Centromeres Off the Hook: Massive Changes in Centromere Size and Structure Following Duplication of CenH3 Gene in Fabeae Species.

In most eukaryotes, centromere is determined by the presence of the centromere-specific histone variant CenH3. Two types of chromosome morphology are generally recognized with respect to centromere organization. Monocentric chromosomes possess a single CenH3-containing domain in primary constriction, whereas holocentric chromosomes lack the primary constriction and display dispersed distribution of CenH3. Recently, metapolycentric chromosomes have been reported in Pisum sativum, representing an intermediate type of centromere organization characterized by multiple CenH3-containing domains distributed across large parts of chromosomes that still form a single constriction. In this work, we show that this type of centromere is also found in other Pisum and closely related Lathyrus species, whereas Vicia and Lens genera, which belong to the same legume tribe Fabeae, possess only monocentric chromosomes. We observed extensive variability in the size of primary constriction and the arrangement of CenH3 domains both between and within individual Pisum and Lathyrus species, with no obvious correlation to genome or chromosome size. Search for CenH3 gene sequences revealed two paralogous variants, CenH3-1 and CenH3-2, which originated from a duplication event in the common ancestor of Fabeae species. The CenH3-1 gene was subsequently lost or silenced in the lineage leading to Vicia and Lens, whereas both genes are retained in Pisum and Lathyrus. Both of these genes appear to have evolved under purifying selection and produce functional CenH3 proteins which are fully colocalized. The findings described here provide the first evidence for a highly dynamic centromere structure within a group of closely related species, challenging previous concepts of centromere evolution.

Comparative genomics of the mimicry switch in Papilio dardanus.

The African Mocker Swallowtail, Papilio dardanus, is a textbook example in evolutionary genetics. Classical breeding experiments have shown that wing pattern variation in this polymorphic Batesian mimic is determined by the polyallelic H locus that controls a set of distinct mimetic phenotypes. Using bacterial artificial chromosome (BAC) sequencing, recombination analyses and comparative genomics, we show that H co-segregates with an interval of less than 500 kb that is collinear with two other Lepidoptera genomes and contains 24 genes, including the transcription factor genes engrailed (en) and invected (inv). H is located in a region of conserved gene order, which argues against any role for genomic translocations in the evolution of a hypothesized multi-gene mimicry locus. Natural populations of P. dardanus show significant associations of specific morphs with single nucleotide polymorphisms (SNPs), centred on en. In addition, SNP variation in the H region reveals evidence of non-neutral molecular evolution in the en gene alone. We find evidence for a duplication potentially driving physical constraints on recombination in the lamborni morph. Absence of perfect linkage disequilibrium between different genes in the other morphs suggests that H is limited to nucleotide positions in the regulatory and coding regions of en. Our results therefore support the hypothesis that a single gene underlies wing pattern variation in P. dardanus.

Epigenetic Histone Marks of Extended Meta-Polycentric Centromeres of Lathyrus and Pisum Chromosomes.

Species of the legume genera Lathyrus and Pisum possess chromosomes that exhibit a unique structure of their centromeric regions, which is clearly apparent during metaphase by the formation of extended primary constrictions which span up to a third of the length of the chromosome. In addition, these species express two different variants of the CenH3 protein which are co-localized in multiple domains along the poleward surface of the primary constrictions. Here, we show that the constrictions represent a distinct type of chromatin differing from the chromosome arms. In metaphase, histone phosphorylation patterns including H3S10ph, H3S28ph, and H3T3ph were observed along the entire constriction, in a way similar to holocentric chromosomes. On the other hand, distribution of phosphorylated H2AT120 was different from that previously reported from either, holocentric and monocentric chromosomes, occurring at chromatin surrounding but not overlapping CenH3 domains. Since some of these phosphorylations play a role in chromatid cohesion, it can be assumed that they facilitate correct chromosome segregation by ensuring that multiple separate CenH3 domains present on the same chromatid are oriented toward the same pole. The constrictions also displayed distinct patterns of histone methylation marks, being enriched in H3K9me2 and depleted in H3K4me3 and H3K27me2 compared to the chromosome arms. Super-resolution fluorescence microscopy revealed that although both CenH3 protein variants are present in all CenH3 domains detected on metaphase chromosomes, they are only partially co-localized while there are chromatin subdomains which are mostly made of only one CenH3 variant. Taken together, these data revealed specific features of extended primary constrictions of Lathyrus and Pisum and support the idea that they may represent an intermediate stage between monocentric and holocentric chromosomes.

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae.

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55-83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes.

Molecular divergence of the W chromosomes in pyralid moths (Lepidoptera).

Most Lepidoptera have a WZ/ZZ sex chromosome system. We compared structure of W chromosomes in four representatives of the family Pyralidae--Ephestia kuehniella, Cadra cautella, Plodia interpunctella, and Galleria mellonella--tracing pachytene bivalents which provide much higher resolution than metaphase chromosomes. In each species, we prepared a W-chromosome painting probe from laser-microdissected W-chromatin of female polyploid nuclei. The Ephestia W-probe was cross-hybridized to chromosomes of the other pyralids to detect common parts of their W chromosomes, while the species-specific W-probes identified the respective W chromosome. This so-called Zoo-FISH revealed a partial homology of W-chromosome regions between E. kuehniella and two other pyralids, C. cautella and P. interpunctella, but almost no homology with G. mellonella. The results were consistent with phylogenetic relationships between the species. We also performed comparative genomic hybridization, which indicated that the W chromosome of C. cautella is composed mainly of repetitive DNA common to both sexes but accumulated in the W chromosome, whereas E. kuehniella, P. interpunctella, and G. mellonella W chromosomes also possess a large amount of female specific DNA sequences, but differently organized. Our results support the hypothesis of the accelerated molecular divergence of the lepidopteran W chromosomes in the absence of meiotic recombination.

Probing the W chromosome of the codling moth, Cydia pomonella, with sequences from microdissected sex chromatin.

The W chromosome of the codling moth, Cydia pomonella, like that of most Lepidoptera species, is heterochromatic and forms a female-specific sex chromatin body in somatic cells. We collected chromatin samples by laser microdissection from euchromatin and W-chromatin bodies. DNA from the samples was amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and used to prepare painting probes and start an analysis of the W-chromosome sequence composition. With fluorescence in situ hybridization (FISH), the euchromatin probe labelled all chromosomes, whereas the W-chromatin DNA proved to be a highly specific W-chromosome painting probe. For sequence analysis, DOP-PCR-generated DNA fragments were cloned, sequenced, and tested by Southern hybridization. We recovered single-copy and low-copy W-specific sequences, a sequence that was located only in the W and the Z chromosome, multi-copy sequences that were enriched in the W chromosome but occurred also elsewhere, and ubiquitous multi-copy sequences. Three of the multi-copy sequences were recognized as derived from hitherto unknown retrotransposons. The results show that our approach is feasible and that the W-chromosome composition of C. pomonella is not principally different from that of Bombyx mori or from that of Y chromosomes of several species with an XY sex-determining mechanism. The W chromosome has attracted repetitive sequences during evolution but also contains unique sequences.

Conserved synteny of genes between chromosome 15 of Bombyx mori and a chromosome of Manduca sexta shown by five-color BAC-FISH.

The successful assignment of the existing genetic linkage groups (LGs) to individual chromosomes and the second-generation linkage map obtained by mapping a large number of bacterial artificial chromosome (BAC) contigs in the silkworm, Bombyx mori, together with public nucleotide sequence databases, offer a powerful tool for the study of synteny between karyotypes of B. mori and other lepidopteran species. Conserved synteny of genes between particular chromosomes can be identified by comparatively mapping orthologous genes of the corresponding linkage groups with the help of BAC-FISH (fluorescent in situ hybridization). This technique was established in B. mori for 2 differently labeled BAC probes simultaneously hybridized to pachytene bivalents. To achieve higher-throughput comparative mapping using BAC-FISH in Lepidoptera, we developed a protocol for five-color BAC-FISH, which allowed us to map simultaneously 6 different BAC probes to chromosome 15 in B. mori. We identified orthologs of 6 B. mori LG15 genes (RpP0, RpS8, eIF3, RpL7A, RpS23, and Hsc70) for the tobacco hornworm, Manduca sexta, and selected the ortholog-containing BAC clones from an M. sexta BAC library. All 6 M. sexta BAC clones hybridized to a single M. sexta bivalent in pachytene spermatocytes. Thus, we have confirmed the conserved synteny between the B. mori chromosome 15 and the corresponding M. sexta chromosome (hence provisionally termed chromosome 15).

Codling moth cytogenetics: karyotype, chromosomal location of rDNA, and molecular differentiation of sex chromosomes.

We performed a detailed karyotype analysis in the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), the key pest of pome fruit in the temperate regions of the world. The codling moth karyotype consisted of 2n = 56 chromosomes of a holokinetic type. The chromosomes were classified into 5 groups according to their sizes: extra large (3 pairs), large (3 pairs), medium (15 pairs), small (5 pairs), and dot-like (2 pairs). In pachytene nuclei of both sexes, a curious NOR (nucleolar organizer region) bivalent was observed. It carried 2 nucleoli, each associated with one end of the bivalent. FISH with an 18S ribosomal DNA probe confirmed the presence of 2 clusters of rRNA genes at the opposite ends of the bivalent. In accordance with this finding, 2 homologous NOR chromosomes were identified in mitotic metaphase, each showing hybridization signals at both ends. In highly polyploid somatic nuclei, females showed a large heterochromatin body, the so-called sex chromatin or W chromatin. The heterochromatin body was absent in male nuclei, indicating a WZ/ZZ (female/male) sex chromosome system. In keeping with the sex chromatin status, pachytene oocytes showed a sex chromosome bivalent (WZ) that was easily discernible by its heterochromatic W thread. To study molecular differentiation of the sex chromosomes, we employed genomic in situ hybridization (GISH) and comparative genomic hybridization (CGH). GISH detected the W chromosome by strong binding of the Cy3-labelled, female-derived DNA probe. With CGH, both the Cy3-labelled female-derived probe and Fluor-X labelled male-derived probe evenly bound to the W chromosome. This suggested that the W chromosome is predominantly composed of repetitive DNA sequences occurring scattered in other chromosomes but accumulated in the W chromosome. The demonstrated ways of W chromosome identification will facilitate the development of genetic sexing strains desirable for pest control using the sterile insect technique.