Development and applications of A‐genome chromosome‐specific oligo pools for peanut genomic relationships revealing and single‐chromosome microdissection

SUMMARY Wild A‐genome species represent critical genetic resources for improving cultivated peanut, an allotetraploid crop of global economic importance. However, lacking accurate identification and localization of individual target chromosomes hinders distant hybridization germplasm innovation and single‐chromosome sequencing in peanut. Here, we developed 10 A‐genome chromosome‐specific oligonucleotide (oligo) probe pools, which enable specific tracing of individual A‐genome chromosomes via single‐chromosome painting without generating signals on chromosomes in other genomes of Arachis species. Combining single‐chromosome painting with fluorescence in situ hybridization (FISH) using repetitive sequence oligonucleotide probes, we established a consensus karyotype for homeologous groups of wild A‐genome species and constructed a phylogenetic tree, providing novel insights into Arachis genome evolution and classification. We further applied these probe pools to detect chromosomal numerical variations in distant hybridization progeny, successfully identifying a hybrid of Arachis duranensis and Arachis ipaensis , and two addition lines from Arachis hypogaea cv. Silihong and A. duranensis . By tracking chromosome A08 with the specific probe pool coupled with microdissection, we achieved the first single‐chromosome isolation in peanut. These findings provide a powerful tool for single‐chromosome sequencing and chromosome engineering‐based breeding.