independent Inheritance of Genes for Dhurrin and Leucoanthocyanidin in a Sorghum Cross

Flag leaves of KS8 sorghum (Sorghum bicolor (L.) Moench] are low in dhurrin (p-hydroxy-(S)-mandelonitrile-p-D-glucosidel content and thus in hydrocyanic acid potential (HCN-p), and they contain little (if any) leucoanthocyanidin (LAC). Comparable leaves of 'Colman' sorghum are intermediate in HCN-p and high in LAC. This study was conducted to investigate the inheritance of HCN-p and LAC in crosses of KS8 X Colman. Flag leaves from field-grown plants of both parents; the Flo KS8 X Colman; the backcross, KS8 X (KS8 X Colman); and the F 2, (KS8 X Colman) selfed, were assayed for both HCN-p and LAC. Assays for HCN-p indicated that the backcross values provided a good fit to 1 intermediate:l low (Xl = 0.05, P = 0.82) and F2 results to 3 intermediate:l low (Xl = 0.96, P =0.33). For LAC, backcross results were 1 LAC +:1 LAC(Xl = 0.20, P = 0.65) and F2 values were 3 LAC+:1 LAC(exact fit). Classification for both traits yielded good fits to 1:1:1:1 for the backcross (Xl = 0.30, P = 0.96) and 9:3:3:1 for the F 2 (Xl = 3.24, P = 0.36). These results indicated that the difference in HCN-p between Colman and KS8 depended primarily on a single gene, the difference in LAC depended largely on a separate single gene, and the HCN-p gene and the LAC gene were not linked. A 1986 REPORT (1) indicated that a single gene difference was primarily responsible for the difference in dhurrin content (expressed as hydrocyanic acid potential, HCN-p) of mature leaves between KS8 (lowHCN-p) and N32 (high-HCN-p) lines of sorghum. Similarly, the large difference in flag-leaf leucoanthocyanidin (LAC) content between 'White Collier' (LAC-) and Colman (LAC+) sorghums depended largely on a single pair of genes (2). Both KS8 and N32 were LAC-, and the HCN-p values for flag leaves of both Colman and White Collier were intermediate between the values for KS8 and N32 (EA. Haskins and H.J. Gorz, 1988, unpublished observations). Therefore, the crosses used in the previous studies were considered inappropriate for investigating the possible linkage of the HCN-p and LAC traits. The objective of the present study was to investigate the segregation of these two traits in a cross of KS8 (low-HCN-p, LAC-) X Colman (intermediate-HCN-p, LAC+). Materials and Methods Colman (male-fertile), AKS8 (cytoplasmic male-sterile), and BKS8(male-fertile, sterility maintainer) sorghum plants weregrown at the Agronomy Farm, Lincoln, NE, during the summer of 1985. The status of Colman with respect to fertilityrestoration was unknown; therefore, to ensure that malefertile F, plants would be produced, BKS8 plants were hand emasculatedand pollinated with Colman pollen. In NovemFA Haskins, Dep, of Agronomy, and H.J. Gorz, USDA-ARS and Dep. ofAgronomy, Nebraska Agric. Res. Div.,Lincoln, NE 68583. Published as Paper no. 8484, Journal Series, Nebraska Agric. Res. Div. Research was conducted under Project no. 12-114. Received 27 Nov. 1987. *Corresponding author. Published in Crop Sci. 28:864-865 (1988) ber, 1985, the F, seed was sent to Puerto Rico (Tropical Agricultural Research Station, USDA-ARS, Mayaguez)where F, plants were self-pollinated to produce F2 seed, and were also used as pollen parents in backcrosses to AKS8. AKS8 X Colman crosses also were made during the summer of 1985 to obtain larger quantities ofseed from which F , plants could be grown for sampling. Plants of BKS8, Colman, the F, (AKS8 X Colman), the backcross [AKS8 X (BKS8 X Colman)], and the F2 [(BKS8 X Colman) selfed] were started in the greenhouse during the spring of 1986 and were transplanted to the Agronomy Farm on 29 May. Plants were placed 0.61 m apart in rows with a 0.76-m spacing. The experiment was planted in four replications, with each replication including one 10-plant row of each parent and the F" two rows of the backcross, and five rows of the F2• Thus, the experiment was designed to include 40 plants of each parent and the F I , 80 backcross plants, and 200 F2 plants. Entries were assigned at random to the 10 rows in each replication. Insufficient plants of both parents and the F, were available for transplanting; therefore, seeded rows of BKS8, Colman and the F, were used in two, three, and one of the replications, respectively. On 30 July, when panicles were emerging from most plants, the blade of the flagleaf was harvested from each plant. One KS8 and 21 F2 plants were not sufficiently advanced to allow positive identification of the flag leaf; for these plants, the blade ofthe youngest leaf with a visible collar was harvested. Midribs were removed from the leaf blades, and the remaining tissue was dried at 70 to 75°C overnight. The dry tissue was ground through a I-mm screen and stored in plastic vials at -18°C prior to extraction for assay. Extraction and assay for HCN-p were conducted as described previously (I) except that dhurrin was hydrolyzed enzymatically rather than with NaOH. The enzyme preparation was an extract made by soaking defatted almond meal (Sigma Chemical Co., St. Louis, MO)' in distilled water (8 mg mL -I) and filtering the suspension through Whatman no. 1 filter paper. To hydrolyze dhurrin, I mL of this filtrate was added to I mL of leaf extract, and the mixture was incubated in a parafilm-capped tube at room temperature for 1.25 h. Following this incubation, 8 mL of 0.1 MNaOH was added, and a l-mL portion of the resulting solution was assayed colorimetrically as described previously (I). The procedure described by Haskins and Gorz (2) was used for extraction and assay of LAC. Absorbance at 540 nm was used as a measure of LAC content. Results and Discussion Segregation for HCN-p Mean HCN-p values for KS8 and Colman were 29 and 135 mg kg-I, respectively, and standard errors were such that these means appeared to be well separated (Table 1). However, ranges in HCN-p for these two parents overlapped slightly, which caused some uncertainty in the classification of backcross and F2 plants as either low (L) or intermediate (I) in HCNp. As shown in the table, with 50 mg kg-I as the dividing line between the classes, only one of 37 KS8 plants was classified as I-HCN-p and only two of 40 Colman plants were classified as L-HCN-p. With this dividing line, 41 of the backcross plants were I-HCNI Names ofproducts are included for the benefit of the reader and do not imply endorsement or preferential treatment by the USDA or the Univ. of Nebraska.