A proposal in partial fulfillment of the requirements for the degree of
Introduction
Wheat (Triticum sp.), an extensively adapted crop, is one of the primitive and primary staple food crops in the world. Wheat is cultivated from temperate; dry to irrigated or rain-fed areas, to warm, humid or cold conditions. Such a high adaptability is attributed to its intricate genome (Acevedo et al., 2002). Wheat cultivation covers more land area compared to other commercial crops and endures to be an essential food grain for humans (Curtis, 2002). Estimations reveal that by 2050, the population of the world will reach 9.1 billion (34 percent greater than today); correspondingly, the food requirement and water consumption will also rise. All these modifications in the environmental conditions bring climate transformation and peril for long-term food security; essentially, agriculture needs to adapt to such reforms (FAO. 2009). This research proposal aims to study physiological and genetic responses of resistant and susceptible spring wheat in the grain-filling stage of development to water stress.
Increasing population raises freshwater demands for daily needs and for producing more grains to feed them. There is an ever-increasing demand for water supply across the world, threatening biodiversity, water supply for the cultivation of food and for other vital human activities. Agriculture requires 70 percent of the available freshwater (900L water is needed to grow 1 kg of cereal grain) (Pimentel et al., 2004). Forest mortality and global climate change are causing global warming responsible for prolonged intense aridity, high temperature or floods. To meet domestic water demands by means of transferal from agriculture brings alarm regarding rural sustainability and food security since regional water utilization is dominated by agriculture (MacDonald, 2010).
The importance of understanding the limitations of plant growth and development under limitations in the availability of water is based on the essential aspect that crops play in the world economy and the everyday lives of people. Plant products, such as wheat, not only affect the stability of the world economy but are also a staple in the diets of people in various countries throughout. In understanding the development of these plants through the study of their physiological and genetic mechanisms, the ability to better maintain this ecological and economic position can be achieved.
Drought is among the most severe abiotic stresses which limit plant growth and crop productivity in agriculture worldwide mainly due to deleterious and adaptive responses of the plants itself (Chaves et al., 2002 and de Oliveira et al., 2013). About 25% of the agricultural throughout the world is affected by drought at present (Li et al., 2011). Both the severity and duration of the stress are critical that losses in crop yield induced by drought probably exceeds that of all other causes (de Oliveira et al., 2013). Selection and development of drought tolerant plant cultivars are a very important aspect of sustainable agricultural production systems in the water-limited areas.
The inclination of plants to keep up a high capability of water in the tissues under drought is called dehydration avoidance, and tolerance that decides plant inclination to survive water inadequacy is called drought resistance (Blum, 2005).
Crop production and yield are limited by drought or water restraints. Crops validate numerous morphological, biochemical; physiological, as well as molecular responses to deal with drought stress. Drought tolerance in the plant is controlled by polygenes; expressed under diverse environmental elements (Nezhadahmadi et al., 2013).
Abayomi and Wright (1999), conducted two experiments to assess the outcomes of water stress applied at diverse growth stages on growth, yield factors and grain produce of Triticum aestivum L, the spring wheat. Water stress influences growth, however, a reduction in yield and better recovery was seen when water stress was introduced at an early vegetative stage (caused reduced tiller production, compensated as late tiller stage after re-watering) compared to late vegetative stage as well as post anthesis phase. Water stress introduced after 32-65 days, reduced spikelet fertility and number of grains in each ear, thereby reducing the total crop yield. The study emphasized that water stress significantly influences yield if induced at the reproductive stage and therefore screening of genotypes tolerant to water stress should be done during the reproductive stage.
Drought is emerging as a new challenge to plant researchers and breeders. Estimations reveal that by 2025, 65 percent of the world population would live in the water-stressed environment; essentially water stress tolerant species are important (Nezhadahmadi et al., 2013). Water stress comprises drought avoidance (encompasses root depth, to utilize available water and lifestyle modifications in plants to use rainfall) and dehydration tolerance (involves the ability of the plant to dehydrate partially and revert to growth when rainfall endures). Factors influencing plant’s response to drought are genotype of plant, stage of drought induction, duration and severity of stress, gene expression, respiration pattern, physiological process of growth, photosynthetic machinery and other environmental factors (Nezhadahmadi et al., 2013).
Certain genes are influenced by drought and express diverse proteins and enzymes under drought stress such as vacuolar acid invertase, dehydrins, late embryo abundant (LEA), glutathione S-transferase (GST), ABA gene expression, protein RAB formation, proline, helicase and carbohydrate form the molecular basis of tolerating drought. Altered gene expression and protein formation is a method by which plants deal with stress milieu (Nezhadahmadi et al., 2013). According to Sivamani et al. (2000), under drought stress, HVA1 gene aids to promote wheat growth, by synthesizing a protein belonging to 3 LEA.
A vital role is played by proline in drought stress, Hong-Bo et al., (2006), considered proline as preventive protein in drought, an anti-drought protein. RUBISCO, the key enzyme of Calvin cycle is also expressed under drought. LEA is preserved in vegetative tissue during the process of seed desiccation. Kam et al. (2007) detected TaRZF70 (RING-H2 Zn-finger), a water stress gene in wheat and found that the gene is downregulated in the root (TaRZF38 and TaRZF70) and up-regulated in the shoot (TaRZF74 and TaRZF59). RD (responsive to desiccation) gene is influenced by drought stress. In wheat, genes responding to drought stress comprise 265 genes identified at junction phase while 146 genes at the seedling stage (Shi et al., 2010).
Drought, abiotic stress, influences photosynthesis of the plant. Wheat cultivation requires arid-agricultural fields; condition of drought stress directly affects wheat cultivation. A study carried out by Hasan et al., (2014), with four wheat cultivars using remobilization of storage, water stress was induced just before flowering stage by withholding irrigation. It was found that seed weight in spike, seed yield, and harvesting index are found to be low in water stress induced before the flowering stage. Moreover, remobilization of stored integrates was reported to be higher in the condition of water stress. The remobilization efficacy was enhanced under the condition of water stress. Environmental conditions influence genotypes competence (Hasan et al., 2014).
Limitations of previous studies on drought stress
However, limited knowledge is available on drought responsive genes and roles they play during drought stress. Most of the studies are being carried out in the wheat seedling stage; however, research reveals that the junction or joining phase (between vegetative and flowering stage) is highly susceptible to drought (Nezhadahmadi et al., 2013). Little information is available for the efficacy of flag leaves function under stress or adaptations facilitating such functioning. The flag leaf of the wheat plant plays very crucial roles in the growth and development of wheat (Ledent & Renerd, 1982). First and foremost the flag leaf is responsible for approximately 75% to 80% of photosynthesis in the wheat plant. Photosynthesis is without an iota of doubt a significant phenomenon towards the processing of food in plants. The emergence of the flag leaf in wheat plant triggers the start of reproductive growth in wheat plants. Before the emergence of the flag leaf only vegetative growth occurs in wheat plants. In addition to contributing towards photosynthesis in wheat plants, flag leaf also plays an important role in the emergence spikes in wheat plant.
Moreover, inconsistent information exists on the physiological, morpho-anatomical, as well as a biochemical aspect of flag leaves under drought. Significantly, very few reports endure on the mechanistic and molecular understanding of recognized adaptations of flag leaf functionality under the condition of drought at the ripening or the filling stage. Moreover, the natural disparity in photosynthetic aptitude presently remained an unexploited genetic reserve for prospective crop enhancement. The present study aims to explore the genome sequence of the tolerant (Alpowa) and sensitive (Idaho) genotypes of these spring wheat at the filling or the ripening stage, the least explored stage and not much literature are available on this subject.
Literature Review
Global warming is increasing. Consequently, the temperature is also increasing, and little water is available for the agriculture (Lobell et al., 2008). The situation is becoming critical due to an exponential increase in the human population. The chief cereal crops encompassing wheat, maize, and rice are required to survive the condition of drought by various modifications involving genetic and physiological adaptations and grab scientific attentiveness (Collins et al., 2008), particularly for the sessile beings, the plants, the source of survival.
Under the condition of drought, plants show enormous adaptation as they have well-defined reproduction and survival strategies. Modifications occur at physiological, cellular and molecular levels by genotype, species and water loss criticality, and stage of development as well as tissue type. Loss of water and condition of drought induces abscisic acid (ABA)-independent as well as ABA-dependent controlling systems comprising signal transduction cascades the protein kinases/phosphatases (for example, Ca2+ dependent protein kinase phosphatases) causing activation of series of transcription factors. These results in alteration of transcription programs for the formation of novel proteins permitting protections against osmotic stresses, triggering the elimination of toxic compounds and boosting functional defense or deficiency of existing proteins (Barnabás et al., 2008).
Freshly produced proteins in response to water insufficiency comprise LEA (late embryogenesis abundant) proteins, sugar transporters, dehydrins, osmolytes, lipid metabolism-related genes, aquaporin, antioxidants proteasome components and heat shock proteins (HSPs) (Barnabás et al., 2008). Numerous sugars (like sucrose, sorbitol or trehalose), amino acids (like proline), sugar alcohols (such as mannitol), and amines like polyamides and glycine betaine gather in plants in the condition of water loss as well as play an important role as osmolytes adjusting cell turgidity as well as stabilize proteins (Seki et al., 2007). Akin to dehydrins, LEA proteins are very hydrophilic and could have a role in shielding already existing proteins from ensuing degradation and unfolding (Mahajan & Tuteja, 2005).
The major reason for drought-induced difficulties is the blockage of photosynthesis since it results in the collection of reactive oxygen species (ROS). To combat such an accumulation of ROS, intricate and dissimilar group of biochemical antioxidants, like glutathione, ascorbic acid, flavonoids, and an enormous amount of enzymes, like catalase (CAT) or peroxidases, usually complement drought responses (Foyer & Noctor, 2009).
It is estimated that adaptation to prime adaptation and domestication milieus for maximum yield brought deprivation of genetic diversity in modern wheat (hexaploid, (Triticum aestivum L.) that empowered the wheat prototypes to deal with the suboptimal environment for growth. On the other hand, wild emmer wheat (a tetraploid, Triticum turgid sap. microcodes has preserved its evolutionary adaptive features and is accordingly, an encouraging entrant for understanding abiotic stress resilience genes that could be subjugated for improving crop (Dubcovsky & Dvorak, 2007).
Identification of new-flanged abiotic stress tolerant genes could be made with the help of microarray. The techniques practice is based on the utilization of a platform for genome-wide investigation of transcripts obtained from wheat species for scrutinizing transformations in the transcriptome of wheat when they are exposed to the condition of drought. The analysis performed with the help of microarray involves testing the hypothesis that a few transcriptome pathways are distinctive to wild type wheat and associated with their ability to tolerate drought.
Rampino et al., (2012) used cDNA-AFLP analysis and identified that novel durum wheat genes 7, 8 and 15 were up-regulated due to the condition of heat, drought or their combination respectively. Furthermore, transcriptome analysis for wheat caryopses administered to water scarcity alone or in combination with heat utilizing 15 k oligonucleotide microarrays disclosed that merely 0.5 % of the examined genes were influenced by drought only and an equivalent heat treatment amplified the proportion to 5–7 percent (Szűcs et al., 2010).
Polyploidy is reported in 70 percent of angiosperms and is responsible for broader adaptability under unfavorable circumstances (Dubcovsky & Dvorak, 2007).
RNA-sequencing methodology, which is also referred to as whole transcriptome shotgun sequencing refers to techniques employed in an attempt to determine the sequence of RNA molecules. RNA-seq methodology has been known to continuously replacing gene expression microarrays in various labs. Normally, mRNA are converted to cDNA which after that is employed as an input to a next generation sequencing library preparation. There are various methods of performing RNA-seq experiment. In fact, there are so many techniques that deciding on the one to use is rather difficult. Without an iota of doubt, RNA-seq is a powerful tool which has been in use for the last few years. RNA sequencing can be used in psoriasis investigations and also in identifying new genes for functional analysis. This is just one of the many uses of RNA-seq. RNA-seq is continuously evolving. The method is not only more robust and sensitive but also cost effective. Therefore, making use of RNA-seq in this research project is prudent. However, although RNA-Seq technology is becoming used in various transcriptomics, there are a lot of challenges experienced in analysis and interpretation of RNA-Seq data due to transcriptome complexity (Nawy, 2013).
Objective and objective significance
Transcriptomic analysis for spike and flag leaf of selected spring wheat varieties to water limitations
Drought tolerant and susceptible varieties of wheat will be selected and screened for transcriptomic using RNA-Seq under the condition of stress. Grain yield is the finale of the procedure of grain filling, closely associated with flag leaf functionalities. The present study will give us information on the functionality of wheat flag leaf under drought to emphasize the necessity for a better understanding of adaptations, particularly at the molecular level, implying the efficient and sensible use of properties for screening wider germplasm pools.
Materials and methods
The imposing of drought levels takes place four days after the commencement of synthesis. At this juncture, wheat was at Feekes growth stage 11.2 (Mealy consistency), suitable for harvesting. To begin, flag leaf weight, head weight, and shoot weight (dry and green leaves) were taken. After this process, the flag leaves, and heads were separately wrapped in aluminum foil. All collected aluminum rolls were placed in liquid nitrogen for ten seconds and put in the freezer at -80 degrees Celsius.
Methods Objective
The most assuring tolerant and sensitive genotypes from present screens will be selected by genetical responses to the conditions of drought. The tolerant (Alpowa) and sensitive (Idaho) genotypes of spring wheat will then be utilized for genome-wide evaluation of transcript changes when the condition of water stress be introduced using the Affymetrix GeneChip® Wheat Genome Array.
RNA extraction and synthesis of cDNA: Trizol reagent protocol
The first step involves homogenizing tissue samples in a polytron homogenizer. It is important to clean the probe before commencing the homogenizing procedure. Therefore, it is important to run the homogenizer for approximately 30sec with treated water, preferably DECP. After that, run the homogenizer with absolute ethanol and eventually with 2ml of triazole in a 5ml tube. It is important to inspect the probe for residual tissue regularly. Once homogenization is done, the sample can be stored in negative seventy for up to one month. However, using a fresh sample for extraction is more preferable. The second step should be allowing the sample to stand for five minutes at room temperature. By giving the sample five minutes, complete dissociation of nucleoprotein complexes is certain. The third step is phase separation. Phase separation is achieved by adding 0.2ml of chloroform 1ml of Trizol used. It is important to cover the sample tightly to avoid any splashing. After adding chloroform, shake the tube vigorously for fifteen seconds. After that allow the tube to stand for five minutes at room temperature. After that, Centrifuge the resulting mixture at 10,000rpm for fifteen minutes at 4 degrees Celsius. Centrifugation facilitates the separation of the mixture into three sections: a red section containing the protein, a white section containing the DNA and a colorless section containing RNA.
DNA quantification
Quantification is also done by SYBR fluorescence method, in the process, DNA samples (25-200ng) are run on 0.8 percent (Barbas et al., 2007).
RNA-Seq library formation
RNA library formation involves capturing the complete transcriptome of the genes that encompasses coding or non-coding, intergenic RNAs or antisense RNAs with the highest degree of integrity. Normally, small RNA transcriptome contains a myriad of RNAs such as small nucleolar RNA, small nuclear RNA, small interfering RNA, microRNA and Piwi-interacting RNA. Current commercial procedures for preparing microRNA libraries take advantage of five phosphates, and three hydroxyl groups contained on the miRNA. The initial step of RNA library preparation entails 3end ligation by making use of 5 adenylated group on the adapter with a blocked three end. The second step involves the use of a 5’RNA adapter which is joined to the 5’phosphate end of microRNA by T4 RNA ligase 1. For this ligation reaction, the only substrates are the 5’phosphate and RNA molecules. The third step involves gradually heating and cooling reverse transcription primer to the three ligated extension and adapter. After that, it is amplified using a pair of primers that contribute towards Illumina flow cell attaching sequence and barcodes. ("Small RNA (miRNA) Sequencing Services", 2016)Currently, the common methods of preparing small RNA-seq methods entail the process of ligation tagging of the 3 and 5 ends of the RNA. However, some of these methods suffer primarily two major setbacks: the methods do not acknowledge 5’ capped and 5’triphosphorylated RNAs and amplification of adaptor dimer reduce the success rate of ligation reaction thus contaminating the sequencing library (Picardi, 2015).
HiSeq Sequencing
HiSeq represents the most advanced sequencer generation that can perform faster sequencing. Hiseq sequencing is a method that involves the use of HiSeq sequencing systems which make use of Illumina's popular terminator-based sequencing by synthesis. Hiseq sequencing combines both HiSeq 2000 and HiSeq 1000systems which are both high-performance sequencing systems. The systems are comprised of human interaction design systems which facilitate easy and effective sequencing workflow. As a result, the Hiseq sequencing method not only sets a new standard for user experience but it has also delivered the highest sequencing output and an even greater data generation rate. This is based on kinetic exclusion amplification and patterned flow cells. HiSeq Systems facilitate individuals and organizations to handle complex sequencing studies at an affordable price. Due to HiSeq systems contain outstanding imaging and scanning technology, systems such as the HiiSeq 2000 make it possible to carry out sequence on five human genomes at 30x coverage simultaneously. The HiSeq systems are one of the easiest customer friendly systems to use. The systems entail a touch screen user interface with step by step instructions which re-geared towards providing utmost help to users ("HISeq Sequencing Systems", 2016). The system also facilitates direct monitoring of any progress by use of any internet enabled phone. Currently, HiSeq systems can handle numerous samples, therefore, making it possible to not only decode larger genomes but also to carry out any sequencing project that crops up. The ongoing technological advancements ascertain there will be greater sequencing capability in the coming future.
Since hexaploid wheat is the predominant variant and comprises 95 percent of the total wheat production, the genome is large and intricate and contains 80 percent repetitive DNA. However, the complete wheat genome sequence is still not available, while using HiSeq sequencing technology 60 percent of the spring wheat is available for wheat landrace Chinese Spring (CS), a whole of 133,090 high confidence genes were interpreted. However, this is not complete, and some of the genes may be missing (Dong et al., 2015; Mayer et al., 2014). Therefore the draft genome needs further improvement. The method will be utilized to perform the sequencing of the transcriptome and to get the genome sequence of the spring wheat the tolerant (Alpowa) and sensitive (Idaho) genotypes.
Quality filtering of sequences: Quality filtering Q30
The consensus amongst independent reads enhances the precision of genome as well as transcriptome analysis, lacking consensus amongst very analogous sequences especially in metagenomic studies signify natural deviation of biological implication. The raw sequence was analyzed using quality-based filtering or the Q-scores (Eren et al., 2013). This will aid in measuring sequencing quality score. The sequencing quality score could be obtained by base Q, the equation helps in calculation Q= -10log10(e), e is known as estimated probability of the base, designated wrong. Higher Q score designates smaller error while lower Q score designates reads as unsuitable, which may enhance the possibility of the false-positive variant. The following relationship between base call accuracy and sequencing quality score will be utilized
(Source: Ewing et al., 1998; Ewing and Green, 1998)
Sequence alignments
A sequence alignment aids in arranging protein sequences in an array with a motive that the residues in the provided column are homologous (same ancestry) or bear a common functional role encompassing nuclear localization signal, catalytic sites, protein-protein interaction sites, or they are superimposable (present in three-dimensional structural alignment either as -helix or as -sheet). This method helps in placing amino acids playing a similar role in the structural homology, evolutionary, functional and sequence similarity, f in the same column (Notredame, 2007). However, many multiple sequence programs are available, which will be selected meticulously on the basis of the sequence obtained and needs an alignment, the software of sequence alignment will be checked to understand the influence of various parameters. The best option will be to use two programs and make a comparison of the results (Gonze, 2015). For this project using the ABA and Base By Base program would be more prudent since they are both effective and free for educational research.
Differential Gene Expression Analysis
Numerous plant genes participating in the cellular signaling and metabolism are yet to be identified, while other genes are being characterized to a certain extent. The gene expression could be demonstrated and explained using various methodologies encompassing microarray and cDNA libraries or SSH (suppression subtractive hybridization), RNA fingerprinting by primed PCR, (DD) differential display, EST (expressed sequence tags), RDA (representational difference analysis), SAGE (serial analysis of gene expression), cDNA-AFLP (cDNA-amplified fragment length polymorphism) and RNA-seq (RNA sequencing) (Casassola, 2013). These procedures will be adopted to perform the differential gene expression analysis of the tolerant (Alpowa) and sensitive (Idaho) genotypes of spring wheat for drought stress. RNA fingerprinting has proved to be more resourceful in differential gene expression analysis and would, therefore, be more suitable to use in this research project.
CUFF links program identify
Advances in RNA-seq or high-throughput cDNA sequencing reveal novel genes as well as splice variants and enumerate genome-wide expression in a single assay. Cufflinks are open-source software, free tools used for gene discovery as well as for comprehensive expression analysis of high-throughput mRNA sequencing. These software facilitate biologists to recognize new genes as well as new splice variants of already known genes (Trapnell et al., 2012).
Expected results
It is expected to observe alterations in genes/pathways related general stress associated genes as well as previously characterized drought-responsive genes (e.g. dehydration-responsive element binding gene, late embryogenesis abundant proteins and dehydrins) and their pathways. Most importantly new candidate genes and their operational pathways are expected during this study to explain the limitations associated with previous studies.
Abstract
In the present era, drought is emerging as the most frequent incidence and with the enormous growth in population, it will be one of the most significant phenomena, which limit crops regarding production and yield. Water is an essential component, and it severely affects vegetative and reproductive stages of the plants. Essentially drought tolerant mechanisms need a thorough understanding. Polygenes control drought stress, application of molecular methods such as molecular markers, mapping strategies, as well as expression patterns of genes are implemented to understand the drought tolerant genes. Genetic improvement and modification of crops for drought tolerance demands a search for promising affiliation of physiological and yield characters as well as utilization of genetic variation in the cultivars for these characteristics. Various studies reveal that drought stress inflicted at various growth stages, influence crop plants. The present research reviews water limitation and its impact on molecular and physiological rejoinders of the tolerant (Alpowa) and sensitive (Idaho) genotypes of spring wheat with the conceivable losses instigated by drought stress.
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