Knowledge of the locations of repeat elements could be very important in the assembly of genome sequences and their assignment to physical chromosomes. Genomic and species relationships among 16 species were investigated using fluorescence in situ hybridization (FISH) with the Am1 and (ACT)₆ probes. The Am1 oligonucleotide probe was particularly enriched in the C genomes, whereas the (ACT)₆ trinucleotide repeat probe showed a diverse distribution of hybridization patterns in the A, AB, C, AC, and ACD genomes but might not be present in the B and D genomes. The hybridization pattern of Avena sativa was very similar to that of A. insularis, indicating that this species most likely originated from A. insularis as a tetraploid ancestor. Although the two FISH probes failed to identify relationships of more species, this proof-of-concept approach opens the way to the use of FISH probes in assigning other signature elements from genomic sequence to physical chromosomes.

Oligo-fluorescence in situ hybridization (FISH) facilitates precise chromosome identification and comparative cytogenetic analysis. Detection of autosomal chromosomes of Hippophaë rhamnoides has not been achieved using oligonucleotide sequences. Here, the chromosomes of five H. rhamnoides taxa in the mitotic metaphase and mitotic metaphase to anaphase were detected using the oligo-FISH probes (AG₃T₃)₃, 5S rDNA, and (TTG)₆. In total, 24 small chromosomes were clearly observed in the mitotic metaphase (0.89–3.03 μm), whereas 24–48 small chromosomes were observed in the mitotic metaphase to anaphase (0.94–3.10 μm). The signal number and intensity of (AG₃T₃)₃, 5S rDNA, and (TTG)₆ in the mitotic metaphase to anaphase chromosomes were nearly consistent with those in the mitotic metaphase chromosomes when the two split chromosomes were integrated as one unit. Of note, 14 chromosomes (there is a high chance that sex chromosomes are included) were exclusively identified by (AG₃T₃)₃, 5S rDNA, and (TTG)₆. The other 10 also showed a terminal signal with (AG₃T₃)₃. Moreover, these oligo-probes were able to distinguish one wild H. rhamnoides taxon from four H. rhamnoides taxa. These chromosome identification and taxa differentiation data will help in elucidating visual and elaborate physical mapping and guide breeders’ utilization of wild resources of H. rhamnoides.

Walnuts are economically important tree species in Sichuan Province (China) that provide heathy nuts. Fluorescence in situ hybridization (FISH) and analyses of an early-fruiting gene fragment and simple sequence repeats (SSRs) were used to distinguish Sichuan walnut cultivars and examine their relationships with Juglans regia L. and Juglans sigillata Dode. Thirty-four small chromosomes were counted in four Sichuan walnut cultivars. In the four cultivars, 5S rDNA was located in the proximal regions of two chromosomes (5 and 6), while (AG₃T₃)₃ was located at both ends of each chromosome. The existence of the signal at both chromosome ends ensured accurate chromosome counts. 5S rDNA and (AG₃T₃)₃ were not effective in identifying Sichuan walnut cultivars. Evolutionary analysis involving 32 early-fruiting nucleotide sequences from Sichuan walnut materials were performed with the maximum likelihood method. There were a total of 602 positions. All positions with gaps and missing data were eliminated, resulting in a final dataset of 562 positions. The ML tree with the highest log likelihood (−1607.82) revealed two obvious groups: one including materials of J. regia, which fruits 1 year after grafting, and another including materials of J. sigillata, which fruits >3 years after grafting. The early-fruiting gene fragment divided 22 walnut materials (10 walnut cultivars and 12 walnut accessions) into two groups, indicating that it was somewhat effective for distinguishing Sichuan walnut cultivars. Furthermore, 22 SSR loci were revealed to identify nine walnut cultivars. Eight cultivars were exclusively discerned by one SSR locus each: Chuanzao 1 [CUJRB307 (116) or CUJRA206a (182)], Chuanzao 2 [JSI-73 (154)], Shuangzao [CUJRB103a (123), CUJRB218 (144), JSI-71 (146), or CUJRA206a (176)], Shimianju [ZMZ11 (138)], Meigupao [CUJRB218 (149), CUJRB103a (151), or CUJRA206a (190)], Muzhilinhe [CUJRB220 (136), ZMZ11 (147), CUJRC310 (156), or JSI-73 (166)], Maerkang [CUJRA124 (154), CUJRB218 (159), or CUJRA123 (182)], Yanyuanzao [CUJRA124 (150) or CUJRA206a (192)]. The Shuling cultivar was identified by the combination of ZMZ11 (148) and other SSR loci, which distinguished and excluded the Chuanzao 1 and Yanyuanzao cultivars. Our results will guide the identification and breeding of Sichuan walnut cultivars.

We report the cytogenetic map for a collection of species in the Oleaceae, and test similarities among the karyotypes relative to their known species phylogeny. The oligonucleotides 5S ribosomal DNA (rDNA), (AGGGTTT)₃, and (TTG)₆ were used as fluorescence in situ hybridization (FISH) probes to locate the corresponding chromosomes in three Oleaceae genera: Fraxinus pennsylvanica, Syringa oblata, Ligustrum lucidum, and Ligustrum × vicaryi. Forty-six small chromosomes were identified in four species. (AGGGTTT)₃ signals were observed on almost all chromosome ends of four species, but (AGGGTTT)₃ played no role in distinguishing the chromosomes but displayed intact chromosomes and could thus be used as a guide for finding chromosome counts. (TTG)₆ and 5S rDNA signals discerned several chromosomes located at subterminal or central regions. Based on the similarity of the signal pattern (mainly in number and location and less in intensity) of the four species, the variations in the 5S rDNA and (TTG)₆ distribution can be ordered as L. lucidum < L. × vicaryi < F. pennsylvanica < S. oblata. Variations have observed in the three genera. The molecular cytogenetic data presented here might serve as a starting point for further larger-scale elucidation of the structure of the Oleaceae genome, and comparison with the known phylogeny of Oleaceae family.