Accurate Chromosome Identification in the Prunus Subgenus Cerasus (Prunus pseudocerasus) and its Relatives by Oligo-FISH

Abstract

A precise, rapid and straightforward approach to chromosome identification is fundamental for cytogenetics studies. However, the identification of individual chromosomes was not previously possible for Chinese cherry or other Prunus species due to the small size and similar morphology of their chromosomes. To address this issue, we designed a pool of oligonucleotides distributed across specific pseudochromosome regions of Chinese cherry. This oligonucleotide pool was amplified through multiplex PCR with specific internal primers to produce probes that could recognize specific chromosomes. External primers modified with red and green fluorescence tags could produce unique signal barcoding patterns to identify each chromosome concomitantly. The same oligonucleotide pool could also discriminate all chromosomes in other Prunus species. Additionally, the 5S/45S rDNA probes and the oligo pool were applied in two sequential rounds of fluorescence in situ hybridization (FISH) localized to chromosomes and showed different distribution patterns among Prunus species. At the same time, comparative karyotype analysis revealed high conservation among P. pseudocerasus, P. avium, and P. persica. Together, these findings establish this oligonucleotide pool as the most effective tool for chromosome identification and the analysis of genome organization and evolution in the genus Prunus.

Karyotype Description and Comparative Chromosomal Mapping of 5S rDNA in 21 species

“…Numerous previous studies showed that the numbers of 5S rDNA FISH signal sites ranged from 1 to 71 (Ali et al 2005, Lan and Albert 2011, Luo et al 2017, Kovács et al 2023, Rodríguez-González et al 2023). Their signal position was found in the chromosome interstitial position, distal position, proximal position, and far away from the chromosome (Amarasinghe and Carlson 1988, Cai et al 2006, Campomayor et al 2021, Wang et al 2022, Rodríguez-González et al 2023). In this study, 5S rDNA was rather diverse and abundant in signal site number (2–18), position (e.g., interstitial, distal, proximal position, occasionally, outside chromosome), and even as intense (e.g., strong, weak, slight).…”

“…For example, four 5S rDNA signal sites in six in Sinapidendron frutescens (Aiton) Lowe (Ali et al 2005), eight in Prunus pseudocerasus (Lindl.) G. Don (Wang et al 2022), 8-19 in Trifolium medium L. (Lukjanová et al 2023), 10 in Brassica juncea (Linnaeus) Czernajew, 12 in Olimarabidopsis cabulica (J. D. Hooker & Thomson) Al-Shehbaz et al, 14 in Brassica napus L. (Ali et al 2005), 15 in Paphiopedilum sukhakulii Schoser & Senghas (Lan and Albert 2011), 16 in Piptanthus concolor Harrow ex Craib (Luo et al 2017), 19 in Paphiopedilum henryanum Braem, 20 in Paphiopedilum druryi (Bedd.) Stein, 22 in Paphiopedilum sangii Braem, 23 in Paphiopedilum tigrinum Koop.…”

“…F.) Stein, and 38 in Paphiopedilum gigantifolium Braem, M.L.Baker & C.O.Baker (Lan and Albert 2011). Notably, there were two 5S rDNA sites on the same chromosome, such as P. concolor (Luo et al 2017), Brassica oleracea L. (Ali et al 2005), O. cabulica (Ali et al 2005), P. primulinum , P. liemianum , P. randsii , P. parishii , Paphiopedilum dianthum T. Tang et F. T. Wang (Lan and Albert 2011), Prunus cerasus L. (Wang et al 2022), and Plantago maxima Juss. ex Jacq.…”

“…Franco (Amarasinghe and Carlson 1988), or in the chromosome proximal position from Brassica rapa L. (Campomayor et al 2021), even as far away the chromosome from Hedysarum setigerum Turcz. (Yurkevich et al 2022), and P. cerasus (Wang et al 2022).…”

Autotetraploid Origin of Chinese Cherry Revealed by Chromosomal Karyotype and In Situ Hybridization of Seedling Progenies

“…In this study, rDNA-FISH signals accurately identified chromosome pairs 3, 4, 5, and 7 in Chinese cherry. In terms of chromosome position, 5S rDNA sites are in proximal regions while 45S rDNA repeats are localized at (sub)-terminal regions on short arms, which is highly conserved in the Rosaceae species including Cerasus [25,38,40]. Interestingly, there is one 5S and one 45S rDNA site co-localized on short arms of chromosome 4 in a haploid set of Chinese cherry.…”