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.     

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.     

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.     

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.     

Effect of the timing of water deficit on growth, phenology and yield of pearl millet (Pennisetum glaucum (L.) R. Br.) grown in Sahelian conditions

Several studies conducted under high input conditions have indicatedlittle susceptibility of pearl millet to water deficit untillearly grain filling, because the losses in main shoot productionwere fully compensated by increased tiller fertility. The presentstudy assessed the impact of water deficits at three developmentstages: prior to flowering (S30), at the beginning of flowering(S45), and at the end of flowering (S60) in pearl millet grownin experimental conditions similar to Sahelian farming conditions.It included a control irrigation treatment simulating the naturaldistribution of rainfall throughout the cropping season. Bothbiomass production and grain yield were severely reduced byS30 and S45, while S60 had no effect. In S30 and S45, the floweringof tillers was delayed or totally inhibited. In both of thesetreatments, the low number of productive tillers did not compensatefor damage to panicle initiation and flowering of the main shoot.All treatments maintained green leaves on the main shoot duringthe grain filling period, and in S30 leaf growth recovered frommid-season drought. These results illustrate how pearl milletmostly escapes drought by matching its phenology to the meanrainfall distribution in the Sahel. In the case of mid-seasondrought, some late productive tillers and the maintenance ofgreen leaf biomass of the main shoots limited, but did not overcome,the yield losses. This study stresses the importance of agro-ecologicalconditions in control treatments, particularly the water regimeand crop density, when assessing crop drought resistance. Key words: Drought resistance strategy, experimental conditions, main shoots, tillers, Pennisetum glaucum     

Patterns of molecular and phenotypic diversity in pearl millet [<Emphasis Type="Italic">Pennisetum glaucum</Emphasis>(L.) R. Br.] from West and Central Africa and their relation to geographical and environmental parameters

Background  

The distribution area of pearl millet in West and Central Africa (WCA) harbours a wide range of climatic and environmental conditions as well as diverse farmer preferences and pearl millet utilization habits which have the potential to lead to local adaptation and thereby to population structure. The objectives of our research were to (i) assess the geographical distribution of genetic diversity in pearl millet inbreds derived from landraces, (ii) assess the population structure of pearl millet from WCA, and (iii) identify those geographical parameters and environmental factors from the location at which landraces were sampled, as well as those phenotypic traits that may have affected or led to this population structure. Our study was based on a set of 145 inbred lines derived from 122 different pearl millet landraces from WCA.     

An integrated genetic map and a new set of simple sequence repeat markers for pearl millet, <Emphasis Type="Italic">Pennisetum glaucum</Emphasis>

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.     

Correlation between germination and adult stage under water stress in some Tunisian autochthonous pearl millet ecotypes (Pennisetum glaucum (L.) R. Br.)

This study compared the behaviour of six Tunisian autochthonous pearl millet ecotypes collected through the Tunisian territory under drought, from germination to maturity. Moderate water stress was found to improve germination due to better root elongation; it did not have any notable consequence on the yield components of pearl millet. When drought is severe, germination is compromised, radicle and epicotyle lengths decrease, the size of the plant is reduced as well as the surface of its flag sheet, and all the components of yield are penalized. This effect varies according to the ecotype's morphology and its geographical origin. In the same way, we could establish important correlations that show that radicle and epicotyle lengths are related to the final size of the pearl millet plant. All this shows that the study of germination is a scientific tool that can have agronomic repercussions, practical and exploitable.     

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.     

Genetic architecture of purple pigmentation and tagging of some loci to SSR markers in pearl millet, Pennisetum glaucum (L.) R. Br

This report describes the construction of integrated genetic maps in pearl millet involving certain purple phenotype and simple sequence repeat (SSR) markers. These maps provide a direct means of implementing DNA marker-assisted selection and of facilitating "map-based cloning" for engineering novel traits. The purple pigmentation of leaf sheath, midrib and leaf margin was inherited together 'en bloc' under the control of a single dominant locus (the 'midrib complex') and was inseparably associated with the locus governing the purple coloration of the internode. The purple panicle was caused by a single dominant locus. Each of the three characters (purple lamina, purple stigma and purple seed) was governed by two complementary loci. One of the two loci governing purple seed was associated with the SSR locus Xpsmp2090 in linkage group 1, with a linkage value of 22 cM, while the other locus was associated with the SSR locus Xpsmp2270 in linkage group 6, with a linkage value of 23 cM. The locus for purple pigmentation of the midrib complex was either responsible for pigmentation of the panicle in a pleiotropic manner or was linked to it very closely and associated with the SSR locus Xpsmp2086 in linkage group 4, with a suggestive linkage value of 21 cM. A dominant allele at this locus seems to be a prerequisite for the development of purple pigmentation in the lamina, stigma and seed. These findings suggest that the locus for pigmentation of the midrib complex might regulate the basic steps in anthocyanin pigment development by acting as a structural gene while other loci regulate the formation of color in specific plant parts.     

Role of seed flow on the pattern and dynamics of pearl millet (<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> [L.] R. Br.) genetic diversity assessed by AFLP markers: a study in south-western Niger

We studied the regional genetic diversity and seed exchange dynamics of pearl millet landraces in south-western Niger. The genetic study was based on AFLP markers. We found significant genetic differentiation between landraces in different geographical areas of south-western Niger. However, the degree of differentiation was low insofar as only 1.9% of the total molecular diversity was due to regional differentiation, suggesting a relatively high gene flow. Anthropologic studies on farming practices have suggested that seed exchanges between farmers on a large geographical scale probably make a considerable contribution to this result. In order to test this hypothesis, the effects of seed exchange on the genetic diversity of landraces was analyzed on seed samples from two distant villages in contrasting areas of south-western Niger. Seeds imported by farmers into the southern village of Sina Koara did not differ significantly from locally grown landraces. By contrast, in the northern village of Alzou, several samples were genetically different from locally grown landraces and closer to southern accessions. These data suggest that the seed flow is preferentially from south to north, i.e. from an area with more favorable rainfall conditions. The potential consequences for the genetic diversity and adaptation of northern pearl millet landraces are discussed.     

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.     

Ability of <Emphasis Type="Italic">Emericella rugulosa</Emphasis> to mobilize unavailable P compounds during Pearl millet [<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.] crop under arid condition

Phosphate solubilizing microorganisms are ubiquitous in soils and could play an important role in supplying P to plants where plant unavailable P content in soil was more. A phosphatase and phytase producing fungus Emericella rugulosa was isolated and tested under field condition (Pearl millet as a test crop) in a loamy sand soil. In the experimental soil 68% organic phosphorous was present as phytin; less than 1% of phosphorous was present in a plant available form. The maximum effect of inoculation on different enzyme activities (acid phosphatase, alkaline phosphatase, phytase, and dehydrogenase) was observed between 5 and 8 weeks of plant age. The depletion of organic P was much higher than mineral and phytin P. The microbial contribution was significantly higher than the plant contribution to the hydrolysis of the different P fractions. A significant improvement in plant biomass, root length, seed and straw yield and P concentration of root and shoot resulted from inoculation. The results suggest that Emericella rugulosa produces phosphatases and phytase, which mobilize P and enhance the production of pearl millet.     

Fertile transgenic pearl millet [<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.] plants recovered through microprojectile bombardment and phosphinothricin selection of apical meristem-, inflorescence-, and immature embryo-derived embryogenic tissues

Pearl millet [ Pennisetum glaucum (L.) R. Br.] is a drought-tolerant cereal crop used for grain and forage. Novel traits from outside of the gene pool could be introduced provided a reliable gene-transfer method were available. We have obtained herbicide-resistant transgenic pearl millet plants by microprojectile bombardment of embryogenic tissues with the bar gene. Embryogenic tissues derived from immature embryos, inflorescences and apical meristems from diploid and tetraploid pearl millet genotypes were used as target tissues. Transformed cells were selected in the dark on Murashige and Skoog medium supplemented with 2 mg/l 2,4-D and 15 mg/l phosphinothricin (PPT). After 3-10 weeks in the dark, herbicide-resistant somatic embryos were induced to germinate on MS medium containing 0.1 mg/l thidiazuron and 0.1 mg/l 6-benzylaminopurine. Plants were transferred to the greenhouse after they were rooted in the presence of PPT and had passed a chlorophenol red assay (the medium turned from red to yellow). Transgenic plants were recovered from bombardments using intact pAHC25 plasmid DNA, a gel-purified bar fragment, or a mixture of pAHC25 plasmid or bar fragment and a plasmid containing the enhanced green fluorescent protein ( gfp) gene (p524EGFP.1). Analyses by the polymerase chain reaction, Southern blot hybridization, GFP expression, resistance to herbicide application, and segregation of the bar and gfp genes confirmed the presence and stable integration of the foreign DNA. Transformed plants were recovered from all three explants, although transformation conditions were optimized using only the tetraploid inflorescence. Time from culture initiation to rooted transgenic plant using the tetraploid inflorescence ranged from 3-4 months. Seven independent DNA/gold precipitations were used to bombard 52 plates, 29 of which produced an average of 5.5 herbicide-resistant plants per plate. The number of herbicide-resistant plants recovered per successful bombardment ranged from one to 28 and the frequency of co-transformation with gfp ranged from 5% to 85%.     

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.     

Quantitative trait loci mapping of heat tolerance in broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">italica</Emphasis>) using genotyping-by-sequencing

Key message

Five quantitative trait loci and one epistatic interaction were associated with heat tolerance in a doubled haploid population of broccoli evaluated in three summer field trials.

Abstract

Predicted rising global temperatures due to climate change have generated a demand for crops that are resistant to yield and quality losses from heat stress. Broccoli (Brassica oleracea var. italica) is a cool weather crop with high temperatures during production decreasing both head quality and yield. Breeding for heat tolerance in broccoli has potential to both expand viable production areas and extend the growing season but breeding efficiency is constrained by limited genetic information. A doubled haploid (DH) broccoli population segregating for heat tolerance was evaluated for head quality in three summer fields in Charleston, SC, USA. Multiple quantitative trait loci (QTL) mapping of 1,423 single nucleotide polymorphisms developed through genotyping-by-sequencing identified five QTL and one positive epistatic interaction that explained 62.1% of variation in heat tolerance. The QTL identified here can be used to develop markers for marker-assisted selection and to increase our understanding of the molecular mechanisms underlying plant response to heat stress.