Mallana Gowdra Mallikarjuna

Genomic Selection for Drought Tolerance Using Genome-Wide SNPs in Maize

Traditional breeding strategies for selecting superior genotypes depending on phenotypic traits have proven to be of limited success, as this direct selection is hindered by low heritability, genetic interactions such as epistasis, environmental-genotype interactions, and polygenic effects. With the advent of new genomic tools, breeders have paved a way for selecting superior breeds. Genomic selection (GS) has emerged as one of the most important approaches for predicting genotype performance. Here, we tested the breeding values of 240 maize subtropical lines phenotyped for drought at different environments using 29,619 cured SNPs. Prediction accuracies of seven genomic selection models (ridge regression, LASSO, elastic net, random forest, reproducing kernel Hilbert space, Bayes A and Bayes B) were tested for their agronomic traits. Though prediction accuracies of Bayes B, Bayes A and RKHS were comparable, Bayes B outperformed the other models by predicting highest Pearson correlation coefficient in all three environments. From Bayes B, a set of the top 1053 significant SNPs with higher marker effects was selected across all datasets to validate the genes and QTLs. Out of these 1053 SNPs, 77 SNPs associated with 10 drought-responsive transcription factors. These transcription factors were associated with different physiological and molecular functions (stomatal closure, root development, hormonal signaling and photosynthesis). Of several models, Bayes B has been shown to have the highest level of prediction accuracy for our data sets. Our experiments also highlighted several SNPs based on their performance and relative importance to drought tolerance. The result of our experiments is important for the selection of superior genotypes and candidate genes for breeding drought-tolerant maize hybrids.

Identification, Characterization, and Functional Validation of Drought-responsive MicroRNAs in Subtropical Maize Inbreds

MicroRNA-mediated gene regulation plays a crucial role in controlling drought tolerance. In the present investigation, 13 drought-associated miRNA families consisting of 65 members and regulating 42 unique target mRNAs were identified from drought-associated microarray expression data in maize and were subjected to structural and functional characterization. The largest number of members (14) was found in the zma-miR166 and zma-miR395 families, with several targets. However, zma-miR160, zma-miR390, zma-miR393, and zma-miR2275 each showed a single target. Twenty-three major drought-responsive cis-regulatory elements were found in the upstream regions of miRNAs. Many drought-related transcription factors, such as GAMYB, HD-Zip III, and NAC, were associated with the target mRNAs. Furthermore, two contrasting subtropical maize genotypes (tolerant: HKI-1532 and sensitive: V-372) were used to understand the miRNA-assisted regulation of target mRNA under drought stress. Approximately 35 and 31% of miRNAs were up-regulated in HKI-1532 and V-372, respectively. The up-regulation of target mRNAs was as high as 14.2% in HKI-1532 but was only 2.38% in V-372. The expression patterns of miRNA-target mRNA pairs were classified into four different types: Type I- up-regulation, Type II- down-regulation, Type III- neutral regulation, and Type IV- opposite regulation. HKI-1532 displayed 46 Type I, 13 Type II, and 23 Type III patterns, whereas V-372 had mostly Type IV interactions (151). A low level of negative regulations of miRNA associated with a higher level of mRNA activity in the tolerant genotype helped to maintain crucial biological functions such as ABA signaling, the auxin response pathway, the light-responsive pathway and endosperm expression under stress conditions, thereby leading to drought tolerance. Our study identified candidate miRNAs and mRNAs operating in important pathways under drought stress conditions, and these candidates will be useful in the development of drought-tolerant maize hybrids.

Understanding Genetic and Molecular Bases of Fe and Zn Accumulation Towards Development of Micronutrient-Enriched Maize

Micronutrient malnutrition is a global problem afflicting billions of people worldwide. The effects are more prevalent in developing countries where people rely upon cereal-based diets that are inherently deficient in micronutrients. Micronutrients are required in less quantity but play critical role in the growth and development of humans. Since human body cannot synthesize micronutrients, they must be made available through diet. Among micronutrients, deficiency of iron (Fe) and zinc (Zn) has profound effects and require urgent attention. Development of micronutrient-rich staple plant foods through plant breeding, a process referred to as “biofortification,” holds promise for sustainable food-based solutions to combat micronutrient deficiency. Maize is the third most important crop of the world, serving as staple food to billions of people in sub-Saharan Africa, Latin America and Asia. The development of Fe- and Zn-rich maize cultivar(s) would therefore have positive effects on health and well-being of humans. Wide variability has been reported for Fe and Zn in maize, which can be explored for genetic improvement of the trait. Genetics of Fe and Zn has been well elucidated, and genes/QTLs governing high Fe and Zn accumulation in maize have been identified. Moreover, by targeting the genes involved in Fe and Zn uptake, transportation and translocation, concentration of the same can be increased in the maize endosperm. Further, manipulating genes for promoter and antinutritional factors, bioavailability of Fe and Zn can be enhanced. Quality protein maize (QPM) genotype reported to have higher concentration of Fe and Zn provides opportunity to develop multinutrient-rich maize through a systematic breeding approach. We discussed here available genetic variation for Fe and Zn and their interactions with environments, relationship among micronutrients and grain yield, summary of research efforts with specific emphasis on mechanism of uptake and translocation, genetic and molecular basis of Fe and Zn accumulation, and the strategies that can be explored to breed for high Fe and Zn maize.

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.

Stability Performance of Inductively Coupled Plasma Mass Spectrometry-Phenotyped Kernel Minerals Concentration and Grain Yield in Maize in Different Agro-Climatic Zones.

Deficiency of iron and zinc causes micronutrient malnutrition or hidden hunger, which severely affects ~25% of global population. Genetic biofortification of maize has emerged as cost effective and sustainable approach in addressing malnourishment of iron and zinc deficiency. Therefore, understanding the genetic variation and stability of kernel micronutrients and grain yield of the maize inbreds is a prerequisite in breeding micronutrient-rich high yielding hybrids to alleviate micronutrient malnutrition. We report here, the genetic variability and stability of the kernel micronutrients concentration and grain yield in a set of 50 maize inbred panel selected from the national and the international centres that were raised at six different maize growing regions of India. Phenotyping of kernels using inductively coupled plasma mass spectrometry (ICP-MS) revealed considerable variability for kernel minerals concentration (iron: 18.88 to 47.65 mg kg–1; zinc: 5.41 to 30.85 mg kg–1; manganese: 3.30 to17.73 mg kg–1; copper: 0.53 to 5.48 mg kg–1) and grain yield (826.6 to 5413 kg ha–1). Significant positive correlation was observed between kernel iron and zinc within (r = 0.37 to r = 0.52, p < 0.05) and across locations (r = 0.44, p < 0.01). Variance components of the additive main effects and multiplicative interactions (AMMI) model showed significant genotype and genotype × environment interaction for kernel minerals concentration and grain yield. Most of the variation was contributed by genotype main effect for kernel iron (39.6%), manganese (41.34%) and copper (41.12%), and environment main effects for both kernel zinc (40.5%) and grain yield (37.0%). Genotype main effect plus genotype-by-environment interaction (GGE) biplot identified several mega environments for kernel minerals and grain yield. Comparison of stability parameters revealed AMMI stability value (ASV) as the better representative of the AMMI stability parameters. Dynamic stability parameter GGE distance (GGED) showed strong and positive correlation with both mean kernel concentrations and grain yield. Inbreds (CM-501, SKV-775, HUZM-185) identified from the present investigation will be useful in developing micronutrient-rich as well as stable maize hybrids without compromising grain yield.

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.

Variable Level of Dominance of Candidate Genes Controlling Drought Functional Traits in Maize Hybrids

Breeding maize for drought tolerance necessitates the knowledge on tolerant genotypes, molecular basis of drought tolerance mechanism, action, and expression pattern of genes. Studying the expression pattern and gene action of candidate genes during drought stress in the hybrids will help in choosing target genes for drought tolerance breeding. In the present investigation, a set of five hybrids and their seven parents with a variable level of tolerance to drought stress was selected to study the magnitude and the direction of 52 drought-responsive candidate genes distributed across various biological functions, viz., stomatal regulation, root development, detoxification, hormone signaling, photosynthesis, and sugar metabolism. The tolerant parents, HKI1105 and CML425, and their hybrid, ADWLH2, were physiologically active under drought stress, since vital parameters viz., chlorophyll, root length and relative water content, were on par with the respective well-watered control. All the genes were up-regulated in ADWLH2, many were down-regulated in HM8 and HM9, and most were down-regulated in PMH1 and PMH3 in the shoots and roots. The nature of the gene action was controlled by the parental combination rather than the parent per se. The differentially expressed genes in all five hybrids explained a mostly non-additive gene action over additivity, which was skewed toward any of the parental lines. Tissue-specific gene action was also noticed in many of the genes. The non-additive gene action is driven by genetic diversity, allele polymorphism, events during gene regulation, and small RNAs under the stress condition. Differential regulation and cross-talk of genes controlling various biological functions explained the basis of drought tolerance in subtropical maize hybrids. The nature of the gene action and the direction of the expression play crucial roles in designing introgression and hybrid breeding programmes to breed drought tolerant maize hybrids.

Genetic variability and correlation of kernel micronutrients among exotic quality protein maize inbreds and their utility in breeding programme

Micronutrient malnutrition is a widespread problem known to affect millions of children and women. However, the adverse effects of micronutrient deficiency can be overcome through self-targeting, cost-effective and sustainable genetic biofortification approach, which is mainly based on staple food crops. Since maize has emerged as a prominent future crop especially for India, developing maize hybrids that are rich in kernel micronutrients will help in reducing the problems of micronutrient malnutrition. Here, we report variability of kernel Fe and Zn in 120 exotic Quality Protein Maize (QPM) inbreds and kernel Mn and Cu in a representative subset of 68 lines. A wide range of genetic variation was found among genotypes for all the kernel micronutrients: Fe (16.6–83.4 ppm), Zn (16.4–53.2 ppm), Mn (1.7–34.8 ppm) and Cu (0.5–9.5 ppm). Higher mean for kernel Fe and Zn in QPM inbreds suggests possible influence of opaque2 gene and/or modifiers present in them. Significant and positive correlation was observed among kernel Fe, Zn, Mn and Cu. Genetic diversity based on all four micronutrients grouped 68 lines into three major clusters with a mean coefficient of genetic dissimilarity of 2.46. Parental combinations were selected from different heterotic pools and maturity groups to develop high-yielding hybrids enriched with micronutrients.

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.

Genetics and Applied Genomics of Quality Protein Maize for Food and Nutritional Security

Maize (Zea mays L.) is an important food and feed crop of the world. Together with rice and wheat, it provides around 40% of the food calories to more than 4.5 billion people in 94 developing countries. It also provides nearly 50% of the dietary protein for humans. In Africa and some of the Asian countries, almost 90% of maize grown is for human consumption and may account for 80–90% of the energy intake. In India, it is the third most important food crop after rice and wheat, both in terms of area and production. India is the fifth largest producer of maize in the world contributing 3% of the total global production. Protein malnutrition is widespread in the developing and underdeveloped countries, where 780 million people are affected by the same. Maize is the leading cereal in terms of production and accounts for 15% of proteins and 20% of calories requirement of the world. Protein malnutrition is caused by lack of access to adequate quantity and better quality protein intake and usually affects children and elderly persons. Maize, however, lacks adequate amounts of the essential amino acids, namely, lysine and tryptophan. Decades of efforts by maize researchers lead to the development of nutritionally superior maize cultivar popularly called as quality protein maize (QPM), which has twice the amount of lysine and tryptophan, thus making its quality as good as casein of milk. The o2 allele along with modifiers for tryptophan and lysine content and grain hardness made QPM agronomically suitable for cultivations. Intensive efforts were made by many workers to understand the genetics, molecular mechanism of QPM modifiers and applied these genomics knowledge to developed MAS-based QPM inbreds and commercial hybrids. All those studies and concerted efforts led to development and utilization of QPM. The area under QPM globally is more than 9.0 million hectares. Several reports were available on positive impact of QPM on children and adults. It has also been demonstrated in poultry and piggery, resulting in increased egg production and egg quality parameters and body mass. The area under QPM and consumption of QPM can be increased significantly by providing policy supports for QPM.