The Best of Both Worlds: Cross Pollination

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The Best of Both Worlds Cross Pollination Nathan
Etsitty1 , Von Mark Cruz2, Candice Gardner23,
Matt Lively3, David Losure3, Barbara Bingaman3,
Lisa Burke3, Jeff Carstens3, Irv Larsen3, Cindy
Clark3, and Carolyn Lawrence2,3,4 1. Northern
Arizona University, Flagstaff, AZ 2. Department
of Agronomy, Iowa State University, Ames, IA 3.
USDA-ARS 4. Department of Development Genetics
Cell Biology, Iowa State University, Ames, IA
Abstract Cross pollination is a breeding
tool that was used by the early Native Americans
to develop wild teosinte into corn, a food crop
which feeds not only the native peoples but is
used by the whole population in todays society
as a food and through its industrial uses (like
producing ethanol). Cross pollination can yield
better plants, or it can yield poorer plants.
This difference is due to the effects of
combining the two parents characteristics.
Whether a particular hybrid is successful depends
mainly upon the demand for the products that can
be made from the hybrid. The process used to
make crosses can be open (by detassling the
females and planting males and females
intermittently) or by controlled hand
pollination. Hand pollination involves bagging
the plants tassels and shoots (young ears), and
crossing only specific individual plants of
interest. At the North Central Regional Plant
Introduction Station, populations of maize
collected from the American Southwest are
conserved and are propagated by non-reciprocal
hand pollination. By finding molecular markers
for identifying the Abnormal 10 chromosome of
maize, its presence can be identified in plant
populations. Subsequently, the markers can be
used to determine the genetic diversity of the
populations.
Materials Methods First, we collected
samples from a corn field at the Plant
Introduction that was planted before our arrival.
Each sample was placed in a small test tube and
put in ice to keep it fresh. Next, the
samples collected were taken back to the lab
where we prepared leaf extracts. This was done
by adding an Extraction solution to the leaf
and putting them in a hot bath to extract the
DNA. Once the sample was ready, we mixed in a
Dilution solution to stop the reaction.
Immediately, the samples were stored in a
refrigerator. I prepared a cocktail by
adding different PCR reagents, then each sample
was added. This was carried out in separate
tubes. After this was done, we took the PCR mix
and put them into a machine (called a
thermocycler) which enabled the amplification of
the SSRs. Following that, a gel plate was
made for the SSRs. Previously used gels were
melted down and poured into a tray where they
were allowed to set. The samples from each
individual tube were transferred on the sample
well on the gels. Then we put the gels in the
electrophoresis machine that would pull the DNA
strands through the gel using an electric
current. Last, pictures of the SSR patterns
on our gels were taken.
Background Teosinte is a tall annual grass
believed to be the ancestor of modern day corn.
Early Native American Indians utilized this plant
and helped transforming it into present day corn.
They were among the first to practice cross
pollination and are said to be early scientist
who experimented with many things but just never
wrote it down. Through generations, they carried
what knowledge was passed to them on and use that
to survive in modern day society. Cross
pollination is a method used to develop new
varieties of plants. The downfall to controlled
cross pollinating is contamination. During
controlled cross pollination, factors are taken
into account to prevent possible contamination.
The outcome for crossing two plants is that the
progeny will have the characteristics that the
plant receives from both parents. This
determines if the new crop will be a certain
color, multicolored, long, or small. A
hybrid is the offspring of two animals or plants
of different races, breeds, varieties, species,
or genera. Hybridization or crossing plays a key
role by allowing a diverse groups of plants with
different backgrounds to be cross pollinated and
keep the plant from dying out. The types of
hybrids that are successful depends upon demand
for the product. Cross pollinating can be
done by the two options listed in the abstract by
using the detassling method or controlled hand
pollination. Cross pollination is simply pollen
that is delivered to a flower of a different
plant. Plants adapted to outcross or
cross-pollinate often have taller stamens than
carpels to better spread pollen to other flowers.
In the instance of maize, the male and female
flowers occur on different locations of the
plant, another method to ensure outcrossing. For
open pollination of maize where crosses are
desired, the detassling method is used by
removing the pollen-producing (or male) tassel
from a corn (maize) plant. Detasseling is done
to breed, or hybridize, two different varieties
of corn the varieties are carefully selected so
that the new variety will exhibit hybrid vigor.
By removing the tassels from all plants of one
variety, all the grain growing on those plants
will be fertilized by the other varietys
tassels. To do the same thing on a
plant-by-plant basis, the hand pollination
process is done by placing a bag over the tassel
to capture the pollen so that it can be crossed
with a selected plant to cross with. Bags are
also placed on ear shoots to protect the female
flower from receiving pollen from other plants
nearby. Native people in the American
Southwest used techniques similar to those used
by plant breeders to propagate their plants- they
only grew plants they wished to cross in close
proximity and separated ones they did not want to
cross. The methods they used ensured that plants
were not contaminated with unwanted pollen,
keeping their sacred germplasm available for the
future generations. The Southwest Accessions of
maize are preserved by the North Regional Central
Plant Introduction Station. Populations of
plants are non-reciprocally crossed for
propagation. The term non-reciprocal means that
any two plants are only crossed with each other
unidirectionally, which is to say that if plant A
is used to pollinate plant B, plant B cannot be
used to pollinate plant A. In our study, we
investigated whether the Southwest Maize
accessions had a variant of chromosome 10 present
which would cause non-reciprocal propagation
techniques to fail to ensure that the genetic
profile of a population was maintained through
many generations. We used amplified fragments of
DNA to determine if a corn plant has normal or
abnormal chromosome 10. This was done by
collecting a sample from the plants to be tested,
then, using a kit to extract the DNA from the
leaves. Next, PCR (Polymerase Chain Reaction)
was carried out by adding a small amount of the
leaf extract to a PCR mix with microsatellite
primers. Amplification of microsatellites was
done using a PCR machine. After PCR, an agarose
gel was prepared with Ethidum Bromide. The PCR
products were carefully loaded into the open
slots on the gel, then placed in a
electrophoresis machine to separates products.
Last, we took pictures of the gel pictures to
help us determine if a variation in
microsatellites (also called a Simple Sequence
Repeat or SSR) is associated with abnormal/normal
chromosome 10. By determining if there are
markers associated with abnormal 10 chromosome,
we may monitor the genetic changes in the
populations of maize and determine if collections
are faithfully preserved through time.
About Me Hello, my name is Nathan Etsitty
and I am a member of the Navajo Nation Tribe
located near the four corners where Arizona,
Utah, Colorado, and New Mexico meet. By
clanship, I am identified as Big Water, Born for
the Bitter Water People, and my grandparents are
Red Streak Running Into the Water and Edge Water
People. Im from a small community known as Many
Farms, Arizona. I recently received my
Associates of Science Degree in Health
Occupations from Diné College. What
interested me in doing this research was to gain
more experience in different fields. The
research itself seemed relevant to a great study
and finding answers to questions that have been
asked for some time like, How can we preserve or
save our crop fields? Without hesitation, I
gladly accepted this research in hopes that my
contribution will be helpful to others in years
to come.
Fig. 3 Activities done during the research and
tools used in the lob.
The Work Place I conducted my work at two
sites 1.) Plant Introduction Station
-This was Monday thru
Wednesday each week. 2.) G223 Agronomy Hall
(Lab) -Thursday Friday
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Fig. 2 The PI station, a corn stalk I worked
with, my work station in the Lab at G223
Agronomy Hall.
1. A1 C1 r1 N10 homozygous (Dawe lab JM 9 sib.
W23) 2. R1 Ab10-I/r1 N10 (Dawe lab JM 2-3 x 1-2
backcrossed 9x to W23) 3. R1-nj N10/r1 N10 (Dawe
lab CL04AI 9-2 W23) 4. R1-st N10/r1 N10 (Dawe
lab CL04AI 13-1 W23) 5. C1 sh1 wx1 R1 K9S-l
Ab10-I homozygous (stock ID 905D, mongrel
background) 6. r1 Ab10-I (stock ID X16B, mongrel
background) 7. R1 Ab10-II / r1 N10 (stock ID
X16F, mongrel background) 8. r1 Ab10-II / r1 N10
(stock ID X16E, mongrel background) 9. R1 N10
homozygous in W22 (Weber lab CL04AI 11-1A) 10.
R1 N10/r1-x1 in W22 (Weber lab CL04AI 19-2) 11.
H99 12. B73 13. Mo17 14. Tx303 15. CO159 16. GT119
11 12 13 14 15 16
Fig. 4 Gel patterns of SSR p-umc2126 and an
artists rendering of a DNA strand.
Acknowledgements I thank the NSF and
USDA-ARS as well as Carolyn Lawrence, Candice
Gardner, Von Mark Cruz, Matt Lively, David
Losure, Barbara Bingaman, Lisa Burke, Jeff
Carstens, Irv Larsen, Cindy Clark, Nina Grant,
Mary De Baca, Aurelio Curbelo, Carmen Sanchez,
the rest of the GWC staff and my family back home.
Conclusion Cross Pollination is a great
breeding tool used by both indigenous peoples and
by modern day breeders. No molecular
markers evaluated served to identify abnormal
chromosome ten.
Fig. 1 Navajo Nation, Various types of corn, and
my mom and myself.