Enzyme diversity in pearl millet (Pennisetum glaucum)

Summary Polymorphism in twelve genes coding for eight enzymes in pearl millet (Pennisetum glaucum (L.) R. Br.): alcohol dehydrogenases (ADH), catalases (CAT), -esterases (EST), glutamate oxaloacetate transaminases (GOT), malate dehydrogenases (MDH), 6-Phosphogluconate dehydrogenases (PGD), phosphoglucoisomerases (PGI) and phosphoglucomutases (PGM), was observed by electrophoresis on 74 cultivated samples and 8 wild samples from West Africa. Six genes: Est A, Adh A, Pgm A, Cat A, Pgi A, Pgd A contain 95% of the total variation. Principal component analyses and discriminant analyses of the 82 samples described by 46 allelic frequencies showed an almost complete separation into 3 groups: wilds, early maturing cultivars and late maturing cultivars. The early group has the highest enzyme diversity, with cultivated millets from Niger showing the most diversity. The high diversity of the early group and its extensive divergence from West-African wild millets suggest, firstly, the existence, elsewhere in Africa of other enzymatically different sources of wild millet, and secondly, the occurrence, prehistorically, of several different domestications. The late group of cultivars has the lowest variability and a relatively low coefficient of differentiation. This relatively homogeneous enzyme structure does not seem to be associated to ecology. A hypothesis is advanced suggesting that West African late-cultivars were derived from a common cultivated early complex. This complex must have been distributed across the Sudanian zone and must have been later sumitted to modifications by limited gene flow with local early maturing cultivars.     

An RFLP-based genetic map of pearl millet (Pennisetum glaucum)

Analysis of a sample of diverse pearl millet genotypes with 200 genomic DNA probes revealed this crop species to be extremely polymorphic. Among these genotypes, 85% of probes detected polymorphism using only two restriction enzymes, with an average pair-wise polymorphism between all of the probe-enzyme combinations of 56%. Two crosses were employed to construct an RFLP-based genetic map. In an intervarietal F₂ population, derived from a single F₁ plant, 181 loci were placed on a linkage map. The total length of this map, which comprised seven linkage groups, was 303 cM and the average map distance between loci was about 2 cM, although a few intervals in excess of 10 cM were present at the ends of a few linkage groups. Very few clones, including those which hybridized to more than one copy, detected more than one locus in the pearl millet genome. The analysis was complicated initially because 83 of the 181 loci mapped to a single linkage group. Analysis of a second cross identified a probable translocation breakpoint in the middle of this large linkage group.     

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     

Analysis of recombination rate in female and male gametogenesis in pearl millet (Pennisetum glaucum) using RFLP markers

Sex as a factor affecting recovered recombination in plant gametes was investigated in pearl millet, Pennisetum glaucum, by using reciprocal three-way crosses [(AxB)xCvCx(A x B)]. The two populations were mapped at 42 loci pre-selected to cover the majority of the genome. No differences in recombination distances were observed at the whole-genome level and only a few individual linkage intervals were found to differ, all in favour of increased recombination through the male. Distorted segregations found in the three-way crosses provide evidence of post-gametic selection for particular gene(s) or chromosome regions. The significance of these results for the design of pearl millet breeding programmes and inheritance experiments, as well as for other experimental strategies, is discussed.     

The ultrastructure of zygotic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of zygotic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Embryogenesis was initially characterized by the presence of a vacuolated egg cell and zygote. The increased presence of Golgi bodies in the zygote suggested it was metabolically more active than the egg cell. The first zygotic division resulted in a densely cytoplasmic apical cell and a highly vacuolated basal cell. The club-shaped proembryo displayed a large amount of endoplasmic reticulum (ER) and ribosomes, very few lipids, and a continuous gradient of vacuoles from the highly vacuolated basal suspensor cells to the densely cytoplasmic apical cells. The embryo had well-defined parts by 8 days after pollination, including shoot and root meristems, coleoptile, scutellum, provascular system, and the first leaf primordium. Large increases in ER, lipids, starch, and vacuoles occurred in the scutellum during the maturation of the embryo, except in the provascular cells. Throughout zygotic embryogenesis, embryo cells were connected by plasmodesmata except where intercellular spaces occurred. Ultrastructural, morphological, and histological observations of zygotic embryogenesis in pearl millet are in agreement with previous reports for other grass species.     

The ultrastructure of somatic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of somatic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Somatic embryogenesis was initiated from zygotic embryo explants cultured 8 d after pollination. Formation of a ridge of tissue began 3–4 d after culture (DAC) by divisions in the epidermal and subepidermal cells of the scutellum. Ridge formation was accompanied by a decrease in vacuoles, lipid bodies, and cell size, and an increase in endoplasmic reticulum (ER). Proembryonic cell masses (proembryoids) formed from the scutellar ridge by 10 DAC. Proembryoid cells had abundant Golgi bodies and ER while the amounts of lipids and starch varied. Somatic embryos developed from the proembryonic masses 13 DAC and by 21 DAC had all the parts of mature zygotic embryos. Although shoot and root primordia of somatic embryos were always less differentiated than those of zygotic embryos, scutellar cells of somatic and zygotic embryos had similar amounts of lipids, vacuoles, and starch. Somatic scutellar epidermal cells were more vacuolated than their zygotic counterparts. In contrast, somatic scutellar nodal cells were smaller and not as vacuolated as in zygotic embryos. Somatic embryogenesis was characterized by three phases of cell development: first, scutellar cell dedifferentiation with a reduction in lipids and cell and vacuole size; second, proembryoid formation with high levels of ER; and third, the development of somatic embryos that were functionally and morphologically similar to zygotic embryos.     

Construction of a BAC library of pearl millet, Pennisetum glaucum

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from pearl millet (Pennisetum glaucum), and used as a resource for the isolation of microsatellite sequences. The library contains a total of 159,100 clones with an average insert size of 90 kb, and corresponds to 5.8 haploid genome equivalents. The BAC library was pooled for screening by the polymerase chain reaction (PCR) as well as robotically gridded on high-density filters. PCR-based screening of a subset of the library (4.7 haploid genome equivalents) using five sequence-tagged site (STS) and six microsatellite markers identified between 2 and 11 positives superpools (5.4 on average). The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be less than 1% by hybridisation with a rice chloroplast probe. Received: 30 January 2000 / Accepted: 16 October 2000     

Associative diazotrophs of pearl millet (Pennisetum glaucum) from semi arid region--isolation and characterization

Diversity of the native diazotrophs associated with the rhizosphere of pearl millet (P. glaucumn), grown in nutritionally poor soils of semi-arid regions was studied with a view to isolate effective nitrogen fixing and plant growth stimulating bacteria with root associative characteristics. The native population varied from 10(3)-10(4) g(-1) of rhizosphere soil after 40 d growth and belonged to genera Azospirillum, Azotobacter and Klebsiella. Another non-diazotrophic root associative group was Pseudomonas sp., which also produced IAA and enhanced plant growth. Some of these rhizobacteria showed high in vitro acetylene reduction activity along with production of indole acetic acid. Out of 11 selected diazotrophs used as seed inoculants, M10B (Azospirillum sp.), M11E (Azotobacter sp.) and M12D4 (Klebsiella sp.) resulted in significant increase in total root and shoot nitrogen at 45 and 60 days of plant growth under pot culture conditions.     

Longevity of pearl millet (Pennisetum glaucum) seeds harvested at different stages of maturity

The storage potential of seeds harvested at weekly intervals after controlled pollination was studied in three diverse cytoplasmic male sterile pearl millet (Pennisetum glaucum) lines. In the first experiment in 1989, a comparison of p₅₀ (time for viability to decline to 50% during storage) among seeds of the line DSA 105A harvested 14, 21, 28, 35 and 42 days after pollination (DAP), and then stored at 35°C with 15% moisture content or 40°C with 13% moisture content, showed that those harvested 35 DAP had the greatest longevity. In the second experiment in 1990, a comparison of p₅₀ within the lines 5141A and L 67A harvested 28, 35 and 42 DAP, and then stored at 40°C with 13% moisture content, showed that seeds of both lines harvested 42 DAP had the greatest longevity. In both the seasons, and in all three lines, maximum seed longevity (p₅₀) was attained one week after physiological maturity (defined as the end of the grain filling period), which is therefore the optimum time of harvest to obtain good quality seeds for conservation.     

Isozymic classification of pearl millet (Pennisetum glaucum,Poaceae) landraces from Niger (West Africa)

The principal landraces of the pearl millet,Pennisetum glaucum (L.)R. Br., from Niger have been analysed for their genetic structure at eight enzyme systems coded by 12 loci and 46 alleles. Three groups have been identified: (1) early-maturing pearl millets, cultivated between 8° and 13°E longitude, including the oases from Aïr mountains; (2) early-maturing millets situated more to the west (1° and 8°E longitude), and (3) late-maturing millets. Group 1 shows the highest isozyme diversity. The differences between the accessions represent 8.8% of the total diversity and the differences between the three groups 4.5%. The accessions from groups 1 and 3 are the least distant. When considering pearl millets from areas outside Niger, the chadian and sudanese millets are enzymatically close to the Niger group 1. The pearl millets from Niger group 2 are close to millets from east Mali, northern Burkina Faso and Senegal, and the Niger group 3 to the late-maturing millets group from West Africa. This study should help breeders to select the landraces for improvement and parents for crosses from cultivars of Niger and introduced germ plasm.     

Genetic control of weediness traits and the maintenance of sympatric crop-weed polymorphism in pearl millet (Pennisetum glaucum)

Pearl millet (Pennisetum glaucum), a diploid outcrossing crop widely grown in semiarid tropics, provides a unique extant material for the study of crop-weed interactive evolution. Co-occurrence of a weedy, shattering type of pearl millet with the cultivated one is the rule in the traditional agro-ecosystem in the Sahel zone of Africa. Selfed progeny of weed-type plants invariably segregated into distinct weed and crop types in an approximately 3:1 ratio. Genetic analysis using a cleaved amplified polymorphic sequence (CAPS) marker strongly suggested that a series of differences between the crop and the weed types are determined by a single putative supergene that has two allelic types, C and W. The crop-type plants are CC homozygotes, and the weed-type plants are CW heterozygotes. WW homozygotes are sterile and rare in the field. Thus, the CW weed plants recurrently arise from crosses between the crop and the weed, as well as from crosses among the weed-type plants. The weed type appears to have a sufficiently high fitness to maintain the W allele in the pearl millet population, resulting in the perpetuation of this unique crop-weed polymorphism.     

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.     

Genetic analysis of short term callus culture and morphogenesis in pearl millet,Pennisetum glaucum

Genetic analysis of five in vitro characters was made through a 5 × 5 diallel analysis using callus derived from immature inflorescence segments of pearl millet (Pennisetum glaucum). The characters studied were: — volume of total callus, — frequency of embryogenic calli, — embryogenic callus volume, — growth rate in terms of increase in fresh weight, and — frequency of regeneration. High heritability values and heterosis were noticed for all these characters except for E callus frequency. Additive gene action was predominant for callus growth rate and frequency of regeneration. Of the five inbreds, IP 1346 (= P5) was found to be the best genetic background for embryogenic callus volume, embryogenic growth rate and frequency of regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.     

The expression and perpetuation of inherent somatic variation in regenerants from embryogenic cultures of Pennisetum glaucum (L.) R. Br. (pearl millet)

Summary Genetic analysis was conducted on the qualitative and quantitative traits of sexual progeny derived from embryogenic cultures of two inbred lines of Pennisetum glaucum (L.) R. Br. (pearl millet). These lines included a genetically stable inbred of Tift 23 BE and a genetic marker line, derived from Tift 23BE, which bore qualitative genetic markers for a dominant purple plant trait (P) and two recessive traits, early flowering (e₁) and yellow stripe (ys). Tissue culture regenerant populations (R₀) and progeny populations (R₁) produced from these plants by selfing showed no qualitative genetic variation when derived from the genetically stable inbred Tift 23BE. In contrast, stably inherited qualitative variation for a number of genetic markers was observed in R₀, R₁, and R₂ progeny of the genetic marker line. In a population of 1,911 plants regenerated over a 12-month period, 0.02% of the population lost or showed reduced expression of the purple plant trait and 92% of plants were chlorophyll deficient. Plants showing reduction or loss of anthocyanin synthesis also flowered later. None of the purple plants showed any significant variation in flowering time. The incidence of chlorophyll deficiency increased with time in culture, 51 % of the progeny regenerated after 1 month were chlorophyll deficient, while 100% of the plants regnerated after 12 months were chlorophyll deficient. Qualitative variation was also observed in control populations of the genetic marker line where 1 plant in a total of 1,010 lacked purple pigmentation and a total of 6% showed chlorophyll variation in the first generation (S₀). The presence of qualitative variation in controls suggests that the inherent variation present in the original explant was expressed and perpetuated in vitro. Quantitative variation was observed for a number of traits in the first sexual cycle (R₁) of the marker line but did not occur in a subsequent generation, suggesting that this variation was epigenetic.     

Somatic embryogenesis in pearl millet (Pennisetum glaucum): Strategies to reduce genotype limitation and to maintain long-term totipotency

Three genotypes of Pearl millet were screened in vitro for induction of embryogenic callus, somatic embryogenesis and regeneration. Shoot apices excised from in vitro germinated seedlings or immature embryos isolated from green house established plants were used as primary explants. The frequency of embryogenic callus initiation was significantly higher in shoot apices in comparison with immature zygotic embryos. Moreover, differences between genotypes were minimal when using shoot apices. Friable embryogenic calli (type II) developed on the initial nodular calli after 1 to 3 months of culture. The frequency of type II callus is related to the composition of the maintenance medium and they were more often found in ageing cultures. The transfer of embryogenic calli onto auxin-free medium was sufficient for inducing somatic embryo development in short-term culture (3 months) while a progressive loss in regeneration potential was observed with increasing time of subcultures. Maturation of embryogenic calli on medium supplemented with activated charcoal, followed by germination of somatic embryos on medium supplemented with gibberellic acid, restored regeneration in long-term cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular evolutionary characterization of an Activator (Ac)-like transposable element sequence from pearl millet (Pennisetum glaucum) (Poaceae)

We present data on the evolution of the Ac/Ds family of transposable elements in select grasses (Poaceae). A defective Ac-like element was cloned from a DNA library of the grass Pennisetum glaucum (pearl millet) and its entire 4531 bp sequence has been determined. When the pearl millet Ac-like sequence is aligned with the maize Ac sequence, it is found that there is approximately 70% DNA similarity in the central region spanning most of maize Ac exon II and all of exon III. In addition, there are two smaller regions of similarity at the Ac terminii. Besides these three major structural similarities, Pennisetum Ac has two large regions, one 5 and one 3, that show little similarity to Zea Ac. Furthermore, most of the sequences corresponding to intron II in maize Ac are absent in pearl millet Ac. Kimura's evolutionary distance between the central region of maize and pearl millet Ac sequences is estimated to be 0.429±0.020 nucleotide substitutions per site. This value is not significantly different from the average number of synonymous substitutions for coding regions of the Adh1 gene between maize and pearl millet, which is 0.395±0.051 nucleotide substitutions per site. If we assume Ac and Adh1 divergence times are equivalent between maize and pearl millet, then the above calculations suggest Ac-like sequences have probably not been strongly constrained by natural selection. Conserved DNA and amino acid sequence motifs are also examined. The level of DNA sequence divergence between maize and pearl millet Ac sequences, the estimated date when maize and pearl millet diverged (25–40 million years ago), coupled with their reproductive isolation/lack of current genetic exchange, all support the theory that Ac-like sequences have not been recently introduced into pearl millet from maize. Instead, Ac-like sequences were probably present in the progenitor of maize and pearl millet and have thus existed in the grasses for at least 25 million years.     

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