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The agricultural sector faces colossal challenges amid environmental changes and a burgeoning human population. In this context, crops must adapt to evolving climatic conditions while meeting increasing production demands. The dairy industry is anticipated to hold the highest value in the agriculture sector in future. The rise in the livestock population is expected to result in an increased demand for fodder feed. Consequently, it is crucial to seek alternative options, as crops demand fewer resources and are resilient to climate change. Pearl millet offers an apposite key to these bottlenecks, as it is a promising climate resilience crop with significantly low energy, water and carbon footprints compared to other crops. Numerous studies have explored its potential as a fodder crop, revealing promising performance. Despite its capabilities, pearl millet has often been overlooked. To date, few efforts have been made to document molecular aspects of fodder-related traits. However, several QTLs and candidate genes related to forage quality have been identified in other fodder crops, which can be harnessed to enhance the forage quality of pearl millet. Lately, excellent genomic resources have been developed in pearl millet allowing deployment of cutting-edge genomics-assisted breeding for achieving a higher rate of genetic gains. This review would facilitate a deeper understanding of various aspects of fodder pearl millet in retrospect along with the future challenges and their solution. This knowledge may pave the way for designing efficient breeding strategies in pearl millet thereby supporting sustainable agriculture and livestock production in a changing world., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: Through the histological, physiological, and transcriptome-level identification of the abscission zone of Pennisetum alopecuroides 'Liqiu', we explored the structure and the genes related to seed shattering, ultimately revealing the regulatory network of seed shattering in P. alopecuroides. Pennisetum alopecuroides is one of the most representative ornamental grass species of Pennisetum genus. It has unique inflorescence, elegant appearance, and strong stress tolerance. However, the shattering of seeds not only reduces the ornamental effect, but also hinders the seed production. In order to understand the potential mechanisms of seed shattering in P. alopecuroides, we conducted morphological, histological, physiological, and transcriptomic analyses on P. alopecuroides cv. 'Liqiu'. According to histological findings, the seed shattering of 'Liqiu' was determined by the abscission zone at the base of the pedicel. Correlation analysis showed that seed shattering was significantly correlated with cellulase, lignin, auxin, gibberellin, cytokinin and jasmonic acid. Through a combination of histological and physiological analyses, we observed the accumulation of cellulase and lignin during 'Liqiu' seed abscission. We used PacBio full-length transcriptome sequencing (SMRT) combined with next-generation sequencing (NGS) transcriptome technology to improve the transcriptome data of 'Liqiu'. Transcriptomics further identified many differential genes involved in cellulase, lignin and plant hormone-related pathways. This study will provide new insights into the research on the shattering mechanism of P. alopecuroides., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: Analyze the evolutionary pattern of DNAJ protein genes in the Panicoideae, including pearl millet, to identify and characterize the biological function of PgDNAJ genes in pearl millet. Global warming has become a major factor threatening food security and human development. It is urgent to analyze the heat-tolerant mechanism of plants and cultivate crops that are adapted to high temperature conditions. The Panicoideae are the second largest subfamily of the Poaceae, widely distributed in warm temperate and tropical regions. Many of these species have been reported to have strong adaptability to high temperature stress, such as pearl millet, foxtail millet and sorghum. The evolutionary differences in DNAJ protein genes among 12 Panicoideae species and 10 other species were identified and analyzed. Among them, 79% of Panicoideae DNAJ protein genes were associated with retrotransposon insertion. Analysis of the DNAJ protein pan-gene family in six pearl millet accessions revealed that the non-core genes contained significantly more TEs than the core genes. By identifying and analyzing the distribution and types of TEs near the DNAJ protein genes, it was found that the insertion of Copia and Gypsy retrotransposons provided the source of expansion for the DNAJ protein genes in the Panicoideae. Based on the analysis of the evolutionary pattern of DNAJ protein genes in Panicoideae, the PgDNAJ was obtained from pearl millet through identification. PgDNAJ reduces the accumulation of reactive oxygen species caused by high temperature by activating ascorbate peroxidase (APX), thereby improving the heat resistance of plants. In summary, these data provide new ideas for mining potential heat-tolerant genes in Panicoideae, and help to improve the heat tolerance of other crops., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: An approach to release 'frozen' variability in apomictic species using sexuality of another species, eventually its utilization in crop improvement and de-novo domestication of crop wild relatives is presented. Pennisetum squamulatum, a secondary gene pool species of pearl millet (P. glaucum), harbours many desirable traits. However, it was neither utilized to improve pearl millet fodder traits nor improvement of its own domestication traits was attempted, due to the complexities of genomes and apomictic reproduction. To overcome this, we followed an Apomixis Mediated Genome Addition (AMGA) strategy and utilized the contrasting reproductive capacities (sexuality and apomixis) of both the species to access the otherwise un-available variability embedded in P. squamulatum. Segregating population of interspecific hybrids exhibited significant variability and heterosis for desired morphological, agronomical, and nutritional traits. Elite apomictic and perennial hybrids were evaluated in breeding trials, and eventually a novel grass cultivar was released for commercial cultivation in India. The performance of newly developed cultivar was superior to other adapted perennial grasses of arid and semi-arid rangelands. Through AMGA, the sexuality of one species was successfully utilized to 'release' the 'frozen' variability embedded in another species. Subsequently, the hybrids representing desirable trait combinations were again 'fixed' utilizing the apomixis alleles from the male parent in a back-and-forth apomixis-sexual-apomixis selection cycle. This study also demonstrated the potential of AMGA to improve crop relatives through genomes introgression as well as de novo domestication of new crops from wild species., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: Pearl millet breeding programs can use this heterotic group information on seed and restorer parents to generate new series of pearl millet hybrids having higher yields than the existing hybrids. Five hundred and eighty hybrid parents, 320 R- and 260 B-lines, derived from 6 pearl millet breeding programs in India, genotyped following RAD-GBS (about 0.9 million SNPs) clustered into 12 R- and 7 B-line groups. With few exceptions, hybrid parents of all the breeding programs were found distributed across all the marker-based groups suggesting good diversity in these programs. Three hundred and twenty hybrids generated using 37 (22 R and 15 B) representative parents, evaluated for grain yield at four locations in India, showed significant differences in yield, heterosis, and combining ability. Across all the hybrids, mean mid- and better-parent heterosis for grain yield was 84.0% and 60.5%, respectively. Groups G12 B × G12 R and G10 B × G12 R had highest heterosis of about 10% over best check hybrid Pioneer 86M86. The parents involved in heterotic hybrids were mainly from the groups G4R, G10B, G12B, G12R, and G13B. Based on the heterotic performance and combining ability of groups, 2 B-line (HGB-1 and HGB-2) and 2 R-line (HGR-1 and HGR-2) heterotic groups were identified. Hybrids from HGB-1 × HGR-1 and HGB-2 × HGR-1 showed grain yield heterosis of 10.6 and 9.3%, respectively, over best hybrid check. Results indicated that parental groups can be formed first by molecular markers, which may not predict the best hybrid combination, but it can reveal a practical value of assigning existing and new hybrid pearl millet parental lines into heterotic groups to develop high-yielding hybrids from the different heterotic groups.

Key Message: Four genetic regions associated with water use traits, measured at different levels of plant organization, and with agronomic traits were identified within a previously reported region for terminal water deficit adaptation on linkage group 2. Close linkages between these traits showed the value of phenotyping both for agronomic and secondary traits to better understand plant productive processes. Water saving traits are critical for water stress adaptation of pearl millet, whereas maximizing water use is key to the absence of stress. This research aimed at demonstrating the close relationship between traits measured at different levels of plant organization, some putatively involved in water stress adaptation, and those responsible for agronomic performance. A fine-mapping population of pearl millet, segregating for a previously identified quantitative trait locus (QTL) for adaptation to terminal drought stress on LG02, was phenotyped for traits at different levels of plant organization in different experimental environments (pot culture, high-throughput phenotyping platform, lysimeters, and field). The linkages among traits across the experimental systems were analysed using principal component analysis and QTL co-localization approach. Four regions within the LG02-QTL were found and revealed substantial co-mapping of water use and agronomic traits. These regions, identified across experimental systems, provided genetic evidence of the tight linkages between traits phenotyped at a lower level of plant organization and agronomic traits assessed in the field, therefore deepening our understanding of complex traits and then benefiting both geneticists and breeders. In short: (1) under no/mild stress conditions, increasing biomass and tiller production increased water use and eventually yield; (2) under severe stress conditions, water savings at vegetative stage, from lower plant vigour and fewer tillers in that population, led to more water available during grain filling, expression of stay-green phenotypes, and higher yield.

Key Message: Linkage analysis confirmed the association in the region of PHYC in pearl millet. The comparison of genes found in this region suggests that PHYC is the best candidate. Major efforts are currently underway to dissect the phenotype-genotype relationship in plants and animals using existing populations. This method exploits historical recombinations accumulated in these populations. However, linkage disequilibrium sometimes extends over a relatively long distance, particularly in genomic regions containing polymorphisms that have been targets for selection. In this case, many genes in the region could be statistically associated with the trait shaped by the selected polymorphism. Statistical analyses could help in identifying the best candidate genes into such a region where an association is found. In a previous study, we proposed that a fragment of the PHYTOCHROME C gene (PHYC) is associated with flowering time and morphological variations in pearl millet. In the present study, we first performed linkage analyses using three pearl millet F2 families to confirm the presence of a QTL in the vicinity of PHYC. We then analyzed a wider genomic region of ~100 kb around PHYC to pinpoint the gene that best explains the association with the trait in this region. A panel of 90 pearl millet inbred lines was used to assess the association. We used a Markov chain Monte Carlo approach to compare 75 markers distributed along this 100-kb region. We found the best candidate markers on the PHYC gene. Signatures of selection in this region were assessed in an independent data set and pointed to the same gene. These results foster confidence in the likely role of PHYC in phenotypic variation and encourage the development of functional studies.

Pearl millet is an important component of food security in the semi-arid tropics and is assuming greater importance in the context of changing climate and increasing demand for highly nutritious food and feed. Molecular tools have been developed and applied for pearl millet on a limited scale. However, the existing tool kit needs to be strengthened further for its routine use in applied breeding programs. Here, we report enrichment of the pearl millet molecular linkage map by exploiting low-cost and high-throughput Diversity Arrays Technology (DArT) markers. Genomic representation from 95 diverse genotypes was used to develop a DArT array with circa 7,000 clones following PstI/BanII complexity reduction. This array was used to genotype a set of 24 diverse pearl millet inbreds and 574 polymorphic DArT markers were identified. The genetic relationships among the inbred lines as revealed by DArT genotyping were in complete agreement with the available pedigree data. Further, a mapping population of 140 F(7) Recombinant Inbred Lines (RILs) from cross H 77/833-2 × PRLT 2/89-33 was genotyped and an improved linkage map was constructed by integrating DArT and SSR marker data. This map contains 321 loci (258 DArTs and 63 SSRs) and spans 1148 cM with an average adjacent-marker interval length of 3.7 cM. The length of individual linkage groups (LGs) ranged from 78 cM (LG 3) to 370 cM (LG 2). This better-saturated map provides improved genome coverage and will be useful for genetic analyses of important quantitative traits. This DArT platform will also permit cost-effective background selection in marker-assisted backcrossing programs as well as facilitate comparative genomics and genome organization studies once DNA sequences of polymorphic DArT clones are available.

Pennisetum squamulatum reproduces by apomixis, a type of asexual reproduction through seeds. Apomixis in P. squamulatum is transmitted as a dominant Mendelian trait, and a genomic region, the apospory-specific genomic region (ASGR), is sufficient for inheritance of the trait. The ASGR is physically large (>50 Mb), highly heterochromatic, hemizygous, and recombinationally suppressed. These characteristics have hindered high-resolution genetic mapping and map-based cloning of apomixis genes. In this study, the long terminal repeat (LTR) regions of ASGR-abundant retrotransposons in the genome of P. squamulatum and ASGR-linked bacterial artificial chromosome clones were identified and sequenced for designing LTR-specific primers. Two hundred and ninety single-dose sequence specific amplified polymorphism (SSAP) markers were generated from 38 primer combinations. The SSAP markers combined with two previous ASGR-mapped markers were used for genetic linkage analysis and construction of a genetic map resulting in the formation of 27 linkage groups at LOD 10, one of which contained >60% of the SSAP markers. After removing identical markers (identical band scoring) on the largest linkage group, 46 markers were finally used for genetic mapping at LOD 10. The markers distributed across 10 different loci covering 19 cM; however, 45 markers were distributed within 9 cM. Six markers were recovered and sequenced. Five markers were successfully converted into sequence characterized amplified regions (SCARs). Segregation of SCAR markers was not always consistent with the SSAP markers of origin suggesting a greater level of error in the SSAP map resulting in an inflated map distance for the ASGR. One SCAR marker (Pst 56-1205-400) detected expression of an ASGR retrotransposon in root, anther, leaf and ovary of P. squamulatum, although sequencing of the RT-PCR product failed to find a functional open reading frame for the transcript.

Cytoplasmic male sterility (CMS) represents an important agricultural trait in pearl millet [Pennisetum glaucum (L.) R. Br.] with a value to the seed industry in facilitating economical hybrid seed production. Among the CMS systems available in millet, the A1 source is the most commonly used for hybrid production, but it can undergo low frequency reversion to fertility. Plant mitochondrial genomes are highly recombinogenic, becoming unstable and prone to ectopic recombination under conditions of tissue culture, somatic hybridization, or interspecific crossing. Similarly, CMS systems prone to spontaneous fertility reversion experience sporadic mitochondrial genome instability. We compared mitochondrial genome configurations between the male-sterile A1 line and fertile revertants of pearl millet to develop a model for millet mitochondrial genome reorganization upon reversion. Relative copy number of a subgenomic molecule containing the CoxI-1-2 junction region, a component of the recombination process for reversion, is amplified tenfold following reversion, relative to the CMS A1 line. We propose that increased copy number of this molecule in a small number of cells or at low frequency triggers a recombination cascade, likely during reproductive development. The proposed recombination process initiates with ectopic recombination through a 7-bp repeat to produce a novel CoxI-3-2 junction molecule and an unstable recombination intermediate. Subsequent intra-molecular recombination stabilizes the intermediate to form a new copy of CoxI accompanied by a deletion. This study furthers the argument that substoichiometric shifting within the plant mitochondrial genome plays an important role in the evolution of the mitochondrial genome and plant reproductive dynamics.

During the last 12,000 years, different cultures around the world have domesticated cereal crops. Several studies investigated the evolutionary history and domestication of cereals such as wheat in the Middle East, rice in Asia or maize in America. The domestication process in Africa has led to the emergence of important cereal crops like pearl millet in Sahelian Africa. In this study, we used 27 microsatellite loci to analyze 84 wild accessions and 355 cultivated accessions originating from the whole pearl millet distribution area in Africa and Asia. We found significantly higher diversity in the wild pearl millet group. The cultivated pearl millet sample possessed 81% of the alleles and 83% of the genetic diversity of the wild pearl millet sample. Using Bayesian approaches, we identified intermediate genotypes between the cultivated and wild groups. We then analyzed the phylogenetic relationship among accessions not showing introgression and found that a monophyletic origin of cultivated pearl millet in West Africa is the most likely scenario supported by our data set.

Genetic diversity of crop species in sub-Sahelian Africa is still poorly documented. Among such crops, pearl millet is one of the most important staple species. In Niger, pearl millet covers more than 65% of the total cultivated area. Analyzing pearl millet genetic diversity, its origin and its dynamics is important for in situ and ex situ germplasm conservation and to increase knowledge useful for breeding programs. We developed new genetic markers and a high-throughput technique for the genetic analysis of pearl millet. Using 25 microsatellite markers, we analyzed genetic diversity in 46 wild and 421 cultivated accessions of pearl millet in Niger. We showed a significantly lower number of alleles and lower gene diversity in cultivated pearl millet accessions than in wild accessions. This result contrasts with a previous study using iso-enzyme markers showing similar genetic diversity between cultivated and wild pearl millet populations. We found a strong differentiation between the cultivated and wild groups in Niger. Analyses of introgressions between cultivated and wild accessions showed modest but statistically supported evidence of introgressions. Wild accessions in the central region of Niger showed introgressions of cultivated alleles. Accessions of cultivated pearl millet showed introgressions of wild alleles in the western, central, and eastern parts of Niger.

Weedy plants with intermediate (domesticated x wild) phenotypes occur in most pearl millet fields in West Africa, even in the absence of wild populations. They are usually found, in high numbers, both inside and outside of drills. Questions pertaining to the evolutionary dynamics of diversity within the pearl millet complex (domesticated-weedy-wild forms) were addressed in this study. The diversity of the different components of this complex sampled in two pearl millet fields in two villages of southwestern Niger was assessed at both molecular (AFLP) and morphological levels. Results show that, in both fields, weedy plants found outside of drills are morphologically distinct from weedy plants found inside drills, despite their close similarity at AFLP markers. The data suggest some introgression from the wild to the weedy population but nevertheless that the gene flow between the parapatric wild and domesticated populations is very low. This challenges the traditional view that regular hybridization between domesticated and wild pearl millets explains the abundance of these weedy plants despite farmers' seed selection. The level of genetic differentiation between fields from the two villages was low when considering domesticated and weedy plants. This could be explained by high gene flow resulting from substantial seed exchanges between farmers. The fact that it is very difficult for farmers to keep their own selected seeds, and the consequent substantial seed exchanges between them, is probably the main factor accounting for the maintenance and dispersal of weedy pearl millets in the region, even in areas where no wild forms have been observed.

This study describes the first detailed linkage maps of two bermudagrass species, Cynodon dactylon (T89) and Cynodon transvaalensis (T574), based on single-dose restriction fragments (SDRFs). The mapping population consisted of 113 F1 progeny of a cross between the two parents. Loci were generated using 179 bermudagrass genomic clones and 50 heterologous cDNAs from Pennisetum and rice. The map of T89 is based on 155 SDRFs and 17 double-dose restriction fragments on 35 linkage groups, with an average marker spacing of 15.3 cM. The map of T574 is based on 77 SDRF loci on 18 linkage groups with an average marker spacing of 16.5 cM. About 16 T89 linkage groups were arranged into four complete and eight into four incomplete homologous sets, while 15 T574 linkage groups were arranged into seven complete homologous sets, all on the basis of multi-locus probes and repulsion linkages. Eleven T89 and three T574 linkage groups remain unassigned. In each parent consensus maps were built based on alignments of homologous linkage groups. Four ancestral chromosomes were inferred after aligning T89 and T574 parental consensus maps using multi-locus probes. The inferred ancestral marker orders were used in comparisons to a detailed Sorghum linkage map using 40 common probes, and to the rice genome sequence using 98 significant BLAST hits, to find regions of colinearity. Using these maps we have estimated the recombinational length of the T89 and T574 genomes at 3,012 and 1,569 cM, respectively, which are 61 and 62% covered by our maps.

A considerable array of genomic resources are in place in pearl millet, and marker-aided selection is already in use in the public breeding programme at ICRISAT. This paper describes experiments to extend these publicly available resources to a single nucleotide polymorphism (SNP)-based marker system. A new marker system, single-strand conformational polymorphism (SSCP)-SNP, was developed using annotated rice genomic sequences to initially predict the intron-exon borders in millet expressed sequence tags (ESTs) and then to design primers that would amplify across the introns. An adequate supply of millet ESTs was available for us to identify 299 homologues of single-copy rice genes in which the intron positions could be precisely predicted. PCR primers were then designed to amplify approximately 500-bp genomic fragments containing introns. Analysis of these fragments on SSCP gels revealed considerable polymorphism. A detailed DNA sequence analysis of variation at four of the SSCP-SNP loci over a panel of eight inbred genotypes showed complex patterns of variation, with about one SNP or indel (insertion-deletion) every 59 bp in the introns, but considerably fewer in the exons. About two-thirds of the variation was derived from SNPs and one-third from indels. Most haplotypes were detected by SSCP. As a marker system, SSCP-SNP has lower development costs than simple sequence repeats (SSRs), because much of the work is in silico, and similar deployment costs and through-put potential. The rates of polymorphism were lower but useable, with a mean PIC of 0.49 relative to 0.72 for SSRs in our eight inbred genotype panel screen. The major advantage of the system is in comparative applications. Syntenic information can be used to target SSCP-SNP markers to specific chromosomal regions or, conversely, SSCP-SNP markers can be used to unravel detailed syntenic relationships in specific parts of the genome. Finally, a preliminary analysis showed that the millet SSCP-SNP primers amplified in other cereals with a success rate of about 50%. There is also considerable potential to promote SSCP-SNP to a COS (conserved orthologous set) marker system for application across species by more specifically designing primers to precisely match the model genome sequence.

Over the past 10 years, resources have been established for the genetic analysis of pearl millet, Pennisetum glaucum (L.) R. Br., an important staple crop of the semi-arid regions of India and Africa. Among these resources are detailed genetic maps containing both homologous and heterologous restriction fragment length polymorphism (RFLP) markers, and simple sequence repeats (SSRs). Genetic maps produced in four different crosses have been integrated to develop a consensus map of 353 RFLP and 65 SSR markers. Some 85% of the markers are clustered and occupy less than a third of the total map length. This phenomenon is independent of the cross. Our data suggest that extreme localization of recombination toward the chromosome ends, resulting in gaps on the genetic map of 30 cM or more in the distal regions, is typical for pearl millet. The unequal distribution of recombination has consequences for the transfer of genes controlling important agronomic traits from donor to elite pearl millet germplasm. The paper also describes the generation of 44 SSR markers from a (CA)n-enriched small-insert genomic library. Previously, pearl millet SSRs had been generated from BAC clones, and the relative merits of both methodologies are discussed.

To analyze quantitative trait loci (QTLs) affecting flooding tolerance and other physiological and morphological traits in Echinochloa crus-galli, a restriction fragment length polymorphism (RFLP) map was constructed using 55 plants of the F(2) population ( E. crus-galli var. praticola x E. crus-galli var. formosensis). One hundred forty-one loci formed 41 linkage groups. The total map size was 1,468 cM and the average size of linkage groups was 35.8 cM. The average distance between markers was 14.7 cM and the range was 0-37.2 cM. Early comparisons to the genetic maps of other taxa suggest appreciable synteny with buffelgrass ( Pennisetum spp.) and sorghum ( Sorghum spp.). One hundred ninty-one F(2) plants were used to analyze QTLs of flooding tolerance, plant morphology, heading date, number of leaves, and plant height. For flooding tolerance, two QTLs were detected and one was mapped on linkage group 24. Other traits, including plant morphology, heading date, number of leaves, and plant height were highly correlated. Three genomic regions accounted for most of the mapped QTLs, each explaining 2-4 of the significant marker-trait associations. The high observed correlation between the traits appears to result from QTLs with a large contribution to the phenotypic variance at the same or nearby locations.

A mapping population of 104 F(3) lines of pearl millet, derived from a cross between two inbred lines H 77/833-2 x PRLT 2/89-33, was evaluated, as testcrosses on a common tester, for traits determining grain and stover yield in seven different field trials, distributed over 3 years and two seasons. The total genetic variation was partitioned into effects due to season (S), genotype (G), genotype x season interaction (G x S), and genotype x environment-within-season interaction [G x E(S)]. QTLs were determined for traits for their G, G x S, and G x E(S) effects, to assess the magnitude and the nature (cross over/non-crossover) of environmental interaction effects on individual QTLs. QTLs for some traits were associated with G effects only, while others were associated with the effects of both G and G x S and/or G, G x S and G x E(S) effects. The major G x S QTLs detected were for flowering time (on LG 4 and LG 6), and mapped to the same intervals as G x S QTLs for several other traits (including stover yield, harvest index, biomass yield and panicle number m(-2)). All three QTLs detected for grain yield were unaffected by G x S interaction however. All three QTLs for stover yield (mapping on LG 2, LG 4 and LG 6) and one of the three QTLs for grain yield (mapping on LG 4) were also free of QTL x E(S) interactions. The grain yield QTLs that were affected by QTL x E(S) interactions (mapping on LG 2 and LG 6), appeared to be linked to parallel QTL x E(S) interactions of the QTLs for panicle number m(-2) on (LG 2) and of QTLs for both panicle number m(-2) and harvest index (LG 6). In general, QTL x E(S) interactions were more frequently observed for component traits of grain and stover yield, than for grain or stover yield per se.