Vijay Gahlaut

Transcription factors involved in drought tolerance and their possible role in developing drought tolerant cultivars with emphasis on wheat ( Triticum aestivum L.)

Key messageTFs involved in drought tolerance in plants may be utilized in future for developing drought tolerant cultivars of wheat and some other crops.AbstractPlants have developed a fairly complex stress response system to deal with drought and other abiotic stresses. These response systems often make use of transcription factors (TFs); a gene encoding a specific TF together with -its target genes constitute a regulon, and take part in signal transduction to activate/silence genes involved in response to drought. Since, five specific families of TFs (out of >80 known families of TFs) have gained widespread attention on account of their significant role in drought tolerance in plants, TFs and regulons belonging to these five multi-gene families (AP2/EREBP, bZIP, MYB/MYC, NAC and WRKY) have been described and their role in improving drought tolerance discussed in this brief review. These TFs often undergo reversible phosphorylation to perform their function, and are also involved in complex networks. Therefore, some details about reversible phosphorylation of TFs by different protein kinases/phosphatases and the co-regulatory networks, which involve either only TFs or TFs with miRNAs, have also been discussed. Literature on transgenics involving genes encoding TFs and that on QTLs and markers associated with TF genes involved in drought tolerance has also been reviewed. Throughout the review, there is a major emphasis on wheat as an important crop, although examples from the model cereal rice (sometimes maize also), and the model plant Arabidopsis have also been used. This knowledge base may eventually allow the use of TF genes for development of drought tolerant cultivars, particularly in wheat.

Identification of Novel SNP in Promoter Sequence of TaGW2-6A Associated with Grain Weight and Other Agronomic Traits in Wheat (Triticum aestivum L.)

TaGW2 is an orthologue of rice gene OsGW2, which encodes E3 RING ubiquitin ligase and controls the grain size in rice. In wheat, three copies of TaGW2 have been identified and mapped on wheat homoeologous group 6 viz. TaGW2-6A, TaGW2-6B and TaGW2-6D. In the present study, using as many as 207 Indian wheat genotypes, we identified four SNPs including two novel SNPs (SNP-988 and SNP-494) in the promoter sequence of TaGW2-6A. All the four SNPs were G/A or A/G substitutions (transitions). Out of the four SNPs, SNP-494 was causal, since it was found associated with grain weight. The mean TGW (41.1 g) of genotypes with the allele SNP-494_A was significantly higher than mean TGW (38.6 g) of genotypes with the allele SNP-494_G. SNP-494 also regulates the expression of TaGW2-6A so that the wheat genotypes with SNP-494_G have higher expression and lower TGW and the genotypes with SNP-494_A have lower expression but higher TGW. Besides, SNP-494 was also found associated with grain length-width ratio, awn length, spike length, grain protein content, peduncle length and plant height. This suggested that gene TaGW2-6A not only controls grain size, but also controls other agronomic traits. In the promoter region, SNP-494 was present in ‘CGCG’ motif that plays an important role in Ca2+/calmodulin mediated regulation of genes. A user-friendly CAPS marker was also developed to identify the desirable allele of causal SNP (SNP-494) for use in marker-assisted selection for improvement of grain weight in wheat. Using four SNPs, five haplotypes were identified; of these, Hap_5 (G_A_G_A) was found to be a desirable haplotype having significantly higher grain weight (41.13g) relative to other four haplotypes (36.33-39.16 g).

Genome Wide Single Locus Single Trait, Multi-Locus and Multi-Trait Association Mapping for Some Important Agronomic Traits in Common Wheat (T. aestivum L.)

Genome wide association study (GWAS) was conducted for 14 agronomic traits in wheat following widely used single locus single trait (SLST) approach, and two recent approaches viz. multi locus mixed model (MLMM), and multi-trait mixed model (MTMM). Association panel consisted of 230 diverse Indian bread wheat cultivars (released during 1910–2006 for commercial cultivation in different agro-climatic regions in India). Three years phenotypic data for 14 traits and genotyping data for 250 SSR markers (distributed across all the 21 wheat chromosomes) was utilized for GWAS. Using SLST, as many as 213 MTAs (p ≤ 0.05, 129 SSRs) were identified for 14 traits, however, only 10 MTAs (~9%; 10 out of 123 MTAs) qualified FDR criteria; these MTAs did not show any linkage drag. Interestingly, these genomic regions were coincident with the genomic regions that were already known to harbor QTLs for same or related agronomic traits. Using MLMM and MTMM, many more QTLs and markers were identified; 22 MTAs (19 QTLs, 21 markers) using MLMM, and 58 MTAs (29 QTLs, 40 markers) using MTMM were identified. In addition, 63 epistatic QTLs were also identified for 13 of the 14 traits, flag leaf length (FLL) being the only exception. Clearly, the power of association mapping improved due to MLMM and MTMM analyses. The epistatic interactions detected during the present study also provided better insight into genetic architecture of the 14 traits that were examined during the present study. Following eight wheat genotypes carried desirable alleles of QTLs for one or more traits, WH542, NI345, NI170, Sharbati Sonora, A90, HW1085, HYB11, and DWR39 (Pragati). These genotypes and the markers associated with important QTLs for major traits can be used in wheat improvement programs either using marker-assisted recurrent selection (MARS) or pseudo-backcrossing method.

A multi-step phosphorelay two-component system impacts on tolerance against dehydration stress in common wheat

Wheat is an important staple crop, and its productivity is severely constrained by drought stress (DS). An understanding of the molecular basis of drought tolerance is necessary for genetic improvement of wheat for tolerance to DS. The two-component system (TCS) serves as a common sensor-regulator coupling mechanism implicated in the regulation of diverse biological processes (including response to DS) not only in prokaryotes, but also in higher plants. In the latter, TCS generally consists of two signalling elements, a histidine kinase (HK) and a response regulator (RR) associated with an intermediate element called histidine phosphotransferase (HPT). Keeping in view the possible utility of TCS in developing water use efficient (WUE) wheat cultivars, we identified and characterized 62 wheat genes encoding TCS elements in a silico study; these included 7 HKs, 45 RRs along with 10 HPTs. Twelve of the 62 genes showed relatively higher alterations in the expression under drought. The quantitative RT-PCR (qRT-PCR)-based expression analysis of these 12 TCS genes was carried out in wheat seedlings of a drought sensitive (HD2967) and a tolerant (Dharwar Dry) cultivar subjected to either dehydration stress or cytokinin treatment. The expression of these 12 genes under dehydration stress differed in sensitive and tolerant genotypes, even though for individual genes, both showed either up-regulation or down-regulation. In response to the treatment of cytokinin, the expression of type-A RR genes was higher in the tolerant genotype, relative to that in the sensitive genotype, the situation being reverse for the type-B RRs. These results have been discussed in the context of the role of TCS elements in drought tolerance in wheat.

Stage-specific reprogramming of gene expression characterizes Lr48-mediated adult plant leaf rust resistance in wheat

Wheat genotype CSP44 carrying a recessive gene Lr48 exhibits adult plant resistance (APR; incompatible reaction) but gives a compatible reaction (susceptibility) at the seedling stage against leaf rust. A comparative gene expression analysis involving cDNA-amplified fragment length polymorphism (cDNA-AFLP) and quantitative PCR (qPCR) was carried out for incompatible and compatible reactions in the genotype CSP44. cDNA-AFLP analysis was conducted using RNA samples that were isolated from flag leaves following inoculation with leaf rust race 77–5 (the most virulent race) and also after mock inoculation. As many as 298 of a total of 493 expressed transcript-derived fragments (TDFs) exhibited differential expression (262 upregulated and 36 downregulated). Of these 298 TDFs, 48 TDFs were eluted from gels, re-amplified, cloned, and sequenced. Forty two of these 48 TDFs had homology with known genes involved in the following biological processes: energy production, metabolism, transport, signaling, defense response, plant-pathogen interaction, transcriptional regulation, translation, and proteolysis. The functions of the remaining six TDFs could not be determined; apparently, these represented some novel genes. The qPCR analysis for 18 TDFs (with known and unknown functions, but showing major differences in expression) was conducted using RNA isolated from the seedlings as well as from the adult plants. The expression of at least 11 TDFs was induced and that of 4 other TDFs attenuated or remained near normal in adult plants following leaf rust inoculations. The remaining three TDFs had non-specific/developmental stage-specific expression. Functional annotation of TDFs that were upregulated suggest that the APR was supported by transient recruitment and reprogramming of processes like perception and recognition of pathogen effector by receptors, followed by CDPK and MAPK signaling, transport, metabolism, and energy release.

QTL mapping for nine drought-responsive agronomic traits in bread wheat under irrigated and rain-fed environments

In bread wheat, QTL interval mapping was conducted for nine important drought responsive agronomic traits. For this purpose, a doubled haploid (DH) mapping population derived from Kukri/Excalibur was grown over three years at four separate locations in India, both under irrigated and rain-fed environments. Single locus analysis using composite interval mapping (CIM) allowed detection of 98 QTL, which included 66 QTL for nine individual agronomic traits and 32 QTL, which affected drought sensitivity index (DSI) for the same nine traits. Two-locus analysis allowed detection of 19 main effect QTL (M-QTL) for four traits (days to anthesis, days to maturity, grain filling duration and thousand grain weight) and 19 pairs of epistatic QTL (E-QTL) for two traits (days to anthesis and thousand grain weight). Eight QTL were common in single locus analysis and two locus analysis. These QTL (identified both in single- and two-locus analysis) were distributed on 20 different chromosomes (except 4D). Important genomic regions on chromosomes 5A and 7A were also identified (5A carried QTL for seven traits and 7A carried QTL for six traits). Marker-assisted recurrent selection (MARS) involving pyramiding of important QTL reported in the present study, together with important QTL reported earlier, may be used for improvement of drought tolerance in wheat. In future, more closely linked markers for the QTL reported here may be developed through fine mapping, and the candidate genes may be identified and used for developing a better understanding of the genetic basis of drought tolerance in wheat.

A Web-resource for Nutrient Use Efficiency related Genes, QTLs, and microRNA in important cereals and model plants

Cereals are the key contributors to global food security. Genes involved in uptake (transport), assimilation and utilization of macro- and micro-nutrients are responsible for their content in grain and straw. Although many cereal genomic databases are available, currently there is no cohesive web-resource of manually curated nutrient use efficiency (NtUE) related genes and QTLs, etc. In this study, we present a web-resource containing information on NtUE related genes/QTLs and the corresponding available microRNAs for some of these genes in four major cereal crops [wheat (Triticum aestivum), rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare)], two alien species (Triticum urartu and Aegilops tauschii) related to wheat, and two model species including Brachypodium distachyon and Arabidopsis thaliana. Gene annotations integrated in the current web-resource were collected from the existing databases and the available literature. The primary goal of developing this web-resource is to provide descriptions of the NtUE related genes and their functional annotation. MicroRNA targeting some of the NtUE related genes and the quantitative trait loci (QTLs) for NtUE related traits are also included. The available information in the web-resource should help the users to readily search the desired information. Web-resource URL http://bioclues.org/NtUE/

­­­ A web resource for nutrient use efficiency-related genes, quantitative trait loci and microRNAs in important cereals and model plants

Cereals are key contributors to global food security. Genes involved in the uptake (transport), assimilation and utilization of macro- and micronutrients are responsible for the presence of these nutrients in grain and straw. Although many genomic databases for cereals are available, there is currently no cohesive web resource of manually curated nutrient use efficiency (NtUE)-related genes and quantitative trait loci (QTLs). In this study, we present a web-resource containing information on NtUE-related genes/QTLs and the corresponding available microRNAs for some of these genes in four major cereal crops (wheat ( Triticum aestivum), rice ( Oryza sativa), maize ( Zea mays), barley ( Hordeum vulgare)), two alien species related to wheat ( Triticum urartu and Aegilops tauschii), and two model species ( Brachypodium distachyon and Arabidopsis thaliana). Gene annotations integrated in the current web resource were manually curated from the existing databases and the available literature. The primary goal of developing this web resource is to provide descriptions of the NtUE-related genes and their functional annotation. MicroRNAs targeting some of the NtUE related genes and the QTLs for NtUE-related traits are also included. The genomic information embedded in the web resource should help users to search for the desired information.

Genome-Wide Mining, Characterization and Development of miRNA-SSRs in Arabidopsis thaliana

Simple Sequence Repeats (SSRs), also known as microsatellites are short tandem repeats of DNA sequences that are 1-6 bp long. In plants, SSRs serve as a source of important class of molecular markers because of their hypervariabile and co-dominant nature, making them useful both for the genetic studies and marker-assisted breeding. The SSRs are widespread throughout the genome of an organism, so that a large number of SSR datasets are available, most of them from either protein-coding regions or untranslated regions. It is only recently, that their occurrence within microRNAs (miRNA) genes has received attention. As is widely known, miRNA themselves are a class of non-coding RNAs (ncRNAs) with varying length of 19-22 nucleotides (nts), which play an important role in regulating gene expression in plants under different biotic and abiotic stresses. In this communication, we describe the results of a study, where miRNA-SSRs in full length pre-miRNA sequences of Arabidopsis thaliana were mined. The sequences were retrieved by annotations available at EnsemblPlants using BatchPrimer3 server with miRNA-SSR flanking primers found to be well distributed. Our analysis shows that miRNA-SSRs are relatively rare in protein-coding regions but abundant in non-coding region. All the observed 147 di-, tri-, tetra-, penta- and hexanucleotide SSRs were located in non-coding regions of all the 5 chromosomes of A. thaliana. While we confirm that miRNA-SSRs were commonly spread across the full length pre-miRNAs, we envisage that such studies would allow us to identify newly discovered markers for breeding studies.