Pearl millet is an important cereal crop worldwide and shows superior heat tolerance. Here, we developed a graph-based pan-genome by assembling ten chromosomal genomes with one existing assembly adapted to different climates worldwide and captured 424,085 genomic structural variations (SVs). Comparative genomics and transcriptomics analyses revealed the expansion of the RWP-RK transcription factor family and the involvement of endoplasmic reticulum (ER)-related genes in heat tolerance. The overexpression of one RWP-RK gene led to enhanced plant heat tolerance and transactivated ER-related genes quickly, supporting the important roles of RWP-RK transcription factors and ER system in heat tolerance. Furthermore, we found that some SVs affected the gene expression associated with heat tolerance and SVs surrounding ER-related genes shaped adaptation to heat tolerance during domestication in the population. Our study provides a comprehensive genomic resource revealing insights into heat tolerance and laying a foundation for generating more robust crops under the changing climate.

Background: Heat and drought are serious threats for crop growth and development. As the sixth largest cereal crop in the world, pearl millet can not only be used for food and forage but also as a source of bioenergy. Pearl millet is highly tolerant to heat and drought. Given this, it is considered an ideal crop to study plant stress tolerance and can be used to identify heat-resistant genes. Results: In this study, we used Pacbio sequencing data as a reference sequence to analyze the Illumina data of pearl millet that had been subjected to heat and drought stress for 48 h. By summarizing previous studies, we found 26,299 new genes and 63,090 new transcripts, and the number of gene annotations increased by 20.18%. We identified 2792 transcription factors and 1223 transcriptional regulators. There were 318 TFs and 149 TRs differentially expressed under heat stress, and 315 TFs and 128 TRs were differentially expressed under drought stress. We used RNA sequencing to identify 6920 genes and 6484 genes differentially expressed under heat stress and drought stress, respectively. Conclusions: Through Pacbio sequencing, we have identified more new genes and new transcripts. On the other hand, comparing the differentially expressed genes under heat tolerance with the DEGs under drought stress, we found that even in the same pathway, pearl millet responds with a different protein.

Summary: Millets are a class of nutrient‐rich coarse cereals with high resistance to abiotic stress; thus, they guarantee food security for people living in areas with extreme climatic conditions and provide stress‐related genetic resources for other crops. However, no platform is available to provide a comprehensive and systematic multi‐omics analysis for millets, which seriously hinders the mining of stress‐related genes and the molecular breeding of millets. Here, a free, web‐accessible, user‐friendly millets multi‐omics database platform (Milletdb, http://milletdb.novogene.com) has been developed. The Milletdb contains six millets and their one related species genomes, graph‐based pan‐genomics of pearl millet, and stress‐related multi‐omics data, which enable Milletdb to be the most complete millets multi‐omics database available. We stored GWAS (genome‐wide association study) results of 20 yield‐related trait data obtained under three environmental conditions [field (no stress), early drought and late drought] for 2 years in the database, allowing users to identify stress‐related genes that support yield improvement. Milletdb can simplify the functional genomics analysis of millets by providing users with 20 different tools (e.g., ‘Gene mapping’, ‘Co‐expression’, ‘KEGG/GO Enrichment’ analysis, etc.). On the Milletdb platform, a gene PMA1G03779.1 was identified through ‘GWAS’, which has the potential to modulate yield and respond to different environmental stresses. Using the tools provided by Milletdb, we found that the stress‐related PLATZs TFs (transcription factors) family expands in 87.5% of millet accessions and contributes to vegetative growth and abiotic stress responses. Milletdb can effectively serve researchers in the mining of key genes, genome editing and molecular breeding of millets.

High-temperature stress negatively affects the growth and development of plants, and therefore threatens global agricultural safety. Cultivating stress-tolerant plants is the current objective of plant breeding programs. Pearl millet is a multi-purpose plant, commonly used as a forage but also an important food staple. This crop is very heat-resistant and has a higher net assimilation rate than corn under high-temperature stress. However, the response of heat resistant pearl millet has so far not been studied at the transcriptional level. In this study, transcriptome sequencing of pearl millet leaves exposed to different lengths of heat treatment (1 h, 48 h and 96 h) was conducted in order to investigate the molecular mechanisms of the heat stress response and to identify key genes related to heat stress. The results showed that the amount of heat stress-induced DEGs in leaves differs with the length of exposure to high temperatures. The highest value of DEGs (8286) was observed for the group exposed to heat stress for 96 h, while the other two treatments showed lower DEGs values of 4659 DEGs after 1 h exposure and 3981 DEGs after 48 h exposure to heat stress. The DEGs were mainly synthesized in protein folding pathways under high-temperature stress after 1 h exposure. Moreover, a large number of genes encoding ROS scavenging enzymes were activated under heat stress for 1 h and 48 h treatments. The flavonoid synthesis pathway of pearl millet was enriched after heat stress for 96 h. This study analyzed the transcription dynamics under short to long-term heat stress to provide a theoretical basis for the heat resistance response of pearl millet.

Background: Newly formed polyploids may experience short-term adaptative changes in their genome that may enhance the resistance of plants to stress. Considering the increasingly serious effects of drought on biofuel plants, whole genome duplication (WGD) may be an efficient way to proceed with drought resistant breeding. However, the molecular mechanism of drought response before/after WGD remains largely unclear. Result: We found that autoploid switchgrass (Panicum virgatum L.) 8X Alamo had higher drought tolerance than its parent amphidiploid 4X Alamo using physiological tests. RNA and microRNA sequencing at different time points during drought were then conducted on 8X Alamo and 4X Alamo switchgrass. The specific differentially expressed transcripts (DETs) that related to drought stress (DS) in 8X Alamo were enriched in ribonucleoside and ribonucleotide binding, while the drought-related DETs in 4X Alamo were enriched in structural molecule activity. Ploidy-related DETs were primarily associated with signal transduction mechanisms. Weighted gene co-expression network analysis (WGCNA) detected three significant DS-related modules, and their DETs were primarily enriched in biosynthesis process and photosynthesis. A total of 26 differentially expressed microRNAs (DEmiRs) were detected, and among them, sbi-microRNA 399b was only expressed in 8X Alamo. The targets of microRNAs that were responded to polyploidization and drought stress all contained cytochrome P450 and superoxide dismutase genes. Conclusions: This study explored the drought response of 8X and 4X Alamo switchgrass on both physiological and transcriptional levels, and provided experimental and sequencing data basis for a short-term adaptability study and drought-resistant biofuel plant breeding.