Evolutionary history of pearl millet (Pennisetum glaucum [L.] R. Br.) and selection on flowering genes since its domestication

The plant domestication process is associated with considerable modifications of plant phenotype. The identification of the genetic basis of this adaptation is of great interest for evolutionary biology. One of the methods used to identify such genes is the detection of signatures of selection. However, domestication is generally associated with major demographic effects. It is therefore crucial to disentangle the effects of demography and selection on diversity. In this study, we investigated selection in a flowering time pathway during domestication of pearl millet. We first used a random set of 20 genes to model pearl millet domestication using approximate Bayesian computation. This analysis showed that a model with exponential growth and wild-cultivated gene flow was well supported by our data set. Under this model, the domestication date of pearl millet is estimated at around 4,800 years ago. We assessed selection in 15 pearl millet DNA sequences homologous to flowering time genes and showed that these genes underwent selection more frequently than expected. We highlighted significant signatures of selection in six pearl millet flowering time genes associated with domestication or improvement of pearl millet. Moreover, higher deviations from neutrality were found for circadian clock-associated genes. Our study provides new insights into the domestication process of pearl millet and shows that a category of genes of the flowering pathway were preferentially selected during pearl millet domestication.     

Agrobacterium tumefaciens-mediated genetic transformation and production of stable transgenic pearl millet (Pennisetum glaucum [L.] R. Br.)

A synthetic gene encoding the antimicrobial peptide magainin has been designed, cloned, and engineered for regulation by the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (nos) terminator. The plant expression cassette was introduced into the vector pSB11-bar (with the glyphosate [Basta®] resistance gene, bar), and the recombinant plasmid was mobilized into Agrobacterium tumefaciens strain LBA4404 for the generation of a super-binary vector pSB111-bar-mag. Magainins, positively charged amphipathic antimicrobial peptides of 21–26 amino acid residues, are potential candidates for the development of disease resistant transgenic plants. Six-wk-old pearl millet (Pennisetum glaucum [L.] R. Br.) calli and A. tumefaciens harboring pSB111-bar-mag were cocultivated in a medium supplemented with 400 μM acetosyringone and 3.3 mM l-cysteine. Out of 3,000 infected calli subjected to selection on phosphinothricin medium, 82 calli showed sectors of healthy growth, resulting in a transformation frequency of 2.73%. Among 13 Basta-tolerant putative transformed plants, eight were fertile and their transgenic nature and expression of the transgene was characterized by Southern and Northern blot analyses, respectively. Subsequent T₁ progenies co-segregated for bar and magainin genes in a 3:1 ratio. Bioassays that challenged the eight transgenic T₁ plant progenies against three highly virulent strains of Sclerospora graminicola, viz., Sg 384, Sg 445, and Sg 492 failed to show resistance. The failure of synthetic magainin gene to confer resistance against downy mildew in pearl millet may be attributed to the complexity of the cell wall and cell membrane of the pathogen.     

Genetic variation in grain-filling ability in dwarf pearl millet [Pennisetum glaucum (L.) R. Br.] restorer lines

The d2 dwarfing gene in pearl millet [Pennisetum glaucum (L.) R. Br.] carries a yield penalty due to an associated reduction in individual grain mass. This reduction, however, varies with genetic background, indicating that it may be possible to select against poor grain filling in d2 dwarfbackgrounds, given an effective measure of grain filling. This study was conducted to assess genetic variability forgrain-filling ability (in contrast to simply grain size),and its relationship to grain yield,indwarf pearl milletrestorer (R) lines. The grain-filling ability (GFA) of an individual R line was defined as the least squares estimate of its effect on individual grain mass in the analysis of variance, following a linear covariance adjustment for grain number. The study was based on 93dwarf hybrids involving31 d2 dwarfR-lines, evaluated over 3 years. Half of the variation in individual grain mass in the 93 hybrids was related to variation in grain number. Covariance adjustment in individual grain mass for grain number resulted in highly significant differences among hybrids and R lines in GFA. The R-line combining ability for GFA accounted for 26% of the variation in the R-line combining ability for yield, compared to 46% for the combining ability for grain number, and just 8% for the combining ability of individual grain mass. The combining ability for GFA was independent of the combining ability for various pre-flowering effects, including grain number, but was related to the combining ability for individual grain mass and harvest index. Improvement in individual grain mass achieved through selection for GFA should translate directly into yield improvement, whereasimprovement by direct selectionfor individual grain mass is less-likely to do so. Received: 9 April 2000 / Accepted: 16 May 2000     

Host plant resistance to grain mould in germplasm accessions of pearl millet (Pennisetum glaucum [L.] R. Br.)

Abstract

The paucity of information on the moulds in Indian pearl millet (Pennisetum glaucum) led to the studies that were conducted at ICRISAT, India to evaluate (a) 447 germplasm accessions of 32 countries for mould reaction in rainy season, (b) threshed grain mould rating (TGMS) and mycoflora on grains of each accession, and (c) mould scores in field and in vitro. Post physiological maturity evaluation showed that 16% of the accessions secured a mould rating of 2. In TGMS, 18% were mould free and 57% secured a rating of 2 on a 1 – 9 scale. Assessment of twenty representative accessions in vitro against individual and mixed conidial suspensions (1 × 10⁽⁶⁾ conidia ml^(?1)) of Fusarium moniliforme, F. pallidoroseum and Curvularia pennisetti indicated significant correlation (r = 0.97) between the overall field and in vitro scores of mixed spores inoculations. The mycoflora for TGMS in blotter test revealed that Fusarium moniliforme, F. pallidoroseum, Curvularia pennisetti, Helminthosporium spp., Alternaria spp. and Colletotrichum spp. to be the major fungi affecting pearl millet grain. It is advisable to harvest panicles at the physiological maturity stage to obtain better quality grains. A strong negative correlation between TGMS and % GS (r = 0.4601) and positive correlation between TGMS and % UGS (r = 0.4654) indicated that, the lesser the threshed grain mould rating higher the % seed germination.     

Hydrous characteristics of pearl millet (Pennisetum glaucum (L.) R. Br.) under drought

The variations in the hydrous characteristics - leaf water potential (psi(F)) and relative water content (TRE) - of plants indicate their hydrous status and also their capacity to incorporate water, particularly in situations of hydrous stress. These characteristics were used to differentiate the behaviour of six autochthonous ecotypes of pearl millet (Pennisetum glaucum (L.) R. Br.) subjected to various hydrous modes. The results showed that the psi(F) and the TRE evolve in the same direction and decrease in the presence of hydrous stress. These hydrous characteristics are also very important, since they condition the yield. The study also showed that the six pear millet ecotypes present two standard behaviours: the first type is characterized by an important fall of the psi(F) and TRE, representing an osmotic adjustment of adaptive type, whereas the second group showed a weak reduction of leaf water potential while maintaining a TRE appreciably higher, which could be the result of an important reduction of the osmotic potential due to an active accumulation of solutes (constitutive osmotic adjustment). Moreover, the genetic variability of ecotypes with respect to the hydrous stress does not seem related to the geographical origin of the millet ecotypes, but rather to their phenology and their morphology.     

Quantitative analysis of ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.)

Ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.) were quantified using morphometric techniques. The total area per cell profile and the cell volume percentage of the whole cell, endoplasmic reticulum (ER), Golgi bodies, mitochondria, nuclei, lipids, plastids, starch grains and vacuoles were measured and comparisons made between three zygotic and three somatic embryo developmental stages. All measurements were taken from scutellar or scutellar-derived cells. Zygotic embryogenesis was characterized by increases in cell size, lipids, plastids, starch, Golgi bodies, mitochondria and ER. Somatic embryogenesis was characterized by two phases of cell development: (1) the dedifferentiation of scutellar cells involving a reduction in cell and vacuole size and an increase in cell activity during somatic proembryoid formation and (2) the development of somatic embryos in which most cell organelle quantities returned to values found in late coleoptile or mature predesiccation zygotic stages. In summary, although their developmental pathways differed, the scutella of somatic embryos displayed cellular variations which were within the ranges observed for later stages of zygotic embryogenesis.     

Quantitative trait loci associated with constitutive traits control water use in pearl millet [Pennisetum glaucum (L.) R. Br.]

There is substantial genetic variation for drought adaption in pearl millet in terms of traits controlling plant water use. It is important to understand genomic regions responsible for these traits. Here, F₇ recombinant inbred lines were used to identify quantitative trait loci (QTL) and allelic interactions for traits affecting plant water use, and their relevance is discussed for crop productivity in water‐limited environments. Four QTL contributed to increased transpiration rate under high vapour pressure deficit (VPD) conditions, all with alleles from drought‐sensitive parent ICMB 841. Of these four QTL, a major QTL (35.7%) was mapped on linkage group (LG) 6. The alleles for 863B at this QTL decreased transpiration rate and this QTL co‐mapped to a previously detected LG 6 QTL, with alleles from 863B for grain weight and panicle harvest index across severe terminal drought stress environments. This provided additional support for a link between water saving from a lower transpiration rate under high VPD and drought tolerance. 863B alleles in this same genomic region also increased shoot weight, leaf area and total transpiration under well‐watered conditions. One unexpected outcome was reduced transpiration under high VPD (15%) from the interaction of two alleles for high VPD transpiration (LG 6 (B), 40.7) and specific leaf mass and biomass (LG 7 (A), 35.3), (A, allele from ICMB 841, B, allele from 863B, marker position). The LG 6 QTL appears to combine alleles for growth potential, beneficial for non‐stress conditions, and for saving water under high evaporative demand, beneficial under stressful conditions. Mapping QTL for water‐use traits, and assessing their interactions offers considerable potential for improving pearl millet adaptation to specific stress conditions through physiology‐informed marker‐assisted selection.     

Phylogeny and origin of pearl millet (Pennisetum glaucum [L.] R. Br) as revealed by microsatellite loci

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.     

Diversity of wild and cultivated pearl millet accessions (Pennisetum glaucum [L.] R. Br.) in Niger assessed by microsatellite markers

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.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Cedric Mariac and Viviane Luong have contributed equally to this work.     

Efficient regeneration of pearl millet (Pennisetum glaucum (L.) R. Br.) from shoot tip cultures

A simple, genotype-independent and efficient method for plant regeneration using shoot tip explants of pearl millet (Pennisetum glaucum) was established. Linsmaier and Skoog (LS) medium supplemented with 2,4-dichloro-phenoxyacetic acid (2.5 mg l(-1)) and kinetin (0. 2 mg l(-1)) was used for induction of embryogenic calli. Development of numerous somatic embryos was observed within 10 days after transferring onto Murashige and Skoog (MS) medium supplemented with 6-benzyl aminopurine (2 mg l(-1)) and indole 3-butyric acid (0. 5 mg l(-1)) under light (16 hr photoperiod). Histological observations confirmed the origin of somatic pro-embryoids and globular embryoids. Regenerated plants established in soil, grew normally and produced fertile seeds. RAPD analysis also revealed genetic uniformity of the regenerants. The short duration of time taken for regeneration (30-35 days) and its high frequency (78-87%) makes this system highly suitable for applications such as genetic transformation.     

Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew

Transgenic pearl millet lines expressing pin gene—exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola—were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T₀, 2T₀, and 3T₀ showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T₁ progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T₁ plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T₁ plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T₂ progenies of 1T₀ conformed to dihybrid segregation of 15 resistant:1 susceptible plants.     

Efficient in vitro plant regeneration from immature zygotic embryos of pearl millet [Pennisetum glaucum (L.) R. Br.] and Sorghum bicolor (L.) Moench

We report here an in vitro culture system that provides reliable, highly efficient regeneration from immature embryos of pearl millet [Pennisetum glaucum (L.) R. Br.] and sorghum [Sorghum bicolor (L.) Moench]. Immature embryos were isolated 10-20 days after pollination and cultured on various L3 media. The influence of different parameters during the callus induction phase was examined with respect to the regeneration rate: (1) the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and various cytokinins; (2) the addition of AgNO₃; (3) the use of maltose or sucrose as a carbon source. Modifications in the phytohormones alone resulted in the regeneration of fertile sorghum plants at high efficiency. Significant increases in the regeneration rates of pearl millet genotypes were achieved by the combination of sucrose as a carbon source and silver nitrate as a potential ethylene inhibitor.     

In vitro morphogenesis of pearl millet [Pennisetum glaucum (L.) R.Br.]: efficient production of multiple shoots and inflorescences from shoot apices

 This report presents a procedure for high-frequency multiple shoot production from cultured shoot apical meristems of pearl millet [Pennisetum glaucum (L.) R. Br.]. Shoot apices from 1-week-old aseptically germinated seedlings were cultured in vitro on MS medium containing various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) with biweekly subculture. A low concentration of 2,4-D coupled with four different concentrations of BA induced the production of adventitious shoots from the enlarged shoot apical meristems. Somatic embryogenesis was also observed at higher concentrations of BA. The use of higher levels of 2,4-D resulted in callusing of shoot apical meristems, while the shoot tips produced many leaves and in vitro flowering in 2,4-D-free media containing BA. All four pearl millet genotypes produced similar results. Fertile pearl millet plants were produced from in vitro-produced multiple shoots. Received: 1 April 1999 / Revision received: 8 July 1999 / Accepted: 17 August 1999     

Water saving traits co-map with a major terminal drought tolerance quantitative trait locus in pearl millet [Pennisetum glaucum (L.) R. Br.]

Low transpiration rates in pearl millet under fully irrigated conditions decrease plant water use at vegetative stage and then increase the water availability during grain filling and finally the terminal drought tolerance. Hundred and thirteen recombinant inbred lines developed from a cross between H77/833-2 and PRLT2/89-33 (terminal drought-sensitive?×?tolerant genotype) were evaluated to map transpiration rate (Tr, a proxy for canopy conductance), organ weights, leaf area and thickness and to study their interactions. Transpiration rate was increased by two H77/833-2 and two PRLT2/89-33 alleles on linkage group (LG) 2, whose importance depended on the vapor pressure deficit. The two H77/833-2 and one PRLT2/89-33 alleles co-mapped to a previously identified major terminal drought tolerance quantitative trait locus (QTL), although in a much smaller genetic interval. The other Tr allele from H77/833-2 also enhanced biomass dry weight and co-located with a formerly identified stover and tillering QTL. Leaf characteristics were linked to two loci on LG7. Plant water use was increased and decreased by different loci combinations for Tr, tillering and leaf characteristics, whose respective importance depended on the environmental conditions. Therefore, different alleles influence plant water use and have close interactions with one another and with the environment, so that different ideotypes for plant water use exist or could be designed from specific allele combinations conferring particular physiological characteristics for specific adaptation to a range of terminal drought conditions.     

Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T₁ progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome.     

Genotype identification and assessment of genetic relationships in pearl millet [Pennisetum glaucum (L.) R. Br] using microsatellites and RAPDs

 The potential of DNA markers such as microsatellites, minisatellites and RAPDs was investigated in pearl millet [Pennisetum glaucum (L.) R. Br] with respect to their abundance and variability. Southern analysis, using 22 different di-, tri-, tetra- and penta-oligonucleotide probes and five minisatellite probes, identified (GATA)₄ as the most useful probe for the detection of multiple polymorphic fragments among pearl millet cultivars and landraces from India. The clustering patterns of pearl millet cultivars and landraces based on (GATA)₄ and RAPD (randomly amplified polymorphic DNA) markers differed. The landraces, representing eight states in India, could not be grouped based on their geographical distribution with the DNA markers. RAPD analysis revealed a high degree of genetic diversity among the cultivars and landraces employed in this study. The probability of an identical match by chance for any two genotypes using (GATA)₄ and RAPDs was 3.02×10^-20 for cultivars and 5.2×10^-9 for landraces. The microsatellite (GATA)₄ and RAPDs provide useful tools for genotype identification and for the assessment of genetic relationships in pearl millet. Received: 19 October 1997 / Accepted: 9 December 1997     

Genetic control of domestication traits in pearl millet (Pennisetum glaucum L., Poaceae)

Quantitative trait loci (QTLs) controlling the morphological differences between pearl millet (Pennisetum glaucum ssp. glaucum) and its wild ancestor (Pennisetum glaucum ssp. monodii, form mollissimum) were investigated in a cultivated/wild F₂ population by means of RFLP markers. The most critical adaptive changes resulting from the domestication process involved the spikelet structure: non-shedding seeds with reduced bracts and bristles and long involucral pedicel. Major differences also concerned characters describing the plant architecture, phenology and spike sizes. Many morphological differences could be attributed to the effect of a small number of loci with relatively large effects. These loci are mainly concentrated on four linkage groups (2, 5, 6 and 7). The loss of shedding ability, due to the absence of a functional abscission layer, is controlled by a single locus on linkage group 6 (al6). Genetic control of the other spikelet traits involved factors with large effects which are located in the region of linkage group 6 close to al6 and to an esterase gene, Esterase-E. Moreover, QTLs with large effects on plant and spike morphology traits such as plant height, number of spikes and weight of the spike were also mapped on linkage groups 6 and 7. This strong linkage of factors in the domestication syndrome may be involved in the maintenance of the phenotypic identity of wild and cultivated populations in sympatry. This result also brings new arguments in the understanding of the domestication process of this allogamous crop. Received: 8 March 1999 / Accepted: 29 April 1999     

Long-term storage of Pennisetum glaucum (L.) R. Br. pollen

Summary Pearl millet [Pennisetum glaucum (L.) R. Br.] pollen has been successfully stored for 2,615 and 2,911 days at -18° and -73 °C, respectively, and continues to be viable. Viability of pollen stored at -73 °C appears to be little affected either by pollen storage moisture contents below 7.2% or by storage in glass vial or zip-lock plastic bag containers. Pollen moisture content appears to be more critical for maintaining viability at -18°C than at -73°C. Glass vials appear to be more desirable for longer term (>3 years) storage at -18°C.