Search

The JC-1 dye is widely used in apoptosis studies to monitor mitochondrial health. The probe was tested in vitro on two established cell lines and peripheral porcine blood lymphocytes after gamma irradiation (IR) to assess its potential in biodosimetric evaluation. In brief, we stained irradiated and non-irradiated cells with the JC-1 dye to determine the existing changes in mitochondrial membrane potential and monitor cell health through flow cytometry. The stage of injury in these cells was evaluated through an irradiated versus non-irradiated ratio (IVNIR), comparing the relative proportion of polarised cells containing red JC-1 aggregates. We observed a decreasing IVNIR as the radiation dose increased (i.e. 0.5; 1; 2; 4; 6; 8 and 10 Gy), performing the analysis at 4, 8 and 24 h after IR in all the tested cells. The results from the JC1-dye test showed that CD4 T lymphocytes were more sensitive to irradiation than other subpopulations.

This study establishes a new experimental approach for retrospective biodosimetric assessment by apoptosis detection ex vivo. For this purpose, we used mononuclear blood leukocytes isolated from the peripheral blood of irradiated Wistar rats and cultured them ex vivo for posterior analysis. Using flow cytometry, we distinguished apoptotic lymphocyte subsets individual biodosimetric potential at different time periods after exposure: B-lymphocytes 6–8 h (0–7 Gy), natural killer cells 24 h (0–7 Gy) and T-lymphocytes 24 h (0–1 Gy). This novel experimental design innovates through the need of a single blood sample from irradiated individuals for a complete biodosimetric assessment.

Salinity remains a major inhibitor of crop production in irrigated and marginal lands. The identification of genes involved in salinity tolerance has been predominantly limited to model plants and crop species. However, plants naturally adapted to highly saline environments can provide key insights into mechanisms of salinity tolerance. Plants of the genusSalicorniagrow in coastal salt marshes, and their growth is even stimulated by NaCl – much can be learnt from them. We generated genome sequences of twoSalicorniaspecies and studied the transcriptomic and proteomic responses ofSalicornia bigeloviito NaCl. Through the generation of subcellular membrane proteomes, we found that SbiSOS1, a homolog of the well-known SALT-OVERLY-SENSITIVE 1 (SOS1) protein, appears to localize to the tonoplast, where it could be involved in mediating Na+translocation into the vacuole to prevent toxicity in the cytosol. We identified 11 proteins of interest which, when expressed in yeast, altered salinity tolerance. One of these proteins, SbiSALTY, substantially improves yeast growth on saline media. Structural characterization using NMR showed it to be an intrinsically disordered protein and to localize to the endoplasmic reticulumin planta, where it could interact with ribosomes and RNA, potentially stabilizing or protecting them during salt stress. The study and understanding of the molecular mechanisms providing high salinity tolerance inS. bigeloviiis likely to provide significant insights for improving salinity tolerance of crop plants.

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emissions and loss of biodiversity1. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value2. Faba bean (Vicia faba L.) has a high yield potential and is well suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has expanded to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, although with substantial copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association analysis to dissect the genetic basis of seed size and hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate the improvement of sustainable protein production across the Mediterranean, subtropical and northern temperate agroecological zones.

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emission and loss of biodiversity. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value. Faba bean (Vicia faba L.) has a high yield potential and is well-suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has grown to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, though with significant copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association (GWA) analysis to dissect the genetic basis of hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate improvement of sustainable protein production across Mediterranean, subtropical, and northern temperate agro-ecological zones.

More than a century has passed since the B chromosomes were first discovered. Today we know much of their variability, morphology, and transmission to plant progeny. With the advent of modern technologies, B chromosome research has accelerated, and some of their persistent mysteries have since been uncovered. Building on this momentum, here we extend current knowledge of B chromosomes in Sorghum purpureosericeum to the sequence level. To do this, we estimated the B chromosome size at 421 Mb, sequenced DNA from flow-sorted haploid pollen nuclei of both B-positive (B+) and B-negative (B0) plants, and performed a repeat analysis on the Illumina raw sequence data. This analysis revealed nine putative B-specific clusters, which were then used to develop B chromosome-specific markers. Additionally, cluster SpuCL4 was identified and verified to be a centromeric repeat. We also uncovered two repetitive clusters (SpuCL168 and SpuCL115), which hybridized exclusively on the B chromosome under fluorescence in situ hybridization and can be considered as robust cytogenetic markers. Given that B chromosomes in Sorghum are rather unstable across all tissues, our findings could facilitate expedient identification of B+ plants and enable a wide range of studies to track this chromosome type in situ., We sequenced plants of the tropical grass Sorghum purpureosericeum carrying B chromosomes. Based on repeat clustering, we assigned nine clusters to B chromosome and developed B-specific PCR and cytogenetic markers.

SummaryThe parasitic genus Cuscuta (Convolvulaceae) is exceptional among plants with respect to centromere organization, including both monocentric and holocentric chromosomes, and substantial variation in genome size and chromosome number. We investigated 12 species representing the diversity of the genus in a phylogenetic context to reveal the molecular and evolutionary processes leading to diversification of their genomes.We measured genome sizes and investigated karyotypes and centromere organization using molecular cytogenetic techniques. We also performed low-pass whole genome sequencing and comparative analysis of repetitive DNA composition.A remarkable 102-fold variation in genome sizes (342–34,734 Mbp/1C) was detected for monocentric Cuscuta species, while genomes of holocentric species were of moderate sizes (533–1,545 Mbp/1C). The genome size variation was primarily driven by the differential accumulation of repetitive sequences. The transition to holocentric chromosomes in the subgenus Cuscuta was associated with loss of histone H2A phosphorylation and elimination of centromeric retrotransposons. In addition, the basic chromosome number (x) decreased from 15 to 7, presumably due to chromosome fusions.We demonstrated that the transition to holocentricity in Cuscuta was accompanied by significant changes in epigenetic marks, chromosome number and the repetitive DNA sequence composition.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

The banana is a staple food crop and represents an important trade commodity for millions of people living in tropical and subtropical countries. The most important edible banana clones originated from natural crosses between diploid Musa balbisiana and various subspecies of M. acuminata. It is worth mentioning that evolution and speciation in the Musaceae family were accompanied by large-scale chromosome structural changes, indicating possible reasons for lower fertility or complete sterility of these vegetatively propagated clones. Chromosomal changes, often accompanied by changes in genome size, are one of the driving forces underlying speciation in plants. They can clarify the genomic constitution of edible bananas and shed light on their origin and on diversification processes in members of the Musaceae family. This article reviews the development of molecular cytogenetic approaches, ranging from classical fluorescence in situ hybridization (FISH) using common cytogenetic markers to oligo painting FISH. We discuss differences in genome size and chromosome number across the Musaceae family in addition to the development of new chromosome-specific cytogenetic probes and their use in genome structure and comparative karyotype analysis. The impact of these methodological advances on our knowledge of Musa genome evolution at the chromosomal level is demonstrated. In addition to citing published results, we include our own new unpublished results and outline future applications of molecular cytogenetics in banana research.

Yam (Dioscorea spp.) is a monocotyledonous herbaceous vine plant grown in the tropics and subtropics. It is a multi-species plant with varied intra- and interspecific ploidy levels. Of the 600 species, 11 are cultivated staple supporting the livelihood of over 300 million people. The paucity of information on ploidy and the genomic constitution is a significant challenge to the crop’s genetic improvement through crossbreeding. The objective of this study was to investigate the ploidy levels of 236 accessions across six cultivated and two wild species using chromosome counting, flow cytometry and genotyping-based ploidy determination methods. Results obtained from chromosome counting and genotyping-based ploidy determination were in agreement. In majority of the accessions, chromosome counting and flow cytometry were congruent, allowing future rapid screening of ploidy levels using flow cytometry. Among cultivated accessions, 168 (71%) were diploid, 50 (21%) were triploid, and 12 (5%) were tetraploid. Two wild species included in the study were diploids. Resolution of ploidy level in yams offers opportunities for implementing successful breeding programmes through intra- and inter-specific hybridization.

The increased risk of acute large-scale radiation exposure of the population underlies the necessity to develop new methods that could provide a rapid assessment of the doses received while using modern high-throughput technologies. At the same time, there is a growing interest in discovering new biomarkers enabling the categorization of irradiated individuals that could be used in epidemiological studies to correlate the estimated absorbed doses with the consequent impact on patients health. The aim of this study was to summarize the current literature on biological dosimetry, specifically ionizing radiation-responsive biomarkers. We briefly describe current knowledge in the field of radiation genomics, metabolomics, and proteomics. Although the majority of studies that provided a plethora of useful information were conducted in animal models, oncological patients remain the crucial experimental model. The authors describe various biological materials that could be potentially used to predict the effect of ionizing radiation. Plasma proteins appear to be ideal for this purpose. Out of many candidate markers, the ferredoxin reductase (FDXR) seems to be promising, as it has been confirmed in several biodosimetric studies at the level of both human gene and protein.

Edible banana cultivars are diploid, triploid or tetraploid hybrids which originated by natural cross hybridization between subspecies of diploid Musa acuminata, or between M. acuminata and diploid M. balbisiana. Participation of two other wild diploid species M. schizocarpa and M. textilis was also indicated by molecular studies. Fusion of gametes with structurally different chromosome sets may give rise to progenies with structural chromosome heterozygosity and reduced fertility due to aberrant chromosome pairing and unbalanced chromosome segregation. Only a few translocations have been classified on the genomic level so far and a comprehensive molecular cytogenetic characterization of cultivars and species of the family Musaceae is still lacking. FISH with chromosome-arm specific oligo painting probes was used for comparative karyotype analysis in a set of wild Musa species and edible banana clones. The results revealed large differences in chromosome structure discriminating individual accessions. These results permitted identification of putative progenitors of cultivated clones and clarified genomic constitution and evolution of aneuploid banana clones, which seem to be common among the polyploid banana accessions. New insights into the chromosome organization and structural chromosome changes will be a valuable asset in breeding programs, particularly in selection of appropriate parents for cross hybridization.HighlightOligo painting FISH revealed chromosomal translocations in subspecies of Musa acuminata (A genome), their intra-specific hybrids as well as in M. balbisiana (B genome) and in interspecific hybrid clones originating from cross hybridization between M. acuminata and M. balbisiana

Background Cultivated grasses are an important source of food for domestic animals worldwide. Increased knowledge of their genomes can speed up the development of new cultivars with better quality and greater resistance to biotic and abiotic stresses. The most widely grown grasses are tetraploid ryegrass species (Lolium) and diploid and hexaploid fescue species (Festuca). In this work, we characterized repetitive DNA sequences and their contribution to genome size in five fescue and two ryegrass species as well as one fescue and two ryegrass cultivars. Results Partial genome sequences produced by Illumina sequencing technology were used for genome-wide comparative analyses with the RepeatExplorer pipeline. Retrotransposons were the most abundant repeat type in all seven grass species. The Athila element of the Ty3/gypsy family showed the most striking differences in copy number between fescues and ryegrasses. The sequence data enabled the assembly of the long terminal repeat (LTR) element Fesreba, which is highly enriched in centromeric and (peri)centromeric regions in all species. A combination of fluorescence in situ hybridization (FISH) with a probe specific to the Fesreba element and immunostaining with centromeric histone H3 (CENH3) antibody showed their co-localization and indicated a possible role of Fesreba in centromere function. Conclusions Comparative repeatome analyses in a set of fescues and ryegrasses provided new insights into their genome organization and divergence, including the assembly of the LTR element Fesreba. A new LTR element Fesreba was identified and found in abundance in centromeric regions of the fescues and ryegrasses. It may play a role in the function of their centromeres.

Background Cultivated grasses are an important source of food for domestic animals worldwide. Better knowledge of their genomes can speed up the development of new cultivars with better quality and resistance to biotic and abiotic stresses. The most widely grown grasses are tetraploid ryegrass species ( Lolium spp.) and diploid and hexaploid fescue species ( Festuca spp.). In this work we characterized repetitive DNA sequences and their contribution to genome size in five fescue and two ryegrass species, as well as one fescue and two ryegrass cultivars. Results Partial genome sequences produced by Illumina technology were used for genome-wide comparative analyses using RepeatExplorer pipeline. Retrotransposons were found to be the most abundant repeat types in all seven grass species. Athila element of Ty3/gypsy family showed the most striking differences in copy number between fescues and ryegrasses. The sequence data enabled the assembly of an LTR element Fesreba, which is highly enriched in centromeric and (peri)centromeric regions in all species. A combination of FISH with a probe specific to Fesreba element and immunostaining with CENH3 antibody showed their colocalization and indicated a possible role of Fesreba in centromere function. Conclusions Comparative repeatome analysis in a set of fescues and ryegrasses provided new insights into their genome organization and divergence, including the assembly of LTR element Fesreba. A new LTR element Fesreba was identified and found abundant in centromeric regions of the fescues and ryegrasses. It may have a role in the function of their centromeres.

Melanoma is the least common form of skin tumor, but it is potentially the most dangerous and responsible for the majority of skin cancer deaths. We suggest that the skin microbiome might be changed during the progression of melanoma. The aim of this study is to compare the composition of the skin microbiota between different locations (skin and melanoma) of a MeLiM (Melanoma-bearing Libechov Minipig) pig model (exophytic melanoma). Ninety samples were used for PCR-DGGE analysis with primers specifically targeting the V3 region of the 16S rRNA gene. The profiles were used for cluster analysis by UPGMA and principal coordinate analysis PCoA and also to calculate the diversity index (Simpson index of diversity). By comparing the obtained results, we found that both bacterial composition and diversity were significantly different between the skin and melanoma microbiomes. The abundances of Fusobacterium and Trueperella genera were significantly increased in melanoma samples, suggesting a strong relationship between melanoma development and skin microbiome changes.

Malignant melanoma may be a life-threatening disease caused by various conditions. Cutaneous bacteria could play a role in melanoma development or regression. The present work aimed to analyze the bacterial species present on the epidermis of piglets with hereditary melanoma. Bacteria isolated by swabs directly from melanomas and healthy epidermis were analysed using MALDI-TOF mass spectrometry. From the total of 290 isolates, 92 were identified, while the extraction by ethanol turned out to be more efficient compared to identification by direct transfer. Staphylococcus sciuri, Staphylococcus cohnii, and Lactococcus lactis were significantly more frequent on healthy skin, whereas Staphylococcus chromogenes, Staphylococcus hyicus, and Enterococcus faecalis have thrived significantly better on melanoma. Overall, the results indicate that the micro-biota of melanoma is different from that of healthy epidermis, so piglet skin bacteria inspections are recommended.

© 2018, The Author(s). Bananas (Musa spp.), including dessert and cooking types, are of major importance in the tropics. Due to extremely high levels of sterility, the diversity of cultivated bananas is fixed over long periods of time to the existing genotypes. This pattern puts banana-based agrosystems at risk. Therefore, assessing the extent of wild and cultivated banana diversity, conserving it and making it available for further use is a priority. We report here the collection of new wild and cultivated banana germplasm in the Autonomous Region of Bougainville, Papua New Guinea. In total, 61 accessions were collected and their names and uses were recorded when possible. Classification was also provided based on the observations made in the field. Three wild specimens were collected. Among the 58 cultivated accessions, we noted that eight were used as ornamental plants, seven were edible varieties of the Fe’i type and two were natural tetraploids from the Musa section. The ploidy was then checked by flow cytometry and the accessions were genotyped with a set of 19 SSR markers. The genotyping results were merged to the dataset from Christelová et al. (Biodivers Conserv 26:801–824, 2017). This joint analysis helped refine or confirm the classification of the collected accessions. It also allowed to identify 10 private alleles and 35 genotypes or Genotype Groups that were not present in the wider dataset. Finally, it shed light on the diversification processes at work in the region, such as the capture of mutations by farmers and the likely occurrence of geneflow within the cultivated genepool. ispartof: Genetic Resources and Crop Evolution vol:65 issue:8 pages:2267-2286 status: published

Key message Fluorescence in situ hybridization with probes for 45 cDNAs and five tandem repeats revealed homoeologous relationships of Agropyron cristatum with wheat. The results will contribute to alien gene introgression in wheat improvement. Abstract Crested wheatgrass (Agropyron cristatum L. Gaertn.) is a wild relative of wheat and a promising source of novel genes for wheat improvement. To date, identification of A. cristatum chromosomes has not been possible, and its molecular karyotype has not been available. Furthermore, homoeologous relationship between the genomes of A. cristatum and wheat has not been determined. To develop chromosome-specific landmarks, A. cristatum genomic DNA was sequenced, and new tandem repeats were discovered. Their distribution on mitotic chromosomes was studied by fluorescence in situ hybridization (FISH), which revealed specific patterns for five repeats in addition to 5S and 45S ribosomal DNA and rye subtelomeric repeats pSc119.2 and pSc200. FISH with one tandem repeat together with 45S rDNA enabled identification of all A. cristatum chromosomes. To analyze the structure and cross-species homoeology of A. cristatum chromosomes with wheat, probes for 45 mapped wheat cDNAs covering all seven chromosome groups were localized by FISH. Thirty-four cDNAs hybridized to homoeologous chromosomes of A. cristatum, nine hybridized to homoeologous and non-homoeologous chromosomes, and two hybridized to unique positions on non-homoeologous chromosomes. FISH using single-gene probes revealed that the wheat-A. cristatum collinearity was distorted, and important structural rearrangements were observed for chromosomes 2P, 4P, 5P, 6P and 7P. Chromosomal inversions were found for pericentric region of 4P and whole chromosome arm 6PL. Furthermore, reciprocal translocations between 2PS and 4PL were detected. These results provide new insights into the genome evolution within Triticeae and will facilitate the use of crested wheatgrass in alien gene introgression into wheat. Electronic supplementary material The online version of this article (10.1007/s00122-018-3148-9) contains supplementary material, which is available to authorized users.

Nuclear genome size (C-value) was determined by flow cytometry for 23 populations of 15 Crepis L. (Asteraceae) taxa originating from Turkey. Genome size of seven taxa are reported for the first tim...

Background Cultivated grasses are an important source of food for domestic animals worldwide. Better knowledge of their genomes can speed up development of cultivars with better quality and resistance to biotic and abiotic stresses. The most widely grown grasses are tetraploid ryegrass ( Lolium ) species and diploid and hexaploid fescues ( Festuca ) species. In this work we characterized repetitive DNA sequences and their contribution to genome size in seven fescue and ryegrass species. Results Partial genome sequences were produced by Illumina technology and used for genome-wide comparative analyses using RepeatExplorer pipeline. Retrotransposons were found to be the most abundant repeat types in all seven grass species. Athila element of Ty3/gypsy family showed most striking difference in copy numbers in nuclear genomes between fescues and ryegrasses. The sequence data enabled the assembly of an LTR element Fesreba, which is highly enriched in centromeric and (peri)centromeric regions in all species. A combination of FISH with a probe specific to Fesreba element and immunostaining with CENH3 antibody showed their colocalization and indicated a possible role of Fesreba in centromere function. Conclusions Comparative analysis of repeatome in a set of fescues and ryegrasses provided new insights into their genome organization and divergence, including the assembly of LTR element Fesreba. The element was abundant in centromeric regions of the fescues and ryegrasses and may have a role in function of their centromeres.

Rye (Secale cereale L.) is an exceptionally climate-resilient cereal crop, used extensively to produce improved wheat varieties via introgressive hybridization and possessing the entire repertoire of genes necessary to enable hybrid breeding. Rye is allogamous and only recently domesticated, thus giving cultivated ryes access to a diverse and exploitable wild gene pool. To further enhance the agronomic potential of rye, we produced a chromosome-scale annotated assembly of the 7.9-gigabase rye genome and extensively validated its quality by using a suite of molecular genetic resources. We demonstrate applications of this resource with a broad range of investigations. We present findings on cultivated rye’s incomplete genetic isolation from wild relatives, mechanisms of genome structural evolution, pathogen resistance, low-temperature tolerance, fertility control systems for hybrid breeding and the yield benefits of rye–wheat introgressions., A chromosome-scale genome assembly of rye inbred line ‘Lo7’ provides insights into its incomplete genetic isolation from wild relatives, mechanisms of genome structural evolution and the yield benefits of rye–wheat introgressions.

We describe complex multiple concentration dependencies for the response of isolated pith tissues to plant biologically active substances. Kale and tobacco stem pith explants were cultured on agar media containing combinations of sucrose, cytokinin [kinetin or benzyladenine (BA)] and auxin [indole-3-acetic acid (IAA) or naphthaleneacetic acid (NAA)]. Absorption of these components by explants and their effects on explant mass, contents of soluble proteins, starch and sugars, and activity of ADP-glucose pyrophosphorylase (AGPase) were studied in relation to their concentration. Up to ten pronounced statistically significant maxima (peaks or waves) were repeatedly detected in the dose–response curves over a concentration range of several logarithmic orders. Slight maxima were observed in the corresponding absorption curves. Pronounced maxima of sucrose absorption were induced by IAA and BA, and those of NAA absorption were induced by sucrose. Both types of multiple maxima (in dose–response and absorption curves) may be due to changes in concentration of intracellular solutes (sugars, auxins and cytokinins), thereby affecting metabolic processes that act as sinks for external solutes and elicit feedback appearance of maxima in absorption curves. Good correspondence between external concentrations at which maxima of different compared curves occur in addition to statistical significance of individual maxima and repeatability of experimental results supports the conclusion that the multiple maxima exhibited are genuine. We consider it possibile that the multiple maxima are associated with endopolyploidy or mixoploidy and/or epigenomic diversity of pith cells that show different sensitivities to biologically active solutes.

Silene latifolia (or white campion) possesses a well-established sex determination system with a dominant Y chromosome in males (the mammalian type). The heteromorphic sex chromosomes X and Y in S. latifolia largely stopped recombination; thus, we can expect a gradual genetic degeneration of the Y chromosome. It is well proven that neither diploid nor polyploid S. latifolia sporophytes can survive without at least one X, so the only life stage possessing the Y as the sole sex chromosome is the male gametophyte (pollen tube), while the female gametophyte seems to be X-dependent. Previous studies on anther-derived plants of this species showed that the obtained plants (largely haploid or dihaploid) were phenotypically and cytologically female. In this paper, we provide molecular evidence for the inviability of plants lacking the X chromosome. Using sex-specific PCR primers, we show that all plantlets and plants derived from anther cultures are female. In studying anther-derived diploid females by sequencing of X-linked markers, we demonstrate that these plants are really homozygous dihaploids. A haploid regenerant plant was sequenced (8× genome coverage) using Illumina technology. Genome data are disposable in the EMBL database as a standard for full genome and X chromosome assembly in this model species. Homozygous dihaploids were back-crossed with males to yield a progeny useful for the study of the evolution of the Y chromosome.

Any project seeking to deliver a plant or animal reference genome sequence must address the question as to the completeness of the assembly. Given the complexity introduced particularly by the presence of sequence redundancy, a problem which is especially acute in polyploid genomes, this question is not an easy one to answer. One approach is to use the sequence data, along with the appropriate computational tools, the other is to compare the estimate of genome size with an experimentally measured mass of nuclear DNA. The latter requires a reference standard in order to provide a robust relationship between the two independent measurements of genome size. Here, the proposal is to choose the human male leucocyte genome for this standard: its 1C DNA amount (the amount of DNA contained within unreplicated haploid chromosome set) of 3.50 pg is equivalent to a genome length of 3.423 Gbp, a size which is just 5% longer than predicted by the most current human genome assembly. Adopting this standard, this paper assesses the completeness of the reference genome assemblies of the leading cereal crops species wheat, barley and rye.

© 2016, The Author(s). Bananas (Musa spp.) are one of the main fruit crops grown worldwide. With the annual production reaching 144 million tons, their production represents an important contribution to the economies of many countries in Asia, Africa, Latin-America and Pacific Islands. Most importantly, bananas are a staple food for millions of people living in the tropics. Unfortunately, sustainable banana production is endangered by various diseases and pests, and the breeding for resistant cultivars relies on a far too small base of genetic variation. Greater diversity needs to be incorporated in breeding, especially of wild species. Such work requires a large and thoroughly characterized germplasm collection, which also is a safe depository of genetic diversity. The largest ex situ Musa germplasm collection is kept at the International Transit Centre (ITC) in Leuven (Belgium) and currently comprises over 1500 accessions. This report summarizes the results of systematic cytological and molecular characterization of the Musa ITC collection. By December 2015, 630 accessions have been genotyped. The SSR markers confirmed the previous morphological based classification for 84% of ITC accessions analyzed. The remaining 16% of the genotyped entries may need field verification by taxonomist to decide if the unexpected classification by SSR genotyping was correct. The ploidy level estimation complements the molecular data. The genotyping continues for the entire ITC collection, including newly introduced accessions, to assure that the genotype of each accession is known in the largest global Musa gene bank. Open Access ispartof: Biodiversity and Conservation vol:26 issue:4 pages:801-824 status: published

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.

With the expansion of next-generation sequencing technology and advanced bioinformatics, there has been a rapid growth of genome sequencing projects. However, while this technology enables the rapid and cost-effective assembly of draft genomes, the quality of these assemblies usually falls short of gold standard genome assemblies produced using the more traditional BAC by BAC and Sanger sequencing approaches. Assembly validation is often performed by the physical anchoring of genetically mapped markers, but this is prone to errors and the resolution is usually low, especially towards centromeric regions where recombination is limited. New approaches are required to validate reference genome assemblies. The ability to isolate individual chromosomes combined with next-generation sequencing permits the validation of genome assemblies at the chromosome level. We demonstrate this approach by the assessment of the recently published chickpea kabuli and desi genomes. While previous genetic analysis suggests that these genomes should be very similar, a comparison of their chromosome sizes and published assemblies highlights significant differences. Our chromosomal genomics analysis highlights short defined regions that appear to have been misassembled in the kabuli genome and identifies large-scale misassembly in the draft desi genome. The integration of chromosomal genomics tools within genome sequencing projects has the potential to significantly improve the construction and validation of genome assemblies. The approach could be applied both for new genome assemblies as well as published assemblies, and complements currently applied genome assembly strategies.

The use of flow cytometry for evaluation of plant chromosomes requires some specialized attention to preparation and instrumentation. This unit deals exclusively with plant cytogenetics and presents an outline of this area as well as methods for accumulation of cells in metaphase, preparation of chromosome suspensions, flow analysis and sorting of chromosomes, and processing of the sorted chromosomes. Each method is described in tremendous detail because in many aspects dealing with plant cells is quite different from dealing with mammalian cells. Supporting histograms are presented as well as a range of special hints on dealing with plant material and a discussion of the utility of sorted chromosomes for plant genome mapping.. Keywords: flow cytometry; plant chromosomes; cell cycle synchronization; chromosome isolation; chromosome sorting; physical gene mapping; chromosome-specific DNA libraries