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.

Further evidence that a terminal drought tolerance QTL of pearl millet is associated with reduced salt uptake.

Earlier, we established that a major drought tolerance QTL on linkage group 2 of pearl millet is also associated with reduced salt uptake and enhanced growth under salt stress. Present study was undertaken to re-assess the performance of drought tolerant (PRLT 2/89-33) and drought sensitive (H 77/833-2) parents along with two QTL-NILs (ICMR 01029 and ICMR 01040), under salinity stress specifically imposed during post-flowering growth stages when plants had developed their ion sinks in full. Time course changes in ionic accumulation and their compartmentalization in different plant parts was studied, specifically to monitor and capture changes conferred by the two alleles at this QTL, at small intervals. Amongst different plant parts, higher accumulation of toxic ion Na+ was recorded in roots. Further, the Na+ concentration in roots of the testcross hybrid of the drought-sensitive parent (H 77/833-2) reached its maximum at ECiw 15 dS m-1 within 24 h after salinity imposition, whereas it continued to increase with time in the testcross hybrids of the drought tolerant parent PRLT 2/89-33 as well as those of its QTL-NILs (ICMR 01029 and ICMR 01004) and reached at its maximum at 120 h stage. Comparison of differential distribution of toxic ions in individual leaves revealed that Na+ ions were not uniformly distributed in the leaves of the drought-tolerant parent and drought-tolerant QTL-NILs; but accumulated preferentially in the older leaves, whereas the hybrid of the drought-sensitive parent showed significantly higher Na+ concentration in all main stem leaves irrespective of their age. Dynamics of chlorophyll and proline concentration variation studied under salt stress at late flowering stages revealed a greater reduction, almost twice, in both leaf chlorophyll and proline concentrations in younger leaves in the hybrids of the sensitive parent as compared to the tolerant parent and QTL NILs. Imposition of salinity stress even at flowering stage affected the yield performance in pearl millet, wherein higher yield was recorded in drought tolerant parent and the two QTL-NILs compared to drought sensitive parent.

Assessment of genetic diversity in the sorghum reference set using EST-SSR markers.

Selection and use of genetically diverse genotypes are key factors in any crop breeding program to develop cultivars with a broad genetic base. Molecular markers play a major role in selecting diverse genotypes. In the present study, a reference set representing a wide range of sorghum genetic diversity was screened with 40 EST-SSR markers to validate both the use of these markers for genetic structure analyses and the population structure of this set. Grouping of accessions is identical in distance-based and model-based clustering methods. Genotypes were grouped primarily based on race within the geographic origins. Accessions derived from the African continent contributed 88.6 % of alleles confirming the African origin of sorghum. In total, 360 alleles were detected in the reference set with an average of 9 alleles per marker. The average PIC value was 0.5230 with a range of 0.1379-0.9483. Sub-race, guinea margaritiferum (Gma) from West Africa formed a separate cluster in close proximity to wild accessions suggesting that the Gma group represents an independent domestication event. Guineas from India and Western Africa formed two distinct clusters. Accessions belongs to the kafir race formed the most homogeneous group as observed in earlier studies. This analysis suggests that the EST-SSR markers used in the present study have greater discriminating power than the genomic SSRs. Genetic variance within the subpopulations was very high (71.7 %) suggesting that the germplasm lines included in the set are more diverse. Thus, this reference set representing the global germplasm is an ideal material for the breeding community, serving as a community resource for trait-specific allele mining as well as genome-wide association mapping.

A paradigm shift towards low-nitrifying production systems: the role of biological nitrification inhibition (BNI).

BACKGROUND: Agriculture is the single largest geo-engineering initiative that humans have initiated on planet Earth, largely through the introduction of unprecedented amounts of reactive nitrogen (N) into ecosystems. A major portion of this reactive N applied as fertilizer leaks into the environment in massive amounts, with cascading negative effects on ecosystem health and function. Natural ecosystems utilize many of the multiple pathways in the N cycle to regulate N flow. In contrast, the massive amounts of N currently applied to agricultural systems cycle primarily through the nitrification pathway, a single inefficient route that channels much of this reactive N into the environment. This is largely due to the rapid nitrifying soil environment of present-day agricultural systems. SCOPE: In this Viewpoint paper, the importance of regulating nitrification as a strategy to minimize N leakage and to improve N-use efficiency (NUE) in agricultural systems is highlighted. The ability to suppress soil nitrification by the release of nitrification inhibitors from plant roots is termed 'biological nitrification inhibition' (BNI), an active plant-mediated natural function that can limit the amount of N cycling via the nitrification pathway. The development of a bioassay using luminescent Nitrosomonas to quantify nitrification inhibitory activity from roots has facilitated the characterization of BNI function. Release of BNIs from roots is a tightly regulated physiological process, with extensive genetic variability found in selected crops and pasture grasses. Here, the current status of understanding of the BNI function is reviewed using Brachiaria forage grasses, wheat and sorghum to illustrate how BNI function can be utilized for achieving low-nitrifying agricultural systems. A fundamental shift towards ammonium (NH4(+))-dominated agricultural systems could be achieved by using crops and pastures with high BNI capacities. When viewed from an agricultural and environmental perspective, the BNI function in plants could potentially have a large influence on biogeochemical cycling and closure of the N loop in crop-livestock systems.

Development of a molecular linkage map of pearl millet integrating DArT and SSR markers.

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.

Terminal drought-tolerant pearl millet [Pennisetum glaucum (L.) R. Br.] have high leaf ABA and limit transpiration at high vapour pressure deficit.

It was previously shown that pearl millet genotypes carrying a terminal drought tolerance quantitative trait locus (QTL) had a lower transpiration rate (Tr; g cm(-2) d(-1)) under well-watered conditions than sensitive lines. Here experiments were carried out to test whether this relates to leaf abscisic acid (ABA) and Tr concentration at high vapour pressure deficit (VPD), and whether that leads to transpiration efficiency (TE) differences. These traits were measured in tolerant/sensitive pearl millet genotypes, including near-isogenic lines introgressed with a terminal drought tolerance QTL (NIL-QTLs). Most genotypic differences were found under well-watered conditions. ABA levels under well-watered conditions were higher in tolerant genotypes, including NIL-QTLs, than in sensitive genotypes, and ABA did not increase under water stress. Well-watered Tr was lower in tolerant than in sensitive genotypes at all VPD levels. Except for one line, Tr slowed down in tolerant lines above a breakpoint at 1.40-1.90 kPa, with the slope decreasing >50%, whereas sensitive lines showed no change in that Tr response across the whole VPD range. It is concluded that two water-saving (avoidance) mechanisms may operate under well-watered conditions in tolerant pearl millet: (i) a low Tr even at low VPD conditions, which may relate to leaf ABA; and (ii) a sensitivity to higher VPD that further restricts Tr, which suggests the involvement of hydraulic signals. Both traits, which did not lead to TE differences, could contribute to absolute water saving seen in part due to dry weight increase differences. This water saved would become critical for grain filling and deserves consideration in the breeding of terminal drought-tolerant lines.

Exploiting rice-sorghum synteny for targeted development of EST-SSRs to enrich the sorghum genetic linkage map.

The sequencing and detailed comparative functional analysis of genomes of a number of select botanical models open new doors into comparative genomics among the angiosperms, with potential benefits for improvement of many orphan crops that feed large populations. In this study, a set of simple sequence repeat (SSR) markers was developed by mining the expressed sequence tag (EST) database of sorghum. Among the SSR-containing sequences, only those sharing considerable homology with rice genomic sequences across the lengths of the 12 rice chromosomes were selected. Thus, 600 SSR-containing sorghum EST sequences (50 homologous sequences on each of the 12 rice chromosomes) were selected, with the intention of providing coverage for corresponding homologous regions of the sorghum genome. Primer pairs were designed and polymorphism detection ability was assessed using parental pairs of two existing sorghum mapping populations. About 28% of these new markers detected polymorphism in this 4-entry panel. A subset of 55 polymorphic EST-derived SSR markers were mapped onto the existing skeleton map of a recombinant inbred population derived from cross N13 x E 36-1, which is segregating for Striga resistance and the stay-green component of terminal drought tolerance. These new EST-derived SSR markers mapped across all 10 sorghum linkage groups, mostly to regions expected based on prior knowledge of rice-sorghum synteny. The ESTs from which these markers were derived were then mapped in silico onto the aligned sorghum genome sequence, and 88% of the best hits corresponded to linkage-based positions. This study demonstrates the utility of comparative genomic information in targeted development of markers to fill gaps in linkage maps of related crop species for which sufficient genomic tools are not available.

An integrated genetic map and a new set of simple sequence repeat markers for pearl millet, Pennisetum glaucum.

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.

Mapping the d1 and d2 dwarfing genes and the purple foliage color locus P in pearl millet.

The d(1) and d(2) dwarfing genes and the P purple foliage color gene were placed on the restriction fragment length polymorphism (RFLP)-based molecular marker linkage map of pearl millet [Pennisetum glaucum (L.) R. Br.] using a mapping population based on a cross of inbred lines IP 18293 (D(1)/D(1), d(2)/d(2), P/P) and Tift 238D1 (d(1)/d(1) D(2)/D(2) p/p). A skeleton genetic linkage map of 562 cM (Haldane function) was constructed using 33 RFLP markers and these three morphological markers. The D(1)/d(1) plant height locus mapped to pearl millet linkage group 1, while the D(2)/d(2) plant height locus and the P/p foliage color locus mapped to pearl millet linkage group 4. Loose genetic linkage was observed between the D(2)/d(2) and P/p loci, with 42% repulsion-phase recombination corresponding to 92 cM (Haldane). This loose linkage of morphological marker loci detected on pearl millet LG4 can likely find use in applied pearl millet breeding programs, as host plant resistances to both downy mildew and rust have previously been identified in this genomic region. Such exploitation of these morphological markers in an applied disease resistance breeding program would require development of appropriate genetic stocks, but the relatively loose genetic linkage between d(2) and P suggests that this should not be difficult.

Mapping and characterisation of QTL x E interactions for traits determining grain and stover yield in pearl millet.

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.

Quantitative trait loci associated with traits determining grain and stover yield in pearl millet under terminal drought-stress conditions.

Drought stress during the reproductive stage is one of the most important environmental factors reducing the grain yield and yield stability of pearl millet. A QTL mapping approach has been used in this study to understand the genetic and physiological basis of drought tolerance in pearl millet and to provide a more-targeted approach to improving the drought tolerance and yield of this crop in water-limited environments. The aim was to identify specific genomic regions associated with the enhanced tolerance of pearl millet to drought stress during the flowering and grain-filling stages. Testcrosses of a set of mapping-population progenies, derived from a cross of two inbred pollinators that differed in their response to drought, were evaluated in a range of managed terminal drought-stress environments. A number of genomic regions were associated with drought tolerance in terms of both grain yield and its components. For example, a QTL associated with grain yield per se and for the drought tolerance of grain yield mapped on linkage group 2 and explained up to 23% of the phenotypic variation. Some of these QTLs were common across stress environments whereas others were specific to only a particular stress environment. All the QTLs that contributed to increased drought tolerance did so either through better than average maintenance (compared to non-stress environments) of harvest index, or harvest index and biomass productivity. It is concluded that there is considerable potential for marker-assisted backcross transfer of selected QTLs to the elite parent of the mapping population and for their general use in the improvement of pearl millet productivity in water-limited environments.

QTL mapping of stay-green in two sorghum recombinant inbred populations.

The stay-green trait is a reported component of tolerance to terminal drought stress in sorghum. To map quantitative trait loci (QTLs) for stay-green, two sorghum recombinant inbred populations (RIPs) of 226 F(3:5) lines each were developed from crosses (1) IS9830 x E36-1 and (2) N13 x E36-1. The common parental line, E36-1 of Ethiopian origin, was the stay-green trait source. The genetic map of RIP 1 had a total length of 1,291 cM, with 128 markers (AFLPs, RFLPs, SSRs and RAPDs) distributed over ten linkage groups. The map of RIP 2 spanned 1,438 cM and contained 146 markers in 12 linkage groups. The two RIPs were evaluated during post-rainy seasons at Patancheru, India, in 1999/2000 (RIP 2) and 2000/2001 (RIP 1). The measures of stay-green mapped were the green leaf area percentages at 15, 30 and 45 days after flowering (% GL15, % GL30 and % GL45, respectively). Estimated repeatabilities for % GL15, % GL30 and % GL45 amounted to 0.89, 0.81 and 0.78 in RIP 1, and 0.91, 0.88 and 0.85 in RIP 2, respectively. The number of QTLs for the three traits detected by composite interval mapping ranged from 5 to 8, explaining 31% to 42% of the genetic variance. In both RIPs, both parent lines contributed stay-green alleles. Across the three measures of the stay-green trait, three QTLs on linkage groups A, E and G were common to both RIPs, with the stay-green alleles originating from E36-1. These QTLs were therefore consistent across the tested genetic backgrounds and years. After QTL validation across sites and verification of the general benefit of the stay-green trait for grain yield performance and stability in the target areas, the corresponding chromosomal regions could be candidates for marker-assisted transfer of stay-green into elite materials.

Mapping quantitative trait loci for downy mildew resistance in pearl millet.

Quantitative trait loci (QTLs) for resistance to pathogen populations of Scelerospora graminicola from India, Nigeria, Niger and Senegal were mapped using a resistant x susceptible pearl millet cross. An RFLP map constructed using F2 plants was used to map QTLs for traits scored on F4 families. QTL analysis was carried out using the interval mapping programme Mapmaker/QTL. Independent inheritance of resistance to pathogen populations from India, Senegal, and populations from Niger and Nigeria was shown. These results demonstrate the existence of differing virulences in the pathogen populations from within Africa and between Africa and India. QTLs of large effect, contributing towards a large porportion of the variation in resistance, were consistently detected in repeated screens. QTLs of smaller and more variable effect were also detected. There was no QTLs that were effective against all four pathogen populations, demonstrating that pathotype-specific resistance is a major mechanism of downy mildew resistance in this cross. For all but one of the QTLs, resistance was inherited from the resistant parent and the inheritance of resistance tended to be the result of dominance or over-dominance. The implications of this research for pearl millet breeding are discussed.

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.

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.