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 TABLE OF CONTENTS PAGE INTRODUCTION. 000000000 oes cote teens 175 Methods......0 00500200 cece center te rrr e eres ern es 176 Observations on trisomic inheritance of R........- 60-0500 sence teers 176 Independence of the R-G linkage group..... 6. - 5-20-5522 sere rt ener ens 186 SUMMARY... 00050 tes Detter ents tence eens 190 ACKNOWLEDGMENTS... 000000 eee tet e t sc te ener esses eens 190 LITERATURE CITED... 020 e eect tte enters tt sss 190 APPENDIX coo ccc eect ne enter teen ere r nner n ese 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 2n-+1 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 2n+1 plants have been obtained from the progeny of one original triploid (McCiintock 1929a). The chromosome-number in the female gametes of a triploid varies from ten to twenty. Trisomic individuals were obtained directly from theF, of a cross triploid X diploid and from the 2n+1 progenies of F, individuals with more than one extra chromosome. Inheritance data obtained from the 2n+1 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, 2n+1 Genetics 16: 175 Mr 1931 176 BARBARA McCLINTOCK AND HENRY E. HILL plants of other cultures which show the smallest chromosome in duplicate in 11-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 parafin. 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 R As has been stated, the 2n+1 individuals of culture 131 were trisomic for the r-g chromosome. This culture arose from a 2n+1 plant which had been selfed. Of 61 individuals examined, 39 (or 63.7 percent) were 2n, 21 (or 34.4 percent) were 2n+1 and 1 (or 1.6 percent) was 2n+2. It can be safely assumed that all the 2n+1 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, 11-chromosome carrying and 10-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 Percent of 2n+-1 individuals resulting from the cross 2nQ X2n+10. 2n9 X2n+1d 2n 2n-+1 Percent 2n-+-1 192, X 224, 261 3 1.13 192, X 2253 83 2 IDENTIFICATION 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 Number 2n +1: 2n individuals from the cross 2n+19 X2nd. CULTURE a * 2n+t 166 11 4 168 1 1 176 5 2 224 2 2 225 14 5 229 10 10 230 14 4 231 17 2 232 9 li Totals 83 41 33.06 percent 2n+1 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 nuclei, 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 n+1. This phenomenon could account for the increased ratio of n to n+1 gametes. The difference in many cases is probably not due to lack of viability of n+1 gametes or 2n+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 2n+1 plants bore almost perfectly filled ears. It is possible, also, that the lowest megaspore, if it contains the extra chromosome, does not function to produce the embryosac but, in a certain percent of the Genetics 16: Mr 1931 178 BARBARA McCLINTOCK AND 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 F, ratios were obtained to determine which factors were inherited on a trisomic and which on a disomic basis. Abundant evidence for trisomic inheritance of rin the 2n+1 progenies of culture 131 (cultures 189, 209, 224, 225, 229, 231) was obtained. Simple ratios may best be considered first. When the K factor for red aleurone is duplex (R&r) the gametic ratio expected from random dis- tribution of the extra chromosome is 2K: 2Kr:1RR:1", ora total of 5k: 1r. Since only the n-carrying pollen grains need be considered, the functioning male gametic ratio is 2R:1r. In the 2n+1 progenies of culture 131 duplex for R the backcross ratios through the pollen were 646R:355r (table 3), TABLE 3 mOXRRro COLORED COLORLESS 19232253 240 91 1929 X 20949 198 141 19219 224, 208 123 Totals 646 355 a fair approximation to 2:1. On the same basis, crosses of 2n Rr 9 X2n +1RRro should give 5R:1r. Table 4 shows a total count of 2102R:435r. A sib cross between two heterozygous 2n individuals gave 290R:88r, or the expected 3:1 ratio (table 7). TABLE 4 RrQXRRroe COLORED | COLORLESS 2251 X 2242 265 | 74 22514 2242 235 | 49 231;X2319 - 382 61 2311423145 419 82 231igX 2314 357 82 23417% 2319 444 87 Totals 2102 435 When R is simplex (Rrr) the expected gametic ratio is 1R:2Rriirri2r. With elimination of the n+1 carrying pollen grains the functioning male IDENTIFICATION OF LINKAGE GROUPS 179 gametic ratio is 1K: 2r. In the progenies of culture 131 only one individual tested was so constituted and gave a backcross ratio through the pollen of 110R:234r (table 5). Conversely a 2:1 ratio is expected in crossing a In Rr with this 2n+1 of constitution Rrr; actual counts showed 348R:182r (table 5). TABLE 5 Crosses involving the simplex (Rrr) individual, 224). mox Rr COLORED COLORLESS. 192; X 224, 110 234 Rr@ XRrrg 2244 224; 182 97 2244224, second ear 166 85 Totals 348 182 RrrQ XRre 2245 2244 160 57 The extra chromosome having thus been shown to carry a factor for the r-z 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 11-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 figures 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 are 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 11-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: Mr 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. Ficurer 1.—Late prophase chromosomes in an 11-chromosome (n+1) 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. 2150. Pra Ficure 2.—The four smallest chromosomes from a normal (n) microspore; late prophase 2150. BERRA Ficure 3.—The five smallest chromosomes from an 11-chromosome (n+1) microspore. Note that the chromosome in duplicate is the smallest chromosome of the set. Compare with figure 2. X 2150. It is clear that the extra chromosome which carries the genes for the r-g linkage group is the smallest chromosome of the haploid complement. The presence in triplicate of the smallest chromosome does not markedly affect the growth or appearance of the plant. One is unable to detect even a moderate difference in comparing 2n+1 with 2n individuals. On IDENTIFICATION OF LINKAGE GROUPS 181 the average, the 2n+1 individuals are somewhat weaker than the 2n individuals. Since genetic material of Zea mays is very heterozygous for growth and morphological characters, it is possible that the morphological changes produced by the presence of this extra chromosome would be recognizable only in fairly homozygous material. In consequence of the inability to recognize 2n-++1 plants with certainty in the field, every plant must be examined cytologically or tested genetically to detect the presence of the extra chromosome. FicurE 4.—Photomicrograph of microspore showing late prophase chromosomes of first nuclear division. The smallest chromosome of the complement, that which carries the genes of the R-G linkage group, is the second chromosome from the left. It is the only chromosome in focus throughout its entire length. 1800. Another 2n+1 plant (1441) not directly related to culture 131, an Fe individual from the cross triploid Xdiploid, was examined cytologically. The examination showed clearly the presence of the smallest chromosome in triplicate. It was therefore necessary to prove that this plant or its 2n+1 progeny would show trisomic inheritance for r. Colored kernels from the cross 144, (2n+1)xACr (2n) were grown. The 2n and 2n+1 F; in- dividuals were backcrossed to r testers (ACr). Pollen of 2n+1 plants placed on r testers gave a total count of 1282 colored to 2451 colorless kernels, a simple trisomic ratio of 1:2 (table 6). Plant 144, probably had the constitution Rrr, since each of the 2n+1 offspring was Rrr. The 2n sibs (culture 232, table 7) showed, in contrast, a good disomic 1:1 ratio. Genetics 16: Mr 1931 182 BARBARA McCLINTOCK AND HENRY E. HILL TABLE 6 ro X Rr COLORED COLORLESS 19412 2322 99 178 19414X 2322 33 75 1945, X 2324 82 153 19415232, 79 184 194, X232s 97 140 19413 2326 78 174 194; X232. 82 167 194, X232n 69 132 194, X232n 104 154 19212 23212 89 199 194, X23212 98 202 19416 23213 52 100 19415X 232i3 99 195 1932 X23216 _ 27 55 1933 X 23216 79 143 193, X232i6 115 200 Totals 1282 2451 TABLE 7 Crosses involving 2n Rr individuals of cultures 189, 225, 231 and 232. Rr BELFED COLORED COLORLESS 22510 274 99 23 his 334 105 Totals 608 204 Rr@ XRro* 2251722516 290 88 RrQ Xrra 22512 1924 184 219 22513 1924 177 187 232, X1945 172 195 2325 X1925 113 104 2327 192, 221 198 23214% 1925 182 168 232, X192¢ 70 73 23210 XK 19413 20 30 23213192 22 22 Totals 1161 1196 mORrd 192; X189As0 132 135 IDENTIFICATION OF LINKAGE GROUPS 183 Still another unrelated trisomic plant (219As) was found which possessed the smallest chromosome in triplicate. Its genic constitution with respect to r was unknown, but its pollen was used on r testers with the thought that it might be heterozygous. The total backcross ratio from the three ears obtained (table 8) was 295R:131r, indicating a duplex (RRr) con- dition in this plant. TABLE & mreXRRrs COLORED COLORLESS 1921. X219A5 192 94 193, X219A5 15 li 1944, X219A5 88 26 Totals 295 131 Thus far only ratios through the pollen have been considered. The ratios are simple and agree with the expectations. The trisomic ratios as ex- pressed through the female resulting from the cross 2n+1? X2no" are complicated by the presence of an excess of 2n over 2n+1 individuals. Possible explanations for this have been given on page 177. As was stated there, if there were random assortment of the three homologous chromo- somes at meiosis 50 percent of the gametes should be n and 50 percent n+1. Actually, only about one-third of the eggs carry the extra chromo- some. This condition materially changes the expected ratio. In a simplex plant (Rrr) the gametic ratio, on the basis of random assortment of the three homologous chromosomes would be 1R:2Rrilrr:2r which would give on backcrossing to rr plants, a phenotypic ratio of iR:tr. If, however, lagging and loss of one of the three chromosomes occur and at random (or if there is also some selection against n+1 megaspores in favor of n) the gametic ratio produced as a result of these occurrences would be 1R:2r and all the gametes would be n. The total gametic ratio, therefore should fluctuate between these two extremes de- pending upon the relative amounts of random distribution of the three homologous chromosomes as compared to lagging and loss that have occurred. It is therefore necessary to know what proportion of the female gametes were produced after random assortment and what proportion after loss. If a 2n+1 simplex plant (rr) is pollinated with pollen from a 2n rr plant the proportion of R-carrying and r-carrying gametes may be directly determined. The amount of lagging, or of lagging plus mega- Genetics 16: Mr 1931 184 BARBARA McCLINTOCK AND HENRY E. HILL spore selection, then, may be computed from the distortion of the 1:1 ratio (expected with no lagging) in the direction of a 1:2 ratio (expected with 100 percent lagging). An approximate measure of the total amount of lagging and loss that has occurred during megasporogenesis in the ovules of the 2n+1 plants of culture 232 (table 9) has been obtained in this man- TABLE 9 RrrQ Xr? COLORED COLORLESS 23241924 137 178 2326 X 19415 130 149 23231925 115 150 2329192, 137 178 23216 X 1925 160 181 Totals 679 836 ner. The 2n+1 plants of culture 232 were all Rrr (see table 6). They were pollinated with pollen from 2n r-testers (ACr). Each ear obtained showed a deviation from a 1:1 ratio expected with random assortment of the three homologous chromosomes toward the 1:2 ratio expected from lagging and loss, or from lagging and loss plus megaspore selection. Of the 1515 kernels obtained 679 were colored (dominant) and 836 were colorless (recessive). 1R:1r=gametic ratio due to random assortment of three homologous chromosomes. 1R:1r:1r =gametic ratio due to lagging and loss. When these two gametic ratios are placed one above the other, it is seen that 679 represents the number of R-carrying gametes produced by both types of distribution, and similarly, 836 the number of r-carrying gametes produced. If 679 represents the number of R-carrying gametes pro- duced, then 679 can be given to a similar proportion of r-carrying gametes. The total number (679+679) is 1358. The difference between 1515, the total number of gametes, and 1358 is 157, which is the number of the remaining recesives gametes (or kernels). It is easy to see by this method that this number (157) represents one-third of the total number of gametes produced as the result of lagging and loss (or lagging and loss plus mega- spore selection). The number 157 is also the amount by which the re- cessives exceed the dominants. Thus, in a population composed of indi- viduals, some of which, taken as a group, represent a 1:1 ratio and others IDENTIFICATION OF LINKAGE GROUPS 185 of which, similarly, represent a 1:2 ratio, the number by which the re- cessives in the total population exceed the dominants should equal one- third of the individuals representing the 1:2 ratio, or, in the present in- stance, one-third of the individuals produced as a result of lagging. The number of colorless kernels (recessives) exceeds the number of colored kernels (dominants) by 157; hence, the total number of gametes produced after loss should be three times 157, or 471. This number repre- sents approximately 30 percent of the total number of kernels. Assuming that the data represent a sufficiently uniform random sample, it can be concluded that approximately 30 percent of the gametes were produced after loss of the extra chromosome through lagging at the first or second meiotic mitosis, or possibly through lagging and loss plus any loss that may have occurred through selective viability of megaspores. If 30 percent of the gametes were produced through such loss, then loss occurred in 30 percent of the ovules. From the remaining 70 per- cent of the ovules half, or 35 percent, should possess n+1 female gametes and consequently 35 percent of the plants from the cross In+1? X2nod should carry the extra chromosome. This, in turn, can well explain the deviation from the expected ratio of 2n-+1 to 2n individuals in the cross In+1X2n (see page 177 and table 2). It is expected that both cytological and genetical evidence will be obtained which will indicate more definitely how much of the deviation is actually due to lagging and loss, and how much may be due to a possible selective viability of n+1 gametes or 2n+1 embryos. Assuming this same amount of loss, deviations from the 5R:1r ratio expected in the cross RRrxXrr (table 10) can be interpreted. Each in- Tas_e 10 RRrQ Xrrm" | COLORED | COLORLESS 20941925 (first ear) 130 33 20941192, (second ear) 207 56 20941933 (tiller ear) 193 52 20958 X 1926 289 72 Totals 819 | 213 dividual cross shows a deviation from the 5:1 ratio in an increase in the number of colorless kernels. over expectancy. The total count of 819 colored: 213 colorless represents a ratio of 3.84:1 instead of 5:1. A ratio Genetics 16: Mr 1931 186 BARBARA McCLINTOCK AND HENRY E. HILL of 3.61:1 would be expected if it were assumed that 30 percent of the female gametes were produced as the result of loss. The ratio due to loss is 2R:1r, as contrasted with the ratio of 5R:17 produced through random assortment of the three homologous chromosomes. These same assumptions are utilized in explaining the deviations ob- served in selfing an individual of the constitution RRr. On the basis of random assortment and no lagging of the three homologous chromosomes, the female gametic ratio should be 2R:2Rr:1RR:1r, the male, because of the elimination of the extra chromosome carrying pollen, 2R:1r7. One would expect, therefore, a 17:1 ratio in F,. On the basis of 30 percent of the gametes being produced as the result of loss of the extra chromosome a 12.8:1 ratio would be expected instead. Table 11 shows the results obtained. TABLE 11 AACCRE? setren coLoRED COLORLESS 2319 first ear 396 41 X?=2.47, P=0.10+ 2319 second ear 188 24 Totals 584 65 X?=7.42, P= >0.01 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 x? 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-+1 embryos did not develop fully on this particular ear. The description of trisomic inheritance of r given above shows the nature of trisomic inheritance in Zea mays with regard to the smallest chromo- some of the haploid set. INDEPENDENCE OF THE R-G 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-g 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, v, gu, Pr, f, @ and a). - 2n+1 individuals heterozygous for these genes have been selfed and backcrossed. TABLE 12 Results of crosses involving ¢-Wz, Su, b, ¥, gun, Pr, @ and a among 2n+1 individuals trisomic for the r-g chromosome. For explanation see page 189. I. Disomic inheritance of ¢ and w, (see appendix). 2In+1 Ce [C or ¢]X2n AcR Cc c 13133 X B346, 78 69 Qn AcRX2n+1 Cc [C or c} 104,88, 194 149 B346, X1315 172 184 W: Ws In+i W.ws {[W,] pollen counts 535 549 2n Ww, pollen counts 633 594 Qn wu, X2n+1 Wwe [We] 192.2253 152 179 II. Disomic inheritance of su. In+1 S.su [su] selfed Su Su 2203 282 110 - 22014 279 97 22014 174 53 22015 223 68 22017 261 90 22017 221 86 2315 318 102 Totals 1758 586 InSuSuX2n+1 S54 [su] 2201 X18 228 98 22514X2 208 73 231; Xo 324 112 231isX9 316 126 231i7X%9 359 139 Totals 1495 548 2n suSuX2n-+ 1 Susu [su] 188 X189Bis 190 169 2n S.su selfed 220, 288 99 220. 269 77 22019 352 112 22510 278 95 231; 420 138 23415 327 112 Totals 1934 633 GeNETIcs 16: Me 1931 188 BARBARA McCLINTOCK AND HENRY E, HILL TasLe 12—(continued) III. Disomic inheritance of b (see page 189). 2n-+1 Bb{d] selfed B 88) 54 19 IV. Disomic inheritance of y (see appendix). 2n+1 Yy [¥ or y] selfed y ¥ 176, 94 36 an YyX2n+1 Vy [y] 19412 X 2322 139 37 1943a X 2324 113 40 19415232, 136 46 1943 X232¢ 102 38 19413 X 232, 128 46 194, 232, 127 40 19419 232)3 143 52 Totals 888 299 an yyX2n+1 Vy [y] 194, X232n 95 89 194, X2321 85 63 19446 2325 77 72 1933 X23216 66 74 Totals 323 298 VY. Disomic inheritance of gn. 2n+1 Gunga [Ga] X2n gngn Gn gu 2297X 201, 113 105 VI. Disomic inheritance of 9. 2n+1 P-p, [P,] selfed P, Pr 2205 149 45 22014 287 89 22014 170 37 22017 228 87 22017 196 78 Totals 1030 356 2n+1 PrP, [Ps]X2n p,p, 220i5 X 2031 129 120 22019 X 2035 110 108 Totals 239 228 2n P,p,X2n-+1 P,p, [Pr] P, Pr 22011 X 220i 8 306 80 2n P,p, selfed 2205 271 116 220s 249 97 22012 362 102 Totals 882 315 2n Pypp X20 pepe 22013 X 203 ; 194 207 IDENTIFICATION OF LINKAGE GROUPS 189 TABLE 12—(continued) VIL. Disomic inheritance of d (see appendix). 2n+1 Dd [D] selfed D d 231, 129 44 2n ddX2n-+-1 Dd [D] 205X231 119 118 2n+1 Dd [D]X2n dd 23112205 170 140 VIII. Disomic inheritance of a (see appendix). 2n+1 Aa [A or a] selfed A a 88; 73 19 2n+1 Aa [A or a]X2n aa 131sx R511; 80 53 2n aaX2n+1 Aa [A ora] 2065 X 189 Beso 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 (s,,) can be used as a single example (see table 12, section II). A 2n+1 plant of culture 131, homozygous for sugary, was crossed with pollen from a 2n starchy plant (S.S,). The Fi 2n and 2n+1 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 genic constitution of the 2n+1 individual would have differed had it been trisomic for this gene. On selfing 2n+1 plants heterozygous for sugary a total of 1758 S, to 586 s, kernels were obtained, precisely a 3:1 ratio. Diploid (2n) sibs upon selfing gave a total count of 1934 S,:633 s, kernels. The two ratios are similar and disomic. If these 2n+1 plants were trisomic for sugary (S.ususu), an ap- proach to a 2:1 ratio would be expected. Similarly, a 2:1 ratio would be expected in the sib crosses 2n (Susu) X2n+1. Here a ratio of 1495 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, w:, ¥, gn, pr and d are sufficiently numerous to need no further explanation. In the case of 6, genetic data are hardly necessary, since the 5-l, linkage group has been associated with another chromosome. The data on the a factor are few but indicate a disomic instead of a trisomic inheritance. In the case of a 2n+1 heterozygous a plant selfed the results (734 :19a) indicate neither a duplex (AAa) 17—:1 ratio nor a simplex (Aaa) 2:1 ratio, but better, a disomic 3:1 ratio. Further, the cross of a heterozygous 2n+1 plant X2n aa would have given, if duplex, 54:1 Gevemcs 16: Mr 1931 190 BARBARA McCLINTOCK AND HENRY E. HILL or if simplex, 14:1+¢. 804 :53a probably represents a 1:1 disomic ratio. In the case of 2n aaX2n-+1 heterozygous a, a simplex constitution would have given a 1:2 ratio and a duplex constitution a Q:1 ratio. 1444:173¢ approaches neither of these but probably represents a 1:1 disomic back- cross ratio. The factor for fine stripe (f) 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 f-), linkage group is carried by a long chromosome. SUMMARY 1. A 2n+1 plant of Zea mays resulting from the cross diploid X 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+1 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+1 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, Wz, Su, 5, V, Su, Pr d anda. ACKNOWLEDGMENTS To Marcus M. RuoapEs and GEORGE W. BEADLE the authors wish to express sincere gratitude for their very generous cooperation in this investigation. Also to LESTER W. Suarp, Rotiins A. EMERSON and MERLE T. JENKINS Many thanks are due for their careful reading and re- vision of the manuscript. LITERATURE CITED McCurntock, B., 1929a A cytological and genetical study of triploid maize. Genetics 14: 180- 222. 1929b Chromosome morphology in Zea mays. 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—s,—Wz, ¥-P1, A —di—c,. By the method of association of linkage groups with particular chromosomes the independence of six of the ten linkage groups (C —s,— Wz, R-g,B-1,,¥—-P1, P—b,, A—di—¢,) has been definitely established.