Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis

Chromosome painting is a powerful technique for chromosome and genome studies. We developed a flexible chromosome painting technique based on multiplex PCR of a synthetic oligonucleotide (oligo) library in cucumber (Cucumis sativus L., 2n = 14). Each oligo in the library was associated with a universal as well as nested specific primers for amplification, which allow the generation of different probes from the same oligo library. We were also able to generate double‐stranded labelled oligos, which produced much stronger signals than single‐stranded labelled oligos, by amplification using fluorophore‐conjugated primer pairs. Oligos covering cucumber chromosome 1 (Chr1) and chromosome 4 (Chr4) consisting of eight segments were synthesized in one library. Different oligo probes generated from the library painted the corresponding chromosomes/segments unambiguously, especially on pachytene chromosomes. This technique was then applied to study the homoeologous relationships among cucumber, C. hystrix and C. melo chromosomes based on cross‐species chromosome painting using Chr4 probes. We demonstrated that the probe was feasible to detect interspecies chromosome homoeologous relationships and chromosomal rearrangement events. Based on its advantages and great convenience, we anticipate that this flexible oligo‐painting technique has great potential for the studies of the structure, organization, and evolution of chromosomes in any species with a sequenced genome.     

Next‐generation sequencing,FISH mapping and synteny‐based modeling reveal mechanisms of decreasing dysploidy in Cucumis

In the large Cucurbitaceae genus Cucumis, cucumber (C. sativus) is the only species with 2n = 2x = 14 chromosomes. The majority of the remaining species, including melon (C. melo) and the sister species of cucumber, C. hystrix, have 2n = 2x = 24 chromosomes, implying a reduction from n = 12 to n = 7. To understand the underlying mechanisms, we investigated chromosome synteny among cucumber, C. hystrix and melon using integrated and complementary approaches. We identified 14 inversions and a C. hystrix lineage‐specific reciprocal inversion between C. hystrix and melon. The results reveal the location and orientation of 53 C. hystrix syntenic blocks on the seven cucumber chromosomes, and allow us to infer at least 59 chromosome rearrangement events that led to the seven cucumber chromosomes, including five fusions, four translocations, and 50 inversions. The 12 inferred chromosomes (AK1–AK12) of an ancestor similar to melon and C. hystrix had strikingly different evolutionary fates, with cucumber chromosome C1 apparently resulting from insertion of chromosome AK12 into the centromeric region of translocated AK2/AK8, cucumber chromosome C3 originating from a Robertsonian‐like translocation between AK4 and AK6, and cucumber chromosome C5 originating from fusion of AK9 and AK10. Chromosomes C2, C4 and C6 were the result of complex reshuffling of syntenic blocks from three (AK3, AK5 and AK11), three (AK5, AK7 and AK8) and five (AK2, AK3, AK5, AK8 and AK11) ancestral chromosomes, respectively, through 33 fusion, translocation and inversion events. Previous results (Huang, S., Li, R., Zhang, Z. et al., 2009 , Nat. Genet. 41, 1275–1281; Li, D., Cuevas, H.E., Yang, L., Li, Y., Garcia‐Mas, J., Zalapa, J., Staub, J.E., Luan, F., Reddy, U., He, X., Gong, Z., Weng, Y. 2011a, BMC Genomics, 12, 396) showing that cucumber C7 stayed largely intact during the entire evolution of Cucumis are supported. Results from this study allow a fine‐scale understanding of the mechanisms of dysploid chromosome reduction that has not been achieved previously.     

An efficient Oligo-FISH painting system for revealing chromosome rearrangements and polyploidization in Triticeae

A chromosome-specific painting technique has been developed which combines the most recent approaches of the companion disciplines of molecular cytogenetics and genome research. We developed seven oligonucleotide (oligo) pools derivd from single-copy sequences on chromosomes 1 to 7 of barley (Hordeum vulgare L.) and corresponding collinear regions of wheat (Triticum aestivum L.). The seven groups of pooled oligos comprised between 10 986 and 12 496 45-bp monomers, and these then produced stable fluorescence in situ hybridization (FISH) signals on chromosomes of each linkage group of wheat and barley. The pooled oligo probes were applied to high-throughput karyotyping of the chromosomes of other Triticeae species in the genera Secale, Aegilops, Thinopyrum, and Dasypyrum, and the study also extended to some wheat-alien amphiploids and derived lines. We demonstrated that a complete set of whole-chromosome oligo painting probes facilitated the study of inter-species chromosome homologous relationships and visualized non-homologous chromosomal rearrangements in Triticeae species and some wheat-alien species derivatives. When combined with other non-denaturing FISH procedures using tandem-repeat oligos, the newly developed oligo painting techniques provide an efficient tool for the study of chromosome structure, organization, and evolution among any wild Triticeae species with non-sequenced genomes.     

Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides

Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a single chromosome of cucumber were identified using a newly developed bioinformatic pipeline and then massively synthesized de novo in parallel. The synthesized oligos were amplified and labeled with biotin or digoxigenin for use in fluorescence in situ hybridization (FISH). We developed three different probes with each containing 23,000–27,000 oligos. These probes spanned 8.3–17 Mb of DNA on targeted cucumber chromosomes and had the densities of 1.5–3.2 oligos per kilobases. These probes produced FISH signals on a single cucumber chromosome and were used to paint homeologous chromosomes in other Cucumis species diverged from cucumber for up to 12 million years. The bulked oligo probes allowed us to track a single chromosome in early stages during meiosis. We were able to precisely map the pairing between cucumber chromosome 7 and chromosome 1 of Cucumis hystrix in a F₁ hybrid. These two homeologous chromosomes paired in 71% of prophase I cells but only 25% of metaphase I cells, which may provide an explanation of the higher recombination rates compared to the chiasma frequencies between homeologous chromosomes reported in plant hybrids.     

Generation of an artificial ring chromosome in Arabidopsis by Cre/LoxP‐mediated recombination

A eukaryotic chromosome consists of a centromere, two telomeres and a number of replication origins, and ‘artificial chromosomes’ may be created in yeast and mammals when these three elements are artificially joined and introduced into cells. Plant artificial chromosomes (PACs) have been suggested as new vectors for the development of new crops and as tools for basic research on chromosomes. However, indisputable PAC formation has not yet been confirmed. Here, we present a method for generating PACs in the model plant Arabidopsis thaliana using the Cre/LoxP and Activator/Dissociation element systems. The successfully generated PAC, designated AtARC1 (A. thaliana artificial ring chromosome 1), originated from a centromeric edge of the long arm of chromosome 2, but its size (2.85 Mb) is much smaller than that of the original chromosome (26.3 Mb). Although AtARC1 contains only a short centromere domain consisting of 180 bp repeats approximately 250 kb in length, compared with the 3 Mb domain on the original chromosome 2, centromere‐specific histone H3 (HTR12) was detected on the centromeric region. This result supported the observed stability of the PAC during mitosis in the absence of selection, and transmission of the PAC to the next generation through meiosis. Because AtARC1 contains a unique LoxP site driven by the CaMV 35S promoter, it is possible to introduce a selectable marker and desired transgenes into AtARC1 at the LoxP site using Cre recombinase. Therefore, AtARC1 meets the criteria for a PAC and is a promising vector.     

Extraordinarily conserved chromosomal synteny of Citrus species revealed by chromosome‐specific painting

Reliable identification of individual chromosomes in eukaryotic species is the foundation for comparative chromosome synteny and evolutionary studies. Unfortunately, chromosome identification has been a major challenge for plants with small chromosomes, such as the Citrus species. We developed oligonucleotide‐based chromosome painting probes for all nine chromosomes in Citrus maxima (Pummelo). We were able to identify all C. maxima chromosomes in the same metaphase cells using multiple rounds of sequential fluorescence in situ hybridization with the painting probes. We conducted comparative chromosome painting analysis in six different Citrus and related species. We found that each painting probe hybridized to only a single chromosome in all other five species, suggesting that the six species have maintained a complete chromosomal synteny after more than 9 million years of divergence. No interchromosomal rearrangement was identified in any species. These results support the hypothesis that karyotypes of woody species are more stable than herbaceous plants because woody plants need a longer period to fix chromosome structural variants in natural populations.     

Chromosomal‐level genomes of three rice planthoppers provide new insights into sex chromosome evolution

The brown planthopper Nilaparvata lugens, white‐backed planthopper Sogatella furcifera, and small brown planthopper Laodelphax striatellus are three major insect pests of rice. They are genetically close; however, they differ in several ecological traits such as host range, migration capacity, and in their sex chromosomes. Though the draft genome of these three planthoppers have been previously released, the quality of genome assemblies need to be improved. The absence of chromosome‐level genome resources has hindered in‐depth research of these three species. Here, we performed a de novo genome assembly for N. lugens to increase its genome assembly quality with PacBio and Illumina platforms, increasing the contig N50 to 589.46 Kb. Then, with the new N. lugens genome and previously reported S. furcifera and L. striatellus genome assemblies, we generated chromosome‐level scaffold assemblies of these three planthopper species using HiC scaffolding technique. The scaffold N50s significantly increased to 77.63 Mb, 43.36 Mb and 29.24 Mb for N. lugens, S. furcifera and L. striatellus, respectively. To identify sex chromosomes of these three planthopper species, we carried out genome re‐sequencing of males and females and successfully determined the X and Y chromosomes for N. lugens, and X chromosome for S. furcifera and L. striatellus. The gene content of the sex chromosomes showed high diversity among these three planthoppers suggesting the rapid evolution of sex‐linked genes, and all chromosomes showed high synteny. The chromosome‐level genome assemblies of three planthoppers would provide a valuable resource for a broad range of future research in molecular ecology, and subsequently benefits development of modern pest control strategies.     

A chromosome‐level genome assembly of the parasitoid wasp Pteromalus puparum

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short‐read, PacBio long‐read and Hi‐C scaffolding technologies was used to develop a high‐quality chromosome‐level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome‐level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338 Mb with a contig N50 of 38.7 kb and a scaffold N50 of 1.16 Mb. Hi‐C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8 Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single‐Copy Orthologs) gene set, supporting a high‐quality assembly and annotation. In total, 40.1% (135.6 Mb) of the assembly is composed of repetitive sequences, and 14,946 protein‐coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase‐related proteins and kynurenine–oxoglutarate transaminases, which have amplified in the P. puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P. puparum. These examples illustrate how chromosome‐level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.     

Construction of a bacterial artificial chromosome library of Endoclita excrescens as a tool for comparative gene mapping in Lepidoptera

Karyotype evolution in one of the most diverse and species‐rich group of insects, moths and butterflies (Lepidoptera), has interesting features that remain to be resolved. Recent studies showed that fluorescence in situ hybridization using bacterial artificial chromosome clones (BAC‐FISH) is an efficient cytogenetic method for identification and gene mapping of lepidopteran chromosomes. Using comparative mapping by BAC‐FISH, extensive synteny of genes was revealed between chromosomes of different lepidopteran species based on Bombyx mori genomic information. However, this comparative mapping has been done only in representatives of advanced groups of Lepidoptera. Here we constructed a BAC library of Endoclita excrescens, which belongs to the primitive lepidopteran family Hepialidae. High molecular weight DNA for the library construction was prepared from the pupae by using a rapid nuclear isolation method known in plants. The BAC clones of E. excrescens contain 66.6 kb inserts on average. The successful application of BAC‐FISH showed that the BAC library of E. excrescens is a useful tool for comparative gene mapping on chromosomes of this species.     

Chromosome rearrangements during domestication of cucumber as revealed by high-density genetic mapping and draft genome assembly

Cucumber, Cucumis sativus L. is the only taxon with 2n = 2x = 14 chromosomes in the genus Cucumis. It consists of two cross‐compatible botanical varieties: the cultivated C. sativus var. sativus and the wild C. sativus var. hardwickii. There is no consensus on the evolutionary relationship between the two taxa. Whole‐genome sequencing of the cucumber genome provides a new opportunity to advance our understanding of chromosome evolution and the domestication history of cucumber. In this study, a high‐density genetic map for cultivated cucumber was developed that contained 735 marker loci in seven linkage groups spanning 707.8 cM. Integration of genetic and physical maps resulted in a chromosome‐level draft genome assembly comprising 193 Mbp, or 53% of the 367 Mbp cucumber genome. Strategically selected markers from the genetic map and draft genome assembly were employed to screen for fosmid clones for use as probes in comparative fluorescence in situ hybridization analysis of pachytene chromosomes to investigate genetic differentiation between wild and cultivated cucumbers. Significant differences in the amount and distribution of heterochromatins, as well as chromosomal rearrangements, were uncovered between the two taxa. In particular, six inversions, five paracentric and one pericentric, were revealed in chromosomes 4, 5 and 7. Comparison of the order of fosmid loci on chromosome 7 of cultivated and wild cucumbers, and the syntenic melon chromosome I suggested that the paracentric inversion in this chromosome occurred during domestication of cucumber. The results support the sub‐species status of these two cucumber taxa, and suggest that C. sativus var. hardwickii is the progenitor of cultivated cucumber.     

Dual‐color oligo‐FISH can reveal chromosomal variations and evolution in Oryza species

Fluorescence in situ hybridization using probes based on oligonucleotides (oligo‐FISH) is a useful tool for chromosome identification and karyotype analysis. Here we developed two oligo‐FISH probes that allow the identification of each of the 12 pairs of chromosomes in rice (Oryza sativa). These two probes comprised 25 717 (green) and 25 215 (red) oligos (45 nucleotides), respectively, and generated 26 distinct FISH signals that can be used as a barcode to uniquely label each of the 12 pairs of rice chromosomes. Standard karyotypes of rice were established using this system on both mitotic and meiotic chromosomes. Moreover, dual‐color oligo‐FISH was used to characterize diverse chromosomal abnormalities. Oligo‐FISH analyses using these probes in various wild Oryza species revealed that chromosomes from the AA, BB or CC genomes generated specific and intense signals similar to those in rice, while chromosomes with the EE genome generated less specific signals and the FF genome gave no signal. Together, the oligo‐FISH probes we established will be a powerful tool for studying chromosome variations and evolution in the genus Oryza.     

Improvement of high‐resolution fluorescence in situ hybridisation mapping on chromosomes of Brassica oleracea var. capitata

The low resolution of chromosome‐based Fluorescence in situ hybridisation (FISH) mapping is primarily due to the structure of the plant cell wall and cytoplasm and the compactness of regular chromosomes, which represent a significant obstacle to FISH. In order to improve spatial resolution and signal detection sensitivity, we provide a reproducible method to generate high‐quality extended chromosomes that are ~13 times as long as their pachytene counterparts. We demonstrate that proteinase K used in this procedure is crucial for stretching pachytene chromosomes of Brassica oleracea in the context of a modified Carnoy's II fixative (6:1:3, ethanol:chloroform:acetic acid). The quality of super‐stretched chromosomes was assessed in several FISH experiments. FISH signals from both repetitive 5S rDNA and single‐copy ARC1 on super‐stretched chromosomes are brighter than those on other different types of chromosome due to enhanced accessibility to targets on stretched pachytene chromosomes. In conclusion, the resulting extended chromosomes are suitable for FISH mapping for repetitive DNA sequences and the localisation of a single‐copy locus, and FISH performed on super‐stretched chromosomes can achieve significantly higher sensitivity and spatial resolution than other chromosome‐based FISH mapping techniques.     

Occasional recombination of a selfish X‐chromosome may permit its persistence at high frequencies in the wild

The sex‐ratio X‐chromosome (SR) is a selfish chromosome that promotes its own transmission to the next generation by destroying Y‐bearing sperm in the testes of carrier males. In some natural populations of the fly Drosophila neotestacea, up to 30% of the X‐chromosomes are SR chromosomes. To investigate the molecular evolutionary history and consequences of SR, we sequenced SR and standard (ST) males at 11 X‐linked loci that span the ST X‐chromosome and at seven arbitrarily chosen autosomal loci from a sample of D. neotestacea males from throughout the species range. We found that the evolutionary relationship between ST and SR varies among individual markers, but genetic differentiation between SR and ST is chromosome‐wide and likely due to large chromosomal inversions that suppress recombination. However, SR does not consist of a single multilocus haplotype: we find evidence for gene flow between ST and SR at every locus assayed. Furthermore, we do not find long‐distance linkage disequilibrium within SR chromosomes, suggesting that recombination occurs in females homozygous for SR. Finally, polymorphism on SR is reduced compared to that on ST, and loci displaying signatures of selection on ST do not show similar patterns on SR. Thus, even if selection is less effective on SR, our results suggest that gene flow with ST and recombination between SR chromosomes may prevent the accumulation of deleterious mutations and allow its long‐term persistence at relatively high frequencies.     

A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome‐arm identification,integration of genetic linkage groups and analysis of major repeat family distribution

We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North–South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome‐specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S‐5.8S‐25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber‐FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA.     

Bacterial artificial chromosome library for genome‐wide analysis of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene‐amplified CHO DR1000L‐4N cell line for genome‐wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC‐FISH). Thirteen BAC‐FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR‐deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165‐kb DNA region containing exogenous Dhfr was cloned from the BAC library using high‐density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome. Biotechnol. Bioeng. 2009; 104: 986–994. © 2009 Wiley Periodicals, Inc.     

Contrasting patterns of X‐chromosome divergence underlie multiple sex‐ratio polymorphisms in stalk‐eyed flies

Sex‐linked segregation distorters cause offspring sex ratios to differ from equality. Theory predicts that such selfish alleles may either go to fixation and cause extinction, reach a stable polymorphism or initiate an evolutionary arms race with genetic modifiers. The extent to which a sex ratio distorter follows any of these trajectories in nature is poorly known. Here, we used X‐linked sequence and simple tandem repeat data for three sympatric species of stalk‐eyed flies (Teleopsis whitei and two cryptic species of T. dalmanni) to infer the evolution of distorting X chromosomes. By screening large numbers of field and recently laboratory‐bred flies, we found no evidence of males with strongly female‐biased sex ratio phenotypes (SR) in one species but high frequencies of SR males in the other two species. In the two species with SR males, we find contrasting patterns of X‐chromosome evolution. T. dalmanni‐1 shows chromosome‐wide differences between sex‐ratio (X^SR) and standard (X^ST) X chromosomes consistent with a relatively old sex‐ratio haplotype based on evidence including genetic divergence, an inversion polymorphism and reduced recombination among X^SR chromosomes relative to X^ST chromosomes. In contrast, we found no evidence of genetic divergence on the X between males with female‐biased and nonbiased sex ratios in T. whitei. Taken with previous studies that found evidence of genetic suppression of sex ratio distortion in this clade, our results illustrate that sex ratio modification in these flies is undergoing recurrent evolution with diverse genomic consequences.     

Chromosomal‐level assembly of Takifugu obscurus (Abe, 1949) genome using third‐generation DNA sequencing and Hi‐C analysis

The Tetraodontidae family are known to have relatively small and compact genomes compared to other vertebrates. The obscure puffer fish Takifugu obscurus is an anadromous species that migrates to freshwater from the sea for spawning. Thus the euryhaline characteristics of T. obscurus have been investigated to gain understanding of their survival ability, osmoregulation, and other homeostatic mechanisms in both freshwater and seawater. In this study, a high quality chromosome‐level reference genome for T. obscurus was constructed using long‐read Pacific Biosciences (PacBio) Sequel sequencing and a Hi‐C‐based chromatin contact map platform. The final genome assembly of T. obscurus is 381 Mb, with a contig N50 length of 3,296 kb and longest length of 10.7 Mb, from a total of 62 Gb of raw reads generated using single‐molecule real‐time sequencing technology from a PacBio Sequel platform. The PacBio data were further clustered into chromosome‐scale scaffolds using a Hi‐C approach, resulting in a 373 Mb genome assembly with a contig N50 length of 15.2 Mb and and longest length of 28 Mb. When we directly compared the 22 longest scaffolds of T. obscurus to the 22 chromosomes of the tiger puffer Takifugu rubripes, a clear one‐to‐one orthologous relationship was observed between the two species, supporting the chromosome‐level assembly of T. obscurus. This genome assembly can serve as a valuable genetic resource for exploring fugu‐specific compact genome characteristics, and will provide essential genomic information for understanding molecular adaptations to salinity fluctuations and the evolution of osmoregulatory mechanisms.     

Fissions,fusions, and translocations shaped the karyotype and multiple sex chromosome constitution of the northeast‐Asian wood white butterfly,Leptidea amurensis

Previous studies have shown a dynamic karyotype evolution and the presence of complex sex chromosome systems in three cryptic Leptidea species from the Western Palearctic. To further explore the chromosomal particularities of Leptidea butterflies, we examined the karyotype of an Eastern Palearctic species, Leptidea amurensis. We found a high number of chromosomes that differed between the sexes and slightly varied in females (i.e. 2n = 118–119 in females and 2n = 122 in males). The analysis of female meiotic chromosomes revealed multiple sex chromosomes with three W and six Z chromosomes. The curious sex chromosome constitution [i.e. W_1–3/Z_1–6 (females) and Z_1–6/Z_1–6 (males)] and the observed heterozygotes for a chromosomal fusion are together responsible for the sex‐specific and intraspecific variability in chromosome numbers. However, in contrast to the Western Palearctic Leptidea species, the single chromosomal fusion and static distribution of cytogenetic markers (18S rDNA and H3 histone genes) suggest that the karyotype of L. amurensis is stable. The data obtained for four Leptidea species suggest that the multiple sex chromosome system, although different among species, is a common feature of the genus Leptidea. Furthermore, inter‐ and intraspecific variations in chromosome numbers and the complex meiotic pairing of these multiple sex chromosomes indicate the role of chromosomal fissions, fusions, and translocations in the karyotype evolution of Leptidea butterflies.     

A chromosome‐level genome assembly of the woolly apple aphid,Eriosoma lanigerum Hausmann (Hemiptera: Aphididae)

Woolly apple aphid (WAA, Eriosoma lanigerum Hausmann) (Hemiptera: Aphididae) is a major pest of apple trees (Malus domestica, order Rosales) and is critical to the economics of the apple industry in most parts of the world. Here, we generated a chromosome‐level genome assembly of WAA—representing the first genome sequence from the aphid subfamily Eriosomatinae—using a combination of 10X Genomics linked‐reads and in vivo Hi‐C data. The final genome assembly is 327 Mb, with 91% of the assembled sequences anchored into six chromosomes. The contig and scaffold N50 values are 158 kb and 71 Mb, respectively, and we predicted a total of 28,186 protein‐coding genes. The assembly is highly complete, including 97% of conserved arthropod single‐copy orthologues based on Benchmarking Universal Single‐Copy Orthologs (busco ) analysis. Phylogenomic analysis of WAA and nine previously published aphid genomes, spanning four aphid tribes and three subfamilies, reveals that the tribe Eriosomatini (represented by WAA) is recovered as a sister group to Aphidini + Macrosiphini (subfamily Aphidinae). We identified syntenic blocks of genes between our WAA assembly and the genomes of other aphid species and find that two WAA chromosomes (El5 and El6) map to the conserved Macrosiphini and Aphidini X chromosome. Our high‐quality WAA genome assembly and annotation provides a valuable resource for research in a broad range of areas such as comparative and population genomics, insect–plant interactions and pest resistance management.     

An extraordinarily stable karyotype of the woody Populus species revealed by chromosome painting

The karyotype represents the basic genetic make‐up of a eukaryotic species. Comparative cytogenetic analysis of related species based on individually identified chromosomes has been conducted in only a few plant groups and not yet in woody plants. We have developed a complete set of 19 chromosome painting probes based on the reference genome of the model woody plant Populus trichocarpa. Using sequential fluorescence in situ hybridization we were able to identify all poplar chromosomes in the same metaphase cells, which led to the development of poplar karyotypes based on individually identified chromosomes. We demonstrate that five Populus species, belonging to five different sections within Populus, have maintained a remarkably conserved karyotype. No inter‐chromosomal structural rearrangements were observed on any of the 19 chromosomes among the five species. Thus, the chromosomal synteny in Populus has been remarkably maintained after nearly 14 million years of divergence. We propose that the karyotypes of woody species are more stable than those of herbaceous plants since it may take a longer period of time for woody plants to fix chromosome number or structural variants in natural populations.