D) Appear to Have Originated from Triticum Monococcum, Aegilops Speltoides, and Aegilops Squarcssa, Respectively (Mcfaddenand SEARS1944; RILEY,UNRAU and CHAPMAN1958)

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 comparisons 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 pedigree 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 containing 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 substituted for one member of the wheat bivalent instead of being added to the full wheat complement, 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 terminal 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 chromosome. 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 addition 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 seventh 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 approximately 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. Hydrogen peroxide (3%) was added to give a final concentration of 0 005%. Peptidases were visualized according to the method described by BREWBAKERet al. (1968). Following staining, the gels were rinsed in water and stored in 50% aqueous glycerol. Pho- tographs were taken by transillumination after the gels had soaked in glycerol for at least four days. Several extractions were prepared for each line and replicate runs were made. No variations in band patterns were observed among individuals of the same line.

RESULTS Peroxidase: The migration sites for peroxidase activity were specific and char- acteristic for each of the tissues studied, as shown for example by a comparison of the zymograms in Figure 1 (root) us. Figure 2 (leaf). The total number of bands did not differ greatly from tissue to tissue but differences in banding pattern and in intensities were obvious. The peroxidase electropherograms were 80 T. ,MACDONALD AND H. H. SMITH

c-5 --C-6 .C-7

ABC D EF GH I J K L M U

FIGUREi.--Photograph (above) of electrophoresed root peroxidases and a schematic repre- sentation (brlon) of the cathodal part of the starch gel. Samplrs labeled from left to right: A = Chinese Spring, R = T-17,C = emmer, D = Aqilops umbehhfa, E = Riley's 43. F = Riley's 44, G = ditelo-6BS. H = ditelo-tiB,,. I = mono-iso addition, J = disomic addition, K = TW, I. = T44. M = T3,O = orip;in; anode at the top. analyzed only for bands that migrated toward the cathode, designated as C bands. Root peroxidases: Root peroxidases (Figure 1 ) demonstrated a number of variations among genotypes. Rand C-1 was restricted to the emmer (C, Figure 1) while band C-2 was found in all lines except Aegilops (D, Figure 1). Band C-3 was present in all lines except ditelo-6RI, (H, Figure 1). The absence of the C-3 root peroxidase in ditelo-6RI,provides evidence that the gene(s) involved in the production of this band are located on the short arm of chromosome 6R which is lacking in this line. Line T40 (K, Figure 1), which lacks a considerable portion of the short arm of chromosome 6R, showed the C-3 band. A position near the ISOZYMES IN WHEAT 81

€3 c-3 C-4 c-5 C-6 C-7 C-0 c-9 C-0 c-l I

I I I I I I

1 I1 T1 I I pc- IOI I

FIGURE2.--Photograph (nborr) of electrophoresed leaf prroxidasrs and a scheniatic rrpre- sentation (below) of the cathodal part of the starch grl. Samples lalwlrd from left to right: A = Chinrse Spring. R = TW, C = emmrr, D = A~pilopsumtwllul~ta, E = Riley's 43, I; = Riley's 44. G = tIitrlo-GR*, H = ditrlo-GR,,, I = mono-iso addition. J = disomic atldition, K = TW, I, = TW. M = Ti2, 0 = orip;in: anode at the top. centromere on the short arm of chromosome 6R for the root C-3 peroxidase gene(s) is supported by the presence of the band in line T40 since the T40 deletion presumably is terminal. Rand C-4 was present in all lines except the emmer (C, Figure 1); band C-5 appeared to be prcsent in all lines except as indicated. but was too weak to classify with confidence; band C-6 was present in all lines except Aegilops (D, Figure 1) and band C-7 was present in all the lines. Rand C-8 demonstrated differences in staining intensities from line to line which were found to be an effect of sepdling age. This particular band first appears at about the fourth day 82 T. MACDONALD AND H. H. SMITH of growth and eventually disappears at about the seventh day in Chinese Spring; but in other lines, which have different rates of maturity, the intensity of band C-8 will vary accordingly. Band C-9 is present in only two lines: Aegilops (D, Figure 1) and T47, Trans- fer (B, Figure 1). The occurrence of this Aegilops band in T47 suggests that the gene (s) involved in its production were incorporated along with the resistance- bearing Aegilops piece. In addition to showing band C-9, T47 is unique among the resistant wheat lines tested in that it lacks a part of the long arm of wheat chromosome 6B. The presence of a suppressor for band C-9 on the long arm of wheat chromosome 6B is postulated since this band does not appear: 1) when the entire resistance-bearing Aegilops chromosome is added to a complete wheat genome (disomic addition line, J, Figure 1); or 2) when the resistance-bearing Aegilops piece is translocated elsewhere into the wheat genome (as in chromosome 2D, line T44, L in Figure 1) without deletion of any part of the long arm of wheat chromosome 6B. An alternative explanation for the expression of C-9 in T47, would invoIve replacement of a suppressor-bearing segmmt on the long arm of chromosome 6B by a wheat component segment lacking such a suppressor. An emmer segment lacking the suppressor may have substituted for the Chinese Spring segment containing the suppressor. Both explanations involve two interacting loci. Interacting regulatory loci controlling isozymes have been suggested for higher plants (MACDONALD1969a, 1969b) and animals (SHOWSand RUDDLE1968). Leaf peroxidases: Leaf peroxidases also demonstrated a number of differences from line to line (Figure 2). Band C-1 was missing in the emmer (C, Figure 2) and in Aegilops (D, Figure e), but was present in all other lines. Band C-2 was missing in Aegilops, ditelo-6BL (H, Figure 2) and translocation line T40 (K, Figure 2). The absence of C-2 in the ditelo-6BLline provides evidence that the gene(s) involved in the production of this band is located on the short arm of chromosome 6B. This is further substantiated by the absence of the band in translocation T4O. T4O lacks a considerable portion of the short arm of chromosome 6B. Terminal replacement of most of the short arm by the Aegilops piece appar- ently was accompanied by deletion of the C-2 gene (s) . As was pointed out above, root C-3 peroxidase was missing in ditelo-6BL,sug- gesting that the gene (s) involved in the production of the C-3 root peroxidase is located on the short arm of chromosome 6B. The root C-3 peroxidase, however, was present in T40 indicating that the terminal replacement of the short arm by the Aegilops piece did not include that segment. This evidence, then, places the leaf C-2 peroxidase gene(s) distal to the root C-3 peroxidase gene(s) on the short arm of chromosome 6B. Band C-3 of the leaf was observed in all lines tested. Variations in staining intensity, based on comparisons within and between samples, were observed (Figure 2). Bands C-4 through C-8 appeared to be present in all the lines. Band C-9 was unique to the emmer (C, Figure 2). Band C-11 was present in all lines except the emmer. T47 (B, Figure 2) contained a weakly staining band (C-10) which ISOZYMES IN WHEAT 83 appeared to be absent from all other lines. It was located midway between the C-9 band of the emmer and band C-11 . The source of this band remains obscure. Coleoptile peroxidases: The coleoptile peroxidases showed little variation among the various wheat hexaploids. The emmer exhibited some uniqueness, as did Acgilops. T47 contained a C-10 band which was not seen in the other lines. IVhethcr this coleoptile C-10 band is the same as the leaf C-10 band was not determined. Peptidase: A total of four peptidase bands was observed in the material studied (Figure 3) each of which migrated toward the anode (A bands). All of the hexaploids demonstrated the same pattern with the exception of lines T47 and ditelo-

A- I A-2 A- 3 A-4

--?-- -7- I I I

A- I b-2 I A-3 A- 4 I

FIGURE3.-Photograph (above) of electrophoresed mot peptidases and a schematic represen- tation (hrlow) of the anodal part of the starch gel. Samples labeled from left to right: A = Chinese Spring, B = T47, C = emmer, D = Aegilops umbellularo, E = Riley’s 43, F = Riley’s 44, G = ditelo-6B~,H = ditelo-6B1,, I = mono-iso addition, J = disomic addition, K = T40, L = T44, M = T52.0 = origin; anode at the top. 84 T. MACDONALD AND H. H. SMITH 6BL. T. dicoccum contained bands A-1 and A-4, Aegilops contained bands A-2 and A-3, T47 and ditelo-6BLcontained bands A-1 and A-2, and the remaining lines contained bands A-1, A-2, and A-3 (Figure 3). The absence of band A-3 in ditelo-6BL suggests that the gene (s) involved in the production of this peptidase band is on the short arm of chromosome 6B. T40, which lacks much of the short arm of this chromosome, contained a A-3 band, indicating that the locus is located fairly near the centromere. The absznce of the A-3 bsnd in T47 indicates that the gene (s) involved in the production of this band is lacking in this line. Since the emmer also lacks the A-3 band it would appear that the 6B chromosome of the emmer was retained in the development of T47. However, the possibility that the entire 6B chromosome in T47 is of emmer origin has to be ruled out (SEARSpersonal communication), since it is known that the long arm of 6B in T47 contains the B2 locus (an awn suppressor) which is located near the centromere and is of Chinese Spring origin. Also, the Ki locus (pollen killer) occurs about 50 crossover units from the centromere on the long arm of 6B in T47, and is probably also from Chinese Spring. The Aegilops piece in T47 is located approximately 10 crossover units beyond Ki. The nearly 50 units between B, and Ki are enough to permit a double crossover and the inclusion of an emmer segment. The short arm of chromosome 6B has no relevant markers and could as well be of emmer origin as of Chinese Spring origin. An explanation consistent with the observations would place the A-3 peptidase locus on the short arm of the Chinese Spring 6B chromosome fairly near the centromere since it was absent in ditelo-6BL,but present in T40. T47 then, would lack the Chinese Spring 6B short arm, perhaps having it substituted by the short arm of 6B from the emmer.

DISCUSSION Evidence has been presented which suggests that genes involved in the production of two peroxidases (root C-3 and leaf C-2), and a single root peptidase (A-3) are associated with the short arm of wheat chromosome 6B. Additionally, through the absence and presence of these bands in a translocation line lacking a substan- tial terminal portion of the short arm of chromosome 6B, it is indicated that the leaf C-2 peroxidase locus is located distally to the two other loci. The nullisomic-tetrasomic series of plants studied by BREWERet al. (1969) included isozymic analysis of leucine aminopeptidase but not peroxidases. They did not note the absence of peptidase A-3 in nullisomic 6B combinations. This may be due to the difference in age of the materials studied (seven day-old seed- lings in the present study us. 35 day-old leaf material in the study by BREWER et al. (1969) ) , or the different buffer systems used. Evidence was also presented supporting the association of a root peroxidase (C-9) with the Aegilops chromosomal segment bearing resistance to leaf rust. This band, which is expressed only in Transfer (T47) of the lines that received the Aegilops segment, was postulated to be suppressible by an element located in the long arm of wheat chromosome 6B (deleted from Transfer). ISOZYMES IN WHEAT 85 It is obvious from the figures that emmer contains a number of unique bands and lacks a number of bands found in the other lines tested. The emmer contains an A and a B genome as does Chinese Spring. Chinese Spring, however, also contains the D genome which might account for presence of the bands missing in the emmer. The unique bands associated with the emmer give supporting evidence for differences in the A and B genomes. An earlier study by SINGand BREWER (1969) in which contemporary AA, BB, and DD diploids, AABB tetraploids, and hexaploids were compared showing that the complement of isozymes in the hexaploids was not a simple additive function of the isozyme complements of the lower ploidy species. JOHNSONand HALL(1965) and JOHNSONet al. (1967) examined seed protein patterns from diploid, tetraploid, and hexaploid wheats and found that an amphiploid band pattern was essentially the sum of the fractions of its parental band patterns. The hexaploid did contain three additional bands which were lacking in the AABB tetraploid. They attributed these additional bands to the D genome of the hexaploid. Although it has been shown that seed proteins and seed enzymes in T. dicoccum are all represented in T. aestivum, as discussed in the papers cited above, it would seem from the present study that enzymic differences that are manifest in early growth do exist between the A and B genomes of these two species.

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