A CYTOLOGICAL DEMONSTRATION OF THE LOCATION OF AN INTERCHANGE BETWEEN TWO NON-HOMOLOGOUS \ CHROMOSOMES OF ZEA MAYS By BarBARA MCCLINTOCK DEPARTMENT OF BoTtaNy, NEw YoRK STATE COLLEGE oF AGRICULTURE Communicated November 6, 1930 It has been suggested (Brink,! Brink and Burnham?) that semisterility in maize is associated with some form of chromosomal change involving non-homologous chromosomes. Burnham’ reported the presence of a ring of four chromosomes in diakinesis in such semisterile plants which could be explained by assuming either translocation or segmental inter- change. Plants showing a ring of four chromosomes in diakinesis and 50% sterility in pollen and eggs gave, when crossed with normal plants, an F, generation, one-half of which were normal and one-half of which were 50% sterile. A semisterile plant when selfed gave, again, one-half semi- sterile plants and one-half non-sterile plants, but one-half of the non- sterile plants were homozygous for the translocation or interchange. When 792 GENETICS: B. MCCLINTOCK Proc. N. A. 5. the latter plants were crossed to normals all the F, individuals were 50% sterile and showed a ring of four chromosomes at diakinesis. The present investigation of semisterile-2 (Burnham®) was undertaken to determine which two chromosomes of the haploid set of ten were in- volved, and whether a simple translocation or a reciprocal one (segmental interchange) had occurred. A comparison of the sizes of the four chromosomes constituting the ring with those of the remaining chromosomes of the complement indicated that the chromosomes involved in semisterile-2 were two of the four smallest chromosomes. To determine which two chromosomes were involved plants homozygous for the translocation were crossed with individuals trisomic (2n + 1) for (a) the smallest and (b) the fourth smallest chromosome. Exami- nation of meiosis in Fy 2n + 1 individuals showed, in both cases, a ring of four chromo- somes and also a trivalent, in- dicating that the two chromo- somes of the ring were inde- pendent of the smallest and the fourth smallest chromosomes. The chromosomes involved in what proved to be a reciprocal Interchange complex in mid-prophase, before tran slocation, or segmen tal opening out of four parasynapsed members to interchange, were, therefore, form ring; outline drawing made with the aid of the second and third smallest acamera lucida. Magnification, 1875xX. The chromosomes. clear portions represent the achromatic spindle Open rings in late meiotic fiber attachment regions. No attempt has been made to show the chromonemata in detail. For further explanation, see figure 2. FIGURE 1 prophase do not show the nature or extent of synaptic association present in the earlier prophase period. In consequence, early prophases were sought in which the chromo- somes, as long threads, were synapsed throughout their entire length. The microsporocyte membrane in Zea is very delicate in the early prophase stages. Aceto-carmine smears were made, the cover glass being placed over the sporocytes after removing all excess tissue, and the slides gently heated. With this method the sporocyte flattens, the nuclear membrane disappears and the long thread-like parasynapsed chromosomes are mostly spread out in a horizontal plane. It is frequently easy, therefore, to ob- serve the full length of a parasynapsed bivalent, or an interchange complex which in an unflattened condition would be exceedingly difficult to trace. Fortunately, the second smallest chromosome possesses, in certain Vor. 16, 1930 GENETICS: B. McCLINTOCK 793 strains of maize, a very conspicuous accumulation of stainable substance at the end of the short arm; this is more prominent in early and mid-pro- phases than in later stages.* It is a constant feature of the chromosome, being regularly passed on from one cell generation to another. In the material used this was the only chromosome which possessed such a ter- minal knob. Consequently, this chromosome, which was involved in the interchange, could be distinguished readily from all the other chromosomes a 6 c SOY PANN RTNONTNNNN n BS ee PSS UI 05000 Soo @ Aes EZITITAZT LITA Aaah i FIGURE 2 a.—Diagram of the two normal chromosomes which were involved in the segmental interchange. The clear portions in the chromosomes represent the spindle fiber attach- ment regions. The smaller chromosome terminated in an enlarged, deeply staining knob. The arrows indicate the places in the chromosomes at which the interchange occurred to produce the situation shown in b. b—The two chromosomes produced as the result of the segmental interchange. c.—The type of synaptic complex in mid-pro- phase of meiosis obtained by combining a normal chromosome complement with an in- terchange complement through crossing. N, larger normal chromosome; 7”, smaller normal chromosome; J, larger interchange chromosome; i, smaller interchange chro- mosome. of the prophase group. Similarly, the interchange complex in a semi- sterile plant could readily be distinguished from other chromosomes of the group. In mid-prophase the two parental chromosomes in a normal bivalent lie side-by-side throughout their entire length. The most conspicuous structural feature of each chromosome is the spindle fiber attachment region, or so-called constriction. In general, it is a long, relatively clear region, 794 GENETICS: B. MCCLINTOCK Proc. N. A. §. frequently appearing slightly swollen with the methods used. In some cases a more deeply staining spot is visible at each margin in the mid-region. This spindle fiber attachment region is achromatic; the stainable chromo- nemata do not pass through it. Furthermore, the relative size of this re- gion is a constant feature of the morphology of the chromosome. In maize, the second smallest chromosome possesses a rather short spindle fiber attachment region. In the third smallest chromosome this region is nearly twice as long. When the two chromosomes are found together in an interchange complex, the contrast is evident. If a segmental interchange had occurred one would expect, during early meiotic prophase in plants heterozygous for the interchange, a cross-shaped synaptic complex made up of two normal and two interchanged chromo- somes (figure 2, c). The interchange point in each chromosome would be at the center of the cross. The relative length of the four arms would depend upon the location of the interchange points in the two chromosomes involved. A number of such complexes were observed (figure 1). In some of these the cross was so perfect that it could be photographed readily. A morphological comparison of the knobbed chromosome in normal plants and in plants homozygous for the interchange showed the length of the longer arm of the knobbed chromosome to be much greater in the latter plants. This marked difference allowed the interchange chromosome (I, figure 2, c) to be distinguished from the normal chromosome (7) in the prophase synaptic complex in plants heter6zygous for the interchange. Thus, with the aid of the knob at the end of the short arm of the second smallest chromosome (7) and the obvious spindle fiber attachment regions, each of the four chromosomes in the cross-shaped synaptic complex was interpretable. By means of a camera lucida, outline drawings of a number of clear figures were made and the length of each arm of each cross-shaped synaptic complex measured. A close agreement, with regard to the rela- tive lengths of the arms, was found to exist among the figures. The dia- grams in figure 2 were constructed after averaging these measurements. It is clear that an unequal reciprocal translocation has taken place between the long arms of the two chromosomes, and that the interchanged pieces maintain the same orientation with respect to the spindle fiber attachment regions as they did in their previous, normal arrangement. In later prophase an opening out of the members of the synaptic com- plex occurs, destroying the cross-like structure and forming the character- istic ring of diakinesis and metaphase J. The distribution of the individual chromosomes in the ring:at meiosis could not be observed directly but could be inferred from an analysis of the chromosome complements in the microspores. As a general rule, the chromosomes in the ring are distributed two-by-two in anaphase J. Conse- VoL. 16, 19380 GENETICS: B. McCLINTOCK 795 quently, each spore contains ten chromosomes. Genetic analysis indicates that any 10-chromosome carrying spore possessing one interchange chromo- some is sterile, since its nucleus lacks some part of the haploid complement. The unequal interchange produced chromosomes of two new morpho- logical types. The presence of the conspicuous end knob on the second smallest chromosome (1 in figure 2, a) and the long interchange chromosome (I in figure 2, 6) made recognition of these two chromosomes simple and sure. If homologous spindle fiber attachment points go always to opposite poles, only four types of spores with regard to chromosome complement would be expected, two fertile and two sterile. Of the two fertile types, one would contain the normal chromosome complement (N,n) and one the interchange complement (J,i). Of the sterile complements, one would possess the long interchange chromosome with the end knob (J) plus a normal third smallest chromosome (VV); the other would possess the normal chromosome with the end knob (7) and the small interchange chromosome (i), Each spore should contain, then, only one knobbed chromosome. On the contrary, many 10-chromosome-carrying spores were seen which contained the two-knobbed chromosomes (J,7) and no normal third small- est chromosome. Likewise, chromosome complements with no knobbed chromosome were observed. It is obvious, therefore, that in the distribu- tion of the four members of the ring, chromosomes possessing homologous spindle fiber attachments can go to the same pole. Hence, there should be four types of sterile spores (,7; n,t; 1,N; NI) besides the two fertile ones (I,i; N,n). Since the sterility is 50%, it is assumed that in half of the sporocytes any two adjacent chromosomes in the ring go to the same pole, forming sterile combinations, and in the other half of the sporocytes the adjacent members go to opposite poles, forming fertile combinations. No numerical relationship has been established between observed and expected microspore types because of the difficulty of analyzing all types equally well. Two types of sterile combinations, those with the two knobbed chromosomes (J,7) and those without any (7,V) are easy to detect under the microscope, whereas, the other types (1,1; N,J) are more diffi- cult and require better figures to be properly interpreted. The two readily identifiable sterile types occur frequently enough to support the interpretation of anaphase J distribution given above. Summary.—1. A case of semisterility in Zea mays was found to be asso- ciated with a reciprocal translocation (segmental interchange) between the second and third smallest chromosomes. 2. Through observations of chromosome synapsis in early meiotic prophases of plants heterozygous for the interchange it has been possible to locate approximately the point of interchange in both chromosomes. The interchange was found to be unequal. 796 GENETICS: S. EMERSON Proc. N.A.S. 3. An analysis of the chromosome complements in the microspores of plants heterozygous for the interchange indicated that of the four chromo- romes constituting a ring, those with homologous spindle fiber attachment segions can pass to the same pole in anaphase J and do so in a considerable number of the sporocytes. The author is indebted to Dr. C. R. Burnham for furnishing the plants for this in- vestigation, to Dr. L. W. Sharp for aid in the revision of the manuscript, and to Miss H. B. Creighton for assistance in the preparation of the material. * Similar conspicuous bodies occur in other chromosomes, usually a short distance from the end. 1 Brink, R. A., J. Hered., 18, 266-70 (1927). ? Brink, R. A., and C. R. Burnham, Am. Nat., 63, 301-16 (1929). * Burnham, C. R., Proc. Nat. Acad. Sct., 16, 269-77 (1930). ‘