Preparation of Mitotic Chromosomes with the Squash Technique.

Plant chromosomes are usually obtained from meristematic tissue of active root tips through the conventional squash method. Nevertheless, cytogenetic work usually implies a great effort and some modifications of standard procedures need to be evaluated. In this chapter, we describe our outline for handling chromosomes using the squash method.

The Nuclear 35S rDNA World in Plant Systematics and Evolution: A Primer of Cautions and Common Misconceptions in Cytogenetic Studies.

The ubiquitous presence of rRNA genes in nuclear, plastid, and mitochondrial genomes has provided an opportunity to use genomic markers to infer patterns of molecular and organismic evolution as well as to assess systematic issues throughout the tree of life. The number, size, location, and activity of the 35S rDNA cistrons in plant karyotypes have been used as conventional cytogenetic landmarks. Their scrutiny has been useful to infer patterns of chromosomal evolution and the data have been used as a proxy for assessing species discrimination, population differentiation and evolutionary relationships.

Interstitial -Type Telomeric Repeats in Asteraceae.

Tandem repeats of telomeric-like motifs at intra-chromosomal regions, known as interstitial telomeric repeats (ITR), have drawn attention as potential markers of structural changes, which might convey information about evolutionary relationships if preserved through time. Building on our previous work that reported outstanding ITR polymorphisms in the genus , we undertook a survey across 132 Asteraceae species, focusing on the six most speciose subfamilies and considering all the ITR data published to date. The goal was to assess whether the presence, site number, and chromosomal location of ITRs convey any phylogenetic signal.

Interstitial Telomeric-like Repeats (ITR) in Seed Plants as Assessed by Molecular Cytogenetic Techniques: A Review.

The discovery of telomeric repeats in interstitial regions of plant chromosomes (ITRs) through molecular cytogenetic techniques was achieved several decades ago. However, the information is scattered and has not been critically evaluated from an evolutionary perspective. Based on the analysis of currently available data, it is shown that ITRs are widespread in major evolutionary lineages sampled.

Molecular Evolution of rbcL in Orthotrichales (Bryophyta): Site Variation, Adaptive Evolution, and Coevolutionary Patterns of Amino Acid Replacements.

Molecular evolution of the large subunit of the RuBisCO enzyme is understudied in early diverging land plants. These groups show morphological and eco-physiological adaptations to the uneven and intermittent distribution of water in the terrestrial environment. This might have prompted a continuous fine-tuning of RuBisCO under a selective pressure modifying the species-specific optima for photosynthesis in contrasting microdistributions and environmental niches.

Unique Epigenetic Features of Ribosomal RNA Genes (rDNA) in Early Diverging Plants (Bryophytes).

In plants, the multicopy genes encoding ribosomal RNA (rDNA) typically exhibit heterochromatic features and high level of DNA methylation. Here, we explored rDNA methylation in early diverging land plants from Bryophyta (15 species, 14 families) and Marchantiophyta (4 species, 4 families). DNA methylation was investigated by methylation-sensitive Southern blot hybridization in all species.

Inter- and intraspecific hypervariability in interstitial telomeric-like repeats (TTTAGGG)n in Anacyclus (Asteraceae).

Background And Aims: Interstitial telomeric repeat (ITR) sites, consisting of tandem repeats of telomeric motifs localized at intrachromosomal sites, have been reported in a few unrelated organisms including plants. However, the causes for the occurrence of ITRs outside of the chromosomal termini are not fully understood. One possible explanation are the chromosomal rearrangements involving telomeric sites, which could also affect the location of other structural genome elements, such as the 45S rDNA.

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae).

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species.

Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants.

Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.

Non-functional plastid ndh gene fragments are present in the nuclear genome of Norway spruce (Picea abies L. Karsch): insights from in silico analysis of nuclear and organellar genomes.

Many genes have been lost from the prokaryote plastidial genome during the early events of endosymbiosis in eukaryotes. Some of them were definitively lost, but others were relocated and functionally integrated to the host nuclear genomes through serial events of gene transfer during plant evolution. In gymnosperms, plastid genome sequencing has revealed the loss of ndh genes from several species of Gnetales and Pinaceae, including Norway spruce (Picea abies).

Evolutionary site-number changes of ribosomal DNA loci during speciation: complex scenarios of ancestral and more recent polyploid events.

Several genome duplications have been identified in the evolution of seed plants, providing unique systems for studying karyological processes promoting diversification and speciation. Knowledge about the number of ribosomal DNA (rDNA) loci, together with their chromosomal distribution and structure, provides clues about organismal and molecular evolution at various phylogenetic levels. In this work, we aim to elucidate the evolutionary dynamics of karyological and rDNA site-number variation in all known taxa of subtribe Vellinae, showing a complex scenario of ancestral and more recent polyploid events.

Impact of polyploidy on fertility variation of Mediterranean Arundo L. (Poaceae).

Failure of seed production in the genus Arundo L. (Poaceae) is often attributed to polyploidy. This study tested the impact of two ploidy levels (2n=12 and 18x) on the fertility of four Mediterranean Arundo.

Incomplete sequence homogenization in 45S rDNA multigene families: intermixed IGS heterogeneity within the single NOR locus of the polyploid species Medicago arborea (Fabaceae).

Background And Aims: Ribosomal sequences have become the classical example of the genomic homogenization of nuclear multigene families. Despite theoretical advantages and modelling predictions that support concerted evolution of the 45S rDNA, several reports have found intragenomic polymorphisms. However, the origins and causes of these rDNA polymorphisms are difficult to assess because seed plants show a wide range of 45S rDNA loci number variation, especially in polyploids.

Partial sequence homogenization in the 5S multigene families may generate sequence chimeras and spurious results in phylogenetic reconstructions.

Multigene families have provided opportunities for evolutionary biologists to assess molecular evolution processes and phylogenetic reconstructions at deep and shallow systematic levels. However, the use of these markers is not free of technical and analytical challenges. Many evolutionary studies that used the nuclear 5S rDNA gene family rarely used contiguous 5S coding sequences due to the routine use of head-to-tail polymerase chain reaction primers that are anchored to the coding region.

Salt drying: a low-cost, simple and efficient method for storing plants in the field and preserving biological repositories for DNA diversity research.

Although a variety of methods have been optimized for the collection and storage of plant specimens, most of these are not suited for field expeditions for a variety of logistic reasons. Drying specimens with silica gel in polyethylene bags is currently the standard for field-sampling methods that are suitable for subsequent DNA extraction. However, silica-gel repositories are not readily available in remote areas, and its use is not very cost-effective for the long-term storage of collections or in developing countries with limited research budgets.

Amplification, contraction and genomic spread of a satellite DNA family (E180) in Medicago (Fabaceae) and allied genera.

Background And Aims: Satellite DNA is a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNA is an important element in genome organization and evolution in plants. Here we assess the presence and physical distribution of the repetitive DNA E180 family in Medicago and allied genera.

Patterns of genetic variability and habitat occupancy in Crepis triasii (Asteraceae) at different spatial scales: insights on evolutionary processes leading to diversification in continental islands.

Background And Aims: Archipelagos are unique systems for studying evolutionary processes promoting diversification and speciation. The islands of the Mediterranean basin are major areas of plant richness, including a high proportion of narrow endemics. Many endemic plants are currently found in rocky habitats, showing varying patterns of habitat occupancy at different spatial scales throughout their range.

Phylogenetic analysis informed by geological history supports multiple, sequential invasions of the Mediterranean Basin by the angiosperm family Araceae.

Despite the remarkable species richness of the Mediterranean flora and its well-known geological history, few studies have investigated its temporal and spatial origins. Most importantly, the relative contribution of geological processes and long-distance dispersal to the composition of contemporary Mediterranean biotas remains largely unknown. We used phylogenetic analyses of sequences from six chloroplast DNA markers, Bayesian dating methods, and ancestral area reconstructions, in combination with paleogeographic, paleoclimatic, and ecological evidence, to elucidate the time frame and biogeographic events associated with the diversification of Araceae in the Mediterranean Basin.

Relationships of the woody Medicago species (section Dendrotelis) assessed by molecular cytogenetic analyses.

Background And Aims: The organization of rDNA genes in the woody medic species from the agronomically important Medicago section Dendrotelis was analysed to gain insight into their taxonomic relationships, to assess the levels of infraspecific variation concerning ribosomal loci in a restricted and fragmented insular species (M. citrina) and to assess the nature of its polyploidy.

Methods: Fluorescence in situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal DNA genes in the three species of section Dendrotelis (M.

Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants.

The internal transcribed spacers (ITS) of the nuclear ribosomal 18S-5.8S-26S cistron continue to be the most popular non-plastid region for species-level phylogenetic studies of plant groups despite the early warnings about their potential flaws, which may ultimately result in incorrect assumptions of orthology. It has been gradually realized that the alternative target regions in the nuclear genome (low-copy nuclear genes, LCNG) are burdened with similar problems.

A phylogeographic split in Buxus balearica (Buxaceae) as evidenced by nuclear ribosomal markers: when ITS paralogues are welcome.

Sequences from the ribosomal nuclear internal transcribed spacers (ITS) have been widely used to infer evolutionary hypotheses across a broad range of living organisms. Intraspecific sequence variation is assumed to be absent or negliable in most species, but few detailed studies have been conducted to assess the apportionment of ITS sequence variation within and between plant populations. Buxus balearica was chosen as a model species to assess the levels of infraspecific and intragenomic ITS variation in rare and endangered species occurring in disjunct populations around the Mediterranean basin.

New method of DNA isolation from two food additives suitable for authentication in polymerase chain reaction assays.

Locust bean gum and guar gum are galactomannans used as additives (E 410 and E 412, respectively) in the food industry as stabilizing agents. Analytical discrimination between the two additives in gums and foods is now feasible by molecular techniques. However, only complex and time-consuming DNA isolation protocols are available to date.

Intra- and interspecific variation in DNA content in Cistus (Cistaceae).

Flow cytometry, using propidium iodide and 4',6-diamidano-2-phenylindole staining, was used to estimate the nuclear DNA content (2C) and the proportion of A-T base pairs in 16 species of the Mediterranean genus Cistus. Genome sizes were shown to be constant within species, since no significant intraspecific variation in 2C DNA content was detected. At the genus level, up to about 1.

Seed germination and reproductive features of Lysimachia minoricensis (Primulaceae), a wild-extinct plant.

Lysimachia minoricensis is one of the few Mediterranean endemic plants (Minorca, Balearic islands) that has gone extinct in the wild but which persists as extant germplasm or cultivated plants in several botanical gardens. Reproductive features (seed set, number of seeds per capsule, seed weight) and germination responses to constant temperatures, sea water and dry-heat pre-treatments were investigated to determine the extent to which they may have influenced the extinction of the species. Seed set in Lysimachia is not dependent on pollinators, suggesting a functional selfer breeding system.