Identification of iron and zinc responsive genes in pearl millet using genome-wide RNA-sequencing approach.

Pearl millet (Pennisetum glaucum L.), an important source of iron (Fe) and zinc (Zn) for millions of families in dryland tropics, helps in eradicating micronutrient malnutrition. The crop is rich in Fe and Zn, therefore, identification of the key genes operating the mineral pathways is an important step to accelerate the development of biofortified cultivars. In a first-of-its-kind experiment, leaf and root samples of a pearl millet inbred ICMB 1505 were exposed to combinations of Fe and Zn stress conditions using the hydroponics method, and a whole-genome transcriptome assay was carried out to characterize the differentially expressed genes (DEGs) and pathways. A total of 37,093 DEGs under different combinations of stress conditions were identified, of which, 7,023 and 9,996 DEGs were reported in the leaf and root stress treatments, respectively. Among the 10,194 unique DEGs, 8,605 were annotated to cellular, biological, and molecular functions and 458 DEGs were assigned to 39 pathways. The results revealed the expression of major genes related to the mugineic acid pathway, phytohormones, chlorophyll biosynthesis, photosynthesis, and carbohydrate metabolism during Fe and Zn stress. The cross-talks between the Fe and Zn provided information on their dual and opposite regulation of key uptake and transporter genes under Fe and Zn deficiency. SNP haplotypes in rice, maize, sorghum, and foxtail millet as well as in Arabidopsis using pearl millet Fe and Zn responsive genes could be used for designing the markers in staple crops. Our results will assist in developing Fe and Zn-efficient pearl millet varieties in biofortification breeding programs and precision delivery mechanisms to ameliorate malnutrition in South Asia and Sub-Saharan Africa.

Identification of Candidate Genes Regulating Drought Tolerance in Pearl Millet.

Pearl millet is an important crop of the arid and semi-arid ecologies to sustain food and fodder production. The greater tolerance to drought stress attracts us to examine its cellular and molecular mechanisms via functional genomics approaches to augment the grain yield. Here, we studied the drought response of 48 inbreds representing four different maturity groups at the flowering stage. A set of 74 drought-responsive genes were separated into five major phylogenic groups belonging to eight functional groups, namely ABA signaling, hormone signaling, ion and osmotic homeostasis, TF-mediated regulation, molecular adaptation, signal transduction, physiological adaptation, detoxification, which were comprehensively studied. Among the conserved motifs of the drought-responsive genes, the protein kinases and MYB domain proteins were the most conserved ones. Comparative in-silico analysis of the drought genes across millet crops showed foxtail millet had most orthologs with pearl millet. Of 698 haplotypes identified across millet crops, MyC2 and Myb4 had maximum haplotypes. The protein-protein interaction network identified ABI2, P5CS, CDPK, DREB, MYB, and CYP707A3 as major hub genes. The expression assay showed the presence of common as well as unique drought-responsive genes across maturity groups. Drought tolerant genotypes in respective maturity groups were identified from the expression pattern of genes. Among several gene families, ABA signaling, TFs, and signaling proteins were the prospective contributors to drought tolerance across maturity groups. The functionally validated genes could be used as promising candidates in backcross breeding, genomic selection, and gene-editing schemes in pearl millet and other millet crops to increase the yield in drought-prone arid and semi-arid ecologies.

Development of a new AgriSeq 4K mid-density SNP genotyping panel and its utility in pearl millet breeding.

Pearl millet is a crucial nutrient-rich staple food in Asia and Africa and adapted to the climate of semi-arid topics. Since the genomic resources in pearl millet are very limited, we have developed a brand-new mid-density 4K SNP panel and demonstrated its utility in genetic studies. A set of 4K SNPs were mined from 925 whole-genome sequences through a comprehensive in-silico pipeline. Three hundred and seventy-three genetically diverse pearl millet inbreds were genotyped using the newly-developed 4K SNPs through the AgriSeq Targeted Genotyping by Sequencing technology. The 4K SNPs were uniformly distributed across the pearl millet genome and showed considerable polymorphism information content (0.23), genetic diversity (0.29), expected heterozygosity (0.29), and observed heterozygosity (0.03). The SNP panel successfully differentiated the accessions into two major groups, namely B and R lines, through genetic diversity, PCA, and structure models as per their pedigree. The linkage disequilibrium (LD) analysis showed Chr3 had higher LD regions while Chr1 and Chr2 had more low LD regions. The genetic divergence between the B- and R-line populations was 13%, and within the sub-population variability was 87%. In this experiment, we have mined 4K SNPs and optimized the genotyping protocol through AgriSeq technology for routine use, which is cost-effective, fast, and highly reproducible. The newly developed 4K mid-density SNP panel will be useful in genomics and molecular breeding experiments such as assessing the genetic diversity, trait mapping, backcross breeding, and genomic selection in pearl millet.