September 2015

A Ray of Sunshine for Sunflower Breeding
How a government agency, an agricultural trade group, and a biotech company came together tobring sunflower breeding into the genomics era.

Introduction
The sunflower, Helianthus annuus, is a large annual crop with a strong
global market for a variety of domestic and industrial uses. Aside fromtasty sunflower seeds and healthy vegetable oil, sunflower products
are used as bird feed, livestock feed, cooking oils, biodiesel fuel, andlatex production. Last year, United States sunflower farms harvested
1.5 million acres valued at nearly 500 million US dollars.1 However,in recent years sunflower acreage in the United States has been
declining and farmers are looking for ways to improve resistance todiseases such as sunflower rust and Sclerotinia stalk rot.2
iCommunity spoke with Denise Thiede, PhD, Vice President of
BioDiagnostics (BDI), and Farhad Ghavami, PhD, Manager ofMolecular Breeding and Genomics Technology at BDI, to discuss how
the company participated in modernizing breeding programs in thesunflower industry.
Q: What is BDI and how is it involved in genomic breeding?
Denise Thiede (DT): BioDiagnostics is a seed testing company
that offers a full range of genomics tests to large and small seedcompanies. It was founded in 1996 by Quentin Schultz, our current
president, who wanted to bring modern approaches to quality
assurance testing. Our business has invested significantly in genetic
analysis platforms. We’ve been building and developing technologies
that will have a significant impact on advances in agriculture.
Q: What are some of the challenges faced by the US
sunflower industry?
DT: Fifteen years ago, large seed companies in the US, such as cornand soybean companies, began focusing their research on transgenic
products and genetic breeding programs. They built a large research
infrastructure that identified markers for native traits and transgenic
traits. This drove the development of many genomic breeding
programs, and ultimately led to improvements in the quality of theirseed products.
However, the sunflower industry rejected transgenics and as a result,none of the modern genetic tools were developed for sunflower
breeding. Over time, the National Sunflower Association (NSA) began
to see that significant improvements were being made in corn andsoybeans by using advanced genomic breeding techniques. While
improvements in drought tolerance and disease resistance wereexpanding the acreage of corn and soy, the acreage of sunflowers wasdecreasing. The sunflower industry and the NSA realized they needed
these genomic tools to protect the future of sunflower farming.

Sunflower fields in North Dakota. The sunflower industry realized
they needed genomic tools to protect the future of sunflower
farming.

Denise Thiede, PhD, Vice President of BioDiagnostics and Farhad
Ghavami, PhD, Manager of Molecular Breeding and Genomics
Technology at BDI.
Q: What role did BDI play in addressing these challenges?
DT: Historically, agricultural research has been funded through theUnited States Department of Agriculture (USDA), which has researchstations around the US. North Dakota is the heart of sunflower country,
so the USDA research unit in Fargo was focusing on sunflowers aspart of their effort to serve local industries. The NSA started talking tothe USDA researchers in Fargo to see how they could facilitate andspeed up their genetic discovery process. The USDA researchers
knew that whole-genome, high-density marker technologies wereneeded to associate markers with the phenotypic traits they had
identified in the field. However, the USDA in Fargo did not have theinfrastructure to develop a high-density array for sunflower.

September 2015
Farhad Ghavami (FG): They were using markers like simple sequence
repeats (SSRs) and restriction fragment length polymorphisms(RFLPs)—marker information that did not provide a dense and robust
set of markers for modern breeding needs.

arrays in 2005/2006, Nimblegen and Affymetrix arrays were available,but Illumina had the best arrays on the market and had proven to be agood platform.

DT: At the same time, BDI was also serving the sunflower industry.
We were doing a lot of quality assurance testing—germination
testing, genetic purity testing. Through discussions at an annual NSA
Research Conference in January 2010, a trifecta came together: theUSDA had the critical phenotypic data and research infrastructure, theNSA had the funding, and BDI had the genomic technology. Throughthis collaboration, BDI helped the US sunflower industry adopt themodern genomic tools that would enable them to be successful intheir breeding programs and maintain production here in the USand abroad.

“A significant advantage of theiSelect array is that it’s highly flexible
in the number of samples that canbe processed.”

Q: How did BDI develop genomic tools for the sunflower industry?
FG: The first step was to sequence the sunflower genome. Crops
such as corn and rice had full reference sequences, but the sunflower
did not. This made finding relevant markers and single nucleotide
polymorphisms (SNPs) difficult. Also, the sunflower genome is largeand complex; it’s ~3.5 Gb, which is larger than the human genome.
Because of its size and complexity, we decided to use restriction
site associated DNA sequencing (RAD-Seq). RAD-Seq is based on
identifying polymorphic variants next to restriction enzyme digestionsites. It can reduce the complexity of the genome by eliminating
duplicates and repetitive sequences.

“BDI helped the US sunflower
industry adopt the modern genomic
tools that would enable them tobe successful in their breeding
programs and maintain production
here in the US and abroad.”
DT: The sequencing project was done in collaboration withFloragenex, which has expertise in RAD-Seq. We provided them with
6 sunflower lines, including oil and confection lines, for the sunflower
sequencing project.3
Q: What platform was used to sequence the sunflower genome?
FG: We used paired-end Illumina sequencing. We started with an early
Illumina sequencing platform, but now RAD-Seq is performed on theHiSeq® or MiSeq® Systems. Floragenex also did the bioinformatics
work to find and refine a SNP set for a whole-genome, high-density
SNP array. A final set of 16,467 SNPs was used to create thesunflower array with the Illumina Infinium® iSelect® platform.
Q: Why did the NSA and BDI decide to use the Infinium iSelectplatform to create the sunflower SNP array?
FG: At that time, we had experience with the Illumina GoldenGate®
platform and really liked it because it could analyze 1536 markers ata time. Then higher-density Infinium arrays, like the MaizeSNP 50K
BeadChip,entered the market with over 10,000 markers per array.
Because GoldenGate had been so successful, it was not difficult toconvince us to use the Infinium arrays. When it came to high-density

DT: We recommended the iSelect array and told the NSA and USDA
that this is the technology that would be most useful to them.
FG: A significant advantage of the iSelect array is that it’s highly
flexible in the number of samples that can be processed. We ran 1100
samples at the beginning of the project, but later were running a few
samples at a time. We were not stuck having to run 96 sample plates.
Running iSelect arrays was easy and we liked it.
Q: What do you and your customers think of the sunflower arraydata quality and data analysis?
FG: For data analysis, Illumina is one of the best companies on themarket. The genotyping error rates are low and the GenomeStudio®
software is robust and easy to use.
Q: What information are you able to provide to your customers withthe sunflower array?
DT: With the iSelect sunflower array, we perform whole-genome
SNP genotyping and provide customers with the final genotype calls.
Our customers get the final results and then make their breeding
decisions based on the genotype data. Some of our customers
are sophisticated users of that genomic data and some are at theearly stages of the learning curve. If they are new users of genomic
data, we provide basic consulting services to help them use thedata appropriately.

“Now we have customers using
those markers to breed for
disease resistance against key
sunflower diseases.”
Q: What discoveries have been made using the sunflower array?
DT: After we had the sunflower array, the next major step was to start
performing large-scale genotyping for the NSA participants. This reallyhelped them gain an understanding of the diversity in their crop lines
as well as the extent to which their lines were related. It gave them
important information, however they still didn’t have good disease
resistance markers. That’s where the sunflower array in combination
with the work being done at the USDA became extremely valuable.
FG: The USDA had a lot of good phenotypic data and we were able
to provide the genotypic data. Using the sunflower array, we merged
the phenotypic and genotypic data through association mappingand found many agronomic traits as well as many disease resistancegenes at the same time.

September 2015
DT: The sunflower array allowed the USDA group to narrow down andlocate the genetic regions associated with disease resistance. Since
then they’ve published many papers about those disease resistancegenes. Now we have customers using those markers to breed for
disease resistance against key sunflower diseases.

NSA SNP ConsortiumThis project was conducted in accordance with the NSA SNP
Consortium, a public-private partnership between the nonprofit
National Sunflower Association, public researchers at USDA, andQ: What are some of the most destructive sunflower diseases?
FG: In North America, sunflower rust is a fungus that has caused
economic losses in sunflower yield and seed quality. Dr. Lili Qi fromthe USDA-ARS recently discovered the R12 rust resistance geneand performed high-density SNP mapping of the R12 gene with thesunflower array.4 We now use these markers every day for customers
who want to perform marker selection for the R12 rust-resistance gene.

“The iSelect sunflower array isvery effective for these breeding
programs because it can scan thewhole genome and find markers
much closer to the disease
resistance genes.”
Sclerotinia stalk rot is another fungus that is extremely destructive
to sunflower crops worldwide. Under certain conditions, it canreduce crop yield by up to 20%. Dr. Brent Hulke at the USDA used
the sunflower array to identify over 180 markers associated with
Sclerotinia head rot, Sclerotinia stalk rot, and Phomopsis stalk canker.
Dr. Hulke found that testing for these markers greatly improves crop
selection and allows them to select sunflower varieties that show thegreatest resistance to disease.
The iSelect sunflower array is very effective for these breedingprograms because it can scan the whole genome and find markers
that are much closer to the disease resistance genes. It’s also easier to
use and more cost-effective than the older SSR/RFLP markers.
Q: Have you seen any changes in the plant breeding industry as aresult of modern genomic breeding techniques?
DT: There’s a lot more complexity in plant breeding today. One of
the challenges is that historically plant breeders are phenotypicallyorganized, or if they use quantitative genetics, it’s more of a statistical
approach rather than a genomic approach. In many companies, there
are traditional plant breeders who know they need to use the newgenomic tools, but they don’t necessarily know how to use them. We
try to help them adopt the new tools. The companies that have thegreatest success are the ones that hire new plant breeders who havethe knowledge and experience to immediately adopt and use modern

private seed companies. It was formed with the intent of having
widespread, economically feasible application of SNP markers forthe advancement of sunflower germplasm. In 2014 the sunflower
SNPs were published in PLOS One.5

genomic tools. Plant breeding is not performed by a single individual
anymore. It’s a breeding team that includes people who can do thebioinformatics and people who can do the field work.
FG: People are shifting from traditional plant breeding to genomicbased breeding. We are in a state of transition. Some people haveadopted this new scheme quickly and can use the new technology.
Some people are waiting and watching to see how successful the new
approaches are. These days, most people involved in plant breeding
are using genomic tools to some degree.
Q: One final question: do you like sunflower seeds?
FG: I love them! Most sunflower seeds are roasted and salted, but I
eat them raw, the way birds do.

References
1.
2.
3.

4.
5.

NSA website (www.sunflowernsa.com/stats/historical-prices-values/).
Accessed August 5th, 2015.
NSA website (www.sunflowernsa.com/magazine/details.asp?ID=989).
Accessed August 4th, 2015.
Pegadaraju V, Nipper R, Hulke Brent, Qi L, and Schultz Q. De novo
sequencing of sunflower genome for SNP discovery using RAD (Restrictionsite Associated DNA) approach. BMC Genomics. 2013;14:556.
Gong L, Hulke BS, Gulya TJ, et al. Molecular tagging of a novel rustresistance gene R12 in sunflower. Theor Appl Genet. 2013;126:93-99.
Talukder ZI, Gong L, Hulke BS, et al. A high-density SNP Map of sunflower
derived from RAD-sequencing facilitating fine-mapping of the rust resistancegene R12. PLoS One. 2014;9:e98628.

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