Table_4_Genome-Wide Analysis of the HSP20 Gene Family and Expression Patterns of HSP20 Genes in Response to Abiotic Stresses in Cynodon transvaalensis.XLSX

African bermudagrass (Cynodon transvaalensis Burtt–Davy) is an important warm-season turfgrass and forage grass species. Heat shock protein 20 (HSP20) is a diverse, ancient, and important protein family. To date, HSP20 genes have not been characterized genome-widely in African bermudagrass. Here, we confirmed 41 HSP20 genes in African bermudagrass genome. On the basis of the phylogenetic tree and cellular locations, the HSP20 proteins were classified into 12 subfamilies. Motif composition was consistent with the phylogeny. Moreover, we identified 15 pairs of paralogs containing nine pairs of tandem duplicates and six pairs of WGD/segmental duplicates of HSP20 genes. Unsurprisingly, the syntenic genes revealed that African bermudagrass had a closer evolutionary relationship with monocots (maize and rice) than dicots (Arabidopsis and soybean). The expression patterns of HSP20 genes were identified with the transcriptome data under abiotic stresses. According to the expression profiles, HSP20 genes could be clustered into three groups (Groups I, II, and III). Group I was the largest, and these genes were up-regulated in response to heat stress as expected. In Group II, one monocot-specific HSP20, CtHSP20-14 maintained higher expression levels under optimum temperature and low temperature, but not high temperature. Moreover, a pair of WGD/segmental duplicates CtHSP20-9 and CtHSP20-10 were among the most conserved HSP20s across different plant species, and they seemed to be positively selected in response to extreme temperatures during evolution. A total of 938 cis-elements were captured in the putative promoters of HSP20 genes. Almost half of the cis-elements were stress responsive, indicating that the expression pattern of HSP20 genes under abiotic stresses might be largely regulated by the cis-elements. Additionally, three-dimensional structure simulations and protein–protein interaction networks were incorporated to resolve the function mechanism of HSP20 proteins. In summary, the findings fulfilled the HSP20 family analysis and could provide useful information for further functional investigations of the specific HSP20s (e.g., CtHSP20-9, CtHSP20-10, and CtHSP20-14) in African bermudagrass.

非洲狗牙根（Cynodon transvaalensis Burtt–Davy）是一种重要的暖季型草坪草与饲草作物。热休克蛋白20（HSP20）是一类分布广泛、起源古老且功能重要的蛋白质家族。截至目前，尚未在非洲狗牙根的全基因组范围内对HSP20基因开展系统鉴定与分析。本研究在非洲狗牙根基因组中鉴定出41个HSP20基因；基于系统发育树与亚细胞定位信息，该家族蛋白可被划分为12个亚家族，其基序组成与系统发育关系高度一致。此外，本研究还鉴定出15对HSP20基因旁系同源对，其中包含9对串联重复基因与6对全基因组复制/片段复制同源基因。不出所料，共线性基因分析结果显示，非洲狗牙根与单子叶植物（玉米、水稻）的进化亲缘关系较双子叶植物（拟南芥、大豆）更为紧密。本研究利用非生物胁迫下的转录组数据，解析了非洲狗牙根HSP20基因的表达模式：基于表达谱分析，HSP20基因可被划分为3个类群（类群I、II、III）。类群I为最大的类群，该类群基因正如预期般在热胁迫下呈现上调表达。类群II中，1个单子叶植物特异性HSP20基因CtHSP20-14在适宜温度与低温条件下维持较高表达水平，但在高温条件下无此特征。此外，1对全基因组复制/片段复制同源基因CtHSP20-9与CtHSP20-10是不同植物物种中最为保守的HSP20基因之一，且在进化过程中似乎受到了极端温度胁迫的正向选择。在HSP20基因的预测启动子区域中共鉴定到938个顺式作用元件，近半数为胁迫响应型元件，这表明非生物胁迫下HSP20基因的表达模式可能主要由这些顺式作用元件调控。此外，本研究还通过三维结构模拟与蛋白质互作网络分析，解析了HSP20蛋白的功能机制。综上，本研究完整完成了非洲狗牙根HSP20基因家族的系统分析，可为后续针对非洲狗牙根中特定HSP20基因（如CtHSP20-9、CtHSP20-10与CtHSP20-14）的功能研究提供有价值的参考依据。