Finger millet RNA-seq reveals differential gene expression associated with tolerance to aluminum toxicity and provides novel genomic resources

Abstract

Eleusine coracana, finger millet, is a multipurpose crop cultivated in arid and semi-arid regions of Africa and Asia. RNA sequencing (RNA-seq) was used in this study to obtain valuable genomic resources and identify genes differentially expressed between Al-tolerant and Al-susceptible genotypes. Two groups of finger millet genotypes were used: Al-tolerant (215836, 215845, and 229722) and Al-susceptible (212462, 215804 and 238323). The analysis of the RNA-seq data resulted in 198,546 unigenes, 56.5% of which were annotated with significant hits in one or more of the following six databases: NR (48.8%), GO (29.7%), KEGG (45%), PlantTFDB (19.0%), Uniprot (49.2%), and NT (46.2%). It is noteworthy that only 220 unigenes in the NR database had significant hits against finger millet sequences suggesting that finger millet’s genomic resources are scarce. The gene expression analysis revealed that 322 genes were significantly differentially expressed between the Al-tolerant and Al-susceptible genotypes, of which 40.7% were upregulated while 59.3% were downregulated in Al-tolerant genotypes. Among the significant DEGs, 54.7% were annotated in the GO database with the top hits being ATP binding (GO:0005524) and DNA binding (GO:0003677) in the molecular function, DNA integration (GO:0015074) and cell redox homeostasis in the biological process, as well as cellular anatomical entity and intracellular component in the cellular component GO classes. Several of the annotated DEGs were significantly enriched for their corresponding GO terms. The KEGG pathway analysis resulted in 60 DEGs that were annotated with different pathway classes, of which carbohydrate metabolism and signal transduction were the most prominent. The homologs of a number of significant DEGs have been previously reported as being associated with Al or other abiotic stress responses in various crops, including carboxypeptidase SOL1, HMA3, AP2, bZIP, C3H, and WRKY TF genes. A more detailed investigation of these and other DEGs will enable genomic-led breeding for Al tolerance in finger millet. RNA-seq data analysis also yielded 119,073 SNP markers, the majority of which had PIC values above 0.3, indicating that they are highly informative. Additionally, 3,553 single-copy SSR markers were identified, of which trinucleotide SSRs were the most prevalent. These genomic resources contribute substantially to the enrichment of genomic databases for finger millet, and facilitate future research on this crop.

Finger millet: a hero in the making to combat food insecurity

“…When assessing gene expression differences between accessions that vary in a morphological trait, pseudoreplication can be used. For example, Brhane et al ( 2022 ) conducted RNA-seq on three aluminum (Al)-tolerant and three Al-susceptible finger millet accessions, and considered the three accessions within each tolerance class as pseudoreplicates. This approach removes a lot of the genotypic variation that is not associated with the trait of interest, similarly to a bulked segregant approach, but requires that the genes and alleles that confer Al tolerance are the same across accessions within a pseudoreplicate.…”

“…Another consideration in RNA-seq analyses of allopolyploids is the possible co-alignment of reads from more than one subgenome. The RNA-seq studies in finger millet published to date conducted transcript assembly in the absence of a reference genome (Brhane et al 2022 ; Hittalmani et al 2017 ; Parvathi et al 2019 ; Zhang et al 2019 ) or relied on heterologous genome assemblies to be used as a reference (Rahman et al 2014 ) . Under both scenarios, A- and B-genome transcripts will likely have collapsed to varying extents during read alignments.…”

Finger millet (Eleusine coracana) improvement: Challenges and prospects—A review

“…Li et al (2021) deployed transcriptomics and proteomics to determine drought response mechanisms and identified 113 associated differentially expressed genes (DEGs) and differentially expressed proteins (DEPs). Brhane, Haileselassie, Tesfaye, Ortiz, Hammenhag, Abreha, Vetukuri, and Geleta (2022) exploited transcriptomics for both marker and trait discovery. They reported differentially expressed genes between Al‐tolerant and Al‐susceptible finger millet and SNPs and SSR markers.…”

Alternative Splicing Analysis Revealed the Role of Alpha-Linolenic Acid and Carotenoids in Fruit Development of Osmanthus fragrans

“…The p-value was FDR-corrected and, in general, a function with a Qvalue ≤ 0.05 is considered significantly enriched. The ORF of unigene was detected using getorf [42], then aligned to the transcription factor protein domain with hmmsearch [43] (data from TF), and then the unigene was characterized according to the transcription factor family characteristics described by PlantTFDB [44]. According to the existing splicing factor (SF) family information of A. thaliana, we compared the homologous genes of O. fragrans to identify the splicing factor family of O. fragrans.…”