In situ hybridisation (ISH) techniques have become one of the most powerful approaches for mapping specific sequences of DNA in plant cytogenetics, including telomere sequences. The basic principles underlying ISH is similar among all the types of experimental variants that have been developed, regardless of whether standard or sophisticated methods were used. However, experimental issues involving the type of the used probes (cloned, synthetic oligonucleotides), probe labeling (nick-translation, PCR-labeling, pre-labeled oligomer), and probe detection (fluorescent, enzymatic) contribute to the sensitivity of ISH approaches [13,14,15,16]. Several technical approaches have been used to date to detect telomeric repeats in plants, including non-Isotopic ISH [11], FISH (fluorescent in situ hybridisation; [6]), PRINS (primer in situ DNA labeling, [17]), PNA-FISH (peptide nucleic acid-FISH, [18]), ND-FISH (non-denaturant-FISH, [19]), PLOPs-FISH (Pre-Labelled Oligomer Probes, [16]), and CO-FISH (chromosome orientation-FISH, [20]). The drawback of molecular cytogenetic methods is that short arrays of telomeric-like sites may be undetectable by ISH [21]. In these cases, DNA sequencing of interstitial chromosomal regions or whole genomes is the best available option [22].

Initially, the location of telomeres in plant chromosomes were identified in Hordeum vulgare and Secale cereale by [11], who also detected interstitial sites. Some years later, a synthetic oligonucleotide (TTTAGGG), representing the canonical Arabidopsis-type repeat was used as a template in PCR and fluorescently labelled to locate telomere repeats in several unrelated flowering plant species [6]. Most of the studies (71.56%) dealing with the cytogenetic mapping of plant telomeres used synthetic oligonucleotide probes for ISH, including the Arabidopsis-type repeat (53.13%; e.g., [7]), the vertebrate-type repeat (TTAGGG) (15.62%; e.g., [23]), other unusual plant-specific telomere sequences (CTCGGTTATGG, TTTTTTAGGG, T₄-₅AGCA, TTCAGG and TTTCAGG; 2.14%, e.g., [24,25,26,27]), and the Tetrahymena-type repeat (TTGGGG; 0.67%, e.g., [23]), while a significantly lower number (28.44%) used cloned sequences involving the Arabidopsis clone (27.90%; e.g., [28]) or other specific telomeric regions (0.54%; e.g., [18]). The most likely reasons explaining the preferential use of synthetic probes over cloned sequences may be related to the more complex technical requirements and higher costs involved in the handling and conservation of clones. Currently, ITR repeats detected in plants are constituted by the Arabidopsis-type (TTTAGGG), vertebrate-type (TTAGGG), and Cestrum-type (T₄-₅AGCA) sequences.

A total 627 species from 330 genera belonging to 79 families (sensu APG IV) have been karyologically analysed to detect telomeric sequences in seed plants [6,7,8,10,11,12,16,17,18,19,20,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185]. These figures sharply contrast with the greater amount of data reported for nuclear ribosomal DNA loci (35S and 5S rDNA families), the most popular chromosomal landmarks used in plant molecular cytogenetics, with data available for 2148 species, 540 genera, and 114 families [186].

The sampling for detecting telomeric sequences is uneven and biased towards the analysis of large groups, with some exceptions (Figure 2).

Whereas in gymnosperms there is a lack of data only for Gnetales, in angiosperms the number of major groups analysed (11) nearly equals those for which there is no data (13). Speciose ordinal groups not sampled to date are few and include Proteales, Vitales, Santalales, and Cornales, which encompass between 14–151 genera and 590–1750 species [187]. Unfortunately, no species from the three most basal lineages of angiosperms (Amboreallales, Nymphaeales, Austrobaileyales) have been analysed. Although the overall diversity of these orders is fairly limited (12 genera and about 175 species; [187]), their key position in the ancestral diversification of flowering plants makes them priority targets for assessing the presence of ITRs.

With the exception of Chloranthales, which exhibits a scarce diversity and has only had three of its analysed, and the monotypic Ginkgoales, all major evolutionary groups sampled had ITRs in their karyotypes (Figure 2 and Figure 3).

However, heterogeneity regarding the distribution at lower taxonomic units is noteworthy. Thus, ITRs occur in only 36 out of 79 sampled families (45.57%), suggesting that disparate results occur within major plant lineages. In gymnosperms, the families with the higher number of ITR occurrences are Podocarpaceae (8 spp.) and Pinaceae (6 spp.), whereas Asteraceae (37 spp.), Fabaceae (21 spp.), and Poaceae (8 spp.) lead among angiosperms. It should be stressed, however, that due to the uneven sampling effort made at different taxonomic levels, these results could be skewed and may not reflect the real values. In this regard, it is worth mentioning that at the family level, the number of species showing ITRs in their karyotypes is strongly correlated to the number of sampled species (Pearson correlation value = 0.994, p < 0.0001).

Overall, lower and similar frequencies of occurrence are attained at the generic and species level. ITRs have been detected in only 88 out of 330 analysed genera (26.67%) and in 149 out of 627 sampled species (23.73%). These results clearly show that although ITRs are widespread in seed plants, their frequency at low taxonomic units is fairly moderate.