Data Sheet 1_Comparative analysis of the NLR gene family in the genomes of garden asparagus (Asparagus officinalis) and its wild relatives.docx

Garden asparagus (Asparagus officinalis), a prominent horticultural crop recognized as the “king of vegetables” in the international market, is usually threatened by severe disease challenges that hinder its sustainable cultivation. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key components of plant immune systems, yet their specific role and evolutionary patterns in A. officinalis and its related species remain poorly characterized. In this study, we conducted a comprehensive analysis of NLR gene distribution patterns, structural features, phylogenetic characterization, and evolutionary dynamics across A. officinalis and two phylogenetically related species, Asparagus kiusianus and Asparagus setaceus, and conducted expression studies after Phomopsis asparagi infection in A. officinalis. Our findings demonstrate that NLR genes in all three species display chromosomal clustering patterns. Phylogenetic reconstruction and N-terminal domain classification categorized these NLRs into three distinct subfamilies, with their promoters containing numerous cis-elements responsive to defense signals and phytohormones. Comparative genomic analysis revealed a marked contraction of the NLR genes from the wild species to the domesticated A. officinalis, with gene counts of 63, 47, and 27 NLR genes identified in A. setaceus, A. kiusianus, and A. officinalis, respectively. Orthologous gene analysis identified 16 conserved NLR gene pairs between A. setaceus and A. officinalis, which are likely the NLR genes preserved during the domestication process of A. officinalis. Pathogen inoculation assays revealed distinct phenotypic responses: A. officinalis was susceptible, while A. setaceus remained asymptomatic. Notably, the majority of preserved NLR genes in A. officinalis demonstrated either unchanged or downregulated expression following fungal challenge, indicating a potential functional impairment in disease resistance mechanisms. In conclusion, our findings suggest that the increased disease susceptibility of domesticated A. officinalis is driven by both the contraction of NLR gene repertoire and the functional reduced or inconsistent induction of retained NLR genes—potentially a consequence of artificial selection favoring yield and quality. This study provides important insights into the evolutionary dynamics of NLR genes within the Asparagus genus and may contribute to future efforts aimed at disease-resistant breeding in A. officinalis.

石刁柏（Asparagus officinalis）是国际市场上享誉“蔬菜之王”的重要园艺作物，却常遭受严重病害威胁，制约其可持续种植。核苷酸结合富亮氨酸重复受体（NLRs）是植物免疫系统的核心组分，然而其在石刁柏及其近缘物种中的具体功能与进化模式仍未得到充分解析。本研究针对石刁柏及其两个系统发育近缘物种——基乌斯天门冬（Asparagus kiusianus）和文竹（Asparagus setaceus），全面解析了NLR基因的染色体分布模式、结构特征、系统发育特征及进化动态，并对石刁柏受芦笋拟茎点霉（Phomopsis asparagi）侵染后的基因表达情况开展了研究。研究结果表明，三个物种的NLR基因均呈现染色体成簇分布的特征。通过系统发育重建与N端结构域分类，这些NLR可被划分为三个独立的亚家族，且其启动子区域含有大量响应防御信号与植物激素的顺式作用元件。比较基因组分析显示，从野生物种到驯化栽培的石刁柏，NLR基因家族发生了显著收缩；其中文竹、基乌斯天门冬与石刁柏中分别鉴定出63、47与27个NLR基因。同源基因分析鉴定出文竹与石刁柏之间存在16个保守的NLR基因对，这些基因大概率是石刁柏驯化过程中保留下来的NLR基因。病原菌接种实验显示二者存在显著的表型差异：石刁柏表现为感病，而文竹无明显病症。值得注意的是，石刁柏中保留的大部分NLR基因在真菌侵染后呈现表达量不变或下调的趋势，表明其抗病机制可能存在功能受损。综上，本研究结果表明，驯化石刁柏的病害易感性提升，是由NLR基因库的收缩以及保留的NLR基因的诱导表达功能减弱或失调共同导致的——这可能是人工选择偏向产量与品质所带来的后果。本研究揭示了天门冬属植物NLR基因的进化动态，可为石刁柏的抗病育种工作提供重要理论参考。