Download 23 development of molecular markers to distinguish cytoplasm

Summaries of Arkansas Cotton Research 2003
DEVELOPMENT OF MOLECULAR MARKERS TO
DISTINGUISH
CYTOPLASM SUBSTITUTION LINES OF COTTON
T. Burke and J. McD. Stewart1
RESEARCH PROBLEM
Traditionally breeders and geneticists alike have used morphological
characteristics or phenotype to distinguish various genotypes/cultivars. However,
with the advent of cytoplasmic capture, phenotyping can become difficult, if not
impossible. Through this process, lines with new cytoplasms (alloplasmic lines)
differ in their cytoplasmic material but have the same nuclear DNA. Since nuclear
DNA is the primary basis of heredity, notable morphological differences may not
be seen, thus making phenotype selection difficult or impossible. In recent years,
molecular measures have provided a sensitive and reliable means to identify
diversity among genotypes. Both geneticists and plant breeders have come to rely
on marker assisted selection (MAS) as a proven and powerful tool for screening
and selection. The objective of this project is to develop molecular markers for
chloroplastic or mitochondrial DNA, as a means to identify different cytoplasm
substitution lines.
BACKGROUND INFORMATION
Genetic diversity is believed to provide a buffer against adverse effects
such as sudden increases in the virulence of pathogens or pests, or rapid changes
in the environment. In the United States, the danger of genetic vulnerability of
major modern crops was illustrated graphically by the epidemic of the Southern
corn leaf blight, which caused a 15 % reduction in corn output in 1970 (Wright,
1996). The majority of corn hybrids at that time shared a common Texas malesterile cytoplasm that was used because it greatly facilitated hybrid seed production.
This cytoplasm, and all hybrids using the cytoplasm, proved to be highly susceptible
to a race of Southern corn leaf blight (Anonymous, 1997). This epidemic came as
a shock to crop breeders and geneticists and exposed the vulnerability of reliance
on a narrow genetic base for important agricultural crops. The result was a
widespread effort to invest in ex situ preservation and research on germplasm
1
Research specialist and professor, respectively, Crop, Soil, and Environmental Sciences
Department, Fayetteville.
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resources. Our greatest opportunity to reduce or minimize genetic vulnerability in
cotton lies in greater and more efficient use of our feral and/or exotic germplasm
(Anonymous, 1997).
In an effort to increase genetic diversity and reduce disease or insect
susceptibility, the cytoplasms of eleven Gossypium species have been introduced
into the G. barbadense (AD2) nuclear genetic background (Stewart, 1990). G.
barbadense was chosen as the nuclear donor because it contains the semigamy
trait that gives rise to haploid male/female chimeric plants. Using this trait, a
completely new nucleus, such as an elite line of upland cotton, can be transferred
into the cytoplasm in one generation rather than through years of successive
backcrossing. Genetic markers are necessary to readily distinguish among these
alloplasmic lines and any new lines that may be developed.
RESEARCH DESCRIPTION
The Gossypium species cytoplasms examined in this study are listed in
(Table 1). Seeds were sown in pots under optimal conditions in a greenhouse at
Fayetteville, Arkansas. DNA was extracted from leaf tissue using the CTAB
miniprep method of Zhang and Stewart (2000). DNA was quantified using a
spectrophotometer to measure absorbance at 260 nm, and then it was diluted to
20ng/:l. Polymerase chain reactions (PCR) were performed using 10 chloroplast
simple sequence repeat (cpSSR) primer pairs, as well as 37 specific primer
combinations spanning 11 chloroplast genes, introns, and spacers. Genes examined
and primers used are listed in (Table 2). Cleaved amplified polymorphism (CAPS)
studies were also conducted on larger fragments (fragments >800 bps). Products
were digested using BamH1, EcoR1, and MSE1, following manufacturer’s
protocols. All products were resolved by electrophoresis through a 2% high
resolution metaphor agarose gel or on a polyacrylamide gel in TAE and TBE buffers,
respectively. All samples were compared to parental (wild type) DNA as a positive
control.
RESULTS AND DISCUSSION
Polymorphisms were found in the matK-cpDNA, rpl16-cpDNA, and
cpSSR#3 PCR products. PCR with the cpSSR primer yielded four groupings. All
the products from the A and B genome, as well as the F1 alloplasmic line, fell into
group number one. Group two included all of the D and C genomes, as well as the
E1 alloplasmic line. E1 and F1 species samples fell into separate groups, suggesting
a labeling error and loss of the introgressed cytoplasm has occurred. Endonuclease
digestion of the matK-cpDNA fragment also yielded two groups. Group-1
encompassed the A, B, and F genomes. Both the D3-d alloplasmic line and wild
species were included in Group-1, while the D8 and D2-2 alloplasmic lines fell into
Group-2 along with the C1 alloplasmic lines. The E1 species also fell into Group1, while the alloplasmic line belonged to Group-2. This further indicates the “E1”
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Summaries of Arkansas Cotton Research 2003
alloplasmic line does not contain the E1 cytoplasm. Also, the pattern of the wild
C1 species did not match that of the “C1” alloplasmic line. Digestion of the rpl16
fragment also yielded two groups, separating the D8 and B1 lines from the remaining
alloplasms in the study. The identity of these lines was confirmed.
The low level of polymorphisms found among the cytoplasms in relation
to the number of primers used and digestions performed can be explained by the
highly conserved nature of chloroplast DNA. Chloroplast DNA is inherited
maternally and, therefore, remains extremely conserved from one generation to
the next. This is also evident in the polymorphisms that were found. Groupings
based on DNA polymorphisms almost always included all of the lines from a specific
genome, and separated only lines of another genome. With the exception of the
D3-d alloplasmic line, no polymorphisms were found with which to distinguish
species cytoplasms within a genomic group. Additional investigation of chloroplast
genes, as well as studies aimed at mitochondrial genes, is needed to find specific
polymorphisms within each genome. After complete examination, fragments will
be cloned, sequenced, and the sequences screened for single nucleotide differences
among cytoplasms. Since mitochondrial sequences are less conserved, studies
will also be performed on the DNA of this organelle using the same techniques
described.
PRACTICAL APPLICATION
The need for genetic markers to distinguish among cytoplasmic
substitution lines is apparent by the lack of morphological diversity between the
lines. Even with the limited data obtained thus far, the assumed cytoplasmic
constitution of three of the alloplasmic lines appears to be incorrect. Observations
relative to the influence of cytoplasm on performance of an alloplasmic line are
meaningless if the line is incorrectly identified.
ACKNOWLEDGMENTS
This research was supported in part by a Cotton Incorporated grant to
enhance cotton genetic diversity, for which we express our deep appreciation.
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LITERATURE CITED
Anonymous. 1997. Cotton Germplasm Status Report.
(http://www.ars-grin.gov/npgs/cgc_reports/cotton97.htm).
Cronn, R.C., R.L. Small, T. Haselkorn, and J.F. Wendel. 2002. Rapid diversification of the cotton genus (Gossypium: Malvaceae) revealed by analysis of
sixteen nuclear and chloroplast genes. Am. J. Botany. 89:707-725.
Small, R.L., R.J. Ryburn, R.C. Cronn, T. Seelanan, and J.F. Wendel. 1998. The
tortoise and the hare: Choosing between non-coasting and plostome and
nuclear Adh. sequences for phylogeny reconstruction in a recently diverged
plant group. American J. of Botany. 85:1301-1315.
Stewart, J.M. 1990. New cytoplasms for cotton. Pp. 55-58. In: D.M.
Oosterhuis (ed.).
Proc. 1990 Cotton Research Meeting. Ark. Agri. Exp. Sta., Special Report 144.
Wright, B.D., 1996. Crop genetics resource policy: Towards a research agenda.
Department of Agricultural and Resource Economics, University of California at Berkley.
Zhang, J.F. and J.M., Stewart. 2000. Economical and rapid method for extracting cotton genomic DNA. J. Cotton Sci. 4:193-201.
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Table 1. List of Gossypium cytoplasmns and alloplasmic lines in this study.
Species
Genome
Origin
arboreum
A2
Indian Subcontinent
tomentosum
A3
Hawaiian Islands
mustelimum
AD4
Northeast Brazil
darwinii
AD5
Galapagos Islands
anomalum
B1
Southwest Africa
sturtiamum
C1
Central Australia
harknessii
D2-2
Baja, California
davidsonii
D3-d
Baja, California
trilobum
D8
West Central Mexico
sticjsuu
E1
Arabian Penninsula
longlicalyx
F1
East Central Africa
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Table 2. Chloroplast genome area and amplifying primers.
1
Genes
Primers
rp116-cpDNA1
F71, RF-int, R1516, R1661
matK-cpDNA1
trnKF, trnKF2, trnKF3,
TrnKF4, trnKR
trnT-trnL-cpDNA1
trnA2, trnB, trnL2
ndhF-cpDNA1
5' Fnew, 536F, 803F, 972F,
3'R, 972R, 1318R
atpB-rbcL spacer2
atpB, rbcL
trnL-trnF spacer2
E,F
trnT-trnL spacer2
A, B, TrnT-I
accD-psaI spacer2
accD-769F, accDI, psai-75R
ndha intron2
ndhA-F, nahA-R, nahA-I
rpI16 intron2
F71,R1661, R1516
rpoCI intron2
5'rpoCI exon, rpoCI exon2
Cronn et al. (2002).
Small et al. (1998).
2
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