Department of Agronomy ASA-CSSA-SSSA 2009 International Annual Meeting, Pittsburgh, PA November 3, 2009 Screening of Gametophytic Incompatibility in Maize with SSR Markers and Pollen-mix Test Moisés D. González 1, Linda Pollak 2, Walter Goldstein 3, and Susana Goggi 1 INTRODUCTION 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 (Ga1 S ) 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, Ga1 S, occurs in the same locus as Ga1 (Nelson 1993). The difference between Ga1 S and Ga1 alleles is that pollination of plants with genotype Ga1 S /Ga1 S 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 Ga1 S alleles have been found only in certain varieties of popcorn, whereas the North American dents and flints tested are ga1/ga1 (Nelson 1993). 1–Iowa State University Department of Agronomy, 2–USDA ARS CICGRU, 3–Michael Fields Agricultural Institute RESEARCH OBJECTIVES  Screen gametophytically compatible and incompatible plants for genetic polymorphism with simple sequence repeat (SSR) markers.  Compare the efficiency of the pollen-mix test and genotyping with SSR markers on backcross progeny of inbred lines and breeding populations. METHODS Phenotypic evaluation of gametophytic incompatibility  Backcrossing was used to introduces Ga1 S from the donor parent Mo508W/Mo506W into the recurrent parents B114, B125, Mo47/MRBC, CH05 015:N12, AR13 035:B73, and AR16 035:S19.  Fifteen plants from each recurrent parent and their respective BC 1 progeny were grown side-by-side. Pollen collected from individual BC 1 plants was divided into approximately two equal amounts; one- half 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).  Classification of individual BC 1 progenies as compatible or incompatible was made according to the number of yellow and purple kernels obtained from individual mix pollinations (Fig. 2). BC 1 plants with the lowest percentage of purple kernels were considered incompatible (genotype Ga1 S /ga1), and their BC 2 progeny were selected. The same procedures were used to test BC 2 obtained from backcrosses of incompatible BC 1 plants. 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 Ga1 S /ga1) and produce yellow kernels. Genotyping with SSR molecular markers  Approximately 100 mg of leaf tissue was harvested from the recurrent parents, the donor parent, the F 1 progeny, and individual BC 2 plants from inbreds and breeding populations that were subjected to pollen mix for DNA extraction.  Twenty nine SSR markers from the maize database were used to screen for genetic polymorphism (Lawrence et al., 2005).  PCR products were stained with ethidium bromide and separated by electrophoresis on 4% high-resolution agarose. RESULTS Fig. 3 Genotyping with SSR marker umc1943 and gametophytic- incompatibility test for seven BC 2 plants from inbred lines. B114 B125 CH05 015:N12 AR13 035:B73 Mo47/MRBC AR16 035:S19 CONCLUSIONS  The SSR marker umc1943 was the only marker that co-segregate with Ga1 S among the parents, and backcross progeny (Fig. 3 and Fig. 4).  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.  Genotyping of backcross progenies with the SSR marker umc1943 results in the identification of heterozygotic individuals with the Ga1 S allele.  Use of DNA markers is more reliable for screening of cross- incompatibility than phenotypic evaluation alone. REFERENCES Emerson RA (1925) A possible case of selective fertilization in maize hybrids. Anat Rec 29:136 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 Mangelsdorf PC, Jones DF (1926) The expression of Mendelian factors in the gametophyte of maize. Genetics 11:423–455 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 Schwartz D (1950) The analysis of a case of cross-sterility in maize. Proc Nat Acad Sci USA 36:719–724 Ziegler KE, Ashman B (1994) Popcorn. In: Hallauer AR (ed) Specialty Corns. CRC Press, Boca Raton, FL, pp 189–223 Acknowledgements We would like to express our appreciation to Penny Meyerholz, Alan Gaul, and Anania Fessehaie. Fig. 4 Genotyping with SSR marker umc1943 and gametophytic- incompatibility test for seven BC 2 plants from breeding populations. Fig. 2 Ears from two different BC 1 plants of the breeding population CH05 015:N12 segregating for Ga1 S after the pollen-mix test. A plant with genotype ga1/ga1 is compatible to pollen from purple stock (a) and a plant with genotype Ga1 S /ga1 is incompatible to pollen from purple stock (b). a b a b BackcrossRecurrent Blue corn