Development of transgenic finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.) resistant to leaf blast disease

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R₀ and R₁ progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R₁ lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.     

Functional markers based molecular characterization and cloning of resistance gene analogs encoding NBS-LRR disease resistance proteins in finger millet (<Emphasis Type="Italic">Eleusine coracana)</Emphasis>

Magnaporthe grisea, the blast fungus is one of the main pathological threats to finger millet crop worldwide. A systematic search for the blast resistance gene analogs was carried out, using functional molecular markers. Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encodes nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from Eleusine coracana. Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from resistant genotypes of finger millet using PCR based approach with primers designed from conserved regions of NBS domain. Attempts were made to identify molecular markers linked to the resistance gene and to differentiate the resistant bulk from the susceptible bulk. A total of 9 NBS-LRR and 11 EST-SSR markers generated 75.6 and 73.5% polymorphism respectively amongst 73 finger millet genotypes. NBS-5, NBS-9, NBS-3 and EST-SSR-04 markers showed a clear polymorphism which differentiated resistant genotypes from susceptible genotypes. By comparing the banding pattern of different resistant and susceptible genotypes, five DNA amplifications of NBS and EST-SSR primers (NBS-05_504, NBS-09₇₁₁, NBS-07₆₈₈, NBS-03₅₀₉ and EST-SSR-04₂₄₁) were identified as markers for the blast resistance in resistant genotypes. Principal coordinate plot and UPGMA analysis formed similar groups of the genotypes and placed most of the resistant genotypes together showing a high level of genetic relatedness and the susceptible genotypes were placed in different groups on the basis of differential disease score. Our results provided a clue for the cloning of finger millet blast resistance gene analogs which not only facilitate the process of plant breeding but also molecular characterization of blast resistance gene analogs from Eleusine coracana.     

A Full-Length Dof1 Transcription Factor of Finger Millet and its Response to a Circadian Cycle

DOF1 (DNA binding with one finger) plays an important role in regulating C/N metabolism in cereals. In order to validate its role in the regulation of nitrogen use efficiency (NUE) and photosynthetic efficiency in finger millet, 5′–3′ RACE PCR was performed to obtain and characterize full-length Dof1 genes of high and low grain protein finger millet genotypes. The full-length DOF1 ORFs were both 1,284 nt long and were 98.8 % similar over 427 amino acids containing the characteristic Dof domain. Comparison of both the EcDof1 protein sequences with the Dof1 of other cereals revealed high sequence similarity to the Dof1 of rice. Southern hybridization carried out using the probe developed from the region encoding the highly variable C-terminal region of EcDof1 showed the presence of four copies of the DOF1 gene in finger millet, which might explain the high NUE and photosynthetic performance of finger millet. Since the genes involved in C/N metabolism are regulated diurnally and play crucial roles in determining grain protein content during grain filling, the diurnal expression of EcDOF1 was assessed in two finger millet genotypes (GE 3885 and GE 1437) with differing grain protein content (13.8 % and 6.15 % respectively). It was found that EcDOF1 exhibited diurnal regulation and peak differential pattern expression with early phasing in GE3885 and late phasing in GE1437. Differential expression of DOF1 might alter the regulation of genes involved in C/N metabolism affecting grain protein composition of finger millet genotypes.     

Alterations in the biosynthesis of proteins and nucleic acids in finger millet (Eleucine coracana) seedlings during water stress and the effect of proline on protein biosynthesis

The biosynthesis of DNA appeared to be unaffected in the water-stressed seedlings of finger millet (Eleucine coracana), but an increase in the synthesis de novo of RNA and proteins was observed during mild water stress. The polyribosome content was also increased in stressed finger millet seedlings. Proline, a solute which accumulates during water stress, enhanced the incorporation of radioactive precursors into proteins; caused an increase in translatability of finger millet messengers in vitro; and stabilized the polyribosomes isolated from normal seedlings. The results emphasize the role of proline in the adaptation of finger millet to the intermittent drought it experiences during cultivation.     

Mapping quantitative trait loci for important agronomic traits in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis>) mini core collection with genomic and genic SSR markers

Allele identification for agro-morphological traits and stress resistance is a major concern across the globe for improving productivity of finger millet. Here, we used 46 genomic and 58 genic simple sequence repeats (SSRs) markers in a set of 66 accessions used to constitute a global mini-core collection for analysing their genetic structure as a population and establishing association among markers and twenty morphological traits including resistance to finger blast. Phenotypic data revealed a wide range of variation for all traits except flag leaf width and flag leaf sheath width. We got amplification of 81 alleles by the 31 genomic SSRs at an average of 2.61 alleles per locus. Polymorphism information content (PIC) values varied from 0.21 to 0.75 and average gene diversity was 0.49. Structure analysis of the population using the genomic SSR data divided the accessions into two clusters where Indian and exotic accessions were grouped in separate clusters. Genic SSRs which were associated with blast resistance genes, amplified 36 alleles at an average of 2 alleles per locus. PIC values ranged from 0.32 to 0.37 and average gene diversity was 0.45. Population structure analysis using data from these SSRs grouped the accessions into three clusters, which broadly correspond to their reaction to blast disease. Twenty-two significant associations were found using the GLM approach for 20 agro-morphological traits both in 2012 and 2014, while, 7 and 5 significant marker-trait associations were identified using MLM in 2012 and 2014 respectively. The SSR markers FMBLEST35 and FMBLEST36 designed from the Pi21 gene sequence of rice were found to be associated with blast disease resistance in finger millet indicating that the gene homologues play a significant role in an important role for neck blast resistance.     

Characterization of Insect Proteinases and Their Inhibition by Finger and Little Millet Inhibitors

Empirical approach was adopted to examine the interaction of a little and finger millet inhibitors with digestive proteinases ofdifferent field and storage pests. Accordingly, gut proteinases of four storage and phytophagous pests were characterized andtheir inhibition by finger millet (Eluesine coracana Gaertneri) and little millet (Panicum sumatrense Roth) inhibitors has beeninvestigated. The Callosobruchus sp showed acid proteinase with pH optimum of 3.5 and Sitophilus oryzae showed pHoptimum of 4.5 apart from their alkaline proteinases with pH 8.5. The proteinases of Tribolium castaneum had wider pHoptimum from 5.5 to 8.5. However, all lepidopteran insect proteinases had pH optima ranging from 8.5 to 10.5. The optimumtemperature was found to be 30 to 40 °C. The inhibitory activities of little and finger millet inhibitors towards the insectproteinases are very low except notable level inhibition of proteinases of some insects. The gut proteinase zymogram ofdifferent insects revealed 2 – 6 isozymes and the inhibitors moderately inhibited all the isozymes of insects tested.     

Flavonoid patterns and systematics in Eleusine

A survey of leaf flavonoids was conducted on Eleusine coracana ssp. coracana and ssp. africana, E. indica, E. multiflora, E. tristachya, E. floccifolia, and E. compressa. Twenty phenolic compounds were detected. Those identified were: orientin, isoorientin, vitexin, isovitexin, saponarin, violanthin, lucenin-1, and tricin. The study revealed a general generic flavonoid pattern except for E. compressa, which occupies an isolated position in Eleusine. Flavonoids of the perennial E. floccifolia and the annuals E. multiflora and E. tristachya are markedly different from those of cultivated E. coracana, suggesting that these species are only distantly related to the crop. The morphologically well defined E. coracana—africana—indica group also forms a unit in respect of flavonoids. Subspecies africana exhibits a higher flavonoid similarity to ssp. coracana (finger millet) than does E. indica. The weedy race of ssp. africana usually combines flavonoids of both the wild and domesticated subspecies. The flavonoid pattern of the dedza race of ssp.africana is identical to that of finger millet, suggesting either a direct origin of the crop from this race, or extensive introgression from the crop into ssp. africana. A lack of qualitative differences in flavonoids between cultivated races of finger millet is indicative of the genetic stability of these compounds. The flavonoid data confirms the domestication of finger millet from ssp. africana.     

Obtaining the transgenic lines of finger millet Eleusine coracana (L.). with dinitroaniline resistance

The current data is dedicated to the study of bioballistic and Agrobacterium-mediated transformation of finger millet with the constructs carrying the mutant α-tubulin gene (TUAml), isolated from R-biotype goosegrass (Eleusine indica L.), for the decision of problem of dinitroaniline-resistance. It was found that 10 μM of trifluralin is optimal for the selection of transgene plants of finger millet. PCR analysis of transformed lines confirmed the transgenic nature of plants. The analysis of seed of T₁ of transgenic lines confirmed heterozygous character of inheritance of trifluralin resistance.     

Development of EST SSRs in Finger Millet (Eleusine coracana ssp coracana) and their Transferability to Pearl Millet (Pennisetum glaucum)

EST-SSR markers were developed using sequence information from 1740 expressed sequence tags (ESTs) of finger millet available in the public domain. A set of 31 SSR markers were synthesized based on di, tri, tetra and penta-nucleotide repeat sequences. These were used for PCR analysis of 11 elite germplasm lines of finger millet of Indian and African origin. Out of 31 SSR markers, amplification products were obtained for 17 primer pairs. Of these nine were found polymorphic with two alleles per locus. These 17 SSR primer pairs were also tested for amplification in three varieties of pearl millet (Pennisetum glaucum) and 11 could be transferred to pearl millet. The informative EST SSR markers developed, can be used in finger millet as well as pearl millet genetic improvement projects.     

Population Structure and Diversity in Finger Millet (Eleusine coracana) Germplasm

A genotypic analysis of 79 finger millet accessions (E. coracana subsp. coracana) from 11 African and five Asian countries, plus 14 wild E. coracana subsp. africana lines collected in Uganda and Kenya was conducted with 45 SSR markers distributed across the finger millet genome. Phylogenetic and population structure analyses showed that the E. coracana germplasm formed three largely distinct subpopulations, representing subsp. africana, subsp. coracana originating from Africa and subsp. coracana originating from Asia. A few lines showed admixture between the African and Asian cultivated germplasm pools and were the result of either targeted or accidental intercrossing. Evidence of gene flow was also seen between the African wild and cultivated subpopulations, indicating that hybridizations among subspecies occur naturally where both species are sympatric. The genotyping, combined with phylogenetic and population structure analyses proved to be very powerful in predicting the origin of breeding materials. The genotypic study was complemented by a phenotypic evaluation. The wild and cultivated accessions differed by a range of domestication-related characters, such as tiller number, plant height, peduncle length, seed color and grain yield. Significant differences in plant architecture and yield were also identified between the Asian and African subpopulations. The observed population structure within cultivated finger millet is consistent with the theory that, after the introduction of finger millet from Africa into India via the trade routes some 3000 years ago, the two germplasm pools remained largely isolated until recent times. The significantly lower diversity present within the Asian subpopulation also suggests that it arose from a relatively small number of founder plants.     

Isolation and characterization of a trypsin inhibitor from finger millet

A trypsin inhibitor was isolated from finger millet (Eleusine coracana) by ammonium sulphate fractionation, chromatography on CM-Sephadex and Sepha     

Comparative analysis of solid-state bioprocessing and enzymatic treatment of finger millet for mobilization of bound phenolics

The present work investigates the probable bioprocessing technique to mobilize the bound phenolics naturally found in finger millet cell wall for enriching it with dietary antioxidants. Comparative study was performed between the exogenous enzymatic treatment and solid-state fermentation of grain (SSF) with a food grade organism Rhizopus oryzae. SSF results indicated that at the 6th day of incubation, total phenolic content (18.64 mg gallic acid equivalent/gds) and antioxidant property (DPPH radical scavenging activity of 39.03 %, metal chelating ability of 54 % and better reducing power) of finger millet were drastically enhanced when fermented with GRAS filamentous fungi. During the enzymatic bioprocessing, most of the phenolics released during the hydrolysis, leached out into the liquid portion rather than retaining them within the millet grain, resulting in overall loss of dietary antioxidant. The present study establishes the most effective strategy to enrich the finger millet with phenolic antioxidants.     

In-silico mining,type and frequency analysis of genic microsatellites of finger millet (Eleusine coracana (L.) Gaertn.): a comparative genomic analysis of NBS–LRR regions of finger millet with rice

In recent years, the increased availability of the DNA sequences has given the possibility to develop and explore the expressed sequence tags (ESTs) derived SSR markers. In the present study, a total of 1956 ESTs of finger millet were used to find the microsatellite type, distribution, frequency and developed a total of 545 primer pairs from the ESTs of finger millet. Thirty-two EST sequences had more than two microsatellites and 1357 sequences did not have any SSR repeats. The most frequent type of repeats was trimeric motif, however the second place was occupied by dimeric motif followed by tetra-, hexa- and penta repeat motifs. The most common dimer repeat motif was GA and in case of trimeric SSRs, it was CGG. The EST sequences of NBS-LRR region of finger millet and rice showed higher synteny and were found on nearly same positions on the rice chromosome map. A total of eight, out of 15 EST based SSR primers were polymorphic among the selected resistant and susceptible finger millet genotypes. The primer FMBLEST5 could able to differentiate them into resistant and susceptible genotypes. The alleles specific to the resistant and susceptible genotypes were sequenced using the ABI 3130XL genetic analyzer and found similarity to NBS–LRR regions of rice and finger millet and contained the characteristic kinase-2 and kinase 3a motifs of plant R-genes belonged to NBS–LRR region. The In-silico and comparative analysis showed that the genes responsible for blast resistance can be identified, mapped and further introgressed through molecular breeding approaches for enhancing the blast resistance in finger millet.     

Comparative analyses reveal high levels of conserved colinearity between the finger millet and rice genomes

Finger millet is an allotetraploid (2n = 4x = 36) grass that belongs to the Chloridoideae subfamily. A comparative analysis has been carried out to determine the relationship of the finger millet genome with that of rice. Six of the nine finger millet homoeologous groups corresponded to a single rice chromosome each. Each of the remaining three finger millet groups were orthologous to two rice chromosomes, and in all the three cases one rice chromosome was inserted into the centromeric region of a second rice chromosome to give the finger millet chromosomal configuration. All observed rearrangements were, among the grasses, unique to finger millet and, possibly, the Chloridoideae subfamily. Gene orders between rice and finger millet were highly conserved, with rearrangements being limited largely to single marker transpositions and small putative inversions encompassing at most three markers. Only some 10% of markers mapped to non-syntenic positions in rice and finger millet and the majority of these were located in the distal 14% of chromosome arms, supporting a possible correlation between recombination and sequence evolution as has previously been observed in wheat. A comparison of the organization of finger millet, Panicoideae and Pooideae genomes relative to rice allowed us to infer putative ancestral chromosome configurations in the grasses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of the protein fractions of finger millet

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) has been employed to resolve protein subunits of finger millet (Eleusine coracana) varieties according to their MW. These studies have established that varietal differences exist in the protein composition of finger millet varieties. The MW distribution of the protein subunits in the albumin-globulin, prolamin and glutelin fractions show many differences between the parental and cross-bred varieties and these differences are greater in the albumin-globulin and glutelin fractions than in the prolamin fraction. The amino acid compositions of the protein fractions show some differences between varieties.