Ramasamy Perumal

Impact of high temperature stress on floret fertility and individual grain weight of grain sorghum: sensitive stages and thresholds for temperature and duration

Sorghum [Sorghum bicolor (L.) Moench] yield formation is severely affected by high temperature stress during reproductive stages. This study pursues to (i) identify the growth stage(s) most sensitive to high temperature stress during reproductive development, (ii) determine threshold temperature and duration of high temperature stress that decreases floret fertility and individual grain weight, and (iii) quantify impact of high daytime temperature during floret development, flowering and grain filling on reproductive traits and grain yield under field conditions. Periods between 10 and 5 d before anthesis; and between 5 d before- and 5 d after-anthesis were most sensitive to high temperatures causing maximum decreases in floret fertility. Mean daily temperatures >25°C quadratically decreased floret fertility (reaching 0% at 37°C) when imposed at the start of panicle emergence. Temperatures ranging from 25 to 37°C quadratically decreased individual grain weight when imposed at the start of grain filling. Both floret fertility and individual grain weights decreased quadratically with increasing duration (0–35 d or 49 d during floret development or grain filling stage, respectively) of high temperature stress. In field conditions, imposition of temperature stress (using heat tents) during floret development or grain filling stage also decreased floret fertility, individual grain weight, and grain weight per panicle.

Simple sequence repeat markers useful for sorghum downy mildew (Peronosclerospora sorghi) and related species

BackgroundA recent outbreak of sorghum downy mildew in Texas has led to the discovery of both metalaxyl resistance and a new pathotype in the causal organism, Peronosclerospora sorghi. These observations and the difficulty in resolving among phylogenetically related downy mildew pathogens dramatically point out the need for simply scored markers in order to differentiate among isolates and species, and to study the population structure within these obligate oomycetes. Here we present the initial results from the use of a biotin capture method to discover, clone and develop PCR primers that permit the use of simple sequence repeats (microsatellites) to detect differences at the DNA level.ResultsAmong the 55 primers pairs designed from clones from pathotype 3 of P. sorghi, 36 flanked microsatellite loci containing simple repeats, including 28 (55%) with dinucleotide repeats and 6 (11%) with trinucleotide repeats. A total of 22 microsatellites with CA/AC or GT/TG repeats were the most abundant (40%) and GA/AG or CT/TC types contribute 15% in our collection. When used to amplify DNA from 19 isolates from P. sorghi, as well as from 5 related species that cause downy mildew on other hosts, the number of different bands detected for each SSR primer pair using a LI-COR- DNA Analyzer ranged from two to eight. Successful cross-amplification for 12 primer pairs studied in detail using DNA from downy mildews that attack maize (P. maydis & P. philippinensis), sugar cane (P. sacchari), pearl millet (Sclerospora graminicola) and rose (Peronospora sparsa) indicate that the flanking regions are conserved in all these species. A total of 15 SSR amplicons unique to P. philippinensis (one of the potential threats to US maize production) were detected, and these have potential for development of diagnostic tests. A total of 260 alleles were obtained using 54 microsatellites primer combinations, with an average of 4.8 polymorphic markers per SSR across 34 Peronosclerospora, Peronospora and Sclerospora spp isolates studied. Cluster analysis by UPGMA as well as principal coordinate analysis (PCA) grouped the 34 isolates into three distinct groups (all 19 isolates of Peronosclerospora sorghi in cluster I, five isolates of P. maydis and three isolates of P. sacchari in cluster II and five isolates of Sclerospora graminicola in cluster III).ConclusionTo our knowledge, this is the first attempt to extensively develop SSR markers from Peronosclerospora genomic DNA. The newly developed SSR markers can be readily used to distinguish isolates within several species of the oomycetes that cause downy mildew diseases. Also, microsatellite fragments likely include retrotransposon regions of DNA and these sequences can serve as useful genetic markers for strain identification, due to their degree of variability and their widespread occurrence among sorghum, maize, sugarcane, pearl millet and rose downy mildew isolates.

Genetic reticulation and interrelationships among citrullus species as revealed by joint analysis of shared AFLPs and species-specific SSR alleles

Thirty-one accessions of Citrullus spp. belonging to Citrullus lanatus var. lanatus , C. lanatus var. citroides and Citrullus colocynthis were subjected to phylogenetic analysis using combined datasets of amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs). Tree topologies inferred by neighbour-joining analysis have resolved the phylogenic relationships among the species with special reference to established taxonomic classification. In this study, we have clearly resolved species boundaries of various taxa of citroides , lanatus and colocynthis into three well-supported clusters. Clustering pattern of principal component analysis with the shared polymorphisms using the subsets of data between any two taxon combinations helped to elucidate the introgression and interrelationships among the species. We report two major groups of C. lanatus taxa, one of which has undergone wide introgressions with the taxa of C. lanatus var. citroides and C. colocynthis . In this paper, we identified 583 AFLP bands that are polymorphic within the var. lanatus of C. lanatus , which is the largest set ever reported. The species-specific diagnostic SSRs and polymorphic AFLPs that are informative within and between the taxa reported in this paper would be immensely useful for future studies of these economically important genera.

Expression of pathogenesis-related protein PR-10 in sorghum floral tissues in response to inoculation with Fusarium thapsinum and Curvularia lunata

Differences in grain mould disease levels among different sorghum varieties grown in the same environment imply that host genes play a role in controlling disease severity. The fungi most often recovered from naturally infected sorghum grain, Fusarium thapsinum and Curvularia lunata, were used to inoculate a set of resistant and susceptible cultivars at anthesis in both field and glasshouse trials. In the field, 12 cultivars were inoculated with a mixture of F. thapsinum and C. lunata and, in the glasshouse, individual panicles from four selected cultivars were inoculated with spore suspensions of C. lunata, F. thapsinum, a mixture of the two or water to serve as a control. Based on grain mould severity ratings and germination tests on the seed from the field trial, Tx2911, Sureno, SC719-11E and SC650-11E displayed a high level of resistance to grain mould. To determine whether resistant and susceptible lines differed in response to the pathogens, PR-10 mRNA levels were measured using real-time reverse transcriptase-polymerase chain reaction. PR-10 is a protein with antifungal properties that has been associated with defence responses in sorghum and other plant species. In field tests, most, but not all, cultivars showed significant induction of normalized relative quantities of PR-10 after dual inoculation with spores of both C. lunata and F. thapsinum. Under glasshouse-controlled conditions, glumes of inoculated plants showed the clear induction of PR-10 mRNA, and the response was greater in resistant (Tx2911 and Sureno) than in susceptible (RTx430 and SC170-6-17) cultivars. Inoculation with spores from a single mould-inducing pathogen generally induced greater responses than when spores were combined. For RTx430, SC170-6-17 and Sureno, the response to C. lunata was greater, whereas Tx2911 showed a stronger response to F. thapsinum. The results indicate that the induction of PR-10 in sorghum glumes may be a factor useful in breeding programmes designed to combine multiple factors for resistance.

Genotypic variation in sorghum [ Sorghum bicolor (L.) Moench] exotic germplasm collections for drought and disease tolerance

Sorghum [Sorghum bicolor (L.) Moench] grain yield is severely affected by abiotic and biotic stresses during post-flowering stages, which has been aggravated by climate change. New parental lines having genes for various biotic and abiotic stress tolerances have the potential to mitigate this negative effect. Field studies were conducted under irrigated and dryland conditions with 128 exotic germplasm and 12 adapted lines to evaluate and identify potential sources for post-flowering drought tolerance and stalk and charcoal rot tolerances. The various physiological and disease related traits were recorded under irrigated and dryland conditions. Under dryland conditions, chlorophyll content (SPAD), grain yield and HI were decreased by 9, 44 and 16%, respectively, compared to irrigated conditions. Genotype RTx7000 and PI475432 had higher leaf temperature and grain yield, however, genotype PI570895 had lower leaf temperature and higher grain yield under dryland conditions. Increased grain yield and optimum leaf temperature was observed in PI510898, IS1212 and PI533946 compared to BTx642 (B35). However, IS14290, IS12945 and IS1219 had decreased grain yield and optimum leaf temperature under dryland conditions. Under irrigated conditions, stalk and charcoal rot disease severity was higher than under dryland conditions. Genotypes IS30562 and 1790E R had tolerance to both stalk rot and charcoal rot respectively and IS12706 was the most susceptible to both diseases. PI510898 showed combined tolerance to drought and Fusarium stalk rot under dryland conditions. The genotypes identified in this study are potential sources of drought and disease tolerance and will be used to develop better adaptable parental lines followed by high yielding hybrids.

Virulence and Molecular Genotyping Studies of Sporisorium reilianum Isolates in Sorghum

Head smut, caused by the fungal pathogen Sporisorium reilianum, has been reported with increasing frequency in the grain sorghum growing areas of Texas. To facilitate analysis of changes in pathogen virulence, four inoculation techniques were examined: soil and teliospore mixture, seed coating, media placement, and syringe injection. Of the four, syringe injection was determined to be the most effective. Inoculations of sorghum host differentials BTx643, BTx7078, BTx635, SC170-6-17 (TAM2571), SA281 (Early Hegari), and Tx414 showed 23 of 32 Texas isolates were race 4. Two isolates from College Station, TX, were classified as race 1, but no race 2 or 3 isolates were found. New, virulent races 5 and 6 were identified among isolates from south Texas. Using 16 amplified fragment length polymorphism (AFLP) primer combinations, genetic diversity was assessed in DNA samples from 49 S. reilianum isolates, including 44 sorghum isolates from Texas, two from Uganda, and one from Mali; and two maize isolates from Mexico. Single-base extensions with EcoRI and MseI primers in the selective amplification increased the number of informative polymorphic bands. High genetic dissimilarity (50%) was observed between isolates originating from maize and those originating from sorghum. The resultant dendrogram, made using cluster analysis, grouped the Texas S. reilianum isolates into four small clusters with ≥82% similarity. Other than for two race 6 isolates from Weslaco, TX, no evidence for geographical or other restrictions on gene flow was evident.

A Pictorial Technique for Mass Screening of Sorghum Germplasm for Anthracnose (Colletotrichum sublineolum) Resistance

Globally, the foliar phase of anthracnose is one of the most destructive diseases of sorghum. In most cases, anthracnose resistance screening relies on the use of a spore suspension. This method is usually conducted after sundown and when there is the possibility of dew formation the following morning. Using a spore suspension for sorghum anthracnose field evaluation in College Station, Texas over five years (1996, 1997, 1999-2001) yielded inconsistent linkage results and failed to identify any closely linked molecular markers. For large scale screening of sorghum germplasm for anthracnose (Colletotrichum sublineolum) resistance, plants are inoculated in the field or in the green house at either 30 d after planting or at the 8-10 leaf-stage. In field inoculation, the use of C. sublineolum-colonized sorghum grains was shown to be the most efficient and effective in identifying resistant sources. For effective, efficient, fast and accurate infection, approximately 10-20 seeds are placed in each plant leaf whorl and it takes about 16.7 kg of colonized grains to cover a 0.4 ha area. In the greenhouse, though colonized grains are equally effective, spray inoculation is preferred for easy and uniform coverage. Using this method of inoculum preparation, spore suspension was extracted and sprayed (106 conidia·ml-1), followed by 10 hr/d misting for 30 sec at 30-45 min interval continuously for a period of one month resulted in effective infection

Population genomics of pearl millet (Pennisetum glaucum (L.) R. Br.): Comparative analysis of global accessions and Senegalese landraces

BackgroundPearl millet is a staple food for people in arid and semi-arid regions of Africa and South Asia due to its high drought tolerance and nutritional qualities. A better understanding of the genomic diversity and population structure of pearl millet germplasm is needed to support germplasm conservation and genetic improvement of this crop. Here we characterized two pearl millet diversity panels, (i) a set of global accessions from Africa, Asia, and the America, and (ii) a collection of landraces from multiple agro-ecological zones in Senegal.ResultsWe identified 83,875 single nucleotide polymorphisms (SNPs) in 500 pearl millet accessions, comprised of 252 global accessions and 248 Senegalese landraces, using genotyping by sequencing (GBS) of PstI-MspI reduced representation libraries. We used these SNPs to characterize genomic diversity and population structure among the accessions. The Senegalese landraces had the highest levels of genetic diversity (π), while accessions from southern Africa and Asia showed lower diversity levels. Principal component analyses and ancestry estimation indicated clear population structure between the Senegalese landraces and the global accessions, and among countries in the global accessions. In contrast, little population structure was observed across in the Senegalese landraces collections. We ordered SNPs on the pearl millet genetic map and observed much faster linkage disequilibrium (LD) decay in Senegalese landraces compared to global accessions. A comparison of pearl millet GBS linkage map with the foxtail millet (Setaria italica) and sorghum (Sorghum bicolor) genomes indicated extensive regions of synteny, as well as some large-scale rearrangements in the pearl millet lineage.ConclusionsWe identified 83,875 SNPs as a genomic resource for pearl millet improvement. The high genetic diversity in Senegal relative to other regions of Africa and Asia supports a West African origin of this crop, followed by wide diffusion. The rapid LD decay and lack of confounding population structure along agro-ecological zones in Senegalese pearl millet will facilitate future association mapping studies. Comparative population genomics will provide insights into panicoid crop evolution and support improvement of these climate-resilient crops.

High-throughput micro-plate HCl–vanillin assay for screening tannin content in sorghum grain†

BACKGROUND The HCI-vanillin assay is a well-accepted method for determining tannin content in sorghum but is limited to small sample sets due to the time-consuming nature of the method. The objective was to develop an accurate and repeatable high-throughput 96-well plate assay for breeders to screen large sample sets of sorghum for tannin content. Validation of the high-throughput assay was tested on 25 sorghums suspected to contain tannin. RESULTS Approximately 30 measurements per day were completed using the conventional assay compared to 224 measurements using the 96-well platform. The correlation between the two tannin assays was 0.98. The coefficient of variation (CV) was 3.54% and 3.21% for the 96-well and conventional method, respectively. The 96-well assay exhibited good repeatability, with the inter-plate CV between 2.77% and 4.85%. CONCLUSION The high-throughput 96-well HCI-vanillin assay exhibited an eightfold increase in the number of measurements completed and was as accurate as the conventional HCI-vanillin assay.

Increased Power To Dissect Adaptive Traits in Global Sorghum Diversity Using a Nested Association Mapping Population

In crop species, adaptation to different agroclimatic regions creates useful variation but also leads to unwanted genetic correlations. Bouchet....... Adaptation of domesticated species to diverse agroclimatic regions has led to abundant trait diversity. However, the resulting population structure and genetic heterogeneity confounds association mapping of adaptive traits. To address this challenge in sorghum [Sorghum bicolor (L.) Moench]—a widely adapted cereal crop—we developed a nested association mapping (NAM) population using 10 diverse global lines crossed with an elite reference line RTx430. We characterized the population of 2214 recombinant inbred lines at 90,000 SNPs using genotyping-by-sequencing. The population captures ∼70% of known global SNP variation in sorghum, and 57,411 recombination events. Notably, recombination events were four- to fivefold enriched in coding sequences and 5′ untranslated regions of genes. To test the power of the NAM population for trait dissection, we conducted joint linkage mapping for two major adaptive traits, flowering time and plant height. We precisely mapped several known genes for these two traits, and identified several additional QTL. Considering all SNPs simultaneously, genetic variation accounted for 65% of flowering time variance and 75% of plant height variance. Further, we directly compared NAM to genome-wide association mapping (using panels of the same size) and found that flowering time and plant height QTL were more consistently identified with the NAM population. Finally, for simulated QTL under strong selection in diversity panels, the power of QTL detection was up to three times greater for NAM vs. association mapping with a diverse panel. These findings validate the NAM resource for trait mapping in sorghum, and demonstrate the value of NAM for dissection of adaptive traits.