The basic methodologies of starch gel electrophoresis of maize isoenzymes have been outlined by Cardy et al. (NCSU mimeo 1317; 1317 rev.). In contrast to the requirements for initial genotyping of lines (relatively few plants run over at least 4 pH systems involving 24 or more isoenzyme loci), the monitoring of hybrid seed lots for contamination with female selfed and other rogue seed necessitates the electrophoretic separation of isozyme extracts from a large number of plants over a reduced number of enzyme loci. Variants showing obvious electrophoretic migration differences between male and female lines of a hybrid can be used to detect rogue seed. Coleoptile segments from 5-day seedlings can be used as the source of enzyme. In addition, embryo samples harvested prior to physiological maturity (2-6 weeks following pollination) can also be used as a source of enzyme extract for many hybrids (Smith, S. in review).
We have made modifications to the methods described by Cardy et al. that allow enzyme extracts from large numbers of plants to be assayed with efficient use of time, economic and labor resources. Modifications involve changes in gel dimensions, reduction in electrode buffer volume, changes in sample grinding procedures, alterations in sample application, use of additional isoenzyme stains, increased flexibility in staining protocol, and use of tissue from developing embryos rather than seedling coleoptile sections when data are required prior to the regular harvest date. These modifications, listed below, allow two full-time technicians to run routinely 2000 individual plants per week (20 samples each of 100 plants) for tests of hybrid versus female selfed seed. Approximately 125 plants can be cut and ground per hour by only one person. These modifications also allow 1200 plants per week to be assayed for other rogue, including outcrossed, genotypes.
Modifications (Gel systems A, B, C, and D are those described by Cardy et al.; slight modifications in pH may be required to obtain best resolution):
1. Gel size i) 23 cm long x 23 cm wide x 1 cm deep; volume 670 ml (B
and 0 gel systems).
ii) 13 cm long x 23 cm wide x I cm deep; volume 550 ml (C gel system). NOTE: C gels cannot be double-stacked as gel shrinkage occurs causing gaps to appear along the upper line of sample application.
2. Electrode tray volume 325 ml (platinum wires are fixed to the tray with banana plug connections to the power cables in order to prevent breakage of soldered connections).
3. Grinding process (leaves not removed unless for GLU1) - multi-plant grinder allows 5 individual plants to be ground simultaneously.
4. Sample application - reduced sample wick size (M. C. Mieth punch #448) and application of samples in 2 parallel rows of 50 to give 100 sample wicks per gel.
5. Additional enzyme systems that can be used, gels (recipes for the
pH 7.0 and pH 7.4 systems are given at the end of this script), and tissues:
|Diaphorase||C, 7.0||embryo, coleoptile|
|ACP4||B||embryo (Kahler, J. Hered. 74:239)|
|Menadione reductase||pH 7.0||coleoptile|
|Hexokinase||C, D, 7.0||embryo|
6. Increased combination of stains that can be used on gels of different
pH thereby maximizing the possibility of staining for two or more loci
with the minimum number of gels (NOTE: "A" gel needs very rarely to be
(PHI may have migrating and staining difficulties in some locations)
7. Use of embryo tissue as source of extract. Developing embryos from 2-6 weeks following pollination can be used adopting the techniques described by Cardy et al. with the modifications outlined herein. GLU1 and CAT3 are not active, and ACP1 cannot routinely be clearly resolved from embryo tissue. However, other additional enzyme stains can be used with embryo extracts (see 5 above). More complete details are presented by Smith, S. (in review).
i) pH 7.0 Tris-citrate system - electrode buffer, 16.4 g Trizma base, 9.1 9 approx. citric acid (adjust to pH 7.0), 1 liter water; gel buffer, 1:14 dilution of electrode buffer.
ii) pH 7.4 Tris-maleic system - electrode buffer, 12.4 g Trizma base, 4.1 g approx. maleic acid (adjust to pH 7.4), 3.7 g Na2 EDTA-2H20, 2.0 9 MgCl2-6H20, 1 liter water; gel buffer, 1:10 dilution of electrode buffer.
Our extensive experience in running electrophoretic tests of seed purity in maize leads us to make the following additional comments. Some constraints apply with regard to the use of some loci in certain circumstances when testing for rogue genotypes. These are the difficulties of detecting migration differences between the alleles Glu1-6 and Glu1-7 and between adjacently migrating variants of the E8 locus. Also the locus Mmm can only be used to detect selfed female plants when the female line is homozygous Mmm-mmm and the male line is homozygous Mmm-MMM, since the heterozygous genotype cannot be distinguished from the homozygous Mmm-MMM. Other constraints are due to alleles encoded by different loci which have an overlapping distribution. Thus, Mdh4-12 obscures the contribution of Mdh5-12, and likewise Mdh2-6 obscures Mdh1-6.
If possible, at least two loci that reveal obvious differences between male and female parents should be used to detect female selfs, thus alleviating most interpretational problems caused by any residual segregation at one or more loci. This rule applies to all hybrids whether they be single, 3-way, 4-way, or modified crosses. In those instances where segregation is seen to cause interpretational problems, 50-100 individuals from parental seed lots should be assayed in order to provide information on levels of segregation that could be expected in parental lots and which might not therefore be considered a problem in hybrid seed purity. Segregation information should be shared with breeders in order to ascertain, in particular, whether the observed levels of segregation are to be expected for each parent in question. Identification of possible outcrosses requires that a greater number of loci be assayed, since in most instances the genotype of contaminating pollen is unknown. An additional complication in the identification of outcrossed individuals is that the alien pollen may be from segregating genotypes, thereby causing the genotypes of outcrossed female plants to differ one from another. Thus, the combined effects of outcrossing and segregation within parental seed lots can confound any interpretation of the causes of genotypic variation which may be recorded within hybrid seed lots. However, even when outcrossing is suspected, it is usually unnecessary to assay Cat3, Enp1, Got1, Got2, and Adh1 since these loci reveal relatively little variation across Canadian and U.S. commercial maize germplasm (Cardy and Kannenberg, Crop Sci. 22:1016-1020; Stuber and Goodman, USDA-ARR-S-16; Smith, S., Crop Sci. in review). Little or no variation is also revealed by Mdh4, Mdh5, Mmm and Pgm1. However, other loci of these same enzyme systems do reveal considerable variability.
S. Smith and H. Weissinger
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