Genotyping by Sequencing Advancements in Barley

Abstract

Barley is considered an ideal crop to study cereal genetics due to its close relationship with wheat and diploid ancestral genome. It plays a crucial role in reducing risks to global food security posed by climate change. Genetic variations in the traits of interest in crops are vital for their improvement. DNA markers have been widely used to estimate these variations in populations. With the advancements in next-generation sequencing, breeders could access different types of genetic variations within different lines, with single-nucleotide polymorphisms (SNPs) being the most common type. However, genotyping barley with whole genome sequencing (WGS) is challenged by the higher cost and computational demand caused by the large genome size (5.5GB) and a high proportion of repetitive sequences (80%). Genotyping-by-sequencing (GBS) protocols based on restriction enzymes and target enrichment allow a cost-effective SNP discovery by reducing the genome complexity. In general, GBS has opened up new horizons for plant breeding and genetics. Though considered a reliable alternative to WGS, GBS also presents various computational difficulties, but GBS-specific pipelines are designed to overcome these challenges. Moreover, a robust design for GBS can facilitate the imputation to the WGS level of crops with high linkage disequilibrium. The complete exploitation of GBS advancements will pave the way to a better understanding of crop genetics and offer opportunities for the successful improvement of barley and its close relatives.

Keywords: SNPs; barley; genotyping by sequencing; restriction enzymes; target enrichment.

Figure 1

Application of GBS approaches for various genetics and plant breeding studies. The high-quality SNPs derived from GBS are being used for diversity analysis, genetic map construction, genome-wide association studies and genomic selection.

Figure 2

Schematic overview of Restriction Enzyme based GBS methodology. (A) Tissue sample collection is followed by DNA isolation from the crop. (B,D,F) DNA digestion by; One enzyme, ApeKI, that makes a cut to produce overhangs (B), classical Two enzyme, a rare cutter PstI and a common cutter MspI produces overhangs (D), and new version of Two enzyme called tGBS, using a different set of restriction enzymes, NspI and BfuCI capable of better digestion produces overhangs in opposite directions (F). (C,E,G) Adapter ligation to the digested sample; One enzyme, ligation of barcode and common adapter (C), classical Two enzyme, ligation of forward barcode and reverse Y adapter (E), tGBS, ligation of barcode and universal oligo. Nucleotide sequence in red possesses the matching bases in selective primer for target specificity (G). (H) Multiplexing; a unique barcode used for each accession allows the pooling of DNA samples before the amplification step. (I) The mixed libraries, uniquely barcoded, and amplified samples will be run in a next-generation sequencer. The sequence reads will be analyzed in various bioinformatic pipelines based on the availability of reference sequence.

Figure 3

Schematic representation of Molecular Inversion Probe to capture a specific target for sequencing. (A) Model structure of a molecular inversion probe (MIP) with a ligation and extension arm connected by a 30 bp linker. Both ligation and extension arms are designed to complement the target region. (B) These complementary arms at the end of linker pairs with the target region, which is followed by gap filling and ligation (Circular DNA molecule). (C) Digestion of exogenous host DNA and probes with the help of exonucleases. (D) Amplification of the captured segments using the universal primers complementary to the linker sequence of MIP along with sample-specific index sequences and sequencer-specific adapters. (E) Libraries will go through the sequencing followed by data curation.

Figure 4

Schematic representation of RNA-Seq to sequence the transcriptome. (A) Poly A tailed messenger RNA (mRNA) is isolated among other RNA types using oligo dT magnetic beads. (B) Double-stranded complementary DNA (cDNA) library is constructed by employing reverse transcriptase on the isolated mRNA. (C) Enzymatic fragmentation of double-stranded cDNA is carried out to construct uniform library followed by end repair and an A nucleotide is added-to facilitate adapter ligation. These fragments are subjected to PCR amplification. (D) The amplicons are sequenced and differentially expressed genes are identified by mapping the reads with a quality reference sequence comparing the contrasting genotypes.

Figure 5

A generalized flowchart for exome capture to isolate the exon variants. It can be broadly divided into two phases viz., target enrichment and DNA sequencing. In enrichment phase high-quality genomic DNA is isolated and specific probes are designed for hybridization followed by capturing of hybridized probes. In sequencing phase, the raw exome sequence data is filtered and aligned to find potential variants.

Figure 6

A schematic representation of DNase Seq to determine chromatin accessibility. (A) Nuclei isolation followed by DNase I digestion is a critical step to collect the chromatin. (B) Targeted fragments will be selected and isolated from gel. (C) Library construction followed by next-generation sequencing and data analysis.

Figure 7

An overall workflow for ChIP Seq to analyze protein interactions with DNA. (A) Isolation of genomic DNA with conserved binding proteins. (B) Crosslinking and DNA fragmentation to access the DNA-protein complex. (C) Immunoprecipitation with protein-specific antibodies to separate the DNA-protein complex. (D) DNA purification is followed by adaptor ligation to prepare the sequencing library. (E) Discovery of DNA biding segments using next-generation sequencing platform. (F) Data analysis and alignment to reference genome will identify targeted DNA sequences that interact with the protein. This figure is adapted from “ChIP sequencing,” by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates.