Exploring the sorghum race level diversity utilizing 272 sorghum accessions genomic resources.

Due to evolutionary divergence, sorghum race populations exhibit significant genetic and morphological variation. A k-mer-based sorghum race sequence comparison identified the conserved k-mers of all 272 accessions from sorghum and the race-specific genetic signatures identified the gene variability in 10,321 genes (PAVs). To understand sorghum race structure, diversity and domestication, a deep learning-based variant calling approach was employed in a set of genotypic data derived from a diverse panel of 272 sorghum accessions. The data resulted in 1.7 million high-quality genome-wide SNPs and identified selective signature (both positive and negative) regions through a genome-wide scan with different (iHS and XP-EHH) statistical methods. We discovered 2,370 genes associated with selection signatures including 179 selective sweep regions distributed over 10 chromosomes. Co-localization of these regions undergoing selective pressure with previously reported QTLs and genes revealed that the signatures of selection could be related to the domestication of important agronomic traits such as biomass and plant height. The developed k-mer signatures will be useful in the future to identify the sorghum race and for trait and SNP markers for assisting in plant breeding programs.

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.

Genetic diversity and population structure in onion (Allium cepa L.) accessions based on morphological and molecular approaches.

Bulb onion is cultivated throughout the world for consumption as vegetable and processed products. Although having high global demand and economic significance, information about genetic diversity and genomic resources is limited. This study investigated the variability of 96 accessions representing seventeen countries. Out of 145 SSR markers, 62 SSRs amplified and 15 SSRs gave consistent polymorphic bands. Fifty three alleles were detected with an average of 3.533 alleles per locus. PIC value ranged from 0.219 (ACM463) to 0.715 (ACM091). Structure and cluster analysis grouped the onion accessions into two clusters. Discriminant analysis of principal components, a tool that maximizes variation between groups while minimizing that within groups, assorted accessions into five clusters. Analysis of molecular variance revealed maximum variation within the populations than among the populations. Highest genetic differentiation (FST = 0.11045; p < 0.001) was observed between Europe and Japan populations whereas the lowest genetic differentiation (FST = 0.05714; p < 0.001) was recorded between India and Japan. Principal component analysis of morphological traits suggested two principal components cumulatively accounting for 74.4% of the total variance. First component (PC1) was positively and strongly correlated with bulbing whereas second component (PC2) had leaf colour with the highest coefficient. Clustering was not on the basis of bulb colour, bulb formation, or flowering but on the basis of geographical origin. Based on clustering, crossing of distantly related accessions can provide an insight about the hybrid vigour of these diverse accessions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01101-3.

Sorghum Pan-Genome Explores the Functional Utility for Genomic-Assisted Breeding to Accelerate the Genetic Gain.

Sorghum (Sorghum bicolor L.) is a staple food crops in the arid and rainfed production ecologies. Sorghum plays a critical role in resilient farming and is projected as a smart crop to overcome the food and nutritional insecurity in the developing world. The development and characterisation of the sorghum pan-genome will provide insight into genome diversity and functionality, supporting sorghum improvement. We built a sorghum pan-genome using reference genomes as well as 354 genetically diverse sorghum accessions belonging to different races. We explored the structural and functional characteristics of the pan-genome and explain its utility in supporting genetic gain. The newly-developed pan-genome has a total of 35,719 genes, a core genome of 16,821 genes and an average of 32,795 genes in each cultivar. The variable genes are enriched with environment responsive genes and classify the sorghum accessions according to their race. We show that 53% of genes display presence-absence variation, and some of these variable genes are predicted to be functionally associated with drought adaptation traits. Using more than two million SNPs from the pan-genome, association analysis identified 398 SNPs significantly associated with important agronomic traits, of which, 92 were in genes. Drought gene expression analysis identified 1,788 genes that are functionally linked to different conditions, of which 79 were absent from the reference genome assembly. This study provides comprehensive genomic diversity resources in sorghum which can be used in genome assisted crop improvement.

Comparative Transcriptome Analysis of Iron and Zinc Deficiency in Maize (Zea mays L.).

Globally, one-third of the population is affected by iron (Fe) and zinc (Zn) deficiency, which is severe in developing and underdeveloped countries where cereal-based diets predominate. The genetic biofortification approach is the most sustainable and one of the cost-effective ways to address Fe and Zn malnutrition. Maize is a major source of nutrition in sub-Saharan Africa, South Asia and Latin America. Understanding systems' biology and the identification of genes involved in Fe and Zn homeostasis facilitate the development of Fe- and Zn-enriched maize. We conducted a genome-wide transcriptome assay in maize inbred SKV616, under -Zn, -Fe and -Fe-Zn stresses. The results revealed the differential expression of several genes related to the mugineic acid pathway, metal transporters, photosynthesis, phytohormone and carbohydrate metabolism. We report here Fe and Zn deficiency-mediated changes in the transcriptome, root length, stomatal conductance, transpiration rate and reduced rate of photosynthesis. Furthermore, the presence of multiple regulatory elements and/or the co-factor nature of Fe and Zn in enzymes indicate their association with the differential expression and opposite regulation of several key gene(s). The differentially expressed candidate genes in the present investigation would help in breeding for Fe and Zn efficient and kernel Fe- and Zn-rich maize cultivars through gene editing, transgenics and molecular breeding.

Structural and Functional Characteristics of miRNAs in Five Strategic Millet Species and Their Utility in Drought Tolerance.

Millets are the strategic food crops in arid and drought-prone ecologies. Millets, by virtue of nature, are very well-adapted to drought conditions and able to produce sustainable yield. Millets have important nutrients that can help prevent micro-nutrient malnutrition. As a result of the adverse effect of climate change and widespread malnutrition, millets have attained a strategic position to sustain food and nutritional security. Although millets can adapt well to the drought ecologies where other cereals fail completely, the yield level is very low under stress. There is a tremendous opportunity to increase the genetic potential of millet crops in dry lands when the genetics of the drought-tolerance mechanism is fully explained. MicroRNAs (miRNAs) are the class of small RNAs that control trait expression. They are part of the gene regulation but little studied in millets. In the present study, novel miRNAs and gene targets were identified from the genomic resources of pearl millet, sorghum, foxtail millet, finger millet, and proso millet through in silico approaches. A total of 1,002 miRNAs from 280 families regulating 23,158 targets were identified using different filtration criteria in five millet species. The unique as well as conserved structural features and functional characteristics of miRNA across millets were explained. About 84 miRNAs were conserved across millets in different species combinations, which explained the evolutionary relationship of the millets. Further, 215 miRNAs controlling 155 unique major drought-responsive genes, transcription factors, and protein families revealed the genetics of drought tolerance that are accumulated in the millet genomes. The miRNAs regulating the drought stress through specific targets or multiple targets showed through a network analysis. The identified genes regulated by miRNA genes could be useful in developing functional markers and used for yield improvement under drought in millets as well as in other crops.

Enhancing ß-Carotene Concentration in Parental Lines of CO6 Maize Hybrid Through Marker-Assisted Backcross Breeding (MABB).

Vitamin A deficiency (VAD) is a global health problem; many people around the world, especially children and pregnant women, are VAD deficient or insufficient. Maize is known as an important source of provitamin A for humans. Hence, enhancement of provitamin A carotenoids (pVAC) in maize varieties through breeding or biofortification is a good option for alleviating VAD in developing countries, especially India. So far, numerous maize hybrids have been developed in India. Among them, CO6, derived from UMI1200 × UMI1230, is a popular maize hybrid and adapted to different agro-climatic zones of India, especially Tamil Nadu, a southern state of India. However, CO6 is deficient for pVAC carotenoid ß-carotene. Thus, the objectives of this study were to increase the ß-carotene concentration in UMI1200 and UMI1230 and generate the ß-carotene enriched hybrids through marker-assisted backcross breeding (MABB). For this purpose, the maize genotype HP467-15 was used as the donor for transferring the ß-carotene gene, crtRB1, into UMI1200 and UMI1230. In the MABB scheme, we used one gene-specific marker (crtRB1 3'TE) and 214 simples sequence repeat (SSR) markers for foreground and background selection, respectively. As a result, six improved lines with recurrent parent genome recovery (RPGR) ranging from 90.24 to 92.42%, along with good agronomic performance, were generated. The ß-carotene concentration of the improved lines ranged from 7.056 to 9.232 µg/g. Furthermore, five hybrid combinations were generated using improved lines and evaluated in a comparative yield trial (CYT) and multi-location trials (MLT) along with the original hybrid CO6 and commercial hybrids. It was revealed that ACM-M13-002 was a superior hybrid with a 7.3-fold increase in ß-carotene concentration and with a comparable yield to CO6. In summary, the improved maize inbreds can be used as possible donors for the development of ß-carotene-rich cultivars in maize breeding programs and the ß-carotene enriched hybrid developed in this study will hold great promise for food and nutritional security.

Multiple Genome Wide Association Mapping Models Identify Quantitative Trait Nucleotides for Brown Planthopper (Nilaparvata lugens) Resistance in MAGIC Indica Population of Rice.

Brown planthopper (BPH), one of the most important pests of the rice (Oryza sativa) crop, becomes catastrophic under severe infestations and causes up to 60% yield loss. The highly disastrous BPH biotype in the Indian sub-continent is Biotype 4, which also known as the South Asian Biotype. Though many resistance genes were mapped until now, the utility of the resistance genes in the breeding programs is limited due to the breakdown of resistance and emergence of new biotypes. Hence, to identify the resistance genes for this economically important pest, we have used a multi-parent advanced generation intercross (MAGIC) panel consisting of 391 lines developed from eight indica founder parents. The panel was phenotyped at the controlled conditions for two consecutive years. A set of 27,041 cured polymorphic single nucleotide polymorphism (SNPs) and across-year phenotypic data were used for the identification of marker-trait associations. Genome-wide association analysis was performed to find out consistent associations by employing four single and two multi-locus models. Sixty-one SNPs were consistently detected by all six models. A set of 190 significant marker-associations identified by fixed and random model circulating probability unification (FarmCPU) were considered for searching resistance candidate genes. The highest number of annotated genes were found in chromosome 6 followed by 5 and 1. Ninety-two annotated genes identified across chromosomes of which 13 genes are associated BPH resistance including NB-ARC (nucleotide binding in APAF-1, R gene products, and CED-4) domain-containing protein, NHL repeat-containing protein, LRR containing protein, and WRKY70. The significant SNPs and resistant lines identified from our study could be used for an accelerated breeding program to develop new BPH resistant cultivars.

Structural, Functional, and Evolutionary Characterization of Major Drought Transcription Factors Families in Maize.

Drought is one of the major threats to the maize yield especially in subtropical production systems. Understanding the genes and regulatory mechanisms of drought tolerance is important to sustain the yield. Transcription factors (TFs) play a major role in gene regulation under drought stress. In the present study, a set of 15 major TF families comprising 1,436 genes was structurally and functionally characterized. The functional annotation indicated that the genes were involved in ABA signaling, ROS scavenging, photosynthesis, stomatal regulation, and sucrose metabolism. Duplication was identified as the primary force in divergence and expansion of TF families. Phylogenetic relationship was developed for individual TF and combined TF families. Phylogenetic analysis clustered the genes into specific and mixed groups. Gene structure analysis revealed that more number of genes were intron-rich as compared to intron-less. Drought-responsive cis-regulatory elements such as ABREA, ABREB, DRE1, and DRECRTCOREAT have been identified. Expression and interaction analyses identified leaf-specific bZIP TF, GRMZM2G140355, as a potential contributor toward drought tolerance in maize. Protein-protein interaction network of 269 drought-responsive genes belonging to different TFs has been provided. The information generated on structural and functional characteristics, expression, and interaction of the drought-related TF families will be useful to decipher the drought tolerance mechanisms and to breed drought-tolerant genotypes in maize.

Marker-assisted introgression of opaque2 allele for rapid conversion of elite hybrids into quality protein maize.

Maize is a valuable source of food and feed worldwide. Maize endosperm protein is, however nutritionally poor due to the reduced levels of two essential amino acids, lysine and tryptophan. In this study, recessive opaque2 (o2) allele that confers enhanced endosperm lysine and tryptophan, was introgressed using marker-assisted backcross breeding into three normal inbred lines (HKI323, HKI1105 and HKI1128). These are the parental lines of three popular medium-maturing single cross hybrids (HM4, HM8 and HM9) in India. Gene-based simple sequence repeat (SSR) markers (umc1066 and phi057) were successfully deployed for introgression of o2 allele. Background selection using genome-based SSRs helped in recovering > 96% of recurrent parent genome. The newly developed quality protein maize (QPM) inbreds showed modified kernels (25-50% opaqueness) coupled with high degree of phenotypic resemblance to the respective recipient lines, including grain yield. In addition, endosperm protein quality showed increased lysine and tryptophan in the inbreds to the range of 52-95% and 47-118%, respectively. The reconstituted QPM hybrids recorded significant enhancement of endosperm lysine (48-74%) and tryptophan (55-100%) in the endosperm. The QPM hybrids exhibited high phenotypic similarity with the original hybrids for morphological and yield contributing traits along with responses to some major diseases like turcicum leaf blight and maydis leaf blight. The grain yield of QPM hybrids was at par with their original versions under multilocation testing. These elite, high-yielding QPM hybrids with improved protein quality have been released and notified for commercial cultivation, and hold significant promise for improving nutritional security.

Development of Biofortified Maize Hybrids through Marker-Assisted Stacking of ß-Carotene Hydroxylase, Lycopene-ε-Cyclase and Opaque2 Genes.

Traditional yellow maize though contains high kernel carotenoids, the concentration of provitamin A (proA) is quite low (<2 µg/g), compared to recommended level (15 µg/g). It also possesses poor endosperm protein quality due to low concentration of lysine and tryptophan. Natural variant of crtRB1 (ß-carotene hydroxylase) and lcyE (lycopene-ε-cyclase) cause significant enhancement of proA concentration, while recessive allele, opaque2 (o2) enhances the level of these amino acids. Development of biofortified maize enriched in proA, lysine and tryptophan thus holds significance in alleviation of micronutrient malnutrition. In the present study, marker-assisted stacking of crtRB1, lcyE and o2 was undertaken in the genetic background of four maize hybrids (HQPM1, HQPM4, HQPM5, and HQPM7) popularly grown in India. HP704-22 and HP704-23 were used as donors, while four elite QPM parents viz., HKI161, HKI163, HKI193-1, and HKI193-2 were used as recipients. CrtRB1 showed severe segregation distortion, while lcyE segregated as per the expectation. Recovery of recurrent parent genome (RPG) among selected backcross progenies ranged from 89 to 93%. Introgressed progenies possessed high concentration of proA (7.38-13.59 µg/g), compared to 1.65-2.04 µg/g in the recurrent parents. The reconstituted hybrids showed an average of 4.5-fold increase in proA with a range of 9.25-12.88 µg/g, compared to original hybrids (2.14-2.48 µg/g). Similar plant-, ear-, and grain- characteristics of improved versions of both inbreds and hybrids were observed when evaluated with their respective original versions. Mean lysine (0.334%) and tryptophan (0.080%) of the improved hybrids were at par with the original versions (lysine: 0.340%, tryptophan: 0.083%). Improved hybrids also possessed similar grain yield potential (6,301-8,545 kg/ha) with their original versions (6,135-8,479 kg/ha) evaluated at two locations. This is the first study of staking crtRB1-, lcyE-, and o2-, favorable alleles in single genetic background. The improved inbreds can be effectively used as potential donor for independent and/or simultaneous introgression of crtRB1, lcyE, and o2 in the future breeding programme. These biofortified maize hybrids, rich in proA, lysine and tryptophan will hold great promise for nutritional security.

Opaque16, a high lysine and tryptophan mutant, does not influence the key physico-biochemical characteristics in maize kernel.

The enhancement of lysine and tryptophan in maize is so far basedon opaque2(o2) mutant, that along with the endosperm-modifiersled to development of Quality Protein Maize[QPM]. Though many mutants improving the endospermic protein quality were discovered, they could not be successfully deployed. Recently discovered opaque16 (o16)mutant enhances the lysine and tryptophan content in maize endosperm. In the present study, the influence of o16 on the endosperm modification was analyzed in four F2 populations, two each segregating for o16 allele alone and in combination with o2. The recessive o16o16 seed endosperm was found to be vitreousphenotypically similar to wild-O16O16. The mutant did not influence the degree of kernel opaqueness in o2o2 genetic background as opaqueness in o2o2/O16O16 and o2o2/o16o16 was similar. Grain hardness of o16o16 was comparable with the normal and QPM maize. The pattern of microscopic organization of proteinaceous matrix and starch granules, and zein profiling of the storage protein in o16o16 were found to be similar with normal maize endosperm, but distinct from the o2o2-soft genotype. The pattern in o2o2/o16o16 was unique and different from o2o2 and o16o16 as well. Here we demonstrated the effects of o16 on physico-biochemical characteristics of endosperm and report of o16 possessing negligible influence on kernel modification and hardness, which holds a great significance in maize quality breeding programme.

Association mapping unveils favorable alleles for grain iron and zinc concentrations in lentil (Lens culinaris subsp. culinaris).

Lentil is a major cool-season grain legume grown in South Asia, West Asia, and North Africa. Populations in developing countries of these regions have micronutrient deficiencies; therefore, breeding programs should focus more on improving the micronutrient content of food. In the present study, a set of 96 diverse germplasm lines were evaluated at three different locations in India to examine the variation in iron (Fe) and zinc (Zn) concentration and identify simple sequence repeat (SSR) markers that associate with the genetic variation. The genetic variation among genotypes of the association mapping (AM) panel was characterized using a genetic distance-based and a general model-based clustering method. The model-based analysis identified six subpopulations, which satisfactorily explained the genetic structure of the AM panel. AM analysis identified three SSRs (PBALC 13, PBALC 206, and GLLC 563) associated with grain Fe concentration explaining 9% to 11% of phenotypic variation and four SSRs (PBALC 353, SSR 317-1, PLC 62, and PBALC 217) were associated with grain Zn concentration explaining 14%, to 21% of phenotypic variation. These identified SSRs exhibited consistent performance across locations. These candidate SSRs can be used in marker-assisted genetic improvement for developing Fe and Zn fortified lentil varieties. Favorable alleles and promising genotypes identified in this study can be utilized for lentil biofortification.

RNAseq revealed the important gene pathways controlling adaptive mechanisms under waterlogged stress in maize.

Waterlogging causes yield penalty in maize-growing countries of subtropical regions. Transcriptome analysis of the roots of a tolerant inbred HKI1105 using RNA sequencing revealed 21,364 differentially expressed genes (DEGs) under waterlogged stress condition. These 21,364 DEGs are known to regulate important pathways including energy-production, programmed cell death (PCD), aerenchyma formation, and ethylene responsiveness. High up-regulation of invertase (49-fold) and hexokinase (36-fold) in roots explained the ATP requirement in waterlogging condition. Also, high up-regulation of expansins (42-fold), plant aspartic protease A3 (19-fold), polygalacturonases (16-fold), respiratory burst oxidase homolog (12-fold), and hydrolases (11-fold) explained the PCD of root cortical cells followed by the formation of aerenchyma tissue during waterlogging stress. We hypothesized that the oxygen transfer in waterlogged roots is promoted by a cross-talk of fermentative, metabolic, and glycolytic pathways that generate ATPs for PCD and aerenchyma formation in root cortical cells. SNPs were mapped to the DEGs regulating aerenchyma formation (12), ethylene-responsive factors (11), and glycolysis (4) under stress. RNAseq derived SNPs can be used in selection approaches to breed tolerant hybrids. Overall, this investigation provided significant evidence of genes operating in the adaptive traits such as ethylene production and aerenchyma formation to cope-up the waterlogging stress.

Genomewide Expression and Functional Interactions of Genes under Drought Stress in Maize.

A genomewide transcriptome assay of two subtropical genotypes of maize was used to observe the expression of genes at seedling stage of drought stress. The number of genes expressed differentially was greater in HKI1532 (a drought tolerant genotype) than in PC3 (a drought sensitive genotype), indicating primary differences at the transcriptional level in stress tolerance. The global coexpression networks of the two genotypes differed significantly with respect to the number of modules and the coexpression pattern within the modules. A total of 174 drought-responsive genes were selected from HKI1532, and their coexpression network revealed key correlations between different adaptive pathways, each cluster of the network representing a specific biological function. Transcription factors related to ABA-dependent stomatal closure, signalling, and phosphoprotein cascades work in concert to compensate for reduced photosynthesis. Under stress, water balance was maintained by coexpression of the genes involved in osmotic adjustments and transporter proteins. Metabolism was maintained by the coexpression of genes involved in cell wall modification and protein and lipid metabolism. The interaction of genes involved in crucial biological functions during stress was identified and the results will be useful in targeting important gene interactions to understand drought tolerance in greater detail.

In Silico Characterization and Functional Validation of Cell Wall Modification Genes Imparting Waterlogging Tolerance in Maize.

Cell wall modification (CWM) promotes the formation of aerenchyma in roots under waterlogging conditions as an adaptive mechanism. Lysigenous aerenchyma formation in roots improves oxygen transfer in plants, which highlights the importance of CWM as a focal point in waterlogging stress tolerance. We investigated the structural and functional compositions of CWM genes and their expression patterns under waterlogging conditions in maize. Cell wall modification genes were identified for 3 known waterlogging-responsive cis-acting regulatory elements, namely, GC motif, anaerobic response elements, and G-box, and 2 unnamed elements. Structural motifs mapped in CWM genes were represented in genes regulating waterlogging stress-tolerant pathways, including fermentation, glycolysis, programmed cell death, and reactive oxygen species signaling. The highly aligned regions of characterized and uncharacterized CWM proteins revealed common structural domains amongst them. Membrane spanning regions present in the protein structures revealed transmembrane activity of CWM proteins in the plant cell wall. Cell wall modification proteins had interacted with ethylene-responsive pathway regulating genes (E3 ubiquitin ligases RNG finger and F-box) in a maize protein-protein interaction network. Cell wall modification genes had also coexpressed with energy metabolism, programmed cell death, and reactive oxygen species signaling, regulating genes in a single coexpression cluster. These configurations of CWM genes can be used to modify the protein expression in maize under waterlogging stress condition. Our study established the importance of CWM genes in waterlogging tolerance, and these genes can be used as candidates in introgression breeding and genome editing experiments to impart tolerance in maize hybrids.

Comparative Analysis of CDPK Family in Maize, Arabidopsis, Rice, and Sorghum Revealed Potential Targets for Drought Tolerance Improvement.

Calcium dependent protein kinases (CDPKs) play significant role in regulation of plant growth and development in response to various stresses including drought. A set of 32 CDPK genes identified in maize were further used for searching of orthologs in the model plant Arabidopsis (72) and major food crops such as rice (78) and sorghum (91). We comprehensively studied the phylogenetic relationship, annotations, gene duplications, gene structure, divergence time, 3-D protein structures and tissue-specific drought induced expression of CDPK genes in all four species. Variation in intron frequency in the studied species was one of the reasons for the functional diversity of CDPK genes to various stress responses. Protein kinase and protein kinase C phosphorylation site domains were the most conserved motifs identified in all species. Four groups were identified from the sequence-based phylogenetic analysis, in which maize CDPKs were clustered in group III. Expression data showed that the CDPK genes were highly expressed in leaf of maize, rice, and sorghum whereas in Arabidopsis the maximum expression was observed in root. The expression assay showed 5, 6, 11, and 9 were the commonly and differentially expressed drought-related orthologous genes in maize, Arabidopsis, rice, and sorghum, respectively. 3-D protein structure were predicted for the nine genes (Arabidopsis: 2, maize: 2, rice: 3, and sorghum: 2) showing differential expression in at least three species. The predicted 3-D structures were further evaluated and validated by Ramachandran plot, ANOLEA, ProSA, and Verify-3D. The superimposed 3-D structure of drought-related orthologous proteins retained similar folding pattern owing to their conserved nature. Functional annotation revealed the involvement of CDPK genes in various pathways such as osmotic homeostasis, cell protection, and root growth. The interactions of CDPK genes in various pathways play crucial role in imparting drought tolerance through different ABA and MAPK signaling cascades. These selected candidate genes could be targeted in development of drought tolerant genotypes in maize, rice, and sorghum through appropriate breeding approaches. Our comparative experiments of CDPK genes could also be extended in the drought stress breeding programmes of the related species.

Mapping Quantitative Trait Loci Controlling High Iron and Zinc Content in Self and Open Pollinated Grains of Pearl Millet [Pennisetum glaucum (L.) R. Br.].

Pearl millet is a multipurpose grain/fodder crop of the semi-arid tropics, feeding many of the world's poorest and most undernourished people. Genetic variation among adapted pearl millet inbreds and hybrids suggests it will be possible to improve grain micronutrient concentrations by selective breeding. Using 305 loci, a linkage map was constructed to map QTLs for grain iron [Fe] and zinc [Zn] using replicated samples of 106 pearl millet RILs (F6) derived from ICMB 841-P3 × 863B-P2. The grains of the RIL population were evaluated for Fe and Zn content using atomic absorption spectrophotometer. Grain mineral concentrations ranged from 28.4 to 124.0 ppm for Fe and 28.7 to 119.8 ppm for Zn. Similarly, grain Fe and Zn in open pollinated seeds ranged between 22.4-77.4 and 21.9-73.7 ppm, respectively. Mapping with 305 (96 SSRs; 208 DArT) markers detected seven linkage groups covering 1749 cM (Haldane) with an average intermarker distance of 5.73 cM. On the basis of two environment phenotypic data, two co-localized QTLs for Fe and Zn content on linkage group (LG) 3 were identified by composite interval mapping (CIM). Fe QTL explained 19% phenotypic variation, whereas the Zn QTL explained 36% phenotypic variation. Likewise for open pollinated seeds, the QTL analysis led to the identification of two QTLs for grain Fe content on LG3 and 5, and two QTLs for grain Zn content on LG3 and 7. The total phenotypic variance for Fe and Zn QTLs in open pollinated seeds was 16 and 42%, respectively. Analysis of QTL × QTL and QTL × QTL × environment interactions indicated no major epistasis.