Comparative cytogenetic analysis of diploid and hexaploid Chenopodium album agg.

Two cytotypes of Chenopodium album, diploid (2n=2x=18) and hexaploid (2n=6x=54), were analysed using flow cytometry and aFisH experiment. The genome size was indicated as 1.795 pg for the diploid and 3.845 pg for the hexaploid plants which suggested genome downsizing in the evolution of hexaploid cytotype. Double FisH with 25s rDNA and 5s rDNA allowed three to five homologue chromosome pairs to be distinguished depending on the cytotype. The variation in size and number of rDNA sites between the polyploid C. album and its putative diploid ancestor indicated that rDNA loci underwent rearrangements after polyploidization. Flow cytometry measurements of the relative nuclear DNA content in the somatic tissue of C. album revealed extensive endopolyploidization resulting in tissues comprising a mixture of cells with a different DNA content (from 2Cto 32C) in varying proportions. The pattern of endopolyploidy was characteristic for the developmental stage of the plant and for the individual organ. Polysomaty was not observed in the embryo tissues however endo- polyploidization had taken place in most tested organs of seedlings. The endopolyploidy in diploid and hexaploid C. album was compared to find any relationship between the pattern of polysomaty and polyploidy level in this species. This revealed that polyploid plants showed a decline in the number of endocycles as well as in the frequency of endopolyploidy cells compared to diploid plants

Molecular cytogenetic studies in Chenopodium quinoa and Amaranthus caudatus

Chenopodium quinoa Wild, and Amaranthus caudatus L„ two plant species from South America, have small and numerous chromosomes. Looking for chromosome markers to distinguish pairs of homologous chromosomes double fluorescence staining, in situ hybridization with 45S rDNA and silver staining were applied. Fluorescent in situ hybridization with 45S rDNA has shown two sites of hybridization occurring on one pair of chromosomes in quinua genome (lines PQ-I. PQ-X). The number of rDNA loci in Amaranthus caudatus L. genome depends on the accession. Kiwicha 3 line has one pair of chromosomes with signals and Kiwicha Molincra cultivar two pairs. All observed rDNA loci were active. After chromomycin/ DAPl staining in all cases, except Kiwicha Molinera cultivar, the CMA, positive bands co-localized with signals of in situ hybridization with rDNA. In Kiwicha Molinera the number of CMA+ bands was higher than the number of 45S rDNA signals after FISH. Genomy dwóch południowoamerykańskich gatunków. Chenopodiuma quinoa Wild, i Amaranthus caudatus L.. charakteryzują się dużą liczbą niewielkich i morfologicznie mało zróżnicowanych chromosomów. Dla opracowania kariotypu tych gatunków niezbędne jest znalezienie cytogenetycznych markerów pozwalających odróżnić pary chromosomów homologicznych. W tym celu zastosowano barwienie fluorescencyjne (CMA,/DAPI) i hybrydyzację in situ z 45S rDNA oraz srebrzenie dla określenia jąderkowej aktywności transkrypcyjnej. Po fluorescencyjnej hybrydyzacji in situ z. 45S rDNA w genomie C. quinoa (linie PQ-I. PQ-8) obserwowano dwa sygnały występujące w jednej parze chromosomów. Liczba loci rDNA w genomie A. caudatus była różna. W linii Kiwicha .3 obserwowano dwa sygnały a u odmiany Kiwicha Molinera cztery. Wszystkie obserwowane loci rDNA są aktywne transkrypcyjnic. We wszystkich przypadkach, za wyjątkiem odmiany Kiwicha Molinera, pozytywne prążki CM A, pokrywały się z sygnałami hybrydyzacji in situ. U Kiwicha Molinera liczba pozytywnych prążków CMA( była większa niż liczba sygnałów po hybrydyzacji z 45S rDNA

Cytogenetic studies of three European species of Centaurea L. (Asteraceae)

Cytogenetic analysis of several populations of Centaurea jacea (2n = 4x = 44), C. oxylepis (2n = 4x = 44) and C. phrygia (2n = 2x = 22) was performed using flow cytometry, differential chromosome staining and FISH. In all species Arabidopsis-type telomeric repeats hybridized only to the terminal part of chromosomes. In C. phrygia three pairs and in C. oxylepis six pairs of chromosomes revealed the hybridization signals of 45S rDNA. Centaurea jacea showed polymorphism in the 45S rDNA loci number, five or six pairs of sites were observed. 5S rDNA loci were located in two pairs of chromosomes in C. phrygia. In C. jacea and C. oxylepis the number and position of 5S rDNA loci were the same: three pairs located interstitially and one terminally. The genome size of the diploid C. phrygia was established as 2.14 pg/2C. The genomes of tetraploid species were nearly two times larger and genome size polymorphism was observed among C. jacea populations

Karyotype analysis of eight cultivated Allium species

The karyotypes of Allium, a genus that comprises many crops and ornamental plants, are relatively poorly studied. To extend our knowledge on karyotype structure of the genus, the chromosomal organization of rRNA genes and CMA/DAPI bands was studied. Fluorescence in situ hybridization using 5S and 35S rDNA probes and banding methods (silver staining and CMA3/ DAPI staining) were used to analyze the karyotypes of eight cultivated Allium L. species. Analyzed Allium taxa revealed three different basic chromosome numbers (x = 7, 8, 9) and three different ploidy levels (diploid, triploid, and tetraploid). The rDNA sites chromosomal organization is reported the first time for the six species (A. moly, A. oreophilum, A. karataviense, A. nigrum, A. sphaerocephalon, A. porrum). The Allium species that were analyzed showed a high level of interspecies polymorphism in the number and localization of the rDNA sites. The fluorescence in situ hybridization patterns of 35S rDNA sites were more polymorphic than those of the 5S rDNA in the diploid species. Several groups of similar chromosomes could be distinguished among the chromosomes that had rDNA sites in the polyploid species. Each of the groups had three chromosomes (triploid A. sphaerocephalon L.) or four chromosomes (tetraploid A. porrum L.) suggesting their autopolyploid origin. In the genomes of four of the analyzed species, only some of the 35S rDNA sites were transcriptionally active. Fluorochrome banding revealed that the CMA3 + bands were associated with the 35S rDNA sites in all of the species that were analyzed, except A. fistulosum L. in which positive CMA3 + bands were detected in the terminal position of all of the chromosome arms. The rDNA sequences, nucleolar organizer regions (NORs), and CMA/DAPI bands are very good chromosome markers that allowed to distinguished from two to five pairs of homologous chromosomes in analyzed Allium species. The karyotypes of the studied species could be clearly distinguished by the number and position of the rDNA sites, NORs, and CMA/DAPI bands, which revealed high interspecific differentiation among the taxa

Genome size variation in Chenopodium quinoa (Chenopodiaceae)

The extent and significance of intraspecific genome size variation were analysed in quinoa (Chenopodium quinoa Willd.), a pseudocereal important for human consumption in the Andean region of South America. Flow cytometry, with propidium iodide as the DNA stain, was used to estimate the genome size of 20 quinoa accessions from Ecuador, Peru, Bolivia, Argentina, Chile and the USA. Limited genome size variation was found among the analysed accessions. The differences between the accessions were statistically significant but the maximum inter-accession difference between the populations with the largest and the smallest genome reached only 5.9%. The largest genome was found in population C4 from Chile (mean 3.077 pg/2C) and the smallest in the Peruvian population P2 (mean 2.905 pg/2C). The variation was not correlated with collection site; however, the quinoa accessions analysed in this study belonged to three distinct geographical groups: northern highland, southern highland and lowland

Tracing the Evolution of the Angiosperm Genome from the Cytogenetic Point of View

Cytogenetics constitutes a branch of genetics that is focused on the cellular components, especially chromosomes, in relation to heredity and genome structure, function and evolution. The use of modern cytogenetic approaches and the latest microscopes with image acquisition and processing systems enables the simultaneous two- or three-dimensional, multicolour visualisation of both single-copy and highly-repetitive sequences in the plant genome. The data that is gathered using the cytogenetic methods in the phylogenetic background enable tracing the evolution of the plant genome that involve changes in: (i) genome sizes; (ii) chromosome numbers and morphology; (iii) the content of repetitive sequences and (iv) ploidy level. Modern cytogenetic approaches such as FISH using chromosome- and genome-specific probes have been widely used in studies of the evolution of diploids and the consequences of polyploidy. Nowadays, modern cytogenetics complements analyses in other fields of cell biology and constitutes the linkage between genetics, molecular biology and genomics

Descending dysploidy and bidirectional changes in genome size accompanied Crepis (Asteraceae) evolution

The evolution of the karyotype and genome size was examined in species of Crepis sensu lato. The phylogenetic relationships, inferred from the plastid and nrITS DNA sequences, were used as a framework to infer the patterns of karyotype evolution. Five different base chromosome numbers (x = 3, 4, 5, 6, and 11) were observed. A phylogenetic analysis of the evolution of the chromosome numbers allowed the inference of x = 6 as the ancestral state and the descending dysploidy as the major direction of the chromosome base number evolution. The derived base chromosome numbers (x = 5, 4, and 3) were found to have originated independently and recurrently in the different lineages of the genus. A few independent events of increases in karyotype asymmetry were inferred to have accompanied the karyotype evolution in Crepis. The genome sizes of 33 Crepis species differed seven-fold and the ancestral genome size was reconstructed to be 1 C = 3.44 pg. Both decreases and increases in the genome size were inferred to have occurred within and between the lineages. The data suggest that, in addition to dysploidy, the amplification/elimination of various repetitive DNAs was likely involved in the genome and taxa differentiation in the genus

Field cress genome mapping: Integrating linkage and comparative maps with cytogenetic analysis for rDNA carrying chromosomes

Field cress (Lepidium campestre L.), despite its potential as a sustainable alternative oilseed plant, has been underutilized, and no prior attempts to characterize the genome at the genetic or molecular cytogenetic level have been conducted. Genetic maps are the foundation for anchoring and orienting annotated genome assemblies and positional cloning of candidate genes. Our principal goal was to construct a genetic map using integrated approaches of genetic, comparative and cytogenetic map analyses. In total, 503 F2 interspecific hybrid individuals were genotyped using 7,624 single nucleotide polymorphism markers. Comparative analysis demonstrated that ~57% of the sequenced loci in L. campestre were congruent with Arabidopsis thaliana (L.) genome and suggested a novel karyotype, which predates the ancestral crucifer karyotype. Aceto-orcein chromosome staining and fluorescence in situ hybridization (FISH) analyses confirmed that L. campestre, L. heterophyllum Benth. and their hybrids had a chromosome number of 2n = 2x = 16. Flow cytometric analysis revealed that both species possess 2C roughly 0.4 picogram DNA. Integrating linkage and comparative maps with cytogenetic map analyses assigned two linkage groups to their particular chromosomes. Future work could incorporate FISH utilizing A. thaliana mapped BAC clones to allow the chromosomes of field cress to be identified reliably

A Chromosome-Scale Assembly of the Garden Orach (Atriplex hortensis L.) Genome Using Oxford Nanopore Sequencing

Atriplex hortensis (2n = 2x = 18, 1C genome size 1.1 gigabases), also known as garden orach and mountain-spinach, is a highly nutritious, broadleaf annual of the Amaranthaceae-Chenopodiaceae alliance (Chenopodiaceae sensu stricto, subfam. Chenopodioideae) that has spread in cultivation from its native primary domestication area in Eurasia to other temperate and subtropical regions worldwide. Atriplex L. is a highly complex but, as understood now, a monophyletic group of mainly halophytic and/or xerophytic plants, of which A. hortensis has been a vegetable of minor importance in some areas of Eurasia (from Central Asia to the Mediterranean) at least since antiquity. Nonetheless, it is a crop with tremendous nutritional potential due primarily to its exceptional leaf and seed protein quantities (approaching 30%) and quality (high levels of lysine). Although there is some literature describing the taxonomy and production of A. hortensis, there is a general lack of genetic and genomic data that would otherwise help elucidate the genetic variation, phylogenetic positioning, and future potential of the species. Here, we report the assembly of the first high-quality, chromosome-scale reference genome for A. hortensis cv. “Golden.” Long-read data from Oxford Nanopore’s MinION DNA sequencer was assembled with the program Canu and polished with Illumina short reads. Contigs were scaffolded to chromosome scale using chromatin-proximity maps (Hi-C) yielding a final assembly containing 1,325 scaffolds with a N50 of 98.9 Mb – with 94.7% of the assembly represented in the nine largest, chromosome-scale scaffolds. Sixty-six percent of the genome was classified as highly repetitive DNA, with the most common repetitive elements being Gypsy- (32%) and Copia-like (11%) long-terminal repeats. The annotation was completed using MAKER which identified 37,083 gene models and 2,555 tRNA genes. Completeness of the genome, assessed using the Benchmarking Universal Single Copy Orthologs (BUSCO) metric, identified 97.5% of the conserved orthologs as complete, with only 2.2% being duplicated, reflecting the diploid nature of A. hortensis. A resequencing panel of 21 wild, unimproved and cultivated A. hortensis accessions revealed three distinct populations with little variation within subpopulations. These resources provide vital information to better understand A. hortensis and facilitate future study

Molecular cytogenetic studies in Chenopodium quinoa and Amaranthus caudatus

Chenopodium quinoa Wild. and Amaranthus caudatus L., two plant species from South America, have small and numerous chromosomes. Looking for chromosome markers to distinguish pairs of homologous chromosomes double fluorescence staining, in situ hybridization with 45S rDNA and silver staining were applied. Fluorescent in situ hybridization with 45S rDNA has shown two sites of hybridization occurring on one pair of chromosomes in qunion genre (lines PQ-1, PQ-8). The number of RDA loci in Amaranth's caudate L. genre depends on the accession. Kiwicha 3 line has one pair of chromosomes with signals and Kiwicha Molinera cultivar two pairs. All observed rDNA loci were active. After chromomycin/DAPI staining in all cases, except Kiwicha Molinera cultivar, the CMA3 positive bands co-localized with signals of in situ hybridization with rDNA. In Kiwicha Molinera the number of CMA+ bands was higher than the number of 45S rDNA signals after FISH