Low Chromosome Number Research Articles

First chromosomal study of Mantophasmatodea: Karyotype of Karoophasma biedouwense (Austrophasmatidae)

We have investigated for the first time the chromosomes of Karoophasma biedouwense, a species belonging to the Mantophasmatodea, a recently discovered order of carnivorous insects. Our study has revealed that males of this species display testes with numerous seminal tubes (follicles), as in other Polyneoptera, and short tubular seminal vesicles embedded in a utricular gland. The karyotype consists of 2n = 12A + X and biarmed, meta/submetacentric chromosomes (fundamental number of arms: FN = 26) with blocks of heterochromatin around centromeres. The autosomes are classified into two size groups, one represented by a single, very large pair of autosomes, the other by five smaller pairs which constitute a continuous series gradually decreasing in size. Among monocentric orders of Polyneoptera, K. biedouwense shares its low chromosome number, 2n = 13, as also found with some Orthoptera (Acridoidea, Grylloidea, Gryllacridoidea). Male meiosis is of the classical pre-reductional type and the X(0) sex determination system is probably an ancestral state. Use of FISH along with an 18S rDNA probe revealed multiple ribosomal clusters, which most likely represent an apomorphic condition. We identified the ancestral insect telomeric sequence (TTAGG)n in the terminal areas of the chromosomes. Currently available data on the polyneopteran orders putatively related to Mantophasmatodea showed a wide variability of cytogenetic characteristics within and between them. The only character allowing some tentative inference to be made on the ancestry of K. biedouwense is its low chromosome number, the karyotypic pattern so far unreported for the Polyneoptera except in certain Orthoptera.

Is post-polyploidization diploidization the key to the evolutionary success of angiosperms?

Advances in recent years have revolutionized our understanding of both the context and occurrence of polyploidy in plants. Molecular phylogenetics has vastly improved our understanding of plant relationships, enabling us to better understand trait and character evolution, including chromosome number changes. This, in turn, has allowed us to appreciate better the frequent occurrence and extent of polyploidy throughout the history of angiosperms, despite the occurrence of low chromosome numbers in some groups, such as in Arabidopsis (A. thaliana was the first plant genome to be sequenced and assembled). In tandem with an enhanced appreciation of phylogenetic relationships, the accumulation of genomic data has led to the conclusion that all angiosperms are palaeopolyploids, together with better estimates of the frequency and type of polyploidy in different angiosperm lineages. The focus therefore becomes when a lineage last underwent polyploidization, rather than simply whether a plant is ‘diploid’ or ‘polyploid’. This legacy of past polyploidization in plants is masked by large-scale genome reorganization involving repetitive DNA loss, chromosome rearrangements (including fusions and fissions) and complex patterns of gene loss, a set of processes that are collectively termed ‘diploidization’. We argue here that it is the diploidization process that is responsible for the ‘lag phase’ between polyploidization events and lineage diversification. If so, diploidization is important in determining chromosome structure and gene content, and has therefore made a significant contribution to the evolutionary success of flowering plants. © 2015 The Authors. Botanical Journal of the Linnean Society published by John Wiley & Sons Ltd on behalf of The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, ●●, ●●–●●.

The evolutionary dynamics of haplodiploidy: Genome architecture and haploid viability.

Haplodiploid reproduction, in which males are haploid and females are diploid, is widespread among animals, yet we understand little about the forces responsible for its evolution. The current theory is that haplodiploidy has evolved through genetic conflicts, as it provides a transmission advantage to mothers. Male viability is thought to be a major limiting factor; diploid individuals tend to harbor many recessive lethal mutations. This theory predicts that the evolution of haplodiploidy is more likely in male heterogametic lineages with few chromosomes, as genes on the X chromosome are often expressed in a haploid environment, and the fewer the chromosome number, the greater the proportion of the total genome that is X‐linked. We test this prediction with comparative phylogenetic analyses of mites, among which haplodiploidy has evolved repeatedly. We recover a negative correlation between chromosome number and haplodiploidy, find evidence that low chromosome number evolved prior to haplodiploidy, and that it is unlikely that diplodiploidy has reevolved from haplodiploid lineages of mites. These results are consistent with the predicted importance of haploid male viability.

Cytogenetic and cytometric analyses in artificial intercytotypic hybrids of the emergent orchid model species Erycina pusilla

AbstractErycina pusilla is considered a potential model organism for orchids, because of several advantageous features, such as short juvenile period, low chromosome number and all year round blooming. Two different chromosome numbers (2n = 10 and 2n = 12) are reported for E. pusilla, which suggests two cytotypes. To reveal the genome homology between these two cytotypes, we generated hybrids from the intercytotypic reciprocal crosses (2n = 10 × 2n = 12 and 2n = 12 × 2n = 10), and applied 4′,6-diamidino-2-phenylindole staining, genomic in situ hybridization and flow cytometry for genomic and cytogenetic analysis. The parental genomes showed high similarity both in genomic composition and content. The hybrids displayed a chromosome number of 2n = 11 in mitotic cells. Moreover, five bivalents and one univalent were observed at meiotic metaphase I stage. We observed meiotic synaptic behavior and found homeologous pairing with unpaired loops between parental chromosome pairing segments. These results demonstrated that chromosome rearrangement events have occurred between parental cytotypes during evolution. This study also illustrated the genome homology and homeologous pairing at pachytene phase, indicating that the chromosome number variation of two cytotypes mainly resulted from chromosome rearrangements, not changes in genomic constitution.

Cytogenetics of Aspidogaster limacoides (Trematoda, Aspidogastrea): karyotype, spermatocyte division, and genome size.

A detailed cytogenetic analysis of the aspidogastrean fluke Aspidogaster limacoides revealed a karyotype consisting of six medium-sized chromosome pairs. The first and the last pairs were two-armed while four remaining were one-armed; 2n = 12, n = 1 m + 1 m - sm + 4a. Fluorescence in situ hybridization with 18S ribosomal DNA (rDNA) probe detected a single cluster of ribosomal genes (NOR) located in pericentromeric regions of the long arms of the third chromosome pair in a site of secondary constriction apparent in meiotic prophase, especially in diplotene. The silver nitrate staining showed only a single active NOR site on one of homologous chromosomes in the majority of spermatogonia and spermatocyte divisions. A course of meiosis corresponded to standard schemes. The nucleolus was apparent in early meiotic spermatocytes and disintegrated by the end of pachytene. For the first time in Aspidogastrea, the genome size was determined. The flow cytometry showed 1.21 pg DNA per haploid nucleus in A. limacoides which is in accordance with relatively low genome sizes of other flukes and tapeworms (Neodermata). A comparison of cytogenetic data available to date in the fluke sister groups Aspidogastrea and Digenea suggests that the lower chromosome number of Aspidogastrea might represent an ancestral condition and their split might have been accompanied by an increase in chromosome number via either chromosome fissions or paleopolyploidy.

Chromosome numbers of selected species of Elatine L. (Elatinaceae)

The paper reports chromosome numbers for 13 taxa of <em>Elatine</em> L., including all 11 species occurring in Europe, namely <em>E. alsinastrum</em>, <em>E. ambigua</em>, <em>E. brachysperma</em>, <em>E. brochonii</em>, <em>E. californica</em>, <em>E. campylosperma</em>, <em>E. gussonei</em>, <em>E. hexandra</em>, <em>E. hungarica</em>, <em>E. hydropiper</em>, <em>E. macropoda</em>, <em>E. orthosperma</em>, <em>E. triandra</em> originating from 17, field-collected populations. For seven of them (<em>E. ambigua</em>, <em>E. californica</em>, <em>E. campylosperma</em>, <em>E. brachysperma</em>, <em>E. brochonii</em>, <em>E. hungarica</em>, <em>E. orthosperma</em>) the chromosome numbers are reported for the first time. With these records, chromosome numbers for the whole section <em>Elatinella</em> Seub. became available. Although 2<em>n</em> = 36 was reported to be the most common and the lowest chromosome number in the genus, our data show that out of thirteen species analyzed, six had 36 chromosomes but five species had 54 chromosomes, and the lowest number of chromosomes was 18. These data further corroborates that the basic chromosome number in <em>Elatine</em> is <em>x</em> = 9.

The first cytogenetic data on Strumigenys louisianae Roger, 1863 (Formicidae: Myrmicinae: Dacetini): the lowest chromosome number in the Hymenoptera of the neotropical region.

In the present study, the first cytogenetic data was obtained for the ant species Strumigenys louisianae, from a genus possessing no previous cytogenetic data for the Neotropical region. The chromosome number observed was 2n = 4, all possessing metacentric morphology. Blocks rich in GC base pairs were observed in the interstitial region of the short arm of the largest chromosome pair, which may indicate that this region corresponds to the NORs. The referred species presented the lowest chromosome number observed for the subfamily Myrmicinae and for the Hymenoptera found in the Neotropical region. Observation of a low chromosome number karyotype has been described in Myrmecia croslandi, in which the occurrence of tandem fusions accounts for the most probable rearrangement for its formation. The accumulation of cytogenetic data may carry crucial information to ensure deeper understanding of the systematics of the tribe Dacetini.

Comparative Analysis of Miscanthus and Saccharum Reveals a Shared Whole-Genome Duplication but Different Evolutionary Fates.

Multiple polyploidizations with divergent consequences in the grass subtribe Saccharinae provide a singular opportunity to study in situ adaptation of a genome to the duplicated state, heretofore known primarily from paleogenomics. We show that allopolyploidy in a common Miscanthus-Saccharum ancestor ∼3.8 to 4.6 million years ago closely coincides in time with their divergence from the Sorghum lineage. Subsequent Saccharum-specific autopolyploidy may have created pseudo-paralogous chromosome groups with random pairing within a group but infrequent pairing between groups. High chromosome number may reduce differentiation among Saccharum pseudo-paralogs by increasing opportunities for recombinations, with the lower chromosome numbers of Miscanthus favoring the return to disomic inheritance. The widespread tendency of plant chromosome numbers to recursively return to a narrow range following genome duplication appears to be occurring now in Saccharum spontaneum based on rich polymorphism for chromosome number among genotypes, with past reductions indicated by condensations of two ancestral chromosomes in Miscanthus (now n = 19) and perhaps as many as 10 in the Narenga-Sclerostachya clade (n = 15).

Characterization, efficient transformation and regeneration of Chirita pumila (Gesneriaceae), a potential evo-devo model plant

An efficient transformation and regeneration system is essential for functional investigation of developmental genes and related elements in the field of evolutionary developmental biology (evo-devo). Chirita pumila D. Don belongs to the Gesneriaceae family, one of the most basal groups in Lamiales sensu lato, and possesses many tractable biological features including annual habit, small plant size, short generation time, abundant offspring and low chromosome number. In addition, C. pumila has cleistogamous flowers with potential cross-pollination, a special phenomenon first reported herein in Gesneriaceae. Parameters affecting shoot induction and genetic transformation have been evaluated, including plant growth regulators, temperature, antibiotic concentration, pre- and co-culture duration, Agrobacterium cell density and infection time. Polymerase chain reaction and β-Glucuronidase (GUS) activity assays of T0 and T1 plants show that the GUS gene has been introduced into the host with stable and universal expression. The applicability of the transformation system in gene function investigation is further confirmed by transforming a GsNST1B gene from Glycine soja. This transformation system provides a valuable platform for deep function analyses of related genes and elements for a wide range of evo-devo studies, especially in the field of floral evolution, which would develop its potential of being a model organism in Lamiales s. l.

EFFECTS OF NAA AND BA ON PLB GROWTH OF PSYGMORCHIS PUSILLA

Psygmorchis (syn. Erycina) pusilla Dodson & Dressler, belonging to the subtribe Oncidiinae, is a small orchid distributed in Latin America. It has the lowest chromosome number (2n=12) in the orchid family, shorter life cycle, which is about one year from germination to flowering, and can be incubated easily through life in vitro. According to these features, P. pusilla has the possibility to be a model plant in the Orchidaceae to research orchid-specific characters, such as flower shape, column, pollinia or protocorm. To establish P. pusilla as a model plant, we investigated PLB growth with varying concentrations of NAA and BA on NDM medium or 1/2 MS medium. The fastest proliferation was found on NDM medium with 0.1 mg L-1 NAA + 1.5 mg L-1 BA. On the other hand, 2.0 mg L-1 BA with any NAA concentration showed lower growth of PLB, but more number of protrusions was produced.

Combining FISH and model-based predictions to understand chromosome evolution in Typhonium (Araceae)

Since the advent of molecular phylogenetics, numerous attempts have been made to infer the evolutionary trajectories of chromosome numbers on DNA phylogenies. Ideally, such inferences should be evaluated against cytogenetic data. Towards this goal, we carried out phylogenetic modelling of chromosome number change and fluorescence in situ hybridization (FISH) in a medium sized genus of Araceae to elucidate if data from chromosomal markers would support maximum likelihood-inferred changes in chromosome numbers among close relatives. Typhonium, the focal genus, includes species with 2n = 65 and 2n = 8, the lowest known count in the family. A phylogeny from nuclear and plastid sequences (96 taxa, 4252 nucleotides) and counts for all included species (15 of them first reported here) were used to model chromosome number evolution, assuming discrete events, such as polyploidization and descending or ascending dysploidy, occurring at different rates. FISH with three probes (5S rDNA, 45S rDNA and Arabidopsis-like telomeres) was performed on ten species with 2n = 8 to 2n = 24. The best-fitting models assume numerous past chromosome number reductions. Of the species analysed with FISH, the two with the lowest chromosome numbers contained interstitial telomeric signals (Its), which together with the phylogeny and modelling indicates decreasing dysploidy as an explanation for the low numbers. A model-inferred polyploidization in another species is matched by an increase in rDNA sites. The combination of a densely sampled phylogeny, ancestral state modelling and FISH revealed that the species with n = 4 is highly derived, with the FISH data pointing to a Robertsonian fusion-like chromosome rearrangement in the ancestor of this species.

Begonia wui-senioris (sect. Platycentrum, Begoniaceae), a new species from Myanmar.

BackgroundThe flora of Myanmar is under-collected compared with all other tropical Asian countries. An unknown Begonia was grown from seeds collected from a limestone hill in Central Myanmar, and compared with potentially allied species.ResultsThe unknown Begonia is rhizomatous, has peltate leaves, 2-locular ovaries, and is evergreen. It is clearly assignable to sect. Platycentrum. Only two other species of Begonia, B. josephii and B. subperfoliata, in Myanmar have peltate leaves, but they are deciduous tuberous plants with 3-locular ovaries and belong to sect. Diploclinium.ConclusionsThorough studies of literature and herbarium materials support the recognition of a new species, Begonia wui-senioris, which is fully described and illustrated. Begonia wui-senioris has the lowest chromosome number (2n = 14) for the genus.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-13) contains supplementary material, which is available to authorized users.

Acute Lymphoblastic Leukemia (ALL) With Low-Hypodiploid/Near-Triploid Karyotype Is a Specific Clinical Entity Characterized By a High TP53 Mutation Frequency

BackgroundB-lineage acute lymphoblastic leukemia (ALL) called B lymphoblastic leukemia/lymphoma by the WHO classification 2008 is subdivided into 5 subgroups defined by a specific translocation (t(9;22)(q34;q11); t(v;11q23); t(12;21)(p13;q22); t(5;14)(q31;q32); t(1;19)(q23;p13.1)). Two additional categories are defined on the basis of chromosome number: ALL with hyperdiploidy and hypodiploid ALL. The former group comprises a well defined genetic subset with blasts containing >50 and usually >66 chromosomes. In contrast, the hypodiploid subgroup is very heterogeneous and comprises ALL with a chromosome number of >46 chromosomes to near-haploid. AimTo characterize this rare subset of ALL with low hypodiploidy in detail with respect to cytogenetic abnormalities, molecular mutations and clinical outcome. Patients and MethodsOut of a total cohort of 878 ALL patients analyzed between 2005 and 2013 we selected a subset of 26 cases harbouring a hypodiploid clone with 1) a chromosome number of ≤40 or 2) a near triploid clone with 56-78 chromosomes that had arisen from a low hypodiploid clone by duplication of the chromosome set. Data on karyotype, array CGH and FISH for rearrangement of BCR-ABL1, ETV6 and MLL was available in all cases. Further, cases were investigated for mutations in NRAS, KRAS, TP53, NOTCH1, FBXW7 and intragenic deletions of IKZF1. For 17 cases data on the immunophenotype was available. Clinical follow-up data was available for 19 pts. Results12/17 pts showed a common B ALL-phenotype, 5 pts were classified as Pro-B-ALL. All cases were negative for t(12;21)(p13;q22)/ETV6-RUNX1, t(9;22)(q34;q11)/BCR-ABL1 and translocations involving the MLL gene. The pattern of chromosomal losses was non-random. One chromosome 3 and 7 was lost in all 26 cases. Other frequently deleted chromosomes were chromosomes 15 (n=24), 17 (n=25), 13, 16 (lost in 23 cases each), 9 (n=18), 4 (n=17), 12 and 20 (lost in 16 cases each), which were all deleted in more than 50% of the cases, confirming a very specific pattern of chromosome losses. Less frequently lost were chromosomes 2 (n=12), 8 (n=11), 14 (n=10), 5 (n=8) and 18 (n=7). Rarely lost were chromosomes 6, 11 (lost in 5 cases each), 1, 10, 19, 22 (lost in 4 cases each) and the sex chromosomes X or Y in 3 cases each. Only chromosome 21 was retained in all cases. In 14 cases with doubling of the low-hypodiploid karyotype we observed the same typical pattern of gains and losses with two copies of typically lost chromosomes and four copies of typically retained chromosomes. In 11/14 pts we detected additional losses of chromosomes resulting in trisomies, most frequently of chromosomes 2 (n=7), 5 and 14 (n=6), respectively. Structural abnormalities in addition to chromosome losses were found in 85% of pts by chromosome banding analysis, none of these was recurrent. The only recurrent submicroscopic deletion detected by aCGH was a 9p deletion encompassing the CDKN2A/B locus (n=7), which was homozygous in 4 pts. As 14 additional pts showed a monosomy 9, only 5 pts did not show a deletion of at least one CDKN2A/B copy. We discovered no mutations in FBXW7, NOTCH1 and KRAS and only one mutation in NRAS. Further, no intragenic deletion of IKZF1 was detected. However, and most interestingly, 24 out of 26 pts (92.3%) showed a mutation within the TP53 gene. In 23/24 pts with TP53 mutation and in both pts with TP53wt chromosome 17 was monosomic. Further, both patients with TP53wt and monosomy 17 showed a remarkably low chromosome number of 25 and 28 chromosomes compared to TP53mut pts who showed at least 32 chromosomes. Outcome was poor: 2-year-overall survival rate of pts treated with intensive chemotherapy (n=19) was only 20%. ConclusionsALL with low hypodiploidy is characterized by a typical pattern of chromosome losses including loss of chromosomes 7, 3, 15, 17, 13 and 16, a very high TP53 mutation frequency and poor outcome. A duplication of the low-hypodiploid karyotype occurs frequently and results in a near triploid karyotype based on the definition by merely counting chromosomes but in fact is a very low tetraploid chromosome set. Our data suggests the introduction of a novel WHO entity within the B lymphoblastic leukaemia/lymphoma group showing low-hypodiploid/very low-tetraploid karyotype and concomitant TP53 mutation. The poor outcome of this specific ALL subset might be related to the high TP53 mutation frequency in combination with loss of the second TP53 allele due to monosomy 17. Disclosures:Patterer:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Weissmann:MLL Munich Leukemia Laboratory: Employment. Poetzinger:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Bellos:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The influence of a substrate including extracellular matrix proteins on karyotypic variability in two cell lines of Indian muntjac skin fibroblasts

The influence of cell-culture conditions on numerical and structural karyotypic variability has been investigated in two Indian muntjac skin fibroblast “markerless” cell lines, M and MT. The cells were cultivated on a substrate consisting of extracellular matrix proteins (ECMs) synthesized by human mesenchymal stem cells (SC5-MSC). The character of cell distribution for the chromosome number of the cell line M changed after cultivation for 1 and 4 days as compared to control cells, which were cultured on a hydrophilic surface without ECM coating. These changes involve a significant decrease in frequency of cells with the modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Many new types of additional structural variants of the karyotype (SVK) appear. The MT cell line, differing from M line in the number of homologous chromosomes, exhibited a character of cell distribution similar to that of the M line for the chromosome number for only 1 day after cultivation on the ECM substrate, but not after 4 days under the same culture conditions, when no difference from the control cells was observed. Further cultivation of MT cells for 8 days did not change the character of cell distribution for the chromosome number relative to the control variant. The observed alterations seem to be due to disturbances in the correct chromosome-segregation process, which were caused by an abrupt shift in the cell-culture conditions. Analysis of the structural karyotypic variability revealed a significant increase in frequency of chromosomal aberrations in the M cell line for 1 and 4 days in culture on the ECM substrate as compared to the control cells. The frequency of dicentric chromosomes (telomeric associations) was increased and constituted more than 50% of all chromosome aberrations. No increase in frequency of chromosome aberrations was observed for MT cells cultured under the same conditions. It can be suggested that the differing by the karyotypic structure, but the genetically identical cell lines have different response to the substrate. In contrast to the M line, in the MT line, a fast normalization of numerical karyotypic characteristics and no enhancement of structural karyotypic variability takes place. This provides a possibility to cultivate an MT cell on the given protein substrate while maintaining a balanced karyotypic structure characteristic of MT cell line.

Phylogeny and chromosomal variations in East Asian Carex, Siderostictae group (Cyperaceae), based on DNA sequences and cytological data

Carex (Cyperaceae) is one of the largest genera of the flowering plants, and comprises more than 2,000 species. In Carex, section Siderostictae with broader leaves distributed in East Asia is thought to be an ancestral group. We aimed to clarify the phylogenetic relationships and chromosomal variations within the section Siderostictae, and to examine the relationship of broad-leaved species of the sections Hemiscaposae and Surculosae from East Asia, inferred from DNA sequences and cytological data. Our results indicate that a monophyletic Siderostictae clade, including the sections Hemiscaposae, Siderostictae and Surculosae, as the earliest diverging group in the tribe Cariceae. Low chromosome numbers, 2n = 12 or 24, with large sizes were observed in these three sections. Our results suggest that the genus Carex might have originated or relictly restricted in the East Asia. Geographical distributions of diploid species are restricted in narrower areas, while those of tetraploid species are wider in East Asia. It is concluded that chromosomal variations in Siderostictae clade may have been caused by polyploidization and that tetraploid species may have been able to exploit their habitats by polyploidization.

A karyological study of four European species of Roncus (Pseudoscorpiones: Neobisiidae)

We present the results of the first study on the karyotypes of four European species of Roncus: Roncus alpinus L. Koch, 1873, Roncus lubricus L. Koch, 1873, Roncus transsilvanicus Beier, 1928 and Roncus sp. The diploid number was 2n = 23 in Roncus sp., 2n = 43 in R. alpinus and R. transsilvanicus and 2n = 45 in R. lubricus. Telocentric autosomes predominate in species with a high chromosome number and metacentric autosomes in Roncus sp. We assume that the ancestral situation for this genus is a high number of chromosomes. A low number of chromosomes is very likely a consequence of centric fusions, which have possibly played a very important role in karyotype evolution in the genus Roncus. All the species analyzed have the X0 sex chromosome system. The X chromosome is metacentric and is the smallest element in the karyotypes of all the species analyzed.

Catalog of Erycina pusilla miRNA and categorization of reproductive phase-related miRNAs and their target gene families

The orchid Erycina pusilla has a short life cycle and relatively low chromosome number, making it a potential model plant for orchid functional genomics. To that end, small RNAs (sRNAs) from different developmental stages of different organs were sequenced. In this miRNA mix, 33 annotated miRNA families and 110 putative miRNA-targeted transcripts were identified in E. pusilla. Fifteen E. pusilla miRNA target genes were found to be similar to those in other species. There were putative novel miRNAs identified by 3 different strategies. The genomic sequences of the four miRNAs that were identified using rice genome as the reference can form the stem loop structure. The t0000354 miRNA, identified using rice genome sequences and a Phalaenopsis study, had a high read count. The target gene of this miRNA is MADS (unigene30603), which belongs to the AP3-PI subfamily. The most abundant miRNA was E. pusilla miR156 (epu-miR156), orthologs of which work to maintain the vegetative phase by repressing the expression of the SQUAMOSA promoter-binding-like (SPL) transcription factors. Fifteen genes in the E. pusilla SPL (EpSPL) family were identified, nine of which contained the putative epu-miR156 target site. Target genes of epu-miR172, also a key regulator of developmental changes in the APETALA2 (EpAP2) family, were identified. Experiments using 5'RLM-RACE demonstrated that the genes EpSPL1, 2, 3, 4, 7, 9, 10, 14 and EpAP2-9, -10, -11 were regulated by epu-miR156 and epu-miR172, respectively.

Global transcriptome analysis and identification of a CONSTANS-like gene family in the orchid Erycina pusilla

The high chromosome numbers, polyploid genomes, and long juvenile phases of most ornamental orchid species render functional genomics difficult and limit the discovery of genes influencing horticultural traits. The orchid Erycina pusilla has a low chromosome number (2n = 12) and flowers in vitro within 1 year, making it a standout candidate for use as a model orchid. However, transcriptomic and genomic information from E. pusilla remains limited. In this study, next-generation sequencing (NGS) technology was used to identify 90,668 unigenes by de novo assembly. These unigenes were annotated functionally and analyzed with regard to their gene ontology (GO), clusters of orthologous groups (COG), and KEGG pathways. To validate the discovery methods, a homolog of CONSTANS (CO), one of the key genes in the flowering pathway, was further analyzed. The Arabidopsis CO-Like (COL) amino acid sequences were used to screen for homologs in the E. pusilla transcriptome database. Specific primers to the homologous unigenes were then used to isolate BAC clones, which were sequenced to identify 12 E. pusilla CO-like (EpCOL) full-length genes. Based on sequence homology, domain structure, and phylogenetic analysis, these EpCOL genes were divided into four groups. Four EpCOLs fused with GFP were localized in the nucleus. Some EpCOL genes were regulated by light. These results demonstrate that nascent E. pusilla resources (transcriptome and BAC library) can be used to investigate the E. pusilla photoperiod-dependent flowering genes. In future, this strategy can be applied to other biological processes, marketable traits, and molecular breeding in this model orchid.

Replication of somatic micronuclei in bovine enucleated oocytes

BackgroundMicrocell-mediated chromosome transfer (MMCT) was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes.MethodsMicronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+)] or not [Micronucleus- injected (−)] to a transgene (50 ng/μl pCX-EGFP) during 5 min. Enucleated oocytes [Enucleated (+)] and parthenogenetic [Parthenogenetic (+)] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (−)] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (−), Parthenogenetic (−) and in vitro fertilized (IVF) embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05).ResultsAll the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had fewer than 15 chromosomes per blastomere (from 1 to 13), while none of the IVF and Parthenogenetic controls showed less than 30 chromosomes per spread.ConclusionsWe have developed a new method to replicate somatic micronuclei, by using the replication machinery of the oocyte. This could be a useful tool for making chromosome transfer, which could be previously targeted for transgenesis.

A fascinating island: 2n = 4

Among Angiosperms, only six species are known to possess the lowest identified chromosome number, i.e. 2n = 4. These plants are the monocotyledons Zingeria biebersteiniana, Colpodium versicolor, Ornithogalum tenuifolium and Rhynchospora tenuis, and the dicotyledons Haplopappus gracilis and Brachyscome dichromosomatica. The low chromosome number may be cytogenetically derived from a different ancestral basic number, characteristic of each genus, by different processes, including tandem fusion or unequal reciprocal translocations with loss of centric fragments. All these plants possess low nuclear 4C DNA contents, ranging from 1.56 pg (R. tenuis) to 8.20 pg (H. gracilis), and they generally display a similar chromosome size and a similar position of the nucleolus organising region (NOR), that is often located in the terminal or subterminal region of the small chromosome pair. All these characteristics could be a consequence of common adaptative mechanisms. Peculiar characteristics within these karyotypes are the holocentric chromosomes of R. tenuis and the presence of B chromosomes in B. dichromosomatica. Plants with a very low chromosome number may be considered to constitute a fascinating “island of interest”; moreover, they represent simple systems helpful for the examination of the structural organisation and evolution of Angiosperm chromosomes.