Plant Chromosomes Research Articles

Plant Chromosome-Specific Probes by Microdissection of a Single Chromosome: Is That a Reality?

Painting plant chromosomes through chromosomal in situ suppression (CISS) hybridization has long been considered impracticable. Seeking to build specific and complex probes from a single microdissected chromosome, we employed human chromosomes as models to standardize all the necessary steps for application in plants. Human metaphases were used to define the adequate conditions for microdissection, chromosome DNA amplification and labeling through degenerate oligonucleotide-primed PCR, and in situ hybridization stringency. Subsequently, these methodologies were applied in the plant species Zea mays (chromosome 1) and Capsicum annuum (chromosome 7 or 8). The high quality of human and plant cytogenetic preparations and the meticulous standardization of each step, especially the most critical ones – microdissection and first round of DNA amplification – were crucial to eliminate the signs of non-specific hybridization and for direct application in plants. By overcoming these challenges, we obtained chromosome-specific probes, which allowed to achieve a clear and uniform painting of the entire target chromosomes with little or no background, evidencing their complexity and specificity. Despite the high amount of ubiquitous repetitive sequences in plant genomes, the main drawback for chromosome painting, we successfully employed our methodology on two plant species. Both have more than 80% repetitive sequences, which is compared to the human genome (66–69%). This is the first time that plant chromosome-specific probes were successfully obtained from a single A mitotic or meiotic microdissected chromosome. Thereby, we assume that chromosome painting through microdissection and CISS hybridization can now be considered a reality in the field of plant cytogenetics.

INDUCED IN VIVO MUTAGENESIS USING COLCHICINE IN HYDROGEN PEROXIDE ON M2 COWPEA, YARDLONG BEAN AND COMMON BEAN

Research on doubling plant chromosome using colchicine has been carried out for years with water as its solvent. In this study, an innovation was carried out by trying to do colchicine treatment in hydrogen peroxide. In this in vivo induced mutagenesis research, colchicine in hydrogen peroxide was administered as mutagen on seeds three species of crops i.e. mutan M2 cowpea, yardlong bean, and common bean by soaking method. The results of this research showed that three times multistep mutant (M3) of cowpea was having mutant characteristics that the shape of its leaflet changed from rhombus to ovate. Besides, both yardlong bean varieties used in this research (BLR and KTR) showed indication of a mutant crop that having compound leaves with more than three leaflets in a stalk. Common bean of PTW variety was not attacked by any aphid but it was attacked by leafminer. Finally, we found “rainbow common bean” from this research.

The Formation of Sex Chromosomes in Silene latifolia and S. dioica Was Accompanied by Multiple Chromosomal Rearrangements.

The genus Silene includes a plethora of dioecious and gynodioecious species. Two species, Silene latifolia (white campion) and Silene dioica (red campion), are dioecious plants, having heteromorphic sex chromosomes with an XX/XY sex determination system. The X and Y chromosomes differ mainly in size, DNA content and posttranslational histone modifications. Although it is generally assumed that the sex chromosomes evolved from a single pair of autosomes, it is difficult to distinguish the ancestral pair of chromosomes in related gynodioecious and hermaphroditic plants. We designed an oligo painting probe enriched for X-linked scaffolds from currently available genomic data and used this probe on metaphase chromosomes of S. latifolia (2n = 24, XY), S. dioica (2n = 24, XY), and two gynodioecious species, S. vulgaris (2n = 24) and S. maritima (2n = 24). The X chromosome-specific oligo probe produces a signal specifically on the X and Y chromosomes in S. latifolia and S. dioica, mainly in the subtelomeric regions. Surprisingly, in S. vulgaris and S. maritima, the probe hybridized to three pairs of autosomes labeling their p-arms. This distribution suggests that sex chromosome evolution was accompanied by extensive chromosomal rearrangements in studied dioecious plants.

A willow sex chromosome reveals convergent evolution of complex palindromic repeats

BackgroundSex chromosomes have arisen independently in a wide variety of species, yet they share common characteristics, including the presence of suppressed recombination surrounding sex determination loci. Mammalian sex chromosomes contain multiple palindromic repeats across the non-recombining region that show sequence conservation through gene conversion and contain genes that are crucial for sexual reproduction. In plants, it is not clear if palindromic repeats play a role in maintaining sequence conservation in the absence of homologous recombination.ResultsHere we present the first evidence of large palindromic structures in a plant sex chromosome, based on a highly contiguous assembly of the W chromosome of the dioecious shrub Salix purpurea. The W chromosome has an expanded number of genes due to transpositions from autosomes. It also contains two consecutive palindromes that span a region of 200 kb, with conspicuous 20-kb stretches of highly conserved sequences among the four arms that show evidence of gene conversion. Four genes in the palindrome are homologous to genes in the sex determination regions of the closely related genus Populus, which is located on a different chromosome. These genes show distinct, floral-biased expression patterns compared to paralogous copies on autosomes.ConclusionThe presence of palindromes in sex chromosomes of mammals and plants highlights the intrinsic importance of these features in adaptive evolution in the absence of recombination. Convergent evolution is driving both the independent establishment of sex chromosomes as well as their fine-scale sequence structure.

MicroRNA-mediated responses to colchicine treatment in barley.

In Hordeum vulgare, nine differentially expressed novel miRNAs were induced by colchicine. Five novel miRNA in colchicine solution showed the opposite expression patterns as those in water. Colchicine is a commonly used agent for plant chromosome set doubling. MicroRNA-mediated responses to colchicine treatment in plants have not been characterized. Here, we characterized new microRNAs induced by colchicine treatment in Hordeum vulgare using high-throughput sequencing. Our results showed that 39 differentially expressed miRNAs were affected by water treatment, including 34 novel miRNAs and 5 known miRNAs; 42 miRNAs, including 37 novel miRNAs and 5 known miRNAs, were synergistically affected by colchicine and water, and 9 differentially expressed novel miRNAs were induced by colchicine. The novel_mir69, novel_mir57, novel_mir75, novel_mir38, and novel_mir56 in colchicine treatment showed the opposite expression patterns as those in water. By analyzing these 9 differentially expressed novel miRNAs and their targets, we found that novel_mir69, novel_mir56 and novel_mir25 co-target the genes involving the DNA repair pathway. Based on our results, microRNA-target regulation network under colchicine treatment was proposed, which involves actin, cell cycle regulation, cell wall synthesis, and the regulation of oxidative stress. Overall, the results demonstrated the critical role of microRNAs mediated responses to colchicine treatment in plants.

Does the chromosomal position of 35S rDNA sites influence their transcription? A survey on Nothoscordum species (Amaryllidaceae).

35S ribosomal DNA (rDNA) sites are the regions where the ribosomal genes 18S, 5.8S and 25S, responsible for the formation of the nucleoli, are found. The fact that rDNA sites have non-random distribution on chromosomes suggests that their positions may influence their transcription. To identify if the preferentially transcribed rDNA sites occupy specific position, six species (nine cytotypes) of the genus Nothoscordum were analyzed using two different techniques to impregnate the nucleolar organizer regions (NORs) with silver nitrate. Both techniques strongly stained NORs, but one of them also stained the proximal region of all chromosomes, suggesting the existence of another group of argentophilic proteins in this region. In species with rDNA sites in acrocentric and metacentric chromosomes, sites located on the short arms of the acrocentric chromosomes were preferentially activated. On the other hand, in species with rDNA sites restricted to the short arms of the acrocentrics, all of them were activated, whereas in those species with sites restricted to the terminal region of metacentric chromosomes, the frequency of active sites was always lower than expected. This indicate that, at least in Nothoscordum, the transcription of an rDNA site is influenced by its chromosomal position, and may explain, at least partially, the strongly non-random distribution of these sites in plant and animal chromosomes.

Genomic Markers Associated with Cold-Hardiness in Brassica rapa L.

Brassica rapa L. is a valuable and widespread species, but its cultivation in risk farming areas requires high-quality cold-hardy varieties to be developed. Mechanisms of the cold stress response in plants involve expression of numerous genes, including ribosomal ones, and are related to plant chromosome variability. FISH- and PCR-based methods were used to study intraspecific chromosome variability in the number and localization of 45S and 5S rDNA clusters and also to examine a set of molecular markers associated with cold-hardiness in winter B. rapa cultivars from high-risk farming areas. Several SSR (Na10-CO3 and BrgMS5339-1) and SCAR (BoCCA1-F/BoCCA1-1R1 and BoCCA1-F/BoCCA1-2R1) markers were identified as suitable for diagnosing cold-resistant and cold-susceptible genotypes in B. rapa. Compared with fodder cultivars, oilseed and leaf cultivars were shown to have more molecular markers associated with cold-hardiness and a higher level of polymorphism for the chromosomal distribution of 45S and 5S rDNAs, including chromosome heteromorphism. Thus, the least cold-resistant genotypes were found to display the lowest level of chromosome variability in the distribution of the 45S and 5S rDNA clusters and vice versa. The findings could be useful for the development of new cold-tolerant B. rapa varieties.

Fern Genomics: Unfurling the Mystery of Plant Chromosome Numbers

Fern Genomics: Unfurling the Mystery of Plant Chromosome Numbers

Evolution of Structural Diversity of Triterpenoids.

Plants have evolved to produce a blend of specialized metabolites that serve functional roles in plant adaptation. Among them, triterpenoids are one of the largest subclasses of such specialized metabolites, with more than 14,000 known structures. They play a role in plant defense and development and have potential applications within food and pharma. Triterpenoids are cyclized from oxidized squalene precursors by oxidosqualene cyclases, creating more than 100 different cyclical triterpene scaffolds. This limited number of scaffolds is the first step towards creating the vast structural diversity of triterpenoids followed by extensive diversification, in particular, by oxygenation and glycosylation. Gene duplication, divergence, and selection are major forces that drive triterpenoid structural diversification. The triterpenoid biosynthetic genes can be organized in non-homologous gene clusters, such as in Avena spp., Cucurbitaceae and Solanum spp., or scattered along plant chromosomes as in Barbarea vulgaris. Paralogous genes organized as tandem repeats reflect the extended gene duplication activities in the evolutionary history of the triterpenoid saponin pathways, as seen in B. vulgaris. We review and discuss examples of convergent and divergent evolution in triterpenoid biosynthesis, and the apparent mechanisms occurring in plants that drive their increasing structural diversity within and across species. Using B. vulgaris’ saponins as examples, we discuss the impact a single structural modification can have on the structure of a triterpenoid and how this affect its biological properties. These examples provide insight into how plants continuously evolve their specialized metabolome, opening the way to study uncharacterized triterpenoid biosynthetic pathways.

Development and application of an optimized drop-slide technique for metaphase chromosome spreads in maize

ABSTRACTChromosome spreads are important for complex molecular cytogenetic studies. An adequate chromosome spreading method for identification and isolation of the maize B chromosome, however, has not been reported. We used the maize inbred lines, B73 and Mo17, the hybrid YD08 line and the landrace DP76 to develop an optimized chromosome spreading method. We investigated the effects of four treatment factors on the quality of metaphase chromosome spreads using a factorial analysis of variance. Optimal conditions for metaphase spreading were identified using regression analysis based on multifactor orthogonal design of four treatment factors with five levels for each factor. We developed optimal conditions for metaphase spreading as follows: nitrous oxide treatment for 2 h, glacial acetic acid fixation for 2 h, enzyme hydrolysis for 6.0 h, and a drop height of 35 cm for cell suspension. We obtained high quality metaphase chromosome spreads with large metaphase areas, large numbers of chromosomes, few chromosome overlaps and high frequency of intact metaphases. Our optimized drop-slide procedure was markedly better than the traditional flame smear technique. We identified 487 B chromosomes in three forms from maize landraces from Southwest China. We found no relation between the C-band number and B chromosome. Single B chromosomes also were isolated directly from a metaphase chromosome drop-slide using a micromanipulator. Our optimized method provides a simple, efficient and reproducible procedure for preparing high quality plant chromosome spreads.

Cytological methods of analysis of haploid plants-regenerants of cabbage (<i>Brassica oleracea</i> L.) obtained <i>in vitro</i>

Relevance Currently, in genetic studies and selection of cabbage cultures, biotechnological methods for creating clean lines — doubled haploids in the culture of anthers and in the culture of isolated microspores are widely used. A common feature of these technologies is that the plants obtained in vitro have different levels of ploidy and along with doubled haploids there are haploid, tetraploid and mixoploid forms. Therefore, the use of new cytological methods of analysis of haploid plants remains an urgent problem. Material and method The aim of this work is to establish the genetic nature of regenerated plants of Brassica oleracea L., obtained from reproductive organs in vitro. Isolated anthers and ovaries of white cabbage were cultivated on solid nutrient media containing mineral salts according to the recipe Murashige and Skoog (MS). The obtained regenerated plants were used to calculate the number of chromosomes in the root meristem, as well as the number of chloroplasts in the cells of the closing stomata of leaves using the new universal method of preparing preparations of plant chromosomes – “SteamDrop”. Results As a result of the research, the dependence of the level of ploidy on the cultivation conditions was studied. It has been shown that plants-regenerants of white cabbage, obtained in vitro from reproductive organs, had a different set of chromosomes (n, 3n, 5n). It was established that the number of chloroplasts in the stomatal cells of regenerated plants was from 9 to 45, while the original donor plants had 18–20.

Nuclear Disposition of Alien Chromosome Introgressions into Wheat and Rye Using 3D-FISH.

During interphase, the chromosomes of eukaryotes decondense and they occupy distinct regions of the nucleus, called chromosome domains or chromosome territories (CTs). In plants, the Rabl’s configuration, with telomeres at one pole of nucleus and centromeres at the other, appears to be common, at least in plants with large genomes. It is unclear whether individual chromosomes of plants adopt defined, genetically determined addresses within the nucleus, as is the case in mammals. In this study, the nuclear disposition of alien rye and barley chromosomes and chromosome arm introgressions into wheat while using 3D-FISH in various somatic tissues was analyzed. All of the introgressed chromosomes showed Rabl’s orientation, but their relative positions in the nuclei were less clear. While in most cases pairs of introgressed chromosomes occupied discrete positions, their association (proximity) along their entire lengths was rare, and partial association only marginally more frequent. This arrangement is relatively stable in various tissues and during various stages of the cell cycle. On the other hand, the length of a chromosome arm appears to play a role in its positioning in a nucleus: shorter chromosomes or chromosome arms tend to be located closer to the centre of the nucleus, while longer arms are more often positioned at the nuclear periphery.

From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas.

In the last years, tremendous progress has been achieved in the field of gene editing in plants. By the induction of single site-specific double-strand breaks (DSBs), the knockout of genes by non-homologous end joining has become routine in many plant species. Recently, the efficiency of inducing pre-planned mutations by homologous recombination has also been improved considerably. However, very little effort has been undertaken until now to achieve more complex changes in plant genomes by the simultaneous induction of several DSBs. Several reports have been published on the efficient induction of deletions. However, the induction of intrachromosomal inversions and interchromosomal recombination by the use of CRISPR/Cas has only recently been reported. In this review, we want to sum up these results and put them into context with regards to what is known about natural chromosome rearrangements in plants. Moreover, we review the recent progress in CRISPR/Cas-based mammalian chromosomal rearrangements, which might be inspiring for plant biologists. In the long run, the controlled restructuring of plant genomes should enable us to link or break linkage of traits at will, thus defining a new area of plant breeding.

The metribuzin herbicide in polycaprolactone nanocapsules shows less plant chromosome aberration than non-encapsulated metribuzin

The excessive use of the metribuzin herbicide is contaminating groundwater and damaging non-target plants and organisms. There is therefore a need for safer formulations of the herbicide. Here we encapsulated metribuzin by interfacial deposition of the pre-formed polymer. Results show that the encapsulation efficiency of metribuzin reached 83.2%. Nanocapsules remained stable in the suspension without any aggregation or decomposition. Nanocapsules containing metribuzin induced less chromosome aberration and higher mitotic index values than the pure metribuzin. We conclude that herbicide encapsulation in polycaprolactone nanocapsules is safer for food and the environment.

Young sex chromosomes in plants and animals

A major reason for studying plant sex chromosomes is that they may often be 'young' systems. There is considerable evidence for the independent evolution of separate sexes within plant families or genera, in some cases showing that the maximum possible time during which their sex-determining genes have existed must be much shorter than those of several animal taxa. Consequently, their sex-linked regions could either have evolved soon after genetic sex determination arose or considerably later. Plants, therefore, include species with both young and old systems. I review several questions about the evolution of sex-determining systems and sex chromosomes that require studies of young systems, including: the kinds of mutations involved in the transition to unisexual reproduction from hermaphroditism or monoecy (a form of functional hermaphroditism); the times when they arose; and the extent to which the properties of sex-linked regions of genomes reflect responses to new selective situations created by the presence of a sex-determining locus. I also evaluate which questions are best studied in plants, vs other suitable candidate organisms. Studies of young plant systems can help understand general evolutionary processes that are shared with the sex chromosomes of other organisms.

Identification of P genome chromosomes in Agropyron cristatum and wheat-A. cristatum derivative lines by FISH

Agropyron cristatum (L.) Gaertn. (P genome) is cultivated as pasture fodder and can provide many desirable genes for wheat improvement. With the development of genomics and fluorescence in situ hybridization (FISH) technology, probes for identifying plant chromosomes were also developed. However, there are few reports on A. cristatum chromosomes. Here, FISH with the repeated sequences pAcTRT1 and pAcpCR2 enabled the identification of all diploid A. cristatum chromosomes. An integrated idiogram of A. cristatum chromosomes was constructed based on the FISH patterns of five diploid A. cristatum individuals. Structural polymorphisms of homologous chromosomes were observed not only among different individuals but also within individuals. Moreover, seventeen wheat-A. cristatum introgression lines containing different P genome chromosomes were identified with pAcTRT1 and pAcpCR2 probes. The arrangement of chromosomes in diploid A. cristatum was determined by identifying correspondence between the P chromosomes in these genetically identified introgression lines and diploid A. cristatum chromosomes. The two probes were also effective for discriminating all chromosomes of tetraploid A. cristatum, and the differences between two tetraploid A. cristatum accessions were similar to the polymorphisms among individuals of diploid A. cristatum. Collectively, the results provide an effective means for chromosome identification and phylogenetic studies of P genome chromosomes.

Cytogenetic and molecular genotyping in the allotetraploid Festuca pratensis \xd7 Lolium perenne hybrids

BackgroundSpecies of the Festuca and Lolium genera, as well as intergeneric Festuca × Lolium (Festulolium) hybrids, are valuable fodder and turf grasses for agricultural and amenity purposes worldwide. Festulolium hybrids can merge in their genomes agronomically important characteristics. However, in polyploid plants, especially in allopolyploids, the hybridization of divergent genomes could contribute to various abnormalities, such as variability in chromosome number, structural rearrangements, and/or disorders in inheritance patterns. Here we studied these issues in allotetraploid Festuca pratensis × Lolium perenne hybrids.ResultsCytogenetic procedures, including fluorescent in situ hybridization, genomic in situ hybridization, and molecular markers – inter-simple sequence repeats (ISSR) were exploited. This cytogenetic approach indicated the dynamics in the number and distribution of ribosomal RNA genes and structural rearrangements for both parental genomes (Festuca and Lolium) in hybrid karyotypes. The separate analysis of F. pratensis and L. perenne chromosomes in hybrid plants (F2-F3 generations of F. pratensis × L. perenne) revealed the asymmetrical level of rearrangements. Recognized structural changes were mainly located in the distal part of chromosome arms, and in chromosomes bearing ribosomal DNA, they were more frequently mapped in arms without this sequence. Based on the ISSR markers distribution, we found that the tetrasomic type of inheritance was characteristic for the majority of ISSR loci, but the disomic type was also observed. Nonetheless, no preference in the transmission of either Festuca or Lolium alleles to the following generations of allotetraploid F. pratensis × L. perenne hybrid was observed.ConclusionOur study reports cytogenetic and molecular genotyping of the F. pratensis × L. perenne hybrid and its following F2-F3 progenies. The analysis of 137 allotetraploid F. pratensis × L. perenne hybrids revealed the higher level of recombination in chromosomes derived from F. pratensis genome. The results of ISSR markers indicated a mixed model of inheritance, which may be characteristic for these hybrids.

Quilting plant chromosomes with CRISPR/Cas9.

Quilting plant chromosomes with CRISPR/Cas9.

Epigenetic consequences of interploidal hybridisation in synthetic and natural interspecific potato hybrids.

Interploidal hybridisation can generate changes in plant chromosome numbers, which might exert effects additional to the expected due to genome merger per se (that is genetic, epigenetic and phenotypic novelties). Wild potatoes are suitable to address this question in an evolutionary context. To this end, we performed genetic (AFLP and single sequence repeart (SSR)), epigenetic (MSAP), and cytological comparisons in: (1) natural populations of the diploid cytotype of the hybrid taxonomic species Solanum×rechei (2n=2×, 3×) and its parental species, the triploid cytotype of Solanum microdontum (2n=2×, 3×) and Solanum kurtzianum (2n=2×); and (2) newly synthesised intraploidal (2××2×) and interploidal (3××2×) S.microdontum×S.kurtzianum hybrids. Aneuploidy was detected in S.×rechei and the synthetic interploidal progeny; this phenomenon might have originated the significantly higher number of methylation changes observed in the interploidal vs the intraploidal hybrids. The wide epigenetic variability induced by interploidal hybridisation is consistent with the novel epigenetic pattern established in S.×rechei compared to its parental species in nature. These results suggest that aneuploid potato lineages can persist throughout the short term, and possibly medium term, and that differences in parental ploidy resulting in aneuploidy are an additional source of epigenetic variation.

Chromosome number and karyomorphologic studies of endemic Astragalus victoriae Podlech - Kirchhoff

This paper is the first report known concerning with chromosome number and morphology of Astragalus victoriae Podlech & Kirchhoff in Turkey. Karyomorphological analyses includes the chromosome length, centromer position, the arms rates (short/long) and asymmetry indices. In the analyses, the chromosome number of A. victoriae was determined as 2n = 2x = 16 and this data was consistent with the suggested chromosomal counts in Index to Plant Chromosome Numbers (IPCN) for the genus. The karyotype of A. victoriae consists of metacentric and submetacentric chromosomes and the species is seen fairly different from in points of chromosome lengths (CVCL: 30.774). The chromosome formula has been determined as 2sm+14m and the species consists of symmetric chromosomes (AI: 2.958, CI: 46).