Background: Hevea brasiliensis, a member of the Euphorbiaceae family, is the major commercial source of natural rubber (NR). NR is a latex polymer with high elasticity, flexibility, and resilience that has played a critical role in the world economy since 1876. Results: Here, we report the draft genome sequence of H. brasiliensis. The assembly spans ~1.1 Gb of the estimated 2.15 Gb haploid genome. Overall, ~78% of the genome was identified as repetitive DNA. Gene prediction shows 68,955 gene models, of which 12.7% are unique to Hevea. Most of the key genes associated with rubber biosynthesis, rubberwood formation, disease resistance, and allergenicity have been identified. Conclusions: The knowledge gained from this genome sequence will aid in the future development of high-yielding clones to keep up with the ever increasing need for natural rubber.

Recent sequencing and software enhancements have advanced our understanding of the evolution of genomic structure and function, especially addressing novel evolutionary biology questions. Yet fragmentary turtle genome assemblies remain a challenge to fully decipher the genetic architecture of adaptive evolution. Here, we use optical mapping to improve the contiguity of the painted turtle (Chrysemys picta) genome assembly and use de novo fluorescent in situ hybridization (FISH) of bacterial artificial chromosome (BAC) clones, BAC-FISH, to physically map the genomes of the painted and slider turtles (Trachemys scripta elegans). Optical mapping increased C. picta’s N50 by ~242% compared to the previous assembly. Physical mapping permitted anchoring ~45% of the genome assembly, spanning 5544 genes (including 20 genes related to the sex determination network of turtles and vertebrates). BAC-FISH data revealed assembly errors in C. picta and T. s. elegans assemblies, highlighting the importance of molecular cytogenetic data to complement bioinformatic approaches. We also compared C. picta’s anchored scaffolds to the genomes of other chelonians, chicken, lizards, and snake. Results revealed a mostly one-to-one correspondence between chromosomes of painted and slider turtles, and high homology among large syntenic blocks shared with other turtles and sauropsids. Yet, numerous chromosomal rearrangements were also evident across chelonians, between turtles and squamates, and between avian and non-avian reptiles.

Sex-determining mechanisms (SDMs) set an individual's sexual fate by its genotype (genotypic sex determination, GSD) or environmental factors like temperature (temperature- dependent sex determination, TSD), as in turtles where the GSD “trigger” remains unknown. SDMs co-evolve with turtle chromosome number, perhaps because fusions/fissions alter the relative position/regulation of sexual development genes. Here, we map 10 such genes via FISH onto metaphase chromosomes in 6 TSD and 6 GSD turtles for the first time. Results uncovered intrachromosomal rearrangements involving 3 genes across SDMs (Dax1, Fhl2, and Fgf9) and a chromosomal fusion linking 2 genes (Sf1 and Rspo1) in 1 chromosome in a TSD turtle (Pelomedusa subrufa) that locate to 2 chromosomes in all others. Notably, Sf1 and its repressor Foxl2 map to Apalone spinifera's ZW chromosomes but to a macro- (Foxl2) and a microautosome (Sf1) in other turtles potentially inducing SDM evolution. However, our phylogenetically informed analysis refutes Foxl2 (but not Sf1) as Apalone's master sex-determining gene. The absence of common TSD-specific or GSD-specific rearrangements underscores the independent evolutionary trajectories of turtle SDMs. Further comparative analyses using more genes from the sexual development network are warranted to inform genome evolution and its contribution to enigmatic turnovers of vertebrate sex determination.

The discovery of sex chromosome systems in non-model organisms has elicited growing recognition that sex chromosomes evolved via diverse paths that are not fully elucidated. Lineages with labile sex determination, such as turtles, hold critical cues, yet data are skewed toward hide-neck turtles (suborder Cryptodira) and scant for side-neck turtles (suborder Pleurodira). Here, we used classic and molecular cytogenetics to investigate Emydura subglobosa (ESU), an unstudied side-neck turtle with genotypic sex determination from the family Chelidae, where extensive morphological divergence exists among XX/XY systems. Our data represent the first cytogenetic description for ESU. Similarities were found between ESU and E. macquarii (EMA), such as identical chromosome number (2n = 50), a single and dimorphic nucleolus organizer region (NOR) localized in a microchromosome pair (ESU14) of both sexes (detected via FISH of 18S rDNA). Only the larger NOR is active (detected by silver staining). As in EMA, comparative genome hybridization revealed putative macro XX/XY chromosomes in ESU (the 4th largest pair). Our comparative analyses and revaluation of previous data strongly support the hypothesis that Emydura's XX/XY system evolved via fusion of an ancestral micro-Y (retained by Chelodina longicollis) onto a macro-autosome. This evolutionary trajectory differs from the purported independent evolution of XX/XY from separate ancestral autosomes in Chelodina and Emydura that was previously reported. Our data permit dating this Y-autosome fusion to at least the split of Emydura around 45 Mya and add critical information about the evolution of the remarkable diversity of sex-determining mechanisms in turtles, reptiles, and vertebrates.

Understanding the evolution of chromatin conformation among species is fundamental to elucidate the architecture and plasticity of genomes. Nonrandom interactions of linearly distant loci regulate gene function in species-specific patterns, affecting genome function, evolution, and, ultimately, speciation. Yet, data from nonmodel organisms are scarce. To capture the macroevolutionary diversity of vertebrate chromatin conformation, here we generate de novo genome assemblies for two cryptodiran (hidden-neck) turtles via Illumina sequencing, chromosome conformation capture, and RNA-seq:Apalone spinifera(ZZ/ZW, 2n= 66) andStaurotypus triporcatus(XX/XY, 2n= 54). We detected differences in the three-dimensional (3D) chromatin structure in turtles compared to other amniotes beyond the fusion/fission events detected in the linear genomes. Namely, whole-genome comparisons revealed distinct trends of chromosome rearrangements in turtles: (1) a low rate of genome reshuffling inApalone(Trionychidae) whose karyotype is highly conserved when compared to chicken (likely ancestral for turtles), and (2) a moderate rate of fusions/fissions inStaurotypus(Kinosternidae) andTrachemys scripta(Emydidae). Furthermore, we identified a chromosome folding pattern that enables “centromere–telomere interactions” previously undetected in turtles. The combined turtle pattern of “centromere–telomere interactions” (discovered here) plus “centromere clustering” (previously reported in sauropsids) is novel for amniotes and it counters previous hypotheses about amniote 3D chromatin structure. We hypothesize that the divergent pattern found in turtles originated from an amniote ancestral state defined by a nuclear configuration with extensive associations among microchromosomes that were preserved upon the reshuffling of the linear genome.

Understanding the evolution and regulation of nucleolar organizing regions (NORs) is important to elucidate genome structure and function. This is because ribosomal gene (rDNA) copy number and activity mediate protein biosynthesis, stress response, ageing, disease, dosage compensation and genome stability. Here, we found contrasting dosage compensation of sex-linked NORs in turtles with male and female heterogamety. Most taxa examined exhibit homomorphic rRNA gene clusters in a single autosome pair (determined by 28S rDNA fluorescence in situ hybridization), whereas NORs are sex-linked in Apalone spinifera, Pelodiscus sinensis and Staurotypus triporcatus. Full-dosage compensation upregulates the male X-NOR (determined via silver staining-AgNOR) in Staurotypus (who lacks Y-NOR) compared with female X-AgNORs. In softshell Apalone and Pelodiscus, who share homologous ZZ/ZW micro-chromosomes, their enlarged W-NOR is partially active (due to 28S rDNA invasion by R2 retroelements), whereas their smaller Z-NOR is silent in females but active in both male-Zs (presumably because the W-NOR meets cellular demands and excessive NOR activity is costly). We hypothesize that R2 disruption favoured W enlargement to add intact 28S-units, perhaps facilitated by reduced recombination during sex chromosome evolution. The molecular basis of the potentially adaptive female Z-silencing is likely intricate and perhaps epigenetic, as non-ribosomal Z genes are active in Apalone females. Yet, Emydura maquarii exhibit identical heteromorphism in their autosomal NOR (R2 invaded 28S-units and the small-autosome NOR is silent), suggesting that the softshell turtle pattern can evolve independent of sex chromosome evolution. Our study illuminates the complex sex chromosome evolution and dosage compensation of non-model systems that challenges classic paradigms.