Genomic-based-breeding tools for tropical maize improvement

Maize has traditionally been the main staple diet in the Southern Asia and Sub-Saharan Africa and widely grown by millions of resource poor small scale farmers. Approximately, 35.4 million hectares are sown to tropical maize, constituting around 59% of the developing worlds. Tropical maize encounters tremendous challenges besides poor agro-climatic situations with average yields recorded <3 tones/hectare that is far less than the average of developed countries. On the contrary to poor yields, the demand for maize as food, feed, and fuel is continuously increasing in these regions. Heterosis breeding introduced in early 90 s improved maize yields significantly, but genetic gains is still a mirage, particularly for crop growing under marginal environments. Application of molecular markers has accelerated the pace of maize breeding to some extent. The availability of array of sequencing and genotyping technologies offers unrivalled service to improve precision in maize-breeding programs through modern approaches such as genomic selection, genome-wide association studies, bulk segregant analysis-based sequencing approaches, etc. Superior alleles underlying complex traits can easily be identified and introgressed efficiently using these sequence-based approaches. Integration of genomic tools and techniques with advanced genetic resources such as nested association mapping and backcross nested association mapping could certainly address the genetic issues in maize improvement programs in developing countries. Huge diversity in tropical maize and its inherent capacity for doubled haploid technology offers advantage to apply the next generation genomic tools for accelerating production in marginal environments of tropical and subtropical world. Precision in phenotyping is the key for success of any molecular-breeding approach. This article reviews genomic technologies and their application to improve agronomic traits in tropical maize breeding has been reviewed in detail

Identification of salt-induced genes from Salicornia brachiata, an extreme halophyte through expressed sequence tags analysis

Salinity severely affects plant growth and development causing crop loss worldwide. We have isolated a large number of salt-induced genes as well as unknown and hypothetical genes from Salicornia brachiata Roxb. (Amaranthaceae). This is the first description of identification of genes in response to salinity stress in this extreme halophyte plant. Salicornia accumulates salt in its pith and survives even at 2 M NaCl under field conditions. For isolating salt responsive genes, cDNA subtractive hybridization was performed between control and 500 mM NaCl treated plants. Out of the 1200 recombinant clones, 930 sequences were submitted to the NCBI database (GenBank accession: EB484528 to EB485289 and EC906125 to EC906292). 789 ESTs showed matching with different genes in NCBI database. 4.8% ESTs belonged to stress-tolerant gene category and approximately 29% ESTs showed no homology with known functional gene sequences, thus classified as unknown or hypothetical. The detection of a large number of ESTs with unknown putative function in this species makes it an interesting contribution. The 90 unknown and hypothetical genes were selected to study their differential regulation by reverse Northern analysis for identifying their role in salinity tolerance. Interestingly, both up and down regulation at 500 mM NaCl were observed (21 and 10 genes, respectively). Northern analysis of two important salt tolerant genes, ASR1 (Abscisic acid stress ripening gene) and plasma membrane H<SUP>+</SUP>ATPase, showed the basal level of transcripts in control condition and an increase with NaCl treatment. ASR1 gene is made full length using 5' RACE and its potential role in imparting salt tolerance is being studied

A high-throughput genome-walking method and its use for cloning unknown flanking sequences

We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5' ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5' end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5' flanking regions/promoters of selected plant genes

Comprehensive evaluation of candidate reference genes for real-time quantitative PCR (RT-qPCR) data normalization in nutri-cereal finger millet [Eleusine Coracana (L.)].

Finger millet (Eleusine coracana L.) is an annual herbaceous self-pollinating C4 cereal crop of the arid and semi-arid regions of the world. Finger millet is a food security crop proven to have resilience to changing climate and scores very high in nutrition. In the current study, we have assessed sixteen candidate reference genes for their appropriateness for the normalization studies in finger millet subjected to experimental regimes and treatments. Ten candidate reference genes (GAPDH, β-TUB, CYP, EIF4α, TIP41, UBC, G6PD, S24, MACP and MDH) were cloned and six (ACT, ELF1α, PP2A, PT, S21 and TFIID) were mined from the NCBI database as well as from the literature. Expression stability ranking of the finger millet reference genes was validated using four different statistical tools i.e., geNorm, NormFinder, BestKeeper, ΔCt and RefFinder. From the study, we endorse MACP, CYP, EIF4α to be most stable candidate reference genes in all 'tissues', whereas PT, TFIID, MACP ranked high across genotypes, β-TUB, CYP, ELF1α were found to be best under abiotic stresses and 'all samples set'. The study recommends using minimum of two reference genes for RT-qPCR data normalizations in finger millet. All in all, CYP, β-TUB, and EF1α, in combination were found to be best for robust normalizations under most experimental conditions. The best and the least stable genes were validated for confirmation by assessing their appropriateness for normalization studies using EcNAC1 gene. The report provides the first comprehensive list of suitable stable candidate reference genes for nutritional rich cereal finger millet that will be advantageous to gene expression studies in this crop

Molecular cloning and characterization of gene encoding for cytoplasmic Hsc70 from Pennisetum glaucum may play a protective role against abiotic stresses

Molecular chaperones (Hsps) have been shown to facilitate protein folding or assembly under various developmental and adverse environmental conditions. The aim of this study was to unravel a possible role of heat-shock proteins in conferring abiotic stress tolerance to plants. We isolated a cDNA encoding a cytoplasmic Hsp70 (PgHsc70) from Pennisetum glaucum by screening heat-stress cDNA library. PgHsc70 cDNA encoding 649 amino acids represents all conserved signature motifs characteristic of Hsp70s. The predicted molecular model of PgHsc70 protein suggests that the N-terminus ATP-binding region is evolutionarily conserved, in comparison to C-terminus peptide-binding domains. A single intron in ATPase domain coding region of PgHsc70 exhibited a high degree of conservation with respect to its position and phasing among other plant Hsp70 genes. Recombinant PgHsc70 protein purified from E. coli possessed in vitro chaperone activity and protected PgHsc70 expressing bacteria from damage caused by heat and salinity stress. Nucleotide sequence analysis of 5' flanking promoter region of PgHsc70 gene revealed a potential heat-shock element (HSE) and other putative stress-responsive transcription factor binding sites. Positive correlation existed between differentially up-regulated PgHsc70 transcript levels and the duration and intensity of different environmental stresses. Molecular and biochemical analyses revealed that PgHsc70 gene was a member of the Hsp70 family and suggested that its origin was from duplication of a common ancestral gene. Transcript induction data, presence of several putative stress-responsive transcription factor-binding sites in the promoter region of PgHsc70 and the presence of a protective in vitro chaperone activity of this protein against damage caused by heat and salinity, when expressed in E. coli, suggest its probable role in conferring abiotic stress tolerance to this plant

Isolation and characterization of expressed sequence tags (ESTs) from subtracted cDNA libraries of Pennisetum glaucum seedlings

Pearl millet (Pennisetum glaucum), used as forage and grain crop is a stress tolerant species. Here we identify differentially regulated transcripts in response to abiotic (salinity, drought and cold) stresses from subtracted cDNA libraries by single-pass sequencing of cDNA clones. A total of 2,494 EST sequences were clustered and assembled into a collection of 1,850 unique sequences with 224 contigs and 1,626 singleton sequences. By sequence comparisons the putative functions of many ESTs could be assigned. Genes with stress related functions include those involved in cellular defense against abiotic stresses and transcripts for proteins involved in stress response signaling and transcription in addition to ESTs encoding unknown functions. These provide new candidate genes for investigation to elucidate their role in abiotic stress. The relative mRNA abundance of 38 selected genes, quantified using real time quantitative RT-PCR, demonstrated the existence of a complex gene regulatory network that differentially modulates gene expression in a kinetics-specific manner in response to different abiotic stresses. Notably, housekeeping and non-target genes were effectively reduced in these subtracted cDNA libraries constructed. These EST sequences are a rich source of stress-related genes and reveal a major part of the stress-response transcriptome that will provide the foundation for further studies into understanding Pennisetum's adaptability to harsh environmental conditions

Biochemical and molecular characterization of stress-induced β-carbonic anhydrase from a C<SUB>4</SUB> plant, Pennisetum glaucum

Genes encoding for many &#946;-carbonic anhydrases and their functions in various developmental processes are well established in lower plants, however, similar studies are limited in higher plants. We report the cloning and characterization of cDNA encoding for a &#946;-carbonic anhydrase (PgCA) from Pennisetum glaucum, a C4 crop plant. cDNA encoding 249 amino acids and its deduced amino acid sequence analysis revealed that is related to other plant &#946;-CA family members with an over all conserved architecture of a typical &#946;-CA protein. Phylogenetic analysis revealed that PgCA is evolutionarily very close to chloroplast &#946;-CA isoform. Signal sequence predicting programs identify a N-terminus putative chloroplast targeting sequence. Heterologous Escherichia coli expression system was utilized to overexpress recombinant PgCA, which showed high thermostability, an alkaline pH optima and dual activity with both reversible CO2 hydration and esterase activities. The &#946;-CAs studied so far possessed only CO2 hydration activity with no detectable esterase activity. Recombinant PgCA esterase activity is inhibited by standard CA inhibitors acetazolamide, methazolamide and azide. Subcellular immunostaining studies revealed a chloroplastic localization of PgCA protein. Expression of PgCA transcript is differentially up regulated in response to various abiotic stresses wherein its accumulation in Pennisetum leaves positively correlated with the intensity and duration of stress. Biochemical and transcript analyses suggest that PgCA may play a significant role in plant's adaptation to different abiotic stresses in addition to the previously recognized role of replenishing the CO2 supply within plant cells

Unraveling regulation of the small heat shock proteins by the heat shock factor HvHsfB2c in barley: its implications in drought stress response and seed development.

The rapid increase in heat shock proteins upon exposure to damaging stresses and during plant development related to desiccation events reveal their dual importance in plant development and stress tolerance. Genome-wide sequence survey identified 20 non-redundant small heat shock proteins (sHsp) and 22 heat shock factor (Hsf) genes in barley. While all three major classes (A, B, C) of Hsfs are localized in nucleus, the 20 sHsp gene family members are localized in different cell organelles like cytoplasm, mitochondria, plastid and peroxisomes. Hsf and sHsp members are differentially regulated during drought and at different seed developmental stages suggesting the importance of chaperone role under drought as well as seed development. In silico cis-regulatory motif analysis of Hsf promoters showed an enrichment with abscisic acid responsive cis-elements (ABRE), implying regulatory role of ABA in mediating transcriptional response of HvsHsf genes. Gene regulatory network analysis identified HvHsfB2c as potential central regulator of the seed-specific expression of several HvsHsps including 17.5CI sHsp. These results indicate that HvHsfB2c is co-expressed in the central hub of small Hsps and therefore it may be regulating the expression of several HvsHsp subclasses HvHsp16.88-CI, HvHsp17.5-CI and HvHsp17.7-CI. The in vivo relevance of binding specificity of HvHsfB2C transcription factor to HSE-element present in the promoter of HvSHP17.5-CI under heat stress exposure is confirmed by gel shift and LUC-reporter assays. Further, we isolated 477 bp cDNA from barley encoding a 17.5 sHsp polypeptide, which was predominantly upregulated under drought stress treatments and also preferentially expressed in developing seeds. Recombinant HvsHsp17.5-CI protein was expressed in E. coli and purified to homogeneity, which displayed in vitro chaperone activity. The predicted structural model of HvsHsp-17.5-CI protein suggests that the α-crystallin domain is evolutionarily highly conserved

Genome-wide Scanning and Characterization of Sorghum bicolor L. Heat Shock Transcription Factors

A genome-wide scanning of Sorghum bicolor resulted in the identification of 25 SbHsf genes. Phylogenetic analysis shows the ortholog genes that are clustered with only rice, representing a common ancestor. Promoter analysis revealed the identification of different cis-acting elements that are responsible for abiotic as well as biotic stresses. Hsf domains like DBD, NLS, NES, and AHA have been analyzed for their sequence similarity and functional characterization. Tissue specific expression patterns of Hsfs in different tissues like mature embryo, seedling, root, and panicle were studied using real-time PCR. While Hsfs4 and 22 are highly expressed in panicle, 4 and 9 are expressed in seedlings. Sorghum plants were exposed to different abiotic stress treatments but no expression of any Hsf was observed when seedlings were treated with ABA. High level expression of Hsf1 was noticed during high temperature as well as cold stresses, 4 and 6 during salt and 5, 6, 10, 13, 19, 23 and 25 during drought stress. This comprehensive analysis of SbHsf genes will provide an insight on how these genes are regulated in different tissues and also under different abiotic stresses and help to determine the functions of Hsfs during drought and temperature stress tolerance

Thermo and pH stable ATP-independent chaperone activity of heat-inducible Hsp70 from Pennisetum glaucum

Heat shock proteins (Hsps) are a class of molecular chaperones that play an essential role in preserving cellular functions under stressful conditions. The over production of recombinant proteins often causes cellular stress that results in aggregation/misfolding of proteins, which sometimes leads to the formation of inclusion bodies. Here we report the cloning and characterization of heat-inducible PgHsp70 from Pennisetum glaucum, a heat and drought tolerant plant that showed stability and chaperone activity at elevated temperatures. The predicted amino acid sequence of PgHsp70 revealed a high homology with Hsp70 from other plants, and the overall 3D structure homology modeling is similar to that of the constitutively expressed bovine cytosolic Heat Shock Cognate (HSC)-70. The purified recombinant protein had an apparent molecular mass of 70 kDa and displayed optimal chaperone activity at 50°C, and pH 8.0. Under these conditions, the T1/2 of PgHsp70 increased from 10 to 15 h in the presence of glycerol. The PgHsp70 exhibited a higher chaperone activity towards glutamate dehydrogenase than alcohol dehydrogenase. The expression of recombinant carbonic anhydrase (CA ) in E. coli in a catalytically active soluble form rather than in inclusion bodies was made feasible by co-expression of PgHsp70. Circular dichroism (CD) studies of the recombinant PgHsp70 did not reveal any discernible changes in the α-helix content, with increase in temperature from 35 to 85°C, thus suggesting a critical role of α-helix content in maintaining the chaperone activity