Altered chromatin conformation and transcriptional regulation in watermelon following genome doubling.

Polyploidy has played a crucial role in plant evolution, development and function. Synthetic autopolyploid represents an ideal system to investigate the effects of polyploidization on transcriptional regulation. In this study, we deciphered the impact of genome duplication at phenotypic and molecular levels in watermelon. Overall, 88% of the genes in tetraploid watermelon followed a >1:1 dosage effect, and accordingly, differentially expressed genes were largely upregulated. In addition, a great number of hypomethylated regions (1688) were identified in an isogenic tetraploid watermelon. These differentially methylated regions were localized in promoters and intergenic regions and near transcriptional start sites of the identified upregulated genes, which enhances the importance of methylation in gene regulation. These changes were reflected in sophisticated higher-order chromatin structures. The genome doubling caused switching of 108 A and 626 B compartments that harbored genes associated with growth, development and stress responses.

In silico genome-wide identification and characterization of the glutathione S-transferase gene family in Vigna radiata.

Plant glutathione S-transferases (GSTs) are integral to normal plant metabolism and biotic and abiotic stress tolerance. The GST gene family has been characterized in diverse plant species using molecular biology and bioinformatics approaches. In the current study, in silico analysis identified 44 GSTs in Vigna radiata. Of the total 44 GSTs identified, chromosomal locations of 31 GSTs were confirmed. The pI value of GST proteins ranged from 5.10 to 9.40. The predicted molecular weights ranged from 13.12 to 50 kDa. Subcellular localization analysis revealed that all GSTs were predominantly localized in the cytoplasm. The active site amino acids were confirmed to be serine in tau, phi, theta, zeta, and TCHQD; cysteine in lambda, DHAR, and omega; and tyrosine in EF1G. The gene architecture conformed to the two-exon/one-intron and three-exon/two-intron organization in the case of tau and phi classes, respectively. MEME analysis identified 10 significantly conserved motifs with the width of 8-50 amino acids. The motifs identified were either specific to a specific GST class or were shared by multiple GST classes. The results of the current study will be of potential importance in the characterization of the GST gene family in V. radiata, an economically important leguminous crop.

Chromosomal distribution patterns of the (AC)10 microsatellite and other repetitive sequences, and their use in chromosome rearrangement analysis of species of the genus Avena.

Fluorescence in situ hybridization (FISH) was used to determine the physical location of the (AC)10 microsatellite in metaphase chromosomes of six diploid species (AA or CC genomes), two tetraploid species (AACC genome), and five cultivars of two hexaploid species (AACCDD genome) of the genus Avena, a genus in which genomic relationships remain obscure. A preferential distribution of the (AC)10 microsatellite in the pericentromeric and interstitial regions was seen in both the A- and D-genome chromosomes, while in C-genome chromosomes the majority of signals were located in the pericentromeric heterochromatic regions. New large chromosome rearrangements were detected in two polyploid species: an intergenomic translocation involving chromosomes 17AL and 21DS in Avena sativa 'Araceli' and another involving chromosomes 4CL and 21DS in the analyzed cultivars of Avena byzantina. The latter 4CL-21DS intergenomic translocation differentiates clearly between A. sativa and A. byzantina. Searches for common hybridization patterns on the chromosomes of different species revealed chromosome 10A of Avena magna and 21D of hexaploid oats to be very similar in terms of the distribution of 45S and Am1 sequences. This suggests a common origin for these chromosomes and supports a CCDD rather than an AACC genomic designation for this species.

Genome-wide identification and expression analysis of the CPP-like gene family in soybean.

Cysteine-rich polycomb-like protein (CPP-like) genes are a group of transcription factors with highly conserved cysteine-rich domains and are widely distributed in animals and plants, but do not present in yeast. Previous studies have shown that members of this family play important roles in the development of reproductive tissue and in the control of cell division in plants. In this study, whole genome identification of soybean CPP transcription factors was performed using bioinformatic methods. The results showed that there were 20 CPP transcription factors in the soybean genome, which encoded for 28 distinct CPP proteins. These transcription factors were distributed on 16 of 20 chromosomes. Phylogenetic relationship analysis showed that expression of CPP gene family members occurred before the differentiation of monocotyledons and dicotyledons. RNA-Seq analysis showed that 5 genes were highly expressed in all tissues, including Glyma10g39080, Glyma01g44670, Glyma101g66920, Glyma02g01540, and Glyma20g28740. One gene (Glyma14g14750) was specifically expressed in young leaves, while 2 genes (Glyma02g01540 and Glyma10g01580) were highly expressed in root nodules. Quantitative reverse transcriptase-polymerase chain reaction analysis revealed that the expression levels of most genes increased in the roots under high temperature stress. Our findings indicate that these genes are not only involved in growth and development, but also in the responses to high temperature stress in soybean roots.

Chromosome number and secondary chromosomal associations in wild populations of Geranium pratense L. from the cold deserts of Lahaul-Spiti (India).

In this work we studied the meiotic chromosome number and details of secondary chromosomal associations recorded for the first time in Geranium pratense L. from the alpine environments in the cold deserts of Lahaul-Spiti (India). All the presently studied individuals of the species existed at 4x level (x = 14). The present chromosome count of n = 28 in the species adds a new cytotype to the already existing diploid chromosome count of 2n = 28 from the Eastern Himalayas and outside of India. Out of the six accessions scored presently four showed normal meiotic course. However, two accessions investigated from Mud, 3800 m and Koksar, 3140 m depicted abnormal meiotic course due to the presence of multivalents and univalents, and secondary associations of bivalents/chromosomes. The secondary chromosomal associations in the species existed among bivalents/chromosomes were noticed in the PMCs at prophase-1 (diakinesis) and persisted till the separation of sister chromatids at M-II. The variation in the number of bivalents/chromosomes involved in the secondary associations at M-I (2-8) and A-I/M-II (2-12) has also been recorded. The occurrence of such secondary associations of bivalents/chromosomes in G. pratense which existed at 4x level indicated the secondary polyploid nature of the species.

Flow cytometric chromosome sorting in plants: the next generation.

Genome analysis in many plant species is hampered by large genome size and by sequence redundancy due to the presence of repetitive DNA and polyploidy. One solution is to reduce the sample complexity by dissecting the genomes to single chromosomes. This can be realized by flow cytometric sorting, which enables purification of chromosomes in large numbers. Coupling the chromosome sorting technology with next generation sequencing provides a targeted and cost effective way to tackle complex genomes. The methods outlined in this article describe a procedure for preparation of chromosomal DNA suitable for next-generation sequencing.

[Critical level of radiation damage of root apical meristem and mechanisms for its recovery in Pisum sativum L].

The dose dependencies of growth and cytogenetical values have been built to determine the critical level of root apical meristem damage induced by cute irradiation in the range from 2 to 20 Gr. We have analyzed the frequencies of aberrant anaphases and the aberration distribution per cell, on the one hand, and the growth of biomass, the survival and regeneration of the root meristem, on the other hand. The critical level of damage to the stem apical meristem and root of seedlings was defined as 44-48% of aberrant anaphase. Exceeding of this level leads to the launch of suicidal program through induction of multiaberrant damages and interphase cell death. It appears that competition of clones of non-aberrant cells, the cells bearing 1 and 2 damages and multiaberrant cells plays the primary role in the mechanisms of recovery. The regeneration provides full or partial restoration of the main root apical meristem. However these local processes are insufficient to restore morphogenesis and survival of seedlings in excess of the critical level damage.

[Chromosomal mutations in Scotch pine (Pinus sylvestris L.) growing in a hydromorphic environment: a first case of a giant chromosome detection].

Chromosomal mutations and mixoploidy have been revealed in the seedlings of Scotch pine (Pinus sylvestris) growing in a hydromorphic environment. A giant chromosome was detected for the first time in gymnosperms.

Reticulate evolution of the hybrid produced artificially by crosses between Osmunda banksiifolia and Osmunda lancea.

Although ferns have been developed by hybridization and chromosome doubling, no natural polyploidy has yet been recorded in Osmundaceae. So, we produced hybrids artificially by crosses between Osmunda banksiifolia (2n = 2x = 44) and Osmunda lancea (2n = 2x = 44), and investigated their sporogenesis. From the O. banksiifolia x O. lancea hybrid with 44 univalent chromosomes, allotetraploids with 44 bivalent chromosomes were produced by chromosome doubling, and allotriploids with 22 univalent chromosomes and 22 bivalent chromosomes were then produced by back crosses. The results show when and how chromosome doubling occurs in hybrids. The success of artificial hybridization between O. banksiifolia and O. lancea, did not, however, reflect any product of natural hybridization between the two species.

Extraordinary tertiary constrictions of Tripsacum dactyloides chromosomes: implications for karyotype evolution of polyploids driven by segmental chromosome losses.

Tripsacum dactyloides (2n = 2x = 36) is an ancient tetraploid species. Here we report that T. dactyloides chromosomes contain an extraordinary tertiary constriction, which causes a radical and distant separation of a terminal segment from the chromosome. The relationships between extraordinary tertiary constriction and segmental chromosome loss as well as karyotype evolution of polyploid species are discussed.

Cytotoxic effects of pesticides in somatic cells of Vicia faba L.

The effects of pesticides (Endosulfan, Dieldrin, Aldrin) on cell division and chromosomal morphology of Vicia faba L. were studied. The results showed that the pesticides are mitode-pressive in higher concentrations and mitopromotor in lower concentrations and induced a variety of chromosomal abnormalities such as stickiness, fragments, chromatid separation, disturbed metaphase, C-mitosis, laggards, precocious movement and late separation where lagging chromosomes were predominant. The concentration of 500 ppm or above, for all the pesticides used in the present study showed pronounced toxic effect. In remaining treatments, although the milotic index was improved but less than that of absolute controls.

[Characteristics of the cytomixis in the pollen mother cells of transgenic tobacco plants (Nicotiana tabacum L.) with mutant phenotype].

This paper represents the frequency characteristics and cytological analysis of cytomixis in the course of male meiosis in transgenic tobacco plants (Nicotiana tabacum L.) with altered flower morphology and male sterility. The influence of cytomixis on qualitative composition of the meiotic products is shoron (cytoplasts and polyads formation). It was established that doubling of chromosomes number increased cytomixis frequency in the investigated plants.

The mismatch repair protein MLH1 marks a subset of strongly interfering crossovers in tomato.

In most eukaryotes, the prospective chromosomal positions of meiotic crossovers are marked during meiotic prophase by protein complexes called late recombination nodules (LNs). In tomato (Solanum lycopersicum), a cytological recombination map has been constructed based on LN positions. We demonstrate that the mismatch repair protein MLH1 occurs in LNs. We determined the positions of MLH1 foci along the 12 tomato chromosome pairs (bivalents) during meiotic prophase and compared the map of MLH1 focus positions with that of LN positions. On all 12 bivalents, the number of MLH1 foci was approximately 70% of the number of LNs. Bivalents with zero MLH1 foci were rare, which argues against random failure of detecting MLH1 in the LNs. We inferred that there are two types of LNs, MLH1-positive and MLH1-negative LNs, and that each bivalent gets an obligate MLH1-positive LN. The two LN types are differently distributed along the bivalents. Furthermore, cytological interference among MLH1 foci was much stronger than interference among LNs, implying that MLH1 marks the positions of a subset of strongly interfering crossovers. Based on the distances between MLH1 foci or LNs, we propose that MLH1-positive and MLH1-negative LNs stem from the same population of weakly interfering precursors.

Estudio de la diversidad citogenetica de Physalis peruviana L. (Solanaceae) / Study of the cytogenetic diversity of Physalis peruviana L. (Solanaceae)

Se caracterizó el cariotipo de cinco ecotipos de Physalis peruviana L., tres silvestres distribuidas en los municipios de Villa de Leyva (Boyacá, Colombia), La Calera y Choachí (Cundinamarca, Colombia) y dos cultivados, uno de ellos nativo, el ecotipo Colombia distribuido en el municipio de Subachoque (Cundinamarca) y uno foráneo, procedente de Kenia, cultivado en el municipio de Paipa (Cundinamarca). Ápices radicales obtenidos a partir de semillas y de hojas en medios de cultivo in vitro enriquecidos con auxinas se emplearon para estandarizar el protocolo de obtención de cromosomas, con las diferentes técnicas de pretratamiento, fijación, hidrólisis y montaje de las muestras. Adicionalmente se evaluó la duración del ciclo celular para establecer la hora mitótica. Se encontró variabilidad genética entre los ecotipos evaluados. Los ecotipos silvestres presentaron una dotación cromosómica 2n=24, el ecotipo Colombia 2n=32 y el ecotipo Kenia 2n=48. Los ecotipos exhibieron también variación en las características morfológicas y anatómicas, que de acuerdo a la literatura, son un reflejo del nivel de ploidía, como altura, área foliar, número de estomas/mm2, número de cloroplastos en las células guarda de los estomas, diámetro de frutos, semillas y contenido de masa seca.

Genome balance in six successive generations of the allotetraploid Festuca pratensis x Lolium perenne.

In the allotetraploid, Festuca pratensis Huds. (2n = 4x = 28) x Lolium perenne L. (2n = 4x = 28) the balance of chromatin, as determined by GISH, changes over successive generations of open pollination in favour of L. perenne. There is extensive recombination between chromosomes of the two parental genomes, as well as substitution of whole Festuca chromosomes by whole Lolium chromosomes. The total number of Lolium chromosomes increased from a mean 14.36 in the F(2) to 16.26 in the F(6), and the total number of Festuca chromosomes decreased correspondingly from a mean of 13.57 to a value of 11.56. The number of recombinant chromosomes and recombination breakpoints per genotype also increased from generation to generation, although the respective values of both characters were higher for Festuca (0.86-8.41 and 1.14-15.22) than for Lolium (0.68-4.59 and 0.68-6.0). The proportion of total genome length contributed by the L. perenne chromatin increased from about 50% in F(2) to 59.5% in F(6). The results are based on the sample of 134 plants studied (26-28 plants per generation), and are discussed in terms of the dominance of Lolium chromosomes over those of Festuca, and possible mechanisms underlying this phenomenon of chromatin substitution.

Complex genome rearrangements reveal evolutionary dynamics of pericentromeric regions in the Triticeae.

Most pericentromeric regions of eukaryotic chromosomes are heterochromatic and are the most rapidly evolving regions of complex genomes. The closely related genomes within hexaploid wheat (Triticum aestivum L., 2n=6x=42, AABBDD), as well as in the related Triticeae taxa, share large conserved chromosome segments and provide a good model for the study of the evolution of pericentromeric regions. Here we report on the comparative analysis of pericentric inversions in the Triticeae, including Triticum aestivum, Aegilops speltoides, Ae. longissima, Ae. searsii, Hordeum vulgare, Secale cereale, and Agropyron elongatum. Previously, 4 pericentric inversions were identified in the hexaploid wheat cultivar 'Chinese Spring' ('CS') involving chromosomes 2B, 4A, 4B, and 5A. In the present study, 2 additional pericentric inversions were detected in chromosomes 3B and 6B of 'CS' wheat. Only the 3B inversion pre-existed in chromosome 3S, 3Sl, and 3Ss of Aegilops species of the Sitopsis section, the remaining inversions occurring after wheat polyploidization. The translocation T2BS/6BS previously reported in 'CS' was detected in the hexaploid variety 'Wichita' but not in other species of the Triticeae. It appears that the B genome is more prone to genome rearrangements than are the A and D genomes. Five different pericentric inversions were detected in rye chromosomes 3R and 4R, 4Sl of Ae. longissima, 4H of barley, and 6E of Ag. elongatum. This indicates that pericentric regions in the Triticeae, especially those of group 4 chromosomes, are undergoing rapid and recurrent rearrangements.

Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes.

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute "molecular drive".

Chromosome organization in wheat endosperm and embryo.

We have analysed the chromosome organization in endosperm and embryo of bread wheat (Triticum aestivum L.), in order to compare these tissues with developing anthers, in which the centromeres associate, and the developing root xylem vessel cells, in which the chromosomes endoreduplicate to become polytene and associate via their centromeres. Both endosperm and embryo showed a typical Rabl configuration and a degree of non-homologous centromere association and the endosperm also showed extensive telomere association. Wheat endosperm is initially triploid and during its development a percentage of the nuclei increase their DNA content to 6C and 12C. 6C nuclei showed twice as many centromeres as 3C nuclei and the centromere number increased further in 12C nuclei. The higher the C-content of a nucleus the more the telomeres associated in endosperm. The vast majority of 12C nuclei showed six rye chromosome arms, although a few showed three associated groups of rye chromosome arms. This means that during endosperm development wheat nuclei show both polyploidization and polytenization.

Identification of individual chromosomes and parental genomes in Brassica juncea using GISH and FISH.

The three diploid (B. nigra, B. oleracea, B. campestris) and three allotetraploid (B. carinata, B. juncea, B. napus) species of Brassica, known as the "U-triangle" are one of the best model systems for the study of polyploidy. Numerous molecular investigations have provided a wealth of new insights into the polyploid origin and changes during the evolution of Brassica, but there are still many controversial aspects of their relationship and evolution. Interpretation of genome changes during evolution requires individual chromosome identification within the genome and clear distinction of genomes within the allotetraploid. The aim of this study was to identify individual chromosomes of B. juncea (genome AABB; 2n = 4x = 36) and to determine their genomic origin. Fluorescence in situ hybridization with 5S and 45S rDNA probes enabled discrimination of a substantial number of chromosomes, providing chromosomal landmarks for 20 out of 36 chromosomes of B. juncea. Additionally, along with double target genomic in situ hybridization, it allowed assignment of all chromosomes to either the A or B genomes.