August 2015

Growing Better Cotton for Future Generations
International collaboration led researchers to develop a cotton genotyping array, enabling
breeders to produce more sustainable and valuable cotton.
Introduction
From the terry cloth in bath towels to the denim in blue jeans, cotton isused to make clothing and linens all over the world. The fiber is spun into
yarn or thread to make textiles and the remaining cottonseed is used to
produce oil, which is then refined and used for cooking. While most of
the cotton supply is grown in China, India, and the United States, cotton
is grown in over 75 countries. It is a hardy crop that can thrive in harshclimates and can be cultivated with little equipment, enabling small-farmholders in developing countries to grow and sell it easily.
Researchers and breeders are using genetic selection to develop new
types of cotton that are more productive and resilient to challenging
environments. Because cotton exists in both diploid and polyploidforms, it is difficult to crossbreed the 2 types of species, making
genetic studies challenging. Until recently, few single nucleotide
polymorphisms (SNPs) had been identified for cotton due to its highly
complex genome. Researchers and breeders knew that certain cotton
species had desirable characteristics, such as drought tolerance
and insect resistance, but didn’t know which genes conferred these
traits. Breeders largely relied on visual inspection and quantitative
assessments of the plants (eg, fiber yield, fiber quality, pest resistance)
to select the best progeny for each breeding cycle, which is atime-consuming process.
Recent research in the lab of David Stelly, PhD at Texas A&M
University is helping to change that. Amanda Hulse-Kemp, PhD, a
postdoctoral research associate in Dr. Stelly’s lab, focused her PhD
research on identifying cotton SNPs and developing a genotyping
tool to help breeders improve cotton crops. Through an international
collaboration with researchers and Illumina, Dr. Hulse-Kemp andher colleagues developed a custom cotton genotyping array: the
CottonSNP63K BeadChip.1 International partners included theCommonwealth Scientific and Industrial Research Organization(CSIRO) in Australia, the Agricultural Research Centre for International
Development (CIRAD) in France, the Council of Scientific and Industrial
Research Organization’s National Botanical Research Institute in India,
and Illumina. The International Cotton SNP Chip Consortium designed
the CottonSNP63K BeadChip to enable researchers and breeders to
identify SNPs associated with valuable traits, leading to higher yieldand hardier, disease-resistant crops.
iCommunity spoke with Dr. Hulse-Kemp to learn about how her work
will transform the way researchers and breeders grow better cotton.
Q: Why did you get involved in cotton genomics?
Amanda Hulse-Kemp (AHK): Agriculture is the basis of society, andplants are a large part of that. By 2050, it’s estimated that the global
population will be approximately 9 billion. With the rapidly increasing
population size, agriculture will play an even more important role.
There’s not only an immediate need to feed people, but also to provide
clothing and linen supplies. For that reason, cotton will be an importantcrop for the future.
For Research Use Only. Not for use in diagnostic procedures.

Amanda Hulse-Kemp, PhD, is a postdoctoral research associate
in the Department of Soil and Crop Sciences at Texas A&M
University. She received the Outstanding Achievement in Graduate
Research Award in 2014 and the Distinguished Graduate Student
Award in 2015 for her work on cotton genomics.
Q: How many different cotton species are you studying?
AHK: Typically, most breeders are only interested in the cultivatedspecies. Our lab is interested in leveraging the diversity found in wild
species. Wild species are unique because they’ve evolved naturally inmultiple environments, so they have many specialized characteristics.
Cultivated species tend to have similar traits, and there isn’tmuch variety.
The 2 main cultivated species that we study are upland cotton(Gossypium hirsutum) and Pima cotton, sometimes called Egyptian
cotton (G. barbadense). Upland cotton produces a large yield ofmoderate-quality cotton and makes up about 95% of the cottongrown worldwide. Pima cotton produces a higher-quality product withlong fibers, which is used to make expensive clothing and linens. The
problem with Pima cotton is that even though it’s of higher quality, it
produces a much lower yield than upland cotton, so the overall value
to the farmers is lower. A goal for many cotton breeders is to develop
an optimal cultivar that combines the high-yielding characteristics of
upland cotton with the high-quality characteristics of Pima. However,both of these species still have limited diversity due to multiple
genetic bottlenecks, which leaves them vulnerable to various pests
and climate changes. That’s where wild cotton species become
incredibly valuable.

August 2015
Q: What approaches have cotton breeders used in the past to
select for traits?
AHK: Historically, cotton breeders have largely relied on visuallyinspecting plants to identify traits. Modern breeding involves qualitativeand quantitative testing, including multi-location replicated testing at
advanced generations to select the best progeny during each breeding
cycle. Today, breeders cross many plants, looking for cotton lines that
perform well or have valuable characteristics. This approach involves
a lot of work, because it’s basically a numbers game. You have to go
through perhaps a million plants to capture that right combination of
traits that makes a great cultivar. Even if you evaluate many lines, you
still might not find exactly what you’re looking for, because a single
significant fault can render a line unusable for production.
Q: When did agricultural researchers start using genomics tostudy cotton?
AHK: We’re at a turning point in using genomics to study cotton. By
integrating array technology into our operations, our lab is working
to demonstrate the ease, affordability, and value of genomic tools sothat others can incorporate them into their research and breeding
programs. Breeders are beginning to discover the value that array
technology can bring to tracking cotton traits. They need a tracking
tool that enables them to look at large numbers of plants at a low
cost per sample. Arrays can help them do that. Also, genomics
can help breeders evaluate and select for genes that affect lowly
heritable or polygenic traits, which are difficult to manipulate using
conventional methods.

For breeders, it’s a numbers
game. They need a tracking toolthat enables them to look at large
numbers of plants at a low cost
per sample. Arrays can help themdo that.
Q: How did your group start the International Cotton SNP
Chip Consortium?
AHK: We started the International Cotton SNP Chip Consortium
as a way to bring researchers together to create a low-cost SNP
genotyping tool. When I started working on cotton genomics in2010, everyone relied on simple sequence repeat (SSR) markers, or
microsatellites. The problem with SSRs is that they’re limited and notnecessarily evenly distributed throughout the genome. The advantage
of SNPs over SSRs is that they have the possibility of existing at any
given base in the genome, so they provide a larger number of more
evenly distributed markers. SNP markers can also be assayed invarious ways, in a high-throughput fashion, and at a low cost per data
point. They’re cheap and reliable.
Dr. Stelly realized this, too. Working with Dr. Sukhwan (Sam) Yang (apostdoctoral researcher at the time), and later with Dr. Allen Van Deynze
and others, he started identifying cotton SNPs. Given the complexitiesof the cotton genome, there were many hurdles to overcome. When I
started my research program, only about 200 SNPs had been identified
and mapped for cotton, so our lab set out to find more markers.
For Research Use Only. Not for use in diagnostic procedures.

The Complex Cotton Genome
Although 2 of the 4 cultivated species of cotton are
diploid (2 sets of chromosomes), the other 2 cultivatedspecies are tetraploid (4 sets of chromosomes). The
tetraploid genome is similar to a deck of 52 playing
cards, with 2 red and 2 black suits (genomes), eachwith 13 cards (chromosomes). Complicating the cotton
genome even further is the fact that both historical andrecent data indicate that whole-genome duplication(polyploidization) preceded formation of the ancestralgenome that gave rise to diploid cotton. Thus, uplandcotton is at least paleo-octaploid, abounding withgenetic redundancy, which makes genetic analysis,
sequencing, and breeding a challenge.

By 2012, our group had developed tens of thousands of markers, andwe connected with Dr. Iain Wilson at CSIRO because his group had
also developed many SNPs. We now had enough to start genotyping
on a large scale. The challenge was getting the price per sample low
enough. So at the International Cotton Genome Initiative meetingin 2012, we distributed an announcement asking if anyone wanted
to be a part of a collaboration to develop a cotton array. The cotton
community was very supportive and interested in the overall goal.
Afterwards, we worked with Illumina to develop an interest list. We
then followed our announcement with a worldwide message asking
if researchers from other countries would like to join the initiative,
contribute to the SNP markers, and purchase the array.
By 2013, the Consortium decided on the size and types of content
on the array. I identified the attributes of the SNP populations,removed duplicates, and trimmed the content down to about 70,000
SNPs. We sent it to Illumina for production in the fall of 2013, andwe were delighted to have such high assay success in conversion topolymorphic markers for the array. The CottonSNP63K BeadChip was
released in early 2014. It has been adopted quickly by researchersand several companies. Now we’re trying to spread the word toother researchers and breeders, so that they too can benefit from the
CottonSNP63K BeadChip.
Q: What were the goals of the International Cotton SNP
Chip Consortium?
AHK: One of the main goals of the Consortium was the developmentof a cluster file for upland cotton and the other tetraploid species,
so that everyone’s CottonSNP63K BeadChip results would bestandardized throughout the cotton community. Dr. Martin Ganal’sgroup at TraitGenetics GmbH contributed their expertise to the cluster
file development.
One of the challenges with working with inbred crops is that mostof them are homozygous, so the heterozygous clusters aren’t
normally captured. Localizing the heterozygous cluster is the mostdifficult type of pattern to identify. Illumina helped us create thebest cluster file possible by representing more varieties to makethe CottonSNP63K BeadChip a more broadly useful tool. With abroad representation of the germplasm, we could identify a large
portion of the clusters by including samples that belong to both theAugust 2015
homozygous and heterozygous clusters. Now anyone, including
breeders, can send in their DNA sample, receive the genotyping
data in 3 days, and immediately use the information to identify whichlines to keep, select, or cross for their program. This tool enables
us to associate the markers on the array with important traits anddetermine if they might be causal alleles or linked to causal regions forbeneficial characteristics.

The unique thing about thecluster file is that it’s designed fortetraploid cotton. Anyone can takea tetraploid sample, put it throughthe file, and get a genotype.
Q: Which cotton species are represented on the CottonSNP63K
BeadChip?
AHK: The CottonSNP63K BeadChip contains markers for G. hirsutum,
G. barbadense, G. tomentosum, G. mustelinum, G. longicalyx, andG. armourianum. The BeadChip is primarily designed for G. hirsutum
breeding, or integrating features of the other species into G. hirsutumlines, because G. hirsutum is the most widely cultivated species.
G. tomentosum and G. mustelinum are tetraploids from Hawaii
and Brazil, respectively. They have some characteristics for disease
resistance and seem to be able to withstand high amounts of rain,
which is especially useful with the heavy rainfall we’ve recently seen
here in Texas. G. mustelinum has been shown to have the highest leaf
concentration of terpenoid aldehydes, which affect insect resistance.
G. tomentosum also has resistance to cotton leaf hopper and a few
other insects. The 2 diploids represented on the BeadChip are
G. longicalyx and G. armourianum. Because it originated in Africa,
G. longicalyx theoretically has some drought resistance features,and has also been shown to have high resistance to the reniform
nematode, which we’re studying in our lab. G. armourianum has
resistance to the white fly, which is a vector for the leaf curl virus, a
new form of which has been devastating to cotton crops, particularly
in Pakistan and India.
Q: How did you select the SNPs to include on the CottonSNP63K
BeadChip?
AHK: Because research on cotton SNPs has been limited and we had
only recently identified the SNPs, we did not have the ability to selectSNPs that were associated with traits. Instead, we chose groups of
SNPs that were known to be genic in origin and others that were not.
By combining both types of SNPs, we assumed that the SNPs would
be more or less evenly distributed across the genome.
The only reference genome for the genus Gossypium available at thetime was from a wild, diploid, Peruvian small tree species. We didn’t
want to bias our SNP selection in the tetraploid species based on a
diploid, so we used a random spread of SNPs based on design scoresfrom each species. Now the community can use the CottonSNP63K
BeadChip to make those associations between SNPs and traits.

Q: How did you validate the CottonSNP63K BeadChip?
AHK: We used 1158 samples to validate the CottonSNP63K
BeadChip, which included many different types of samples so that we
could populate each of the genotype clusters. Because cotton is atetraploid species, it is likely that these genotyping assays evaluate 2
positions in the genome, because there would be 2 identical positionsin the different subgenomes. As a result, it required manual validationof the cluster positions. Because they had previous experience
analyzing polyploid data, our collaborators at TraitGenetics manually
positioned or changed the position of the clusters in the cluster filewhere the automated clustering algorithm was insufficient due tothe polyploidy. Through validation, we’re making sure that we havesamples that represent the 3 different cluster types to distinguish a
successful polymorphic marker from a monomorphic or intergenomic
marker. The segregating markers were further validated using 2
mapping populations.
Q: How will the genetic maps and the cluster file that you createdbenefit cotton researchers and breeders?
AHK: The unique thing about the cluster file is that it’s designed fortetraploid cotton. Anyone can take a tetraploid sample, put it throughthe file, and get a genotype; it doesn’t require any modifications todetermine the true genotype. Also, this is the first time that cotton
researchers have an intraspecific genetic map that links into linkage
groups that represent chromosomes. Before, you would not haveenough markers to associate them into the correct number ofcotton chromosomes. Our group is also developing a cluster file for
diploid cotton.

Illumina has been really helpful
throughout the whole process.
If we had a question, they knew
the answer.
Q: What was the value of having a consortium when you weredeveloping the CottonSNP63K BeadChip?
AHK: The International Cotton SNP Chip Consortium was instrumental
in developing the CottonSNP63K BeadChip. Everyone in theConsortium was fabulous and helpful. We had breeders, molecular
geneticists, public agencies, representatives from companies, and a
representative from the commodity organization Cotton Incorporated.
They provided ideas and input on what their groups were interested
in. Their input was instrumental in helping us make the CottonSNP63K
BeadChip relevant to people in different areas of cotton research.
Because the CottonSNP63K BeadChip was so marketable, many
samples were reserved in the initial buy-in, reducing per-sample costs
for consortium members. This enabled us to process many samples
cost-effectively. Otherwise, we could never have afforded to run all the
SNPs on our own.

For Research Use Only. Not for use in diagnostic procedures.

August 2015
Q: What was your experience with Illumina in developing
the Consortium?
AHK: Illumina has been really helpful throughout the whole process.
I’m pretty sure that we would not have made it this far so quickly
without input from Illumina. If we had a question, they knew theanswer. They helped us with any technical difficulties and streamlined
the process from formation to application. Illumina continues tosupport the Consortium by distributing BeadChips so we can run
them in our lab, enabling us to get quotes to collaborators or send
and receive samples from collaborators easily. The fact that you can
use the array technology anywhere or send it to a service provider
is useful because many of the breeders who will want to use the
CottonSNP63K BeadChip don’t have the technology or capability to
run the array themselves.

project can be a cultivar line instead of fine mapping that someone
else will work on during their doctoral research. The impact on student
projects is enormous. They can now do about 10 times the amount ofwork that they could do before with the same time and effort. It’s muchcheaper and faster to use arrays.

Almost everyone who works on
cotton will use the CottonSNP63K
BeadChip in the future.

Using array technology, graduate
students can now do about 10
times the amount of work thatthey could before with the sametime and effort.

Q: How did the International Cotton SNP Chip Consortium affectyour doctoral research?
AHK: While not initially a part of my doctoral research proposal,
developing the CottonSNP63K BeadChip became a large part of
my PhD research. Dr. Stelly helped me gain valuable experience byhaving me work directly with the consortium members and participate
in all the meetings and teleconferences. Not only have I used the
CottonSNP63K BeadChip, but everyone in our lab is already using it,
and almost everyone who works on cotton will use it in the future.
Developing a tool that molecular researchers and breeders can use
and that incorporates lines produced in the public and private sectors
is remarkable. I’ve met many people through the Consortium that I
never would have otherwise. I think it’s valuable to know that there isa tool out there that you can use to assay the same position at a pointin the cotton genome, and that your research can easily be correlatedwith someone else’s research.
Q: Do you have any advice for other graduate students studying
cotton genomics?
AHK: Using array technology can significantly speed up graduate
research. As Dr. Stelly likes to emphasize, students can benefit
enormously because array technology expedites their progress, giving
them a “quantum leap” forward and changing the timeline and scopeof their research.
Most research at universities is conducted by graduate students andpostdoctoral fellows with limited time. For many graduate students
today, their whole doctoral project is making a high-density map orfine mapping a small region. They don’t know where many of the traits
they’re dealing with are located. With an array, you can quickly run amapping population and determine which regions you’re interested
in within a month rather than years. So, the final output of your PhD

Q: What are the next steps in your research?
AHK: We’re now working on the diversity analysis with the inbred linesthat were included on the CottonSNP63K BeadChip for validation anddevelopment of the cluster file. Researchers can use those results
to find out how close their lines are, how many markers they have
between the parents, and things like that. They will then be able toassociate the markers on the array with actual phenotypes, leading tostreamlined development of superior cultivars.

Q: Where do you see the future of cotton genomics research goingfrom here?
AHK: The CottonSNP63K BeadChip will allow a revolution in cotton
breeding as long as we can get it into the breeders’ hands. This willallow for faster development of varieties so cotton farmers will bebetter equipped to deal with problems like climate change, drought,
and disease. The disease-resistant, higher-quality lines will enable
growers to obtain a higher yield per acre, which theoretically will
generate additional income, something that will benefit all cotton
producers in the future.

References
1.

Hulse-Kemp AM, Lemm J, Plieske J, et al. Development of a 63K SNP
array for cotton and high-density mapping of intraspecific and interspecificpopulations of Gossypium spp. G3 (Bethesda). 2015;5:1187-1209.

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U.S. and/or other countries. Pub. No. 1370-2015-011 Current as of 20 August 2015