October 2013

Seeking Sustainable Protein Sources for Asia andSub-Saharan Africa
ICRISAT researchers are using Illumina sequencing to develop disease-resistantpigeonpea varieties.

Recognizing that its technology could
play a critical role in alleviating global
hunger, malnutrition, and poverty, Illumina
created the Agricultural Greater Goodinitiative. Each year, Illumina awards
Greater Good grants to agriculturalresearch organizations that are focusedon identifying and breeding plants and animals that will increase
the sustainability, productivity, and nutritional density of crop andlivestock species. Under the grants, Illumina sequencing andgenotyping reagents are provided free of charge.

ICRISAT2013 Illumina Greater Good
Initiative Award Winner

Introduction
Founded in 1972, the International Crops Research Institute for theSemi-Arid Tropics (ICRISAT)1 is a non-profit, non-political organization
conducting agricultural research for development in Asia and subSaharan Africa. It’s an area of the world that is in desperate needof new farming tactics and tools, with more than 25% of its 2 billion
people living in poverty and hunger.
According to Rajeev Varshney, Ph.D., Research Program DirectorGrain Legumes and Director, Center of Excellence in Genomics atRajeev Varshney, Ph.D., is Research Program Director-Grain Legumes andDirector, Center of Excellence in Genomics at ICRISAT.

ICRISAT, one of the organization’s objectives is to apply scientificinnovations to dryland agriculture. This farming method is designed forregions of scant rainfall, focusing on water-retentive tillage methods
and the selection of suitable crops, rather than relying on irrigation.
“The objective of ICRISAT is to develop superior varieties of crops that
can thrive in drought conditions,” Dr. Varshney said. “There’s not muchvariety within the few crops that are cultivated in Asia and Africa. While
breeders have been successful in developing new, hardier crops using
traditional approaches, it wasn’t until 2005 that we really started toleverage genomics. Molecular breeding enables us to make strides indeveloping crops that are truly sustainable in this region.”
The pigeonpea (Cajanus cajan) is a hardy drought-tolerant legumefrom India that is used for food, livestock feed, soil erosion
amendment, and as a wind break. It’s an excellent protein source,
and is often referred to as the “poor man’s meat.” India produces 90%of the pigeonpea grown in the world and is also its largest importer.
While pigeonpea is important for food security in India, it’s an incomegenerating crop in Africa, particularly Kenya, Tanzania, and Rwanda.
In these countries, farming pigeonpea enables families to send theirchildren to school, something that would be otherwise impossible.
“When I joined ICRISAT, we had just 10 molecular markers inpigeonpea,” Dr. Varshney said. “These were simple sequence repeatsor SSRs. We had no genomic resources such as advanced molecularmarkers or single nucleotide polymorphisms (SNPs), and no mapping
populations. However, when you have a crop like pigeonpea, which
has narrow genetic diversity in the germplasm, even if you have
10,000 SNPs, there are only about 200 to 300 that will be polymorphic
in germplasm. For the last several years, we have been collaboratingwith different partners to develop genomic resources in pigeonpea
using next-generation sequencing (NGS).”

Pigeonpea (Cajanus cajan) is an excellent protein source, and is often
referred to as the “poor man’s meat.”.

August 2013
In 2011, the ICRISAT team, Beijing Genomics Institute (BGI), andother partners used Illumina sequencers to generate a draft genomeof pigeonpea2. The Greater Good initiative award ICRISAT received in2013 enabled it to accelerate projects to enhance pigeonpea genetic
diversity and support development of superior varieties.
Upon receiving the Greater Good Initiative award, the ICRISAT team
had several rounds of discussions with its stakeholders about therange of problems afflicting pigeonpea crops. “We realized we couldn’t
tackle all of them in a single go,” Dr. Varshney said. “We decided touse the Greater Good award to support development of pigeonpea
varieties resistant to sterility mosaic disease (SMD) and Fusarium wilt
(FW). These diseases negatively impact pigeonpea yield and quality,and have spread to farms throughout India, Uganda, Kenya, andTanzania.”“The award has also enabled us to focus our research funds on
developing pigeonpea varieties with higher yield, protein content, andearly maturity,” said Dr. Varshney. “In general it takes about 140 to 180
days for a pigeonpea crop to be harvest-ready. In India, we would liketo grow two crops in about 120 days. This would ensure greater foodsecurity and support income generation, enabling farmers to make theshift away from subsistence farming.”
Pigeonpea is grown in marginal environments where water is scarce.
There it is also exposed to several biotic and abiotic stresses, reducing
crop productivity to >1 ton per hectare. “In the face of climate change,
our goal is to establish climate-resistant pigeonpea varieties,” saidDr. Varshney. “Molecular breeding enables us to develop plants thatcan withstand drought conditions and environmental stresses, andultimately generate much higher yields3”.
By leveraging NGS systems, such as Illumina MiSeq® and HiSeq®
systems, ICRISAT intends to enhance the genetic base of pigeonpea.
“The great thing about the HiSeq system is that we can generate
a huge amount of sequence data in a very cost-effective manner.
We use the MiSeq system for smaller projects, such as pathogen(bacteria, fungi) resequencing. We’re in the process of optimizing
genotyping-by-sequencing and hope to be employing that soon.”

Molecular breeding will enable ICRISAT to increase the allelic diversity
in the cultivated gene pool from landraces (traditional pigeonpea
breeding populations), the gene bank, and existing wild varieties. It’sdeveloping introgression libraries in the genetic background of thecultivated varieties, performing extension phenotyping, and developing
specialized genetic stock called MAGIC (Multi-parents Advanced
Generation Inter-Cross). “We are making some complex two-, four-,and eight-way crosses, creating varieties that possess different
haplotype blocks,” Dr. Varshney said. “The set of 500 MAGIC lines wedeveloped possess a huge amount of genetic diversity, because weare reshuffling the haplotype blocks.”“We’re using three different approaches to identify genomic regions
responsible for various economically important traits,” Dr. Varshney
added. “The first is through linkage mapping, second is genomewide association studies (GWAS), and the third is nested association
mapping (NAM). For GWAS analysis, we have already completed theresequencing of 300 plant lines at 5–10× coverage.”This work also supports capacity building. “The research is being
conducted by eight Ph.D. students, two from Africa (Uganda andTanzania) and six from India,” Dr. Varshney said. “Through this work,
we are developing the next generation of scientists.”
By the end of this three- to five-year project, ICRISAT will havemolecular markers associated with specific pigeonpea traits. “Thesemarkers will enable breeders to develop new varieties with improved
traits in less time. In general, it takes 8 to 10 years to develop thevarieties, but we believe that we can reduce this time to 4 to 6 years,”
Dr. Varshney added.

Reference1.

http://www.icrisat.org/index.htm#

2.

Varshney RK, Chen W, Li Y, Bharti AK, Saxena RK, et al. (2012) Draft
Genome sequencing of pigeonpea (Cajanus cajan), an orphan legume cropof resource-poor farmers. Nat Biotechnol 30: 83–89.

3.

Varshney RK, Ribaut J-M, Buckler ES, Tuberosa R, Rafalski A andLangridge P (2012) Can genomics boost productivity of orphan crops?
Nat Biotechnol 30: 1172–1176.

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