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Evolution of duplicate pathways
in polyploid Gossypium
Introduction
The genus Gossypium (Cotton) is believed to have
originated between five to fifteen million years ago.
Evidence indicates that 2 genome groups, known as the A& D-genome, developed in different geographic regions
before combining about one to two million years ago to
form the modern polyploid form of cotton. The modern
polyploid form of cotton (Gossypium hirsutum) feeds a
$12 billion international industry. A comparison of the A& D-genomes contained in polyploid cotton reveals a
duplication of genes coding for the anthocyanin pathway.
The anthocyanin pathway is a linear pathway found
among many of the higher land plants. The flavonoid
metabolites produced by the genes of the pathway are
known to protect the plant from microorganisms,
herbivores and ultra-violet light. Also, these metabolites
produce pigments in various tissues of the plant, which
aides in the identification of phenotype (visible) changes.
Based on our knowledge, we are trying to understand
how the anthocyanin pathway has been affected by
combining the A- and D-genomes to form the AD-genome
of polyploid Gossypium.
Jim Kollman1, Spencer Mesick1, Lex Flagel1, and Jonathan F. Wendel1
1Department
of Ecology, Evolution and Organismal Biology at Iowa State University, Ames, Iowa
From the phylogenetic
trees that were made we
interpreted CHS, CHI and
DFR to be single copy
genes for Gossypium. The
F3H, ANS and UF3GT
genes appear to have
possible ancient and/or
recent duplications for
Gossypium.
Figure 1. A-genome originated in Africa
while the D-genome originated in Mexico.
Rausher, Mark D., Richard E. Miller, and Peter Tiffin
(1999) Patterns of Evolutionary Rate Variation
Among Genes of the Anthocyanin Biosynthetic
Pathway. Molecular Biology and Evolution, Vol.
16
Rausher, Mark, Yingqing Lu, and Kyle Meyer (2008)
Variation in Constraint Versus Positive Selection
as an Explanation for Evolutionary Rate Variation
Among Anthocyanin Genes Journal of Molecular
Evolution, Vol. 67
Figure 5. Phylogenetic tree of the
CHI gene.
Figure 4. Anthocyanin pathway.
Winkel-Shirley, Brenda (2001) Flavonoid Biosynthesis. A
Colorful Model for Genetics, Biochemistry, Cell `
Biology, and Biotechnology. Plant Physiology,
Vol. 126
Parks, C.R., W. L. Ezell, D.E. Williams, D.L. Dreyer
(1975) The Application of Flavonoid Distribution
to Taxonomic Problems in the Genus Gossypium.
Bulletin of the Torrey Botanical Club
Results
We were able to find DNA sequences
of the following genes for the
anthocyanin pathway: CHS (Chalcone
Synthase), CHI (Chalcone Isomerase),
F3H (Flavanone 3-Hydroxylase), DFR
(Dihydroflavanol Reductase), ANS
(Anthocyanidin Synthase), UF3GT
(UDP glucose flavonoid 3-oxyglucosyltransferase).
References
Koes, Ronald, Walter Verweij, and Francesca
Quattrocchio (2005) Flavonoids: A Colorful
Model for the Regulation and Evolution of
Biochemical Pathways. Trends in Plant Science,
Vol. 10
Grotewold, Erich (2006) The Genetics and Biochemistry
of Floral Pigments. The Annual Review of Plant
Biology, Vol. 57
Figure 2. The A- and D-genome
formed the AD-genome.
Materials and methods
Identified genes present in the anthocyanin pathway under
investigation, and found their DNA sequences by
performing BLAST searches on the NCBI (National
Center for Biotechnology Information) website. Created
phylogenetic trees of the DNA sequences to make sure
that they were the only genes responsible for the
anthocyanin pathway. Ordered primers for the genes
CHS, CHI, F3H and UF3GT. Used primers and PCR
protocol to amplify a specific gene for both the A- and Dgenomes. Each gene was then transformed into bacteria
cells and the cells grew into colonies on petri dishes
overnight. Colonies that appeared to contain our genes
were tested using gel electrophoresis to confirm that the
bacteria contained our gene. The colonies containing our
gene were saved, cloned and sequenced to see if our
bacteria contain our gene of interest.
Acknowledgments
Figure 6. Gel electrophoresis of PCR
products for the F3H (lanes 2 & 3)
and UF3GT (lanes 6 & 7) genes.
Gel electrophoresis allowed
us to isolate and identify our
amplified PCR products for
each gene.
Figure 7. Example X-gal/IPTG
plate.
Figure 8. Gel electrophoresis of plasmid screen for the
F3H and UF3GT genes.
We used ligation and
transformation protocols
to grow our PCR product
in E. coli.
The plasmid screen helped us identify genes that had
been properly transformed into the E. coli cells.
According to our interpretation of our plasmid screens,
we successfully isolated the following genes for both
the A- and D-genome: CHS, CHI, F3H and UF3GT.
Conclusions
Figure 3. An overview of the protocols used to
amplify and sequence a specific gene from the
anthocyanin pathway.
It is our understanding that the anthocyanin
pathway contains six main genes that can
be found bolded in figure four. Out of the
six genes we successfully identified and
isolated all four genes (CHS, CHI, F3H,
UF3GT) for which we designed primers.
According to our phylogenetic trees, of
these four isolated genes, only CHS and
CHI appear to be single copy genes in
Gossypium. Currently, all four isolated
genes are waiting to be sequenced in order
to confirm their identity and copy number.
Future goals
It is our hope that other researchers will continue our project and
soon find the answers to our important, and unanswered questions:
 How many genes in the genome encode each step of the
pathway?
 Which copies of each gene are used at each step?
 How many genes have been retained since polyploidy?
 Have the duplicate copies diverged in their function? When did
they diverge? Before or after polyploidy?
 How are these genes expressed in different tissues and under
different environmental conditions?
 How have any of these changes affected metabolite levels and
total fitness of the plant?
We would like to thank the NSF Plant Genomics
Research Program (grant # DBI-0638418),
especially Adah Leshem-Ackerman and Jonathan
Wendel for the opportunity to participate in the
program. We are very grateful to Lex Flagel for
his mentorship, patience, and willingness to set
aside his other projects to guide us through this
project. Thank you, also, to the other members of
the Wendel Lab who were so supportive, helpful,
and such graceous hosts: Armel Salmon,
Dharminder Pathak, Lei Gong, Ying Bao,
Corrinne Grover, Guanjing Hu and Kara Grupp.
For further information
Please contact Spencer Mesick, Jim
Kollman, Lex Flagel or Jonathan
Wendel through Wendel Lab,
Department of Ecology, Evolution and
Organismal Biology at Iowa State
University, Ames, Iowa.