Improvement of wheat for rust resistance through genomic tools

Charu Lata*, Sneha Adhikari, Pramod Pramod, O.P. Gangwar, Subodh Kumar

ICAR-Indian Institute of Wheat and Barley research Institute,

Regional Station, Flowerdale- Shimla (171002)

Summary
Wheat (Triticum aestivum L.) is the second most important food crop ensuring nutrition to ever-
growing population of the world. It occupies approximately 215 M ha area and is one of the
largest grown cereal in the world. Global demand for wheat has overtaken its production that’s
why stocks are continuously under pressure globally. The rust diseases (stripe, leaf and stem
rust) of wheat are major threat to sustainable wheat production in the wheat growing countries
of the world because of their air borne nature and faster evolution of new virulent pathotypes.
Consequently resistant wheat varieties become susceptible within a short time interval of 6-7
years. Several strategies are being undertaken to meet this challenge, including preventing yield
losses through more effective rust resistance. Moreover, to achieve this goal, traditional
breeding practices must be supplemented with recent advance technologies based on recent
benefits from wheat genome sequencing. Sequencing and alignment of wheat genome is the
significant landmark in the area of wheat genomics which uncovers the opportunities to identify
candidate genes, marker trait associations and promote genomic selection (GS) studies.
Advancements in next-generation sequencing platforms and bioinformatics tools have
transformed the field of wheat genomics. In this article we focus on the potential of genomic
research strategies to control the killer pathogen Puccinia and how these technologies have been
adopted to fight against wheat rust and gaining rust resistance. Genomic tools make accessible
identification and characterization of effective rust resistant genes from diverse sources, and
deployment into pre-breeding lines and future wheat varieties.

Keywords- Triticum aestivum, Puccinia, Genomics, NGS technologies, Crop Improvement

Introduction
Wheat, the second most important cereal food crop after rice throughout the world, is the most
consumed and traded crop. All over the world wheat is the major source of calories in human
diet and supplies 20 % of the dietary proteins. The growing demand for wheat world over has
necessitated to increase the wheat production and more so to curb the losses due to pests and
pathogens especially rusts. There should be a balance of demand and supply but in case of
wheat production demand has outstripped always, consequently global stocks of wheat grains
are under constrains (http://www.icarda.org).
Puccinia is a pathogen of global importance and well documented to cause significant yield loss
in wheat growing countries. Stem (black), leaf (brown), and stripe (yellow) rusts, caused by
fungal pathogens Puccinia graminis f. sp. tritici (Pgt), P. triticina (Ptr), and P. striiformis f. sp.
tritici (Pst), respectively, are the most important wheat diseases, causing significant yield
reduction (McIntosh et al., 1995; Bhardwaj et al., 2019). Wheat stem rust, a devastating fungal
disease known as polio of agriculture, has spread from Africa to South and Central Asia, the
Middle East and Europe, with calamitous losses in the production of wheat
[www.independent.co.uk]. Due to its air borne nature and faster evolution of new virulent
pathotypes, resistant wheat varieties become susceptible consequently within a short time
interval of 5-7 years. Stripe and stem rusts affect more and cause 100% loss in yield, while leaf
rust result in 50% loss under severe rust epidemics (Bhardwaj et al., 2019). According to
Huerta-Espino et al. (2014) although, leaf rust causes less loss as compared to other two rusts
but occurs more regularly and in more worldwide regions as compare to the other two rusts. As
of its regular and widespread nature, the damage by leaf rust is greater than stem or stripe rusts
of wheat individually (Kolmar, 1996). There are mainly two effective approaches for
controlling the loss due to rust are; 1. chemical control and 2. genetic control (Asad et al.,
2012). Among them second is environmentally safe approach and followed by breeders, enables
reduction in the use of pesticides and sustainable greater benefits associated (Bariana et al.,
2013). Utilization of multiple rust resistance genes (major and minor genes) in wheat genotypes
is most eco-friendly approach; it could increase the resistance and durability as well as reducing
the evolution of pathogen.
The branch of molecular biology covered the structure, function, evolution, and mapping of
genomes is called genomics. Genomics target to understand the structure of the genome,
including the mapping genes and sequencing the DNA. Nowadays, genomics come up with new
set of tools and techniques which allow the analysis of the whole genome, and represents a
hypothetical shift, by direct study of the genotype and its correlation with the phenotype (Tester
et al., 2010). The field of genomics and its applications develop quickly which can helped in
crop improvement for various biological stresses (including wheat rust). Combination of
conventional techniques with genomic tools and approaches is leading to new genomics-based
approaches to find out solutions for rust resistance in wheat. Next generation sequencing (NGS)
technologies provides high density genetic maps, high collections of markers, high-throughput
genotyping tools, new experimental populations, are strengthening pillars of genomics and its
application for rust resistance in wheat crop (M Perezet al., 2012). Modern advancements in
genomics are accelerating the process of crop improvement for disease resistance through
several ways viz. marker assisted selection, association mapping, gene pyramiding, genomic
selection and ‘breeding by design’ etc. ( Lorenz et al., 2011). Gene-specific DNA markers are
required for pyramiding of rust resistance genes in wheat varieties, but complexity and
enormous genome size make a challenge to draft wheat genome sequences as well as
development of robust DNA markers and (Borrill et al., 2015). Sequence and alignment of
complex wheat genome tells a story of advancement of NGS technologies. As a result PCR-
based markers, including Simple Sequence Repeat (SSR), Sequence Tagged Site (STS),
Cleaved Amplified Polymorphic Sequences (CAPS), and SNP-based KASP (Kompetitive
Allele-Specific PCR) markers have revolutionized wheat improvement programs. In the present
article we enlighten some applications of genomics which could be utilized for gaining rust
resistance in wheat.

Resistance Genes: Identification and Mapping

Genetic resistance can be classified in three groups: 1. monogenic resistance in which resistance
governed by a single gene 2. oligogenic resistance, in this category resistance governed by few
genes and 3. Polygenic resistance in which number of genes involved and resistance is governed
through additive effects. Several approaches are used to postulate the presence of rust
resistance genes in wheat cultivars and breeding lines. In traditional methods plants are screened
against pathogen isolates and further comparison to the resistance/susceptibility reactions is
done with a set of near isogenic lines possessing the commonly occurring genes and wheat
cultivars. The lines having known resistance genes and fixed pattern of resistance/ susceptibility
are identified. These postulations based on the theory of gene for-gene relationship which says
that for each resistance gene in the host, there is a corresponding and specific avirulence gene in
the pathogen and vice -versa (Flor, 1956). Two types of combinations are products of these
reactions; compatible and incompatible which gives rise to susceptible and resistant responses,
respectively. Till date 80 leaf rust resistance genes, 60 stem rust resistance and 83 stripe rust
resistance genes have been catalogued (McIntosh et al., 2017) by crossing bi-parental
populations of wheat (Li et al., 2020). Nature has wonder in their lap and having immense
reservoir of genetic variation. Wild progenitors and related species of wheat contain several
economic traits along with the resistance to biotic stresses and breeders have always deployed
the wellness of nature through interspecific hybridization. Possible application of genomics is to
access genome sequence of wheat and pathogen and mine for the virulence genes in pathogen
and graph possible target genes in wheat and vice -versa. Sequences of previously identified
gens and aligned the sequences for conserved region and homology approaches in landraces as
well as contemporary wheat varieties could find new resistance genes through bioinformatic
tools. Advancement of high throughput genotyping platforms for example i-select 90K SNP
Infinium array (Wang et al., 2014), DArTSeq (www. diversityarrays.com), and genotyping-by-
sequencing approaches (Poland and Rife, 2012) speed up to find the discovery of rust resistance
genes in wheat. Discovery and mapping of rust resistance genes gives rise to robust linked
markers and accelerated the breeder-friendly strategies for wheat improvement.

Mining and isolation of rust resistance genes with genomic tools

Genomic composition of wheat is allo-hexaploid (2n = AABBDD = 42) including diploid
genomes of three inter-related plant genus (Marcussen et al., 2014) and evolved about 8,000 to
10,000 years ago by hybridization between Triticum turgidum and Aegelops tauschii and
became T. aestivum. The sequence alignment of the wheat genome is influential landmark
which makes genomics possible to identify candidate genes, marker trait associations and
enhanced breeding values in genomic selection (GS) studies. The basic information about a
gene and its target can be quickly obtained by using high-throughput genomic platforms and
functional annotation of protein coding genes can be determined by whole genome sequence.
The sequence of relevant coding genes can also be quickly obtained by means of gene
annotation and further used for isolation as well cloning of genes. Investigation and
understanding the structure and evolution of polyploid crop genomes is significant application
of NGS technology. Discovery of huge numbers of genome-wide SNP markers in wheat is
another most important and rapid impact in the area of wheat crop improvement. High
throughput genotyping platforms have illustrated their role in the estimation of genetic
diversity, assembly of the high-density genetic maps, dissecting polygenic traits, with improved
understanding with interactions through GWAS (genome-wide association studies), QTL
mapping, identification and isolation of R genes. Application of genomic tools has expedited the
identification, mapping, cloning and pyramiding of rust resistance genes /alleles in elite lines for
wheat improvement.

Next Generation Sequencing (NGS) technologies

Advancements in NGS technologies have revolutionized genomics studies in almost every crop
plant. Wheat genome size is approximately 16GB which is about five times larger than the
human genome as well as more complex, having high percentage of junk DNA and redundant in
nature. But the NGS technologies make available faster and cheaper means to generate wheat
sequence data as compared to previous sequencing technologies. Combination of both
sequencing technologies and bioinformatics tools makes easy applications of NGS in wheat
improvement programs for rust resistance. Discovery of rust resistance and susceptibility genes
in wheat plant and their possible target genes in rust pathogens could be achieved through
sequencing and bioinformatics tools. Up- regulation, down regulation and suppression of
candidate genes governs resistance in wheat plant to rust pathogens (Panwar et al., 2018)
Allelic variations can be discovered by NGS technologies and genotype-by-sequencing
approaches that covers whole genome by thousands of markers (Liu H. et al., 2014).
Investigation and understanding the structure and evolution of polyploid crop genomes is
significant application of NGS technology. Discovery of huge numbers of genome-wide SNP
markers in wheat is another most important and rapid impact in the area of wheat crop
improvement.

Bioinformatics analysis
NGS platforms are facilitating sequencing projects, and produce huge sequences of DNA, RNA
and proteins, but massive sequences brought new challenges, as more than millions DNA RNA
and protein reads have to be aligned, analyzed and characterized to obtain relevant information.
Genetic maps and expression information produced at a genomic scale through sequencing of
genome by NGS technologies have to be processed in order to gain fruitful information which
can utilized by researchers for crop improvement (M Perezet al., 2012). Therefore, it is
necessary utilization of bioinformatics algorithms and software along with to develop new
bioinformatics tools, which enables the analyses of huge amounts of genome-wide data. Some
databases are given in Table I, for general purpose to make the significant biological
information available to the researchers.
Table I. List of some important bioinformatics databases and repositories of genomic
information, which helps to access necessary information in plants and pathogens (wheat and
rust fungi).

Database Description URL

MAS Wheat Database for MAS in wheat https://maswheat.ucdavis.edu/
GrainGenes A Database for Triticeae and https://wheat.pw.usda.gov/GG3/
Avena
CPGR Phytopathogen genomic resource http://cpgr.plantbiology.msu.edu/
Tair Arabidopsis information resource http://www.arabidopsis.org/
NCBI Biomedical and genomical http://www.ncbi.nlm.nih.gov/
information
Genbank General public sequence repository http://www.ncbi.nlm.nih.gov/genbank/
EMBL General public sequence repository http://www.ebi.ac.uk/embl/
GOLD Repository of genomes databases http://genomesonline.org/cgi-
bin/GOLD/bin/gold.cgi
UniProt Protein sequences and functional http://www.uniprot.org/
information
DDBJ General public sequence repository http://www.ddbj.nig.ac.jp
Gene Index Transcriptome repository http://compbio.dfci.harvard.edu/tgi/
Project
Plantgdb Genomic plant database http://www.plantgdb.org
CropNet Genomic plant database http://ukcrop.net/
Phytozome Genomic plant database http://www.phytozome.net/

DNA based markers

DNA markers based techniques are neutral/free from environmental changes and developmental
stage of the plant because the changes in DNA sequences are free from these factors (Collard et
al., 2005). DNA markers are deployed to find out allelic variations between organisms or
species. In wheat improvement these markers have been extensively deployed to find out allelic
variation in rust resistance/susceptibility governing loci. Applications of genomic tools
supplemented the efficiency of the DNA marker technology. Conventional genetic markers
RFLPs (Anderson et al., 1992) and SSRs (Röder et al., 1998) were used to start the mapping of
genes in wheat. Afterward, more PCR-based markers (CAPS, RGAP, AFLP, EST, STS and
RAPD) also implemented for gene mapping in relation to disease resistance (Bariana et al.,
2001). Identification and mapping of variations in the single base pair is possible after Sanger
sequencing techniques (Wang et al., 1998) and these sequencing approaches have helped to
substitute gel-based marker with robust and bi-allelic SNP markers. Marker assisted selection
enabled rapid detection for the presence of the gene as well as gene pyramiding strategies.
Restriction fragment length polymorphism (RFLP), Sequence Characterized Amplified Region
(SCAR), Sequence Tagged Site (STS), Simple Sequence Repeat (SSR), and Cleaved Amplified
Polymorphic Sequences (CAPS) are several DNA based markers extensively explored as
important tools for wheat improvement for disease resistance (Babu et al., 2020).
Conclusion
Genomics has emerged as a powerful technique in the modern era with advancements in
sequencing platforms. Wheat improvement programs have been accelerated with the application
of genomic tools and breeding for rust resistance is an essential part. Information produced
through genomics and bioinformatics tools and its applications enabled the identification,
mapping, cloning and pyramiding of rust resistance genes /alleles in elite lines and utilization of
this information for wheat crop improvement. Evolution of gene-specific DNA markers could
be possible by using genomic tools, which are essential to introgress the rust resistance genes in
the good agronomic background. Discovery of major and minor genes, characterization and
identification of these genes, inrtgression of these genes in contemporary wheat varieties could
be significant output of genomics and bioinformatics tools in future. Considerable future
challenge would be to identify efficient resistance genes by using genomic tools and their
deployment to achieve rust resistance in wheat crop.
 Disclosure statement
No potential conflict of interest was reported by the authors.

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