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. By using these protocols, high-quality chromosome spreads are obtained, which allow chromosome counting, building karyotypes, and assessing chromosomal landmarks, and enable genome mapping by fluorochrome banding and in situ hybridization techniques.

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. The correct interpretation of rDNA markers in plant taxonomy and evolution is not free of drawbacks given the complexities derived from the lability of the genetic architecture, the diverse patterns of molecular change, and the fate and evolutionary dynamics of the rDNA units in hybrids and polyploid species. In addition, the terminology used by independent authors is somewhat vague, which often complicates comparisons. To date, no efforts have been reported addressing the potential problems and limitations involved in generating, utilizing, and interpreting the data from the 35S rDNA in cytogenetics. This review discusses the main technical and conceptual limitations of these rDNA markers obtained by cytological and karyological experimental work, in order to clarify biological and evolutionary inferences postulated in a systematic and phylogenetic context. Also, we provide clarification for some ambiguity and misconceptions in terminology usually found in published work that may help to improve the usage of the 35S ribosomal world in plant evolution.

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. However, their presence has been detected in only 45.6% of the analysed families, 26.7% of the sampled genera, and in 23.8% of the studied species. The number of ITR sites greatly varies among congeneric species and higher taxonomic units, and range from one to 72 signals. ITR signals mostly occurs as homozygous loci in most species, however, odd numbers of ITR sites reflecting a hemizygous state have been reported in both gymnosperm and angiosperm groups. Overall, the presence of ITRs appears to be poor predictors of phylogenetic and taxonomic relatedness at most hierarchical levels. The presence of ITRs and the number of sites are not significantly associated to the number of chromosomes. The longitudinal distribution of ITR sites along the chromosome arms indicates that more than half of the ITR presences are between proximal and terminal locations (49.5%), followed by proximal (29.0%) and centromeric (21.5%) arm regions. Intraspecific variation concerning ITR site number, chromosomal locations, and the differential presence on homologous chromosome pairs has been reported in unrelated groups, even at the population level. This hypervariability and dynamism may have likely been overlooked in many lineages due to the very low sample sizes often used in cytogenetic studies.

Interstitial Arabidopsis-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 Anacyclus, 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. We conducted fluorescent in situ hybridization (FISH) using an Arabidopsis-type telomeric sequence as a probe on karyotypes obtained from mitotic chromosomes. FISH signals of ITR sites were detected in species of subfamilies Asteroideae, Carduoideae, Cichorioideae, Gymnarhenoideae, and Mutisioideae, but not in Barnadesioideae. Although six small subfamilies have not yet been sampled, altogether, our results suggest that the dynamics of ITR formation in Asteraceae cannot accurately trace the complex karyological evolution that occurred since the early diversification of this family. Thus, ITRs do not convey a reliable signal at deep or shallow phylogenetic levels and cannot help to delimitate taxonomic categories, a conclusion that might also hold for other important families such as Fabaceae.