements in spinach after its divergence from ancestral Chenopodiaceae, coinciding with high repeat content in the spinach genome. Population genomic analyses provide insights into spinach genetic diversity and population differentiation. Genome-wide association studies of 20 agronomical traits identify numerous significantly associated regions and candidate genes for these traits. Domestication sweeps in the spinach genome are identified, some of which are associated with important traits (e.g., leaf phenotype, bolting and flowering), demonstrating the role of artificial selection in shaping spinach phenotypic evolution. This study provides not only insights into the spinach evolution and domestication but also valuable resources for facilitating spinach breeding.

The Tetragnatha kauaiensis Genome Sheds Light on the Origins of Genomic Novelty in Spiders.

Spiders (Araneae) have a diverse spectrum of morphologies, behaviors, and physiologies. Attempts to understand the genomic-basis of this diversity are often hindered by their large, heterozygous, and AT-rich genomes with high repeat content resulting in highly fragmented, poor-quality assemblies. As a result, the key attributes of spider genomes, including gene family evolution, repeat content, and gene function, remain poorly understood. Here, we used Illumina and Dovetail Chicago technologies to sequence the genome of the long-jawed spider Tetragnatha kauaiensis, producing an assembly distributed along 3,925 scaffolds with an N50 of ∼2 Mb. Using comparative genomics tools, we explore genome evolution across available spider assemblies. Our findings suggest that the previously reported and vast genome size variation in spiders is linked to the different representation and number of transposable elements. Using statistical tools to uncover gene-family level evolution, we find expansions associated with the sensory perception of taste, immunity, and metabolism. In addition, we report strikingly different histories of chemosensory, venom, and silk gene families, with the first two evolving much earlier, affected by the ancestral whole genome duplication in Arachnopulmonata (∼450 Ma) and exhibiting higher numbers. Together, our findings reveal that spider genomes are highly variable and that genomic novelty may have been driven by the burst of an ancient whole genome duplication, followed by gene family and transposable element expansion.

Comparative Mitogenomic Analysis and the Evolution of Rhizoctonia solani Anastomosis Groups.

Mitochondria are the major energy source for cell functions. However, for the plant fungal pathogens, mitogenome variations and their roles during the host infection processes remain largely unknown. Rhizoctonia solani, an important soil-borne pathogen, forms different anastomosis groups (AGs) and adapts to a broad range of hosts in nature. Here, we reported three complete mitogenomes of AG1-IA RSIA1, AG1-IB RSIB1, and AG1-IC, and performed a comparative analysis with nine published Rhizoctonia mitogenomes (AG1-IA XN, AG1-IB 7/3/14, AG3, AG4, and five Rhizoctonia sp. mitogenomes). These mitogenomes encoded 15 typical proteins (cox1-3, cob, atp6, atp8-9, nad1-6, nad4L, and rps3) and several LAGLIDADG/GIY-YIG endonucleases with sizes ranging from 109,017 bp (Rhizoctonia sp. SM) to 235,849 bp (AG3). We found that their large sizes were mainly contributed by repeat sequences and genes encoding endonucleases. We identified the complete sequence of the rps3 gene in 10 Rhizoctonia mitogenomes, which contained 14 positively selected sites. Moreover, we inferred a robust maximum-likelihood phylogeny of 32 Basidiomycota mitogenomes, representing that seven R. solani and other five Rhizoctonia sp. lineages formed two parallel branches in Agaricomycotina. The comparative analysis showed that mitogenomes of Basidiomycota pathogens had high GC content and mitogenomes of R. solani had high repeat content. Compared to other strains, the AG1-IC strain had low substitution rates, which may affect its mitochondrial phylogenetic placement in the R. solani clade. Additionally, with the published RNA-seq data, we investigated gene expression patterns from different AGs during host infection stages. The expressed genes from AG1-IA (host: rice) and AG3 (host: potato) mainly formed four groups by k-mean partitioning analysis. However, conserved genes represented varied expression patterns, and only the patterns of rps3-nad2 and nad1-m3g18/mag28 (an LAGLIDADG endonuclease) were conserved in AG1-IA and AG3 as shown by the correlation coefficient analysis, suggesting regulation of gene repertoires adapting to infect varied hosts. The results of variations in mitogenome characteristics and the gene substitution rates and expression patterns may provide insights into the evolution of R. solani mitogenomes.

Estimation of Genome Size in the Endemic Species Reseda pentagyna and the Locally Rare Species Reseda lutea Using comparative Analyses of Flow Cytometry and K-Mer Approaches.

Genome size is one of the fundamental cytogenetic features of a species, which is critical for the design and initiation of any genome sequencing projects and can provide essential insights in studying taxonomy, cytogenetics, phylogenesis, and evolutionary studies. However, this key cytogenetic information is almost lacking in the endemic species Reseda pentagyna and the locally rare species Reseda lutea in Saudi Arabia. Therefore, genome size was analyzed by propidium iodide PI flow cytometry and compared to k-mer analysis methods. The standard method for genome size measures (flow cytometry) estimated the genome size of R. lutea and R. pentagyna with nuclei isolation MB01 buffer were found to be 1.91 ± 0.02 and 2.09 ± 0.03 pg/2 °C, respectively, which corresponded approximately to a haploid genome size of 934 and 1.022 Mbp, respectively. For validation, K-mer analysis was performed on both species’ Illumina paired-end sequencing data from both species. Five k-mer analysis approaches were examined for biocomputational estimation of genome size: A general formula and four well-known programs (CovEST, Kmergenie, FindGSE, and GenomeScope). The parameter preferences had a significant impact on GenomeScope and Kmergenie estimates. While the general formula estimations did not differ considerably, with an average genome size of 867.7 and 896. Mbp. The differences across flow cytometry and biocomputational predictions may be due to the high repeat content, particularly long repetitive regions in both genomes, 71% and 57%, which interfered with k-mer analysis. GenomeScope allowed quantification of high heterozygosity levels (1.04 and 1.37%) of R. lutea and R. pentagyna genomes, respectively. Based on our observations, R. lutea may have a tetraploid genome or higher. Our results revealed fundamental cytogenetic information for R. lutea and R. pentagyna, which should be used in future taxonomic studies and whole-genome sequencing.