MUTATION IN MAIZE Barpara McCLintock The origin of instability at a number of loci of known genic action in maize chro- mosomes has been described in previous Year Books, and the nature of the unstable expression discussed. In some of these cases, instability appeared when Ds, a trans- posable chromosomal unit, was inserted next to the locus. The original and the subsequent mutations were shown to be expressions of changes induced at the locus by Ds. Ds is known to produce chromatin alterations. It was suspected, therefore, that the mutations it induces may be ac- companied by some form of chromatin al- teration or reorganization. If so, no two mutation-inducing events need produce ex- actly the same type of change at the locus, even though the resulting modification effects the same change in expression of the known genetic factor. It was decided, therefore, to examine a number of Ds- induced mutations in order to determine Whether or not differences could be de- tected among them. For this purpose, a number of independently occurring muta- tions at a selected locus were required. The two cases, described in Year Book No. 51 (1951-1952), in which Ds was in- serted into the short arm of chromosome g just to the left of Shi were selected because each has provided a large num- ber of newly induced mutations to shi (shrunken endosperm). OricIn oF THE MUTANTS All the mutants used in this study ap- peared in the progeny derived from crosses of plants homozygous for C, ds, shi, and bz, and carrying no Ae factor, to plants having Ac and one of the following con- stitutions: (1) J Ds Shi Bze/I Ds Shi Bz, (2) I Ds Shi Bz/I Ds Shi bz, or (3) I Ds Shi Bz/C ds shi bz. I (inhibitor of aleu- rone color, dominant in action to C, which produces aleurone color) is located approx- imately four crossover units to the left of 228 Shi. Bz (allele of bz, recessive mutant which alters the color of the aleurone layer and the plant tissues to a bronze shade) is located approximately two crossover units to the right of S/:. The frequency of occurrence of germinal mutations to sh: among the kernels on the ears resulting from the above-described crosses was recorded in Year Book No. 51. On the ears produced by the first two of these crosses, a few completely colored, Shi kernels were present. Such kernels were expected for the following reasons. In the two cases under consideration, most of the alterations produced by Ds result in a di- centric chromatid and a reciprocal acen- tric fragment. The position of break in the short arm of chromosome g is at the locus of Ds, just to the left of Shi. The acentric fragment, carrying I, is subsequently lost during a mitotic cycle. If such an event occurs in a premeiotic, meiotic, or gameto- phytic nucleus, gametes may be formed that are deficient for a segment of chromo- some g extending from the locus of Ds to the end of the arm. Previous studies had shown that female gametophytes having such deficiencies are viable and functional, and that kernels in which J is absent can be produced from them. In the crosses de- scribed here, such kernels should be com- pletely colored. Plants derived from them should have a long terminal deficiency of the short arm of one of their chromosomes g. As will be shown later, this proved true of only some of these plants. Morphologi- cally normal chromosomes 9 were present in the others, but the action of J carried by the chromosome received from the female parent was inhibited. Cuance IN AcTION oF GENES LocaTED to THE Ricut oF Ds An analysis of plants derived from some of the kernels carrying newly induced shy CARNEGIE INSTITUTION OF WASHINGTON mutants that appeared on the ears from cross 3 listed above was given in Year Book No. 51. The mutants appearing on the ears from crosses 1 and 2, however, arc of particular importance, because neither crossing over in a heterozygote nor con. tamination could account for the mutan: phenotype. Forty-nine plants carrying newly induced alterations at the locus «1 Sh, have been examined. All were derived from kernels that were I shi in phenotype. Sixteen were selected from the ears pro. duced by cross 1. Ten of these kernel were completely colorless, indicating the absence of either Ds or Ac. In four others. areas exhibiting either the C Bz or the C bz phenotype were present, indicatin: the presence of both Ds and de. In the remaining two kernels, areas showing only the C bz phenotype appeared, indicatins the absence or inhibition of Bz. Nine / s/ kernels were selected from the ears pre duced by cross 2. Five of them were com pletely colorless, and four had areas 0! both the C Bz and the C bz phenotypes. From the ears produced by cross 3, mine teen I shi kernels were selected th. showed areas of the C Bz and the C /- phenotypes, and five additional kernels were selected that showed areas only the C bz phenotype. Subsequent study showed that in thir: seven of the forty-nine plants derived from the selected I shi kernels, Bz was pres! in the 1 chromosome and unmodified 1 action. The remaining twelve plants wc’ all bronze (62) in phenotype. As will b: shown later, this phenotype appeared 1 some of these plants because the locus « Bz had been altered by the same event th.’ had altered the SA; locus. COMPARISONS BETWEEN shi MutAxT sim Modification affecting only Sh. >' larities and differences among the thirts: DEPARTMENT OF GENETICS veven cases of mutation to sf: in which the action of Bz was unaffected by the mu- iation-inducing event will be considered arst. In all cases, the chromosome carrying the new shi mutation was transmitted nor- mally through the male and female game- tophytes, and the homozygotes were viable. Ds was present in each, and its position was mot detectably altered by the event that produced the mutation to shi. Rever- sons to Sh: occurred in nine of the thirty- seven cases. In two of them the frequency of reversion was high, and in both cases it was Ac-controlled. The reversions to Shi were not associated with loss or change in location of Ds. The behavior of the shi mutants differed not only with regard to the occurrence of reversions, and to fre-. quencies of reversions when they did occur, but also with regard to frequency of cross- ing over between the locus of shi and the loci of its nearest known neighbors, J and Bz. In one case consistent results have been obtained in tests extending over three successive generations. When this shi is present, crossing over between [ and shy is increased about 50 per cent, in comparison with standard frequencies, and crossing over between shi and Bz is increased ap- proximately 300 per cent. In several other cases, the mutation to shi resulted in marked reductions in crossing over. In a backcross test involving one such case, no crossing over between sh: and Bz was ob- served in a total of 3156 tested gametes; crossing over between I and shi was slightly reduced, but that between Bz and a marker, Wx, located to the right of Bz, was unaltered. In all cases where a modi- fication in crossing over was expressed, it was confined to the vicinity of the sii locus. It may be concluded that the described dissimilarities among these sii mutants reflected dissimilarities in the primary mu- 229 tation-inducing events, even though all were initiated by Ds. Simultaneous modification of the action of Sh, and its neighboring gene, Bz. As was stated above, twelve of the forty-nine plants derived from J shi kernels produced from crosses 1 to 3 exhibited the reces- sive bronze phenotype. Four of the ker- nels from which these plants arose had appeared on ears produced by cross 1, three came from ears produced by cross 2, and five from ears produced by cross 3. (These last five were selected because of the ap- pearance of C bz areas in the aleurone layer and the complete absence of any de- tectable Bz action, and also because in four of them the endosperm was noticeably de- fective.) Only in the four cases derived from cross 1 was it certain that the Sii and Bz loci had been altered by the same event. The female parent in this cross was homozygous for both SA: and Bz; whereas one of the chromosomes 9 in the female parents used in the other two crosses car- tied the known recessive, bz. In the re- maining eight of the twelve plants, the presence of this recessive in the I sAy-car- rying chromosome could account for the bronze phenotypic expression, the mu- tation-inducing event having altered only the Sh: locus. Evidence obtained from subsequent study of one of the three cases derived from cross 2 and four of the five cases derived from cross 3 suggests, how- ever, that a longer segment within the chromosome had been altered, and that it extended from the locus of Ds to or be- yond that of Bz or its allele bz. This evi- dence will appear in the following dis- cussion. One of the four bronze plants obtained from the I shi kernels of cross 1 was com- pletely male- and female-sterile, and there- fore the modification responsible for the bronze phenotype could not be examined further. Cytological examination of the 230 other three plants revealed no structural alteration in their chromosomes 9. Marked differences were noted, however, in genetic behavior. In one of them, the J-carrying chromosome behaved as though the known recessives, sh and bz, were present, in that it was normally transmitted through the male and female gametophytes, and the kernels homozygous for it were normal in appearance. Also, no reversions to Shi or Bz were observed. The behavior of the I-carrying chromosome in the other two plants was decidedly aberrant. It suggested that a segment of the chromosome, includ- ing the SA: and Bz loci, was either absent or greatly modified. In one of these cases, transmission of the chromosome was nearly normal through the female gametophyte but markedly reduced through the pollen grain. Kernels homozygous for the modi- fied segment were not produced, and most of the kernels heterozygous for it were ab- normal in appearance—smaller than nor- mal and showing few to many wrinkled regions. In the second of these two cases, the chromosome was transmitted at a re- duced rate through the female gameto- phyte and was not transmitted through the pollen. Most of the kernels heterozygous for this segment were defective, and in many of them the embryo appeared to be dead. In both cases, Ds was present in the chromosome having the modified segment. Although its exact position has not yet been determined, in both cases it was either adjacent to or a component of the genetically modified segment. No rever- sions to Shi or to Bz were found in either case. The genetic behavior of the J-carrying chromosome in two of the three bronze plants grown from the I sh, kernels from cross 2 was normal. It resembled that of a chromosome carrying the known reces- sives shi and bz. Ds was present in the chromosome in each of these plants, and CARNEGIE INSTITUTION OF WASHINGTON not noticeably altered in location. The behavior of the J chromosome in the third plant was similar, except that somatic rc. versions to Bz occurred and they were Ac-controlled. The frequency of reversion was low, and no germinal mutations ap. peared in tests of several thousand gametes. No reversions to SA: were observed. Ip the event that they did occur, with as low a frequency as the Bz reversions, detection would be difficult. Only germinal rever- sions to SAi, or those occurring in very early development of the kernel, can be recognized with certainty. Ds was present in this chromosome, and again its location had not been altered noticeably by the event that produced the changed pheno. typic expressions of Shi and Bz. It was close to shi and to the left of the mutable bz locus. In one of the five bronze plants derived from I sh, kernels produced by cross 3, no aberrant behavior of the [carrying chro- mosome was observed. Normal transmis. sion occurred through the male and female gametophytes, and the kernels heterozy- gous and homozygous for the chromosome were normal in appearance. Ds was pres- ent in this chromosome. No reversions tv either Sh: or Bz were seen. In the four remaining plants, the I chromosome be- haved as if it had a modified segment, the modification being similar to that in the two cases described above. Here, agai. there was a reduction of gametophyt transmission of the chromosome carryiny the modified segment. In one plant, tran» mission through the female gametophy'c was normal, and that through the pollen grain only slightly reduced. The heter” zygous kernels were normal in appe'"” ance, but no homozygotes were obtains. Another plant showed nearly norm.t! transmission through the female gamet” phyte, but markedly reduced transmissicn through the pollen grain. No homo?}- DEPARTMENT OF GENETICS gous kernels were obtained, but the hetero- zygotes were normal in appearance. The behavior in the remaining two plants was similar to that in the plant just described, except that most of the kernels heterozy- gous for the modified segment were de- fective—small kernels, with few to many wrinkled areas. Cytological examination did not reveal a detectable structural modi- Gcation in the short arm of chromosome 9 in any of the plants that were heterozygous for these modified segments. Again, Ds was present in all the chromosomes with modified segments, and it appeared to be located adjacent to or to be a component of the modified segment. No somatic re- version to either SA: or Bz was observed. Although it is clear from the descriptions above that alterations affecting both the locus of Shi and that of Bz had occurred in at least nine of these twelve cases, and that homozygotes exhibiting the recessive expression could be obtained from two of them, it is not yet possible to state whether or not deficiency was responsible for the recessive expression, except in the one case where reversion to Bz occurred. In that case, deficiency of Bz is excluded. Marked deleterious effects are produced by some of these alterations, even in the heterozygote; and this situation has not previously been encountered in studies of heterozygous de- ficiencies in maize. Although deficiency cannot be excluded as the cause of the re- cessive phenotypic expression in every case, there is at present no direct evidence of it in any one case. CHANGE IN AcTION oF Grnes LocaTED TO THE Lert of Ds The previous sections have considered changes in expression of known genes lo- cated to the right of Ds, namely, Séx and Bz. Changes in action of I, located four crossover units to the left of Ds, may also 231 occur. Evidence of this has been obtained from plants derived from the colored ker- nels appearing on the ears of crosses 1 and 2. As was explained earlier, the appear- ance of such colored kernels on these ears was anticipated; and plants derived from them were expected to have a chromosome 9 with a long terminal deficiency of the short arm, extending from the locus of Ds to the end of the arm. It was considered necessary to test this expectation. Conse- quently, some of the colored kernels were selected and plants were grown from them. Eighteen plants were obtained from the colored kernels of cross 1 and two from those of cross 2. Owing to the pressure of other work, time was not found to ex- amine the chromosomes of these plants. In order to obtain seed for later examina- tion, each plant was self-pollinated and also crossed by plants homozygous for C, shi, and bz. The types of kernels appear- ing on the ears of three of the plants de- rived from colored kernels produced by -cross t and one of the two plants derived from such kernels from cross 2 were quite unexpected. They indicated that the ap- pearance of color in the original kernel from which each plant arose was not due to a terminal deficiency, but rather to some more localized modification within chro- mosome g that had affected the action of I. Since the kernel types and the frequencies of types were the same on the ears of all four plants, they can be considered collec- tively. On the self-pollinated ears, a few completely colorless, Sh. kernels appeared. There were 87 of them among a total of 1224 kernels, and the percentages on the four ears were 4.6, 7.7, 7.7, and 9.0. The remaining kernels were all colored: 604 were Shi, and nearly all of these were very lightly colored; 533 were s/i, and these had the depth of color that is produced by three doses of C. On the ears resulting from the backcross, no colorless kernels were 232 present. The Shi and sf; classes of kernels appeared in equal frequencies. Nearly all the Shi kernels were lightly colored, and nearly all the sh: kernels showed the color associated with three doses of C. It was ob- vious that either a pollen-transmissible de- ficiency of the J locus was present in these plants, or the action of J had been modified in such a way that it was no longer able to inhibit color development in the pres- ence of C. Plants were grown from the different classes of kernels on the self-pollinated ear and the backcross ear of the four excep- tional plants. The seedlings arising from the completely colorless, Shi class of ker- nels from the self-pollinated ears of all four plants were normal in morphology. The chlorophyll coloring in the leaves, on first emergence, appeared to be only slightly lighter than normal. As the seedlings aged, the color faded until the leaves were a very light yellow, after which the seed- lings died. Seedlings arising from the other classes of kernels were normal in all respects. Cytological examinations were made of the chromosomes g in some of the plants arising from the light-colored, S41 kernels derived from the self-pollinated and the backcross ears, in order to deter- mine if any detectable structural altera- tion was present in the short arm of one of them. No evidence of structural change was noted in any case. It may be concluded that the completely colorless, Sh: kernels on the self-pollinated ears—those that gave rise to seedlings with modified chlorophyll expression—repre- sented the homozygotes in all four cases. I normally inhibits color expression in the aleurone layer of the kernel when C is also present in the nucleus; and in kernels homozygous for I no color develops. In the cases described above, complete sup- pression of color did not occur in the pres- ence of C, although kernels homozygous CARNEGIE INSTITUTION OF WASHINGTON for the modified J were completely color. less. In these cases, not only the action of I had been altered, but also that of some previously unknown factor associated with chlorophyll development or stability; and this alteration was similarly expressed in the homozygous seedlings in all four cases. It is evident from the foregoing discus. sion that genetic material located to the left of Ds had been modified. Ds was known to be present in the chromosome showing modified action of J in three ot the four cases, although its position has not yet been determined. Tests of its presence and location in all these cases are now under way. The progeny obtained from three others of the plants derived from colored kernels produced by cross 1 resembled that of the four plants just described, with the excep tion that no colorless, Si: kernels appeared on the self-pollinated ears. In other words. no homozygotes were produced on thes ears. Cytological examination of the plants derived from the light-colored, Sh: kernc!s showed no detectable alteration in the short arm of chromosome g. It remains to be determined in what way the alteration affecting the J locus in these three cases differs from that in the four cases described above. Tests for this purpose are now being carried out. In the remaining thirteen plants derive! from colored kernels, the expected det! ciency of a segment of the short arm © chromosome g was found. It included «1~ proximately the distal one-third of the arm. from the locus of Ds to the end of the arm: Cytological examination revealed that in ten of these cases the deficiency had bee? produced by a Ds event that resulted in « dicentric chromosome g and an acentri< fragment, the latter including the Jocus “ I. In the remaining three cases, 4 tr! location had occurred. A segment from: another chromosome had been translocates! DEPARTMENT OF GENETICS to the short arm of chromosome g at the locus of Ds. A segment of the short arm, from Ds to the end of the arm, had. subse- quently been lost to the nucleus, with the result that J was deleted. From the cases described in this and previous sections, it is evident that Ds can initiate changes affecting the action of genes on either side of it, and that the effect may be localized, close to Ds, or may spread along the chromosome. As was mentioned earlier, it is difficult to determine the maximum extent of this effect, because the more extended altera- tions include genetic materials whose ab- sence or modified action adversely affects gametic transmission and viability, even in the heterozygote. MeEIoTIc SEGREGATION AND MuTATION It has been demonstrated, with regard to the Ac-controlled mutable loci, that the time of occurrence of mutation during the development of a tissue is a reflection of the dose of Ac present in the nucleus: the higher the dose, the later the time of occurrence of mutation. Somatic segrega- tions of Ac occur, and result in the forma- tion of sister cells whose nuclei differ with respect to Ac—its presence or absence, and the dose. The progenies of the two sister cells reflect these segregations by showing either no mutations or altered times of mu- tation. In other words, control of mutation at these loci is an expression of the constitu- tion of the nucleus with regard to Ac, and this constitution can be altered as a conse- quence of somatic segregation. In genetic studies, somatic segregations have not been fully examined or appreciated. Meiotic segregations, on the other hand, have pro- vided the key information for an interpre- tation of genetic mechanisms in general. When plants are heterozygous for one or more genetic factors, meiotic segregations 233 of chromosomes accomplish abrupt changes in nuclear constitution. The resulting spores or gametes differ with regard to these factors. The orderliness of meiotic segregation gives rise to definite ratios of particular genetic constitutions among spores and gametes. It is this orderliness that allows us to make deductions about the constitution of the parent. Mutation at a mutable locus may occur whenever a particular genetic constitution is present in the nucleus. Such constitu- tions may arise from somatic segregation or from meiotic segregation. If mutation occurs in all cells having a particular con- stitution, then meiotic segregation in heter- ozygous individuals may produce spores or gametes among which a definite fraction will undergo mutation at the mutable locus. When the heterozygous individual is crossed to one that can serve to test the gametic constitutions, definite ratios of mu- tant to nonmutant individuals should ap- pear in the progeny. Two cases of instability, designated a:”"™ and ai", which arose at the A locus in chromosome 2 have given evidence of mu- tation that occurs in spores or gametophytes receiving particular genetic constitutions as a consequence of meiotic segregation. Neither a? nor ai" is Ac-controlled. Both undergo a number of different types of mutation in the somatic tissues. Two main classes of mutation occurring at a” are recognized in the somatic tissues, but mutations that occur in the spore or game- tophyte, as a result of particular genetic constitutions arising from meiotic segrega- tions, belong to only one of these classes. Similarly, somatic mutations occurring at a." are diverse in type, but those occur- ring in the spore or gametophyte as a re- sult of particular genetic constitutions of the nucleus created by meiotic segregations are of only one type. Although more ex- tensive data indicating the relation be- 234 tween meiotic segregation and mutation are available for a,""* than for ai™7, the latter will be used to illustrate the nature of the evidence. This is because a factor has been found, located in chromosome 6, that actuates mutation at ai” in the spores receiving it. The location of the fac- tor influencing mutation at a:”°* in spores has not yet been determined. The original state of a1" allowed many mutations to occur early in the develop- ment of the plant and endosperm tissues. Each resulted in production of anthocya- nin pigmentation; and many grades of ex- pression were recognized, each resulting from a particular mutation. When many mutations occur early in the development of a plant, it is not easy to recognize the nature of control of mutation at the muta- ble locus, or to describe accurately its in- heritance pattern. Therefore, in crosses of the original plant carrying a.”* to plants homozygous for the stable recessive, ai, a search was made for kernels exhibiting changes in state of a”, in the hope of finding a state of ai"* in which mutation would occur late in the endosperm tissues. Delay of mutation beyond the meiotic stages would allow a more accurate analy- sis to be made of the inheritance pattern of the mutable locus and of its mutation-con- trolling system. Several kernels did exhibit such a change in state of the mutable locus. In them, the mutations were expressed by dots of the full 4: color and by small areas that were pale in color. Five such kernels were removed from four different ears, and plants were grown from them in the greenhouse during the winter of 1950-1951. Each plant was self-pollinated and crossed to plants homozygous for the stable reces- sive, a1. In tests of all five plants, the in- heritance behavior was the same. The ra- tio of kernel types on the ears suggested autonomous control of mutation at a:"7. One case will be considered below. CARNEGIE INSTITUTION OF WASHINGTON On the self-pollinated ear of this green. house plant, there were 243 kernels. Among them, 7 carried a germinal muta. tion that produced pale aleurone color; 14; were variegated, with dots of A: and smal} pale areas, like the kernel from which the plant arose; and 55 were completely color. less. Since this plant was ai""/ai in con. stitution, the observed ratio of kernel types was that to be expected if mutation at @," is autonomously controlled. This plant was used as the pollen parent in a cross to a plant homozygous for a, and again the ex- pected ratio of kernel types appeared: « kernels with germinal mutations to palc aleurone, 181 kernels with dots of full J. and small pale areas, and 165 completch colorless kernels. The plant carrying a:”'' was heterozygous for Y (Y, yellow endo- sperm, dominant to y, white endosperm). and the plant to which it had been crossed was homozygous for the recessive, y. On the self-pollinated ear, all 7 of the kernels carrying germinal mutations to pale alcu- rone were Y, and on the backcross ear $ of them were Y. No significance was at- tached to this relationship when the cars were first examined, as the total number of kernels with germinal mutations was small. Kernels were selected from both the sclt- pollinated ear and the backcross ear, and plants were grown from them in the sum: mer of 1951. Tests of the plants arising from the kernels having germinal mut: tions indicated that the mutation was com- pletely stable in subsequent generations. Plants were obtained from variegated ker- nels of both the Y class and the y class that had appeared on the ears derived from selt- pollination and backcrossing. These, 1" turn, were self-pollinated and crossed tv plants homozygous for a1. It was desired to introduce the recessive sh2 (shrunken endosperm) into plants carrying 4.” " because shz2 is known to be very closely DEPARTMENT OF GENETICS linked to the A: locus (they are only a quarter of a crossover unit apart). There- fore, some of the plants derived from the variegated kernels, all of which were ho- mozygous for Shz, were crossed either to or by plants homozygous for a and she and also for y. Because of the pressure of other work, time was not taken to examine the ratio of kernel types on the resulting ears. Variegated kernels that were nonyel- low (yy) were selected from two of these ears, however, and plants were grown from them in the summer of 1952. These were again crossed reciprocally to plants homo- zygous for a and she, and all the kernels on the resulting ears were examined. The examination revealed an extraordinary sit- uation with regard to the relative number of kernels carrying germinal mutations. On most of these ears, such mutations ap- peared in approximately 50 per cent of all kernels in the Sho class—that is, the class in which the a:"* locus was represented. In the first culture, consisting of eight plants, six plants showed this phenomenon. The sixteen ears produced by these six ~ plants yielded 3793 kernels, which could be separated into the following classes: full A, color expression, Sh2—3 kernels; pale color, Sh2—1043 kernels; variegated (dots of full A: color and small areas of pale color), Sh2—906; colorless, Sh2—103 pale color, sho—1; variegated, she—1; colorless, shz—1829. Three ears were obtained from each of four plants, and two apiece were obtained from the remaining two plants. On each ear, the ratio of pale-colored to variegated kernels in the Shz class was close to 1:1. The numbers were as follows: plant 1, 83 to 79, 73 to 66, and 27 to 24; plant 2, 127 to 107, 124 to 97, and 22 to 22; plant 3, 54 to 56, 44 to 45, and 59 to 505 plant 4, 15 to 11, 115 to 76, and 91 to 803 plant 5, 102 to 92, and 42 to 51; plant 6, 45 to 31, and 20 to 19. A single ear was ob- tained from each of the remaining two 235 plants in this culture. On one of them, there were 104 pale, Sho kernels and 211 variegated, She kernels—an approximate 1:2 ratio. On the other there were 5 pale, Sho kernels and 136 variegated, She ker- nels; no segregation-like ratio was ex- hibited on this ear. In the second culture, nine plants were crossed by plants homozygous for a: and sho. On thirteen ears produced by seven of these nine plants, the very same type of 1:1 ratio in the She class of kernels was ob- tained. Among a total of 5157 kernels, the following types appeared: pale, Sho—1 368; variegated, Sh2—1216; colorless, S2—10; pale, shz—1; variegated, shz—3} colorless, she—2559. On the ear of one of the re- maining two plants in this culture there were 72 pale, Shz kernels and 157 varie- gated, She kernels, or a 1:2 ratio of kernel types in the Sh class. Three ears were ob- tained from the ninth plant; and, in sharp contrast with the situation described above, the ratios were not the same on all three ears. There were 48 pale, She to 78 varie- gated, Shz on one ear; 48 pale, She to 160 variegated, Shz on the second ear; and 18 pale, Shz to 85 variegated, Sfz on the third ear. When the plants in the two cultures were used as pollen parents in the recipro- cal cross, a majority of the resulting ears likewise exhibited unit ratios of She ker- nels having germinal mutations to those having none. Of the total of seventeen plants in the two cultures, seven were used as pollen parents. Five of them, which had given a 1:1 ratio when used as female par- ents, also produced this ratio when used as male parents. The numbers of the differ- ent types of kernels among a total of 2527 were: pale, Sh2—672; variegated, Sh2—591; colorless, Sh2—15; pale, she—1; colorless, sho—1248. One of the two remaining plants had shown a 1:2 ratio when used as a female parent. This same ratio appeared 236 when it was used as a male parent, with the following distribution of kernel types: pale, Sh2—55; variegated, Sh2—97; color- less, Sh2—2; variegated, shz—1; colorless, shy—161. The seventh plant, when used as a female parent, had shown no segrega- tion-like ratio (5 pale, Sh2 to 136 varie- gated, Sh). When it was used as a male parent, however, a 1:3 ratio appeared on the resulting ear: full 41, S42—1; pale, CARNEGIE INSTITUTION OF WASHINGTON examined in order to determine whether or not they presented similar segregation- like ratios. Some of the plants were Y/) a, well as a." /a1 in constitution, and five of them had been backcrossed to plants ho. mozygous for ai, she, and y. The ears pro- duced by four of these five plants showed a striking linkage of phenotypes. On the ears of three plants, germinal mutations to pale appeared with a high frequency TABLE 3 PHENOTYPES OF KERNELS APPEARING ON EARS PRODUCED BY THE CROSS OF PLANTS HOMOZYGOUS Foi 4,1, Shy, AND Y BY PLANTS WHOSE CONSTITUTIONS WERE 2,"1 Sho/a, Shy; Y/y (part I), or a," Sho/a, She; y/y (part IE) PHENOTYPES OF KERNELS Color in aleurone PLANT PARENTAGE Colorless aleurone NUMBER TN CROSS Pale Variegated SS ——__ Total Y 4 Y y Y 4 Total Part I: 6046B-1.......... a 107 20 12 89 228 119 113 232 6046B-3..........- 9 32 1 58 95 186 93 88 8 6046C-2........... 2 28 7 55 86 176 75 103 178 6047A-1........... 2 30 31 173 162 396 209 228 437 6047A-3........... g 48 82 76 47 253 115 146 261 Part II: 6047C-1........... 9 20 174 194 163 6047C-3........000. g 92 97 189 203 6047C-4.......0.... 9 58 184 242 251 6047B............ g 4 398 402 391 6047B............ ao 5 482 487 483 Shz—57; variegated, Sh2—168; colorless, Shz—3; colorless, shzo—216. The 1:1 ratio of kernel types in the Shz class on the backcross ears obtained dur- ing the summer of 1952 suggested that the a,""*-carrying plants were heterozygous for a factor that induces mutation to pale in the haploid spore receiving it. As was stated earlier, the kernels from which these plants arose had been produced on ears of plants grown the previous summer, whose kernel types had not yet been examined. The kernel types of these summer-1951 plants, and those of their sibs, were now among kernels of the Y class and with a low frequency among kernels of the y class. The ear produced by the fourth plant showed the reciprocal linkage relationship. Part I of table 3 illustrates this. The plants in culture 6046 of table 3 came from varic- gated kernels with yellow endosperm: from the self-pollinated ear of a plant grown in the greenhouse the previous Wil” ter. Those in culture 6047 were derived from variegated kernels appearing on the ear resulting from a cross of this plant © one homozygous for a: and y. The + plants of this culture came from kernels DEPARTMENT OF GENETICS with yellow endosperms, whereas the B and C plants came from kernels with white endosperms, The B plant was derived from a variegated kernel that exhibited a newly altered state of a:”"*. Instead of dots of full A; color in the endosperm and siza- ble streaks in the plant, this state produces only specks of deep A: color in the endo- sperm and small streaks in the plant. A 1:1 ratio of pale to variegated in the colored class appeared in crosses of only three of the plants listed in table 3, two cases in part I and one in part II. The linkage of pale kernels to Y and of varie- gated kernels to y in the cross involving plant 6046B-1, and the reciprocal linkage relationship exhibited in the cross involv- ing plant 6047A-3, are both very definite. These linkages suggest that a heritable fac- tor, located in chromosome 6, controls the occurrence of mutation at the a" locus in those spores receiving it as a conse- quence of meiotic segregation. It is also evident, as the data from the crosses in- volving plants 6046B-3 and 6046C-2 indi- cate, that mutation need not occur in all the spores receiving this factor. Until fur- ther information is available, it may not be profitable to consider why this should be so. It can be mentioned, however, that the study of segregation-like ratios involving the a1" * locus has provided information that may apply to a”. On the ears of held-grown plants, sharp segregation ra- tios appeared. On the ears of plants grown in the greenhouse, segregation ratios were usually absent and germinal mutations were infrequent; in some cases, none were seen. The greenhouse plants came from kernels of the very same ears that provided 237 kernels for the field-grown plants. This would suggest that certain physiological conditions within the plant, in addition to the genetic constitution of the nucleus, in- fluence the occurrence of mutation, and that these conditions are in some way en- vironmentally or nutritionally controlled. It should be stated that the two cultures grown in the summer of 1952 which pro- duced so many ears showing an approxi- mate 1:1 ratio of She kernel types were de- rived from kernels appearing on the ears of two of the crosses recorded in table 3. The plants in one of these cultures were grown from variegated kernels with white endosperms, chosen from an ear produced by the cross involving plant 6047A-3. Plants in the other culture came from the variegated kernels on an ear produced by plant 6047C-4. With regard to these ratios, one further point is of interest. It may be recalled that many of the ears showed a slight excess of kernels with germinal mu- tations. Results of a study of a" have -helped to make this understandable, also, They suggest that the excess represents mutations that occurred in a few cells prior to meiotic segregation. Spores derived from such cells would carry a pale muta- tion regardless of whether or not they had received the mutation-inducing factor as a consequence of meiotic segregation. It is now known that particular changes in genetic constitution of nuclei, whether arising as a consequence of meiotic segre- gation, as described above, or through so- matic segregation, as previously mentioned, influence the occurrence of mutations at a number of mutable loci.