Transcript Poster2009

ASA-CSSA-SSSA 2009 International Annual Meeting, Pittsburgh, PA
November 3, 2009
Department of Agronomy
Screening of Gametophytic Incompatibility in Maize with SSR Markers and Pollen-mix Test
1
González ,
2
Pollak ,
3
Goldstein ,
1
Goggi
Moisés D.
Linda
Walter
and Susana
1–Iowa State University Department of Agronomy, 2–USDA ARS CICGRU, 3–Michael Fields Agricultural Institute
97 bp
85 bp
M
W
B1
25
Ga
1S
F1 1
2
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3
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5
6
03
5:
S1
9
2
85 bp
72 bp
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Plant number
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S
5
W a1 H0 F 1 2 3
M G C
1
2
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Plant number
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 Progenies of B114, and AR16 035:S19 that amplified two bands with
marker umc1943 had significant lower percentages of purple
kernels.
 Genotyping with the SSR marker umc1943 and the pollen-mix test
resulted in different genotypic scores among the progeny of
parents B125, Mo47/MRBC, CH05 015:N12, and AR13 035:B73.
Purple kernels
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4
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Plant number
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 Genotyping of backcross progenies with the SSR marker umc1943
results in the identification of heterozygotic individuals with the
Ga1S allele.
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 Use of DNA markers is more reliable for screening of crossincompatibility than phenotypic evaluation alone.
CH05 015:N12
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REFERENCES
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Yellow kernels
Purple kernels
Percentage of total
kernels per ear
Emerson RA (1925) A possible case of selective fertilization in maize hybrids. Anat Rec
29:136
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80
60
40
20
0
102 bp
87 bp
Lawrence CJ, Seigfried TE, Brendel V (2005) The maize genetics and genomics database.
The community resource for access to diverse maize data. Plant Physiol 138:55–58
1
F1 1
2
3
4
5
Plant number
6
Mangelsdorf PC, Jones DF (1926) The expression of Mendelian factors in the
gametophyte of maize. Genetics 11:423–455
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Nelson OE (1993) The gametophyte factors of maize. In: Freeling M, Walbot V (eds) The
maize handbook. Springer–Verlag, New York, NY, pp 496–503
03
5:
B7
3
M
W
G
a1 S
AR
13
100
80
60
40
20
0
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Fig. 3 Genotyping with SSR marker umc1943 and gametophyticincompatibility test for seven BC2 plants from inbred lines.
2
1
N
5:
1
0
 PCR products were stained with ethidium bromide and separated by
electrophoresis on 4% high-resolution agarose.
Purple kernels
CONCLUSIONS
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0
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 Twenty nine SSR markers from the maize database were used to
screen for genetic polymorphism (Lawrence et al., 2005).
Purple kernels
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a
 Approximately 100 mg of leaf tissue was harvested from the recurrent
parents, the donor parent, the F1 progeny, and individual BC2 plants
from inbreds and breeding populations that were subjected to pollen mix
for DNA extraction.
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 The SSR marker umc1943 was the only marker that co-segregate
with Ga1S among the parents, and backcross progeny (Fig. 3 and
Fig. 4).
Yellow kernels
Genotyping with SSR molecular markers
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1
Mo47/MRBC
Ga1S
Classification of individual BC1 progenies as compatible or
incompatible was made according to the number of yellow and purple
kernels obtained from individual mix pollinations (Fig. 2). BC1 plants
with the lowest percentage of purple kernels were considered
incompatible (genotype Ga1S/ga1), and their BC2 progeny were
selected. The same procedures were used to test BC2 obtained from
backcrosses of incompatible BC1 plants.
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Percentage of total
kernels per ear
F1 1
Fig. 2 Ears from two different BC1 plants of the breeding population
CH05 015:N12 segregating for Ga1S after the pollen-mix test. A
plant with genotype ga1/ga1 is compatible to pollen from purple
stock (a) and a plant with genotype Ga1S/ga1 is incompatible to
pollen from purple stock (b).
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100
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60
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20
0
1
b
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7
100 bp
85 bp
a
4
B125
Yellow kernels
Fig. 1 Backcrossing and evaluation of gametophytic incompatibility in
maize. The purple corn stock has genotype ga1/ga1 and
produce dark kernels whereas the backcross progenies
segregate for the gene (ga1/ga1 and Ga1S/ga1) and produce
yellow kernels.
3
Yellow kernels
AR13 035:B73
2 3
4
5
6
Schwartz D (1950) The analysis of a case of cross-sterility in maize. Proc Nat Acad Sci
USA 36:719–724
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Yellow kernels Purple kernels
95 bp
75 bp
Ziegler KE, Ashman B (1994) Popcorn. In: Hallauer AR (ed) Specialty Corns. CRC Press,
Boca Raton, FL, pp 189–223
Percentage of total
kernels per ear

3
4
5
Plant number
Blue corn
Phenotypic evaluation of gametophytic incompatibility
Fifteen plants from each recurrent parent and their respective BC1
progeny were grown side-by-side. Pollen collected from individual
BC1 plants was divided into approximately two equal amounts; onehalf was used for self pollination, and the remainder was used for
backcrossing a single plant in the recurrent parent. Approximately the
same amount of pollen from a purple corn stock was applied
simultaneously to the silks of plants that where self-pollinated to
produce a mixture of two kinds of pollen on the silks (Fig. 1).
2
2
Purple kernels
96 bp
84 bp
1
Compare the efficiency of the pollen-mix test and genotyping
with SSR markers on backcross progeny of inbred lines and
breeding populations.
Backcrossing was used to introduces
from the donor parent
Mo508W/Mo506W into the recurrent parents B114, B125,
Mo47/MRBC, CH05 015:N12, AR13 035:B73, and AR16 035:S19.
F1 1
7
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METHODS

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Yellow kernels
Blue corn
b

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Percentage of total
kernels per ear

Screen gametophytically compatible and incompatible plants for
genetic polymorphism with simple sequence repeat (SSR)
markers.
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Recurrent
RESEARCH OBJECTIVES

3
Percentage of total
kernels per ear
Backcross
2
B114
Percentage of total
kernels per ear
M
W
Ga
1S
B1
14
F1 1
M
W
Ga
1S
M
o4
7/
M
RB
C
Some varieties of popcorn (Zea mays L.) have the ability to reject fertilization by pollen
from dent and flint corn (Nelson 1993). This trait, known as gametophytic incompatibility,
is controlled by a single dominant gene (Ga1S) and modifier genes (Emerson 1925). The
most important application of cross-incompatibility in maize is to prevent or minimize
contamination by pollen from transgenic corn. The screening of gametophytic
incompatibility is normally made by crossing plants with a mix of its own pollen and pollen
from a stock that produces dark color pigmentation in the kernels (Ziegler and Ashman
1994). However, two limitations of this approach are that selection of incompatible
progenies must be delayed until harvesting, and sometimes, the expression of
pigmentation in seeds is not uniform.
The most-studied gametophyte gene in maize is the gametophyte 1, Ga1, on
chromosome four. Among Ga1 and ga1 gametes produced from a heterozygous plant,
Ga1 gametes fertilize more ovules than ga1 gametes on plants with either genotype
Ga1/Ga1 or Ga1/ga1 (Mangelsdorf and Jones 1926). However, if the same mixture of
pollen grains is used to pollinate plants with the genotype ga1/ga1, selective fertilization
does not occur, and both kinds of gametes have equal probability of fertilizing the ovules.
In the absence of pollen grains with the Ga1 allele, pollen with the ga1 allele leads to
seed set in plants with either homozygous or heterozygous genotype. A third allele, Ga1S,
occurs in the same locus as Ga1 (Nelson 1993). The difference between Ga1S and Ga1
alleles is that pollination of plants with genotype Ga1S/Ga1S with pollen of genotype
ga1/ga1 results in gametophytic incompatibility even in the absence of Ga1 pollen grains
(Schwartz 1950). However, if the pollen has the same genotype as the female plant, full
seed set can occur. The Ga1 and Ga1S alleles have been found only in certain varieties
of popcorn, whereas the North American dents and flints tested are ga1/ga1 (Nelson
1993).
M
W
Ga
1S
AR
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RESULTS
INTRODUCTION
AR16 035:S19
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20
0
Acknowledgements
We would like to express our appreciation to Penny Meyerholz, Alan Gaul, and Anania Fessehaie.
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Plant number
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Fig. 4 Genotyping with SSR marker umc1943 and gametophyticincompatibility test for seven BC2 plants from breeding populations.