Telomere-to-telomere gap-free genome assembly of a male donkey and the identification of novel SVs associated with functional genes

Previous assemblies of the donkey genome remain with gaps and structural errors, and a complete donkey genome will greatly facilitate genetic research related to donkeys. In the present study, a 2.78-Gb telomere-to-telomere gap-free donkey genome (CAU_T2T_donkey) was assembled, including a 29.78-Mb Y chromosome, aided by ONT and trio-binning approach. CAU_T2T_donkey corrected the structural errors of previous assemblies and added a total of 153.8-Mb previously unresolved regions and 354 genes to the reference genome EquAss-T2T_v2. We identified a 1.9-Mb PAR on CAU_T2T_donkey- chromosome Y, and added 17.1Mb regions and 75 new genes to the chromosome Y of the previous reference genome ASM1607732v2. Multi-copy genes, such as TSPY, L1RE, ETY, HSFY, and ETSTY were also identified in CAU_T2T_donkey- chromosome Y. Totally 6 types of repetitive sequences in centromeric regions were identified, and the features of the centromeric regions were revealed, and satellite-free centromeres were identified. We aligned HiFi long-read sequences of donkeys from six breeds against CAU_T2T_donkey and identified SVs in previously unresolved regions, and some of the novel SVs were located in functional genes, such as AOX1 (Chr4:DEL61), ASIC2 (Chr13:INS954), and Twist2 (Chr19:DEL98).