FUNCTIONAL GENOMICS OF Oryza sativa:

LARGE-SCALE GENE DISCOVERY AND

APPLICATIONS TO CROP IMPROVEMENT

Project submitted to:

College of Basic Science and Humanities (CBSH), Odisha
University of Agriculture and Technology (OUAT), Bhubaneswar
for the Degree of
Bachelor of Science in Botany

Submitted by:
MONALI PATRA
ROLL NO -BS-21-124

Under the supervision of:

Dr. Jakesh Mohapatra
 Certificate

This is to certify that the content of this project entitled, "Functional genomics
of Oryza sativa: large-scale gene discovery and applications to crop
improvement" by Monali Patra (BS-21-124), student of +3 final year, is the
bona fide work of her, submitted to Department of Botany, College of Basic
Science and Humanities, Odisha University of Agriculture and Technology,
Bhubaneswar, for consideration in partial fulfilment of the requirement of CBCS,
for the Degree of Bachelor of Science (2021-24).

The original research work was carried out by her and successfully completed
under my supervision in the academic year 2021-24. Based on the declaration
made by her I recommend this project report for evaluation.

Signature Signature
HEAD OF THE DEPARTMENT SUPERVISOR
Dr. (Mrs.) Lakshmi Singh Dr. Jakesh Mohapatra
Department of Botany Department of Botany
CBSH, BHUBANESWAR CBSH, BHUBANESWAR

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 Declaration
I hereby declare that the project entitled "Functional genomics of Oryza sativa:
large-scale gene discovery and applications to crop improvement" is an outcome
of my own efforts and is a record of original work done by me under the guidance
of Dr. Jakesh Mohapatra. The project is submitted to the Department of Botany,
College of Basic Science and Humanities, Odisha University of Agriculture and
Technology, Bhubaneswar, for partial fulfilment of the Degree of Bachelor of
Science (2021-2024).

I further declare that the work reported in this project has not been previously
submitted to any other institute or university for any other degree or diploma.

Monali Patra
BS-21-124
+3, 3rd year, 6th Semester
Botany (honours)

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 Acknowledgement
I would like to express my special thanks of gratitude to my teacher and
supervisor Dr Jakesh Mohapatra for providing me with necessary guidance and
encouragement. His valuable insights, advice and suggestions have been crucial
in shaping the project’s direction and content. I am immensely thankful to him
for spending his valuable time to guide me throughout this research project. It
would not have been possible without him.

I would also like to extend my acknowledgement to CBCS for promoting

innovation and creativity among students by including such project in our
curriculum. This project has not only taught me new scientific concepts but has
also helped me enhance my knowledge other than college textbooks.

I am grateful to my friends who contributed ideas and perspective that enriched

the project. Moreover, I am thankful to my parents for their unwavering support.
Their belief in my abilities kept me focused and motivated throughout the project
work.

I wish to express my sincere gratitude to all those individuals for mentoring and
supporting me in completing this project work within limited time frame.

Monali Patra
BS-21-124
+3, 3rd year, 6th Semester
Botany (honours)

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 Abstract
Rice has an old and rich cultivation history and is more than just a food crop. With immense
economic value, a highly self-pollinated crop, rich in genetic diversity, and one of the most
significant crops for global food security and a relatively small genome size among cereals
makes it a focal point for scientific investigations. Rice genomes have become a valuable
genomic model among cereals for functional genomics study.

Rice breeding is an on-going task for feeding the ever-increasing population. With the rapid
accumulation of knowledge on rice genomics, a plethora of innovative technologies have been
used for rice improvement, yielding new insights on rice breeding methods. Rice genomics has
been a transformative influence on rice biological research and contributes significantly to rice
breeding, making rice a good model plant for studying crop sciences.

Thanks to the rapid development of functional genomic technologies, over 2000 genes
controlling important agronomic traits have been cloned, and their molecular biological
mechanisms have also been partially characterized.

Since the completion of rice reference genome sequences, tremendous progress has been
achieved in understanding the molecular mechanisms on various rice traits; e.g. many of the
genes control agriculturally useful traits such as yield, grain quality, resistances to biotic and
abiotic stresses, and nutrient – use efficiency, thus have potential utility in crop genetic
improvement

These achievements made in functional genomics research will greatly facilitate the
development of Green Super Rice.

This research work will cover advances in rice functional genomics research in past years,
including a summary of functional genomic platforms, new developments in this field and the
sequencing and functional research of genome of rice will be briefly reviewed. Future
challenges and prospects of rice functional genomics research is also discussed.

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 Contents

1. Introduction
2. Functional genomics: An overview
3. Functional genomics of Oryza sativa
4. Recent development of platforms for rice functional genomics research
5. Procedure for rice functional genomics research
6. Genes and their regulatory networks for important agronomic traits
7. Implementation of rice functional genomics research to crop improvement
8. Challenges faced by rice functional genomics research
9. Perspectives on future rice genomics research
10. Conclusion
11. References

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 1. Introduction
Rice: An Introduction
▪ Scientific Name: Oryza sativa (Asian rice) or Oryza glaberrima (African rice)
▪ Family: Poaceae
▪ Number of Chromosomes:12
▪ Genome Size: 420 Mb (Megabase)
▪ Subspecies: Japonica or Sinica, Indica, Javanica now known as (tropical
japonica)
▪ Pendulous inflorescence
▪ Edible part: Grain (caryopsis)

Why Genomic Research in Rice Important?

Rice (Oryza sativa), an important cereal, provides staple food for more than half of the global
population. Thereafter, Rice cultivation is one of the most important agricultural activities on
earth, with nearly 90% of it being produced in Asia. It supplies more than 50% of calories
consumed by the world population. Nevertheless, the growth in annual rice production is no
longer keeping pace with the increasing number of human consumers. Also, various factors
like Climate change and food security have a strong impact on the survival of rice crops.
Therefore, the breeding of rice varieties with high yields, high quality, and high stress
resistance to ensure food security remains an on-going task. From the rice that acquires 2.5
Tons of irrigation water to produce just a kilogram of polished rice, we must revolutionize rice
breeding to become more resilient to global warming and extreme climate change.
The Green Revolution, started more than four decades ago in response to the food crisis in
many parts of the developing world. Although the world food supply has doubled since the
onset of the green revolution, the task of providing food for an additional 2.5 billion people
would have to be accomplished without a significant increase in the area under cultivation. In
fact, the problem is compounded by the loss of land to urban development, degradation of
productive land, and the decreasing yield of several conventional varieties which contributed
immensely towards the green revolution. Besides, every year all over the world, crops worth
several million dollars are lost due to damage caused by various biotic and abiotic stresses.
Although over the years plant breeders have contributed significantly to increased food
production, the time has come to synchronize plant breeding with biotechnology leading to
precision breeding for rice improvement by providing breeding lines with desired traits such

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as resistance against pests and pathogens, tolerance to salinity and drought as well as improved
nutritional quality.
Translation of functional genomics into innovations will contribute significantly to fast-
tracking new rice varietal development.
At present the emphasis of rice functional genomics research is discovery of new genes and
annotation of gene function. The availability of a complete and high-quality map-based
sequence s provides required ingredients for functional genomics and molecular breeding
programs to improving rice productivity. Two remarkable breakthroughs in rice breeding-
dwarf breeding and hybrid rice breeding, have dramatically enhanced grain yield and has set
the trend for rice breeding throughout the world.

2. Functional genomics: an overview

Genomics
Genomics is an interdisciplinary field or the branch of molecular biology concerned with the
structure, function, evolution, and mapping of genomes. A genome is an organism's complete
set of DNAs, including all its genes as well as its hierarchical, three-dimensional structural
configuration.

It attempts to understand the content, organization, function, and evolution of genetic

information contained in whole genome.

It focuses on studying all the DNA of an organism i.e. its genome. Such work includes
identifying and characterising all genes and functional animals in an organism’s genome as
well as how they interact.

In contrast to genetics, which refers to the study of individual genes and their roles in
inheritance, genomics aims at the collective characterization and quantification of all genes of
an organism, their interrelations and influence on the organism.

Genomics can be broadly defined as the understanding of all genes and their encoded biological
function in an organism in totality. It has emerged as an integrative discipline that provides the
means to understand biological complexity and to solve difficult practical problems.

Genomics also involves the sequencing and analysis of genomes through uses of high
throughput DNA sequencing and bioinformatics to assemble and analyse the function and
structure of entire genomes. Advances in genomics have triggered a revolution in discovery-
based research to facilitate understanding of even the most complex biological systems.

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Genome Sequencing Projects

For the past decade, genome sequencing projects, either completed or begun, have produced
such excitement in biological sciences that many young students have been attracted to the
discipline of genomics.

The genome of the first eukaryote — Saccharomyces cerevisiae — was completed (Williams,
1996), followed by several initiatives to sequence other model organisms — Drosophila,
nematode, Arabidopsis, and human. Most of the microorganisms whose genomes have been
completely sequenced are problematic pathogens, such as Haemophilus influenzae. A rough
draft of the human genome was completed by the Human Genome Project (sequenced the entire
genome for one specific person) in early 2001.

The advent of these technologies resulted in a rapid intensification in the scope and speed of
completion of genome sequencing projects.

After an organism has been selected, genome projects involve three components: the
sequencing of DNA, the assembly of that sequence to create a representation of the original
chromosome, and the annotation and analysis of that representation.

What is functional genomics?

Functional genomics is a field of molecular biology that deals with understanding the gene
function and interaction to be able to establish a relationship between the organism’s genome
and its phenotype.

It is a major branch of genomics concerned with sequencing the genomes of various organisms.
The knowledge of full genomes has created the possibility for the field of functional genomics,
mainly concerned with patterns of gene expression during various conditions.

Functional genomics is largely experiment based with a focus on gene function at the whole
genome level using high throughput approaches. The most important tools here are microarrays
and bioinformatics.

It attempts to make use of vast wealth of data produced by genomics to describe gene (and
protein) function and interactions.

It focuses on the dynamic expression of gene products in a specific context at a specific

developmental stage or during a disease.

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Functional genomics is used to better understand various processes related to genomic
sequence, gene regulation, transcription, translation, gene expression, protein- protein
interaction encoded protein function including study of coding and non-coding transcription,
protein translation and attempts to answer the question about function of DNA at levels of gene,
RNA transcripts and protein products.

Goals and Applications of Functional Genomics

The goal of functional genomics is to understand the function of larger numbers of genes or
proteins, eventually all components of a genome. A more long-term goal is to understand the
relationship between an organism’s genome and its phenotype. It helps in the understanding of
dynamic properties of an Organism at cellular levels. It provides more complete picture of how
biological function arises from information encoded in an Organism genome.

Application:

▪ Agriculture:

o Sequencing crop plant genomes

o functional genomics being used to improve crops that are resistant to biotic and
abiotic stress and have improved nutritional quality

▪ Disease diagnosis and drugs discovery

▪ Gene expression study and genetic discovery for useful traits

▪ Used to develop new source of bioenergy; researchers using functional genomics to

engineer microorganisms that can produce biofuels more efficiently.

▪ Functional genomics being used to study the impact of environmental pollutants on

organisms. By understanding how genes are affected by pollutants, researchers can
develop new methods for environmental remediation.

3. Functional genomics of: Oryza sativa

History
Although rice is such an important food crop, it has only a relatively short history as a model
organism for biological investigation. Before the 1980s, rice research was mostly done by
countries where rice is the staple crop and where sufficient infrastructure existed to conduct

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research. This limited most of the rice research to a few countries or international agricultural
research centres. There was hardly any private interest in rice research. In many respects, rice
was an “orphan” crop in terms of research investment. This picture changed in the mid-80s
when advances made in plant molecular biology and gene transfer techniques were seen as
promising avenues to convert biological discoveries to applications. The Rockefeller
Foundation, a long-time supporter of international agricultural development, made a strategic
decision to support the application of molecular biology to solve rice production problems. The
beginning of the International Rice Biotechnology Program in 1985 launched the “molecular
revolution” in rice science. For the next 16 years (1985 – 2001), we witnessed the blossoming
of basic research in rice. There was a growing sense that many biological problems limiting
rice production required a comprehensive understanding of multiple genes and their
interactions with the environment.

Rice, with the smallest genome size among major cereals and crop plants, is seen as the obvious
target for genomics research. The Rockefeller Foundation sponsored a meeting in 1997 at the
International Congress of Plant Molecular Biology in Singapore to discuss the merits and
feasibility of sequencing the entire rice genome. At that time, physical mapping using YAC
and BAC libraries was progressing rapidly, producing the critical resources for sequencing.
However, with the available sequencing technologies at that time, completing the rice genome
would cost about $200 million over 10 years, a formidable undertaking for any individual
institute or program. Thus, a sensible way to achieve the task was to launch an international
collaboration. This led to the formation of an International Rice Genome Sequencing Project
(IRGSP) in 1998 under the coordination of the Rice Genome Program of Japan. The IRGSP
represents a consortium of multiple countries collaborating to sequence the rice genome. In the
initial stage, over 10 countries pledged to take part to sequence individual chromosomes. The
IRGSP was a unique undertaking that has galvanized government commitment and it
subscribed to the principle of releasing all sequence information to the public. Since then, rice
genome sequencing has been the topic of scientific drama and excitement involving the private
and public.

The public initiative has been largely a key factor to leverage the contribution of rice genome
data from the private sector. Together, this has led to the availability of genome sequence of
two rice subspecies (japonica and indica), a favourable situation for public researchers working
not only on rice but on other plant species.

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With a genetic blueprint in place, we ask, what are the new opportunities to understand gene
functions and their interactions? What are the most efficient approaches to find genes of
agronomic importance? Why is international collaboration necessary for gene discovery in
rice? How can we turn gene discovery into practical products?

Oryza sativa as a Model Genetic System

Rice is a model plant for genomics research of monocotyledons that means it has become a
reference of monocot plant for functional genomic research and it has emerged as a model in
plant science research because of its many positive attributes for genetic studies.

Rice has a simple genetic system (diploid and disomic inheritance) and one of the most
amenable transformation systems among grasses.

The small genome size and relatively low amount of repetitive sequences make rice a tractable
system for whole-genome sequencing.

Other than these technical advantages, an important attribute is the great store of genetic
diversity in the genus Oryza.

With a long history of cultivation and selection, and a large and heterogeneous geographical
niche, the wealth of phenotypic and genetic variation in all aspects of plant life, particularly
genes controlling agronomically important traits, is enormous in rice. Below, we highlight the
genetic diversity available in rice that is relevant for functional genomics research.

Rice Genome

Genome size of rice is 430Mb nucleotide and this consists to about 32,000 to 50,000 genes,
spread into 12 chromosomes.

Rice (Oryza sativa L.) is the first crop to have its genome completely sequenced (in 2002).

Rice is a perfect model plant for plant genomics research because of its relatively small genome
size, well-established and highly efficient transformation system, extensive genetic resources,
and synteny with the sequences of other cereal crops.

A complete, high-quality rice reference genome sequence serves as the basis for genomic
analysis, providing an unprecedented opportunity to systematically characterize the molecular
functions of genes.

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As the model cereal crop, large scale sequencing of rice genome plays quite important role
both in theoretical research and practical application in rice breeding, leading to another way
to resolve world food crisis.

The most widely cultivated rice, O. sativa, has two subspecies — indica and japonica —
that provide the main gene pool for rice breeding. There is a third minor group called
“tropical japonica” (also called javanica) that is common in Southeast Asia. Japonica rice is
generally adapted to the temperate area, whereas indica rice is planted in the tropics. Indica
rice is the most widely grown type, constituting about 80% of world rice production. the main
agronomic characteristics of the main rice types is summarized in Table I. There is much
overlap in agronomic characteristics between the two subspecies

Now that the genome sequence of these two subspecies is available, this will open up
opportunities to understand the molecular basis of japonica and indica sterility as well as hybrid
heterosis.

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Table II lists some of the species that have been used in rice improvement programs or genetic
studies (Brar and Khush, 2002).

It illustrates the diversity of the domesticated and wild germplasm that forms the basis for gene
discovery.

There are examples of useful genes that have been extracted from wild germplasm for breeding.
One well-known example is the grassy stunt resistance gene obtained from O. nivara. This
resistance gene has been used in nearly all rice cultivars developed for the tropical lowland and
it remains effective in conditioning resistance to the dominant strain of the grassy stunt virus
until now (Khush, 1977). The first cloned disease resistance gene, Xa21, was also originally
introgressed from the wild rice O. longistaminata from an accession in Mali, Africa. After many
years of genetic and breeding work, Xa21 was put into an agronomically acceptable
background that has facilitated its molecular cloning and use in rice production.

Rice genomics studies can be classified into three stages: structural genomics, functional
genomics, and quantitative genomics. Structural genomics refers primarily to genome
sequencing for the construction of a complete map of rice genome sequence. This is

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fundamental for rice genetics and molecular biology research. Functional genomics (gene
identification and functional characterization) aims to decode the functions of rice genes.
Quantitative genomics is large-scale sequence- and statistics-based research to define the
quantitative traits (quantifying the genetic effects) and genetic features of rice populations.

Procedure for rice functional genomics research: Tools and techniques

Approaches for Gene Identification: Newly Developed Tools for Gene Identification

Since the completion of whole-genome sequencing in rice, various functional genomics

platforms have been established in the past decade, including collection of germplasm
resources and generation of mutant libraries, full-length cDNA libraries, gene expression
microarrays, and RNA-sequencing (RNA-seq) technologies for expression profiling. Platforms
of metabolomics, proteomics, and phenomics have also been gradually established and
improved, and corresponding platforms of bioinformatics analysis and databases have also
been set up in rice.

4. Resources For Rice Functional Genomics Research

Genetic stocks harbouring the traits of interest and associated variation are the essential
materials for revealing biological function of a sequence. A range of genetic stocks can be used
to identify or validate gene function. These include mutants, segregating mapping populations,
backcross lines, well-characterized breeding lines and traditional genetic germplasm.

Rice Genetic Germplasm/Germplasm Resource

Rice is rich in germplasm resources, including naturally occurring and artificially modified
germplasms. Rice gene banks exist to conserve genetic diversity by collecting large numbers
of rice samples, aiming to represent the broad range of diversity within rice species.

Mutant Generation:

Various techniques are employed to generate rice plants with mutations in specific genes. This
can involve techniques like mutagenesis (insertional mutagenesis, chemical/physical
mutagenesis, or gene-editing mutagenesis) where random mutations are induced throughout
the genome, or transposon insertion, where a transposable element is inserted into a specific
gene, disrupting its function, and creating rice plants with mutations in specific genes. Multiple
rice mutant libraries generated by Insertional mutagenesis based on transfer DNA (T-DNA)

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insertions and transposon or retrotransposon tagging, Ds/dSpm tagging, Tos17 tagging, and
chemical/irradiation mutagenesis have been developed.

To find the gene function encoded by each gene or set of genes requires special kinds of genetic
resources and methodologies. The platform has three basic components: genetic resources,
phenotypic/biological evaluation, and genomic tools. These three components are integrated
by bioinformatics. Furthermore, data production eventually leads to the discovery and
validation of gene functions.

Rice functional genomics research involves a series of procedures that aim to study the genetic
makeup and molecular functions of rice The methodology of rice functional genomics research
involves a comprehensive approach to understanding how genes influence rice's biological
functions. Rice functional genomics research employs a multi-step process. Here's a
breakdown of the key steps:

Genome sequencing: Scientists sequence the rice genome to identify genes and their locations.
This allows them to identify all the genes present and their precise locations on the
chromosomes. This involves determining the complete set of genes in the rice genome, which
is achieved through a combination of techniques such as shotgun sequencing, BAC (bacterial
artificial chromosome) sequencing, and EST (expressed sequence tag) sequencing.

Gene annotation: Once the genome sequence is available, the next step is to annotate the
genes, which involves identifying the gene structure, function, and other regulatory elements
such as promoters and enhancers. This is typically done using computational tools and
algorithms.

Phenotypic analysis to observe the physical and biochemical effects of mutations: The rice
plants with mutations are then carefully studied to observe any physical or biochemical changes
in their characteristics (phenotypes) compared to non-mutated plants to see how altered genes
affect rice growth, development, or stress tolerance. These phenotypic changes can provide
clues about the function of the mutated genes.

Gene expression profiling to measure gene activity levels

Scientists measure the gene expression levels of various genes in both mutant and non-mutant
plants to determine how mutations influence gene activity. This helps them understand how
the mutations affect the activity of different genes and how these changes might be linked to
the observed phenotypic variations.

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Gene expression profiling techniques such as microarray and RNA-seq are used to analyze the
expression of thousands of genes under different conditions, providing insights into gene
function and regulation.
Transcriptome and proteome analysis: To further understand the molecular functions of
genes, researchers analyze the transcriptome (the set of RNA molecules in a cell) and proteome
(the set of proteins in a cell). Techniques such as RNA-seq and mass spectrometry are used to
study gene expression and protein abundance, respectively.
Functional genomics approaches: Researchers use various functional genomics approaches
to study gene function in rice, such as gene knockout (using CRISPR/Cas9), overexpression,
and gene editing techniques.

Genetic and molecular marker development: Developing genetic and molecular markers is
essential for studying the genetics of rice. These markers can be used for genetic mapping,
breeding, and genomic selection. Techniques such as EST-based marker development, SSR
(simple sequence repeat) marker development, and genome-wide association studies (GWAS)
are used to develop and validate markers.
QTL mapping and genome-wide association studies: To study the genetic basis of traits
such as yield, quality, and stress resistance in rice, researchers perform QTL mapping
(identifying regions of the genome with significant genetic variation) and GWAS. These
studies help in identifying specific genes and genetic factors responsible for these traits.

Bioinformatic analysis to interpret vast datasets and identify candidate genes: The vast
amount of data collected throughout the research process is analysed using powerful
computational tools. Researchers use computational tools to analyse vast datasets and identify
genes potentially responsible for the observed phenotypic changes. This bioinformatic analysis
helps identify genes that are differentially expressed between mutant and non-mutant plants
and statistically associates them with the observed phenotypic changes.

Functional validation and characterization: Once the genes and genetic factors responsible
for specific traits are identified, researchers need to validate their function and characterize
them further. This may involve overexpressing the genes in transgenic plants or knocking out
the genes using gene editing techniques.

Gene overexpression or silencing to validate gene function: Through techniques like gene
overexpression or gene silencing, scientists confirm if the identified genes are indeed
responsible for the observed phenotypes. Researchers use techniques like gene overexpression
or gene silencing to confirm the function of the identified genes. Overexpression involves

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introducing extra copies of a gene to see if it enhances the associated trait, while silencing
involves suppressing gene expression to observe if the corresponding trait is affected. These
confirmatory steps solidify the link between specific genes and the phenotypic characteristics
of rice plants.

Genetic engineering and breeding: Using the knowledge gained from functional genomics
research, researchers can develop improved rice varieties with enhanced traits such as yield,
quality, and stress resistance. This involves genetic engineering techniques like CRISPR/Cas9
and traditional plant breeding methods.

Throughout the entire process, bioinformatics tools and computational biology approaches are
used to analyze and interpret the vast amounts of data generated in rice functional genomics
research. These tools help in identifying patterns, relationships, and functional elements within
the genome.

In summary, rice functional genomics research involves a combination of genome sequencing,

gene annotation, expression profiling, transcriptome and proteome analysis, functional
genomics approaches, genetic and molecular marker development, QTL and GWAS,
functional validation and characterization, genetic engineering, and bioinformatics analysis.
This multidisciplinary approach helps in understanding the genetic makeup and molecular
functions of rice, ultimately leading to the development of improved rice varieties for better
food security and agricultural practices.

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Figure 1. Schematic diagram illustrating the procedure for rice functional genomics
research.

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Figure 2. An overview of functional genomics approach through diagrammatic
representation

5. Implementation of rice functional genomics research to crop

improvement: Genes underlying important agronomic traits
in rice and Green Super Rice Breeding
Rice functional genomics research is aimed at exploring the genes and molecular regulatory
networks of agronomically important traits and applying them in varietal improvement, for
traits like yield, quality, disease and pest resistance, nutrient-use efficiency (NUE), abiotic
stress resistance, and reproductive development.
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At present, a total of 2294 rice functional genes have been identified.

Critical rice genes underlying the important agronomic traits are briefly summarized and genes
newly coupled with their regulatory pathways are emphasized that have a profound impact on
rice functional genomics and crop improvement.

Features for some of the genes is discussed briefly, especially those showing potential
applications in crop genetic improvement.

Yield

Rice grain yield is a complex trait determined by the number of panicles, number of grains per
panicle, and grain weight. The formation of grain yield is mainly dependent on the genes that
control tillering and panicle development.

MOC1 encodes a plant-specific GRAS family protein, functioning to initiate axillary buds, and
promotes tiller outgrowth in rice. Some genes involved in the biosynthesis and signaling of
strigolactones, such as D3, D10, and D27, also influence the tiller number of rice.

The number of grains per panicle is determined by length of panicle development and the rate
of spikelet formation. For example, LAX1, LAX2, and SPA together enhance the formation of
lateral meristems (Tabuchi et al., 2011), and FZP is required for the establishment of floral
meristem identity by inhibiting the formation of axillary meristems.

Other genes such as LAZY1, LPA1, and PROG1 participate in the regulation of rice tiller angle
and influence rice plant architecture.

Important progress has also been made in identification of genes related to grain size/weight.
For example, GS3 is a major QTL for grain length and weight.

The yield of rice is primarily determined by the number of effective panicles, the number of
grains, and grain weight.

Grain quality

Rice grain quality consists of appearance, cooking, milling, and nutritional quality. It is
conceivable that better grain quality means higher profits for rice growers. To date, many vital
genes underlying grain quality have been harnessed extensively for variety improvement. For
example, Waxy (Wx), encoding granulebound starch synthase I, is responsible for amylose
synthesis that determines eating and cooking quality (ECQ). Grain shape is a yield trait as well
as a factor that determines the appearance quality of rice grains. Grain length gene GS3 and

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width gene GW5 are the major genes for grain shape, and play important roles in the regulation
of the rate of head rice. Eating and cooking quality of rice grain is generally determined by
three physicochemical indices: amylase content, gel consistency, and gelatinization
temperature (GT). The waxy gene regulates the amylose content in the endosperm, and
ALK/ssiia and RSR1 control the GT. The rice grain fragrance is mainly controlled by Badh2,
which encodes glycine betaine aldehyde dehydrogenase. Rice grain nutritional quality mainly
comprises the grain protein content (GPC), and contents of fats, amino acids, vitamins, and
other micronutrients.

Heading/maturity date

Heading date is an important trait that determines the adaptability of rice varieties to different
geographic regions and planting seasons, and it usually affects the grain yield. Rice is a
facultative short-day (SD) plant with diverse photoperiod sensitivity (PS) and possesses
complicated flowering pathways to regulate heading date.

Resistance to biotic stress

Biotic stress refers to the damage caused to crops by other living organisms, such
as bacteria, viruses, fungi, parasites, beneficial and harmful insects, weeds, and cultivated
or native plants. The damage caused by these various living can reduce the yield of the
cultivated plants and affect the quality of the harvested products. There is greater difficulty in
controlling biotic stresses in an experimental context compared to abiotic stress.

Losses to pests and disease in crop plants continue to pose a significant threat to agriculture
and food security. Agriculture became increasingly reliant on synthetic chemical pesticides to
provide control of pests and diseases. This reliance on chemical control is becoming
unsustainable. Pesticides tend to have a limited lifespan due to the emergence of resistance in
the target pests, and in many cases to have negative impacts on biodiversity, and on the health
of agricultural workers and even consumers.

Resistance to diseases

The yield and quality of rice are severely threatened by pathogens and insect herbivores
throughout the growing season. The most destructive rice diseases include rice blast (caused
by the fungus Magnaporthe oryzae), bacterial leaf blight (caused by the hemibiotrophic
bacterium Xoo), bacterial leaf streak (caused by X. oryzae pv. oryzicola (Xoc), sheath blight
(caused by the necrotrophic fungus Rhizoctonia solani), and rice false smut (caused by the
fungal pathogen Ustilaginoidea virens) resulting in serious yield losses worldwide.

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Functional genomics research of rice disease resistance has been mainly focused on the
resistance against bacterial blight caused by the bacterial pathogen Xanthomonas oryzae pv.
oryzae and blast caused by the fungal pathogen Magnaporthe oryzae.

To date, at least 40 QTLs for bacterial blight resistance have been reported in rice.

Resistance to Insect Pests

The major insect pests of rice include plant hoppers, leafhoppers, striped stem borer, yellow
stem borer, leaf folders, and gall midge. Several insect-resistant germplasms have been found
from both cultivated and wild rice species. Great progress has been made in identifying
resistance genes to brown planthopper (BPH), with 31 genes genetically mapped. Among these
genes, Bph3 (Bph17), Bph14, Bph26 (bph2), and bph29 have been cloned through map-
based cloning. Bph14 is the first cloned gene conferring resistance to BPH.

Resistance to abiotic stress

Abiotic stress is the negative impact of non-living factors on the organisms such as temperature,
sunlight, wind, salinity, flooding and drought. Plants are exposed to many stress factors, such
as drought, high salinity, which reduce the yield of the cultivated plants or affect the quality of
the harvested products. As a sessile organism, rice is able to cope with abiotic stresses such as
extreme temperatures, drought, and ionic stress (soil salinity and heavy metals).

Due to the implications of climate change and elevated threat of abiotic stresses (i.e. drought
and heat) are likely to contribute to increased plant pathogen susceptibility.

In the past decades, more than 100 stress-responsive genes and QTLs have been identified in
rice through either the forward or reverse genetics approach. Protein kinases are involved in
the mediation of abiotic stress responses.

OsMAPK5 was functionally characterized as a stress-responsive MAPK (mitogen-activated

protein kinase) that positively regulates the tolerance to abiotic stresses such as drought, salt,
and cold but negatively regulates the tolerance to biotic stresses such as fungi and bacteria.

Transcription factors also play important roles in regulating abiotic stress tolerance of rice.

Nutrient use efficiency

To date, great progress has been achieved in uncovering the mechanisms of how rice senses
and responds to external nutrients. Rice absorbs and transports ammonium nitrogen and nitrate
nitrogen through ammonium transporters (AMTs) and nitrate transporters (NRTs),

23
respectively. There are more than 80 NRT1/ PTR, four NRT2, and two NAR2 members in rice,
but the functions for only a few of them have been clearly characterized.

On the one hand, excessive use of chemical fertilizers composed mainly of the macronutrients
nitrogen (N) and phosphorus (P) has made an outstanding contribution to global food security.
On the other hand, it has also given rise to ever-growing environmental pollution. Hence, the
cultivation of crops with high nutrient use efficiency (NUE) not only guarantees food security
but also promotes agricultural sustainability.

Reproductive development

Male sterility is a major subject of research on reproductive development in rice for both basic
biology and breeding application.

Cytoplasmic male sterility (CMS), a condition under which a plant is unable to produce
functional pollen, is widespread among higher plants. In plants, during the reproductive
development process, lipid metabolism plays an important role in pollen wall and anther cuticle
development. Any disturbance in these two lipid layers (anther cuticle and pollen exine) results
in pollen abortion, which will lead to male sterility. CMS systems represent a valuable tool in
the production of hybrid seed in self-pollinating crop species, including maize, rice, cotton, and
a number of vegetable crops. Hybrid seeds are widely used in vegetable crops for advantages
such as high yield and improved resistance, which require the participation of male (stamen)
and female (pistil) reproductive organs. Male- or female-sterile plants are commonly used for
production of hybrid seeds or seedless fruits in vegetables.

A brief overview of the key genes identified in recent years that are relevant to hybrid rice
breeding, are discussed below.

There are several major types of cytoplasmic male sterility (CMS): CMS-WA (wild abortive),
CMS-HL (Honglian), and CMS-BT (Boro II). Six CMS genes (orf79, orfH79, WA352a,
WA352b, WA352/WA352c, and WA314) from the mitochondrial genome and four Rf genes
(Rf1a/Rf5, Rf1b, Rf4, and Rf6) from the nuclear genome have been identified in CMS/ Rf
systems (Tang et al., 2017). Luo et al. (2013a) identified a CMS-WA gene WA352, which
interacts with nuclear-encoded mitochondrial protein COX11 and thereby triggers pollen
abortion, resulting in male sterility. Further study indicated that two fertility-restorer genes,
Rf3 and Rf4, restore the fertility through inhibiting the expression of WA352 (Luo et al.,
2013a). ORFH79 impairs mitochondrial function and then confers male sterility in CMS-HL.

24
Green super rice (GSR)

Green Super Rice (GSR), proposed by Prof. Qifa Zhang provides a promising strategy for
sustainable agriculture and will be boosted by the breakthrough achievements of rice functional
genomics.

To realize ‘‘resource-saving and environment-friendly’’ modern agriculture, a notion of green

super rice (GSR) was proposed, such that the new rice varieties should possess resistances to
multiple insects and diseases, high water and NUE, and high yield and good grain quality. An
international coordinated project named ‘‘RICE2020’’ in rice functional genomics research
was proposed with the mission to decode the rice genome and apply the findings of functional
genomics research to developing GSR. Rice functional genomics research has provided
abundant gene resources for developing GSR.

For example,

▪ Xa21 and xa13 can be used to prevent rice bacterial leaf blight

▪ Pigm and Bsr-d1 confer broad spectrum resistance to rice blast and can be used in
disease resistance breeding

▪ Brown planthopper resistance gene Bph14 was identified in Oryza minuta indicating
that there are useful genes in wild rice.

▪ Other useful alleles, such as brown planthopper resistance gene BPH3, salt tolerance
gene HKT2, submerge tolerance gene Sub1, and high-temperature tolerance gene
OsTT1, were identified from local varieties. These genes have great potentials to be
used in rice breeding for stress tolerance.

▪ Genes for nitrogen-use efficiency NRT1.1B and DEP1 and cold tolerance gene
COLD1 can be applied to enhance the cold tolerance and nitrogen-use efficiency in
rice breeding.

These findings presented a novel strategy for developing high-yield rice and improving the
other important agronomic traits through manipulating the regulatory modules identified in
rice, thus further contributing to the GSR breeding.

25
 6. Challenges faced by rice functional genomic research
Rice functional genomics research plays a crucial role in ensuring global food security.
However, despite significant advancements, several challenges persist:
Gene function annotation: Accurately predicting gene function based on DNA sequence data
remains a challenge.
Gene redundancy and complex interactions: Many traits are controlled by multiple genes with
overlapping functions, making it difficult to isolate the effects of individual genes.
Environmental influences: Gene expression and function can be significantly influenced by
environmental factors, complicating the analysis of gene function in controlled laboratory
settings.
Translating research to breeding: Effectively integrating functional genomics knowledge into
traditional breeding programs to create new rice varieties with desired traits remains a
challenge. Uncertainties about the edited plant’s performance in natural environment
conditions might exist.

Extensive allelic variation associated with agronomically important traits has long been held
as central to rice breeding. To date, approximately 20 genomes of Oryza species and thousands
of cultivar varieties have been sequenced, but most genomic data come from indica and
japonica. For this reason, some valuable variations that exist in other Oryza species or other
subgroups of O. sativa are inevitably ignored. Hence, to capture all variations, focus on
variations in other Oryza species and other subgroups of O. sativa should also be made. For
this purpose, we should further consolidate all the pan-genome works to encompass more
varieties from different subgroups of O. sativa or their progenitors. Meanwhile, we should
improve the annotation quality of the rice pan-genome by using more powerful algorithms and
integrating more data.

De novo domestication: Rice domestication provides a model system for studying adaptation
and is of substantial interest for crop improvement. On one hand, domestication of rice has
enriched its yield-related traits, such as high yield, ideotype, and easy harvest. On the other
hand, it has also led to a genetic bottleneck that has reduced genetic diversity and stress
resistance. In light of this, an efficient transgenic and genome editing system, as well as a high
quality genome assembly, will be critical for de novo domestication.

26
 7. Perspectives on future rice genomics research
As mentioned earlier, rice has been firmly established as a model organism for both basic and
applied research on plant genomics. Previous reviews have summarized advances in some
aspects of rice genomics, but this review chronologically and comprehensively summarizes
progress in rice genomics over the past 20 years and presents many rich and contemporary
views on important scientific problems based on the latest research findings. In the future, the
main task of rice genomics is still to dissect the function of every gene in the rice genome and
even of every nucleotide in the DNA sequence. Rapid technological advances are making it
possible to cut the Gordian knot in rice genomics, providing insight into rice adaptability to the
environment, rice plasticity, and rice regeneration. In addition, accumulated multi-omics data
have provided fresh and integrated views in rice genomics research. Studies designed to solve
genetic mysteries will be further strengthened by the application of more omics technologies.
Meanwhile, increasing knowledge from basic research will be conducive to undertaking
applied research on rice genomics. For example, understanding the genomic architecture of
rice heterosis will hasten the process of hybrid rice breeding through whole-genome genomic
selection and hybrid performance prediction. In addition, more new technologies such as
genome editing and machine learning approaches will be integrated to revolutionize methods
of rice breeding. In the end, we firmly believe that the rapid development of rice genomics will
accelerate the process of breeding super rice with high yield, high quality, high stress
resistance, and high NUE, so as to cope with the challenges of population growth and climate
change.

27
 8. Conclusion
Recognizing the rapid progress in plant genomics, this chapter does not attempt to provide a
comprehensive review of the past and on-going work related to rice genomics. Rather, we
provide a snapshot of the current status of the field and focus on the framework needed to make
gene discovery relevant to plant improvement. Rice is special because of its botanical history
and biological attributes. The completion of the rice genome sequence is a major milestone in
plant science; however, for the sequence information to be useful, it must be converted into a
format that allows investigation with a variety of genetic materials. Functional genomics
concerns the development of a resource platform by which the function of every gene and its
interaction with other genes can be characterized and understood in an efficient manner. The
excitement in functional genomics lies in the creative application of high-throughput tools to
probe into the biological complexity of genetic networks and biological processes. In the post-
genome-sequencing era, we may save efforts in gene cloning in the conventional sense, but
there is no substitute for detailed studies involving genetics, physiology, and biochemistry if
we are to understand the biological functions of genes. Because rice is a major food crop in
many societies and cultures, discovering rice genes and having access to the knowledge
inevitably has a social impact. The yield increase in the major cereals has largely come from
genetic improvement of the plants supported by improved agronomic practices.

In this chapter, we have highlighted the resources available and being developed to accelerate
gene discovery and we also speculated on the future adoption of new knowledge and tools in
plant improvement programs. Developing countries have a unique role to play in rice functional
genomics because they are rich in genetic resources and agronomic knowledge of the rice crop.
The benefits of engaging developing countries in gene discovery are obvious but this requires
the will and efforts to support the partnerships. The power of bringing together a full range of
expertise in basic and applied research is enormous. The move toward more international
collaboration in rice functional genomics is very encouraging. Success in this effort will rest
upon the continuing goodwill and determination of the research community to sustain the spirit
of collaboration to channel gene discovery into practical crop improvement.

28
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