Karyotype analysis plays a central role in cytogenetics, helping to reveal evolutionary processes and species relationships. Traditional chromosome identification has relied on genomic DNA, repetitive sequences, or bacterial artificial chromosome (BAC) probes, but these approaches often suffer from signal instability and limited cross-species applicability. Advances in genome sequencing and synthetic oligonucleotide technology have created new possibilities for high-resolution fluorescence in situ hybridization (FISH). However, the high costs of synthesizing large oligo libraries remain a barrier, especially in crops with many chromosomes. These challenges have particularly hindered cytogenetic research in longan and lychee, two commercially important tropical fruits. Due to these problems, there is a pressing need to develop cost-effective oligo-based tools for detailed chromosomal research.
Researchers from Guangxi University, Fujian Agriculture and Forestry University, Minjiang University, and the Chinese Academy of Tropical Agricultural Sciences report a new oligo-based barcode system for chromosome identification in longan and lychee. The study, published (DOI: 10.1093/hr/uhae278) on September 28, 2024, in Horticulture Research, demonstrates how the cost of oligo synthesis can be halved by merging DNA sequences and applying multiplex PCR labeling. Using this strategy, the team mapped 36 distinct chromosome regions, corrected genome assembly errors, and clarified evolutionary differences between longan and lychee, providing a new resource for tropical fruit genetics.
The team began by screening over one million 50-nt oligonucleotides from the longan genome using the Chorus2 pipeline. To reduce costs, they merged pairs of oligos into 100-nt synthetic sequences, achieving an efficient pool of 91,550 probes tagged with three primers. This design reduced synthesis costs by half compared to conventional approaches. Using FISH, the probes generated 36 distinct fluorescent signals, enabling unambiguous identification of all 15 chromosomes in both longan and lychee.Comparative analysis revealed structural differences, particularly on chromosome 2, where lychee showed evidence of inversion or transposable element expansion compared to longan. The oligo-barcodes also exposed discrepancies between genome assemblies and cytological observations, especially in the placement of 35S rDNA loci. Longan showed six loci on chromosomes 12–14, while lychee displayed ten loci across five chromosomes, some of which were missing from published assemblies. Phylogenetic analysis indicated that longan and lychee diverged about 10.1 million years ago, while rambutan diverged earlier at 13.6 million years, explaining why the probes failed to produce signals in rambutan. Together, these results confirm that cost-effective oligo-based probes can resolve both evolutionary relationships and genome assembly issues in Sapindaceae fruit crops.
“Our study demonstrates that synthetic oligo barcodes can provide a powerful and affordable alternative for cytogenetic research,” said corresponding author Fan Yu. “By reducing synthesis costs and enabling reliable chromosome identification, this method offers new opportunities to study genome organization and evolution in longan, lychee, and other fruit crops. Importantly, the system also helps identify errors in genome assemblies, which is essential for ensuring the accuracy of genomic resources. This approach can be readily adapted to other plant species, providing broad value for plant genetics and breeding research”.
The development of this oligo-based barcode system provides plant scientists with a reliable toolkit for chromosome identification and genome verification. For breeders, accurate karyotype information can guide hybridization strategies, trait mapping, and the detection of structural variations. For evolutionary biologists, the system clarifies divergence patterns among closely related Sapindaceae species, such as longan and lychee. Importantly, the cost-reduction strategy makes this approach accessible for research groups working on non-model crops. By improving both accuracy and affordability, this method strengthens the foundation for genomic studies, conservation efforts, and the development of improved cultivars of economically significant tropical fruits.
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References
DOI
10.1093/hr/uhae278
Original Source URL
https://doi.org/10.1093/hr/uhae278
Funding information
This work was supported by Natural Science Foundation of Hainan Province Youth Fund Project (320QN322); Young Talent Start-up Fund of South Subtropical Crop Research Institution CATAS; The Natural Science Foundation of Fujian Province, China (2021 J011048); The Guangxi Natural Science Foundation (2024GXNSFBA010352); The Sugarcane Research Foundation of Guangxi University (Grant No. 2022GZB007); and the fellowship of China Postdoctoral Science Foundation (2022MD723761).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.