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Next-Generation Sequencing in Vegetable Crops

Meenu Kumari1*, Tanya Barpanda2, Meghana Devireddy3, Ankit Kumar Sinha3, R. S. Pan1 and A. K. Singh1

1ICAR-Research Complex for Eastern Region, RS, Ranchi, India

2Orissa University of Agriculture & Technology, Odisha, India

3ICAR-Indian Agricultural Research Institute, New Delhi, India

Abstract

The last few decades have witnessed revolutionary advances in all biological disciplines in DNA sequencing technologies at a fraction of the cost with respect to traditional sequencing. There are two methods of crop breeding, i.e., conventional approach through hybridization followed by selection and marker-assisted selection (MAS). Limitations of conventional approach like long periods of selection to fix a trait in the breeding population, environmental effect, and low efficiency for complex and less heritable traits lead breeders to choose MAB. It necessitates the use of different molecular markers based on the availability of information on linkage of traits with markers. However, MAB efficiency was convenient to explore traits that are governed by few numbers of quantitative trait loci (QTLs), whereas for complex traits like yield, quality, biotic stress, and abiotic stress, which are governed by a large number of minor QTLs, Marker-Assisted Selection is not effective. The next-generation sequencing (NGS) approach opened an era of data science where millions of bases are being sequenced in one round and extremely reduced the time and cost of sequencing. In this chapter, we describe the status, recent development, and application of NGS in vegetable crops for their utilization in practical improvement approaches.

Keywords: MAB, QTL, GBS, transcriptomics, GBS, NGS

1.1 Introduction

The next-generation sequencing (NGS) approach has exemplary development in the field of life sciences and shifted sequencing studies from “model organism” to “every organism” with the power of high-throughput NGS technology. NGS techniques became commercially available in 2005, and since then, there has tremendous development at an astonishing rate in this field like the evolutionary process to address important and unexplored questions of plant systems. These approaches can be broadly divided into three main categories: sequencing by synthesis, sequencing by ligation, and single-molecule sequencing. However, emerging technologies made it possible without library preparation, like the use of quantum dot (qdot)-derived fluorescence resonance energy transfer (FRET) to detect fluorescently labeled nucleotide incorporation. Another sequencing approach is nanopore sequencing, where chemical or electronic properties of bases (DNA or RNA) can be analyzed directly while passing through nanopores. The NGS evolution trend with the advancement of technologies has been depicted in Figure 1.1. This has exemplary development in the field of life sciences and shifted sequencing studies from “model organism” to “pan organism” with the power of high-throughput NGS technology.

Figure 1.1 Timeline of next-generation sequencing technology.

With the advancement of high-throughput sequencing platforms, data have been generated for millions of plant species and, therefore, understanding of plant system at the nucleotide level has been enriched in horticultural crops like fruits, vegetables, spices, plantations, etc.

1.2 Next-Generation Sequencing Approach in Genomics

1.2.1 Solanaceous

Among vegetables, the tomato was a pioneer in identifying the genetic basis of quantitative traits and in the map-based cloning of genes and quantitative trait loci (QTLs) [1]. Through the creation of extensive molecular marker libraries [2], genetic and physical maps [3], and mapping populations [4], tomato has served as a model plant for improvement and inheritance studies since the early 1990s. The Tomato Genome Consortium started the project called “SOL-100” associated with NGS-based sequencing of 100 different species of Solanaceae family and relating their sequences to the reference genome (solgenomic.net).

The tomato cultivar “Heinz 1706” is estimated to have a genome size of approximately 900 Mb with a simple structure composed of two main components: pericentromeric heterochromatin with repetitive sequences, occupying 75% of the whole genome, and distal euchromatin comprising the remaining 25% (220 Mb). It was sequenced through the BAC-by-BAC approach, resulting in the sequencing of 117 Mb of euchromatic regions precisely [5]. The main reason behind choosing this particular cultivar was because of its well-characterized HindIII BAC library available at that time [6]. In 2008, 30,800 BAC clones were selected, pooled, and short gun sequencing was done using the Sanger method of sequencing to accelerate the sequencing progress (The Tomato Genetic Consortium.2012). The sequences were congregated into contigs that elucidated 540 Mb of the genome. In 2009, the emerging NGS platforms paved the way for the sequencing consortium to plan for whole genome sequencing, which was earlier confined to only sequencing of euchromatin regions. Different variants of NGS technologies like 454/Roche GS FLX, Illumina Genome Analyser, and SOLiD sequencing were used. Using Sanger data, a de novo sequencing of “Heinz 1706” was assembled. Assemblies were generated by independent programs like Newbler and CABOG, and merged later on. Read-mapping and base error correction resulted in more accurate data where one base calling error per 29.4 kb and one indel error per 6.4 kb were obtained [7, 8]. Two BAC-based physical maps were used to connect the resulting high-quality scaffolds, and a high-density genetic map was used to anchor them [9] as well as with introgression line mapping and genomewide BAC-FISH (Fluorescence In Situ Hybridization). The final assembly of tomato genome consisted of 760 Mb involving of 91 scaffolds. Most of the gaps identified after aligning the scaffolds with 12 chromosomes were confined to pericentromeric regions [7]. The consortium also sequenced the LA1589 accession of wild S. pimpinellifolium to explore the diversity it possesses through variation with the reference genome of “Heinz 1706”. It was performed by Illumina technology using the whole-genome short gun sequencing approach. An assembly of 739 Mb was congregated, which, when compared with reference genome, showed the nucleotide divergence of only 0.6%, indicating the high level of similarity among two species (The Tomato Genome Consortium 2012). The first resequencing in tomato was attempted on 4 S. lycopersicum and 4 S. l. cerasiformae genotypes through the Illumina GAIIx platform, which generated a total of 4 million unique SNPs, 1,686 putative copy-number variations (CNV), and almost 1,28,000 InDels [10]. These variations can be utilized for QTL and gene mapping. Long-read technologies made further improvements in sequencing high-quality reference genomes including S. pimpinellifolium accessions like LA2093, LA1589 [11], LA1670 [12], S. l. var. cerasiformae acc. LA1673 [12], S. l cv. Moneyberg [13], S. l cv. Heinz 1706 [14], and S. lycopersicoides acc. LA2951 [15]. A total of 100 tomato accessions were sequenced using nanopore technology to detect variations and de novo assemblies were released for 14 reference genomes [16]. A total of 2,38,490 structural variants (mostly insertions and deletions) were discovered. The functional analysis allowed the linking of these variants to three major traits important for domestication and improvement targets: smoky flavor (not preferable among consumers), sb1 (for branching patterns), and fw3.2 (major QTL for fruit mass).

Capsicum annuum cv. CM334 was sequenced with high genome coverage and is considered as a reference for genome sequence of pepper [17]. The genome size was estimated to be 3.48 Gb, which is very high when compared to its fellow crops of same family. An increase in the genome size of pepper is supposed to be because of long terminal repeats of retrotransposons. In addition, CM334 genome has 76.4% transposable elements. This study also provided deeper insights into pepper pungency causing capsaicinoid biosynthesis pathway. For a better understanding of evolution and domestication, another cultivar of C. annuum var. glabriusculum (Zunla-1 and wild Chiltepin) was sequenced using Illumina technology through the whole-genome short gun approach [18]. They found 1104 target genes responsible in the capsaicinoid biosynthesis pathway, which indicates miRNAs for regulation. Resequencing data of 20...