Abstract
Plant breeding and biotechnology have been advanced by the advancements in omics technologies in the last two decades. First-generation DNA sequencing revolutionized the way we study plants. Whole-genome sequencing of many crop species was accelerated by the development of next-generation sequencing, which in turn leads to a blooming explosion in omics-based studies in plant science. More than 700 plant species have already been fully sequenced by now. Transcriptomics, proteomics, and metabolomics studies paved the way to the understanding of plant responses to environmental conditions. These extensive omics studies produced a vast number of data sets which forced the development of new bioinformatics and statistical tools. In the last few years, the integration of these different omics technologies became more meaningful to reflect the phenotypic differences. Omics profiling of different species or genotypes led to the understanding of species evolution, diversification, and natural variation. These studies identified novel quantitative trait loci that can be converted into molecular markers for future breeding purposes. This chapter explains the evolution of sequencing technologies and how they revolutionized the omics practices in plant science.
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2.1 Introduction
Plants are exposed to many different environmental stressors during their life cycle. Depending on the species or genotype, these biotic and abiotic stress conditions can hinder plant growth and development and lead to yield penalties. Tolerance or resistance mechanisms have been studied extensively for years to characterize individual genes, proteins, and metabolites involved in these mechanisms. The development of DNA sequencing approaches facilitated the characterization of genomic regions leading to the whole-genome sequencing (WGS) of different species. Advancements in nucleic acid sequencing accelerated the WGS studies. At present, more than 1,000 plant genome assemblies are accessible in GenBank, even though many of them have low quality. WGS approach was extended to the sequencing of RNAs, proteins, and metabolites. Therefore, a new scientific discipline was required to study the genes, transcripts, proteins, and metabolites holistically.
The Greek terms “ome” and “omics” are expressions derived from the suffix -ome which implies “whole,” “all,” or “complete.” Genome, transcriptome, proteome, and metabolome are the expressions generated by adding the suffix with the terms of the gene, transcript, protein, and metabolite, respectively. Genomics, transcriptomics, proteomics, and metabolomics/lipidomics are the areas of studies that are referred to as omics. As the collective and high-throughput analyses, omics technologies integrated through robust systems biology, bioinformatics, and computational tools aim to study the mechanism, interaction, and function of cell populations, tissues, organs, and the whole organism at the molecular level (Nalbantoglu and Karadag 2019).
The approach toward omics studies has evolved since next-generation sequencing (NGS) technologies are generated. The outputs of next-generation sequencing brought about brand-new approaches to gene regulation and the data on crop genomes. It serves a potential to be used in plant breeding within metagenomic and agrigenomic researches. Gene regulation mechanisms, genes taking part in the plant defense system against pathogens, and abiotic stress factors in the whole plant or at a cellular scale can be revealed via RNA sequencing. The genotypes of lots of single-nucleotide polymorphisms (SNPs) are also determined with the methods developed within NGS. Additionally, molecular markers required for investigating genetic relationships among breeding materials, detailed genetic mapping of targeted genes, and genome-wide associations are developed with the methods called genotyping-by-sequencing (GBS) and whole-genome resequencing. Determination of the genotypes of the required genetic materials enables improving the selection of individuals that resist abiotic stressors and increases the efficiency in agriculture (Vlk and Řepková 2017).
Nowadays, the omics terminology is adapted to other fields of study, including ionomics that deals with ionic changes, methylomics studying the methylation changes in nucleic acids, and toxicogenomics. Here in this chapter, we first describe the evolution of sequencing techniques and give examples of each omics technology in plant science.
2.2 First-Generation Sequencing
The sequencing technologies that give rise to decoding and sequencing the genomes of the organisms are based on the discovery of the DNA, which is a double-helix structure consisting of bases (adenine, thymine, cytosine, and guanine). The first laboratory methods used in the interpretation of the DNA sequences in terms of the letters of A, T, C, G, and N representing an ambiguity were generated by Sanger et al. from Cambridge University in 1977 and Maxam et al. from Harvard University in 1980 (Kchouk et al. 2017).
The first-generation sequencing technique was further improved by the Maxam-Gilbert method, which enables sequencing the DNA with chemical degradation of the fragments at specific bases with reagents such as formic acid, dimethyl sulfate, and hydrazine (Maxam et al. 1977). In this method, the strands of the DNA fragments are denatured, and the phosphate groups at the 5’ ends of the denatured DNA strands are removed with phosphatase to identify the fragments on the gel after the radioactive isotopes of phosphorus. The radioactively labeled DNA fragments are exposed to chemical reactions in four different tubes in the presence of distinct base-specific chemical reagents. Each of the reagents results in base modification, removal of the base, and phosphodiester cleavage of the DNA strand at that site. Guanine cleavage is induced by DMS + piperidine, while the cleavage of guanine and adenine requires DMS + formic acid + piperidine. Hydrazine piperidine causes cytosine cleavage, and sodium chloride + hydrazine piperidine facilitates the cleavage of cytosine and thymine. At the end of the reactions in the four distinct tubes, labeled fragments with various sizes are separated by electrophoresis (Saraswathy and Ramalingam 2011). The polyacrylamide gel contains urea which prevents the formation of secondary structures in the single-stranded DNA. Then, the DNA sequence is determined by using autoradiography. This sequencing method does not involve DNA cloning. On the other hand, the development of the Sanger sequencing method is more applicable compared to the Maxam-Gilbert method due to its greater simplicity, higher accuracy, and lower radioactivity (Kulski 2016).
As mentioned above, Sanger sequencing developed by Frederick Sanger in 1977 is expressed as the chain termination, dideoxynucleotide, or the sequencing by synthesis method in which one strand of the DNA is used to identify the sequence (Kchouk et al. 2017). In this technique, dideoxynucleotides (ddNTPs) are used which are the analogs of the monomers of the DNA molecules, deoxyribonucleotides (dNTPs), lacking 3′ hydroxyl groups required for the extension of the DNA strands (Heather 2015). The integration of the ddNTPs to the elongating DNA prevents the process to be terminated successfully as the subsequent base cannot be incorporated into the strand. Thus, the DNA fragments with different sizes and the ddNTP molecules at their ends as the analogs of the related bases are obtained. Chain termination reactions are conducted in four different tubes. Each tube contains a different type of ddNTP and the common reaction components including dNTP mix, template DNA, radiolabeled primer, and DNA polymerase. Radioactive isotopes of the phosphorus (32P or 33P) enable identifying the DNA sequence. The tubes contain a small percentage of ddNTP (about 1%). The polyacrylamide gel with urea is also used, and the DNA sequence is determined in autoradiography (Sanger et al. 1977). The bands of the DNA fragments separated regarding their sizes on the gel slab are displayed with an imaging system, either of X-ray or UV light. The Sanger sequencing was firstly used to sequence the phiX174 genome (5374 bp) and the bacteriophage λ genome (48501 bp). The speed and accuracy of the sequencing were improved with the automatic sequencing machine based on capillary electrophoresis developed by Applied Biosystems in 1995. The genetic materials of varying plant species such as Arabidopsis (The Arabidopsis Genome Initiative 2000), rice (Goff et al. 2002), and soybean (Schmutz et al. 2010) and human genome were also sequenced with Sanger sequencing. The Sanger sequencing has been used for three decades and is still preferred in single or low-throughput DNA sequencing. On the other hand, Sanger sequencing is considered to be time-consuming and expensive. The limited analysis speed also reduces the efficiency besides the inability to decode the complex genomes with the Sanger sequencing (Kchouk et al. 2017).
2.3 Next-Generation Sequencing
Following the domination of Sanger sequencing for 30 years, NGS was developed as a high-throughput DNA sequencing technology considered within the second- and third-generation sequencing methods (Kulski 2016). By this method, a high number of simultaneous sequencing reactions become feasible, and the cost of sequencing is lowered due to the developments in detection systems, microfluidics, and integrating the sequencing reactions to minimized dimensions (Türktaş et al. 2015; Kulski 2016). Increased scalability and speed of generating data paved the way for advanced studies on biological systems besides the decrease in time for obtaining gigabase-sized sequences from years to days or hours via NGS (Noman et al. 2017).
NGS enables carrying out studies on genetic approaches in plant breeding and biotechnology, evolution, discovering genetic markers, gene expression profiling via mRNA sequencing, and de novo draft genome sequences within the relevant method of NGS applications such as WGS, exome sequencing (exome-seq), RNA sequencing (RNA-seq), and methylation sequencing (methyl-seq) (Türktaş et al. 2015; Low et al. 2019). The NGS platforms with 99% accuracy rates may also detect nucleotides with errors. Although the current NGS methods are highly accurate, they are still prone to errors. Even the accuracies of more than 99% may accumulate hundreds of thousands of errors in the sequencing of large genomes since NGS platforms generate high amounts of output. The number of times a nucleotide is sequenced is referred to as “coverage” or “depth” (Sims et al. 2014). Coverage may also be used to refer to the percentage of target bases that have been sequenced a specific number of times. Coverage varies depending on the type of NGS and the research application. More coverage tends to be used when in search for a variant that is less common (<1%) in a sample. For example, whole-genome sequencing generally requires approximately 30x coverage as this will detect 98% of heterozygous single-nucleotide variants identified in a microarray. The coverage can be calculated by the Lander-Waterman equation (Sims et al. 2014).
2.4 Second-Generation Sequencing
To overcome the limitations of the first-generation sequencing tools that were used for three decades such as Sanger sequencing, brand new sequencing methods were developed (Kchouk et al. 2017). Second-generation sequencing methods enable sequencing multiple DNA fragments simultaneously that facilitate assembly and determination of complex genomic regions, methylation detection, and gene isoform detection (Muhammad et al. 2019).
Millions of short fragments are read in parallel, the speed of the sequencing process is increased, electrophoresis is not required for detecting the output and the cost is reduced within the second-generation sequencing methods (Kchouk et al. 2017). Template libraries of randomly fragmented DNA or complementary DNA (cDNA) obtained from reverse transcription are generated with shotgun sequencing by ligating the linker or adapter sequences with the DNA molecules rather than performing cloning via a host cell (Kulski 2016). In second-generation sequencing, the read length of these technologies is shorter than the first generation; therefore, amplification is necessary for signal detection (Kang et al. 2019). A solid surface or beads are used in the library amplification process in the presence of miniaturized emulsion droplets or arrays, while the nucleotides to be sequenced are detected via luminescence or changes in electrical charge (Kulski 2016). These sequencing methods are classified in two, namely, sequencing by ligation (SBL) and sequencing by synthesis (SBS), and the sequencing platforms used are Roche/454 established in 2005, Illumina/Solexa in 2006, and the ABI/SOLiD (Sequencing by Oligonucleotide Ligation and Detection) in 2007 (Kchouk et al. 2017; Meera et al. 2019).
2.5 Pyrosequencing Technology
Pyrosequencing also known as 454 technology was the first second-generation technology developed in 2005. In this technology, the main principle is to determine the base with chemical luminescence. The pyrosequencing method is different from the Sanger sequencing since the nucleotide incorporation is performed in the presence of DNA polymerase, ATP sulfurylase, luciferase, and apyrase enzymes which are kinetically well-balanced (Ramon et al. 2003). PCR amplification and pyrosequencing of the query DNA fragments are utilized to carry out real-time sequencing (Rothberg et al. 2008). In the pyrosequencing method, adapter molecules provide the DNA molecules that have been previously fragmented to bind the agarose beads after attaching the DNA fragments. The agarose beads with DNA fragments are mixed with Taq polymerase and buffer solution before being introduced to an oil-water emulsion to induce emulsion PCR (emPCR). The DNA fragments are then amplified in the presence of dNTP and adapters considered as primers (Saraswathy and Ramalingam 2011). The nucleotides are formed and tested in terms of their inclusion in a DNA template which occurs by the release of pyrophosphate (PPi) proportional to the amount of the nucleotides (Ramon et al. 2003). ATP sulfurylase is the enzyme that uses pyrophosphate in ATP synthesis by converting it to ATP in the presence of adenosine 5’ phosphosulfate. Production of oxyluciferin from luciferin is facilitated by luciferase driven by ATP. Light emission from the oxyluciferin formed previously providing chemical luminescence takes place as a result. The number of nucleotides is associated with the amount of light emitted providing the determination of the base sequence. The emitted light is illustrated with peaks having heights proportional to the number of nucleotides in a program after it is spotted with a charge-coupled device (CCD) camera. As the apyrase enzyme degrades the excess ATP and dNTP, another pyrosequencing cycle initiates with the integration of the subsequent dNTP, and the complementary strand of the DNA is constructed. A cyclic nucleotide dispensation order (NDO) is utilized to decode an unknown sequence with pyrosequencing. In this method, one of the dNTPs is recruited to the DNA template where the rest of the dNTPs are degraded in the presence of apyrase after each cycle of dNTP dispensation. Non-cyclic NDOs are also generated with the order of nucleotide dispensation and the heights of the peaks in the program in case the DNA sequence is known (Ramon et al. 2003).
Besides the disadvantages such as high cost and low accuracy of reading, the 454 technology can read long sequences (around 700 bp). In addition, the sequences are expected to be smaller than the outputs of the other second-generation sequencing methods, and homopolymers would be sequenced with lower accuracy (Saraswathy and Ramalingam 2011).
2.6 Illumina Technology
After developing Illumina sequencing in 2006, Solexa commercialized it as Illumina/Solexa Genome Analyzer. The platforms developed by this company, namely, MiSeq, NextSeq 500, and HiSeq 2500, can put forward 15 Gb, 120 Gb, and 1000 Gb of sequencing data in each run while their maximum read lengths are 2×300 bp, 2×150 bp, and 2×125 bp, respectively. In addition, the NovaSeq 6000 System is declared to present output up to 6 Tb and 20B reads in less than 2 days. It is also claimed that the Illumina sequencing technology has been used in generating more than 90% of the sequencing data of the world as being the most remarkable technology in the NGS market (Krishna et al. 2019).
As being a sequencing by synthesis (SBS)-based technology, cluster generation involves the fragmentation of DNA molecules and ligation of the fragments with short adapter oligo at both ends. This aids connection and amplification of fragments on a flow cell where sequencing reactions take place. There are microfluidic channels on the flow cell called lanes. Oligonucleotide sequences are attached in each lane and are complementary to the adapters. These complementary oligos form a cluster that is called polony since the appearance of each PCR-amplified DNA fragment looks like a bacterial colony (Turcatti et al. 2008). The flow cell surface that is used for the immobilization of the templates for sequencing enables increased stability of DNA and accessibility of enzymes to the DNA. It also reduces the non-specific binding of the fluorescently labeled nucleotides. One thousand copies of a template with a diameter of one micron or less are generated within solid-phase amplification. Single-molecule cluster densities reaching the order of 10 million per square centimeter are obtained by different methods including photolithography and mechanical spotting.
In Illumina technology, the PCR amplification of the DNA fragments is performed using the adapter sequence as a primer, and each type of dNTP is labeled with different types of fluorescent labels. In each sequencing cycle, only a single-labeled dNTP is introduced to the nucleic acid chain, and thus each type of dNTP signal helps the detection of base calling, while the signal length helps the identification of the number of the attached dNTPs. Fluorescently labeled nucleotides with a reversible terminator are used in Illumina sequencing. Therefore, the polymerization terminates in the presence of the nucleotide label. The fluorescent label is screened to determine the base after each dNTP incorporation. The dye is then removed from the 3’ end by the enzymes for the subsequent nucleotide to be incorporated, and the next cycle begins. Even though the sequences generated after the process are short, large data can be generated accurately and fast (Turcatti et al. 2008). As a technology displaying an error rate below 1%, Illumina sequencing is claimed to be one of the most accurate NGS technologies. The incorporation bias is reduced by the natural competition in the presence of reversible terminator-bound dNTPs that are single, separate molecules. In each cycle, the measurements of the intensities of the signals induce the base calls which are the reasons behind significantly reduced raw error rates compared to the alternative technologies. Imaging the clusters on the flow cell surface is the most time-consuming step of the process besides the nucleotide incorporation phase facilitated by the enzymes. The substitution of a nucleotide located in a specified position in the genome which is named as single-nucleotide substitution is the error taking place most frequently (Turcatti et al. 2008).
Within the resequencing approaches, the sequences are allowed to be aligned to a reference in the Illumina data collection software. The full range of data collection, processing, and analysis modules to streamline collection and analysis of data with minimal user intervention is enabled with this software that was generated with the help of leading researchers. The open format of the software with simple application program interfaces also provides accessing data at various stages of processing and analysis.
2.7 Ion Torrent Technology
Ion Torrent technology is based on an SBS process similar to Illumina technology. DNA fragments are amplified by an emulsion PCR (emPCR) on beads that are washed over a picowell plate, and each nucleotide is added later on to release pyrophosphate (Heather 2015). Each ion chip contains a liquid flow chamber which helps the influx and efflux of nucleotides (Merriman et al. 2012). A complementary metal-oxide-semiconductor technology is used to detect the difference in pH caused by the release of protons (H+ ions) during polymerization (Rothberg et al. 2011). The bottom of each chip is covered with millions of pH microsensors. The pH change is not specific to nucleotide types, and each type of dNTP is released in a fixed order. According to the measurement of pH change, the sequence is determined (Merriman et al. 2012). This technology allows for very rapid sequencing during the actual detection phase (Glen et al. 2011). The error rate of the Ion Torrent technology is higher than the Illumina since the indels are the major error in this technology. This technology cannot detect homopolymer sequences of identical nucleotide stretch such as TTTTTT due to the loss of signal as multiple matching dNTPs incorporate (Loman et al. 2012). If the DNA template has a homopolymeric region, pH change should be proportional to the attached nucleotide number. Instead, as the attached nucleotides increase in a homopolymer, the expected pH change decreases gradually. In addition, the lengths of the sequence read obtained in one experiment of Ion Torrent are various rather than being the same. The sequence reads from both ends of a fragment cannot be obtained with the current generation of Torrent devices (Lahens et al. 2017).
2.8 Third-Generation Sequencing
Several biological limitations such as assembly and determination of complex genomic regions, gene isoform detection, and methylation detection are not eliminated by the second-generation sequencing technologies because of the short read lengths, even though they present developments outstripping Sanger sequencing (Rhoads and Au 2015). Third-generation sequencing is presented as a promising technology to eliminate the mentioned limitations. The length of the read is also improved to tens of thousands of bases from tens of bases per read within the third-generation sequencing approaches, besides the decrease in time required for sequencing from days to hours and elimination of sequencing biases resulting from the PCR amplification process (Lu et al. 2016).
Unlike second-generation sequencing, third-generation sequencing technologies do not require the sample amplification step and can sequence a single DNA molecule. Also, they may produce more than 10 Kb reads and thus produce highly precise de novo assemblies and contiguous genome reconstruction even at the regions of high content of repetitive elements. These technologies include Pacific Biosciences, Helicos System (Helicos single-molecule sequencing), and Oxford Nanopore Technologies (ONT). The first commercial NGS implementation was the Helicos System that utilized single-molecule fluorescent sequencing. However, the Helicos Biosciences company filed for bankruptcy in 2012.
2.9 Pacific Biosciences Technology
Pacific Biosciences developed the PacBio RS II sequencer and the single-molecule, real-time (SMRT) sequencing system based on the properties of zero-mode waveguides (Schade et al. 2010). PacBio sequencing enables closing the gaps in reference assemblies and determination of structural variation in genomes with the highly contiguous de novo assemblies. The mutations that are related to the diseases can be spotted, and extended repetitive regions are sequenced by using relatively long reads. Additionally, isoforms of genes, novel genes, and isoforms of annotated genes can be determined with PacBio transcriptome sequencing as the whole transcripts and relatively long fragments are sequenced. Base modifications such as methylation can also be spotted with PacBio sequencing. Moreover, cost-effective and scalable hybrid sequencing strategies are generated to utilize short reads in relation to long reads (Rhoads et al. 2015).
SMRT sequencing is a method carried out in cells with 150,000 ultra-microwells at a zeptoliter scale. Each well contains a DNA polymerase molecule stabilized at the bottom with a nanostructure including a biotin-streptavidin system called zero-mode waveguides (ZMWs) (Kulski 2016). DNA chains pass through the DNA polymerase, and complementary binding nucleotides promote the detection of the sequences via the signals from fluorescence labels that are attached to the end phosphate groups, which are generated by them (Rhoads et al. 2015). Long reads with high accuracy are obtained with a circulating structure (SMRTbell) constructed by the adaptors. In this technology, first, the sequencing templates are annealed. The complex consisting of template-primer-polymerase is immobilized to the 150,000 ZMWs. After the labeled nucleotides interact with the polymerase, the end phosphate group is cleaved, and the fluorescent signal is detected simultaneously and recorded with a CCD camera. Because the wavelength of the visible light is more than the diameter of a ZMW, the light reflected through the glass bottom reaches the bottom 30 nm of the ZMW. Therefore, the reduction in background noise and the detection of a recruited nucleotide are facilitated with the detection volume.
As the nucleotides are integrated and detected simultaneously rather than the second-generation technologies in which the nucleotides are added in order, the sequencing is completed faster. The accuracy of sequencing 900 bp read length has increased from 99.3% to 99.9% by circularizing the template and sequencing it multiple times by using SMRTbell (Travers et al. 2010; Koren et al. 2013). The drawbacks of SMRT are the high cost, the need for the high amount of DNA samples, and the error rate of 10–15%, which is mostly caused by indels.
2.10 Oxford Nanopore Technology
The third-generation sequencing technology developed in 2005 by Oxford Nanopore Technologies Ltd. enables simultaneous analysis of native DNA or RNA sequences at any length in fully scalable formats from pocket to population scale. It uses a nanometer-level channel in a membrane, and it determines the base sequence by the potential difference changing between the membranes passing through a single-stranded DNA (ssDNA). In this technology, the leader and the hairpin adapters are used. Each adapter is ligated to one end of the double-stranded DNA (dsDNA). The leader adapter is denoted as the Y adapter since it has a Y-shaped structure, while the hairpin adapter is called the HP adapter. Sequencing starts at the single-stranded 5’ end of the Y adapter, followed by the template strand, then the HP adapter, and the complementary strand. Helicase enzyme translocates along dsDNA to ssDNA, and the hairpin protein makes each base of ssDNA pass through the nanopore at a constant rate, and so base calling may then be performed. Each type of dNTP causes different electrical potential changes that are read, and base sequences are determined (Branton et al. 2010). In this technology, sample preparation is minimal, and long read lengths can be generated in the Kb range compared to the second-generation sequencing technologies. Also, amplification and ligation steps are not required before sequencing. However, the optimization of the speed of DNA translocation through the nanopore should be needed to obtain the accurate measurement of the ionic current changes and to decrease the high error rates of base calling (Stoddart et al. 2009). Thus, the current error rates (roughly around 98%) are very high; therefore it cannot compete with existing sequencing technologies. Moreover, the low depth of coverage obtained with this technology is a possible barrier to accurate eukaryotic genome sequencing at the moment.
2.11 Genomics
Shotgun sequencing was used for some of the early plant genomes including Arabidopsis, soybean, poplar, and papaya (Michael and Van Buren, 2015). The sequence and genetic structure of plant genomes are determined with an extensive sequencing method called whole-genome sequencing (WGS). In early sequencing projects that focus on WGS, the genomes of strawberry (Shulaev et al. 2011) and wheat (Brenchley et al. 2012) were randomly fragmented, and elements with varying sizes are obtained. The reads obtained from the sequencing process were assembled with bioinformatic tools after the bacterial artificial chromosome (BAC)-end sequencing is performed. De novo projects also utilize WGS besides the resequencing attempts. Preparing a draft of unknown plant genomes is managed with the whole DNA or mRNA de novo sequencing even though the process is time-consuming (Türktaş et al. 2015). Despite the possibility of determining locations of the contigs or scaffolds with low accuracy, and missing several genes while generating draft genomes, the presence of genome information enables analyses with high throughputs and characterization of genes (Sarethy and Saharan 2021). Later, WGS approach was used to generate the draft genomes of einkorn (Ling et al. 2013), wheat, and A. tauschii (Jia et al. 2013). Moreover, resequencing is considered to be useful in transcriptome profiling and detecting SNPs to generate molecular markers. For instance, WGS enabled the construction of the reference genome of potato and discovering SNPs to compare a homozygous doubled-monoploid line with its heterozygous diploid line (The Potato Genome Sequencing Consortium 2011). WGS of many different crop and vegetable species has been completed in the last decade. Although the second-generation sequencing resulted in many lower-quality assemblies, a massive extension WGS of different plant species, especially of the crops, leads to a revolution in plant genomics.
One thousand one hundred twenty-two plant genome assemblies are deposited in GenBank, representing 631 land plant species. The advancements in the long-read sequencing markedly improved the NGS data quality; therefore the number of plant genome assemblies has increased dramatically in the past 20 years. Almost 60% of the plant genome assemblies have been sequenced in the last 3 years alone. Model plants and some crops were the first species whose genomes were fully sequenced. But now, any plant species can be sequenced due to a steady decline in sequencing costs.
The exons considered to be the coding region for the protein synthesis in the genome of an organism are called the exome. Even though they involve the total of the sequences inducing the generation of proteins taking part in phenotypic regulation, they are insufficient to decode fully the mechanisms behind the gene regulation. To enlighten the molecular background of the diseases and phenotypic traits, exome sequencing was introduced as an essential genetic tool. Exome sequencing helps with the identification of genes (whole exome, genes responsible for a disease, or class of genes), determination of phenotypic traits, identification of exome SNPs, and further computational and statistical applications to identify the signals of diseases (Hashmi et al. 2015).
WGS of different populations of the same plant species showed a high degree of genomic variation within the species; therefore it was obvious that single reference genomes no longer can represent the diversity within a species. This observation led to the advancement of the pan-genome concept, which was first developed in bacteria in 2015 (Tettelin et al. 2005). Pan-genomes can distinguish the primary genes that are present in all individuals and variable genes that are found in some individuals but absent in others. Hence, it symbolizes the genomic diversity within the species. Pan-genomes can be curated by three different methods, each with its benefits and disadvantages over the others (Bayer et al. 2020). The first pan-genome study in plants was a comparison of WGS of wild soybean relatives (Li et al. 2014). Another study in rice compared the genomes of three accessions (Schatz et al. 2014). At present, more than 8,000 studies reported pan-genome comparisons in plants (Bayer et al. 2020). These studies have impacts on understanding the biological significance of genotypic variances at loci linked with tolerance and resistance, developmental processes, and yield enhancement.
The transposable elements in the plant genome are high in copy number since their segmental or tandem duplication takes place frequently. Therefore, an extended amount of repetitive elements is found in plant genomes. Autopolyploid or allopolyploid character of the genome or the age of ploidization affects the progress of the sequencing as ploidy is considered a challenge. To eliminate the obstacles caused by the complexity of the genome, library sequencing of fragmented genome elements is executed by using restriction enzymes or obtaining the sequences without using enzymes (Vlk and Řepková 2017). Variations or significant polymorphisms in the genome are considered to be useful in pre-breeding attempts with resequencing projects. The reference genomes of the desired plant are also intended to be generated within various projects. They are considered as providing information about the structure and function of the genome and the genome assembly patterns of the related species together with molecular markers and candidate genes that can be used in further studies (Vlk and Řepková 2017).
Epigenetic changes such as chromatin modifications, transposable element inactivation, paramutation, transgene silencing, and co-suppression are investigated with the sequencing approaches in detail in various plant species. The changes in gene expression and chromatin-based expressional responses generated against environmental stimuli prove the importance of epigenetic studies in plants (Köhler and Springer 2017). Traditional methods used in epigenetic studies involve bisulfite conversion, methylation-sensitive restriction enzymes, and antibodies specific to 5-methylcytosine. Microarray-based methods were also started to be combined with these methods to carry out a genome-wide analysis of DNA methylation (Buck and Lieb 2004). Recently, NGS technologies paved the way for epigenetic studies (Vlk and Řepková 2017). Therefore, the studies of applied epigenetics cause new opportunities for crop improvement. It has been suggested that varietal selection of crops is associated with variability caused by epigenetic mechanisms (Rodríguez López and Wilkinson 2015; Crisp et al. 2016; Fortes and Gallusci 2017; Gallusci et al. 2017). The potential to develop crop performance and energy use efficiency was shown in Brassica napus via an epigenetic selection of isogenic lines (Hauben et al. 2009). Organ-specific epigenetic modifications were determined in maize by Illumina sequencing technology (Wang et al. 2009a). The expression levels of genes are regulated by epigenetic mechanisms in response to plant development and biotic and abiotic stresses, and this affects the phenotype of plants (Kumar 2018).
DNA methylation, histone modifications, and small RNA molecules are the major epigenetic mechanisms affecting the expression levels of genes (Rodríguez López and Wilkinson 2015). DNA methylation is an important chromatin modification that can be inherited in animals and plants. It has been recently suggested that methylation of the promoter and the gene coding region has different effects on gene expression (Wang et al. 2015a, b). The methylation of the promoter region of a gene is related to the repression of transcription (Kass et al. 1997). On the other hand, the methylation of the gene coding region is found with an intermediate expression level in plants. It was shown that it can be involved in reducing erroneous transcription by reducing intron retention by single-cell transcriptome sequencing data from Arabidopsis root quiescent center cells (Horvath et al. 2019). Furthermore, it can enhance the gene expression in certain gene families (Dubin et al. 2015; Anastasiadi et al. 2018). DNA methylation which targets cytosines in varying sequence patterns such as CG, CHG, and CHH can be revealed efficiently with NGS after treating the DNA with sodium bisulfite. Even though mostly the transposons are methylated as being primary targets for epigenetic silencing, the relation between the transposon polymorphism and DNA methylation variation is not easily described because they are highly repetitive and result in large insertion/deletion polymorphisms in the genome. The connection between transposon methylation and transposon insertions was studied using whole-genome bisulfite sequencing data sets by Daron and Slotkin (2017). Also, bisulfite conversion and Illumina sequencing were used together for the identification of the methylated genomic regions in tomato, and it was suggested the ripening of tomato fruits was under the control of epigenetic regulation along with hormonal control (Zhong et al. 2013).
2.12 Functional Genomics
Biological investigations were focused on genes and proteins in vitro during the early 1990s. However, as technologies improved and evolved, the approach shifted to research on different molecular aspects, viz., structural genomics, transcriptomics analysis, proteomics, and metabolomics. For instance, a multidisciplinary approach involving integrative analysis is crucial to study the complexity of plant-microorganism interactions (Sarethy and Saharan 2021).
2.13 Transcriptomics
The complete set of transcripts in a cell, and their quantity, for a specific developmental stage or condition, is called transcriptome. It is essential for understanding the functional elements of the genome and the molecular regulations of cells and tissues and also for revealing disease and development. The ultimate goals of transcriptomics are to determine all species of transcript such as mRNAs, small RNAs, and non-coding RNAs for revealing the transcriptional structure of genes and the changes in expression levels of each transcript during development and under different conditions (Wang et al. 2009). Several technologies have been developed for transcriptomics, including hybridization- or sequence-based approaches. Commercial high-density oligo microarrays and custom-made microarrays with fluorescently labeled cDNA are the important techniques used in hybridization-based approaches. Furthermore, specialized microarrays have been used for some specific purposes such as the detection of spliced isoforms.
They are high-throughput, relatively inexpensive, and high sensitivity by lowering the detection threshold of the transcriptional level of the less represented genes of a mixture, thus facilitating the analysis of thousands of genes in the same reaction (Kerr et al. 2000). However, they have several limitations such as existing knowledge about genome sequence, high background levels due to cross-hybridization, and complicated normalization methods. Microarrays have been widely used to produce global expression profiles under abiotic stresses in plant species (Kayıhan and Eyidoğan 2019). For instance, the AtH1 Arabidopsis GeneChip from Affymetrix has been employed to study transcriptome changes in Arabidopsis under salt stress. Accordingly, approximately 35% of the genome (∼8000 genes) exhibited expression changes under salt or other abiotic stresses (El Ouakfaoui and Miki 2005). Changes in gene expression caused by drought stress by using microarrays have been suggested by several research groups. For the first time in the literature, Ozturk et al. (2002) found that genes encoding jasmonate-responsive, late embryogenesis abundant, and ABA-responsive proteins were upregulated in barley seedlings exposed to drought. Also, it was found by microarray that changes in the expressions of 300 genes were revealed in spring and winter wheat under cold stress (Gulik et al. 2005). Furthermore, microarray technology provided comprehensive data for K+ deficiency in plants, and this showed a more integrative point of view considering all aspects of K+ management in plants (Kayıhan and Eyidoğan 2019). Kayihan et al. (2017) and Öz et al. (2009) performed the microarray experiments in wheat and barley cultivars exposed to excess boron, respectively. They suggest that WRKY transcription factors, genes related to jasmonate biosynthesis, glutathione S transferase, and NIP4;1 can have a role in boron tolerance mechanisms in cereals. Also, global gene expression analyses were performed in Arabidopsis thaliana exposed to high B and low B conditions (Kasajima and Fujiwara 2007). They identified novel high B-induced genes including heat shock protein and multidrug and toxic compound extrusion (MATE) family transporter genes. On the other hand, microarrays have been widely used for transgenic plants such as maize, canola, cotton, tomato, and soybean events (Leimanis et al. 2006; Xu et al. 2007; Schmidt et al. 2008; Zhou 2008; Kim 2010; Feng 2013).
The transcript levels of genes depending on their changes under different conditions are important information, and they can reflect the functions and transcriptional regulation relationships of genes. Modern omics technologies play an important role in better understanding gene expression. The best approach for the characterization of candidate transcripts that are responsible for many biological functions is transcriptome study. NGS technology provides us a powerful tool to reveal the transcriptional landscape of investigated tissue(s) at special developmental stage(s) because it can easily obtain transcriptome data from different plant tissue(s) and developmental stage(s). RNA-seq approach that uses NGS techniques is used for analysis and study of the entire transcriptome, and this approach provides an insight on the expression level of transcripts. Genes expressed within a defined period of time from a particular tissue or cell can be found by RNA-seq. There are some universal steps for this approach. RNA fragments are converted to a cDNA library by reverse transcriptase, and from both ends of cDNA fragments, cDNA library fragments are ligated to adapter molecules. Then adaptor attached library fragments are sequenced. Through cDNA sequencing, transcriptomes are studied deeply and efficiently. For plant transcriptomes, Illumina technology has generally better coverage. Reference genome and de novo assembling are two types of the assembly methods. For large NGS data of complex genomes without a reference genome, de novo assembly is useful (Wang et al. 2009). De novo transcriptomes are provided by some bioinformatic tools such as TRAPID (Van Bel et al. 2003) and Trinity (Brain et al. 2013). RNA-seq data is used for the development of molecular markers (Trick et al. 2009) and gene characterization (Dassanayake et al. 2009).
RNA-seq has successfully assisted in identifying several genes responsible for biotic and abiotic stress responses in various plant species. A large number of genes related to developmental stages were identified by RNA-seq in cucumber via 454 pyrosequencing (Ando et al. 2012). A combination of microarray and Roche technology was used to identify genes that were linked to the quality of cotton fibers (Nigam et al. 2014). To find genes associated with drought tolerance, RNA-seq analysis was performed in Populus euphratica Oliv. grown in arid or semi-arid regions using the Roche 454-GS FLX System (Tang et al. 2012). Likewise, sequencing red clover with Illumina technology discovered genes related to drought tolerance and determined the increase in metabolites such as pinitol, proline, and malate in leaves (Yates et al. 2014). The transcriptome of soybean (Fan et al. 2012), cotton (Xu et al. 2013a, b), and halophyte grass (Yamamoto et al. 2015) was sequenced to explore a molecular mechanism of salt tolerance in these plants. In addition, a whole-genome study was performed in soybean using Illumina technology, which examines the function of the plant-specific family of NAC transcription factors during development and dehydration stress (Le et al. 2011). Ion Torrent technology has been used in transcriptome analysis of finger millet, a hardy grain known for its tolerance to salinity, drought, and disease (Rahman et al. 2014). Transcriptome profiling of jatropha roots was carried out to elucidate molecular reactions to waterlogging (Juntawong et al. 2014). As the third generation, Pacific Biosciences’ SMRT technology was used to investigate the interaction of Xanthomonas oryzae pv. oryzicola and its host, Oryza sativa L., by whole-genome sequencing of the pathogen and RNA-seq of the host under attack (Wilkins et al. 2015). Illumina sequencing was used to obtain responsible herbicide resistance genes for Lolium rigidum Gaudin (Gaines et al. 2014) and for copper tolerance (Wang et al. 2015a, b). In sweet potatoes (Ipomoea batatas L.), biotic stress resistance analyses of catalase genes were performed using NGS technologies, and it was found that a positive response to IbCAT2 may play an important role in stress responses (Yong et al. 2017). In tomatoes, an abiotic stress tolerance identification study was conducted to understand the plant responses and genetic regulatory networks involved in abiotic stress responses (Chaudhary et al. 2019). In plants, RNA-seq technology has been used to determine the patterns of differentially expressed genes between hybrids and their parents to understand the genetic basis of heterosis (Zhai et al. 2013; Hansey et al. 2012; Sexane et al. 2014). Accordingly, gene expression for allopolyploid heterosis was predominant in the emerging hexaploid wheat dominance (Swanson-Wagner et al. 2006), but over-dominance was the key element for nicotine biosynthesis in tobacco (Tian et al. 2018). Dominance and over-dominance effects were shown by heterotic genes in connection with ear development earlier in maize inforescence (Ding et al. 2014). In the chrysanthemum, two characteristics of flowering – the initial flowering time and the flowering duration – are regulated by the presence of two pairs of main genes (Zhang et al. 2011).
MicroRNAs (miRNAs) are the key regulators at the post-transcriptional level in eukaryotic organisms. They regulate the expression levels of genes in response to development and various stress responses in plants. They are complementary with the target mRNAs and are highly conserved. Up till now, several technical approaches have been used to identify and verify the miRNAs. These are in silico prediction based on conserved sequences, to create miRNA libraries and to follow this with cloning and sequencing and finally the sequencing of miRNAs. In silico prediction was applied in rice (Bonnet et al. 2004). Cadmium-responsive miRNAs and their target genes in Raphanus sativus L. roots were identified by Illumina sequencing technology (Xu et al. 2013a, b). Also, circular RNAs were identified by transcriptome analysis by means of SMRT technology by Pacific Biosciences, and it was found that they had an important role in the function of miRNA and transcriptional control (Lu et al. 2015). On the other hand, long non-coding RNAs (lncRNAs), which are longer than 200 bp and do not encode any protein product, are another important regulatory mechanism associated with gene silencing, flowering time regulation, and abiotic stress responses (Wang et al. 2014; Zhang et al. 2014). These molecules were identified in crops, such as wheat (Xin et al. 2011), rape mustard (Yu et al. 2013), apple (Celton et al. 2014), and poplar (Shuai et al. 2014) by tiling array, EST analyses, and RNA-seq.
2.14 Proteomics
Proteomics is one of the growing fields of biological research with an immersive positive impact on plant science. Proteomics is a term that refers to the comprehensive identification and quantitative study of protein expression in an organism, cell, tissue, or organelle at a certain time and under specific conditions (Tan and Chen et al. 2011). Understanding proteome profiles provides a direct connection between genomic and transcriptomic regulation and phenotype. Since the first plant proteomic study in maize (Touzet et al. 1996), exponential progress has been made in different crop species although the full potential of plant proteomics has yet to be realized. Recent advances in new or improved technologies, protocols, or workflows have opened up new possibilities for high-throughput proteome analysis and reduced protein assessment errors.
Two-dimensional polyacrylamide gel electrophoresis and differential in-gel electrophoresis (DIGE) have been used in many early proteomic studies to separate the proteins. However, its resolution is not enough to ensure reproducibility and sensitivity (Rabilloud et al. 2010). Therefore, chromatographic separation followed by mass spectrometry (MS) is now routinely employed in proteomic studies. There are some deviations of chromatographic separation techniques such as high-pressure liquid chromatography (HPLC) and gas chromatography (GC). After proper separation of protein mixtures, they can be identified by single or double MS systems. Sometimes samples can be ionized by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) before identification in MS. This technique uses a laser energy-absorbing matrix to create ions from large molecules with minimal fragmentation (Jurinke et al. 2004).
Genomics and proteomics have developed separately into two different disciplines, thus limiting the cross talk between scientists in the two fields, limiting the integration of useful information from both fields into a single data modality. However, depending on the encoded genomic variants, mutations, or post-transcriptional modifications at the nucleotide level, the final expressed sequence of a protein may vary. NGS can be used to capture and correctly decipher these variants. Single-nucleotide polymorphisms (SNPs) and small insertion-deletion (INDELs) can be characterized using NGS, and these sequence variants can be easily translated in silico into different proteoforms that can be added to existing protein databases (Hernandez et al. 2014). As a result of the merging of genomics and proteomics, a new field known as proteogenomics has emerged (Jaffe et al. 2004; Nesvizhskii et al. 2014; Low et al. 2016; Sheynkman et al. 2016; Ruggles et al. 2017). The expression of a gene, for example, can be determined at the level of mRNAs and proteins in each allelic form using proteogenomics (Wingo et al. 2017). Exon-exon splice junctions, on the other hand, allow for the analysis of alternatively spliced proteomes. Moreover, proteogenomics has been increasingly used to understand the adaptive diversification of plant species and populations (Voelckel et al. 2017).
Tens of studies have been completed on proteomic analysis of various plant species under different developmental stages, abiotic or biotic stress conditions, at different tissues, organs, and cells (Reviewed by Tan et al. 2017; Mustafa and Komatsu et al. 2021; Smythers et al. 2021). Recently the Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/) was released to solve critical questions about the Arabidopsis thaliana proteome (van Wijk et al. 2021). It includes around 0.5 million unique peptides and 17,858 unique proteins at the highest confidence level.
2.15 Metabolomics
Metabolomics is the large-scale study of small molecules, also known as metabolites, in cells, biofluids, tissues, or organisms. The metabolome refers to these small molecules and their interactions within a biological system. Metabolomics is a powerful approach because, unlike other omics approaches, metabolites and their concentrations directly reflect the underlying biochemical activity and state of cells and tissues. As a result, metabolomics is the most accurate representation of the phenotype. Advancements in chromatographic separation and MS allowed for unbiased, high-throughput screening and characterization of the metabolites to study the metabolic pathways and phytochemicals to complement the other omics approaches (Lee et al. 2012; Kang et al. 2013; Kin et al. 2013; Lee et al. 2015). Because of the metabolome complexity, functional characterization of metabolites is a challenging strategy in plants (Chen et al. 2013; Lee et al. 2019). Moreover, plants within the same family generally produce the same or similar metabolites since the metabolic pathways are highly conserved in plant families, which make it easier to study the metabolites in the same family in different species (Ntie-Kang et al. 2013). Plant metabolomics studies can explain the spatiotemporal differences of some essential metabolites in different plant species, which are affected by environmental factors together with genetic determinants (Lee and Lee et al. 2015; Son et al. 2016). In general, genetic factors, nutritional status, and geo-climatic conditions all influence the chemical composition of different plant parts (Dias et al. 2016).
Currently, MS or nuclear magnetic resonance (NMR) spectrometry is used in many metabolomics studies. Some studies use gas chromatography (GC)-MS for the separation and analysis of volatile compounds. However, studying all metabolites is a big challenge since the combination of multiple metabolomics methods is required for this purpose. Many metabolomics studies have been completed in different crop species (Reviewed by Kumar et al. 2017; Sharma et al. 2018; Fernandez et al. 2020). Recent efforts in plant metabolomics science focus on natural variations of metabolites (Reviewed by Sun et al. 2021). These efforts determined the type of natural variations reflecting the metabolomics changes in a given plant family or taxon (Hu et al. 2014; Kusano et al. 2015; Albrecht et al. 2016; Zhen et al. 2016; Yang et al. 2018a, b; Fang et al. 2019). Later, these natural variations were used to select for the genotypes with superior metabolic profiles (e.g., Zhen et al. 2016) and link a specific metabolite or metabolic pathway to a genomic region via the identification of metabolite QTLs (mQTLs) (Chen et al. 2018a, b; Shi et al. 2020; Jamaloddin et al. 2021) or metabolome-based GWAS (mGWAS) (Luo 2015; Fang and Lou 2019; Chen et al. 2020; Wei et al. 2021).
Similar studies have recently been employed in the determination of ionic changes in different plant species (Yang et al. 2018a, b; Pita-Barbosa et al. 2019; Ali et al. 2021; Singh et al. 2022). Comparative metabolomics and ionomics studies revealed the evolutionary divergence of metabolic pathways and how they are conserved in some species or genotype for enhancing the adaptation to a specific condition (Dos Santos et al. 2017; Mawalagedera et al. 2019; Deng et al. 2020; Rastogi et al. 2020). We are now at the beginning of a new phase in plant metabolism research, in which integrative genomics and metabolomics approaches are used (Rai et al. 2017). The supremacy of genomics and transcriptomics should be integrated with metabolomics and proteomics studies to identify novel genes controlling the metabolism.
2.16 Multi-omics
Transcriptomics, proteomics, and metabolomics studies can represent the overall changes in transcripts, proteins, and metabolites, respectively (Aizat et al. 2018); however, a more diverse overall approach is needed to combine and compare large data sets to understand the complex biological systems such as the interactome. Multi-omics data generation and acquisition have become an essential part of modern molecular biology and biotechnology to study the biological pathways under different conditions because of recent advancements in NGS, proteomics, and metabolomics technologies as well as computational and statistical tools (Fondi and Liò 2015; Fabregat et al. 2018). Advancements in systems biology, the computational and mathematical analysis, and modeling of complex biological systems led to a more accurate understanding of complex biological systems. Systematic multi-omics integration (MOI) is essential for systems biology in plant science. MOI in plants has been a difficult task since the genomes of many non-model plant species are not well-annotated, the metabolic processes are diverse, and the interactome is massive (Jamil et al. 2020).
The earliest examples of MOI studies were very successful to demonstrate the power of the integrative omics approach to identify potential candidate genes, proteins, or metabolites for further functional characterization. For example, correlation analysis of transcriptomic and metabolomic data from the potato tubes led to the identification of novel transcript-metabolite pairs that can be further characterized in the future (Urbanczyk-Wochniak et al. 2003). In another study, transcriptomic and metabolomic data were integrated to understand the interactions of sulfur and nitrogen metabolisms and the involvement of secondary metabolites in Arabidopsis thaliana (Hirai et al. 2004). Since then, the MOI has been extensively used by plant scientists for functional characterization of unknown genes and to understand the behavior of complex systems under different conditions. Several different online software have been developed to integrate multi-omics data, such as MapMan (Thimm et al. 2004), and reviewed by Fondi and Lio (2015). The systems biology approach has been integrated extensively in different plant species (Rai et al. 2019).
In contrast to these advancements, some hurdles have slowed the utilization of the systems biology approach, particularly in crop species. These include the incomplete transcriptome, proteome, and metabolome data sets or their total unavailability. Current software is not designed to integrate different omics data sets to describe the phenome. Machine learning and artificial intelligence should yet to be incorporated into this software. The metabolome or ionome can be easily influenced by the environmental changes so that the extensive data generated by metabolomics and ionomics may not be readily integrated with the date of transcriptomics and genomics. Therefore, the results obtained at the levels of transcriptome and genome may not be fully reflected at the metabolome or even phenotype (do Amaral and Souza 2017). Therefore, there are lots of complex and dynamic processes working in parallel in the cell.
2.17 Single-Cell Technologies
The sequencing of a single-cell genome or transcriptome to obtain genomic, transcriptome, or other multi-omics information to show cell population distinctions and cellular evolutionary relationships is referred to as single-cell sequencing technologies (Wen et al. 2018). The molecular insight into tissue and/or time point/developmental groupings using bulk techniques, which average over many cells, has been gained. However, the inherent biases introduced by averaging over different cell populations limit these approaches. Bulk averaging can, in some cases, lead to qualitatively wrong conclusions, a phenomenon known as Simpson’s dilemma (Trapnell et al. 2015). Single-cell technologies have the advantages of detecting heterogeneity among individual cells, distinguishing a small number of cells, and outlining cell maps when compared to standard sequencing technology (Wen et al. 2018). Single-cell genomic approaches offer a potent set of tools for identifying cellular heterogeneity, as well as the formation and differentiation of cell types in complex tissues.
Due to its expensive cost, early single-cell sequencing was not widely used (Wen et al. 2018). High-throughput single-cell transcriptomics has become an accessible and powerful tool for unbiased profiling of complex and heterogeneous systems, thanks to recent improvements in cost and throughput (Klein et al. 2015; Zilionis et al. 2017; Macosko et al. 2015) and the availability of fully commercialized workflows (Zheng et al. 2017). These data sets can be utilized in concert with novel computational approaches to uncover cell types and states (Shekhar et al. 2016; Villani et al. 2017a, b), recreate developmental pathways, make destiny decisions (Trapnell et al. 2014; Welch et al. 2016), and spatially model complex tissues (Satija et al. 2015; Achim et al. 2015).
The emergence of omics techniques has quickly revolutionized our perspectives on plant biology, thanks to the advancement of sequencing technologies. The cellular diversity inside a tissue or organism, on the other hand, is far more complex than can be assessed using bulk analysis, which can only produce population-averaged results (Gawad et al. 2016). As sequencing technologies advanced, allowing smaller and smaller samples, eventually allowing single-cell analysis, the traditional consensus from bulk-based omics studies was questioned (Shapiro et al. 2013). Characterizing the single-cell genome is of significant interest because each cell undergoes a unique chain of DNA synthesis and damage repair events. In a single-cell sequencing-based investigation, there are numerous basic processes. The first step is the preparation of a cell lysate. Plant cell isolation and lysis, unlike animal models, are hampered by the natural cell wall, requiring the use of specific techniques. Single-cell whole-genome amplification (WGA) must be performed once the plant cell lysate has been generated. Single-cell genomics and epigenomic technologies are both based on single-cell WGA; however, single-cell epigenomics is more diverse due to the addition of sample preprocessing procedures for capturing various epigenomic features such as bisulfite conversion for DNA methylation (Smallwood et al. 2014) and proximity DNA ligation for chromatin conformation (Nagano et al. 2013). Single-cell sequencing technologies have been used to investigate the cell heterogeneity that underlies several bulk omics characteristics, such as genomic variation, DNA methylation, and chromatin accessibility, in a variety of animal models (Huang et al. 2015; Kelsey et al. 2017). In recent years, they have been advanced greatly in terms of sensitivity and throughput. These developments have made it possible to profile cell-specific genomic variants and epigenomic characteristics in plant models for the first time, and they hold a great promise for answering a wide range of plant biological problems at the single-cell level (Stuart and Satija et al. 2019). Recently, multiple experimental protocols, including the Assay for Transposase-Accessible Chromatin with High-Throughput Sequencing (ATAC-seq) (Buenrostro et al. 2015), single-cell combinatorial indexing ATAC-seq (sci-ATAC-seq) (Cusanovich et al. 2015), single-cell transposome hypersensitivity site sequencing (scTHS-seq) (Lake et al. 2018), plate-based scATAC-seq protocol (Chen et al. 2018a, b), and droplet-based single-cell combinatorial indexing ATAC-seq (dsci-ATAC-seq) (Lareau et al. 2019), have been developed to profile genome-wide chromatin accessibility in single cells. Very recently, the use of single-nucleus RNA sequencing (sNucRNA-seq) and single-nucleus assay for transposase-accessible chromatin sequencing (sNucATAC-seq) technologies on Arabidopsis roots was reported, and it was suggested that the differential chromatin accessibility is a critical mechanism to regulate gene activity at the cell-type level (Farmer et al. 2021). Furthermore, single-cell resolution maps of open chromatin in the Arabidopsis root to address the issue of tissue heterogeneity and to detect likely endoreduplication events were provided by single-cell ATAC-seq (Dorrity et al. 2021).
2.18 Single-Cell Transcriptomics
Differential gene expression is largely responsible for the development of multiple cell types and cell-specific functions in multicellular organisms. The transcriptome of individual cells is frequently profiled using single-cell RNA sequencing (scRNA-seq). scRNA-seq (single-cell RNA sequencing) is a next-generation sequencing technology that generates gene expression data from thousands of single cells. This large data collection can be used to answer questions like how many different cell kinds are present in a sample and how common each cell type is. The recent development of single-cell RNA sequencing (scRNA-seq) has deepened our understanding of the cell as a functional unit, revealing new populations of cells with distinct gene expression profiles previously hidden within gene expression analyses performed on bulk cells and providing new insights based on gene expression profiles of hundreds to hundreds of thousands of individual cells (Ziegenhain et al. 2017; Macosko et al. 2015).
Single-cell RNA sequencing has been particularly useful in gaining insight into tissue cellular heterogeneity and identifying previously unknown cell types (Artegiani et al. 2017; Villani et al. 2017a, b; Glass et al. 2017). Single-cell technologies can also be used to identify subpopulations within a known cell type by looking for differences in gene expression patterns within the cell population (Artegiani et al. 2017; Shalet and Satija et al. 2013). Furthermore, these technologies can effectively isolate the signal from rare cell populations, which would otherwise be lost in the output of RNA sequencing on a bulk cell population (Shalet and Satija et al. 2014; Grün et al. 2015; Mahata et al. 2014; Torre et al. 2018). Besides that, the technology can be used to infer potentially useful markers for cell types that lack known markers, such as cell surface proteins. Because single-cell sequencing is driven by cell clustering based on differentially expressed genes, the genes that drive the clustering can be studied as potential unique markers for the cell population of interest (Artegiani et al. 2017; Zhao and Gao et al. 2017). Finally, single-cell sequencing can be used to investigate cell lineage and differentiation regulation. A population of stem cells, for example, can be induced to differentiate, and single-cell sequencing at various time points can provide “snapshots” of the differentiation process. The trajectories that cell flows to reach each terminally differentiated state, as well as the key genes that are differentially regulated at each branch point, can then be inferred using these snapshots (Artegiani et al. 2017; Treutlein et al. 2014; Trapnell et al. 2014; Qiu et al. 2017).
Biological tissue samples are frequently used as an input material for single-cell experiments. In the first phase, a single-cell suspension is created by digesting the tissue in a process known as single-cell dissociation. Cells must be isolated to profile the mRNA in each one separately. Depending on the experimental protocol, single-cell isolation is done differently. Droplet-based approaches focus on catching each cell in its microfluidic droplet, whereas plate-based methods separate cells into wells on a plate. Multiple cells can be captured together (doublets or multiplets), non-viable cells can be captured, or no cell can be captured at all (empty droplets/wells) in both circumstances. Droplet-based approaches rely on a low concentration flow of input cells to manage doublet rates; hence empty droplets are typical. Each well or droplet includes the chemicals required to break down cell membranes and perform library construct. The process of capturing intracellular mRNA, reverse-transcribed to cDNA molecules and amplified, is known as library construction. The mRNA from each cell can be labeled with a well- or droplet-specific cellular barcode, while the cells go through this process in isolation. Moreover, captured molecules are labeled with a unique molecular identifier (UMI) in many experimental protocols. To enhance the probability of being measured, cellular cDNA is amplified before sequencing.
Cellular cDNA libraries are labeled with cellular barcodes and, depending on the protocol, UMIs after library formation. For sequencing, these libraries are pooled together (multiplexed). Read data is generated by sequencing and is subjected to quality control, grouping based on assigned barcodes (demultiplexing), and alignment in reading processing pipelines. Read data can be further demultiplexed for UMI-based methods to produce counts of captured mRNA molecules (count data). However, analyzing and utilizing the large amounts of data created by single-cell RNA sequencing research is difficult and requires knowledge of the experimental and computational pathways that go from the preparation of input cells to the production of interpretable data. Single-cell gene expression analysis was previously limited to a few select transcripts from a few individual cells. Modern single-cell sequencing platforms like as Fluidigm C1, Drop-Seq, Chromium 10X, SCI-Seq, and many others have been developed during the past decade thanks to high-throughput sequencing and high-yield cell separation approaches. At any given time, these technologies can define the transcriptional profile of hundreds to thousands of single cells. All rely on the use of DNA barcodes to label mRNA molecules during reverse transcription and/or later processes, allowing the transcripts to be indexed back to their individual cells of origin. Despite the fact that each technique has its own manner of separating cells and labeling mRNA molecules, they all use the same computational pipelines to represent transcriptional profiles.
Single-cell gene expression analyses have not been widely used in plants to date, owing to the presence of the plant cell wall, which makes it difficult to separate and acquire individual cells. Although there is recognition of the potential benefit of large-scale single-cell transcriptome studies in plants, single-cell gene expression studies in plants have so far been limited to a small number of cells (Lieckfeldt et al. 2008; Brennecke et al. 2013; Efroni et al. 2015; Frank and Scanlon 2015; Efroni and Birnbaum 2016; Libault et al. 2017). Several groups have recently used single-cell transcriptomics to plants with high throughput (Denyer et al. 2019; Efroni et al. 2015; Efroni et al. 2016; Jean-Baptiste et al. 2019; Kubo et al. 2019; Nelms et al. 2019; Ryu et al. 2019; Shulse et al. 2019; Zhang et al. 2019). Plant studies using single-cell RNA-seq have primarily focused on the well-studied and understood Arabidopsis root system (Denyer et al. 2019; Jean-Baptiste et al. 2019; Ryu et al. 2019; Shulse et al. 2019; Zhang et al. 2019). The Arabidopsis root, in particular, is a useful organ for scRNA-seq because it has a relatively small number of cells and cell types, and methods for isolating individual cells by protoplasting are available (Birnbaum et al. 2005; Bruex et al. 2012; Efroni et al. 2015; Li et al. 2016). Even in this highly tractable and well-understood system with many known marker genes and cell types, these landmark studies revealed a slew of new and more robust cell-type marker genes and begun to characterize the transition states that give rise to developmental trajectories (Denyer et al. 2019; Jean-Baptiste et al. 2019; Ryu et al. 2019; Shulse et al. 2019; Zhang et al. 2019). Recently, Qing et al. (2020) performed the scRNA-seq on root tips of two agronomically important rice cultivars and identified more than 20,000 single cells. Using integration analysis of two cultivars, most of the major cell types were identified, and novel cell-type-specific marker genes for both cultivars were characterized. In addition, they found well-conserved cell types between the two rice cultivars associated with specific regulatory programs, including phytohormone signaling, biosynthesis, and response. To identify the effects of tissue heterogeneity, Dorrity et al. (2021) applied scATAC-seq and scRNA-seq to Arabidopsis roots separately. They identified thousands of differentially accessible sites using scATAC-seq results and the entirety of a cell’s regulatory landscape and its transcriptome using scRNA-seq. To define the endoreduplication, cell division, and developmental progression, they integrated the scATAC-seq and scRNA-seq data and characterized cell type-specific motif enrichments of transcription factor family analysis and linked the expression of family members to changing accessibility at specific loci, resolving direct and indirect effects that shape expression (Dorrity et al. 2021).
2.19 Conclusion
The omics technologies have been tremendously developed since their first-time introduction in plant science, which was flowed by exponential studies in different plant taxa. At present, at least one research study on plant omics is published every day. These omics studies have generated extensive data such that the pace of software development to analyze this much of data cannot meet the demand. In the past, individual studies of genomics, transcriptomics, or metabolomics were enough to make a judgment about the plant species or genotypes. However, now the focus has shifted from generating high-throughput biological data sets to the integration of these data sets to derive biological meaning out of it. These data sets are valuable for future efforts in establishing models that can describe plant adaptation, cultivation, and production. Novel approaches such as artificial intelligence and machine learning will be required in the near future to get the most out of these data sets and predict the future scenarios, especially under ongoing climate crises.
In the near future, plant biologists will focus on understanding the interactome of different metabolisms in the plant and how these interactions are affecting the phenome. They will utilize integrated omics technologies together with genome editing and speed breeding. Identification of novel genomic, proteomic, or metabolomic markers will be very useful in screening different plant genotypes, wild relatives, or breeding lines to find and develop new cultivars highly adapted to the changing climate with higher yields and nutritional quality.
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Aksoy, E., Yılmaz, H., Kayıhan, C. (2022). The Revolution of Omics Technology in Plant Science. In: Prakash, C.S., Fiaz, S., Fahad, S. (eds) Principles and Practices of OMICS and Genome Editing for Crop Improvement. Springer, Cham. https://doi.org/10.1007/978-3-030-96925-7_2
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