Evaluation location: Wisconsin, United States
Ref. Theor Appl Genet (1989) 78:119-128. Plant material and sample prep. 753 C. sativus and 4 C. hardwickii currently available PIs were obtained from the Regional Plant Introduction Station, Ames, IA in March 1987. Initially, a random sample of cotyledons was taken from the collection to determine the most appropriate buffer system for electrophoresis of 49 enzymes. Subsequently, a random array of 16% of the accessions was evaluated for variation using these enzymes. The remainder of the collection was then surveyed for potential polymorphisms based on information obtained in this initial evaluation. Cotyledons of 15 individuals from each PI were harvested from 7 to 14-day-old seedlings germinated and grown in vermiculite. Approximately 0.01 g of cotyledonary tissue from each seedling was ground in 0.1 ml of buffer solution containing 0.67g/l TRIS base and 7.02 g/l of TRIS-HCl, pH 7.1, and centrifuged at 100 x g for 5 min. Individuals were sampled and either used immediately or stored at -70 C before horizontal starch gel electrophoresis using the techniques of May et al. (1979) and May (1980). Gels consisted of either 42 g or 56 g of a 1:1 mixture of hydrolyzed potato starch (Sigma Co., St. Louis, Mo.) and Connaught starch (Connaught Laboratories, Willowdale, Ontario, Canada) dissolved in either 300 ml or 400 ml of buffer, respectively. Gel and electrode buffers described by Clayton and Tretiak (1972), Ridgeway et al. (1970), and two by Selander et al. (1971) were used and are referred to as C (pH 6.1 gel and electrode), R (pH 8.5 gel, 8.1 electrode), S-4 (pH 6.7 gel, 6.3 electrode), respectively (Table 1). Filter paper wicks (3 x 8 mm, Schleider and Schuell No. 470) were dipped into the homogenized cotyledon tissue and loaded on the gels. A wick dipped in red food coloring (SCM Corporation, Westlake, Ohio in water, propylene glycol, and artificial colors) was placed at the end of each gel to monitor the rate of movement of the front. To standardize the relative mobilities of the electromorphs (bands), extracts of the C. sativus inbred processing cucumber line GY-14a were loaded on each gel and band mobilities were recorded in relation to GY-14a bands. An electric potential of 125-250 V (<=75 mA) was applied to the gels until the marker dye migrated to within 30 mm of the end of the gel. A gel was sliced horizontally into four or more sections by drawing monofilament thread through it. Modified staining solutions of Allendorf et al. (1977), Brewer (1970), and Shaw and Prasad (1970) were used to visualize banding patterns of 40 enzyme systems (Table 1). Evaluation of germplasm. Isozyme banding pattern were recorded for analysis. The inheritance of the allozymes used in this study are not known. However, based on information from other organisms (Markert 1978; Neale et al. 1984; Chiang et al. 1987), it is possible to assign provisionary genetic bases to the observed eletromorphs. Nomenclature follows a modified form (Staub et al. 1985, 1987) previously described by Richmond (1972), such that loci coding for enzymes (uppercase) are designated by the first letter being uppercase and the rest lowercase. If an enzyme is coded by multiple loci, these are designated by hyphenated numerals and are numbered from most cathodal to most anodal. Alleles of a given locus are numbered from most cathodal to most anodal and enclosed in parenthesis. The most common allele of a locus was designated 100, and all other alleles were assigned a value based on the mobility (mm) of their homomeric protein product relative to that of allele 100. For example, an allele of MDH-3 which had a mobility of 2 mm greater than the most common allele was assigned the designation MDH-3(2)-102. Analytical procedures. Three types of multivariate analyses were utilized to depict affinities and dissimilarities among individual PIs and PIs grouped by geographic region. PIs were placed into 45 groups (regions) according to the source country indicated by the NCRPIS.