Modeling transposable elements dynamics during polyploidization in plants.

In this work we study the proliferation of transposable elements (TEs) and the epigenetic response of plants during the process of polyploidization. Through a deterministic model, expanding on our previous work on TE proliferation under epigenetic regulation, we study the long-term TE distribution and TE stability in the subgenomes of both autopolyploids and allopolyploids. We also explore different small-interfering RNA (siRNA) action modes on the subgenomes, including a model where siRNAs are not directed to specific genomes and one where siRNAs are directed - i.e. more active - in subgenomes with higher TE loads. In the autopolyploid case, we find long-term stable equilbria that tend to equilibrate the number of active TEs between subgenomes. In the allopolyploid case, directed siRNA action is fundamental to avoid a "winner takes all" outcome of the competition between the TEs in the different subgenomes. We also show that decaying oscillations in the number of TEs occur naturally in all cases, perhaps explaining some of the observed features of 'genomic shock' after hybridization events, and that the balance in the dynamics of the different types of siRNA is determinant for the synchronization of these oscillations.

Physiological and Molecular Modulations to Drought Stress in the Brassica Species.

Climate change, particularly drought stress, significantly impacts plant growth and development, necessitating the development of resilient crops. This study investigated physiological and molecular modulations to drought stress between diploid parent species and their polyploid progeny in the Brassica species. While no significant phenotypic differences were observed among the six species, drought stress reduced growth parameters by 2.4% and increased oxidative stress markers by 1.4-fold. Drought also triggered the expression of genes related to stress responses and led to the accumulation of specific metabolites. We also conducted the first study of perfluorooctane sulfonic acid (PFOS) levels in leaves as a drought indicator. Lower levels of PFOS accumulation were linked to plants taking in less water under drought conditions. Both diploid and polyploid species responded to drought stress similarly, but there was a wide range of variation in their responses. In particular, responses were less variable in polyploid species than in diploid species. This suggests that their additional genomic components acquired through polyploidy may improve their flexibility to modulate stress responses. Despite the hybrid vigor common in polyploid species, Brassica polyploids demonstrated intermediate responses to drought stress. Overall, this study lays the framework for future omics-level research, including transcriptome and proteomic studies, to deepen our understanding of drought tolerance mechanisms in Brassica species.

Loss of Heterozygosity Spectrum Depends on Ploidy Level in Natural Yeast Populations.

The appearance of genomic variations such as loss of heterozygosity (LOH) has a significant impact on phenotypic diversity observed in a population. Recent large-scale yeast population genomic surveys have shown a high frequency of these events in natural isolates and more particularly in polyploids. However, the frequency, extent, and spectrum of LOH in polyploid organisms have never been explored and are poorly characterized to date. Here, we accumulated 5,163 LOH events over 1,875 generations in 76 mutation accumulation (MA) lines comprising nine natural heterozygous diploid, triploid, and tetraploid natural S. cerevisiae isolates from different ecological and geographical origins. We found that the rate and spectrum of LOH are variable across ploidy levels. Of the total accumulated LOH events, 8.5%, 21%, and 70.5% of them were found in diploid, triploid, and tetraploid MA lines, respectively. Our results clearly show that the frequency of generated LOH events increases with ploidy level. In fact, the cumulative LOH rates were estimated to be 9.3 × 10-3, 2.2 × 10-2, and 8.4 × 10-2 events per division for diploids, triploids, and tetraploids, respectively. In addition, a clear bias toward the accumulation of interstitial and short LOH tracts is observed in triploids and tetraploids compared with diploids. The variation of the frequency and spectrum of LOH events across ploidy level could be related to the genomic instability, characterizing higher ploidy isolates.

Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures.

Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8'-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches.

Endoreplication-Why Are We Not Using Its Full Application Potential?

Endoreplication-a process that is common in plants and also accompanies changes in the development of animal organisms-has been seen from a new perspective in recent years. In the paper, we not only shed light on this view, but we would also like to promote an understanding of the application potential of this phenomenon in plant cultivation. Endoreplication is a pathway for cell development, slightly different from the classical somatic cell cycle, which ends with mitosis. Since many rounds of DNA synthesis take place within its course, endoreplication is a kind of evolutionary compensation for the relatively small amount of genetic material that plants possess. It allows for its multiplication and active use through transcription and translation. The presence of endoreplication in plants has many positive consequences. In this case, repeatedly produced copies of genes, through the corresponding transcripts, help the plant acquire the favorable properties for which proteins are responsible directly or indirectly. These include features that are desirable in terms of cultivation and marketing: a greater saturation of fruit and flower colors, a stronger aroma, a sweeter fruit taste, an accumulation of nutrients, an increased resistance to biotic and abiotic stress, superior tolerance to adverse environmental conditions, and faster organ growth (and consequently the faster growth of the whole plant and its biomass). The two last features are related to the nuclear-cytoplasmic ratio-the greater the content of DNA in the nucleus, the higher the volume of cytoplasm, and thus the larger the cell size. Endoreplication not only allows cells to reach larger sizes but also to save the materials used to build organelles, which are then passed on to daughter cells after division, thus ending the classic cell cycle. However, the content of genetic material in the cell nucleus determines the number of corresponding organelles. The article also draws attention to the potential practical applications of the phenomenon and the factors currently limiting its use.

Chromosome painting reveals the genomic structure of three polyploid species of Ipomoea.

Cultivated sweetpotato [Ipomoea batatas (L.) Lam.] from the family Convolvulaceae is a hexaploid species with 2n = 6x = 90 and has been controversial regarding its nature as an autopolyploid arising within a species or an allopolyploid forming between species. Here, we developed oligonucleotide-based painting probes for two chromosomes of I. nil, a model diploid Ipomoea species. Using these probes, we revealed the pairing behavior of homoeologous chromosomes in I. batatas and its two possible polyploid ancestral species, tetraploid I. tabascana (2n = 4x = 60) and hexaploid I. trifida (2n = 6x = 90). Chromosome painting analysis revealed a high percentage of quadrivalent formation in zygotene-pachytene cells of I. tabascana, which supported that I. tabascana was an autotetraploid likely derived by doubling of structurally similar and homologous genomes rather than a hybrid between I. batatas and I. trifida (2x). A high frequency of hexavalent/bivalent and tetravalent pairing was observed in I. trifida (6x) and I. batatas. However, the percentage of hexavalent pairing in I. trifida (6x) was far higher than that in I. batatas. Thus, the present results tend to support that I. trifida (6x) is an autohexaploid, while I. batatas is more likely to be a segmental allohexaploid.

Aneuploids and its increment on diversity of Lippia alba polyploid complex: genetic aspects and origin.

BACKGROUND: Aneuploidy is associated with add or lack of individual chromosomes. The knowledge regarding aneuploidy is still rare in wild and tropical populations. Lippia alba is a tropical polyploid complex naturally formed, with 2x, 3x, 4x, 6x, and aneuploid individuals. The species presents pharmacological and medicinal importance, due to its essential oil compounds, which are related to the ploidal level. Considering the singularity of aneuploids emergence and stability, we proposed to investigate putative cytotypes involved in the aneuploids formation. METHODS AND RESULTS: Molecular, cytogenetic, reproductive, and chemical approaches were adopted. The results showed that the aneuploids possibly have independent origin considering the genetic, chemical and karyotypical profiles. The chemical composition of aneuploids is related to genetic similarity. The aneuploid origin may involve 2x and 3x cytotypes being possible to rise four scenarios of crosses to explain that. CONCLUSIONS: The results, in general, contribute to the comprehension of the origin of aneuploids and highlight the genetic profile of these accessions as a key element on the understanding of the chemical profile of L. alba accessions.

Integrated transcriptome and miRNA sequencing approaches provide insights into salt tolerance in allotriploid Populus cathayana.

MAIN CONCLUSION: Some salt-stress responsive DEGs, mainly involved in ion transmembrane transport, hormone regulation, antioxidant system, osmotic regulation, and some miRNA jointly regulated the salt response process in allotriploid Populus cathayana. The molecular mechanism of plant polyploid stress resistance has been a hot topic in biological research. In this study, Populus diploids and first division restitution (FDR) and second division restitution (SDR) triploids were selected as research materials. All materials were treated with 70 mM NaCl solutions for 30 days in the same pot environment. We observed the growth state of triploids and diploids and determined the ratio of potassium and sodium ions, peroxidase (POD) activity, proline content, and ABA and jasmonic acid (JA) hormone content in leaves in the same culture environment with the same concentration of NaCl solution treatment. In addition, RNA-seq technology was used to study the differential expression of mRNA and miRNA. The results showed that triploid Populus grew well and the K+ content and the K+/Na+ ratio in the salt treatment were significantly lower than those in the control. The contents of ABA, JA, POD, and proline were increased compared with contents in diploid under salt stress. The salt-stress responsive DEGs were mainly involved in ion transport, cell homeostasis, the MAPK signaling pathway, peroxisome, citric acid cycle, and other salt response and growth pathways. The transcription factors mainly included NAC, MYB, MYB_related and AP2/ERF. Moreover, the differentially expressed miRNAs involved 32 families, including 743 miRNAs related to predicted target genes, among which 22 miRNAs were significantly correlated with salt-stress response genes and related to the regulation of hormones, ion transport, reactive oxygen species (ROS) and other biological processes. Our results provided insights into the physiological and molecular aspects for further research into the response mechanisms of allotriploid Populus cathayana to salt stress. This study provided valuable information for the salt tolerance mechanism of allopolyploids.

Plastome-based phylogenomics elucidate relationships in rare Isoëtes species groups from the Neotropics.

The genus Isoëtes is globally distributed. Within the Neotropics, Isoëtes occurs in various habitats and ecosystems, making it an interesting case study to address phylogenetic and biogeographic questions. We sequenced and assembled plastomes and ribosomal DNA (rDNA) sequences to reconstruct phylogenetic relationships in Isoëtes from tropical regions in the Neotropics. The ploidy level of nine taxa was established to address the potential source of phylogenetic incongruence in the genus. Node ages were estimated using MCMCTree. The ancestral range estimates were conducted in BioGeoBEARS. Plastome-based phylogenies were congruent throughout distinct matrices and partition schemes, exhibiting high support for almost all nodes. Whereas, we found incongruences between the rDNA and plastome datasets. Chromosome counts identified three diploids, five tetraploids and one likely hexaploid among Neotropical species. Plastome-based node age estimates showed that the radiation of the crown Isoëtes group occurred at 20 Ma, with the diversification of the tropical American (TAA) clade taking place in the Pleistocene at 1.7 Ma. Ancestral range estimates showed that the ancestor of the TAA clade may have evolved first in the dry diagonal area in South America before reaching more humid regions. In addition, the colonization of the Brazilian semiarid region occurred three times, while the occupation of the Cerrado and Amazon regions occurred twice and once, respectively. Our study showed a large unobserved diversity within the genus in warm-dry regions in the Neotropics. Plastomes provided sufficient genomic information to establish a robust phylogenetic framework to answer evolutionary questions in Isoëtes from the Neotropics.

Unravelling the complex story of intergenomic recombination in ABB allotriploid bananas.

BACKGROUND AND AIMS: Bananas (Musa spp.) are a major staple food for hundreds of millions of people in developing countries. The cultivated varieties are seedless and parthenocarpic clones of which the ancestral origin remains to be clarified. The most important cultivars are triploids with an AAA, AAB or ABB genome constitution, with A and B genomes provided by M. acuminata and M. balbisiana, respectively. Previous studies suggested that inter-genome recombinations were relatively common in banana cultivars and that triploids were more likely to have passed through an intermediate hybrid. In this study, we investigated the chromosome structure within the ABB group, composed of starchy cooking bananas that play an important role in food security. METHODS: Using SNP markers called from RADSeq data, we studied the chromosome structure of 36 ABB genotypes spanning defined taxonomic subgroups. To complement our understanding, we searched for similar events within nine AB hybrid genotypes. KEY RESULTS: Recurrent homologous exchanges (HEs), i.e. chromatin exchanges between A and B subgenomes, were unravelled with at least nine founding events (HE patterns) at the origin of ABB bananas prior to clonal diversification. Two independent founding events were found for Pisang Awak genotypes. Two HE patterns, corresponding to genotypes Pelipita and Klue Teparod, show an over-representation of B genome contribution. Three HE patterns mainly found in Indian accessions shared some recombined regions and two additional patterns did not correspond to any known subgroups. CONCLUSIONS: The discovery of the nine founding events allowed an investigation of the possible routes that led to the creation of the different subgroups, which resulted in new hypotheses. Based on our observations, we suggest different routes that gave rise to the current diversity in the ABB cultivars, routes involving primary AB hybrids, routes leading to shared HEs and routes leading to a B excess ratio. Genetic fluxes took place between M. acuminata and M. balbisiana, particularly in India, where these unbalanced AB hybrids and ABB allotriploids originated, and where cultivated M. balbisiana are abundant. The result of this study clarifies the classification of ABB cultivars, possibly leading to the revision of the classification of this subgroup.

Differences between diploid donors are the main contributing factor for subgenome asymmetry measured in either gene ratio or relative diversity in allopolyploids.

Subgenome asymmetry (SA) has routinely been attributed to different responses between the subgenomes of a polyploid to various stimuli during evolution. Here, we compared subgenome differences in gene ratio and relative diversity between artificial and natural genotypes of several allopolyploid species. Surprisingly, consistent differences were not detected between these two types of polyploid genotypes, although they differ in times exposed to evolutionary selection. The estimated ratio of shared genes between a subgenome and its diploid donor was invariably higher for the artificial allopolyploid genotypes than those for the natural genotypes, which is expected as it is now well-known that many genes in a species are not shared among all individuals. As the exact diploid parent for a given subgenome is unknown, the estimated ratios of shared genes for the natural genotypes would also include difference among individual genotypes of the diploid donor species. Further, we detected the presence of SA in genotypes before the completion of the polyploidization events as well as in those which were not formed via polyploidization. These results indicate that SA may, to a large degree, reflect differences between its diploid donors or that changes occurred during polyploid evolution are defined by their donor genomes.

ChIP-cloning analysis uncovers centromere-specific retrotransposons in Brassica nigra and reveals their rapid diversification in Brassica allotetraploids.

Centromeres are indispensable functional units of chromosomes. The evolutionary mechanisms underlying the rapid evolution of centromeric repeats, especially those following polyploidy, remain unknown. In this study, we isolated centromeric sequences of Brassica nigra, a model diploid progenitor (B genome) of the allopolyploid species B. juncea (AB genome) and B. carinata (BC genome) by chromatin immunoprecipitation of nucleosomes containing the centromere-specific histone CENH3. Sequence analysis detected no centromeric satellite DNAs, and most B. nigra centromeric repeats were found to originate from Tyl/copia-class retrotransposons. In cytological analyses, six of the seven analyzed repeat clusters had no FISH signals in A or C genomes of the related diploid species B. rapa and B. oleracea. Notably, five repeat clusters had FISH signals in both A and B subgenomes in the tetraploid B. juncea. In the tetraploid B. carinata, only CL23 displayed three pairs of signals in terminal or interstitial regions of the C-derived chromosome, and no evidence of colonization of CLs onto C-subgenome centromeres was found in B. carinata. This observation suggests that centromeric repeats spread and proliferated between genomes after polyploidization. CL3 and CRB are likely ancient centromeric sequences arising prior to the divergence of diploid Brassica which have detected signals across the genus. And in allotetraploids B. juncea and B. carinata, the FISH signal intensity of CL3 and CRB differed among subgenomes. We discussed possible mechanisms for centromeric repeat divergence during Brassica speciation and polyploid evolution, thus providing insights into centromeric repeat establishment and targeting.

FitTetra 2.0 - improved genotype calling for tetraploids with multiple population and parental data support.

BACKGROUND: Genetic studies in tetraploids are lagging behind in comparison with studies of diploids as the complex genetics of tetraploids require much more elaborated computational methodologies. Recent advancements in development of molecular techniques and computational tools facilitate new methods for automated, high-throughput genotype calling in tetraploid species. We report on the upgrade of the widely-used fitTetra software aiming to improve its accuracy, which to date is hampered by technical artefacts in the data. RESULTS: Our upgrade of the fitTetra package is designed for a more accurate modelling of complex collections of samples. The package fits a mixture model where some parameters of the model are estimated separately for each sub-collection. When a full-sib family is analyzed, we use parental genotypes to predict the expected segregation in terms of allele dosages in the offspring. More accurate modelling and use of parental data increases the accuracy of dosage calling. We tested the package on data obtained with an Affymetrix Axiom 60 k array and compared its performance with the original version and the recently published ClusterCall tool, showing that at least 20% more SNPs could be called with our updated. CONCLUSION: Our updated software package shows clearly improved performance in genotype calling accuracy. Estimation of mixing proportions of the underlying dosage distributions is separated for full-sib families (where mixture proportions can be estimated from the parental dosages and inheritance model) and unstructured populations (where they are based on the assumption of Hardy-Weinberg equilibrium). Additionally, as the distributions of signal ratios of the dosage classes can be assumed to be the same for all populations, including parental data for some subpopulations helps to improve fitting other populations as well. The R package fitTetra 2.0 is freely available under the GNU Public License as Additional file with this article.

Chromosome painting in meiosis reveals pairing of specific chromosomes in polyploid Solanum species.

Analysis of chromosome pairing has been an important tool to assess the genetic similarity of homologous and homoeologous chromosomes in polyploids. However, it is technically challenging to monitor the pairing of specific chromosomes in polyploid species, especially for plant species with a large number of small chromosomes. We developed oligonucleotide-based painting probes for four different potato chromosomes. We demonstrate that these probes are robust enough to monitor a single chromosome throughout the prophase I of meiosis in polyploid Solanum species. Cultivated potato (Solanum tuberosum, 2n = 4x = 48) is an autotetraploid. We demonstrate that the four copies of each potato chromosome pair as a quadrivalent in 66-78% of the meiotic cells at the pachytene stage. Solanum demissum (2n = 6x = 72) is a hexaploid and has been controversial regarding its nature as an autopolyploid or allopolyploid. Interestingly, no hexavalent pairing was observed in meiosis. Instead, we observed three independent bivalents in 83-98% of the meiotic cells at late diakinesis and early metaphase I for the four chromosomes. These results suggest that S. demissum has evolved into a cytologically stable state with predominantly bivalent pairing in meiosis.

Molecular cytogenetic characterisation of Elytrigia ×mucronata, a natural hybrid of E. intermedia and E. repens (Triticeae, Poaceae).

BACKGROUND: Interspecific hybridisation resulting in polyploidy is one of the major driving forces in plant evolution. Here, we present data from the molecular cytogenetic analysis of three cytotypes of Elytrigia ×mucronata using sequential fluorescence (5S rDNA, 18S rDNA and pSc119.2 probes) and genomic in situ hybridisation (four genomic probes of diploid taxa, i.e., Aegilops, Dasypyrum, Hordeum and Pseudoroegneria). RESULTS: The concurrent presence of Hordeum (descended from E. repens) and Dasypyrum + Aegilops (descended from E. intermedia) chromosome sets in all cytotypes of E. ×mucronata confirmed the assumed hybrid origin of the analysed plants. The following different genomic constitutions were observed for E. ×mucronata. Hexaploid plants exhibited three chromosome sets from Pseudoroegneria and one chromosome set each from Aegilops, Hordeum and Dasypyrum. Heptaploid plants harboured the six chromosome sets of the hexaploid plants and an additional Pseudoroegneria chromosome set. Nonaploid cytotypes differed in their genomic constitutions, reflecting different origins through the fusion of reduced and unreduced gametes. The hybridisation patterns of repetitive sequences (5S rDNA, 18S rDNA, and pSc119.2) in E. ×mucronata varied between and within cytotypes. Chromosome alterations that were not identified in the parental species were found in both heptaploid and some nonaploid plants. CONCLUSIONS: The results confirmed that both homoploid hybridisation and heteroploid hybridisation that lead to the coexistence of four different haplomes within single hybrid genomes occur in Elytrigia allopolyploids. The chromosomal alterations observed in both heptaploid and some nonaploid plants indicated that genome restructuring occurs during and/or after the hybrids arose. Moreover, a specific chromosomal translocation detected in one of the nonaploids indicated that it was not a primary hybrid. Therefore, at least some of the hybrids are fertile. Hybridisation in Triticeae allopolyploids clearly and significantly contributes to genomic diversity. Different combinations of parental haplomes coupled with chromosomal alterations may result in the establishment of unique lineages, thus providing raw material for selection.

Meiotic abnormalities affect genetic constitution and pollen viability in dicots from Indian cold deserts.

BACKGROUND: Meiotic abnormalities lead to morphological and genetic variations which caused not only to evolution but also intraspecific reproductive barriers. During present study of detailed meiotic course in dicotyledonous plants sampled from Indian cold deserts, various meiotic abnormalities have been detected. For this, the plant materials fixed in Carnoy's fixative and studied detailed meiotic course by standard squash method in 1% acetocarmine. RESULTS: Meiotic abnormalities have been presently detected in 71 species which include multiple associations in diploids (Achillea millefolium L.), multivalents and univalents in polyploids (4 species), cytomixis (40 species), chromosome stickiness (20 species), nonsynchronous disjunction of bivalents (32 species), interbivalent connections (15 species), synaptic mutants (2 species), syncyte meiocytes (2 species), abnormal spindles (7 species), and fusion of pollen grains (1 species), laggards and chromatin bridges, hypo-, hyperploid PMCs, monads, dyads, triads, tetrads with micronuclei and polyads. CONCLUSIONS: Consequently, variable sized apparently fertile pollen grains and considerable amount of sterile pollen grains are resulted as end products which lead to different genetic constitution (aneuploids and polyploids) and curtailed sexual reproductive success in these species.

Effects of polyploidization on the contents of photosynthetic pigments are largely population-specific.

The contents of photosynthetic pigments are an important indicator of many processes taking place in the plant body. Still, however, our knowledge of the effects of polyploidization, a major driver of speciation in vascular plants, on the contents of photosynthetic pigments is very sparse. We compared the contents of photosynthetic pigments among natural diploids, natural tetraploids, and synthetic tetraploids. The material originated from four natural mixed-cytotype populations of diploid and autotetraploid Vicia cracca (Fabaceae) occurring in the contact zone between the cytotypes in Central Europe and was cultivated under uniform conditions. We explored whether the contents of pigments are primarily driven by polyploidization or by subsequent evolution of the polyploid lineage and whether the patterns differ between populations. We also explored the relationship between pigment contents and plant performance. We found very few significant effects of the cytotype on the individual pigments but many significant interactions between the cytotype and the population. In pair-wise comparisons, many comparisons were not significant. The prevailing pattern among the significant once was that the contents of pigments were determined by polyploidization rather than by subsequent evolution of the polyploid lineage. The contents of the pigments turned out to be a useful predictor of plant performance not only at the time of material collection, but also at the end of the growing season. Further studies exploring differences in the contents of photosynthetic pigments in different cytotypes using replicated populations and assessing their relationship to plant performance are needed to assess the generality of our findings.

Genotyping by sequencing can reveal the complex mosaic genomes in gene pools resulting from reticulate evolution: a case study in diploid and polyploid citrus.

BACKGROUND AND AIMS: Reticulate evolution, coupled with reproductive features limiting further interspecific recombinations, results in admixed mosaics of large genomic fragments from the ancestral taxa. Whole-genome sequencing (WGS) data are powerful tools to decipher such complex genomes but still too costly to be used for large populations. The aim of this work was to develop an approach to infer phylogenomic structures in diploid, triploid and tetraploid individuals from sequencing data in reduced genome complexity libraries. The approach was applied to the cultivated Citrus gene pool resulting from reticulate evolution involving four ancestral taxa, C. maxima, C. medica, C. micrantha and C. reticulata. METHODS: A genotyping by sequencing library was established with the restriction enzyme ApeKI applying one base (A) selection. Diagnostic single nucleotide polymorphisms (DSNPs) for the four ancestral taxa were mined in 29 representative varieties. A generic pipeline based on a maximum likelihood analysis of the number of read data was established to infer ancestral contributions along the genome of diploid, triploid and tetraploid individuals. The pipeline was applied to 48 diploid, four triploid and one tetraploid citrus accessions. KEY RESULTS: Among 43 598 mined SNPs, we identified a set of 15 946 DSNPs covering the whole genome with a distribution similar to that of gene sequences. The set efficiently inferred the phylogenomic karyotype of the 53 analysed accessions, providing patterns for common accessions very close to that previously established using WGS data. The complex phylogenomic karyotypes of 21 cultivated citrus, including bergamot, triploid and tetraploid limes, were revealed for the first time. CONCLUSIONS: The pipeline, available online, efficiently inferred the phylogenomic structures of diploid, triploid and tetraploid citrus. It will be useful for any species whose reproductive behaviour resulted in an interspecific mosaic of large genomic fragments. It can also be used for the first generations of interspecific breeding schemes.

Retroelements and DNA Methylation Could Contribute to Diversity of 5S rDNA in Agave L.

Agave L. is a genus of economic importance, and many of the 166 species in the American plant genus Agave L. inhabit high-stress environments, which makes the genus promising for facing global climate change. However, sustainable use of economically important species without interfering with their ecology and evolution requires generating knowledge about the factors responsible for their genetic variation and diversity and, on this basis, their adaptation and speciation. Few genetic studies exploring the evolutionary relationships, speciation processes, genetic variability and diversity within species of Agave are currently available. Analyses of rDNA loci have been performed with the purpose of determining the genetic variability and diversity of the genus Agave, and these loci have been used as genetic markers of ploidy. However, the factors involved in the diversity of 5S rDNA regions in Agave have not yet been studied in depth. Our study explored the possible mechanisms of genetic (retroelements) and epigenetic (DNA methylation) diversity in 5S rDNA regions in Agave. We characterized the 5S rDNA gene tandem in species of the genus with different ploidy numbers and determined the levels of methylation in 13 haplotypes of 5S rDNA and in four non-transcribed spacers (NTS). Our results showed highly dynamic methylation with a high percentage in haplotypes and NTS of 5S rDNA regions in Agave. The characterization of the 5S rDNA tandem array in Agave revealed vestigial remains of the Cassandra terminal-repeat retrotransposon in miniature (TRIM). Our analysis supported previous results suggesting that in species of Agave L., regulation and diversity of 5S rDNA regions are controlled by coordinated genetic and epigenetic events, which will vary according to the species and the level of ploidy. The artificial pressure to which some agave crops are subjected may affect the mechanisms of evolution of gene 5S rDNA.