Optimizing speed breeding and seed/pod chip based genotyping techniques in pigeonpea: A way forward for high throughput line development.

BACKGROUND: The challenge of pigeonpea breeding lies in its photosensitivity and seasonal specificity. This poses a problem to the breeder, as it restricts to single generation advancement in a year. Currently, the cross to cultivar gap is twelve to thirteen years resulting in a limited number of varietal releases over the past six decades. Shortening the breeding cycle was need of the hour, unlikely achieved by conventional breeding. To overcome these hindrances speed breeding was a necessary leap. An experiment was planned to optimize the speed breeding coupled with single seed descent and seed or pod chip-based genotyping to shorten the breeding cycle in pigeonpea at ICRISAT, Hyderabad. Monitored photoperiod, light wavelength, temperature and crop management regime were the indicators attributing to the success of speed breeding. RESULT: A photoperiod of 13 h: 8 h: 13 h at vegetative: flowering and pod filling stages is ideal for shortening the breeding cycle. Broad spectrum light (5700 K LED) hastened early vegetative growth and pod formation. Whereas far-red (735 nm) light favoured early flowering. A significant difference between the photoperiods, genotypes as well as photoperiod x genotype interaction for both days to flowering and plant height was noted. CONCLUSION: The optimized protocol serves as a road map for rapid generation advancement in pigeonpea. Deploying this protocol, it is possible to advance 2-4 generations per year. The breeding cycle can be reduced to 2-4 years which otherwise takes 7 years under conventional breeding. Single Seed Descent and seed or pod chip-based genotyping for early generation marker assisted selection, strengthened the precision of this technique aiding in high throughput line development.

Glutaredoxin regulation of primary root growth is associated with early drought stress tolerance in pearl millet.

Seedling root traits impact plant establishment under challenging environments. Pearl millet is one of the most heat and drought tolerant cereal crops that provides a vital food source across the sub-Saharan Sahel region. Pearl millet's early root system features a single fast-growing primary root which we hypothesize is an adaptation to the Sahelian climate. Using crop modeling, we demonstrate that early drought stress is an important constraint in agrosystems in the Sahel where pearl millet was domesticated. Furthermore, we show that increased pearl millet primary root growth is correlated with increased early water stress tolerance in field conditions. Genetics including genome-wide association study and quantitative trait loci (QTL) approaches identify genomic regions controlling this key root trait. Combining gene expression data, re-sequencing and re-annotation of one of these genomic regions identified a glutaredoxin-encoding gene PgGRXC9 as the candidate stress resilience root growth regulator. Functional characterization of its closest Arabidopsis homolog AtROXY19 revealed a novel role for this glutaredoxin (GRX) gene clade in regulating cell elongation. In summary, our study suggests a conserved function for GRX genes in conferring root cell elongation and enhancing resilience of pearl millet to its Sahelian environment.

Pearl millet is a staple food for over 90 million people living in regions of Africa and India that typically experience high temperatures and little rainfall. It was domesticated about 4,500 years ago in the Sahel region of West Africa and is one of the most heat and drought tolerant cereal crops worldwide. In most plants, organs known as roots absorb water and essential nutrients from the soil. Young pearl millet plants develop a fast-growing primary root, but it is unclear how this unique feature helps the crop to grow in hot and dry conditions. Using weather data collected from the Sahel over a 20-year period, Fuente, Grondin et al. predicted by modelling that early drought stress is the major factor limiting pearl millet growth and yield in this region. Field experiments found that plants with primary roots that grow faster within soil were better at tolerating early drought than those with slower growing roots. Further work using genetic approaches revealed that a gene known as PgGRXC9 promotes the growth of the primary root. To better understand how this gene works, the team examined a very similar gene in a well-studied model plant known as Arabidopsis. This suggested that PgGRXC9 helps the primary root to grow by stimulating cell elongation within the root. Since it is well adapted to dry conditions, pearl millet is expected to play an important role in helping agriculture adjust to climate change. The findings of Fuente, Grondin et al. may be used by plant breeders to create more resilient and productive varieties of pearl millet.

Genotype-by-environment interactions for starch, mineral, and agronomic traits in pearl millet hybrids evaluated across five locations in West Africa.

Introduction: Pearl millet is a staple cereal grown in the harshest environments of arid and semi-arid regions of Asia and sub-Saharan Africa. It is the primary source of calories for millions of people in these regions because it has better adaptation to harsh environmental conditions and better nutritional traits than many other cereals. By screening the pearl millet inbred germplasm association panel (PMiGAP), we earlier reported the best genotypes with the highest concentration of slowly digestible and resistant starch in their grains. Methods: In the current study, we tested these 20 top-performing pearl millet hybrids, identified based on starch data, in a randomised block design with three replications at five locations in West Africa, viz. Sadore and Konni (Niger), Bambey (Senegal), Kano (Nigeria), and Bawku (Ghana). Phenotypic variability was assessed for agronomic traits and mineral traits (Fe and Zn). Results and discussion: Analysis of variance demonstrated significant genotypic, environmental, and GEI effects among five testing environments for agronomic traits (days to 50% flowering, panicle length, and grain yield), starch traits (rapidly digestible starch, slowly digestible starch, resistant starch, and total starch), and mineral trait (iron and zinc). Starch traits, such as rapidly digestible starch (RDS) and slowly digestible starch (SDS), showed nonsignificant genotypic and environmental interactions but high heritability, indicating the lower environmental influence on these traits in the genotype × testing environments. Genotype stability and mean performance across all the traits were estimated by calculating the multi-trait stability index (MTSI), which showed that genotypes G3 (ICMX207070), G8 (ICMX207160), and G13 (ICMX207184) were the best performing and most stable among the five test environments.

Genotype-by-Environment Interaction Analysis of Metabolites in Pearl Millet Genotypes with High Concentrations of Slowly Digestible and Resistant Starch in Their Grains.

Genotype × environment interactions (GEIs) should play an important role in the selection of suitable germplasm in breeding programmes. We here assessed GEI effects on pearl millet (Pennisetum glaucum L.) genotypes, selected to possess a high concentration of slowly digestible starch (SDS) and resistant starch (RS) in their grains. Entries were grown in a randomized complete block design with three replications at locations in Bawku-Ghana, Sadore-Niger, Bamako-Mali, Konni-Nigeria, and Gampella-Burkina Faso across West Africa. Harvested grains from these locations were metabolomically profiled using flow injection ionization-high-resolution mass spectrometry (FIE-HRMS). A total of 3144 mass features (m/z) (1560 negative ion mode and 1584 positive ion mode) were detected, of which, 475 m/z were linked to metabolites be involved in starch, antioxidant and lipid biosynthesis, and vitamin metabolism. Combined ANOVA revealed that the GEI was significantly evident for 54 health-benefiting metabolites, many associated with sugar, especially galactose, metabolism. Additive main effects and multiplicative interaction (AMMI) analysis examined genotype variation and GEI effects, which, when combined with principal component analysis (PCA), found that m/z 171.14864 (positive ionisation, propenyl heptanoate) accounted for 89% of the GEI variation along PC1. The AMMI-based stability parameter (ASTAB), modified AMMI stability value (MASV), and modified AMMI stability index (MASI) were then applied to identify stable and high-performing genotypes for all the health-benefiting metabolites. Similarly, the best-linear-unbiased-prediction (BLUP)-based stability estimation was also performed using the harmonic mean of genotypic values (HMGV), relative performance of genotypic values (RPGV), and harmonic mean of relative performance of genotypic values (HMRPGV), to identify genotype rankings across multiple environments. The multi-trait stability index (MTSI) was calculated and found that the genotypes G1 (ICMH-177111) and G24 (ICMX-207137) were the most stable and were the best mean performers across 52 health-benefiting metabolic traits. These findings demonstrate the potential of G × E assessments on the delivery of health-benefiting metabolite-rich grains in future varieties and hybrids of pearl millet.

Genetic diversity analysis of East African sorghum (Sorghum bicolor [L.] Moench) germplasm collections for agronomic and nutritional quality traits.

Breeding for climate-resilient, high-yielding, and nutrient-rich sorghum cultivars is essential for sustainable food systems and enhanced livelihoods in sub-Saharan Africa. Therefore, this study aimed to determine the genetic diversity among East African sorghum germplasm collections through agronomic and nutritional quality traits to select promising lines for direct production or breeding. A collection of 348 sorghum germplasm was field evaluated at two locations in Uganda using an augmented design, and grain iron (Fe) and zinc (Zn) contents were profiled. Data were collected on 20 sorghum agro-morphological traits and Fe and Zn compositions. A significant (P ≤ 0.05) variation was detected amongst the test genotypes for all the assessed traits, suggesting the presence of sufficient genetic diversity for selection. High heritability (H2 > 0.60) and genetic advance as percent of the mean (GA >20%) were computed for grain yield, Zn content, and selected agronomic traits, ensuring genetic gains through selection. A significant positive correlation was recorded between Fe and Zn concentrations (r = 0.32, P < 0.001), allowing simultaneous selection for the two nutrient compositions. Cluster analysis based on phenotypic traits resolved the test sorghum genotypes into four distinct genetic groups. Six genotypes with superior agronomic traits and high Fe and Zn contents were identified for production or potential parents for quality breeding. Overall, the current study found considerable genetic variation among East African sorghum germplasm collections for strategic conservation and breeding in Uganda or similar agro-ecologies.

A New Formulation for Strigolactone Suicidal Germination Agents, towards Successful Striga Management.

Striga hermonthica, a member of the Orobanchaceae family, is an obligate root parasite of staple cereal crops, which poses a tremendous threat to food security, contributing to malnutrition and poverty in many African countries. Depleting Striga seed reservoirs from infested soils is one of the crucial approaches to minimize subterranean damage to crops. The dependency of Striga germination on the host-released strigolactones (SLs) has prompted the development of the "Suicidal Germination" strategy to reduce the accumulated seed bank of Striga. The success of aforementioned strategy depends not only on the activity of the applied SL analogs, but also requires suitable application protocol with simple, efficient, and handy formulation for rain-fed African agriculture. Here, we developed a new formulation "Emulsifiable Concentration (EC)" for the two previously field-assessed SL analogs Methyl phenlactonoate 3 (MP3) and Nijmegen-1. The new EC formulation was evaluated for biological activities under lab, greenhouse, mini-field, and field conditions in comparison to the previously used Atlas G-1086 formulation. The EC formulation of SL analogs showed better activities on Striga germination with lower EC50 and high stability under Lab conditions. Moreover, EC formulated SL analogs at 1.0 µM concentrations reduced 89-99% Striga emergence in greenhouse. The two EC formulated SL analogs showed also a considerable reduction in Striga emergence in mini-field and field experiments. In conclusion, we have successfully developed a desired formulation for applying SL analogs as suicidal agents for large-scale field application. The encouraging results presented in this study pave the way for integrating the suicidal germination approach in sustainable Striga management strategies for African agriculture.

Combining ability studies of grain Fe and Zn contents of pearl millet (Pennisetum glaucum L.) in West Africa.

Micronutrient malnutrition is a major challenge in Africa, where half a million children die each year because of lack of micronutrients in their food. Pearl millet is an important food and fodder crop for the people living in the Semi-Arid regions of West Africa. The present study was conducted to determine the stability, combining ability, and gene action conditions of the high level of Fe and Zn content in grain and selected agronomic traits. Hence, eight genotypes were selected based on the availability of grain Fe and Zn contents and crossed in a full diallel mating design. Progenies from an 8 × 8 diallel mating along with the parents were evaluated in an alpha lattice design with three replications in three locations for two years. The parental lines Jirani, LCIC 9702 and MORO, had positive significant general combining ability (GCA) effects for grain Fe concentration, while Jirani and MORO had positive significant GCA effects for grain Zn concentration. For the specific combining ability (SCA), among the 56 hybrids evaluated, only the hybrids LCIC 9702 × Jirani and MORO × ZANGO had positive significant SCA effects for grain Fe concentration across locations, and for grain Zn concentration, the hybrids Gamoji × MORO, LCIC 9702 × Jirani, and ICMV 167006 × Jirani had positive significant SCA effects. The reciprocal effects were significant for grain Zn concentration, grain yield, flowering time, plant height, test weight, and downy mildew incidence, suggesting that the choice of a female or male parent is critical in hybrid production. Grain Fe and Zn concentration, flowering time, plant height, panicle length, panicle girth, panicle compactness, and downy mildew incidence were found to be predominantly under additive gene action, while grain yield and test weight were predominantly under non-additive gene action. A highly positive correlation was found between grain Fe and Zn concentrations, which implies that improving grain Fe trait automatically improves the grain Zn content. The stability analysis revealed that the hybrid ICMV 167006 × Jirani was the most stable and high-yielding with a high level of grain Fe and Zn micronutrients.

Metabolite Diversity and Metabolic Genome-Wide Marker Association Studies (Mgwas) for Health Benefiting Nutritional Traits in Pearl Millet Grains.

As efforts are made to increase food security, millets are gaining increasing importance due to their excellent nutritional credentials. Among the millets, pearl millet is the predominant species possessing several health benefiting nutritional traits in its grain that are helpful in mitigating chronic illnesses such as type-2 diabetes and obesity. In this paper, we conducted metabolomic fingerprinting of 197 pearl millet inbred lines drawn randomly from within the world collection of pearl millet germplasm and report the extent of genetic variation for health benefitting metabolites in these genotypes. Metabolites were extracted from seeds and assessed using flow infusion high-resolution mass spectrometry (FIE-HRMS). Metabolite features (m/z), whose levels significantly differed among the germplasm inbred lines, were identified by ANOVA corrected for FDR and subjected to functional pathway analysis. A number of health-benefiting metabolites linked to dietary starch, antioxidants, vitamins, and lipid metabolism-related compounds were identified. Metabolic genome-wide association analysis (mGWAS) performed using the 396 m/z as phenotypic traits and the 76 K SNP as genotypic variants identified a total of 897 SNPs associated with health benefiting nutritional metabolite at the -log p-value ≤ 4.0. From these associations, 738 probable candidate genes were predicted to have an important role in starch, antioxidants, vitamins, and lipid metabolism. The mGWAS analysis focused on genes involved in starch branching (α-amylase, ß-amylase), vitamin-K reductase, UDP-glucuronosyl, and UDP-glucosyl transferase (UGTs), L-ascorbate oxidase, and isoflavone 2'-monooxygenase genes, which are known to be linked to increases in human health benefiting metabolites. We demonstrate how metabolomic, genomic, and statistical approaches can be utilized to pinpoint genetic variations and their functions linked to key nutritional properties in pearl millet, which in turn can be bred into millets and other cereals crops using plant breeding methods.

Identification of High-Yielding Iron-Biofortified Open-Pollinated Varieties of Pearl Millet in West Africa.

Pearl millet is a predominant food and fodder crop in West Africa. This study was carried out to test the newly developed open-pollinated varieties (OPVs) for field performance and stability for grain yield, grain iron (Fe), and grain zinc (Zn) contents across 10 locations in West Africa (i.e., Niger, Nigeria, Mali, Burkina Faso, Senegal, and Ghana). The test material consisted of 30 OPVs, of which 8 are Fe/Zn biofortified. The experiment was conducted in a randomized complete block design in three replications. ANOVA revealed highly significant variability for grain yield and micronutrient traits. The presence of genotype × environment (G × E) indicated that the expressions of traits are significantly influenced by both genetic and G × E factors, for grain Fe and Zn contents. Days to 50% flowering and plant height showed less G × E, suggesting these traits are largely under genetic control. The genotypes CHAKTI (46 days), ICTP 8203 (46 days), ICMV 177002 (50 days), ICMV 177003 (48 days), and Moro (53 days) had exhibited early flowering across locations leading to early physiological maturity. CHAKTI (1.42 t/ha yield; 62.24 mg/kg of grain Fe, 47.29 mg/kg of grain Zn) and ICMP 177002 (1.19 t/ha yield, 62.62 mg/kg of grain Fe, 46.62 mg/kg of grain Zn) have performed well for grain yield and also for micronutrients, across locations, compared with the check. Additive Main Effect and Multiplicative Interaction (AMMI) ANOVA revealed the highly significant genotypic differences, the mean sum of squares of environment, and its interaction with the genotypes. Based on the AMMI stability value (ASV), the most stable genotype is SOSAT-C88 (ASV = 0.04) for grain yield and resistance to downy mildew; mean grain yield and stability rankings (YSI) revealed that the genotypes CHAKTI, SOSAT-C88, and ICMV IS 99001 were high yielding and expressed stability across regions. The strong correlation (r = 0.98∗∗) of grain Fe and Zn contents that merits Fe-based selection is highly rewarding. CHAKTI outperformed over other genotypes for grain yield (71% higher), especially with early maturing varieties in West Africa, such as GB 8735, LCIC 9702, and Jirani, and for grain Fe (16.11% higher) and Zn (7% higher) contents across locations, and made a candidate of high-iron variety to be promoted for combating the micronutrient malnutrition in West and Central Africa (WCA).

Identifying Anti-Oxidant Biosynthesis Genes in Pearl Millet [Pennisetum glaucum (L.) R. Br.] Using Genome-Wide Association Analysis.

Pearl millet [Pennisetum glaucum (L.) R Br.] is an important staple food crop in the semi-arid tropics of Asia and Africa. It is a cereal grain that has the prospect to be used as a substitute for wheat flour for celiac patients. It is an important antioxidant food resource present with a wide range of phenolic compounds that are good sources of natural antioxidants. The present study aimed to identify the total antioxidant content of pearl millet flour and apply it to evaluate the antioxidant activity of its 222 genotypes drawn randomly from the pearl millet inbred germplasm association panel (PMiGAP), a world diversity panel of this crop. The total phenolic content (TPC) significantly correlated with DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity (% inhibition), which ranged from 2.32 to 112.45% and ferric-reducing antioxidant power (FRAP) activity ranging from 21.68 to 179.66 (mg ascorbic acid eq./100 g). Genome-wide association studies (GWAS) were conducted using 222 diverse accessions and 67 K SNPs distributed across all the seven pearl millet chromosomes. Approximately, 218 SNPs were found to be strongly associated with DPPH and FRAP activity at high confidence [-log (p) > 3.0-7.4]. Furthermore, flanking regions of significantly associated SNPs were explored for candidate gene harvesting. This identified 18 candidate genes related to antioxidant pathway genes (flavanone 7-O-beta-glycosyltransferase, GDSL esterase/lipase, glutathione S-transferase) residing within or near the association signal that can be selected for further functional characterization. Patterns of genetic variability and the associated genes reported in this study are useful findings, which would need further validation before their utilization in molecular breeding for high antioxidant-containing pearl millet cultivars.

Genome sequence and genetic diversity analysis of an under-domesticated orphan crop, white fonio (Digitaria exilis).

BACKGROUND: Digitaria exilis, white fonio, is a minor but vital crop of West Africa that is valued for its resilience in hot, dry, and low-fertility environments and for the exceptional quality of its grain for human nutrition. Its success is hindered, however, by a low degree of plant breeding and improvement. FINDINGS: We sequenced the fonio genome with long-read SMRT-cell technology, yielding a ∼761 Mb assembly in 3,329 contigs (N50, 1.73 Mb; L50, 126). The assembly approaches a high level of completion, with a BUSCO score of >99%. The fonio genome was found to be a tetraploid, with most of the genome retained as homoeologous duplications that differ overall by ∼4.3%, neglecting indels. The 2 genomes within fonio were found to have begun their independent divergence ∼3.1 million years ago. The repeat content (>49%) is fairly standard for a grass genome of this size, but the ratio of Gypsy to Copia long terminal repeat retrotransposons (∼6.7) was found to be exceptionally high. Several genes related to future improvement of the crop were identified including shattering, plant height, and grain size. Analysis of fonio population genetics, primarily in Mali, indicated that the crop has extensive genetic diversity that is largely partitioned across a north-south gradient coinciding with the Sahel and Sudan grassland domains. CONCLUSIONS: We provide a high-quality assembly, annotation, and diversity analysis for a vital African crop. The availability of this information should empower future research into further domestication and improvement of fonio.

Genomic diversity in pearl millet inbred lines derived from landraces and improved varieties.

BACKGROUND: Genetic improvement of pearl millet is lagging behind most of the major crops. Development of genomic resources is expected to expedite breeding for improved agronomic traits, stress tolerance, yield, and nutritional quality. Genotyping a breeding population with high throughput markers enables exploration of genetic diversity, population structure, and linkage disequilibrium (LD) which are important preludes for marker-trait association studies and application of genomic-assisted breeding. RESULTS: Genotyping-by-sequencing (GBS) libraries of 309 inbred lines derived from landraces and improved varieties from Africa and India generated 54,770 high quality single nucleotide polymorphism (SNP) markers. On average one SNP per 29 Kb was mapped in the reference genome, with the telomeric regions more densely mapped than the pericentromeric regions of the chromosomes. Population structure analysis using 30,208 SNPs evenly distributed in the genome divided 309 accessions into five subpopulations with different levels of admixture. Pairwise genetic distance (GD) between accessions varied from 0.09 to 0.33 with the average distance of 0.28. Rapid LD decay implied low tendency of markers inherited together. Genetic differentiation estimates were the highest between subgroups 4 and 5, and the lowest between subgroups 1 and 2. CONCLUSIONS: Population genomic analysis of pearl millet inbred lines derived from diverse geographic and agroecological features identified five subgroups mostly following pedigree differences with different levels of admixture. It also revealed the prevalence of high genetic diversity in pearl millet, which is very useful in defining heterotic groups for hybrid breeding, trait mapping, and holds promise for improving pearl millet for yield and nutritional quality. The short LD decay observed suggests an absence of persistent haplotype blocks in pearl millet. The diverse genetic background of these lines and their low LD make this set of germplasm useful for traits mapping.

Response to early drought stress and identification of QTLs controlling biomass production under drought in pearl millet.

Pearl millet plays a major role in food security in arid and semi-arid areas of Africa and India. However, it lags behind the other cereal crops in terms of genetic improvement. The recent sequencing of its genome opens the way to the use of modern genomic tools for breeding. Our study aimed at identifying genetic components involved in early drought stress tolerance as a first step toward the development of improved pearl millet varieties or hybrids. A panel of 188 inbred lines from West Africa was phenotyped under early drought stress and well-irrigated conditions. We found a strong impact of drought stress on yield components. This impact was variable between inbred lines. We then performed an association analysis with a total of 392,493 SNPs identified using Genotyping-by-Sequencing (GBS). Correcting for genetic relatedness, genome wide association study identified QTLs for biomass production in early drought stress conditions and for stay-green trait. In particular, genes involved in the sirohaem and wax biosynthesis pathways were found to co-locate with two of these QTLs. Our results might contribute to breed pearl millet lines with improved yield under drought stress.

Antinutritional factors in pearl millet grains: Phytate and goitrogens content variability and molecular characterization of genes involved in their pathways.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is an important "orphan" cereal and the most widely grown of all the millet species worldwide. It is also the sixth most important cereal in the world after wheat, rice, maize, barley, and sorghum, being largely grown and used in West Africa as well as in India and Pakistan. The present study was carried out in the frame of a program designed to increase benefits and reduce potential health problems deriving from the consumption of pearl millet. The specific goal was to provide a database of information on the variability existing in pearl millet germplasm as to the amounts of phytate, the most relevant antinutrient compound, and the goitrogenic compounds C-glycosylflavones (C-GFs) accumulated in the grain.Results we obtained clearly show that, as indicated by the range in values, a substantial variability subsists across the investigated pearl millet inbred lines as regards the grain level of phytic acid phosphate, while the amount of C-GFs shows a very high variation. Suitable potential parents to be used in breeding programs can be therefore chosen from the surveyed material in order to create new germplasm with increased nutritional quality and food safety. Moreover, we report novel molecular data showing which genes are more relevant for phytic acid biosynthesis in the seeds as well as a preliminary analysis of a pearl millet orthologous gene for C-GFs biosynthesis. These results open the way to dissect the genetic determinants controlling key seed nutritional phenotypes and to the characterization of their impact on grain nutritional value in pearl millet.