Sample and Library Preparation for PacBio Long-Read Sequencing in Grapevine.

PacBio long-read sequencing is a third-generation technology that generates long reads up to 20 kilobases (kb), unlike short-read sequencing instruments that produce up to 600 bases. Long-read sequencing is particularly advantageous in higher organisms, such as humans and plants, where repetitive regions in the genome are more abundant. The PacBio long-read sequencing uses a single molecule, real-time approach where the SMRT cells contain several zero-mode waveguides (ZMWs). Each ZMW contains a single DNA molecule bound by a DNA polymerase. All ZMWs are flushed with deoxy nucleotides with a fluorophore specific to each nucleotide. As the sequencing proceeds, the detector detects the wavelength of the fluorescence and the nucleotides are read in real-time. This chapter describes the sample and library preparation for PacBio long-read sequencing for grapevine.

Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

Genome Wide Identification and Annotation of NGATHA Transcription Factor Family in Crop Plants.

The NGATHA (NGA) transcription factor (TF) belongs to the ABI3/VP1 (RAV) transcriptional subfamily, a subgroup of the B3 superfamily, which is relatively well-studied in Arabidopsis. However, limited data are available on the contributions of NGA TF in other plant species. In this study, 207 NGA gene family members were identified from a genome-wide search against Arabidopsis thaliana in the genome data of 18 dicots and seven monocots. The phylogenetic and sequence alignment analyses divided NGA genes into different clusters and revealed that the numbers of genes varied depending on the species. The phylogeny was followed by the characterization of the Solanaceae (tomato, potato, capsicum, tobacco) and Poaceae (Brachypodium distachyon, Oryza sativa L. japonica, and Sorghum bicolor) family members in comparison with A. thaliana. The gene and protein structures revealed a similar pattern for NGA and NGA-like sequences, suggesting that both are conserved during evolution. Promoter cis-element analysis showed that phytohormones such as abscisic acid, auxin, and gibberellins play a crucial role in regulating the NGA gene family. Gene ontology analysis revealed that the NGA gene family participates in diverse biological processes such as flower development, leaf morphogenesis, and the regulation of transcription. The gene duplication analysis indicates that most of the genes are evolved due to segmental duplications and have undergone purifying selection pressure. Finally, the gene expression analysis implicated that the NGA genes are abundantly expressed in lateral organs and flowers. This analysis has presented a detailed and comprehensive study of the NGA gene family, providing basic knowledge of the gene, protein structure, function, and evolution. These results will lay the foundation for further understanding of the role of the NGA gene family in various plant developmental processes.

Application of Genome Editing in Tomato Breeding: Mechanisms, Advances, and Prospects.

Plants regularly face the changing climatic conditions that cause biotic and abiotic stress responses. The abiotic stresses are the primary constraints affecting crop yield and nutritional quality in many crop plants. The advances in genome sequencing and high-throughput approaches have enabled the researchers to use genome editing tools for the functional characterization of many genes useful for crop improvement. The present review focuses on the genome editing tools for improving many traits such as disease resistance, abiotic stress tolerance, yield, quality, and nutritional aspects of tomato. Many candidate genes conferring tolerance to abiotic stresses such as heat, cold, drought, and salinity stress have been successfully manipulated by gene modification and editing techniques such as RNA interference, insertional mutagenesis, and clustered regularly interspaced short palindromic repeat (CRISPR/Cas9). In this regard, the genome editing tools such as CRISPR/Cas9, which is a fast and efficient technology that can be exploited to explore the genetic resources for the improvement of tomato and other crop plants in terms of stress tolerance and nutritional quality. The review presents examples of gene editing responsible for conferring both biotic and abiotic stresses in tomato simultaneously. The literature on using this powerful technology to improve fruit quality, yield, and nutritional aspects in tomato is highlighted. Finally, the prospects and challenges of genome editing, public and political acceptance in tomato are discussed.

Molecular Evolution and Diversification of Proteins Involved in miRNA Maturation Pathway.

Small RNAs (smRNA, 19-25 nucleotides long), which are transcribed by RNA polymerase II, regulate the expression of genes involved in a multitude of processes in eukaryotes. miRNA biogenesis and the proteins involved in the biogenesis pathway differ across plant and animal lineages. The major proteins constituting the biogenesis pathway, namely, the Dicers (DCL/DCR) and Argonautes (AGOs), have been extensively studied. However, the accessory proteins (DAWDLE (DDL), SERRATE (SE), and TOUGH (TGH)) of the pathway that differs across the two lineages remain largely uncharacterized. We present the first detailed report on the molecular evolution and divergence of these proteins across eukaryotes. Although DDL is present in eukaryotes and prokaryotes, SE and TGH appear to be specific to eukaryotes. The addition/deletion of specific domains and/or domain-specific sequence divergence in the three proteins points to the observed functional divergence of these proteins across the two lineages, which correlates with the differences in miRNA length across the two lineages. Our data enhance the current understanding of the structure-function relationship of these proteins and reveals previous unexplored crucial residues in the three proteins that can be used as a basis for further functional characterization. The data presented here on the number of miRNAs in crown eukaryotic lineages are consistent with the notion of the expansion of the number of miRNA-coding genes in animal and plant lineages correlating with organismal complexity. Whether this difference in functionally correlates with the diversification (or presence/absence) of the three proteins studied here or the miRNA signaling in the plant and animal lineages is unclear. Based on our results of the three proteins studied here and previously available data concerning the evolution of miRNA genes in the plant and animal lineages, we believe that miRNAs probably evolved once in the ancestor to crown eukaryotes and have diversified independently in the eukaryotes.

A Low-Cost High-Throughput Method for Plant Genomic DNA Isolation.

Many of the functional genomics methods require isolation of genomic DNA from large population of plants. The selection of DNA isolation protocols depends on several factors such as choice of starting material, ease of handling, time and labor required for isolation, the final quantity as well as the quality of genomic DNA. We outline here a high-throughput method of DNA extraction from different plant species including cereal crops. The protocol can be used for extraction of DNA in single tubes as well as for large formats in 96-well plates. The protocol includes steps for eliminating interfering secondary products such as phenolics. This protocol can be applied for high-throughput isolation of DNA for varied applications such as TILLING, mapping, fingerprinting, etc. as a cost-effective protocol compared to commercial kits.

Next-generation sequencing (NGS)-based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population.

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next-generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty-five genes belonging to carotenoids and folate metabolism were PCR-amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600-bp amplicons were directly sequenced in a non-overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128-fold pooling. An evaluation of six different software programs (camba, crisp, gatk unified genotyper, lofreq, snver and vipr) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.