Saba Parvez ¹ Maryam Asif ¹ Alizay Ahmad ¹ Iqra Javaid ¹ Muhammad Zaman Rasheed ¹ Aqsa Sarwar ¹ Fozia Saleem ¹ Romana Iftikhar ¹ Rao Sohail Ahmad Khan ¹ Sehar Nawaz ¹ Nada H. Aljarba ² Saad Serfraz ^1*

• ¹ Evolutionary biology lab, CABB, University of Agriculture Faisalabad, 38000, Pakistan, Faisalabd, Pakistan
• ² Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia

The Oleaceae family, encompassing key genera such as Fraxinus (ash trees), Olea (olives), Jasminum (jasmine), Syringa (lilac), and Forsythia, plays a crucial ecological and economic role.Despite their importance, the evolutionary dynamics and immune system adaptations of their NLR (Nucleotide binding leucine-rich repeats) gene family remain largely unexplored. This study employs high-throughput comparative genomics to investigate NLR gene evolution across the Oleaceae family. The genus Fraxinus is widely distributed across both the New and Old Worlds, with 23 distinct species analyzed in this study. Our results reveal a predominant strategy of gene conservation in the evolution of the NLR gene family across these species. Geographical adaptation has played a significant role, particularly in Old World ash tree species, which exhibit dynamic patterns of gene expansion and contraction within the last 50 million years. Notably, genes acquired from an ancient whole genome duplication event (~35 Mya) have been retained across Fraxinus lineages. In contrast, the genus Olea (olives) has undergone extensive gene expansion driven by recent duplications and significant birth of novel NLR gene families. These differences in NLR gene evolution likely enhance Olea's ability to recognize diverse pathogens through recent expansions, while Fraxinus maintains specialized immune responses through conserved genes, with potential trade-offs in pathogen adaptation and energy efficiency. In terms of NLR distribution, all species of the Oleaceae family show an enhanced pseudogenization of TIR-NLRs and expansion in CCG10-NLR. However, the comparative RNA-seq expression analysis in olive suggests that partial NLR genes, despite their incomplete structure, have significant expression and may play important roles in plant immune responses. To the best of our knowledge, this is the first study that has investigated NLR gene evolution in the Oleaceae family to identify genomic resources for improving the resistance of tree species with global importance for the economy and food security.