Mapping grain iron and zinc content QTLs in an Iniadi-derived immortal population of pearl millet

Pearl millet is a climate-resilient nutritious crop requiring low inputs, and is capable of giving economic returns in marginal agro-ecologies. In this study, we report large effect iron (Fe) and zinc (Zn) content QTLs using DArT arrays and SSRs to generate a genetic linkage map using 317 RIL population derived from (ICMS 8511-S1-17-2-1-1-B-P03 ? AIMP 92901-S1-183-2-2-B-08) cross. The base map (7 LGs) of 196 loci was 964.2 cM (Haldane). AIMP 92901-S1-183-2-2-B-08 is a high grain Fe and Zn line, an Iniadi parent tracing its origin to the Togolese Republic, West Africa. QTL analysis revealed a large number of QTLs for grain iron (Fe) and zinc (Zn) content. The concentration of grain Fe in the RIL population ranged between 20 and 131 ppm, and Zn from 18-110 ppm. A total of 19 QTLs for Fe and Zn were detected, of which 11 were for Fe and 8 were for Zn. The portion of observed phenotypic variance explained by different QTLs for grain Fe and Zn concentrations varied between 9.0-31.9% (cumulative 74%) and 9.4-30.4% (cumulative 65%), respectively. Three large effect QTLs for both minerals were co-mapped in this population - one on Linkage group (LG) 1 and the remaining two on LG7. The favourable alleles for QTLs of both the mineral micronutrients were contributed by the male parent (AIMP 92901-deriv-08). Three putative epistasis interactions were observed for Fe while single digenic interaction was for Zn. The reported QTLs may be useful in marker-assisted selection programs for seed and restorer parent breeding and population improvement programs in pearl millet.authorsversionPeer reviewe

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

Novel SSR Markers from BAC-End Sequences, DArT Arrays and a Comprehensive Genetic Map with 1,291 Marker Loci for Chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes

Genomic-based-breeding tools for tropical maize improvement

Maize has traditionally been the main staple diet in the Southern Asia and Sub-Saharan Africa and widely grown by millions of resource poor small scale farmers. Approximately, 35.4 million hectares are sown to tropical maize, constituting around 59% of the developing worlds. Tropical maize encounters tremendous challenges besides poor agro-climatic situations with average yields recorded <3 tones/hectare that is far less than the average of developed countries. On the contrary to poor yields, the demand for maize as food, feed, and fuel is continuously increasing in these regions. Heterosis breeding introduced in early 90 s improved maize yields significantly, but genetic gains is still a mirage, particularly for crop growing under marginal environments. Application of molecular markers has accelerated the pace of maize breeding to some extent. The availability of array of sequencing and genotyping technologies offers unrivalled service to improve precision in maize-breeding programs through modern approaches such as genomic selection, genome-wide association studies, bulk segregant analysis-based sequencing approaches, etc. Superior alleles underlying complex traits can easily be identified and introgressed efficiently using these sequence-based approaches. Integration of genomic tools and techniques with advanced genetic resources such as nested association mapping and backcross nested association mapping could certainly address the genetic issues in maize improvement programs in developing countries. Huge diversity in tropical maize and its inherent capacity for doubled haploid technology offers advantage to apply the next generation genomic tools for accelerating production in marginal environments of tropical and subtropical world. Precision in phenotyping is the key for success of any molecular-breeding approach. This article reviews genomic technologies and their application to improve agronomic traits in tropical maize breeding has been reviewed in 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

Allelic variations for lycopene-ε-cyclase and β-carotene hydroxylase genes in maize inbreds and their utilization in β-carotene enrichment programme

Vitamin A deficiency is a global health problem and can be effectively alleviated through crop biofortification. Quantification of carotenoids using high-performance liquid chromatography is expensive and time-consuming, thereby posing a challenge in the selection of genotypes with high provitamin A. Favourable alleles possessing rare genetic variation in lycopene-ε-cyclase (lcyE) and β-carotene hydroxylase (crtRB1) genes are associated with higher accumulation of provitamin A, especially β-carotene; and selection of these alleles holds immense promise in reducing large-scale phenotypic assays. Screening of a diverse set of 385 maize inbred lines of indigenous and exotic origin detected the presence of two alleles (amplicon size: 250 and 650 bp) of lcyE and three alleles (amplicon size: 296, 543 and 875 bp) of crtRB1 in the inbred panel. Favourable alleles of both the genes were rare among the traditional maize germplasm; 3.38% of the inbreds possessed the favourable allele (650 bp) of lcyE, while 3.90% inbreds had the favourable allele (543 bp) of crtRB1. Five inbreds (1.3%) with favourable alleles of both the genes were found. Inbreds with favourable alleles of crtRB1 and lcyE serve as rich genetic resources for effective utilization in the maize biofortification programme

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

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

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

Abstract 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

Development of β-carotene rich maize hybrids through marker-assisted introgression of β-carotene hydroxylase allele.

Development of vitamin A-rich cereals can help in alleviating the widespread problem of vitamin A deficiency. We report here significant enhancement of kernel β-carotene in elite maize genotypes through accelerated marker-assisted backcross breeding. A favourable allele (543 bp) of the β-carotene hydroxylase (crtRB1) gene was introgressed in the seven elite inbred parents, which were low (1.4 µg/g) in kernel β-carotene, by using a crtRB1-specific DNA marker for foreground selection. About 90% of the recurrent parent genome was recovered in the selected progenies within two backcross generations. Concentration of β-carotene among the crtRB1-introgressed inbreds varied from 8.6 to 17.5 µg/g - a maximum increase up to 12.6-fold over recurrent parent. The reconstituted hybrids developed from improved parental inbreds also showed enhanced kernel β-carotene as high as 21.7 µg/g, compared to 2.6 µg/g in the original hybrid. The reconstituted hybrids evaluated at two locations possessed similar grain yield to that of original hybrids. These β-carotene enriched high yielding hybrids can be effectively utilized in the maize biofortification programs across the globe