Oligonucleotide (Oligo)-based fluorescence in situ hybridization (FISH) represents a highly effective methodology for identifying plant chromosomes. Longan is a commercially significant fruit species, yet lacking basic chromosomal markers has hindered its cytogenetic research. In this study, we developed a cost-effective oligo-based system for distinguishing chromosomes of longan (Dimocarpus longan Lour., 2n = 2x = 30). For this system, each synthesized oligo contained two chromosome-specific sequences that spanned a distance of over 200 kb, and a PCR-based flexible amplification method coupled with nested primers was used for probe labeling. The use of these oligo-based barcodes enabled the marking of 36 chromosomal regions, which allowed for the unambiguous distinction of all 15 chromosomes in both longan and lychee (Litchi chinensis Sonn., 2n = 2x = 30) species. Based on the identification of individual chromosomes, we constructed karyotypes and detected genome assembly errors involving the 35S ribosomal RNA gene (35S rDNA) in longan and lychee. Developing oligo-based barcodes offers considerable promise for advancing cytogenetic research in longan, lychee, and their related species. Furthermore, this cost-effective synthesis system can be referred to the development of new oligo libraries among other species.

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