A PCR-based assay for the wx-c allele of maize
--Dave Zaitlin

Many elite maize dent inbred lines have nearly isogenic counterparts that have been "converted" to the waxy kernel phenotype by introgression of the recessive wx gene through conventional backcross breeding. The efforts by many industrial corn breeders to accomplish this have been driven by the increasing market for waxy grain as animal feed and by the industrial demand for cornstarch composed solely of amylopectin. To effect a rapid conversion of Agrigenetics starchy inbreds to waxy, we required a simple, precise and very fast assay that would allow us to distinguish the mutant gene from the wildtype gene. Additionally, the assay of choice had to be essentially non-destructive, as we wanted to perform it on very young maize seedlings, and had to be able to unequivocally identify Wx/wx individuals. While not as inexpensive as I/KI staining of kernels, the polymerase chain reaction (PCR) meets all of the above criteria.

Wessler and Varagona (PNAS 82:4177-4181, 1985) used molecular techniques to determine the structure of the Wx locus in a collection of wx mutants. This study identified twelve different genetic events, both deletions and insertions, that can give rise to the waxy phenotype in maize. Discussions with commercial maize breeders revealed a general uncertainty as to the particular allele(s) of wx carried in commercial wx inbreds. However, both George Sprague and Sue Wessler believed that wx-c, which originated in China, was a possible candidate since it does not revert to Wx and has been known in the U.S. for over 80 years. While in Dr. Wessler's laboratory Ron Okagaki discovered a small deletion, undetectable by conventional Southern blotting, that almost certainly accounts for the mutant phenotype in plants carrying wx-c. Dr. Wessler generously provided me with the (unpublished) position of this lesion on the Wx gene sequence of Klosgen et al. (Mol. Gen. Genet. 203:237-244, 1986). The 30bp deletion spans nucleotides 1606 to 1636 (relative to the start of transcription) and encompasses the exon7-intron7 border, which apparently disrupts the normal in vivo splicing pattern and produces a non-functional mRNA.

The 500bp of sequence surrounding the 30bp deletion averages 51% G+C, unlike other regions of Wx which can exceed 85%. Two primers which matched this G+C composition were designed to direct in vitro amplification across the position of the gene sequence encompassing the deletion. Primers wx#2 (5'-ACCTCAAGAGCAACTACCAG-3'; nucleotides 1567-1576) and wx#3 (5'-CTTCATCCAGTTGATCTTCCGG-3'; nucleotides 2000-1979) were synthesized and purified at Epicentre Technologies in Madison, WI. A typical 50µl amplification reaction contained 200-250ng of maize DNA, the primers at a concentration of 1µM each, dATP, dCTP, dGTP and TTP at 0.2mM each, 1 unit of Taq DNA polymerase, and the buffers and salts as specified by the enzyme supplier (Perkin-Elmer Cetus). Sequence amplification was effected by cycling the reactions 30 times at 94 C for 1 minute, 55 C for 1.5 minutes and 72 C for 3 minutes. As predicted from the gene sequence, a DNA fragment of 433 base pairs was amplified from genomic DNA isolated from the inbreds C123, W64A, Oh43, Va35, and MBS501. DNA from a maize stock carrying the wx-c reference allele (from O. E. Nelson, OE) and C123wx, W64Awx, Oh43wx and MBS501wx directed synthesis of a 403bp fragment in the PCR, however. Mixing experiments and amplifications with DNA extracted from known Wx/wx heterozygotes showed that the 433 and 403bp fragments are clearly separable on agarose gels of 1.2-1.5% (w/v) in standard Tris-acetate electrophoresis buffers. The notion that wx-c has been widely incorporated into commercial dent corn inbreds is also supported by the finding that DNA from plants homozygous for the mutant waxy alleles wx-B, wx-B1, wx-B6, wx-K, wx-M, and wx-Stonor (see Wessler, S and Varagona, 1985) all support amplification of the 433bp fragment in the PCR.

Coupled with a technique for extracting total DNA from very small amounts of plant tissue (Zaitlin, DeMars and Ma, Genome, in press, 1993) the PCR-based assay for detection of wx-c should be valuable for accelerated conversions of starchy inbreds to waxy. The breeder can simply screen 10-20 seedlings after each backcross and save those that are Wx/wx for the next generation. Fewer individuals need to be carried along and, after 4 or 5 cycles, conventional RFLP can be used to identify the plants that most closely resemble the recurrent parental line. These are then selfed and the resulting seedlings subjected to PCR to find the wx/wx plants. 

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