C. T. Hash

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.

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

Abstract  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.

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.

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.

Development and mapping of Simple Sequence Repeat markers for pearl millet from data mining of Expressed Sequence Tags

BackgroundPearl millet [Pennisetum glaucum (L.) R. Br.] is a staple food and fodder crop of marginal agricultural lands of sub-Saharan Africa and the Indian subcontinent. It is also a summer forage crop in the southern USA, Australia and Latin America, and is the preferred mulch in Brazilian no-till soybean production systems. Use of molecular marker technology for pearl millet genetic improvement has been limited. Progress is hampered by insufficient numbers of PCR-compatible co-dominant markers that can be used readily in applied breeding programmes. Therefore, we sought to develop additional SSR markers for the pearl millet research community.ResultsA set of new pearl millet SSR markers were developed using available sequence information from 3520 expressed sequence tags (ESTs). After clustering, unigene sequences (2175 singlets and 317 contigs) were searched for the presence of SSRs. We detected 164 sequences containing SSRs (at least 14 bases in length), with a density of one per 1.75 kb of EST sequence. Di-nucleotide repeats were the most abundant followed by tri-nucleotide repeats. Ninety primer pairs were designed and tested for their ability to detect polymorphism across a panel of 11 pairs of pearl millet mapping population parental lines. Clear amplification products were obtained for 58 primer pairs. Of these, 15 were monomorphic across the panel. A subset of 21 polymorphic EST-SSRs and 6 recently developed genomic SSR markers were mapped using existing mapping populations. Linkage map positions of these EST-SSR were compared by homology search with mapped rice genomic sequences on the basis of pearl millet-rice synteny. Most new EST-SSR markers mapped to distal regions of linkage groups, often to previous gaps in these linkage maps. These new EST-SSRs are now are used by ICRISAT in pearl millet diversity assessment and marker-aided breeding programs.ConclusionThis study has demonstrated the potential of EST-derived SSR primer pairs in pearl millet. As reported for other crops, EST-derived SSRs provide a cost-saving marker development option in pearl millet. Resources developed in this study have added a sizeable number of useful SSRs to the existing repertoire of circa 100 genomic SSRs that were previously available to pearl millet researchers.

Genomic regions associated with grain yield and aspects of post-flowering drought tolerance in pearl millet across stress environments and tester background

A pearl millet mapping population from a cross between ICMB841 and 863B was studied for DNA polymorphism to construct a genetic linkage map, and to map genomic regions associated with grain and stover yield, and aspects of drought tolerance. To identify genomic regions associated with these traits, mapping population testcrosses of 79 F3 progenies were evaluated under post-flowering drought stress conditions over 2 years and in the background of two elite testers. A significant genotype × drought stress treatment interaction was evident in the expression of grain and stover yield in drought environments and in the background of testers over the 2 years. As a result of this, genomic regions associated with grain and stover yield and the aspects of drought tolerance were also affected: some regions were more affected by the changes in the environments (i.e. severity and duration of drought stress) while others were commonly identified across the drought stress environments and tester background used. In most instances, both harvest index and panicle harvest index co-mapped with grain yield suggesting that increased drought tolerance and yield of pearl millet that mapped to these regions was achieved by increased partitioning of dry matter from stover to the grains. Drought stress treatments, years and testers interactions on genomic regions associated with grain and stover yield of pearl millet are discussed, particularly, in reference to genetic improvement of drought tolerance of this crop using marker-assisted selection.

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

Abstract.A mapping population of 104 F3 lines of pearl millet, derived from a cross between two inbred lines H 77/833-2 × 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 × season interaction (G × S), and genotype × environment-within-season interaction [G × E(S)]. QTLs were determined for traits for their G, G × S, and G × 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 × S and/or G, G × S and G × E(S) effects. The major G × S QTLs detected were for flowering time (on LG 4 and LG 6), and mapped to the same intervals as G × 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 × 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 × E(S) interactions. The grain yield QTLs that were affected by QTL × E(S) interactions (mapping on LG 2 and LG 6), appeared to be linked to parallel QTL × 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 × E(S) interactions were more frequently observed for component traits of grain and stover yield, than for grain or stover yield per se.

Opportunities for marker-assisted selection (MAS) to improve the feed quality of crop residues in pearl millet and sorghum

Cereal crop residues (straw, chaff, etc.) are important components of maintenance rations for ruminant livestock in many parts of the world. They are especially important in small-holder crop-livestock production systems in the sub-humid, semi-arid, and arid tropics and subtropics where most of the world’s poorest livestock producers and consumers are found. Taking as examples tropically adapted cereals in the crop improvement mandate of the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), namely pearl millet [Pennisetum glaucum (L.) R. Br.] and sorghum [Sorghum bicolor (L.) Moench], this paper explores opportunities for using marker-assisted crop breeding methods to improve the quality and quantity of cereal crop residues for use as ruminant livestock feedstuffs. In the case of pearl millet, ICRISAT has been heavily involved with several UK-based collaborating research institutes, in development and initial application of the molecular genetic tools for marker-assisted breeding. We have obtained some useful experience in quantitative trait loci (QTL) mapping and marker-assisted selection (MAS) for stover yield, foliar disease resistance, and in vitro estimates of the nutritive value of various stover fractions for ruminants. In sorghum, ICRISAT has focused on initiating a large-scale high-throughput marker-assisted backcrossing program for the stay-green component of terminal drought tolerance—a trait that is likely to be associated not only with more stable grain and stover yield, but which is also expected to contribute to maintenance of ruminant nutritional value of stover produced under drought stress conditions. Conventional and marker-assisted breeding for foliar disease resistance is recommended for dual-purpose cereal improvement, or indeed for improvement of the nutritional value of residues for any crop in which these are used as feedstuffs for ruminant livestock. Practical problems faced and proposed ways of dealing with these are discussed.

Integration of gene-based markers in a pearl millet genetic map for identification of candidate genes underlying drought tolerance quantitative trait loci

BackgroundIdentification of genes underlying drought tolerance (DT) quantitative trait loci (QTLs) will facilitate understanding of molecular mechanisms of drought tolerance, and also will accelerate genetic improvement of pearl millet through marker-assisted selection. We report a map based on genes with assigned functional roles in plant adaptation to drought and other abiotic stresses and demonstrate its use in identifying candidate genes underlying a major DT-QTL.ResultsSeventy five single nucleotide polymorphism (SNP) and conserved intron spanning primer (CISP) markers were developed from available expressed sequence tags (ESTs) using four genotypes, H 77/833-2, PRLT 2/89-33, ICMR 01029 and ICMR 01004, representing parents of two mapping populations. A total of 228 SNPs were obtained from 30.5 kb sequenced region resulting in a SNP frequency of 1/134 bp. The positions of major pearl millet linkage group (LG) 2 DT-QTLs (reported from crosses H 77/833-2 × PRLT 2/89-33 and 841B × 863B) were added to the present consensus function map which identified 18 genes, coding for PSI reaction center subunit III, PHYC, actin, alanine glyoxylate aminotransferase, uridylate kinase, acyl-CoA oxidase, dipeptidyl peptidase IV, MADS-box, serine/threonine protein kinase, ubiquitin conjugating enzyme, zinc finger C- × 8-C × 5-C × 3-H type, Hd3, acetyl CoA carboxylase, chlorophyll a/b binding protein, photolyase, protein phosphatase1 regulatory subunit SDS22 and two hypothetical proteins, co-mapping in this DT-QTL interval. Many of these candidate genes were found to have significant association with QTLs of grain yield, flowering time and leaf rolling under drought stress conditions.ConclusionsWe have exploited available pearl millet EST sequences to generate a mapped resource of seventy five new gene-based markers for pearl millet and demonstrated its use in identifying candidate genes underlying a major DT-QTL in this species. The reported gene-based markers represent an important resource for identification of candidate genes for other mapped abiotic stress QTLs in pearl millet. They also provide a resource for initiating association studies using candidate genes and also for comparing the structure and function of distantly related plant genomes such as other Poaceae members.

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.