Using in silico predicted ancestral genomes to improve the efficiency of paleogenome reconstruction

Paleogenomics is the nascent discipline concerned with sequencing and analysis of genome‐scale information from historic, ancient, and even extinct samples. While once inconceivable due to the challenges of DNA damage, contamination, and the technical limitations of PCR‐based Sanger sequencing, following the dawn of the second‐generation sequencing revolution, it has rapidly become a reality. However, a significant challenge facing ancient DNA studies on extinct species is the lack of closely related reference genomes against which to map the sequencing reads from ancient samples. Although bioinformatic efforts to improve the assemblies have focused mainly in mapping algorithms, in this article we explore the potential of an alternative approach, namely using reconstructed ancestral genome as reference for mapping DNA sequences of ancient samples. Specifically, we present a preliminary proof of concept for a general framework and demonstrate how under certain evolutionary divergence thresholds, considerable mapping improvements can be easily obtained.     

Complete chloroplast genomes of Saccharum spontaneum,Saccharum officinarum and Miscanthus floridulus (Panicoideae: Andropogoneae) reveal the plastid view on sugarcane origins

Sugarcane (Saccharum hybrid cultivar) ranks among the world's top 10 food crops and annually provides 60–70% of the sugar produced worldwide. Despite its economic importance there has been no large-scale systematics study of genus Saccharum and the existing model of sugarcane origins has remained largely unchallenged for almost 50 years. For the first time, we have assembled the complete plastid genomes of Miscanthus floridulus (first report for this genus), Saccharum spontaneum and Saccharum officinarum allowing us to elucidate the phylogenetic origins of Saccharum s.s. species. We demonstrate that Saccharum s.s. is divided into four species, with S. spontaneum diverging from the remainder of the genus about 1.5 million years ago and S. robustum diverging 750,000 years ago. Two separate lineages, one leading to S. officinarum and the other leading to modern hybrid cultivars diverged from S. robustum 640,000 years ago. These findings overturn all previous hypotheses on sugarcane origins, demonstrating that sugarcane's antecedents could not have arisen by human action. All modern cultivars share a common Polynesian origin, whereas Old World canes, S. barberi and S. sinense, cluster as a distinct S. officinarum lineage. This makes modern cultivars a distinct species of genus Saccharum, and we formally propose the name Saccharum cultum for the ancestor of all lineages currently classified as Saccharum hybrid cultivars.     

Production of polyhydroxybutyrate in sugarcane

We report here the production of the bacterial polyester, polyhydroxybutyrate (PHB), in the crop species sugarcane ( Saccharum spp. hybrids). The PHB biosynthesis enzymes of Ralstonia eutropha [β-ketothiolase (PHAA), acetoacetyl-reductase (PHAB) and PHB synthase (PHAC)] were expressed in the cytosol or targeted to mitochondria or plastids. PHB accumulated in cytosolic lines at trace amounts, but was not detected in mitochondrial lines. In plastidic lines, PHB accumulated in leaves to a maximum of 1.88% of dry weight without obvious deleterious effects. Epifluorescence and electron microscopy of leaf sections from these lines revealed that PHB granules were visible in plastids of most cell types, except mesophyll cells. The concentration of PHB in culm internodes of plastidic lines was substantially lower than in leaves. Western blot analysis of these lines indicated that expression of the PHB biosynthesis proteins was not limiting in culm internodes. Epifluorescence microscopy of culm internode sections from plastidic lines showed that PHB granules were visible in most cell types, except photosynthetic cortical cells in the rind, and that the lower PHB concentration in culm internodes was probably a result of dilution of PHB-containing cells by the large number of cells with little or no PHB. We discuss strategies for producing PHB in mitochondria and mesophyll cell plastids, and for increasing PHB yields in culms.     

Complete nucleotide sequence of the sugarcane (Saccharum officinarum) chloroplast genome: a comparative analysis of four monocot chloroplast genomes.

The complete nucleotide sequence of the chloroplast genome of sugarcane (Saccharum officinarum) has been determined. It is a circular double-stranded DNA molecule, 141,182 bp in size, and is composed of a large single copy of 83,048 bp, a small single copy of 12,544 bp, and a pair of inverted repeat regions of 22,795 bp each. A comparative analysis among monocots showed that the sugarcane chloroplast genome was very similar to maize but not to rice or wheat. Between sugarcane and maize at the rps16-trnQ (UUG) region, however, a length polymorphism was identified. With regard to insertions/deletions equal to or longer than 5 bp, a total of 53 insertion and 31 deletion events were identified in the sugarcane chloroplast genome. Of the 84 loci identified, a pair of direct repeat sequences was located side by side in a tandem fashion in 47 loci (56.0%). A recombination event during plant evolution is discussed at two sites between the sugarcane and tobacco chloroplast genomes.     

Mixed signals from hybrid genomes

Admixture results from interbreeding between individuals from different populations or species that were previously genetically isolated from each other (Fig.  1 ). Identifying admixture events in the genome is not always a straightforward task, because the genetic signature left behind fades with time as recombination events fragment the genomic segments introduced during the interbreeding event. Additionally, when the genetic architecture of populations or species that admix is not very different (e.g. they coalesce to a common ancestor recently), admixture signatures may be difficult to detect. Ignoring the effects of admixture can, however, pose severe problems for population genetic analyses that rely on the distribution of polymorphic markers across the genome. In this issue of Molecular Ecology, Bosse et al. ( 2014 ) analyse genomic data from modern pigs to understand hybridization processes that occurred between domestic pigs from European and Asiatic origin, and between pigs and wild boars. Their results are interesting regarding the fine‐scale distribution of admixture across the pig genome, and the way in which this admixture biases estimates of the effective population size in European domestic pigs. The implications of these results are significant, as they serve as a cautionary note on genomic analyses that depend on the distribution of polymorphic variants in potentially admixed populations.     

Reconstruction of full-length LINE-1 progenitors from ancestral genomes

Sequences derived from the Long INterspersed Element-1 (L1) family of retrotransposons occupy at least 17% of the human genome, with 67 distinct subfamilies representing successive waves of expansion and extinction in mammalian lineages. L1s contribute extensively to gene regulation, but their molecular history is difficult to trace, because most are present only as truncated and highly mutated fossils. Consequently, L1 entries in current databases of repeat sequences are composed mainly of short diagnostic subsequences, rather than full functional progenitor sequences for each subfamily. Here, we have coupled 2 levels of sequence reconstruction (at the level of whole genomes and L1 subfamilies) to reconstruct progenitor sequences for all human L1 subfamilies that are more functionally and phylogenetically plausible than existing models. Most of the reconstructed sequences are at or near the canonical length of L1s and encode uninterrupted ORFs with expected protein domains. We also show that the presence or absence of binding sites for KRAB-C2H2 Zinc Finger Proteins, even in ancient-reconstructed progenitor L1s, mirrors binding observed in human ChIP-exo experiments, thus extending the arms race and domestication model. RepeatMasker searches of the modern human genome suggest that the new models may be able to assign subfamily resolution identities to previously ambiguous L1 instances. The reconstructed L1 sequences will be useful for genome annotation and functional study of both L1 evolution and L1 contributions to host regulatory networks.     

Spatio-temporal characterization of polyhydroxybutyrate accumulation in sugarcane

We report here the results from a glasshouse trial of several transgenic sugarcane ( Saccharum spp. hybrids) lines accumulating the bacterial polyester polyhydroxybutyrate (PHB) in plastids. The aims of the trial were to characterize the spatio-temporal pattern of PHB accumulation at a whole-plant level, to identify factors limiting PHB production and to determine whether agronomic performance was affected adversely by PHB accumulation. Statistical analysis showed that a vertical PHB concentration gradient existed throughout the plant, the polymer concentration being lowest in the youngest leaves and increasing with leaf age. In addition, there was a horizontal gradient along the length of a leaf, with the PHB concentration increasing from the youngest part of the leaf (the base) to the oldest (the tip). The rank order of the lines did not change over time. Moreover, there was a uniform spatio-temporal pattern of relative PHB accumulation among the lines, despite the fact that they showed marked differences in absolute PHB concentration. Molecular analysis revealed that the expression of the transgenes encoding the PHB biosynthesis enzymes was apparently coordinated, and that there were good correlations between PHB concentration and the abundance of the PHB biosynthesis enzymes. The maximum recorded PHB concentration, 1.77% of leaf dry weight, did not confer an agronomic penalty. The plant height, total aerial biomass and culm-internode sugar content were not affected relative to controls. Although moderate PHB concentrations were achieved in leaves, the maximum total-plant PHB yield was only 0.79% (11.9 g PHB in 1.51 kg dry weight). We combine the insights from our statistical and molecular analyses to discuss possible strategies for increasing the yield of PHB in sugarcane.     

Stomatal control of transpiration from a developing sugarcane canopy

Abstract. Stomatal conductance of single leaves and transpiration from an entire sugarcane (Saccharum spp. hybrid) canopy were measured simultaneously using independent techniques. Stomatal and environmental controls of transpiration were assessed at three stages of canopy development, corresponding to leaf area indices (L) of 2.2, 3.6 and 5.6. Leaf and canopy boundary layers impeded transport of transpired water vapour away from the canopy, causing humidity around the leaves to find its own value through local equilibration rather than a value determined by the humidity of the bulk air mass above the canopy. This tended to uncouple transpiration from direct stomatal control, so that transpiration predicted from measurement of stomatal conductance and leaf-to-air vapour pressure differences was increasingly overestimated as the reference point for ambient vapour pressure measurement was moved farther from the leaf and into the bulk air. The partitioning of control between net radiation and stomata was expressed as a dimensionless decoupling coefficent ranging from zero to 1.0. When the stomatal aperture was near its maximum this coefficient was approximately 0.9, indicating that small reductions in stomatal aperture would have had little effect on canopy transpiration. Maximum rates of transpiration were, however, limited by large adjustments in maximum stomatal conductance during canopy development. The product of maximum stomatal conductance and L. a potential total canopy conductance in the absence of boundary layer effects, remained constant as L increased. Similarly, maximum canopy conductance, derived from independent micrometeorological measurements, also remained constant over this period. Calculations indicated that combined leaf and canopy boundary layer conductance decreased with increasing L such that the ratio of boundary layer conductance to maximum stomatal conductance remained nearly constant at approximately 0.5. These observations indicated that stomata adjusted to maintain both transpiration and the degree of stomatal control of transpiration constant as canopy development proceeded.     

The dynamic nature of eukaryotic genomes

Analyses of diverse eukaryotes reveal that genomes are dynamic, sometimes dramatically so. In numerous lineages across the eukaryotic tree of life, DNA content varies within individuals throughout life cycles and among individuals within species. Discovery of examples of genome dynamism is accelerating as genome sequences are completed from diverse eukaryotes. Though much is known about genomes in animals, fungi, and plants, these lineages represent only 3 of the 60-200 lineages of eukaryotes. Here, we discuss diverse genomic strategies in exemplar eukaryotic lineages, including numerous microbial eukaryotes, to reveal dramatic variation that challenges established views of genome evolution. For example, in the life cycle of some members of the "radiolaria," ploidy increases from haploid (N) to approximately 1,000N, whereas intrapopulation variability of the enteric parasite Entamoeba ranges from 4N to 40N. Variation has also been found within our own species, with substantial differences in both gene content and chromosome lengths between individuals. Data on the dynamic nature of genomes shift the perception of the genome from being fixed and characteristic of a species (typological) to plastic due to variation within and between species.     

Sink strength regulates photosynthesis in sugarcane

The relationship in sugarcane (Saccharum spp.) between photosynthetic source tissue and sink material was examined through manipulation of the sink:source ratio of field-grown Saccharum spp. hybrid cv. N19 (N19). To enhance sink strength, all leaves, except for the third fully expanded leaf, were enclosed in 90% shade cloth for varying periods of time. Variations in sucrose, glucose and fructose concentrations were measured and the effects of shading on the leaf gas exchange and fluorescence characteristics recorded. Changes in carbon partitioning caused by shading were examined based on the uptake and translocation of fixed 14CO2. Following a decline in sucrose concentrations in young internodal tissue and shaded leaves, significant increases in the CO2-saturated photosynthetic rate (Jmax), carboxylation efficiency (CE) and electron transport rate were observed in unshaded leaves after 8 d of shading treatment. It was concluded that up-regulation of source-leaf photosynthetic capacity is correlated with a decrease in assimilate availability to acropetal culm sink tissue. Furthermore, a significant relationship was revealed between source hexose concentration and photosynthetic activity.     

Stomatal and hydraulic conductance in growing sugarcane: stomatal adjustment to water transport capacity*

Abstract Stomatal conductance per unit leaf area in well-irrigated field- and greenhouse-grown sugarcane increased with leaf area up to 0.2 m² plant ¹, then declined so that maximum transpiration per plant tended to saturate rather than increase linearly with further increase in leaf area. Conductance to liquid water transport exhibited parallel changes with plant size. This coordiantion of vapour phase and liquid phase conductances resulted in a balance between water loss and water transport capacity, maintaining leaf water status remarkably constant over a wide range of plant size and growing conditions. The changes in stomatal conductance were not related to plant or leaf age. Partial defoliation caused rapid increases in stomatal conductance, to re-establish the original relationship with remaining leaf area. Similarly, pruning of roots caused rapid reductions in stomatal conductance, which maintained or improved leaf water status. These results suggest that sugarcane stomata adjusted to the ratio of total hydraulic conductance to total transpiring leaf area. This could be mediated by root metabolites in the transpiration stream, whose delivery per unit leaf area would be a function of the relative magnitudes of root system size, transpiration rate and leaf area.     

Genomic origin and organization of the allopolyploid Primula egaliksensis investigated by in situ hybridization

Background and Aims: Earlier studies have suggested that the tetraploid Primula egaliksensis(2n = 40) originated from hybridization between the diploidsP. mistassinica (2n = 18) and P. nutans (2n = 22), which werehypothesized to be the maternal and paternal parent, respectively.The present paper is aimed at verifying the hybrid nature ofP. egaliksensis using cytogenetic tools, and to investigatethe extent to which the parental genomes have undergone genomicreorganization. Methods: Genomic in situ hybridization (GISH) and fluorescent in situhybridization (FISH) with ribosomal DNA (rDNA) probes, togetherwith sequencing of the internal transcribed spacer (ITS) regionof the rDNA, were used to identify the origin of P. egaliksensisand to explore its genomic organization, particularly at rDNAloci. Key Results: GISH showed that P. egaliksensis inherited all chromosomes fromP. mistassinica and P. nutans and did not reveal major intergenomicrearrangements between the parental genomes (e.g. interchromosomaltranslocations). However, karyological comparisons and FISHexperiments suggested small-scale rearrangements, particularlyat rDNA sites. Primula egaliksensis lacked the ITS-bearing heterochromaticknobs characteristic of the maternal parent P. mistassinicaand maintained only the rDNA loci of P. nutans. These resultscorroborated sequence data indicating that most ITS sequencesof P. egaliksensis were of the paternal repeat type. Conclusions: The lack of major rearrangements may be a consequence of theconsiderable genetic divergence between the putative parents,while the rapid elimination of the ITS repeats from the maternalprogenitor may be explained by the subterminal location of ITSloci or a potential role of nucleolar dominance in chromosomestabilization. These small-scale rearrangements may be indicativeof genome diploidization, but further investigations are neededto confirm this assumption.     

Hydraulic architecture of sugarcane in relation to patterns of water use during plant development*

Hydraulic conductance was measured on leaf and stem segments excised from sugarcane plants at different stages of development. Maximum transpiration rates and leaf water potential (Ψ_L) associated with maximum transpiration were also measured in intact plants as a function of plant size. Leaf specific hydraulic conductivity (L_sc) and transpiration on a unit leaf area basis (E) were maximal in plants with approximately 0.2 m² leaf area and decreased with increasing plant size. These changes in Fand L_sc were nearly parallel, which prevented φ_L in larger plants from decreasing to levels associated with substantial loss in xylem conductivity caused by embolism formation. Coordination of changes in E and leaf hydraulic properties was not mediated by declining leaf water status, since φ_L increased with plant size. Hydraulic constrictions were present at nodes and in the node-leaf sheath-leaf blade pathway. This pattern of constrictions is in accord with the idea of plant segmentation into regions differing in water transport efficiency and would tend to confine embolisms to the relatively expendable leaves at terminal positions in the pathway, thereby preserving water transport through the stem.     

生长前期叶面喷施乙烯利对甘蔗茎细胞几种酶活性的影响

在甘蔗分蘖初期用乙烯利进行叶面喷施处理 ,并在不同的时期分别对蔗茎细胞质和细胞壁的几种代谢关键酶活性进行测定 ,结果表明 :适当浓度的乙烯利处理提高了整个生长期甘蔗茎细胞质的过氧化物酶活性、生长前中期甘蔗茎细胞质和细胞壁的 Ca2 +-ATP酶、细胞质的 Mg2 +-ATP酶活性和生长后期细胞质的多酚氧化酶活性 ,较高浓度的乙烯利处理普遍提高整个生长期甘蔗茎细胞质和细胞壁的 Ca2 +-ATP酶活性、细胞壁的 Mg2 +-ATP酶活性和旺盛生长期间细胞壁的过氧化物酶活性     

Characterization of a proton translocating ATPase and sucrose uptake in a tonoplast-enriched vesicle fraction from sugarcane

A sucrose gradient fraction was used to characterize the tonoplast ATPase from storage tissue of the sugarcane plant ( Saccharum sp. var. H57–5175). Marker enzyme analyses and characterization of low-density vesicles isolated on a sucrose gradient were consistent with a highly enriched tonoplast fraction. ATPase and proton transport activities were both substantially inhibited by nitrate (80%), but very little by vanadate (10%), indicating a high titer of tonoplast compared to plasma-membrane vesicles in the fraction. Sensitivity toward other inhibitors, as well as ion effects, correlated closely among ATPase and proton translocation activities. Although the vesicles in this fraction showed good proton translocating activity there was no indication that ATP stimulated sucrose uptake in this tonoplast population.     

Characterization of ancestral Fe/Mn superoxide dismutases indicates their cambialistic origin

Superoxide dismutases (SODs) are critical metalloenzymes mitigating the damages of the modern oxygenated world. However, the emergence of one family of SODs, the Fe/Mn SOD, has been recurrently proposed to predate the great oxygenation event (GOE). This ancient family lacks metal binding selectivity, but displays strong catalytic selectivity. Therefore, some homologues would only be active when bound to Fe or Mn, although others, dubbed cambialistic, would function when loaded with either ion. This posed the longstanding question about the identity of the cognate metal ion of the first SODs to emerge. In this work, we utilize ancestral sequence reconstruction techniques to infer the earliest SODs. We show that the “ancestors” are active in vivo and in vitro. Further, we test their metal specificity and demonstrate that they are cambialistic in nature. Our findings shed light on how the predicted Last Common Universal Ancestor was capable of dealing with decomposition of the superoxide anion, and the early relationship between life, oxygen, and metal ion availability.     

Isolation of specific receptors from Xanthomonas albilineans cell walls for lectins-like glycoproteins of sugarcane

Abstract

Two families of sugarcane glycoproteins differing in their molecular mass have been isolated from sugarcane stalks. These glycoproteins specifically bind to cell wall receptors of Xanthomonas albilineans, a sugarcane pathogen, producing bacterial agglutination. Bound glycoproteins can be desorbed from bacterial cell walls by galactitol, a component of the glycosidic moiety of the sugarcane protein. This indicates that sugarcane glycoproteins bind through their glycosidic rest to the peptide moiety of the bacterial receptor. Several cell wall receptors have been isolated by affinity chromatography and separated by capillary electrophoresis.     

Characterisation of an ethyl methanesulfonate-derived drought-tolerant sugarcane mutant line

Sustainable sugarcane production in areas prone to frequent and severe drought can be achieved by creating resilient sugarcane varieties. In this study, a unique mutant line, M9.2, was generated from a drought susceptible commercial sugarcane cultivar, N19, through the exposure of callus cells to the ethyl methanesulfonate mutagen and subsequent in vitro osmotic selection on polyethylene glycol. The study aimed to characterise the M9.2 mutant, in comparison with the parental genotype, in terms of its physiological and biochemical performance and proteome profile when exposed to moderate (14 days without water) and severe (21 days without water) water deficit stress in glasshouse pot trials. In comparison to the parental counterparts, the mutant plants were able to sustain the quantum efficiency of photosystem II (Fv/Fm) throughout the stress. Under mild stress, the mutant plants displayed elevated stomatal conductance, high concentrations of proline, accumulated less H₂O₂ and phenotypically displayed limited wilting and no visible signs of leaf senescence. Under severe stress, the mutant plants accumulated less malondialdehyde and more antioxidant enzymes (superoxide dismutase and catalase) than the parental line. Differential protein expression was also observed according to two-dimensional difference gel electrophoresis patterns of proteins expressed in the M9.2 mutant versus the parental plants during moderate stress. Analysis revealed proteins related to photosynthesis (pyruvate orthophosphate dikinase, un-fragmented ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and chlorophyll a/b-binding protein 3) and carbohydrate metabolism (sucrose synthase) were up-regulated in the mutant. Differentially expressed proteins were further linked to energy metabolism, methylation homeostasis and DNA repair. This study characterises the new M9.2 mutant with beneficial drought-tolerant traits which have the potential to be exploited in future sugarcane breeding programmes.     

Effects of a recent founding event and intrinsic population dynamics on genetic diversity in an ungulate population

Maintenance of genetic diversity has recently become a management goal for a number of species, due to its importance for present and future population viability. Genetic drift, primarily through differential reproductive success and inbreeding, can accelerate the loss of genetic diversity in recently recovered populations. We attempt to quantify the consequences of these factors on the genetic diversity contained in a small, recently founded wood bison (Bison bison athabascae) population by examining the genetic variation in this conservation herd, the calves born therein, and its large source population. The Hook Lake Wood Bison Recovery Project was initiated to found a disease-free herd of wood bison containing a representative amount of the genetic diversity present in the Wood Buffalo National Park metapopulation. Levels of diversity in the Hook Lake Wood Bison Recovery Project founders are higher than in previous salvage attempts. To examine the effects of differential reproductive success on this population, we monitored parentage of the calves born in the Hook Lake Wood Bison Recovery Project for 3 years since the founders reached sexual maturity. Two of the male founders sired over 90% of the offspring born in this population, which has led to a reduction in diversity in their calves. Monitoring of reproductive success, and incorporation of selective breeding strategies will be required to reduce the rate at which genetic diversity is lost from this small, isolated population. These steps should occur in other recovery projects, particularly when a small number of individuals are capable of dominating reproduction.