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3 Opportunity and Challenges for Whole‐Genome Resequencing‐based Genotyping in Plants
Surbhi Kumawat1, Gaurav Raturi1, Pallavi Dhiman1, Sreeja Sudhakarn1, Nitika Rajora1, Vandana Thakral1, Himanshu Yadav1, Gunashri Padalkar1, Yogesh Sharma1, Vinaykumar Rachappanavar2, and Manish Kumar2
1 Department of Agriculture Biotechnology, National Agri-Food Biotechnology Institute (NABI), Mohali, Punjab, India
2 Department of Seed Science and Technology, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India
3.1 Introduction
Recent advancements in next‐generation sequencing (NGS) have speed up genomics and transcriptome research across the board, especially in plant science (Rana et al. 2019; Ratnaparkhe et al. 2020). Genomics is a key for the better understanding of whole‐genome involving the DNA sequences determined in the order in which they are present in a genome which indeed in the sequential exploration of the evolution of genome structure and interpretation of the molecular phylogeny. It involves the combination of various technologies including recombinant DNA, sequencing methods of DNA, and bioinformatics to assemble, analyze and assign the structure and function of the plant genome. Earlier, the Sanger method was commonly used for nucleic acid sequencing in genomics but in 2005 the development of NGS brought sequencing platforms that are fast and cost‐effective enabling the excavating of characteristics unique to particular species in terms of gene structure as shown in Figure 3.1 (Unamba et al. 2015; Loera‐Sánchez et al. 2019). It also enables us to understand the mechanism lying behind the process of gene expression as well as secondary metabolism. Further recent advances in the NGS platforms bring the gratifying efforts in de‐novo whole‐genome/transcriptomic sequencing and also for the developments of markers based on genome‐wide sequences (Vasupalli et al. 2020). The most common platforms of NGS include Illumina/Solexa, ABI/SOLiD, Helicos, and 454/Roche (Unamba et al. 2015).
Earlier techniques for gene expression analysis include northern blotting in which specific RNA are detected and quantified (Goda and Minton 1995). Another technique involves the hybridization of antisense RNA with the complementary known target sequence thereby preventing the digestion of target sequence by single‐strand RNAase resulting in the specific and accurate quantification of the target sequence. But the major drawbacks associated with this technique include it requires prior knowledge about the target sequences. With the advancements in technologies, transcriptomics came into existence which analyses all RNA molecules (tRNA, rRNA, mRNA, and other noncoding RNA) transcribed in an organism. Microarray is a widely employed technique to analyze the transcriptome. In a single experiment, this technique can measure the expression level of a thousand genes but there are several issues associated with its sensitivity and reproducibility.
Figure 3.1 Diagrammatic representation of various high‐throughput‐sequencing methods used in assessments of genetic diversity. Methods mentioned