Comparative Karyotype Analysis Research Articles

Comparative karyotype analysis of the red brocket deer (M. americana sensu lato and M. rufa) complex: evidence of drastic chromosomal evolution and implications on speciation process.

Chromosomal rearrangements are often associated with playing a role in the speciation process. However, the underlying mechanism that favors the genetic isolation associated with chromosomal changes remains elusive. In this sense, the genus Mazama is recognized by its high level of karyotype diversity among species with similar morphology. A cryptic species complex has been identified within the genus, with the red brocket deer (Mazama americana and Mazama rufa) being the most impressive example. The chromosome variation was clustered in cytotypes with diploid numbers ranging from 42 to 53 and was correlated with geographical location. We conducted an analysis of chromosome evolution of the red brocket deer complex using comparative chromosome painting and Bacterial Artificial Chromosome (BAC) clones among different cytotypes. The aim was to deepen our understanding of the karyotypic relationships within the red brocket, thereby elucidating the significant chromosome variation among closely related species. This underscores the significance of chromosome changes as a key evolutionary process shaping their genomes. The results revealed the presence of three distinct cytogenetic lineages characterized by significant karyotypic divergence, suggesting the existence of efficient post-zygotic barriers. Tandem fusions constitute the main mechanism driving karyotype evolution, following a few centric fusions, inversion X-autosomal fusions. The BAC mapping has improved our comprehension of the karyotypic relationships within the red brocket deer complex, prompting questions regarding the role of these changes in the speciation process. We propose the red brocket as a model group to investigate how chromosomal changes contribute to isolation and explore the implications of these changes in taxonomy and conservation.

Karyotypes and Physical Mapping of Ribosomal DNA with Oligo-Probes in Eranthis sect. Eranthis (Ranunculaceae).

A comparative karyotype analysis of four species of yellow-flowered Eranthis sect. Eranthis, i.e., E. bulgarica, E. cilicica, E. hyemalis, and E. longistipitata from different areas, has been carried out for the first time. All the studied specimens had somatic chromosome number 2n = 16 with basic chromosome number x = 8. Karyotypes of the investigated plants included five pairs of metacentric chromosomes and three pairs of submetacentric/subtelocentric chromosomes. The chromosome sets of the investigated species differ mainly in the ratio of submetacentric/subtelocentric chromosomes, their relative lengths, and arm ratios. A new oligonucleotide probe was developed and tested to detect 45S rDNA clusters. Using this probe and an oligonucleotide probe to 5S rDNA, 45S and 5S rDNA clusters were localized for the first time on chromosomes of E. cilicica, E. hyemalis, and E. longistipitata. Major 45S rDNA clusters were identified on satellite chromosomes in all the species; in E. cilicica, minor clusters were also identified in the terminal regions of one metacentric chromosome pair. The number and distribution of 5S rDNA clusters is more specific. In E. cilicica, two major clusters were identified in the pericentromeric region of a pair of metacentric chromosomes. Two major clusters in the pericentromeric region of a pair of submetacentric chromosomes and two major clusters in the interstitial region of a pair of metacentric chromosomes were observed in E. longistipitata. E. hyemalis has many clusters of different sizes, localized mainly in the pericentromeric regions. Summarizing new data on the karyotype structure of E. sect. Eranthis and previously obtained data on E. sect. Shibateranthis allowed conclusions to be formed about the clear interspecific karyological differences of the genus Eranthis.

Chromosomal aberrations in Ukrainian red-spotted dairy breed cows when using different breeding methods

Aim. The aim of this study was a comparative analysis of karyotypes of purebred, inbred, and crossbred cows of the Ukrainian red-spotted dairy breed in order to detect their degree of chromosomal aberrations. Methods. The cyto- genetic study involved 156 cows of the Ukrainian red-spotted dairy breed from two research farms of M.V. Zubets Institute of Animal Breeding and Genetics, the NAAS. Cultures of peripheral lymphocytes, stimulated with phytohemagglutinin (PHA, Difco, USA) were used according to the method of Moorhead et al. (1960). The chromosomal preparations were stained with 2 % Giemsa solution (Giemsa, Merk) and analyzed under an Axiostar plus (Carl Zeiss, Germany) light microscope at 1,000 times magnification. Metaphase slides were photographed with a digital camera Olympus D-460 ZOOM. The biometric processing of the study results was conducted by ANOVA using Microsoft Excel 2010. Results. Genomic mutations (aneuploidy and polyploidy) and structural aberrations of chromosomes were found in the karyotypes of the investigated purebred, inbred, and crossbred cows. The total share of aberrant cells varied from 11.4 ± 0.39 % in purebred, 11.8 ± 0.40 % in inbred to 17.30 ± 0.85 % in crossbred cows. In two animals the Robertson translocation, rob (1 : 29) was found. A statistically significant difference between purebred, inbred, and crossbred animals was found regarding cells with aneuploidy and structural chromosomal aberrations. Conclusions. The karyological analysis yielded data showing changes in genetic structure that can have an influence on fertility and production in different breeding programs.

Comparative Cytogenetics and Fluorescent Chromosome Banding in Five Indian Species of Dipcadi Medik

The genus Dipcadi Medik. (Subfamily: Scilloideae) has a narrow distribution in India and several overlapping morphological traits make the genus taxonomically challenging at the species level. Cytogenetic characterization can provide additional taxonomic data and can be used to evaluate genetic diversity at the species level. We have accomplished comparative karyotype analysis and fluorescence banding patterns using 4′-6-Diamidino-2-phenylindole (DAPI) and Chromomycin A3 (CMA) in five Indian species for the first time. The karyotypes of D. concanense and D. goaense exhibited similar fluorochrome banding profiles. However, D. montanum, D. ursulae and D. erythraeum differ distinctly in their karyotypes. In all taxa, CMA+ve/DAPI−ve or DAPI0 (GC-rich) constitutive heterochromatin was located at the constriction region or terminal satellite of the nucleolar chromosome. DAPI+ve/CMA−ve or CMA0 (AT-rich) heterochromatin dominates in D. montanum, D. ursulae and D. erythraeum. However, D. erythraeum shows a distinct variation in fluorochrome banding pattern from all other species. The distribution of CMA and DAPI bands is a reflection of heterochromatin composition and variations acquired by different species. This characterization can be used to assess phylogenetic relationships in the understudied genus Dipcadi and may serve as a basis for other genomic analyses and evolutionary studies.

Comparative karyotype analysis of eight Cucurbitaceae crops using fluorochrome banding and 45S rDNA-FISH.

To have an insight into the karyotype variation of eight Cucurbitaceae crops including Cucumissativus Linnaeus, 1753, Cucumismelo Linnaeus, 1753, Citrulluslanatus (Thunberg, 1794) Matsumura et Nakai, 1916, Benincasahispida (Thunberg, 1784) Cogniaux, 1881, Momordicacharantia Linnaeus, 1753, Luffacylindrica (Linnaeus, 1753) Roemer, 1846, Lagenariasicerariavar.hispida (Thunberg, 1783) Hara, 1948 and Cucurbitamoschata Duchesne ex Poiret, 1819, well morphologically differentiated mitotic metaphase chromosomes were prepared using the enzymatic maceration and flame-drying method, and the chromosomal distribution of heterochromatin and 18S-5.8S-26S rRNA genes (45S rDNA) was investigated using sequential combined PI and DAPI (CPD) staining and fluorescence in situ hybridization (FISH) with 45S rDNA probe. Detailed karyotypes were established using the dataset of chromosome measurements, fluorochrome bands and rDNA FISH signals. Four karyotype asymmetry indices, CVCI, CVCL, MCA and Stebbins' category, were measured to elucidate the karyological relationships among species. All the species studied had symmetrical karyotypes composed of metacentric and submetacentric or only metacentric chromosomes, but their karyotype structure can be discriminated by the scatter plot of MCA vs. CVCL. The karyological relationships among these species revealed by PCoA based on x, 2n, TCL, MCA, CVCL and CVCI was basically in agreement with the phylogenetic relationships revealed by DNA sequences. CPD staining revealed all 45S rDNA sites in all species, (peri)centromeric GC-rich heterochromatin in C.sativus, C.melo, C.lanatus, M.charantia and L.cylindrica, terminal GC-rich heterochromatin in C.sativus. DAPI counterstaining after FISH revealed pericentromeric DAPI+ heterochromatin in C.moschata. rDNA FISH detected two 45S loci in five species and five 45S loci in three species. Among these 45S loci, most were located at the terminals of chromosome arms, and a few in the proximal regions. In C.sativus, individual chromosomes can be precisely distinguished by the CPD band and 45S rDNA signal patterns, providing an easy method for chromosome identification of cucumber. The genome differentiation among these species was discussed in terms of genome size, heterochromatin, 45S rDNA site, and karyotype asymmetry based on the data of this study and previous reports.

Bulked Oligo-FISH for Chromosome Painting and Chromosome Barcoding.

Recently developed bulked oligo-FISH is a highly versatile method, which is applicable in any plant species with an assembled genome sequence. This technique allows in situ identification of individual chromosomes, large chromosomal rearrangements, comparative karyotype analysis, or even the reconstruction of the three-dimensional organization of the genome. The method is based on the identification of thousands of short oligonucleotides, unique to specific genome regions, which are synthesized in parallel, fluorescently labeled and used as probes for FISH. In this chapter, we propose a detailed protocol for amplification and labeling of single-stranded oligo-based painting probes from so-called MYtags immortal libraries, the preparation of mitotic metaphase and meiotic pachytene chromosome spreads, and a protocol for the fluorescence in situ hybridization procedure using the synthetic oligo probes. The proposed protocols are demonstrated for banana (Musa spp.).

Accurate Chromosome Identification in the Prunus Subgenus Cerasus (Prunus pseudocerasus) and its Relatives by Oligo-FISH.

A precise, rapid and straightforward approach to chromosome identification is fundamental for cytogenetics studies. However, the identification of individual chromosomes was not previously possible for Chinese cherry or other Prunus species due to the small size and similar morphology of their chromosomes. To address this issue, we designed a pool of oligonucleotides distributed across specific pseudochromosome regions of Chinese cherry. This oligonucleotide pool was amplified through multiplex PCR with specific internal primers to produce probes that could recognize specific chromosomes. External primers modified with red and green fluorescence tags could produce unique signal barcoding patterns to identify each chromosome concomitantly. The same oligonucleotide pool could also discriminate all chromosomes in other Prunus species. Additionally, the 5S/45S rDNA probes and the oligo pool were applied in two sequential rounds of fluorescence in situ hybridization (FISH) localized to chromosomes and showed different distribution patterns among Prunus species. At the same time, comparative karyotype analysis revealed high conservation among P. pseudocerasus, P. avium, and P. persica. Together, these findings establish this oligonucleotide pool as the most effective tool for chromosome identification and the analysis of genome organization and evolution in the genus Prunus.

Comparative analysis of karyotypes of Вovidae family from the evolutionary aspect

Aim. The aim of the study was to analyze chromosome sets of specific species of agricultural animals of Bovidae family using the techniques of differentiated G- and Ag-banding of chromosomes and to demonstrate the role of their variability in the evolution. Methods. The culture of lymphocytes and chromosome preparations were pre- pared by the method of Moorhead et al (1960). The preparations of metaphase chromosomes were analyzed by the G-banding methods of Barch M J et al (1997) and by Ag-banding method of Zhu JJ et al (2019). The chromosomal aberrations were classified according to the recommendations of the International System for Chromosome No- menclature of Domestic Bovids (2001). The dendrogram of phylogenetic relations between mammalian species was built using “STATISTICA 6.1”. Results. The comparative study of the chromosomal sets of cattle Bos taurus, domestic sheep Ovis aries and river buffalo Bubalus bubalis bubalis was conducted using G- and Ag-banding methods. The homology of the structural organization of chromosomes and the evolutionary changes in karyotypes of Bovidae family were analyzed, highlighting their sex chromosomes and the chromosomes with specific local- ized groups of gene linkage. A considerable homology of chromosome sites was found in the representatives of the investigated species by the G-banding profiles. To study the phylogenetic interrelations, the index of the number of active nucleolus organizer regions (NOR) in the chromosomes during the metaphase stage was used. Conclu- sions. The comparison of the morphology of chromosomes of agricultural animals of Bovidae family, Bos taurus, Ovis aries, and Bubalus bubalis bubalis confirms the association between the homology of some chromosomal sites and the formation of metacentric chromosomes due to the linkage of acrocentric ones. The species-specific morphological differences in sex chromosomes of the investigated animals were found in terms of the length and presence of pericentric inversions. The phylogenetic relations between the species of Bovidae family demonstrate that the value of distances, determined based on the variability of the number of active NOR, reflects the degree of their phylogenetic similarity.

Comparative Karyotype Analysis of Parasitoid Hymenoptera (Insecta): Major Approaches, Techniques, and Results.

A comprehensive review of main approaches, techniques and results of the chromosome study of parasitic wasps is given. In this group, the haploid chromosome number ranges from n = 3 to 23. Distribution of parasitic wasp species by the chromosome number is bimodal, with two obvious modes at n = 6 and 11. Karyotype analysis based on routinely stained preparations of mitotic chromosomes can be used to identify members of taxonomically complicated parasitoid taxa and to distinguish between them. Morphometric study effectively reveals subtle differences between similar chromosome sets of parasitic wasps. If combined with meiotic analysis and/or cytometric data, information on mitotic karyotypes can highlight pathways of the genome evolution in certain parasitoid taxa. C- and AgNOR-banding as well as staining with base-specific fluorochromes detected important interspecific differences within several groups of parasitic wasps. Fluorescence in situ hybridization (FISH) is successfully used for physical mapping of various DNA sequences on parasitoid chromosomes. These techniques demonstrate that heterochromatic segments are usually restricted to pericentromeric regions of chromosomes of parasitic wasps. Haploid karyotypes carrying one or two nucleolus organizing regions (NORs) are the most frequent among parasitoid Hymenoptera. In combination with chromosome microdissection, FISH could become a powerful tool exploring the genome evolution of parasitic wasps. Perspectives of the comparative cytogenetic study of parasitoid Hymenoptera are outlined.

Advances in the Molecular Cytogenetics of Bananas, Family Musaceae.

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.

CONVENTIONAL AND G-BANDING KARYOTYPE VARIATIONS OF THREE DUCK BREEDS OCCURRING IN EGYPT

In the present work, the chromosome variation and comparative analysis of conventional and G-banding karyotypes between three duck breeds (Pekin, Soudani and Baladi) occurring in Egypt were carried out. Almost all examined cells of the three studied duck breeds showed 7 macro-chromosome pairs and Z, W and 64 microchromosomes (less than 1 micron in size and so called microchromosomes). Some of karyological parameters such as chromosome length, q and p arm lengths, arm ratio, Intra chromosomal asymmetry degree (AsD) and karyotypic asymmetry (AsK) were calculated. Results showed notable differences of the karyotype characteristics between the three studied duck breeds. Likewise,the studies macrochromosomes showed three different categories of karyotypic formulas were obtained (1m+7sm+1st for Baladi, 3m+6sm for Pekin and 9sm for Sudani) including sex chromosomes. Into AsD and AsK parameters also varied among the studied breeds. There was visible variation in the G- banding patterns and their constructed physical maps of the seven pairs of autosomes and sex chromosomes between the three studied duck breeds.

Cytogenetic evidence supports Avena insularis being closely related to hexaploid oats.

Cytogenetic observations, phylogenetic studies and genome analysis using high-density genetic markers have suggested a tetraploid Avena species carrying the C and D genomes (formerly C and A) to be the donor of all hexaploid oats (AACCDD). However, controversy surrounds which of the three extant CCDD tetraploid species—A. insularis, A. magna and A. murphyi—is most closely related to hexaploid oats. The present work describes a comparative karyotype analysis of these three CCDD tetraploid species and two hexaploid species, A. sativa and A. byzantina. This involved the use of FISH with six simple sequence repeats (SSRs) with the motifs CT, AAC, AAG, ACG, ATC and ACT, two repeated ribosomal sequences, and C genome-specific repetitive DNA. The hybridization pattern of A. insularis with oligonucleotide (AC)10 was also determined and compared with those previously published for A. sativa and A. byzantina. Significant differences in the 5S sites and SSR hybridization patterns of A. murphyi compared to the other CCDD species rule out its being directly involved in the origin of the hexaploids. In contrast, the repetitive and SSR hybridization patterns shown by the D genome chromosomes, and by most of the C genome chromosomes of A. magna and A. insularis, can be equated with the corresponding chromosomes of the hexaploids. Several chromosome hybridization signals seen for A. insularis, but not for A. magna, were shared with the hexaploid oats species, especially with A. byzantina. These diagnostic signals add weight to the idea that the extant A. insularis, or a direct ancestor of it, is the most closely related progenitor of hexaploid oats. The similarity of the chromosome hybridization patterns of the hexaploids and CCDD tetraploids was taken as being indicative of homology. A common chromosome nomenclature for CCDD species based on that of the hexaploid species is proposed.

Molecular cytogenetics of Hedysarum gmelinii Ledeb. and H. setigerum Turcz. (Fabaceae) from populations of Southern Siberia

For the first time, a comparative karyotype analysis of closely related species Hedysarum gmelinii andH. setigerum (Hedysarum section Multicaulia) grown in Southern Siberia, has been performed by molecular cytogeneticmarkers. Chromosome numbers in karyotypes of these species were specified – 2n = 4х = 32. In some accessions, additionalB chromosomes were revealed. FISH analyses indicated high similarities in chromosome morphology and also patternsof chromosomal distributions of 45S and 5S rDNA clusters in karyotypes of H. gmelinii and H. setigerum, which confirmsthe close relationship between their genomes.

The use of molecular cytogenetic methods in the investigation of distant potato hybrids

This paper reviews the results of studies of interspecies hybridization, polyploidization, as well as phylogenetic relationships ofSolanumspecies and members of closely related taxa by such molecular cytogenetic techniques as genomic (GISH) and fluorescent (FISH) DNA-DNAin situhybridization. The latter was used to determine the genomic composition and origin of wild species of thePetotasection, while the FISH technique was used for detecting intergenomic collinearity. The combination of these two types of research made possible a comparative analysis of karyotypes and genomes, thus allowing a better understanding of the meiotic interchromosomal interactions in hybrids. This review primarily focuses on the studies of wild allopolyploid potato species and artificially created intergeneric and interspecific hybrids of the genusSolanumand their offspring.

Development of a set of chromosome-specific oligonucleotide markers and karyotype analysis in the Japanese morning glory Ipomoea nil

The phylogenetic relationships for Ipomoea species are incongruent in previous studies. Comparative karyotype analysis can provide valuable information for phylogenetic relationships among species. A reliable and efficient system for chromosome identification is the foundation for karyotype analysis. However, for the Ipomoea species, individual chromosomes were not identified in all previous reported karyotypes due to their small size, high number and similar morphology. Fluorescence in situ hybridization (FISH) using oligonucleotides (oligos) as probes is a new strategy for chromosome identification and karyotype analysis. Here, we developed the first set of oligo-based probes based on the reference genome of Ipomoea nil, a model species in the genus Ipomoea. In all, we developed four oligo-FISH probes. By a combined use of four oligo probes, sequential FISH analysis were conducted on the same metaphase cells with each round including two probes, which permitted simultaneous identification of all I. nil chromosomes and anchoring 15 pseudochromosomes to individual cytological chromosomes. Moreover, 45S and 5S rDNA were mapped to specific chromosomes. A karyotype based on individually identified chromosomes was established, which was the first FISH-based molecular cytogenetic karyotype of I. nil. Our study has created the basis for studying chromosome variation and evolution in genus Ipomoea by comparative karyotype analysis using this set of oligo probes.

Karyological, Molecular and Cytogenetical Studies on Tree Plants in V.N. Sukachev Institute of Forest

Aim. The aim of the article is to find out the karyological features and conduct molecular and cytogenetic analysis on tree plants for biological diversity studies, solve problems of taxonomics, evolutionary and population genetics. Methods. Classic methods with acetohematoxylin staining of slides and new method of fluorescent in situ hybridization (FISH) had been used. Results . More than 250 populations and provenances of representatives of different genera of gymnosperms from the families Pinaceae, Cupressaceae, Ephedraceae and tree angiosperms from the families Rosaceae, Aceraceae, Polygonaceae, Nitrariaceae, Rhamnaceae, Fabaceae, Ericaceae were studied. Investigations had been conducted in natural populations and during the introduction, in optimal and extreme conditions, in disturbed ecosystems, botanical gardens and parks; in addition, various intraspecific forms and unique trees had been studied. The variability of chromosome numbers and a wide range of chromosomal mutations had been revealed. Use of fluorescence in situ hybridization (FISH) with the 45S and 5S ribosomal RNA gene probes and DAPI staining allowed to identify of homologous chromosome pairs in the karyotypes of conifers and to facilitate the comparative karyotype analysis of those species. Analysis of the chromosome numbers of woody angiosperms showed a significant role of polyploidy in their evolution. Conclusions. The studies on karyotypes in tree species showed their diversity, variability of chromosome numbers, higher level of chromosomal anomalies and their wide spectrum in extreme conditions, under introduction and also in cultivars and abnormal form of trees. The use of molecular cytogenetic markers made it possible to obtain new information on the structure of conifer chromosomes. The role of polyploidy in the evolution of woody angiosperms was shown.

Comparative Analysis of Chromosome Karyotype of Three Varieties of the Characteristic Tea plants

ZY, HYZ and CH-1 are three characteristic tea varieties with different shoot colors. However, there is a lack of karyotype analysis of these characteristic tea varieties. In this experiment, the differences of cytological parameters of three characteristic tea varieties, and the control variety FDDB, were analyzed. The results showed that significant differences of the average arm ratio, relative length, karyotype asymmetry index and the karyotype formula were found among the three characteristic tea varieties as well as the control variety FDDB. Moreover, only ZY’s karyotype type was 2B, while the three other varieties all were 2C. Among them, HYZ was the most evolved and closed to FDDB, while CH-1 and ZY were very different from FDDB and evolved more primitive than HYZ. HYZ was the most evolved, and ZY was relatively primitive. PCA analysis indicated that the notable differences were found between FDDB and two characteristic tea varieties (ZY and CH-1), while HYZ is relatively close to FDDB. This findings revealed the variation of chromosomes among different color varieties at the cytological level, and provided a certain theoretical basis for the study of genetic variation and diversity of tea resources.

Karyological and cytogenetic research of the conifers of boreal zone by classic and new methods

Aim. Establishing of karyological features and conducting of cytogenetic analysis on conifer plants for biological diversity studies, solving of problems of taxonomics, evolutionary and population genetics. Methods. Classic methods with acetohematoxylin staining of slides and fluorescent in situ hybridization (FISH). Results. More than 150 populations and provenances of representatives of different conifer genera from the Pinaceae and Cupressaceae families were studied. The studies were carried out in natural populations and during the introduction, in optimal and extreme conditions, in disturbed ecosystems, botanical gardens and parks; in addition, various intraspecific forms have been studied. The variability of chromosome numbers and a wide range of chromosomal mutations have been revealed. Fluorescence in situ hybridization (FISH) with the 45S and 5S ribosomal RNA gene probes and DAPI staining allows to identify of homologous chromosome pairs in the karyotypes of conifers and to facilitate the comparative karyotype analysis of these species. Conclusions. The studies of chromosomes in species of the Pinaceae and Cupressaceae families showed a karyotypic diversity and chromosomal anomalies in extreme conditions and under introduction. The use of molecular cytogenetic markers made it possible to obtain new information on the structure of conifer chromosomes.
 Keywords: chromosomes, nucleolar loci, chromosome mutations, Pinaceae, Cupressaceae.

Phenotypic, biochemical and genomic variability in generations of the rapeseed (Brassica napus L.) mutant lines obtained via chemical mutagenesis.

The phenotypic, biochemical and genetic variability was studied in M2-M5 generations of ethyl methansulfonat (EMS, 0.2%) mutagenized rapeseed lines generated from canola, ‘00’, B. napus cv. Vikros. EMS mutagenesis induced extensive diversity in morphological and agronomic traits among mutant progeny resulted in selection of EMS populations of B. napus- and B. rapa-morphotypes. The seeds of the obtained mutant lines were high-protein, low in oil and stabilized in contents of main fatty acids which make them useful for feed production. Despite the increased level of various meiotic abnormalities revealed in EMS populations, comparative karyotype analysis and FISH-based visualization of 45S and 5S rDNA indicated a high level of karyotypic stability in M2-M5 plants, and therefore, the obtained mutant lines could be useful in further rapeseed improvement. The revealed structural chromosomal reorganizations in karyotypes of several plants of B. rapa-type indicate that rapeseed breeding by chemical mutagenesis can result in cytogenetic instability in the mutant progeny, and therefore, it should include the karyotype examination. Our findings demonstrate that EMS at low concentrations has great potential in rapeseed improvement.

Phenotypic and molecular cytogenetic variability in calendula (Calendula officinalis L.) cultivars and mutant lines obtained via chemical mutagenesis

The morphological, meiotic and chromosomal variability were studied in two cultivars of Calendula officinalis L. and their mutant lines obtained though chemical mutagenesis using diethyl sulphate (DES) (0.04%, 0.08%) and dimethyl sulphate (DMS) (0.025%, 0.05%). The studied cultivars displayed different sensitivity to DMS and DES mutagens. More M1 plants with morphological changes were observed in C. officinalis cv. ‘Zolotoe more’ than in cv. ‘Rajskij sad’. DMS and DES at low concentrations had positive effects on main agro-metrical traits in both cultivars including plant height, inflorescence diameter and number of inflorescences per plant. Dose-dependent increase in number of various meiotic abnormalities was revealed in both mutant lines. Comparative karyotype analysis and FISH-based visualization of 45S and 5S rDNA indicated a high level of karyotype stability in M1 and M2 plants. Seed treatments with DMS and DES at certain concentrations resulted in higher yields of inflorescences in M1 plants compared to the control. In M2 generation, dose-dependent reduction in the yields of inflorescences was observed. Our findings demonstrate that DMS and DES at low concentrations have great potential in calendula mutation breeding.