Nitrate signals determine the sensing of nitrogen through differential expression of genes involved in nitrogen uptake and assimilation in finger millet

In order to understand the molecular basis of high nitrogen use efficiency of finger millet, five genes (EcHNRT2, EcLNRT1, EcNADH-NR, EcGS, and EcFd-GOGAT) involved in nitrate uptake and assimilation were isolated using conserved primer approaches. Expression profiles of these five genes along with the previously isolated EcDof1 was studied under increased KNO₃ concentrations (0.15 to 1,500 μM) for 2 h as well as at 1.5 μM for 24 h in the roots and shoots of 25 days old nitrogen deprived two contrasting finger millet genotypes (GE-3885 and GE-1437) differing in grain protein content (13.76 and 6.15 %, respectively). Time kinetics experiment revealed that, all the five genes except EcHNRT2 in the leaves of GE-3885 were induced within 30 min of nitrate exposure indicating that there might be a greater nitrogen deficit in leaves and therefore quick transportation of nitrate signals to the leaves. Exposing the plants to increasing nitrate concentrations for 2 h showed that in roots of GE-3885, NR was strongly induced while GS was repressed; however, the pattern was found to be reversed in leaves of GE-1437 indicating that in GE-3885, most of the nitrate might be reduced in the roots but assimilated in leaves and vice-versa. Furthermore, compared with the low-protein genotype, expression of HNRT2 was strongly induced in both roots and shoots of high-protein genotype at the least nitrate concentration supplied. This further indicates that GE-3885 is a quick sensor of nitrogen compared with the low-protein genotype. Furthermore, expression of EcDof1 was also found to overlap the expression of NR, GS, and GOGAT indicating that Dof1 probably regulates the expression of these genes under different conditions by sensing the nitrogen fluctuations around the root zone.     

Spatial distribution pattern analysis of Dof1 transcription factor in different tissues of three <Emphasis Type="Italic">Eleusine coracana</Emphasis> genotypes differing in their grain colour,yield and photosynthetic efficiency

In the present study Dof1 gene of finger millet was cloned and sequenced. In silico analysis reveals 61% identity with the Sorghum bicolor and 57% identity with the Oryza sativa Dof1 sequence. A comparative analysis of gene sequences from different crops and three finger millet genotypes {Brown (PRM-1), Golden (PRM-701) and White (PRM-801)} differing in grain colour, yield and photosynthetic efficiency showed a high degree of sequence identity of Dof1 sequence gene ranging from 22 to 70% as evident from distance matrix of the built phylogenetic tree showing two major clusters. A total of five conserved motifs were observed in Dof1 sequences of different cereals. Motif 1 with multilevel consensus sequence CKNCRRYWTKGGAMRNVPVG contains zinc finger Dof domain. Motif 3 and motif 5 contains protein kinase phosphorylation site. Motif 2 contains Dof domain and zinc finger N-glycosylation site while motif 4 is involved in Zinc finger type profiling. Further, we studied the spatial distribution of Dof1 gene in three vegetative tissues (root, stem and flag leaf) as well as four stages of developing spikes (S1, S2, S3 and S4) of the three finger millet genotypes using qualitative and quantitative PCR based approaches. Physiological parameters (plant height, leaf area, chlorophyll content, SPAD value and photosynthetic efficiency) at the time of flowering was found to be highest in white (PRM-801) genotype followed by golden (PRM-701) and brown (PRM-1) genotype. Semi-quantitative RT-PCR and quantitative real-time PCR analysis revealed that the expression of Dof1 is highest in leaves and lowest in roots, which suggests its role in regulation of photosynthesis-related genes and carbon skeleton synthesis. Also at grain maturity stage, expression of Dof1 was higher in white (PRM-801) genotype followed by golden (PRM-701) and brown (PRM-1) genotype. The result is suggestive of Dof1 role in the accumulation of grain protein and yield attribute through regulation of key enzymes involved in source to sink relationship during grain filling stage.     

Simple sequence repeat (SSR) analysis in relation to calcium transport and signaling genes reveals transferability among grasses and a conserved behavior within finger millet genotypes

Being an excellent source of calcium, finger millet crop has nutraceutical importance. Mineral accumulation, being a polygenic trait, becomes essential to target potential candidate genes directly or indirectly involved in the regulation of calcium transport and signaling in cereals and might have influence on grain calcium accumulation. In view of this, genic microsatellite markers were developed from the coding and non-coding sequences of calcium signaling and transport genes viz. calcium transporters (channels; ATPases and antiporters), calcium-binding proteins and calcium-regulated protein kinases available in rice and sorghum. In total, 146 genic "simple sequence repeat" (SSR) primers were designed and evaluated for cross-transferability across a panel of nine grass species including finger millet. The average transferability of genic SSR markers from sorghum to other grasses was highest (73.2 %) followed by rice (63.4 %) with an overall average of 68.3 % which establishes the importance of these major crops as a useful resource of genomic information for minor crops. The transfer rate of SSR markers was also correlated with the phylogenetic relationship (or genetic relatedness) of the species. Primers with successful amplification in finger millet were further used to screen for polymorphism across a set of high and low calcium containing genotypes. The results reveal a conserved behavior across the finger millet genotypes indicating that the mineral transport and the storage machinery largely remain conserved in plants and even SSR variations in them remain suppressed during the course of evolution. Single nucleotide polymorphism and differential expression patterns of candidate genes, therefore, might be a plausible reason to explain variations in grain calcium contents among finger millet genotypes.     

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.     

Comparative genomics and association mapping approaches for opaque2 modifier genes in finger millet accessions using genic,genomic and candidate gene-based simple sequence repeat markers

Identification of alleles responsible for opaque2 modifiers (Opm) influencing tryptophan content in finger millet is a major aim for further improvement of the quality of the locally adapted finger millet germplasm. Since there is little genome sequence information available, comparative genomics plays a very important role in identification of genes/quantitative trait loci (QTLs) linked to the Opm genes using simple sequence repeat (SSR) markers. In the present study, a total of 74 genic SSRs were developed and then used for genetic diversity and population structure analysis of a global collection of 190 finger millet genotypes. The 74 SSRs yielded 133 scorable alleles and the polymorphism information content values varied from 0.186 to 0.707, with an average of 0.408. The gene diversity was in the range of 0.208–0.752, with an average of 0.501. The SSRs developed from the aspartate kinase2 gene of the lysine pathway showed more polymorphism than the other candidate genes. The 74 genic SSR loci grouped the 190 finger millet genotypes into three major clusters based on their tryptophan content, using both phylogenetic clustering and population structure analysis by STRUCTURE software. Association mapping for Opm was done using 120 (74 genic and 46 genomic) SSR loci for identification of QTLs linked to Opm influencing tryptophan content, and found two QTLs for tryptophan and one QTL for protein content. The QTLs for tryptophan content were associated with the genic marker OM5 at a P value of 0.009 and explained 11 % of phenotypic variance (R ²). The OM5 marker was designed from the 27-kDa γ-zein gene of Opm, which influences the tryptophan content to a large extent, whereas the genomic marker FM8 was linked at a P value of 0.004 and explained 9 % of R ². The QTLs for protein content were associated with the genic SSR marker FMO2EST1, which was designed from the RISBZ1 gene of rice and was linked at a P value of 0.002 and explained 9 % of R ². The 220-bp allele of SSR locus OM5 was found to be present mostly in the high tryptophan-containing genotypes such as exotic genotypes, and among the Indian genotypes it was present in NW Himalayan genotypes. The markers linked to the QTLs for Opm found in the present study can be further used for cloning of the full-length gene, for fine mapping and in the marker-assisted breeding programmes for introgression of alleles into locally well-adapted germplasm.     

Development and molecular characterization of genic molecular markers for grain protein and calcium content in finger millet (Eleusine coracana (L.) Gaertn.)

Finger millet (Eleusine coracana (L.) Gaertn), holds immense agricultural and economic importance for its high nutraceuticals quality. Finger millets seeds are rich source of calcium and its proteins are good source of essential amino acids. In the present study, we developed 36 EST-SSR primers for the opaque2 modifiers and 20 anchored-SSR primers for calcium transporters and calmodulin for analysis of the genetic diversity of 103 finger millet genotypes for grain protein and calcium contents. Out of the 36 opaque2 modifiers primers, 15 were found polymorphic and were used for the diversity analysis. The highest PIC value was observed with the primer FMO2E33 (0.26), while the lowest was observed FMO2E27 (0.023) with an average value of 0.17. The gene diversity was highest for the primer FMO2E33 (0.33), however it was lowest for FMO2E27 (0.024) at average value of 0.29. The percentage polymorphism shown by opaque2 modifiers primers was 68.23 %. The diversity analysis by calcium transporters and calmodulin based anchored SSR loci revealed that the highest PIC was observed with the primer FMCA8 (0.30) and the lowest was observed for FMCA5 (0.023) with an average value of 0.18. The highest gene diversity was observed for primer FMCA8 (0.37), while lowest for FMCA5 (0.024) at an average of 0.21. The opaque2 modifiers specific EST-SSRs could able to differentiate the finger millet genotypes into high, medium and low protein containing genotypes. However, calcium dependent candidate gene based EST-SSRs could broadly differentiate the genotypes based on the calcium content with a few exceptions. A significant negative correlation between calcium and protein content was observed. The present study resulted in identification of highly polymorphic primers (FMO2E30, FMO2E33, FMO2-18 and FMO2-14) based on the parameters such as percentage of polymorphism, PIC values, gene diversity and number of alleles.     

Association mapping of agro-morphological characters among the global collection of finger millet genotypes using genomic SSR markers

Identification of alleles responsible for various agro-morphological characters is a major concern to further improve the finger millet germplasm. Forty-six genomic SSRs were used for genetic analysis and population structure analysis of a global collection of 190 finger millet genotypes and fifteen agro-morphological characters were evaluated. The overall results showed that Asian genotypes were smaller in height, smaller flag leaf length, less basal tiller number, early flowering and early maturity nature, small ear head length, and smaller in length of longest finger. The 46 SSRs yielded 90 scorable alleles and the polymorphism information content values varied from 0.292 to 0.703 at an average of 0.442. The gene diversity was in the range of 0.355 to 0.750 with an average value of 0.528. The 46 genomic SSR loci grouped the 190 finger millet genotypes into two major clusters based on their geographical origin by the both phylogenetic clustering and population structure analysis by STRUCTURE software. Association mapping of QTLs for 15 agro-morphological characters with 46 genomic SSRs resulted in identification of five markers were linked to QTLs of four traits at a significant threshold (P) level of ≤0.01 and ≤0.001. The QTL for basal tiller number was strongly associated with the locus UGEP81 at a P value of 0.001 by explaining the phenotypic variance (R ²) of 10.8 %. The QTL for days to 50 % flowering was linked by two SSR loci UGEP77 and UGEP90, explained 10 and 8.7 % of R ² respectively at a P value of 0.01. The SSR marker, FM9 found to have strong association to two agro-morphological traits, flag leaf width (P—0.001, R ²—14.1 %) and plant height (P—0.001, R ²—11.2 %). The markers linked to the QTLs for above agro-morphological characters found in the present study can be further used for cloning of the full length gene, fine mapping and their further use in the marker assisted breeding programmes for introgression of alleles into locally well adapted germplasm.     

A multi-environmental study of recent breeding progress on nitrogen use efficiency in wheat (Triticum aestivum L.)

Key message

By comparing 195 varieties in eight trials, this study assesses nitrogen use efficiency improvement in high and low nitrogen conditions in European winter wheat over the last 25 years.

Abstract

In a context where European agriculture practices have to deal with environmental concerns and nitrogen (N) fertiliser cost, nitrogen use efficiency (NUE) has to be improved. This study assessed genetic progress in winter wheat (Triticum aestivum L.) NUE. Two hundred and twenty-five European elite varieties were tested in four environments under two levels of N. Global genetic progress was assessed on additive genetic values and on genotype × N interaction, covering 25 years of European breeding. To avoid sampling bias, quality, precocity and plant height were added as covariates in the analyses when needed. Genotype × environment interactions were highly significant for all the traits studied to such an extent that no additive genetic effect was detected on N uptake. Genotype × N interactions were significant for yield, grain protein content (GPC), N concentration in straw, N utilisation, and NUE. Grain yield improvement (+0.45 % year^?1) was independent of the N treatment. GPC was stable, thus grain nitrogen yield was improved (+0.39 % year^?1). Genetic progress on N harvest index (+0.12 % year^?1) and on N concentration in straw (?0.52 % year^?1) possibly revealed improvement in N remobilisation. There has been an improvement of NUE additive genetic value (+0.33 % year^?1) linked to better N utilisation (+0.20 % year^?1). Improved yield stability was detected as a significant improvement of NUE in low compared to high N conditions. The application of these results to breeding programs is discussed.     

Strain-specific salt tolerance and chemotaxis ofAzospirillum brasilense and their associative N-fixation with finger millet in saline calcareous soil

Three salt-tolerantAzospirillum brasilense strains were isolated from the roots of finger millet grown in saline calcareous soil and characterized. The effect of various salts on growth and N₂ase activity of these strains was tested and strain STR1 was found more tolerant at higher concentrations of Cl^-, SO₄ ² and HCO₃ ^-. Bicarbonate was found to be the most toxic. The content and concentrations of root exudates of finger millet genotypes were different and chemotaxis to sugars, amino acids, organic acids and root exudates was strain specific. Under salt stress, significant interactions between strains and genotypes of finger millet resulted in different responses of N₂ase activity, endo- and exorhizospheric population, dry weight of root, shoot and grain yield.     

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.