Micronutrient malnutrition, especially deficiency of two mineral elements, iron [Fe] and zinc [Zn] in the developing world needs urgent attention. Pearl millet is one of the best crops with many nutritional properties and is accessible to the poor. We report findings of the first attempt to mine favorable alleles for grain iron and zinc content through association mapping in pearl millet. An association mapping panel of 130 diverse lines was evaluated at Delhi, Jodhpur and Dharwad, representing all the three pearl millet growing agro-climatic zones of India, during 2014 and 2015. Wide range of variation was observed for grain iron (32.3–111.9 ppm) and zinc (26.6–73.7 ppm) content. Genotyping with 114 representative polymorphic SSRs revealed 0.35 mean gene diversity. STRUCTURE analysis revealed presence of three sub-populations which was further supported by Neighbor-Joining method of clustering and principal coordinate analysis (PCoA). Marker-trait associations (MTAs) were analyzed with 267 markers (250 SSRs and 17 genic markers) in both general linear model (GLM) and mixed linear model (MLM), however, MTAs resulting from MLM were considered for more robustness of the associations. After appropriate Bonferroni correction, Xpsmp 2261 (13.34% R2-value), Xipes 0180 (R2-value of 11.40%) and Xipes 0096 (R2-value of 11.38%) were consistently associated with grain iron and zinc content for all the three locations. Favorable alleles and promising lines were identified for across and specific environments. PPMI 1102 had highest number (7) of favorable alleles, followed by four each for PPMFeZMP 199 and PPMI 708 for across the environment performance for both grain Fe and Zn content, while PPMI 1104 had alleles specific to Dharwad for grain Fe and Zn content. When compared with the reference genome Tift 23D2B1-P1-P5, Xpsmp 2261 amplicon was identified in intergenic region on pseudomolecule 5, while the other marker, Xipes 0810 was observed to be overlapping with aspartic proteinase (Asp) gene on pseudomolecule 3. Thus, this study can help in breeding new lines with enhanced micronutrient content using marker-assisted selection (MAS) in pearl millet leading to improved well-being especially for women and children.

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Biofortification of lines of pearl millet (Pennisetum glaucum (L.) R.Br.) with increased iron (Fe) and zinc (Zn) will have great impact because pearl millet is an indispensable component of food and nutritional security of inhabitants of arid and semi-arid regions. The aim of the present study was to assess the stability of Fe and Zn content in recombinant inbred lines (RILs) developed for grain Fe and Zn content, and to use these lines in developing micronutrient-rich pearl millet hybrids. A mapping population consisting of 210 RILs along, with parents and checks, was assessed in three consecutive years (2014–16) under rainfed conditions at the same experimental location in an alpha design with two repetitions. Significant differences were observed in genotype, environment and genotype × environment interaction mean squares for all variables, particularly grain micronutrients. The first two principal components of an interaction principal component analysis cumulatively explained 100% of the total variation; respective contributions of the first and second components were 64.0% and 36.0% for Fe, and 58.1% and 41.9% for Zn. A positive and moderately high correlation (0.696**) between Fe and Zn contents suggests good prospects of simultaneous improvement for both micronutrients. Among the 210 RILs, RIL 69, RIL 186, RIL 191, RIL 149 and RIL 45 were found to be more stable with higher mean micronutrient content, additive main effects and multiplicative interaction stability value (ASV) and genotype selection index (GSI) under rainfed condition. These RILs are promising and can be tested further for their combining ability for yield as well as grain micronutrient content for developing superior biofortified, heterotic pearl millet hybrids.

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Pearl millet is an important staple food crop of poor people and excels all other cereals due to its unique features of resilience to adverse climatic conditions. It is rich in micronutrients like iron and zinc and amenable for focused breeding for these micronutrients along with high yield. Hence, this is a key to alleviate malnutrition and ensure nutritional security. This study was conducted to identify and validate candidate genes governing grain iron and zinc content enabling the desired modifications in the genotypes. Transcriptome sequencing using ION S5 Next Generation Sequencer generated 43.5 million sequence reads resulting in 83,721 transcripts with N50 of 597 bp and 84.35% of transcripts matched with the pearl millet genome assembly. The genotypes having high iron and zinc showed differential gene expression during different stages. Of which, 155 were up-regulated and 251 were down-regulated while during flowering stage and milking stage 349 and 378 transcripts were differentially expressed, respectively. Gene annotation and GO term showed the presence of transcripts involved in metabolic activities associated with uptake and transport of iron and zinc. Information generated will help in gaining insights into iron and zinc metabolism and develop genotypes with high yield, grain iron and zinc content.

Pearl millet is a climate-resilient, nutritious crop with low input requirements that could provide economic returns in marginal agro-ecologies. In this study, we report quantitative trait loci (QTLs) for iron (Fe) and zinc (Zn) content from three distinct production environments. We generated a genetic linkage map using 210 F6 recombinant inbred line (RIL) population derived from the (PPMI 683 × PPMI 627) cross using genome-wide simple sequence repeats (SSRs). The molecular linkage map (seven linkage groups) of 151 loci was 3,273.1 cM length (Kosambi). The content of grain Fe in the RIL population ranged between 36 and 114 mg/Kg, and that of Zn from 20 to 106 mg/Kg across the 3 years (2014–2016) at over the three locations (Delhi, Dharwad, and Jodhpur). QTL analysis revealed a total of 22 QTLs for grain Fe and Zn, of which 14 were for Fe and eight were for Zn on three consecutive years at all locations. The observed phenotypic variance (R2) explained by different QTLs for grain Fe and Zn content ranged from 2.85 (QGFe.E3.2014–2016_Q3) to 19.66% (QGFe.E1.2014–2016_Q3) and from 2.93 (QGZn.E3.2014–2016_Q3) to 25. 95% (QGZn.E1.2014–2016_Q1), respectively. Two constitutive expressing QTLs for both Fe and Zn co-mapped in this population, one on LG 2 and second one on LG 3. Inside the QTLs candidate genes such as Ferritin gene, Al3+ Transporter, K+ Transporters, Zn2+ transporters and Mg2+ transporters were identified using bioinformatics approaches. The identified QTLs and candidate genes could be useful in pearl millet population improvement programs, seed, restorer parents, and marker-assisted selection programs.

Biofortification of pearl millet (Pennisetum glaucum (L.) R. Br.) with improved iron (Fe) and zinc (Zn) will have great impact as it is an indispensable component of nutritional security of inhabitants of arid and semi-arid regions. Ten genotypes along with checks were evaluated in RBD in six locations during kharif, 2016 under rainfed conditions. Significant differences were observed in genotype, environment and genotype × environment interaction mean squares for grain Fe and Zn contents, indicating differential nutrient accumulation by the genotypes. The first two principal components obtained in AMMI analysis were significant and cumulatively explained the total variation were 81.47 % for Fe and 73.97 % for Zn. A positive and moderately high correlation (r=0.6) between Fe and Zn contents suggests good prospects of simultaneous improvement for both micronutrients. Among the ten genotypes, PPMI 953 was found to be more stable with high mean Fe (90 ppm) and Zn (59 ppm) contents. On crossing with designated A lines of pearl millet, the line PPMI 953 found to be restorer for A1 system with complete fertility restoration of F1 panicle of the cross, ICMA(1) 863 x PPMI 953 under bagged condition and resulting F1 with 78-84% fertility measured by seed setting % under bag. The F2 individuals showed 9:7 fertility-sterility ratio (χ 2 value=0.002, P value=0.964). The promising line, PPMI 953 may be used as source for further genetic improvement with respect to grain micronutrient content or can be directly used as male parent in development of high iron pearl millet hybrids.

Pearl millet is an important staple food crop of poor people which is rich in micronutrients like iron and zinc and amenable for focused breeding for these micronutrients along with high yield. Transcriptome sequencing using ION S5 Next Generation Sequencer generated 43.5 million sequence reads resulting in 83,721 transcripts with N50 of 597 bp and 84.35% of transcripts matched with the pearl millet genome assembly. The genotypes having high Fe and Zn showed differential gene expression during different stages. Of which, 155 were up-regulated and 251were down-regulated while during flowering stage and milking stage 349 and 378 transcripts were differentially expressed, respectively. Gene annotation and GO term showed the presence of transcripts involved in metabolic activities associated with uptake and transport of iron and zinc. In the present study, the 83,721 transcripts were also examined for identification of SSRs. A total of 4,327 SSRs were identified with dominance of tri-nucleotide SSRs in comparison to di-nucleotide SSRs. These EST-SSRs can be used in molecular breeding, genetic diversity analysis and determination of heterozygosity of the allelic loci. Information generated will help in gaining insights into iron and zinc metabolism and develop genotypes with high yield, grain iron and zinc content.