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.

Genomic evidence for asymmetric introgression by sexual selection in the common wall lizard.

Strongly selected characters can be transferred from one lineage to another with limited genetic exchange, resulting in asymmetric introgression and a mosaic genome in the receiving population. However, systems are rarely sufficiently well studied to link the pattern of introgression to its underlying process. Male common wall lizards in western Italy exhibit exaggeration of a suite of sexually selected characters that make them outcompete males from a distantly related lineage that lack these characters. This results in asymmetric hybridization and adaptive introgression of the suite of characters following secondary contact. We developed genomewide markers to infer the demographic history of gene flow between different genetic lineages, identify the spread of the sexually selected syndrome, and test the prediction that introgression should be asymmetric and heterogeneous across the genome. Our results show that secondary contact was accompanied by gene flow in both directions across most of the genome, but with approximately 3% of the genome showing highly asymmetric introgression in the predicted direction. Demographic simulations reveal that this asymmetric gene flow is more recent than the initial secondary contact, and the data suggest that the exaggerated male sexual characters originated within the Italian lineage and subsequently spread throughout this lineage before eventually reaching the contact zone. These results demonstrate that sexual selection can cause a suite of characters to spread throughout both closely and distantly related lineages with limited gene flow across the genome at large.

Long interspersed nuclear elements (LINEs) show tissue-specific, mosaic genome and methylation-unrestricted, widespread expression of noncoding RNAs in somatic tissues of the rat.

We report strong somatic and germ line expression of LINE RNAs in eight different tissues of rat by using a novel ~2.8 kb genomic PstI-LINE DNA (P1-LINE) isolated from the rat brain. P1-LINE is present in a 93 kb LINE-SINE-cluster in sub-telomeric region of chromosome 12 (12p12) and as multiple truncated copies interspersed in all rat chromosomes. P1-LINEs occur as inverted repeats at multiple genomic loci in tissue-specific and mosaic patterns. P1-LINE RNAs are strongly expressed in brain, liver, lungs, heart, kidney, testes, spleen and thymus into large to small heterogeneous RNAs (~5.0 to 0.2 kb) in tissue-specific and dynamic patterns in individual rats. P1-LINE DNA is strongly methylated at CpG-dinucleotides in most genomic copies in all the tissues and weakly hypomethylated in few copies in some tissues. Small (700-75 nt) P1-LINE RNAs expressed in all tissues may be possible precursors for small regulatory RNAs (PIWI-interacting/piRNAs) bioinformatically derived from P1-LINE. The strong and dynamic expression of LINE RNAs from multiple chromosomal loci and the putative piRNAs in somatic tissues of rat under normal physiological conditions may define functional chromosomal domains marked by LINE RNAs as long noncoding RNAs (lncRNAs) unrestricted by DNA methylation. The tissue-specific, dynamic RNA expression and mosaic genomic distribution of LINEs representing a steady-state genomic flux of retrotransposon RNAs suggest for biological role of LINE RNAs as long ncRNAs and small piRNAs in mammalian tissues independent of their cellular fate for translation, reverse-transcription and retrotransposition. This may provide evolutionary advantages to LINEs and mammalian genomes.

When barcoding fails: Genome chimerization (admixing) and reticulation obscure phylogenetic and taxonomic relationships.

DNA barcoding is based on the premise that the barcode sequences can distinguish individuals (strains) of different species because their sequence variation between species exceeds that within species. The primary barcodes used in fungal and yeast taxonomy are the ITS segments and the LSU (large subunit) D1/D2 domain of the homogenized multicopy rDNA repeats. The secondary barcodes are conserved segments of protein-encoding genes, which usually have single copies in haploid genomes. This study shows that the analysis of barcode sequences fails to reconstruct accurate species trees and differentiate species when the organisms have chimeric genomes composed of admixed mosaics of different origins. It is shown that the type strains of 10 species of the pulcherrima clade of the ascomycetous yeast genus Metschnikowia cannot be differentiated with standard barcodes because their intragenomic diversity is comparable to or even higher than the interstrain diversity. The analysis of a large group of genes of the sequenced genomes of the clade and the viability and segregation of the hybrids of ex-type strains indicate that the high intragenomic barcode differences can be attributed to admixed genome structures. Because of the mosaic structures of the genomes, the rDNA repeats do not form continuous arrays and thus cannot be homogenized. Since the highly diverse ITS and D1/D2 sequences of the type strains form a continuous pool including pseudogenes, the evolution of their rDNA appears to involve reticulation. The secondary barcode sequences and the nonbarcode genes included in the analysis show incongruent phylogenetic relationships among the type strains, which can also be attributed to differences in the phylogenetic histories of the genes.

Genome analysis of Salmonella enterica serovar Typhimurium bacteriophage L, indicator for StySA (StyLT2III) restriction-modification system action.

Bacteriophage L, a P22-like phage of Salmonella enterica sv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems of Salmonella. We report the complete genome sequences of bacteriophage L cI-40 13-am43 and L cII-101; the deduced sequence of wildtype L is 40,633 bp long with a 47.5% GC content. We compare this sequence with those of P22 and ST64T, and predict 72 Coding Sequences, 2 tRNA genes and 14 intergenic rho-independent transcription terminators. The overall genome organization of L agrees with earlier genetic and physical evidence; for example, no secondary immunity region (immI: ant, arc) or known genes for superinfection exclusion (sieA and sieB) are present. Proteomic analysis confirmed identification of virion proteins, along with low levels of assembly intermediates and host cell envelope proteins. The genome of L is 99.9% identical at the nucleotide level to that reported for phage ST64T, despite isolation on different continents ∼35 years apart. DNA modification by the epigenetic regulator Dam is generally incomplete. Dam modification is also selectively missing in one location, corresponding to the P22 phase-variation-sensitive promoter region of the serotype-converting gtrABC operon. The number of sites for SenLTIII (StySA) action may account for stronger restriction of L (13 sites) than of P22 (3 sites).

The Interspecific Fungal Hybrid Verticillium longisporum Displays Subgenome-Specific Gene Expression.

Hybridization is an important evolutionary mechanism that can enable organisms to adapt to environmental challenges. It has previously been shown that the fungal allodiploid species Verticillium longisporum, the causal agent of verticillium stem striping in rapeseed, originated from at least three independent hybridization events between two haploid Verticillium species. To reveal the impact of genome duplication as a consequence of hybridization, we studied the genome and transcriptome dynamics upon two independent V. longisporum hybridization events, represented by the hybrid lineages "A1/D1" and "A1/D3." We show that V. longisporum genomes are characterized by extensive chromosomal rearrangements, including between parental chromosomal sets. V. longisporum hybrids display signs of evolutionary dynamics that are typically associated with the aftermath of allodiploidization, such as haploidization and more relaxed gene evolution. The expression patterns of the two subgenomes within the two hybrid lineages are more similar than those of the shared A1 parent between the two lineages, showing that the expression patterns of the parental genomes homogenized within a lineage. However, as genes that display differential parental expression in planta do not typically display the same pattern in vitro, we conclude that subgenome-specific responses occur in both lineages. Overall, our study uncovers genomic and transcriptomic plasticity during the evolution of the filamentous fungal hybrid V. longisporum and illustrates its adaptive potential. IMPORTANCEVerticillium is a genus of plant-associated fungi that includes a few plant pathogens that collectively affect a wide range of hosts. On several occasions, haploid Verticillium species hybridized into the stable allodiploid species Verticillium longisporum, which is, in contrast to haploid Verticillium species, a Brassicaceae specialist. Here, we studied the evolutionary genome and transcriptome dynamics of V. longisporum and the impact of the hybridization. V. longisporum genomes display a mosaic structure due to genomic rearrangements between the parental chromosome sets. Similar to other allopolyploid hybrids, V. longisporum displays an ongoing loss of heterozygosity and more relaxed gene evolution. Also, differential parental gene expression is observed, with enrichment for genes that encode secreted proteins. Intriguingly, the majority of these genes display subgenome-specific responses under differential growth conditions. In conclusion, hybridization has incited the genomic and transcriptomic plasticity that enables adaptation to environmental changes in a parental allele-specific fashion.

Quantifying and Characterizing Distributive Conjugal Transfer in Mycobacterium smegmatis.

Horizontal gene transfer (HGT) in prokaryotes disseminates genetic information throughout a population and can facilitate adaptation and evolution of the species. Mycobacteria utilize an atypical method of conjugation called distributive conjugal transfer (DCT), which results in mosaic genomes and the potential for accelerated evolution beyond that enabled by the more classical oriT-mediated conjugation. The following is a description of the basic DCT protocol, some possible variations of the assay, and examples of downstream applications to better understand mycobacterial functions.

Evidence for multiple recombination events within foot-and-mouth disease viruses circulating in West Eurasia.

Phylogenetic studies on foot-and-mouth disease viruses (FMDVs) circulating in the West Eurasian region have largely focused on the genomic sequences encoding the structural proteins that determine the serotype. The present study has compared near-complete genome sequences of FMDVs representative of the viruses that circulate in this region. The near-complete genome sequences (ca. 7,600 nt) were generated from multiple overlapping RT-PCR products. These amplicons were from FMDVs belonging to serotypes O, A and Asia-1, including members of the O-PanAsia-II and the A-Iran05 lineages, and of Group-II and Group-VII (Sindh-08) within serotype Asia-1, which are currently predominant and widespread in West Eurasia. These new sequences were analysed together with other sequences obtained from GenBank. Comparison of different regions of the FMDVs genomes revealed evidence for multiple, inter-serotypic, recombination events between FMDVs belonging to the serotypes O, A and Asia-1. It is concluded from the present study that dramatic changes in virus sequences can occur in the field through recombination between different FMDV genomes. These analyses provide information about the ancestry of the serotype O, A and Asia-1 FMDVs that are currently circulating within the West Eurasian region.

Evidence for suppression of immunity as a driver for genomic introgressions and host range expansion in races of Albugo candida, a generalist parasite.

How generalist parasites with wide host ranges can evolve is a central question in parasite evolution. Albugo candida is an obligate biotrophic parasite that consists of many physiological races that each specialize on distinct Brassicaceae host species. By analyzing genome sequence assemblies of five isolates, we show they represent three races that are genetically diverged by ∼1%. Despite this divergence, their genomes are mosaic-like, with ∼25% being introgressed from other races. Sequential infection experiments show that infection by adapted races enables subsequent infection of hosts by normally non-infecting races. This facilitates introgression and the exchange of effector repertoires, and may enable the evolution of novel races that can undergo clonal population expansion on new hosts. We discuss recent studies on hybridization in other eukaryotes such as yeast, Heliconius butterflies, Darwin's finches, sunflowers and cichlid fishes, and the implications of introgression for pathogen evolution in an agro-ecological environment.