Determination of the Chromosomal Location of a Glutamate Oxaloacetate Transaminase Structural Gene Using Triticum-agropyron Translocations1

The glutamate oxaloacetate transaminase (GOT) zymogram phenotypes of a series of 15 translocation lines, a chromosome addition line and a chromosome substitution line were determined. In each of the translocation lines a segment of the long arm of Triticum aestivum chromosome 3D has been replaced by a portion of an Agropyron elongatum homoeologue. Evidence was obtained that the products of the T . aestivum GOT-3 triplicate structural gene set randomly dimerize with the product of the homoeologous A. elongatum gene. Each translocation chromosome was found to carry either Got-D3 or Got-Ag3. By correlating the zymogram phenotype expressed by each translocation line with the observed frequency of meiotic pairing of each 3D/3Ag translocation chromosome with telocentric-3D1, it was shown that Got-D3 is located in the proximal portion of 3DL, slightly more than 4.3 crossover units from the centromere. The results of this genetic study confirm and extend earlier conclusions derived from cytogenetic studies as to the physical nature of the various 3D/3Ag chromosomes. TRUCTURAL genes for a number of isozymes have been linked to specific chromosome arms of hexaploid wheat (Triticum aestiuum L. em. Thell., 2n = 42, genomes A, B, and D) by zymogram analyses of appropriate aneuploid strains (BARBER et al. 1968,1969; HART 1970,1973,1975; HART and LANGSTON 1975; NISHIKAWA and NOBUHARA 1971). The available aneuploid strains of the cultivar “Chinese Spring” allow an examination of the phenotypic effects of from zero to four doses of each chromosome and of most chromosome arms (SEARS l954,1966a, 1966b). The structural gene linkages have been obtained by the demonstration that the level of expression of a specific gene product varies concordantly with the dosage of a specific chromosome arm. This type of genetic analysis accordingly employs as genetic variation genetic differences between Cooperative investigations of the Department of Plant Sciences, Texas A&M University, College Station, Texas and the Crops Research Division, Agricultural Research Service, US. Department of Agriculture, Columbia, Missouri. Technical article No. 12223 of the Texas Agricultural Experiment Station. Geneticist, Agricultural Research Service, U S . Department of Agriculture. Genetics 83: 49-61 May, 1976. 50 G. E . HART, D. E . M C M I L L I N A N D E. R. SEARS the A, B, and D genomes 01 the species rather than differences between allelic forms of genes. This paper reports the determination of an approximate gene-centromere genetic distance for a glutamate oxaloacetate transaminase (GOT: E.C.2.6.1.1.) structural gene, Got-D3, located in the long arm of chromosome 3D. Hexaploid wheat expresses several genetically-independent GOT systems (HART 1974, 1975). Got-D3 is one of three members of a homoeologous set of structural genes which encode the isozymes of the GOT-3 system (HART 1975). The genetic distance between gene and centromere is readily determined by means of telocentric chromosomes (SEARS 1962, 1966b), provided allelic variation is available. In the absence of a variant form of Got-D3, we have used as genetic variation a difference between Got-D3 and Got-Ag3, a homoeologous Agropyron elongutum gene. The genetic distance was determined by zymogram analysis of a series of 15 translocation lines in which segments of 3DL of substantially differing lengths have been replaced by portions of 3AgL, an A. elongatum homoeologue (SEARS 1972a, 1973). The results of this study confirm and extend SEARS’ earlier conclusions (1973) as to the physical nature of the 3D/3Ag chromosomes. This paper also reports recent cytological studies of five other translocation chromosomes of similar derivation and a zymogram analysis of lines containing these chromosomes. Earlier studies of variation in proteins and enzymes associated with segmental interchanges between hexaploid wheat and related species have been reported by BHATIA and SMITH (1966), UPADHYA (1968), BARBER et al. (1969), and MACDONALD and SMITH (1 972). MATERIALS A N D METHODS The pedigree and characteristics of the 15 homozygous 3D/3Ag translocation lines used in this study have been described (SEARS 3972a, 1973). 3Ag, an Agropyron elongatum (2n=70, x=7) chromosome which conditions resistance to a leaf-rust fungus, Puccinia reconditu, is substituted for wheat chromosome 3D in the line designated TAP 67. A substitution line derived from TAP 67 by a cross and three backcrosses to “Chinese Spring” was made monosomic for chromosome 5B, and was then used as the female parent in a cross with “Chinese Spring” nullisomic-5B, tetrasoniic-5D, in order to obtain nulli-5B, tri-5D, mono-3D, mona-3Ag plants. Meiotic pairing between 3D and 3Ag was made possible in these plants due to the absence of Ph, the chromosome 5B locus which prevents homoeologous pairing. Pairing of 3D with 3Ag occurred in about 30% of the meiocytes of the plants, as a consequence of which numerous 3D/3Ag translocation chromosomes carrying leaf-rust resistance were recovered. Fifteen disomic lines, each homozygous for a different 3D/3Ag translocation chromosome, were subsequently derived. Tests of these lines indicated that in each a segment of the long arm of the Agropyron chromosome 3Ag (3AgL, formerly designated 3Aga) had replaced a segment of the long arm of wheat chromosome 3D (3DL, formerly 3Da). Five additional lines containing other translocation chromosomes (described in SEARS (1 973) and in the RESULTS section of this paper) derived from this same sequence of crosses were also analyzed with the zymogram technique. Four of the five l inecthose carrying chromosomes 10, 12, 13, and 20were homozygous for the translocation chromosome while the fifth carried chromosome 9 in the heterozygous condition. Translocation chromosome 12, which is composed of segments of both 3B and 3D of wheat as well as 3Ag, was substituted for chromosome 3D in the CHROMOSOMAL LOCALIZATION OF GOi-D3 51 line examined. In each of the other four lines, the translocation chromosome was substituted for the wheat chromosome involved in the translocation. Also determined were the GOT-3 zymogram phenotypes of a chromosome substitution line (carrying 20“+1”3Ag(3D)) derived from TAP 67 by a cross and three backcrosses to “Chinese Spring” and of a chromosome addition line (carrying 21 “+1”3Ag) derived from TAP 67 by a cross and five backcrosses to “Chinese Spring”. The Agropyron accession whose chromosome is contained in these lines is no longer available. Extracts were obtained for electrophoresis from shoots of nine-day-old etiolated seedlings grown in moist paper toweling at 23”. Preparation of the tissue extracts, disc acrylamide gel electrophoresis, and staining for GOT activity were performed as described by HART (1975). A minimum of three plants of each line was analyzed.