VARIATION ASSOCIATED WITH AN AEGILOPS UMBELLULATA CHROMOSOME SEGMENT INCORPORATED IN WHEAT. 11. PEROXIDASE AND LEUCINE AMINOPEPTIDASE ISOZYMES1 T. MACDONALD2 AND H. H. SMITH Department of Biology, Brookhaven National Laboratory, Upton, New York 11973 Manuscript received September 8, 1970 Revised copy received April 21,1972 ABSTRACT Zymograms were analyzed of a number of Triticum aestivum derivatives which incorporated a segment of the Aegilops umbellulata chromosome bearing resistance to leaf rust. Evidence has been presented which suggests that genes involved in the production of two peroxidases and a single peptidase are located on the short arm of wheat chromosome 6B. One peroxidase isozyme, attributed to the presence of the Aegilops segment, was seen in only one of the resistant lines (Transfer) and it was postulated that this peroxidase band was present in a suppressed state in a number of lines. Possible differences in the A and B genomes of T.aestivum and T. dicoccum were discussed. €€Ecommon hexaploid wheat (Triticum aestivum) has twenty-one pairs of Tchromosomes which have been classified into seven homoeologous (related) groups, each of three pairs representing three distinct genomes (SEARS1954, 1966; RILEY and CHAPMAN1966). The three genomes of the hexploid (A, B, and D) appear to have originated from Triticum monococcum, Aegilops speltoides, and Aegilops squarcssa, respectively (MCFADDENand SEARS1944; RILEY,UNRAU and CHAPMAN1958). A number of studies have been carried out in which com- parisons among zymograms of the proposed diploid and tetraploid progenitor species and the hexaploid have been made (JOHNSONand HALL1965; JOHNSON, BARNHARTand HALL1967; SINGand BREWER1969). Electrophoretic examination of a nullisomic-tetrasomic series of hexaploid wheat, developed by SEARS(1966), has led to the demonstration of a gene (or genes) for alkaline phosphatase on chromosomes 4B and 4D (BREWER,SING and SEARS1969), and, more recently, HART(1970a, 1970b) has demonstrated that genes involved in the production of alcohol dehydrogenase (ADH) are located on the beta arm of chromosome 4A and on chromosomes 4B and 4D. The same material was used by SHEPHERD(1968) to locate genes for seed-protein bands on particular chromosomes. He found that fourteen of the seventeen major gliadin proteins were dependent, at least in part, on homoeologous groups 1 and 6. Using This research was carried out at Bmokhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission. The fmst paper in this series appeared in Nature 211: 1425-1426 (1966). The general content of this present paper was first presented at a panel meeting on “Mutation Breeding for Disease Resistance” sponsored by the International Atomic Energy Agency, Vienna, Austria, October 8, 1970. Present address: Department of Biological Sciences, Lowell Technological Institute, hell, Massachusetts 01854. Genetics 72: 77-86 September, 1972. 78 T. MACDONALD AND H. H. SMITH similar material, BARBERet al. (1968,1969) and BARBER,DRISCOLL and VICKERY (1968) have reported evidence which suggests that each of the homoeologues of group 3 is involved in the production of certain fast-migrating esterases. Zymograms have also been used to investigate variation in proteins and enzymes associated with known chromosome segments. BHATIAand SMITH (1966) reported variation in the protein patterns associated with a chromosome segment from Aegilops umbellulata which had been transferred to the hexaploid wheat variety Chinese Spring by SEARS(1956), and which contained the leaf rust (Puccinia triticina Eriks.) resistance locus. Extra bands found in Transfer seed and leaf extracts were attributed to the presence of the Aegilops segment, while the absence of a single band in Transfer which was present in Chinese Spring was attributed to either the deletion of the terminal portion of chromosome 6B, brought about by substitution by the Aegilops segment, or suppression of the structural locus coding for this band by incorporation of the Aegilops segment. Using the same two lines UPADHYA(1 968) demonstrated the presence, in Trans- fer, of a cathodal peroxidase isozyme which was missing in Chinese Spring. The presence of this peroxidase was attributed to the presence of the Aegilops segment. Neither of these two studies included Aegilops umbellulata, or the tetraploid emmer wheat used by SEARS(1956) in the development of Transfer. The pedi- gree of Transfer is such that a considerable number of genes from the tetrapoid emmer wheat may persist. It is quite possible, therefore, that the extra bands found in Transfer are due to the emmer genes. The present study examined a number of Chinese Spring derivatives contain- ing the Aegilops segment, Aegilops umbellulata, the emmx line used in the development of the translocation lines, and two ditelocentric lines involving both arms of chromosome 6B. The aim of the study was to determine whether genes involved in the production of peroxidases or leucyl-P-naphthylamide hydrolyzing peptidases are associated with the incorporated Aegilops segment. MATERIALS AND METHODS SEARS’now classic paper (1956) describes the pedigree of the translocation stocks used in the present study. Aegilops umbellulata Zhuk. was crossed with an emmer (Triticum dicoccum; n = 14, genomes A and B) to produce an amphiploid which was then crossed with common spring wheat, T. aestivum vulgare var. Chinese Spnng. In a second backcross to Chinese Spring, a plant was recovered which was resistant to leaf rust and showed the full wheat complement plus an Aegdops chromosome (2II1l1). Among the selfed progeny of this monosomic addition type W~S found one resistant plant with an added pair of Aegzlops chromosomes (the disomic addition line of the present study), and another resistant plant that had an added isochromosome for the long arm of the Aegilops chromosome (the mono-iso addition line of the present study), in addition to the full wheat complement. In SEARS’experiments the mono-iso addition line was increased and irradiated prior to an- thesis. The pollen was used to pollinate Chinese Spring which resulted in the production of 132 resistant offspring. Twelve plants had translocated part-wheat, part-Aegilops chromosomes sub- stituted for one member of the wheat bivalent instead of being added to the full wheat comple- ment, and thus had 2II1. Five of these showed high pollen transmission and four of them are included in the present study. The four translocation lines are T40 (replacement of most of the short arm of chromosome ISOZYMES IN WHEAT 79 6B with the resistance-bearing portion of the Aegilops chromosome (Aegilops piece) ) ; T44 (a transfer of the Aegilops piece to chromosome 2D) ; T47 designated as Transfer, CI 13296, (a ter- minal substitution of the long arm of chromosome 6B by the Aegilops piece) ; and T52 (a transfer of the Aegilops piece to an unidentified chromosome). Two additional lines involving a leaf rust resistance locus derived from Aegilops umbellulata and incorporated into Chinese Spring were included in the study. These two lines were obtained through the courtesy of Dr. RALPHRILEY and presumably involved the same Aegilops chromo- some. They are here referred to as RILEY'S 43 and 44 addition lines and involve a single and double addition of a modified Aegilops chromosome (A,) to the full wheat compliment. The ad- dition chromosome (A,) is composed of the short arm, the centromere, and much of the long arm of the resistance-bearing Aegilops chromosome, (A), which has an attached small piece of wheat ID chromosome in a terminal position. Chinese Spring, the translocation lines, the disomic addition line, the mono-iso addition line, T. dicoccum (of the emmer line used by SEARS),and two ditelocentric lines involving both arms of chromosome 6B were generously supplied by Dr. E. R. SEARS.We have obtained and grown Aegilops umbellulata from three sources: that of Dr. RALPH RILEY,collection SPI 116294 which was used by Dr. E. R. SEARS,and collection PI 276W4 supplied by Dr. J. C. CRADDOCK,U.S. Department of Agriculture, Beltsville. Peroxidase zymograms were made of all three sources; however, since so few seeds were available of the first two, we used only PI 276994 for the analyses described in this paper. Although we detected no major differences among peroxidases of these three seed sources of Aegilops umbellulafa the comparisons were necessarily cursory because of the noted scarcity of material in two of them. Seeds of the above stocks were sown in Petri dishes on moistened filter paper. They were grown in the dark for the first four days, then transferred to controlled conditions of 27 t 1" C under continuous illumination provided by cool white fluorescent lamps that gave 150 to 2001 ft.-c. at culture level. Tissues (coleoptile, first leaf, and root) were separated and extracted on the sev- enth day of growth in Tris-glycine buffer (0.1 M pH 7.5; 250 mg tissue/0.25 ml buffer) at room temperature. Samples for electrophoresis were absorbFd ontc filtei paper wicks which were added to the freshly ground extracts and then transferred to the sample slot of the gel. The electrophoretic methods described by BREWBAKERet al. (1968) were followed with slight modification. Starch gels (14%) were prepared with 9 parts 0.05 M Tris-0.05 M glycine buffer (pH 8.2) and one p3rt 0.1 M borate-0.05 M glycine buffer (pH8.2). The electrode chambers contained only the borate-glycine buffer. Electrophoresis was carried out at 4°C for approxi- mately 5 hr, or until the brown borate front had migrated 7-8 cm beyond the origin. A voltage of 6-8 V per cm of gel surface was applied. Peroxidases were visualized by incubating the electrophoresed gels at room temperature in a solution made up in the following manner: 250 mg o-dianisidine (3,3'-dimethoxybenzidine) were dissolved in 14.0 ml 95% ethanol then 28 ml 0.2 M acetate buffer (pH 4.8) were added and the mixture was made up to 200 ml by the addition of water.