A Simple Method to Test Genetic Allelism in Nearly Sterile Interspecific Plant Hybrids

Abstract

The comparison of electrophoretic isozyme patterns of multimeric enzymes in leaf and pollen extracts from the same individual has been used to distinguish allozymes and isozymes. This is possible because in genetic heterozygotes leaf tissue exhibits both homomers and heteromers whereas pollen shows only homomers. I show that pollen of nearly sterile interspecific plant hybrids contains isozymes that can be examined in the same way. This permits a test to determine whether genes encoding particular isozymes in the parental species have remained allelic; i.e., located at corresponding positions on homologous chromosomes. Such a test is illustrated by examining cytosolic PGI in two classic hybrid combinations in Clarkia. The evidence shows that the coding PGI genes of the parental species are located on different chromosomes (or at different sites on the same chromosome), possibly resulting from reciprocal translocation as proposed by Lewis and Raven. The ability to determine whether genes in different species segregate as alleles or not will likely be useful in studying the extent of gene linkage conservation and as a supplement to RFLP and other DNA-directed maps. Formal genetic analysis is generally restricted to crosses between individuals of a single species because fertile hybrids are necessary to produce an F2 or other segregating progeny. However, for genes encoding multimeric enzymes expressed in pollen and detectable by electrophoretic assays, I show here that tests of allelism can be carried out between species even when their hybrids have very low fertility. This new test is an extention of previous results in which the electrophoretic isozyme pattern of extracts from leaves or other somatic tissues is compared with that of pollen from the same individual (Weeden and Gottlieb 1979). The test depends on the fact that following meiosis the haploid cells that develop into pollen receive one or the other allele at every locus, but not both. Thus if a particular locus is heterozygous for alleles encoding allozymes having different electrophoretic mobilities, leaf tissue exhibits both homomers and heteromers, but only homomers are synthesized in the pollen because any single grain receives only a single allele. For a dimeric enzyme in heterozygous individuals, extracts from a mass of pollen exhibit two homodimers but not the electrophoretically intermediate heterodimer. Its absence is apparent when patterns from the pollen and leaves are compared. In contrast, if the two homodimers are encoded by two different homozygous loci, then both leaf tissue and pollen show the same three-banded pattern. The pollen-leaf comparison has proved useful to test allelism of electrophoretic variants of numerous enzymes within plant species in which formal breeding studies cannot be readily carried out (Crawford 1990). The allelism of variants in different species can also be tested by the pollen-leaf comparison because inviable pollen often characteristic of interspecific hybrids contains many functional enzymes detectable by electrophoresis. In this report, I demonstrate the use of the pollen-leaf test to assess allelism of electrophoretic variants in two classical interspecific hybrids in Clarkia: C. franciscana x amoena and C. amoena x rubicunda (Lewis and Raven 1958a, 1958b). MATERIALS AND METHODS Plants. Interspecific hybrids were produced by crossing Clarkia amoena subsp. huntiana (Jepson) Lewis & Lewis x C. rubicunda (Lindley) Lewis & Lewis and x C. franciscana Lewis & Raven. Clarkia amoena subsp. huntiana was collected in Lake Co., near Manning Creek Bridge on Rt. 175, west of the junction with Rt. 29 (LDG 9025), and in Marin Co., on Lucas Valley Rd., 5.8 miles east of Nicasio Schoolhouse (LDG 714). Clarkia rubicunda was collected in Marin Co., at Ft. Barry (LDG BAR). Clarkia franciscana was represented by two populations, the type locality at The Presidio in San Francisco Co., and a newly discovered population in the foothills above