THE CYTOLOGICAL IDENTIFICATION OF T CHROMOSOME ASSOCIATED WITH THE R LINKAGE GROUP IN ZEA MAYS BARBARA McCLINTOCK AND HENRY E. HILL Cornell University, Ithaca, New York Received March 1. 1930 T-ABLE OF CONTENTS INTRODUCTION ................... hlethods .............................. Observations on trisomic inheritance of R. ... Independence of the R-G linkage group ....... SUmI.~RS ............................... .iCKNOWLEDGMEXTS ...................... LITERATURE CITED ...................... ;\PPEXDIS ............................ . . PAGE 175 176 176 186 190 190 190 190 INTRODUCTION Genetic investigations with Zea mays have established ten linkage groups. Likewise, cytological investigations have revealed the presence of ten morphologically identifiable chromosomes composing the haploid complement (MCCLINTOCK 1929b). It is the aim of the present investi- gation to correlate a particular linkage group with a particular chromo- some. The method employed has been to obtain 2nfl plants trisomic for different members of the haploid complement, and then, by means of trisomic inheritance, to determine which chromosome carries a particular group of genes. The 2nf 1 plants have been obtained from the progeny of one original triploid (MCCLINTOCK 1929a). The chromosome-number in the female gametes of a triploid varies from ten to twenty. Trisomic individuals were obtained directly from theFr of a cross triploid X diploid and from the 2nf 1 progenies of FI individuals with more than one extra chromosome. Inheritance data obtained from the 2nfl plants of culture 131 sug- gested that in this culture the r-g carrying chromosome was present in triplicate. An effort was therefore made to test the validity of this inter- pretation and to verify the genetic inference that the nine other linkage groups are independent of the r-g chromosome. The evidence indicates that the smallest chromosome of the haploid complement carries the genes of the r-g linkage group. Thus, 2n + 1 plants of culture 131 showing trisomic inheritance for r have the smallest chromo- some in duplicate in their 11-chromosome microspores. Likewise, 2nfl (hi~~rcs 16: li5JIr 1931 176 BARBARA McCLINTOCK AND HENRY E. HILL plants of other cultures which show the smallest chromosome in duplicate in ll-chromosome microspores have given trisomic inheritance for r in later genetic investigations. METHODS Root-tips were fixed in a chromic-acetic-formalin mixture and sectioned in paraffin. The aceto-carmine smear method was used for sporocytes which previously had been fixed in an acetic-absolute alcohol mixture. The microspores were fixed in an acetic-absolute alcohol mixture, stained with carmine and cleared with chloral hydrate. For morphological studies of the chromosomes, late prophase stages of the division of the microspore nucleus were found most valuable, since the chromosomes at this stage are longer, their constrictions are more obvious and the relative length of their arms is more readily determined than in the contracted metaphase stage. From comparative studies of the most easily distinguishable chromosomes of the complement it is clear that the morphology as shown by the prophase and metaphase microspore figures is essentially similar to that shown in the root tips. The presence of only the haploid complement and the ease of observation in the micro- spore favored the use of this stage for cytological studies. OBSERVATIONS ON TRISOMIC INHERITANCE FOR K As has been stated, the 2n+l individuals of culture 131 were trisomic for the r-g chromosome. This culture arose from a 2n+l plant which had been selfed. Of 61 individuals examined, 39 (or 63.7 percent) were 2n, 21 (or 34.4 percent) were 2n+l and 1 (or 1.6 percent) was 2n+2. It can be safely assumed that all the 2n+l plants of this culture were trisomic for the same chromosome, since the normal rate of non-disjunction in Zea mays is very low. As a result of the distribution of the members of the trivalent at meiosis, 1 l-chromosome carrying and lo-chromosome carrying gametes are formed. In a normal pollination, the extra chromosome carrying pollen grains do not function well in competition with the n-carrying pollen grains (see table 1). For genetic investigations, therefore, it is necessary to TABLE 1 Percenl of 2n+l individuals resulting from th cross Zn 0 XZn+l cf. 2n? x2n+k? 2n 2n+1 Percent 2n+1 1921x224 261 3 1.13 1928x225s 83 2 2.35 IDENTIFICATIOS OF LINKAGE GROUPS 177 consider the functioning of 11-chromosome carrying gametes only in the case of the female. On the basis of random distribution of the three similar chromosomes at meiosis one should expect half of the eggs to carry the extra chromosome, but actually only about one-third of the eggs carried it (see above and table 2). This discrepancy can be explained on the basis of irregularities at meiosis. Nine bivalents and one tri- valent are found at metaphase I only in approximately two-thirds of the sporocytes; in the other sporocytes there are ten bivalents and one uni- valent. When the extra chromosome appears thus as a univalent its TABLE 2 aTumber Zn+l : 2n ittdividuals from the cross Zn+f Q XZnd. CTx.TCRE 166 168 176 224 225 229 230 231 232 - _- -_ I 2n . -____ 11 1 5 2 14 10 14 17 9 - _- -- I 2nfl 4 1 2 2 5 10 4 2 11 Totals 83 41 33.06 percent 2n+l behavior is very irregular (MCCLINTOCK 1929a). It may not go into the spindle figure but remain in the cytoplasm. It may be found in an ab- normal position in the spindle. Again, the univalent may lag in the central part of the spindle with or without showing evidence of. a separation of its split halves. If the halves should be included in the two telophase I r&lei, they would probably lag in the second meiotic mitosis. In all of these cases a loss of the univalent will occur in meiosis, with the formation of all n-carrying nuclei instead of half n-carrying and half nf 1. This phenomenon could account for the increased ratio of n to nfl gametes. The difference in many cases is probably not due to lack of viability of n+ 1 gametes or 2nf 1 plants, since in many ears of Zea mays the regular- ity of row and kernel position allows undeveloped kernels to be readily de- tected. Some 2nfl plants bore almost perfectly filled ears. It is possible, also, that the lowest megaspore, if it contains the extra chromosome, does not fuuction to produce the embryosac but, in a certain percent of the GENETICS 16: XI 1931 178 BARBARA McCLINTOCK ASI, HENRY E. HILL cases, one of the megaspores that contains the haploid complement func- tions instead. The trisomic individuals of culture 131 were crossed so that their prog- enies were heterozygous for at least one factor of every linkage group. Backcross and FZ ratios were obtained to determine which factors were inherited on a trisomic and which on a disomic basis. Abundant evidence for trisomic inheritance of r in the 2nf 1 progenies of culture 131 (cultures 189, 209, 224, 225, 229, 231) was obtained. Simple ratios may best be considered first. When the R factor for red aleurone is duplex (RRr) the gametic ratio expected from random dis- tribution of the extra chromosome is 2R:2Ry: 1 KR: lr, or a total of 5R: 1~. Since only the n-carrying pollen grains need be considered, the functioning male gametic ratio is 2R: 1~. In the 2n+ 1 progenies of culture 131 duplex for R the backcross ratios through the pollen were 646R:3.5 (table 3), ~9 XRRrS 1928X2253 192gX209,g 19210x224~ Totals COLORED COWRLEES 240 91 198 141 20s 123 646 35.5 a fair approximation to 2: 1. On the same basis, crosses of 2n KY P X2n +lRRr $ should give 5X: Iv. Table 4 shows a total count of 2102R:135. A sib cross between two heterozygous 2n individuals gave 290R:88v, or the expected 3 : 1 ratio (table 7). RrO XRRr8 22511X2242 225,tX224$ 2317x2319 231*4x2319 23116)<231!J 23117x2313 Totals - _- - TABLE 4 CoLORED COLORLEsa 26s 74 235 49 3s2 61 419 82 357 s2 444 Si 2102 . I 435 When R is simplex (Rrr) the expected gametic ratio is 1 R : 2 RY : lrr : 2~. -With elimination of the n+l carrying pollen grains the functioning male IDENTIFIChTIO?U' OF LIhKiGE GROUPS 179 gametic ratio is 1 R : 2~. In the progenies of culture 131 only one individual tested was so constituted and gave a backcross ratio through the pollen of 110X: 23-b (table 5). Conversely a 2: 1 ratio is expected in crossing a Sn Rr with this 2nfl of constitution Rrr; actual counts showeh.348R: 182r (table 5). TABLE 5 Crosses imohing Ihe simpler (R77) iudividzral, 224~. Rr 0 X Rrrc? 2244X2241 lS2 97 224, X 2243 second ear 166 85 Totals 348 182 Rrr Q XRr$ 2243x224, 160 57 The extra chromosome having thus been shown to carry a factor for the r-2 linkage group, cytological examinations were made in order to determine which of the ten chromosomes of the haploid complement it was. Since the ten chromosomes are all morphologically distinguishable, it was only necessary to examine the 1 l-chromosome carrying microspores and see which chromosome was present in duplicate. Observations on diakinesis had already indicated that the chromosome involved was either the smallest or the next to the smallest. The methods devised at the time of the investigation for the observa- tion of the late prophase chromosome made it somewhat difficult to obtain ligures with all of the chromosomes lying perpendicular to the optical axis. Some good figures were obtained, however (figure 1). Many figures were found with all but one or two chromosomes lying flat. Since the differences in size between the four smallest and the six largest chromo- somes arc obvious in the later prophase stage almost regardless of the posi- tion of the chromosomes in the nucleus, it is comparatively easy to know when the four smallest chromosomes are lying flat, and hence to obtain accurate figures (figure 2). In the 1 l-chromosome microspores in which one chromosome is present in duplicate it is easy to determine whether it belongs to the group of four small chromosomes or to the group of six large ones. When it belongs to the former group one observes five small GENETICS 16: hlr 1931 180 BARBARA McCLINTOCK AND HENRY E. HILL instead of four small chromosomes, and the frequency of figures with all five chromosomes lying in the desired plane is sufficiently high to make accurate comparisons of the chromosomes for the purpose of determining which of them is present in duplicate. Such a case is shown in figure 3. FIWRE l.-Late prophase chromosomes in an ll-chromosome (n+l) microspore. The arrows indicate the duplicated chromosomes of the haploid complement; these are the r-g carrying chromosomes. At this stage the chromosomes are frequently very angular. X2150. FIGURE 2.-The four smallest chromosomes from a normal (n) microspore; late prophase X2130. FIGURE 3.-The five smallest chromosomes from an llO.Ol Determinations of goodness of fit by means of the X? method indicate a significant deviation on the total counts. A full ear of well developed kernels of unmistakable classification resulted upon selfing the first ear. A x2 determination on this ear alone gives a fit well within the probability. The second ear on this plant produced by selfing was poorly filled, with many kernels underdeveloped. It is possible that in this ear the color in some of the kernels did not develop. This possibility is supported by the fact that some kernels on this ear showed the presence of color by only a slight degree of mottling. It is possible, also, that some 2n+l embryos did not develop fully on this particular ear. The description of trisomic inheritance of Y given above shows the nature of trisomic inheritance in Zea nzqs with regard to the smallest chromo- some of the haploid set. INDEPENDENCE OF THE RG LINKAGE GROUP The method of trisomic inheritance is a convenient means of determin- ing with certainty the independence of linkage groups. Evidence obtained from both cytological and genetical observations indicates that the r-,o linkage group is independent of all the other nine linkage groups estab- IDENTIFICATION OF LINKAGE GROUPS 187 lished genetically. At least one factor of each linkage group has been tested (c and w+, sU, b, J!, gll, p,,f, d and a). 2nfl individuals heterozygous for these genes have been selfed and backcrossed. TABLE 12 yes& of crosses involving c-x'z, ss, b, y, gl,, p,, d and a among 2nfl individuals lrisonzic for the r-g chromosome. For erplanatiolt see page 189. I. Disomic inheritance of c and w, (see appendix). 2nfl Cc [C or c]XZn AcR C - 131aaXB3461 78 2n AcRXZn+l Cc [C or c] 1042X88, 194 B346iX131s 172 W, - 2nfl W,u, [W,] pollen counts 535 2n W.w, pollen counts 633 2n xw, X2n+l IV+, [IV?] 192sX2253 152 II. Disomic inheritance of s,,. Znfl Sasu [s,] selfed ST` 2203 282 22014 279 22014 174 22015 223 22011 261 22013 221 2319 318 Totals 1758 2nS,s,X2nfl SJ,, [sI,] 22011x1s 228 22514x2 208 231; X9 324 23116X9 316 23117x9 359 Totals 1495 2n s,,s,X2n+ 1 .Sus, [x.] 188X 189B,8 190 2n Susu selfed 2206 288 2208 269 22012 352 22Slo 278 2316 420 231i6 327 Totals 1934 km'1cs16: .\Ir 1931 c - 69 149 184 WT - 549 594 179 su 110 97 53 68 90 86 102 36 98 73 112 126 139 548 169 99 77 112 95 138 112 633 BARBARA McCLINTOCK AND HENRY E. HILL TABLE 12-(conlinued) III. Disomic inheritance of b (see page 189). 2n+l Bb[b] selfed B 881 S4 ,IV. Disomic inheritance of y (see appendix). 2n+l Yy [Y or y] selfed I' 1766 94 2n YyXZnSl Yy [y] 19412x2322 139 1943aX232, 113 1941~x232, 136 1943 X232( 102 1941xX232a 128 194, X2328 127 19419x232~ 143 b 19 3: 37 40 46 38 46 40 S2 -~ Totals 588 299 2n yyXZn+l Yy [y] 194, X232,, 9S 89 1945 X2321~ 85 63 1941~x232~ 77 72 1933 X232,6 66 74 Totals 323 298 V. Disomic inheritance of grl. 2nSl Gzm [GlJXZn gngz1 Gt1 611 2297X2011 113 105 VI. Disomic inheritance of p,. 2n+l P& [PT] selfed Pr Pr 2205 149 45 22014 287 89 22014 170 57 22017 228 87 22017 196 78 Totals 1030 356 2n+l PrPT P,IXh PA 22015 X 2031 129 120 22019X2038 110 108 Totals 239 228 2x1 P4%XZn+l P,p, [PA p, PI 22011X22018 306 80 2n P,p, selfed 2205 271 116 2208 249 97 2201% 362 102 Totals 2n P,p, X 2n prpr 2201~x203 SS2 315 194 207 IDENTIIkICATION OF LINKAGE GROUPS 189 TABLE 12-(c0tiinwd) VII. Disomic inheritance of d (see appendix). 2nfl Dd [DJselfed D d 2319 129 44 2n ddX2nfl Dd [D] 2OSX231g 119 118 2nfl Dd [D]XZn dd 2311~x205 170 140 VIII. Disomic inheritance of a (see appendix). 2nfl Aa [A or a] selfed A 881 73 1: 2nfl Au [A or a]XZn aa 1316XRSlla SO 53 2n aaX2n+l Aa [A or a] 2066X 189&o 144 173 It is needless to discuss every cross represented in table 12, for the re- sults are self explanatory. The crosses involving the recessive sugary gene (.r,,) can be used as a single example (see table 12, section II). A 2n+l plant of culture 131, homozygous for sugary, was crossed with pollen from a 2n starchy plant (S,S,,). The FL 2n and 2n+l individuals were selfed and backcrossed to test for trisomic or disomic inheritance. In each section the type of cross is indicated. The symbol of the gene placed in brackets indicates how the genie constitution of the 2n+l individual would have differed had it been trisomic for this gene. On selfing 2n+ 1 plants heterozygous for sugary a total of 17.58 S,, to 586 sU kernels were obtained, precisely a 3: 1 ratio. Diploid (2n) sibs upon selfing gave a total count of 1934 S,,: 633 sU kernels. The two ratios are similar and disomic. If these 2nfl plants were trisomic for sugary (SusUsU), an ap- proach to a 2 : 1 ratio would be expected. Similarly, a 2: 1 ratio would be expected in the sib crosses 2n (S,L) X2nfl. Here a ratio of 1493 S,:548 sU kernels was obtained, a 3 : 1 instead of a 2 : 1 ratio. It is therefore con- cluded that the linkage group including s,, is independent of the r-g linkage group and must be associated with another chromosome. The data on factors c, We, y, gll, pr and d are sufficiently numerous to need no further explanation. In the case of h, genetic data are hardly necessary, since the b-l, linkage group has been associated with another chromosome. The data on the n factor are few but indicate a disomic instead of a trisomic inheritance. In the case of a 2n+l heterozygous a plant selfed the results (73A : 19~) indicate neither a duplex (AAa) 17 - : 1 ratio nor a simplex (AU) 2: 1 ratio, but better, a disomic 3: 1 ratio. Further, the cross of a heterozygous 2nfl plantx 2n au would have given, if duplex, 54 : la ~~WGXS 16: Mr ,931 190 BARBARA hlcCLINTOCK AND HENRY E. HILL or if simplex, L4 : l+a, SOA : 53a probably represents a 1: 1 disomic ratio. In the case of 2n aaX2n+l heterozygous (I, a simplex constitution would have given a 1: 2 ratio and a duplex constitution a 2: 1 ratio. 144-4 : 173~ approaches neither of these but probably represents a 1: 1 disomic back- cross ratio. The factor for fine stripe (`j) did not segregate sharply in the seedling stage so that backcross counts were not sufficiently reliable. Cytological evidence from Doctor BRINK'S material indicates that the ~`-5~ linkage group is carried by a long chromosome. SUMMARY 1. A 2n+l plant of Zea gnays resulting from the cross diploidx triploid and its 2n + 1 progenies were found to give trisomic inheritance for r. 2. In these plants the smallest chromosome is present in triplicate. 3. Two unrelated 2n+l individuals were found to be trisomic for the smallest chromosome of the haploid set. These plants. upon later testing, gave trisomic inheritance for r. 4. In 2n+l individuals one-third of the eggs carry the extra chromo- some. In a normal pollination the extra chromosome-carrying pollen grains function only in a small percentage of the cases. 5. Plants trisomic for the r-g linkage group have given disomic inheri- tance for c, XI,, sU, b, T, RU, p,, d and a. ACKNOWLEDGMENTS To MARCUS M. RHO~DES and GEORGE W. BEADLE the authors wish to express sincere gratitude for their very generous cooperation in this investigation. Also to LESTER W. SHARP, ROLLINS A. EMERSON and MERLE T. JENKINS many thanks are due for their careful reading and re- vision of the manuscript. LITERATURE CITED MCCLIXTOCK, B., 1929a .\ cytological and genetical study of triploid maize. Genetics 14: lSO- 222. 1929b Chromosome morphology in Zeu rrrays. Science 69: 629. APPENDIX During the time this paper was in press the following linkage groups were found to be associated with chromosomes other than the r-g carrying chromosome: C-sh-wz, Y-P1, A -&l-c,. By the method of association of linkage groups with particular chromosomes the independence of six of the ten linkage groups (C--sIL--wz, R-g, B-l,, Y-PI, P-b,, A -&-c,) has been definitely established.