Telomere-to-telomere gap-free genome assembly and chromosome segment substitution lines facilitated gene identification in wild rice

Abstract The abounding variations hidden in wild rice (Oryza rufipogon) provided potential reservoirs of useful genes for rice breeding. However, the genome of wild rice has not yet been comprehensively assessed. In this study, we constructed the telomere-to-telomere (T2T) genome assembly and annotation of wild rice Y476 with a total length of 421.1 Mb and 38,055 predicted protein-coding genes, which features evident improvements in the continuity and quality of sequence compared with the existing wild rice genomes. Of Y476 genome assembly, 128 Mb and 5,984 novel genes were not found in ‘Nipponbare’ reference, leading to 690 gene families expanded and 247 gene families contracted, including disease resistance and grain regulation gene family. Two sets of chromosome segments substitution lines (CSSLs) were constructed, using Y476 as the donor parent and cultivated rice as the recurrent parents, covering the whole genome of Y476, providing materials for introducing novel genes and identification of their function. Combining the T2T reference genome and CSSL population, 254 QTLs associated with agronomic traits, biotic and abiotic stresses were identified. A receptor-like kinase gene associated with rice blast resistance was identified, a 7.8-kb insertion was found in the intron of this gene which increased its expression level. Using CRISPR/Cas9 technology and transcriptomic data, we confirmed that this wild rice allele improves rice blast resistance via regulating OsMADS26 expression. Collectively, our findings provided not only a T2T gap-free reference genome but also a highly efficient platform for novel gene identification of O. rufipogon, and therefore, have important implications for wild rice utilization.