Mapping QTL for grain yield and other agronomic traits in post-rainy sorghum [Sorghum bicolor (L.) Moench].

Sorghum, a cereal of economic importance ensures food and fodder security for millions of rural families in the semi-arid tropics. The objective of the present study was to identify and validate quantitative trait loci (QTL) for grain yield and other agronomic traits using replicated phenotypic data sets from three post-rainy dry sorghum crop seasons involving a mapping population with 245 F9 recombinant inbred lines derived from a cross of M35-1 × B35. A genetic linkage map was constructed with 237 markers consisting of 174 genomic, 60 genic and 3 morphological markers. The QTL analysis for 11 traits following composite interval mapping identified 91 QTL with 5-12 QTL for each trait. QTL detected in the population individually explained phenotypic variation between 2.5 and 30.3 % for a given trait and six major genomic regions with QTL effect on multiple traits were identified. Stable QTL across seasons were identified. Of the 60 genic markers mapped, 21 were found at QTL peak or tightly linked with QTL. A gene-based marker XnhsbSFCILP67 (Sb03g028240) on SBI-03, encoding indole-3-acetic acid-amido synthetase GH3.5, was found to be involved in QTL for seven traits. The QTL-linked markers identified for 11 agronomic traits may assist in fine mapping, map-based gene isolation and also for improving post-rainy sorghum through marker-assisted breeding.

Identification and validation of genomic regions that affect shoot fly resistance in sorghum [Sorghum bicolor (L.) Moench].

Shoot fly is one of the most important pests affecting the sorghum production. The identification of quantitative trait loci (QTL) affecting shoot fly resistance enables to understand the underlying genetic mechanisms and genetic basis of complex interactions among the component traits. The aim of the present study was to detect QTL for shoot fly resistance and the associated traits using a population of 210 RILs of the cross 27B (susceptible) × IS2122 (resistant). RIL population was phenotyped in eight environments for shoot fly resistance (deadheart percentage), and in three environments for the component traits, such as glossiness, seedling vigor and trichome density. Linkage map was constructed with 149 marker loci comprising 127 genomic-microsatellite, 21 genic-microsatellite and one morphological marker. QTL analysis was performed by using MQM approach. 25 QTL (five each for leaf glossiness and seedling vigor, 10 for deadhearts, two for adaxial trichome density and three for abaxial trichome density) were detected in individual and across environments. The LOD and R (2) (%) values of QTL ranged from 2.44 to 24.1 and 4.3 to 44.1%, respectively. For most of the QTLs, the resistant parent, IS2122 contributed alleles for resistance; while at two QTL regions, the susceptible parent 27B also contributed for resistance traits. Three genomic regions affected multiple traits, suggesting the phenomenon of pleiotrophy or tight linkage. Stable QTL were identified for the traits across different environments, and genetic backgrounds by comparing the QTL in the study with previously reported QTL in sorghum. For majority of the QTLs, possible candidate genes were identified. The QTLs identified will enable marker assisted breeding for shoot fly resistance in sorghum.

Identification of quantitative trait loci for resistance to shoot fly in sorghum [Sorghum bicolor (L.) Moench].

The shoot fly is one of the most destructive insect pests of sorghum at the seedling stage. Deployment of cultivars with improved shoot fly resistance would be facilitated by the use of molecular markers linked to QTL. The objective of this study was to dissect the genetic basis of resistance into QTL, using replicated phenotypic data sets obtained from four test environments, and a 162 microsatellite marker-based linkage map constructed using 168 RILs of the cross 296B (susceptible) x IS18551 (resistant). Considering five component traits and four environments, a total of 29 QTL were detected by multiple QTL mapping (MQM) viz., four each for leaf glossiness and seedling vigor, seven for oviposition, six for deadhearts, two for adaxial trichome density and six for abaxial trichome density. The LOD and R (2) (%) values of QTL ranged from 2.6 to 15.0 and 5.0 to 33%, respectively. For most of the QTL, IS18551 contributed resistance alleles; however, at six QTL, alleles from 296B also contributed to resistance. QTL of the related component traits were co-localized, suggesting pleiotropy or tight linkage of genes. The new morphological marker Trit for trichome type was associated with the major QTL for component traits of resistance. Interestingly, QTL identified in this study correspond to QTL/genes for insect resistance at the syntenic maize genomic regions, suggesting the conservation of insect resistance loci between these crops. For majority of the QTL, possible candidate genes lie within or very near the ascribed confidence intervals in sorghum. Finally, the QTL identified in the study should provide a foundation for marker-assisted selection (MAS) programs for improving shoot fly resistance in sorghum.

Identification of quantitative trait loci for agronomically important traits and their association with genic-microsatellite markers in sorghum.

The identification of quantitative trait loci (QTLs) affecting agronomically important traits enable to understand their underlying genetic mechanisms and genetic basis of their complex interactions. The aim of the present study was to detect QTLs for 12 agronomic traits related to staygreen, plant early development, grain yield and its components, and some growth characters by analyzing replicated phenotypic datasets from three crop seasons, using the population of 168 F(7) RILs of the cross 296B x IS18551. In addition, we report mapping of a subset of genic-microsatellite markers. A linkage map was constructed with 152 marker loci comprising 149 microsatellites (100 genomic- and 49 genic-microsatellites) and three morphological markers. QTL analysis was performed by using MQM approach. Forty-nine QTLs were detected, across environments or in individual environments, with 1-9 QTLs for each trait. Individual QTL accounted for 5.2-50.4% of phenotypic variance. Several genomic regions affected multiple traits, suggesting the phenomenon of pleiotropy or tight linkage. Stable QTLs were identified for studied traits across different environments, and genetic backgrounds by comparing the QTLs in the study with previously reported QTLs in sorghum. Of the 49 mapped genic-markers, 18 were detected associating either closely or exactly as the QTL positions of agronomic traits. EST marker Dsenhsbm19, coding for a key regulator (EIL-1) of ethylene biosynthesis, was identified co-located with the QTLs for plant early development and staygreen trait, a probable candidate gene for these traits. Similarly, such exact co-locations between EST markers and QTLs were observed in four other instances. Collectively, the QTLs/markers identified in the study are likely candidates for improving the sorghum performance through MAS and map-based gene isolations.

Exploration and mapping of microsatellite markers from subtracted drought stress ESTs in Sorghum bicolor (L.) Moench.

Molecular variation within defined genes underlying specific biochemical or physiological functions provide candidate gene-based markers which show very close association with the trait of interest and thus should enable to design superior genotypes. We explored microsatellite loci in a total of 9,892 subtracted drought stress ESTs of sorghum (6,295 after flowering ESTs and 3,597 before flowering ESTs) available in the NCBI dbEST database. Analysis of 9,892 ESTs identified 221 non-redundant ESTs with SSRs, from which 109 functional SSRs were developed. Among them 62 EST-microsatellites (56.8%) exhibited polymorphism for at least one sorghum genotype among the five tested and yielded a total of 161 alleles, with an average of 2.59 alleles per marker. We present a microsatellite linkage map using a RIL population derived from the cross 296B and IS18551. The map contains 128 microsatellite loci distributed over 15 linkage groups, and spanning a genetic distance of 1,074.5 cM. The map includes map positions of 28 drought EST-microsatellites developed and seven new genomic-SSRs, and are distributed throughout the map. The developed EST markers include genes coding for important regulatory proteins and functional proteins that are involved in stress related metabolism. The drought EST-microsatellites will have applications in functional diversity studies, association studies, QTL studies for drought, and other agronomically important traits in sorghum, and comparative genomics studies between sorghum and other members of the Poaceae family.

Development of genic-microsatellite markers for sorghum staygreen QTL using a comparative genomic approach with rice.

The already available comprehensive genome sequence information of model crops along with the transcriptomic resource from other crops provides an excellent opportunity for comparative genome analysis. We studied the synteny between each of the four major sorghum staygreen quantitative trait loci (QTL) regions with that in the rice genome and attempted to increase marker density around the QTL with genic-microsatellites from the sorghum transcriptomic resource using the rice genome as template. For each of the sorghum QTL regions, the reported RFLP markers were compiled, used for sequence similarity searches against the rice genome which identified syntenous regions on rice chromosome 1 for Stg1 and Stg2 QTL, on chromosome 9 for Stg3 QTL, and on chromosome 11 for Stg4 QTL. Using the Gramene genome browsing tool, 869 non-redundant sorghum expressed sequence tags (ESTs) were selected and 50 genic-microsatellites (18, 12, 15, and 5, for Stg1, Stg2, Stg3, and Stg4 QTL, respectively) could be developed. We could experimentally establish synteny of the Stg1, Stg2, Stg3, and Stg4 QTL regions with that of the rice genome by mapping ten polymorphic genic-microsatellite markers (20%) to the positions of the staygreen QTL. The simple strategy demonstrated in the present study could readily be extrapolated to other cereals of the Poaceae family. The markers developed in this study provide a basis for the isolation of genes underling these QTL using an association study or map-based gene isolation approach, and create an additional option for MAS of the staygreen trait in sorghum.

In vitro culture methods in sorghum with shoot tip as the explant material.

Twenty-four diverse genotypes of sorghum were evaluated for response to callus induction and plant regeneration with two media viz., MS and NBKNB using shoot tips as the start material to identify a model genotype. None of the genotypes tested showed promising results. Therefore, alternative methods of in vitro pathways using shoot meristem isolated from shoot tips were explored. Shoot apical meristems were isolated and were induced to multiple shoots or multiple shoot buds pathway by manipulation of thidiazuron (TDZ), 6-benzyl adenine (BAP) and 2, 4-dichlorophenoxy acetic acid (2, 4-D). Choice of the pathway whether large-scale multiplication of shoots or production of target tissues for transformation can be exercised based on the needs and applications. A simple procedure, for large scale handling of shoot tips is described in detail. Electron microscopic studies revealed that meristems isolated from 7-day-old seedlings are superior because of possessing greater number of transformation competent cells.

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus).

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T(1) plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1-8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt delta-endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.

Simple and economical assay systems for evaluation of phosphinothricin resistant transgenics of sorghum, Sorghum bicolor. (L.) Moench., and pearl millet, Pennisetum glaucum (L.) R. Br.

Five simple and rapid methods for evaluation of sorghum and pearl millet transgenics resistant to herbicide phosphinothricin (used as selectable marker) were studied. For rapid in vitro selection, three assays (establishment of sensitivity curves for embryogenic calli, determination of lethal doses for seed germination, and a rapid screening of cut young leaves based on the colour change of the medium) were established. For rapid screening of transgenic progeny, effects of in vivo Basta leaf spray and dip tests were studied at three different morphological stages. For all the above assays, LD50, and LD100 values were higher for pearl millet than sorghum. However, in both the crops, genotype effect was not significant. The assays standardized in the study were found to be effective for rapid, economical and mass-scale identification and characterization of transgenic plants of sorghum and pearl millet.

Construction of a combined sorghum linkage map from two recombinant inbred populations using AFLP, SSR, RFLP, and RAPD markers, and comparison with other sorghum maps.

Sorghum [ Sorghum bicolor (L.) Moench] is an important crop in the semi-arid tropics that also receives growing attention in genetic research. A comprehensive reference map of the sorghum genome would be an essential research tool. Here, a combined sorghum linkage map from two recombinant inbred populations was constructed using AFLP, SSR, RFLP and RAPD markers. The map was aligned with other published sorghum maps which are briefly reviewed. The two recombinant inbred populations (RIPs) analyzed in this study consisted of 225 (RIP 1) and 226 (RIP 2) F(3:5) lines, developed from the crosses IS 9830 x E 36-1 (RIP 1) and N 13 x E 36-1 (RIP 2), respectively. The genetic map of RIP 1 had a total length of 1,265 cM (Haldane), with 187 markers (125 AFLPs, 45 SSRs, 14 RFLPs, 3 RAPDs) distributed over ten linkage groups. The map of RIP 2 spanned 1,410 cM and contained 228 markers (158 AFLPs, 54 SSRs, 16 RFLPs) in 12 linkage groups. The combined map of the two RIPs contained 339 markers (249 AFLPs, 63 SSRs, 24 RFLPs, 3 RAPDs) on 11 linkage groups and had a length of 1,424 cM. It was in good agreement with other sorghum linkage maps, from which it deviated by a few apparent inversions, deletions, and additional distal regions.

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.

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

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