April 2014

Breeding Climate-Resilient Cowpea for Improved
Food Security in West Africa
Illumina high-density genotyping expected to accelerate marker-assisted breeding.

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.

2014 Illumina Greater Good
Initiative Award Winner

Introduction
The cowpea {Vigna unguiculata (L.) Walp} is an important source ofprotein in Sub-Saharan Africa, where it is well adapted to the hot,drought-prone climate of the region. Recognized as the black-eyed
pea in the United States, this ancient legume was domesticated by
African gatherers, cultivators, and farmers from its wild forms datingback to Neolithic times. The modern-day cowpea is grown mainly bysmallholder farmers in semi-arid Sub-Saharan Africa where rainfall
is unpredictable. The threat of climate change and the expansionof desert areas is fueling efforts by the USAID* Feed the Future
program to tap into the cowpea’s genetic diversity and maximize theenvironmental tolerance of this important food source.

The cowpea plant and seed diversity make it an essential part of thenutritional profile for inhabitants of West Africa.

*U.S. Agency for International Development that works to end extreme
global poverty.

In West Africa, the cowpea growing season begins with the firstseasonal rains around April and ends with the harvest in October. The
plant is an essential part of the nutritional profile for inhabitants of theregion. As with other legumes, cowpea seeds are rich in the essential
amino acids lysine and tryptophan. They support a balanced dietwhen combined with cereals such as sorghum and millet, which arerich in methionine. Cowpea is also rich in folic acid, which is importantfor fetal development. All parts of the cowpea plant are eaten. The
leaves can be cooked like spinach and the immature pods are eaten
as fresh vegetables. The mature dry seeds are processed into a
nutritive flour, or cooked and eaten whole. The residual dry matter after
pod harvesting can be stored and used as animal feed1.
As one of the most drought- and heat-tolerant crops in the region,
cowpea plays an important role in breaking the “hungry time,” which
can occur toward the end of each off-season when food reserves
may become depleted. Seeds produced during the annual growing
season must last throughout the year until the next cowpea crop. If
the previous harvest was poor or the rains arrive late, the hungry time
can become more severe, causing malnutrition and starvation thatincreases fatalities from malaria and other diseases 2.
Timothy J. Close, Ph.D., professor of genetics in the Department ofBotany and Plant Sciences at University of California, Riverside (UCR),
is interested in the adaptation of crop plants to environmental stresses.
Dr. Close, a plant geneticist with extensive experience in genome
resource development, is now engaged in the breeding activities ofcowpea, with a specific interest in adaptation to droughtprone environments.

Timothy J. Close, Ph.D., is professor of genetics in the Department of
Botany and Plant Sciences at University of California, Riverside.

April 2014
“The central objective is to identify more climate-resilient cowpea
varieties for each production environment,” said Dr. Close. “Wemake crosses between parents carrying favorable traits and look forprogeny with a more optimal combination than either parent. It’s allabout using the normal reproductive system of the cowpea plant andthe genetic diversity of cowpea that nature has provided. Our work
is not about the creation of transgenic plants, also known as GMOs.
The use of genotyping during breeding helps to keep pre-determinedcrossing and progeny selection plans on track. It gives us the ability
to recognize and remove off-track individuals arising from unintended
outcrosses, while minimizing the size of breeding populations and thenumber of generations required to achieve the intended outcomes.
The overall goal is to improve the nutrition and livelihood of the people
in West Africa by developing hardier cowpea varieties.”

Drawing upon Nearly 40 Years of Cowpea
Genetic Research“The cowpea breeding program at UCR was started nearly 40 yearsago by Professor Emeritus Anthony E. Hall,” Dr. Close said. “Heestablished strong, long-lived connections with West African cowpea
breeders and initiated training programs and partnerships, several ofwhich are still in place today. Those of us who comprise the current
cowpea team are proud to continue the cowpea legacy.”
The core UCR cowpea team has expertise in agronomy, agriculturalbiology, seed systems, general genetics, molecular genetics, andcomputer sciences. Professor Philip A. Roberts, Ph.D., in theDepartment of Nematology, has been a leader in the UCR cowpea
team for nearly 30 years. He currently directs field-level decisions andbrings practical agronomy, plant pathology, nematology knowledge,
and decades of experience in the African context. Bao-Lam Huynh,
Ph.D., handles a range of day-to-day operations spanning genotype
data analysis, genotype-informed advice on crossing and selection
for their African partners, and management of their local field andgreenhouse assistants. The USAID Innovation Lab for Climate Resilient
Cowpea project under the USAID Feed the Future program alsobenefits from the expertise of Professor Stefano Lonardi, Ph.D., in theDepartment of Computer Sciences.
Dr. Close interacts regularly with each of these team members, alongwith numerous students and other staff, to coordinate the use of
informatics tools for genetic marker development and the deploymentof markers to guide the breeding activities. “We have several excellent
students at UCR whose contributions should not be overlooked.
Mitchell Lucas is a graduate student in the Genetics, Genomics andBioinformatics program whose Ph.D. thesis research includes recent
publications on the genetics of heat tolerance and thrips resistance incowpea. Seyed Mirebrahim is a Ph.D. student in Computer Sciences
who has been pivotal in the development of the new Illumina iSelect®
custom genotyping assay, working closely with Professor Lonardi andme, along with Programmer Steve Wanamaker. There are quite a few
more people at UCR who are part of the cowpea team, but there isnot sufficient space in my brief comments to do justice to everyone.”The UCR team also works with partners in Africa who are instrumental
in delivering the improved varieties to small-holder farmers. “Dr. NdiagaCisse in Senegal, Dr. Issa Drabo in Burkina Faso, Dr. Ibrahim Atokple
in Ghana, and Drs. Ousmane Boukar and Christian Fatokun in Nigeria
are the leading cowpea breeders in these four West African nations.
We have additional partners in Mozambique, China, and other nations,and I wish I had the time to acknowledge them all.”Leveraging Cowpea Natural Genetic Variationand Market-Assisted Breeding
The breeding team leverages natural genetic variation within cowpea.
“Cowpea offers a multitude of wonderful targets with extensive genetic
variability,” said Dr. Close. For example, the team recently identified
single nucleotide polymorphisms (SNPs) that mark five quantitative
trait loci (QTL) for heat tolerance during reproductive development.
According to Dr. Close, cowpea pollen won’t develop properly when
the night temperature is 34°C (93°F) or higher3. “At sustained elevatednighttime temperatures, cowpea pollen is not shed from the anthers,
impeding pollination and thus preventing pod development andseed production,” explained Dr. Close. This was shown in cowpea
more than 30 years ago by now-retired Professors Hall and Dareleen
DeMason and their students at UCR, and is a common phenomenonin plants. The opportunity for cowpea breeding lies in the fact thatsignificant genetic variation exists for tolerance or susceptibility at
an intermediate night temperature of 28°C (82°F), which is relevantin production environments. “We’re starting with a species that isalready well adapted to hot, drought-prone areas relative to most othercrop plants,” Dr. Close said. “But, there is still a range of tolerance
within cowpea. We’re trying to combine the best heat-tolerant anddrought-tolerant alleles within individuals that also have favorable seedcharacteristics, along with insect and pathogen resistance.”
The team applies marker-assisted breeding, which involves matchinga trait of interest to a corresponding set of tightly linked DNA markers.
The genetic markers, or SNPs, are used to determine the positionof major loci in the genome that have the strongest influence on atrait. “Having a state-of-the-art genetic marker system is essential totracking and combining (pyramiding) several traits simultaneously inour breeding activities,” Dr. Close said. “This may seem like it needs amiracle to succeed, but the reality of marker-assisted breeding is thatit actually does work for multiple traits. Cowpea has a simple diploid
genetic system that can inbreed or outcross, and there is ample
genetic variation. With an improved genotyping capability it becomesa matter of using good math to extract relationships between excellent
phenotype data that African breeders can provide and volumes ofgenetic data generated using SNP genotyping. The fact that nature
has provided us with such a simple genetic system in cowpea
is a bonus.”
For example, Dr. Close’s team compared progeny derived from across of two parents that differed in their heat tolerance. They locatedfive QTL regions of the genome that contribute significantly to heat
resistance. “We have also identified one major and one minor QTL
responsible for resistance to a California aphid biotype. We can markthese positions and recognize the resistant and sensitive allelic variantsby virtue of a set of tightly linked markers.”

Greater Good Award Enables Increased
Genotyping Density and a Simple Workflow
The cowpea team uses high-density genotyping to map traits andmark favorable and unfavorable haplotypes**, leading the way toward
new varieties. They previously developed an Illumina GoldenGate®
SNP assay for cowpea and used it to mark traits such as bacterial
blight resistance, aphid resistance, Macrophomina resistance,
drought-induced senescence, heat tolerance during reproductive
development, thrips resistance, and leaf shape. “The GoldenGate
assay gave our genetic mapping a tremendous boost, enabling us** A group of markers inherited together.

April 2014
to map more traits and at a much finer level of resolution than waspossible before,” said Dr. Close. “However, there have been several
instances when our nearest marker was a few centimorgan awayfrom a trait determinant. It was clear that we simply didn’t haveenough markers packed together to define a clear haplotype. We
don’t need to know exactly which gene controls a trait. However, forbreeding we need enough markers packed together around favorable
and unfavorable alleles so that we can reliably track haplotypes.”“The Illumina Agriculture Greater Good Award enables us to reachhigher heights than would have been possible otherwise,” Dr. Closeadded. “The Infinium custom iSelect 60K genotyping BeadChip should
provide about 40 times as many markers and 40 times the resolving
power as the 1536-plex GoldenGate assay that we developed
previously. We are excited about progressing to 60,000 bead types
with the iSelect assay. We anticipate about 40,000 technically
successful SNP assays using the same workflow for DNA preparationand data analysis that we used with the GoldenGate assay.”“The goal in designing the iSelect 60k BeadChip assay has been toannotate every known gene in the cowpea genome with two markers
so we can detect up to four haplotypes,” Dr Close said. “We’ll also
scatter markers at various positions throughout the genome in nongenic regions, ensuring that we have positional coverage. Not every
attempted bead type will succeed, but overall the numbers are verymuch in favor of achieving a level of resolution sufficient to mark every
major allele with a reliable haplotype. The iSelect 60k assay design
will also support genome-wide association mapping and mapbased cloning of trait determinants. We are confident that the higher
resolution will provide the needed increase in accuracy of marker-trait
associations to meet our USAID Feed the Future project objectives.”

Data Analysis Training with GenomeStudio®
According to Dr. Close, the Agricultural Greater Good Award has
stimulated consortium interest around a higher density, off-the-shelf
genotyping assay, expanding the consortium group and building
momentum for future studies. “The iSelect assay offers a simple
workflow, including easy data analysis and export tools in theGenomeStudio software,” Dr. Close said. “The portability of relativelysmall, digested datasets and software enables us to put more raw
forms of data into the hands our partners, just in case the lessprocessed data may be useful.”
Through UCR’s partnership under the USAID Innovation Lab for
Climate Resilient Cowpea4, Dr. Close’s team extracts DNA fromleaves sent by breeder partners in Burkina Faso, Ghana, Senegal, andNigeria. After genotyping with the iSelect assay, the data are packaged
in the GenomeStudio software, and exported to spreadsheets thatare formatted for other analytical tools. The Agricultural Greater Good
Initiative is providing a license for the GenomeStudio software to each

African partner site. “The GenomeStudio interface is informatics-lightand thus easy for all of us to use,” said Dr. Close. “It doesn’t require
an informatics specialist, and this simplicity is valuable at each breeder
location. Knowing how, and when, to use GenomeStudio will help
our partners understand what happens to their samples, and what
we routinely do with the data. This familiarity will build confidence inthe whole genotyping workflow. We anticipate that our West African
host country partners will become local sources of expertise regarding
genotyping and cowpea genetics, which will further secure
cowpea’s future.”

Sowing New Seeds
Climate-resilient cowpea varieties that will eventually result from
intensified breeding activities will be distributed through existing
networks in the partner countries. Favorable progeny from breeding
programs typically progress through standard stages of seedmultiplication and distribution. This begins with breeders seed, thenproceeds through carefully controlled increases to produce foundation
seed, then further increased for larger-scale production ascertified seed.
“Our breeder partners are engaged in their local seed systems, andwell linked to local outreach groups and networks,” said Dr. Close.
“They have experience with adoption of new varieties. Cowpea isan inbreeding crop, so the mature seeds taken from a plant will begenetically identical to the plant from which they were collected, aslong as there has been no cross-pollination in the field. This meansthat farmers don’t need to buy certified seed every year. However, theaccumulation of outcrossed progeny will eventually cause collected
seeds to diverge genetically from the original variety.”
Understandably, there is resistance to buying a new seed variety andthe farmers need data that convinces them of its higher value. “Byusing the iSelect assay, we can provide detailed visual displays of thegenotypes of known varieties. This type of knowledge can help assurethat seeds which progress through the seed system will be what they
are supposed to be.”References1.

https://www.hort.purdue.edu/newcrop/afcm/cowpea.html

2.

http://www.fao.org/ag/agp/agpc/doc/publicat/cowpea_cisse/cowpea_cisse_e.htm

3.

http://feedthefuture.gov/article/feed-future-innovation-labs

4.

Lucas MR, Huynh B, Diop N, Roberts PA, Close TJ (2013) Markers forbreeding heat tolerant cowpea. Mol Breeding 31: 529-536.

A unit for measuring genetic linkage.

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Pub. No. 1370-2014-002 Current as of 08 April 2014