1. Ze 36 9° 10. ll. ea MODE Ci OP[RATICN OF A S¥SULM OF CONYROLLING Locusts Th MAT Introduction . m=) Crigin of a, m1 Outline of motte of operation of the Spina, system States of a Detection of the Spm element and its mode of operation Responses of diffe ent states of a, mt to the same Spm element Transpositicn of Spm Stability of Mutants types of Spm elements Modifier efement in the Spm system Conclusions Mode 6f 6peration of a System of Controlling “lements in liaize,. General descriptions of controlling elements in maivse anc of their / modes of operation have been given in a number of reports annearing in recent yesrs (for literature citations, see MNeClintock, 1956b). It is the purpcese of t is report to consider some of the experimental methods that nave been ermloyed to discover the mode of operation of the system of controlling elements which has been referred to hn otier publications as the Spm (Suppressor-mutator)-a, "1 system, Controlling elements may be- defined as those unit components, carried in the chromosomes, that serve to control gene action both with regard to type and degree and to the . . i uw tissues or parts of a tissue térewe this will OCCUr, The different controlling elements are recognized by means of their distinctive modes of control of gene action, regardless of the primary type of action of the gene substance itself, They exnibit Mendelian inheritance patte-ns and their locations in the chromosome complement may be determined by use of ordinary genetic techniques, lowever, they may undergo cnange in location witnin the chromcsome complenent, appearing at new locations and disappearin from previously determined locations, without losing their distinctive properties in the vrocess, This process has been termed transposition and the several m-tnods thet have been used to detect such transposition were presented previously (McClintock, 1956b). For many years, efforts of the author were concentrated on analysis of the system com-:osed of the two controlling elements designated Ac 7 (Activator) and Ds (Dissociation), This system wes chosen for extended w w& studies because both elements @&-teis—-seseem could be identified readily ane regardless of the location in tne chromosome comolement that each might OCCUDYe Thus, change in location of these elements, and the effects produced when one of them is inserted at the locus of a known gene, could be detected and subsequently analvsed, Altogether, the operation of this system at eight Bh6wn gene loci has been examined, Also, two or more independent insertions of one of these elements at four of these eight gene loci were detected and the consequencd of this examined in each case, it was concluded from these studies that this system should be able to operate - at any gene locus provided tiat the effects of i+s-operation at a particular sees locus is not letnal, Most important, however, is the realization that the mode of control of gene action may be predicted in advance ,for it will Aotoyentd My T follow the rules that eee see the operation of this system, Knowledge mn of the mode of operation of the Ac-Ds system has been useful in guiding experiments aimed at revealing the mode of operation of other control svstem his applics particularly to methods for identifying controlling elements and for detecting their transpositions, It has also provided a model for recognizing and subsequently appraising the chanres in state of the affcetec gene locus, Such changes must be recognized if confusion in designing experiments and interpreting results is to be avoided, Some years ago, a large number of different variegates appeared in the progeny of individual -lants with a history of having been subjected to the breakage-fusion-bridge cycle in their early development (McClintock, 1951). ‘At the time tuese variegates appeared, it was realized thet it would not be possible to examine cacn of them. Therefore, only a few amone the many that appeared were selectdd for subsequent study. The Ac-Ds system was discovered among one of tiese selected cases; and as the mode of operation - of t.is system became apparent, attention was focused mainly upon it. Stugy of the other selected cascs either wase discontinued or it was sharply curtaihe » (3 Wot 2 » s until adequate time could be found for @ detailed examination, The system responsible for control of gene action in one of these latter cases is now the sufficiently unde»steod to allow conclusicns to be draw regarding/tynes of controlling elements that are involved in it and their modes of operation, at both a,™1 This is the system 1 mu . . : a ; : ; and a, » mentioned in previous reports. In this report, attention will sa m1 be given mani y to ay ° Cat pe Ti ‘rigin of ay i a ss Ties spe . : The history of origin of ay i from a modification tnat occured at a standard A, locus is as important for an appreciaticn of the controlling elements involved in this system and their modes of opcvaticon as were the histories of origin of modified gene action that appesred in the Ac-Ds carrying cultures for an appreciation of the presence and mode of operation. m= of the elements of that system, The modified A, locus, designated a 3 was the tiird recognized case of change in gene action in a sequence which commenced with an alteration ata previously unidentified gene locus concern with chlorophyll producticn, The plants having this first member of the Fs) sequence exnibited variegation for chlorophyll pigmentation. this varierat was one or toose ceteimaisdy selected among the many that appcared in the Original cultures, as described above, In the early stages of examination of tois variegate, a number of plants in one culture were self-pollinated,. On the ear produced by one of these plants, some kernels appeared that exnibited variegation for anthocyanin pigmentation, Spots of deep pigmentation appeared in a colorless background, The plants derived from them also exhibited variegation for anthocyanin pigmentation, Subsecuent tests of these vlants and their progeny indicated that an alteration had eccurred at the standard Ao locus in one chromoscme 5 of the parent vlant, This altered locus wis given the designation aml, Tests were then m1 undertaken to examine the changes in expression of gene action at ap and te » detormine ths factors i-veolved in control of tris, In the course of touis study, 2 number of plant s in a culture in weich the system responsible | for control of gene action at ae’ was vresent, were used as pistillate pare in crosses with plants that were homozygous for tne stand: rd recessive, ay» in chromosove 3, and for the standard Aj locus in chrovosome 5, On one of the ears this cross produced, a single kernel was found that ex’ bited igment spots of antnocyanin/in a non-pigmented background. A plant was frown from tois kernel and t is slant, in turn, exhibited variegation for antno- wan oe cyanin pigmentation, As~epocted, fests crosses | i tI sm dq: egret bats indicated the presence in it of op det AL locus, and. +, Ula Loewe m1 AOWL cove, chang was thereupon designated a) . It was evident that . wll Aad CLvuvilo, ot the standard Ay locus in one chrom some 3 in the pals Run Teor plore peerghuud parent plant, ae late in development of one of tre ears GF t is pliant, for A only one kernel on tis ear exhibited altered ay action, Studies aimed at determining the components of the system responsible for control of gene action at both a,™t 1 and ase were contined but initially only on a limited scale, Only recently has time been found to examine tis more completely. It is now knogh that the same system of control of gene actior operates at both a mt and ast Very likely this same system also opercted to control action of tne gene thet was associated with crbkoropryll production,--tne initial variegate in the seruence, This nostuhate is based cs on the patterns of varioration that were ex .ibited and upon vance behavior for study of this case was discontinued some years ago- and therefore direct tests to determine identity of control systems cannot now be made, No Cul Lng ' tho anade) Oporaligy of hp Oy 0 Set * caipemieernee . — 9 — In order to learn of the system that is responsible for cort+ol of gene Me» | m= action at a, and Bo » 2 large number of different types of tests were conducted, The résults obtained from each are consistent witn one another on the basis of the eventually determined modes of operation of the componen of tnis system, In order to com ly with space requirements, only a selecte set of tests ee be gien here, These are chosen in order to illustrate t Salient features of toe mode of operation of this svsten and they will be - : as ; : Mme) 4 4 eas m=] confined to studics conducted with ay . Those conducted with a., a will be given in a separate report. From examination of the Ac-Ds system, it was learned that inse=tion of the Ds element at the locus of a gene initiated the vrimary modification tho wy that brouggt this “gene under the control of the Ds-Ab system, In many A L wusiol cases, the action of the gene was noticably altered by this event and A detection of the Potues, of Ds at the locus could be mede shortly after A Wor dn rrntod tha - recluse it eecurred, Subsequent chat the locus resultfs from the effects Ac exerts on the Ds element, The consequence of this is either renaval of Ds from the locus of the gene or a modification at the locus, induced by Ds, that effects a chnanre in its organization,--a chanre in state of the locus, ‘ Both types of events can give rise to recognizable chanrses in action of the oe re m=1 . : gene substance, With regard to a,” ~, insertion of a particular * ecntrolling element at the standard Asclocus is considered to have occurred ang to have been responsible for the initial chanse in gene action, Like Ds, it is this element that directly controls the tyne of gene action that smo eoet gine will occur at the Ay locus and the times of change in tris aetsen that may A A occur subsequently. It appears to be the sane element thet is present at m1 * m= 1 m= ao . This conclusion is based on the response bBth ef ay and a. at to the presence of an ind&@vendently located element designated Suppressor- Wenn Spam ve pat wo dustin © das wy Mock ean mutator (Spm). All action of the gene se ee tb ethan end -es Rovul or plo -2yeopt ia Utd Ulbs, is_suppressed when Spm is omesont in the—nuctet ofr Srartb—excert—=n Oo the element r siding at eiticr m1 Ludivol te wtdlcatoy aye lo a,™t or ao that allowg the gene substance to become active ebteee—in- Geb wy, be ozuens Cnty gs 2 in Ue descondanty, fhe particular type of expressicn of the- 7 A gene that these modifications effect is thereaftor maintained eithor in the wes we tonslglin thon, Uat the Spm presence or the absence of the Spm element, m= 1 . , , , m=1 element is cormlementary to the elerent located at ay and a5 . It serves to activate it and as a consequence of t is, stable mutations -are produced, In t is resvect, it resombles Ac in the Ds-Ac system. tne genic meyostance However,/at bota ay and ap ot may be active to some extent when Spm is not present in the nucleus, whe it is removed, either through meiotic Ssegreraticn or by means of somatic transposition, anthocyanin pigment may h mo - ert ot Apre Med Mea ] appear in the kernels and plants nevine either a or a@ and Lts i iP a, y oa: yaa & 2 ee 1 2 Fas distribution is uniform, In other words, there is no variecation, The tyme and intensity of pigmentation is an exp ession of the particular state jw me wea DULY, oR Adon a . m= = . . ea 4 of ettaexr the ay 1 or a. locus tnat is present, these states and “A their origins will be considered snortly. When Spm is returned to the : . . m-1 m=-1 . nuclei by ancropriate Crosses, wl fene action at ay and a, is suppres en 5 c ed except in tiose cells where mucation-producine events occur, thus, Spm serves not only to activate the complementary elenent residing at m= aad ay and a5 and the eby conditionsag stable mutations ct these two loci, but it also must act upon this element in yet another manner for its presence results in suppression of lmown potentials for rene action, excent in t ose cells whore mutation=producing events occur, these two S-O-Otiiapy different aspeets of the mode of control of the Spm element on the elenent m~1 m=1 - dual : residing at ay and Bo APLesponsidle for its besee designa cody , eo Suppressoremutator, nowever, this seemingly dual action may be the € expressicn of only one process rather than of two unrelated processes, as wil be indicated later, Uo sive, ot Que All examinations of the effect of a controlling element at a gene ptwn must be conducted witr the affected locus, Since, in addition to stable mutations, the controlling clement may also initiate structural or organi-+ 3 gz zational modifications of the locus thot alter its subse uent expression,- that is, chanre the state of the locus,--it is necessary to consider States of the affected Locus and their origins before detailed results of — An Mea] experiments ean—be presented, Wita ay » 4 changed state is readily recognized by the appearance of an individual kernel or plant that exhibits an altered response of the locus to the oresence and absence of Spm, Fomexeuipie, When the controlling element first oneted the locus of Ais it effected a particular tyve of modification in $*9 structure ov organization ,of the locus, In the absence of Spm, some gene action occurred, Kernels were lightly but uniformly pigmented as were the plants derived from them, In the presence of Spm, however, all gene action was suppressed except in some cells where mutation-producing m=1 events occurred at thelocus of ay that allowed the gene substance to aud be active in the descendants of t-is cell. Eeeh such event did not OE an the same degree or type of gene action but many of them restored ne full or near full Ay type activity. Many of these events occurred yng . bo relatively early in plant and kernel development, -Umidke Ac, vwarli¢ats A. . , Tron, * a = wf. ay . 7 . in dose of opm hat.*no effect on altering the time of cecurrence. ofthese ) Ou wa Tr sdageet WOU Sey nibfor» Yon that 0) he. LO alan ocevied) CVEGRES« Ammer of \rutation-pnoducing events oeewmpmed-in germinal cells and this made it possible to examine the nature of the mutation in the ues wittel T Love next generation, Another tyroe of change af ay also occurred ina A few of tne serminal cells. These resulted in altered expressions of the locus both in the presence and absence of Spm, Some of tnem were detected initially in individual kernels in the progeny of the original —_- Me] “ay 0si«@eaarpyiing, plant, When this plant was crossed to vlants trat were -homozygous for the standard recessive, ass wnich does not regs ond to Spm , .. meL but responds, instead to Dt, the maguxkkyxwRxkermekx - EEEXEXXEXAN a, Humeikkindxayxxxxkeems locus in the majority of kernels that received it oe «os ; oe m~J . was unmodified. Among the kernels receiving the unmodified a locus anc 1 also Spm, the variegation pattern was much tre same, Thers we e many pigmented areas, Sy oa Oe Le eS ctx R me] large 3 isumm indicating early occurring mutation-producing OnB.oe Aove Rania pacaniTH Ota a eventS. any of these exhibited the full A A A A Onter However, an occasional kernel appeared that exhibited a quite different 1 type of vigmentation, r pattern of pigmented arsas, Pwo kernels) were found bnong several | ‘ thousand that had only small pigmented areas. ree were uniformly . Nove oldoune Loup dnt OMAR us awed, ann Secu ; distributed pad the intensity of pigment in quem was either quite, light nue “the otha de ally thp puceutay Ott: ibe, db bee ili or very dark, Anotoar kernel appeared that had a numbe? of large b+ ovigmented arsas as well as some small arens but the intensity of the 11 pigment in all of them was b qe , It ranged from very faint in some arcxzs to excrecciiin | i in ot erse excepticnal Plants were gpown from the three described/k>rnels, Tests conducted with them and their progeny indicated tirat the pettern of variegation ar : 1 ; “ m= exhibited in the presence of Spm was, heritable, The altered ay locus,-- the changed svite of tne locus,--in each case responded in its own particular way to the presence of Spm and also to its absence, In the mo 1 ose=aee in the first described koarnel absence of Spm, the state of ay . ‘ m . . produces tumpxmkemenk deeply pigmented kernels and clants. +e t in the second described kernel gives rise to very lightly pismented kernels but 4 * my a a m=] a 2 iy ratner darkly pigmented plants, The state of 5 present in the tvird described kernel produces no pigment in either plant or kernel in the bsen e § < ayy + m1 , Whe . a: absence of Spm, subsequently, other states of a, heve=bean isolated, ach is distinguishable from the other by the tyoes of mutetion and the Jind repder wild Spam io \rewet time and frequency of their cecurrence im the=presemrencéutipm as well as A Wades, L2.r TMA Zee ope’ y the tyne of expression of the gene suvstance thet ss in the absence of Sr ath Comets, q2ue Oebion iy OOF proarcad wa The Prteornp {Spa nergy wa Tapp ex0la (ord Heber Le eendasdt) aula” Spies No relationship was noted anong the different states betwoen the out {rb uth control of time o* occur ence of mutation in the presence of Spm and that A of tyre of gene action thet occurs in its absence, ‘igure 1 illustrates the distinctiveness of several of the states of amy due ahle flere U He range dpruutd Bhar ii eriysercet Oey, Thy pursue, aettd Vokes of a, Le m-1 , . . : The interréty of a state of ay is maintained in heterozygous plants and t is apolics to plants touat carry a different state in each of their chromesonmes 3.6 In such plants, each state resp:nds to Spm in its own predictable way anc the variegation patterns each vroduces will be way . — 7 ml, expressed in the plant or kernel tissues. “igure 2 illustrates this. tne kernels in the phosographs carry the state shown in — of figure 1 in one chromosome 3 and the state shown in « of figure 1 in the homologue, Lhe - dna The Aiutaliqn ty po pattern of mutation produced by each state OY readily recognized in these kernels, In the plants, normal segregation of the two states occurs at meiosis and each may be recovered in the expected proportion from the gametes that these plants produce, states of at are maintained unchansed in the absence of Spm, In its presence, however, new states may arise and the frequency of occurronce- of this in germinal cells is related to the time of occurrence of mutation- producing events that a particulsr state exnibits, If the staze vroduces some early occurring mutations, then new statcs of By may acpear in the germ cells, and the frequency of tis is proportional to the freaveney of occurrence of tnese carly mutations. If, on the other hand, the state is one in wich mutaticns occur only late in development of the plant om ~ a kepned, tnen few or no altered states may be recovered in the gametes of 13 these olants. The mutation-producins events at ay » regardless of state, give prise to stability of expression of tre locus. tne particular teve of gene action the mutational event produceg continues to be expressed in subsecuent generations both in the presence and in the absence of Spm. This suggests that the change resnonsible for these mutations may ewe removed the controlling element from the locus or it may ewe resulted in its inactivation with regard to Spm, Since the chanrses in state arise at tre very same developmental p riod as the mutation-inducing events occur, it is conelvded teat they represent a modification at the locus induced by tne controlling element residing there dhat did not result either in its wh removal or inactivation, In other words, the element responded to the presence of Spm at the appointed time and in the appointed cell¢ but the cons quence of this was not the usual one,-mifef, the mutation-producing event. Instead, the responding element itself was eith x modified or it induced some reorganizaticn at the locus that modified its capacity to + four d oremmune 7p “mutation On Welhao respond to Spm with regard to vime Witten tits rdeleecocun, the trpes of dink alno wid Aare mutations it can induce e=tedet—as the trpe of gene action that can occur in tne absence of Spm, Te is as evident in this case as it is with Ds that a chanre in state is one of the consequences of the response of the of the controlling element residing at a gene locus to the independently located clement of the sysvtem,--Spm in this case and Ac in the case of Ds, Qe toe Dery faad Sun Lhe dvd) Uo smydt aT o ates pomeeny Pk z per td In the early examination of ay » no evidence of the presence of the A Y=. “ . m . Spm element wee detected, This was because the original a, carrying plant had a number of Spm elements located at various »nositions in the OL Oyrtorute Warde all fhromosonie complenent. All, of tne gametes it produced nad Spm elements 2n- them, Since the dose of Spm has no anpreciable e*veect on the pattern Ma of mutation produced by ay » differences in number of Spm elements ina kernel or plant is not made directly evident by tris means as it is with Ac, Basu by ma It was only after several generations of crossing of ay 4 Pees tO ay le ut wean To tester stocks wnich did not have Spm, thet e,ratos of varierated to uniformly pale léeht colored kernels appesred on the test cross ears, These ratios re . MeL . . indicaved the presence in the a, carrying plants of an inderendently located element that is associated with control of at expression, They also indicated that the number and the location of this element was not the same in all tested nlants,. In the meantime, several different states mel . _: : og . . of ay had been isolated on tre basis of the altered variegation patterns - that appeared in individual kernels on several of tne Gkest testeross ears, as described cxcrlier, In successive geverations of crossing of m-1 , ; plants carrying these different svates of a to the a, tester stocks, the same kinds of ratios of variegated to non-variegated kernels also began to anne r, It was then assumed that the uniformly vignented m= ; . (non-variegated) kernels and plents carried a5 but not tne indevendently located elenent, On the oteer hand, this element was assumed to be present in tnose kernels and plants that snowed pigmented areas in a non- pigmented backegrcund, On this interpretation, tie indenendently located element was exerting a suppressor-mutator tyve of control of gene action at aw, Since the dose of t is element obviously did not affect the ; . 7 : : m=1 pattern of mutational events, cortrol of tois must reside at the a locus itself, the type de -end¢ing uron gne state of the locus, It was evicent that the phenot pes of the nonvarieratei ke:nels anc plants also m= 1 reflected tre state of the a5 locus that was present in trem, The hypothesis stated above was subject to test. le it were correct, evidence in supvort of the following four statements s>culd be obtained; (1) All variegated kernels and vlants carry at least one Spm element. (2) No Spm element is present in the non-variegated class of kernels . | mud outel . - and whants.( Germinal mutations, deseribed earlier, are excluded fron this class.) (3) The ay™1l locus in tee non-variegated kerneks and plants is capable - 16 nucleus, (.) The type of response b6 Spm and the prenot»pes ppoduced in its . . 4 Lo, , Mee] absence is a function of the state of the a Locus. A large body of evidence in support of these statements is now availavle and it has been obtained by various types of test, only a few of wich ; y3 ’ * “need be outlined here, In order to facilitate identification of the pr=sence or absence of Spm in a particular plant, so-callscd Spm tester stocks were developed, = 7 § : me=1 These stofck nave either one or the otner of the two states of a, snown cate gah) in = and - of figure 1) dul Prese states were selected for the following reasons, In the first place, wnen Spm is present, very few germinal mutaticns or cnanges in state occur. Therefore, nearly all of the cer ve ck mM gametes produced by plants carrving these states of ay and also Spm A , as m1 ae aa . nave an unmodified a5 locus in them, eedondly, the pattern of variega- tion each produces in the presence of Spm is distinctive and non-obseuring. : etV2B-V) Loirdly, in the absence of Spm, one of these states,- figure 1, gives rise a to darkly pigmented kernels and tis is a useful character in some tests, The tester stocks were made homozygous for one or the other of these two of resvonding to Spm if this element is subsequently intrceduced into a tn he . , . . . statese 4ney were also made homozygous for eithsr Sho or sho, located L7 Yi . very close of a7 ( about one-quarter of a percent crossing over occurs between them). An example of one serics of tests will illustrate some of the metiods employed to detcrmine Spm constitutions of indi¥idual plants, The di lks of a : : M~ : two ears of a variegated nlant carrying a and Sh in one chromosome 3 1 2 and a, and sh, in the homologue and also Y¥ in one chromosome 6 and y in its homologue, recieved pollen from a «lant that was homozygous for a, sh, and ‘ eessah-vy Mo J "| . < t 5 7 > , Ye The state of ay in tne pistillate plant was that snown in ~ of nn figure 1. From tais cross, the two ears vroduced a total of 7lL5 kernels. There were 181 Sho kernels in wnich the aleurone layor was unifornly and rather darkly pigmented; 69 of these were ¥ and 112 were y. the aleurone layer in anothsr 1388 Sh, kernels exhibited a number of spots of the full A, tyoe pigment in a colorless background and 117 of these weve ¥ and Yl were Ve The aleurone layer in the remaining Sh. kernels was comeletely colorles 2 and the stascn in its endosperm was y. Among the 375 sh. kernels on tnese- two cars, the aleurone lay-r in 373 of them was totally colorless; 186 of ae o mm s . 4 1, s these kernels were Y¥ and 107 were y. “he remaining 2 sno kernels ex ibited spots of full Ay type vigment in a colorless background, The pnenotzpe of the starch in one of them was Y¥ and that in the otner was y. Since 1é aml as colsely linked to Sho, neariy all of tne Sh, class of kernels on 1 Te a — a 44 these two ears ghould carry an ay 1 locus and nsarly all of the sh, kernels should be homozygous for the standard recessive, Bs The close Linkage of a, to Sho is obvious for only 1 Sh, kernel in the total of 370 was completely colorless and only 2 sho kernels in a total of 375 had pigment in the aleurone layer, On the basis of the interpretation given above, the uniformly pigmented kernels should have no Spm in them whereas those exnibiting spots— of the full A, type pigment in a colorless background should have tis element. From the ratio of tnese two classes among the sho kernels (181 to 187) it could be concluded that the variegated pistillate parent plant had one Spm. The ratio of Y¥ to y in cach of these two classes indicated that this Spm element was carried in the ¥© bearing chromosome) ti was then necessary to determine wnether or net these conclusions were valid, For t is purpose, 10h plants were grown from various tyves of kernels on these two ears and tests were conducted with them, The phenotyvoes of the selected kernels were as follows: 11 uniformly pigmented Sho Y, 13 uniformly pigmented, Shp y ‘17 variegated Sh, Y, 8 variegated Sho y, 1 variegated shy Y, 30 colorless shop Y and 2h. colorless sho ye All 2h. plants derived from the uniformly vigmented kernels were themsleves Ye sO unifornly vigmented,. All ef=the 26 plants derived from the variegated kernels showed small streaks of the Ay typecbigment in a non-pigmented background. And, as expected, all 5) plants derived from the colorless shy class of kernels lacked anthocyanin pigment, Hach »lant was tnen tested for presence or absence of Spm by crossing it with a plant in an Spm tester stock, To illustrate how the tester stocks can serve to reveal the oresence er absence of Spm, those tests conducted with the 5l, plants derived from the colorless, sho class of kernels will be considered first, this is a completely objective test since the presence or absence,in any one of tnem fhe silks of of Spm could not be assumed on the basis of »henotypic expressions, / 6ne 1 or more ears of cac oF these plants received pollen from plants that were eu) ema) horicargous for either state - or - of figure 1, for Sh and for y; these pollen parents weve uniformly pigmented indicating the abcence of Spm in them according to the stated hypothesis, If the plant being tested has no Spm, then all of the kernels on the resulting ear will be uniformly colored, Lp, nowever, tne plant being tested carries Spm, then it should be present : * . . : : 1 m= in some of its gametes, Following introducintion of the a 1 locus from 1 the male parent, the presence of Spm in those kernels that received it from the female waront should be revealed by the anvn-argnee in them of small, 20 Tha unt Sam suet deeply vigmented spots in a colorless background due to activation-ef-—the att locus be-tire—Spm—etement, In those kernels that did not receive Spm, the aleurone layer should be uniforrily pigmented, Among the 30 plants derived from the colorless, sho, Y class of kernels, it could be det -rmined on this basis that 15 had a single Spm element and 15 had no Spm. In 13 of the 15 plants that had Spm, linkage of it with Y was evicent (A, table 2) but in the 2 remaining plants, no linkace of Spm with Linkage of ¥ was evident (B, table 2), (The reason for the absence of/Spm iwithe Y Latin, in these 2 plants will be considered im ton. It need only be mentioned here that this is not unexpected, ) Among the 2h vlants derived from the colorless, sho, y class of kernels, 6 had a single Spm element (C, table 2),and 18 had no Spm, Hach of the 2h plants derived from the uniformly pigmented k: rnels was tesved for presence or absence of Spm in the described menner and in none of them was Spm found to be present, All of tne plants derived from the variegated kornels stowed stroaks of the A, t:we pigment in a non-pigmented background and the test crosses indicaced 1 2 PLE the oresence of Spm in cach of them, In 16 of the 17 plants derived from the variegated Sho Y class of kernels, one Spm element was present and & 2 3 t in 15 of these plants, it was linked with Y (D, table 2). In one ef—sRese el plant, however, ho evidence of linkage of the single Spm element with Y was noted (E, table 2). In the remaining plent tn this’ group, 2 Spm elenents wero present, neither of which was linked with ¥ (Fy table 2), More than one test cross ear wis obtained from 11 of these 17 plants and the number and locetion of the Spm element was the same in the cells producing all sars except for one plant. In tnis plant, one Spm element Was present in the cells producing the main ear and it was linked with ¥ (G1, table 2). In the cells that produced the tiller ear, however, a sincle Spm element was present but it showed no Binkage with Y (G-2, table < (xin all 8 plants derived from the variegated sh, y class of kernels, one Spm. was present (H, table 2). One Spm element was also p-esent in the plant derived from the variegated shy Y kernel. “his plant was used as a nollen \) parent in crosses with plants having different constitutions: homozygous Lso for: ana/y (01 for a, and sho/and having no Spm, homozygous for aeveral diiferent states of A Tee 1 * “ (3) ay but asving no Spm, and be pdbeutertoeteeeeeescre A =p Te] Ca ewewe a) Sh/a, sho; ¥/3s no Spm, - In this third group, . . m= ye plants with different states of aj were represented, All tests indicate ' s We in one sho corrying chromosome A 4 : Tee the presence in thepcllen parent of a4 i or lewind uy a ’ 3 and of _oP™, in the Y carrying chromoso: "ey Orel rect hin Spa DEMME nate ‘ink vil, he Meise hdfoup tales A Ql The tests described above were conducted with the progeny of a ai ngle plant in a culture. There were 19 variegated plants in tnis culture, Hach was derived from a variegated kernel that appsared on an ear of a varieg:ted plant that was amt Sho/ a, Sho, V/y, wx/wx (chromosome 9), pr/pr (chromosome 5) in constitution when vollen of a plant homozygous for ay» sho, y, Pr, and Wx and having no Spm had been vlaced on tne silks of this ear, All Yernels appearing on t is ear vere Sho and the distribution of phenotypes among them were as follows: 28 uniformly dark pale Y, 7 uniformly dark pale y, 55 variegated (spots of deep pigmentation in a colorless background) Y, 86 variegated y, 75 colorless Y, and 103 colorless Ve Among the kernels showing anthocyanin pigment, the ratio of uniformly dark pale colored kernels to variegated kernels indicated tne vresence of at leasce 2 and vossibly 3 Spm elements in the pistillate pavent nlant and ons of these anpeared to be linked with Y, The silks of ears of 9 plants derived from the variegated ¥ class of kernels on t.is ear and of 10 vlants derived from the variegated y class received pollen from vlants that were honozysous for ass sho; and y3 and hod no Spm, The ratio of kernel tvpes if . Oona ak ee “Hat ‘| Keeraty appearing on the resulting ears produced b> sach of these 9 plants is A entered in table 3, In this table, the 9 plants are rlaced in four groups, A to D, according to the assumed constition of Spm in esch that the ratio of _ “A Ut 23 kernel types suggested, The 6 plants in A of t:is table vere assumed to have a single Spm element located in the Y bearing chromosome, Progeny from l. of these 6 plants were grown and again tested for Spm, Those derived from plant 6629A-1, line 1 of A of table 3, were considered separate or above, The total number of progeny,plants in this group that were tested i a A prada ote epg SUM | jh Wg Oryre and theiw omteins are entered in the last line of A of table 3, These A tests verified the presence of Spm in the varkegated kernels and rlants and 2ts absence in the uniformly pigmented kernels and plants. ‘They also verified thes assumed Spm constitution and location in the pistillate Yowlter parent oleant. Only those tests conducted with the 116 plants derived A “pom the cbkbirless Sho class of kernels will be summarized nore, Among the 56 vlanits derived from the aj sho Y class of kernels, 32 carried Som and alt ned no Spm, In 30 of the 32 plants having Spm, linkage of it with Y was expressed, I, table 2. In two PTANGE yp Ee Som elethhent wes not appear - , \ vhosts Usbe TT \ linked with Y, ae menticned earlier, (B, table 3)\, Among the 60 plants Single Spm element 9 devived from the ay sh, y class of kernels, 17 hed (J, tacle 2) and 43 had no Spm Whotlee , Ruse 1 DI voc : location in the 2 In order to verify the plants entered in B of table 2, tests of some of the progeny of both of them wore conducted, An ear of one of these plants had been self-pollineted 2h fal) a eae ’ Me and another ear of this olant hed been used in tne cross with an By 1 Sh, ¥Y, no Spm tester vlant, Kee Progeny from botn of these ears were arain tested for Spm constitution and location, The silks of ears of 8 vlants dsrived from ay sho kernels in the ¥ class on the self-pollinated ear Mel gh 5 Ys no Spm tester plants, From the D.orxe Q oh O received vollen from the a, kernel types on the resulting ears it could be concluded that +we a plants wore Y/Y in constitution and that one of them had no Spm whereas the other had 1 Spm (199 pale colored kernels : 169 variegated kernels on the test cross ear), the remaining 6 plants were Y/y. One Spm was present in l of them but it was not linked with Y (K, table 2), An Spm element homologous app: ared to be carried at allelic positions in a p&ir of/chromosones in the renaioing 2 plants (Ly table 2), seventeen vlants derived from the Ae variegated Sho Y class of kernelsgon testcross ear of touis same parent . a A . ( dyn Bk yO ze plant weve crossed by plants in the Spm tester stocks. In 16 of these 17 plants, one Spm was present and on none of the esrs produced by 15 of them was their any evidence of linkage of Spm with ¥ (M, table 2), iowever, the ratio of kernel types appearing on the test cross ear of one of them suggested such linkage (N, table 2), The remaiving plant had 2 Spm elements, neither of wich was linked with Y (O, table 2), 25 Tests of Spm constitution in 9 plants derived from the variepated Sho Y kernels on the ear produced by tne test cross with the other plant entcred in B of table 2, suggested that the Spm elenent in this plant nad been carried in the Y bearing chromosome but at a new location that was farther removed fem ¥, seven of the 9 progeny plants had a single Spm thoy peodead sp element and in all of the test cross ears, loose linkage of +6 with Y was A expressed (P, table 2), One plant of the 9 had 2 Spm elements (@, tarle 2) and xa the remaining plant had 3 Spm elements (R, table 2). Further examoles of the progeny test method of determining Spm constitution and location will not be given here, It should be mentioned, however, that such tests were conducted with the indicated progeny of the plants entered in B, C, and D of table 3, and also with the progeny of avery, bdel oun) tne 10 olber plants of this same culture, Also, @ummaer=-e sueht esis S 4 ay introduced by the pollen parent. Again, if the amhtyebiegs element “showed linkage with a genetic factor ameng-bee-ke-ne}s on one ear, The Urn We wash RA tected usuatiy shened thee same linkage on the other ear and an example of tris A J is given in S$ of table 2, In tris test, the main ecr of an a,/8,» ¥/y plant received pollen from on? 8S the Spm tester stocks. The kernel types on the ear tris cross produced, §-1, table 2, indicated the presence im the pistillate parent of an Spm element carried in the * chromosome, The silks of a tiller ear of this same plant received vollen from a plant ioe ree “ Wbo oS that was homozygous for state -, figure 1, and also for y. The pollen parent was uniformly pigmented indicating the absence of Spm in it, The kernel types on the ear this cross produced are entered in §-2 or table 2, The. ratio of variegated to non-variegated kernels in the Y and y classes was mucn the same on both ears, Thus, it could be coneluded that the Spm element, carried in the Y chromosome of the vistillate plant, was capable me] of activating either state of ay e Pollen from the same collection tesS was used in the latter cross was also placed on the silks of a plant homozygous for a, and for y, but in which Spm was known to be absent. All of the 29) kernels on tht ear this cross produced were uniformly lightly pigmented and all were y. This test confirmed the absence of Spm in the pollen parents 29 Another tyne of test tnat was emplbyed to indicate the response of different states of at to the same Spm element, utilized tne pollen of plants that were homozygous for a, and in wich Spm element was present. The troes of test conducted with two such plants, number 6861-1 and 6861-7, abe illustrated in table lL. Both of these plants wore homozygous for a, and sh, i , —e Om C1 3—BPeSOR >i. pe ; 7 Rekexmkankx The silks of an ear of each plant received pollen from the a, 7+ tester stock that carries state 5718. Wekop prowl the tyoes of kernels on the resulting ear are entered in A of table ho, Athao To These ratios indicate the presence of "Spm in exch plant, Both plants A were used as pollen parents in crosses with plants that were homozygous for state 5719A-1 but in which no Spm was present, The types of kernels on the ears resulting from these crosses are entered in Bof table lt, Again, al: 1 ratio of variegated (Spm) to non-variegated (no Spm) kernels appearmd on these ears, indicating the presence of 1 Spm in eacn of tne two pollen parents, These same two plants were also used as pollen parents in + 1. I~ crosses with plants that were a, 1 Sho/ ay sh in constitution and had no _ m an Ti2\ Spm. the state of ay -l in these plants was that wich gives no antnocyani A pigmentation in the kernel and plant in the absence of Spm (state 5720, = Bd figure 1) but mixes many mutations to the lower alleles of AL in its prescne Other ears cf these same plants received pollen from plants that were na - . ~ m p : honmogygous for ay and sh., but had no Spm, +he trvpes of kernsls appearing a on the ears resulting from esch of these two trpes of cross ave entered in Cof table h . Again, it is evident that plants 6861-1 and -7 cach have + m one Spm element that is capable of acting on this state of qa ™1, he 1 results obtained from the described tests are tnrose to be expected if the Spm element in vlants 6861-1 and -7 is canable of acting on different Me) states of ay : The same type of test as that juct described was conducted with az sip/a, sh, plants having more than one Spm element and the ratio of kernel types on the test cross ears was that expected if each of the Spm elements present in the ay sh,/a, sh. plant was capable of ; nh one ; m=] acting on each of tre states of ay ° Still other ty-es of test were conductsd to determine the cavacity of a Spm element to act on different states of amt, One of them utilized the pollen of a plant that was amt sho/a, sh, in which a single Spm element 2 was vresent at a known location in the chromosome com-lement, When such a plant was used as a pollen varent in crosses to plants that were amt Sh/ ay sho and having no Spm but among which different states of amt were represented, the t.-es of kernels on the resulting ears clearly indicated thi capacity of the Spm element in tne male parent to act not onivy upon the stat: 31 of the amt locus delivered by the male parent, but also upon the stete of i the at locus delivered by the female parent, Again, when »lants . m=] : es ca ryving Spm that were ay Sh,/ ay Shoy among which different states of Mod : 1. . ea. ay were represented, were used as vistillate parents in crosses with a 1 , TM 7 . plant that was homozygous for one state of a, 1 and also for sh, but in waich no Spmwas present, activation of the a state celivered by the mabe parent by the Spm element delivered by the female parent was indicateé Imall tests of this tyne, Detailed consideration of the various troes of test mentioned above Cannot be given here, ilowever, all of them cleerly established the Similarity of the Spm element durried in the many diffe vent tested plants, and regardless of its number or its location in the chromosome complement of a given plant, They also established the atbility of th#& 5pm elenent m=1 ; er + 2: to act upon any of the selected states of ay and they indicated that control of type of gene action in the absence of Spm and control o* the as well as its type in the presence of Spm is solely time and frecuency of occurrence of mutation Amex eeKxR ake a function of the m=] stace of ay e pe - voles lonely we augp oh Opfoweiuees heat b> 14 plued Table-2 Sydo dt Rarwels bane £ ' Wc yaa i sg NAPOe A end hte wo Sat Une ined ores cg Vase Care: a md (Stu to stilutioy & nO. cd C No per) telhemay puiwlate |paraiZ. | plaut 1 4 | 4 Tole! | A Ash aay Y Span 4 + 13 613 | 1467 1336] SIS | HHS a nos Iq; ISpea a 4b 47 lab} 16 435° Cc "eT ag)qy [1 Spas b - | ae] — Ite | 24 2 Q Sholay Y Sa I + 1s bLS | 1156 | 108D | 638 | 3539 “B » ONE [Spe 42) WY 3) 35 156 aa woul ow 12 Spey 45 | 5% WY | Ws 369 Bai fw PSpu)ge fromrcon | uD | TH "| 4 LIb G-a 8 PVs pa tiler J V22 | 1249 Hy | ial 5 1b H * uf gly [\Spa | 8 — $64 i $52 VT)6 x Qt lla! “S| abe 3D Dob | AGIA | WHTA | 1335] TTID J now wale \Spu "1 - 3HES - SASl | OTHE K soe PQ fv Span 4 HTT} 500 | 455 ; Heal 1994 L wow fo | Sp Bp} & 3 4H | 259) 29) | 547. Wy woy fo fsa fos IS3b | 16bH | 1544 | 1654 | 639% nN q'shy) ajar Bia} gs 67) | bt qa $2 b | 303 | O vt wal Spay] | $2 75 | 203 | ol 5 6] P sty diye 7 531 | 113 | bas | 543 | ayes Q i pg Spf 5\ TI TO | 19S 467 R n Hy 3 8p] 1b 1s Q5 | id | 2b S-t faye | Qhe | f+ nin to) ; b 130 4) 13 | 4b S-2 as Cen ton) bd \4D Vax} 43 acm She | dalla Sly Q ox Tables a, Ara, ok, AY) Nos pea a] a. Ysw}4+ _ Rliaut Bo. net AD<— — Rhewot \pe s& Ker vel “gpaooS Aeep Color wm Cetadvo lob qowel | Sh \\ ; bb 29 R-1 WT 7 43 37 Sb 36 137 ae 3b los 49 sen feene we Ta us4 Us) 264 aT 43. (geste 23 theey 0 Or rn ve cee acts ——=— sy galt 153 As” 122 > soem ac ee mh eg | ake be A. AA Jar ohae Spa Fx aim ah | 0, az Cotate 5715); no Spe 77 Phono A owed odin dy pi a Vawt wlio YS 2 ‘gah Le Oe ae Nee Toft b%6\-1 AD WT4 Ho3 646\-1 139 14% LET BQ Sha] Qi She Cato ST19R-1) F Aw CxbI-1 ancl CB6I-72 4A, [ays Spur FT Me Spe a NasTighbe Ror baw gat Total Wi Qvut doy 29. ttt TP _ wnt to bso "95 — 6861 1g6 ae 9% Proud lho 4. Yoruba S67 AIe © Qcmishe Catotesr20) /Qyake wpa g % Ayiehr]Q, ele 1Spuu (le 6361-1 NOY 6I- 1) Quel Oh lade wo ynct Coir Qroo | a Morava Lah on ee a ‘4Gted A | | __ Aaa Oetepbee Leder’ | . 2. Shi aha. | Sir “Ah Tole le peal Fb I-1 53D a | Ys O | 1926 5 Soto © Ww Qc Lavah Wo Span 74 TS foo eo | Oe i ag : lo pau uA GOI-T. 4b 1 16 _ 149% bo - > —~#.. — ’ ; ti - 7 ce een 338 bb $ oboe 10 Way Qi di olan, Tbosgan mt th WT ; D O oo DRO m1} Stability of mutants produced by a, Mutation producing events may occur at a, mi not only in somatic cells of the plant and in the endosperm cells of the kernel, but also in ancestor cells of the gametes, that is, in the Sporogenous or gametophytic ag hh”r cells, The frequency of their occurrence in these cells, and the pnenotypic A exoression that will result from toils is related directly to the state of the at locus itself (see table 1), With most states, the majority of germinal mutations modify the locus in such a way that it is subsequently capable of acting much like the standard Ay locus, however, all skates give rise to some germinal mutations that express a ruch reduced capacity e120) for pigment production, State -, figure 1, produces almost exclusively this latter type of mutant, When plants carrving a met and Spm are some kernels on the resulting ear may exhibit a modified phenotype and this is the consecvence A crossed by plants that are homozygous for ays Mea] Such of a germinal mutation at ay ° Teese kernels are uniformly pigmented usually anc the intensity of this/differs markedly from that arpearing in the kernels Soe? hae having an ummodified a, m1 locus and no Spm, Kernels exhibiting phenotvpes 4 A Ongena rom —errried—rertortems were removed from seme—etctire @ rs and the plants grown from them were exarnined for anthocyanin distribution and Wage tested for presence or absence of Spm, It was found that may these kernels gave rise to plants that were uniformly pigmented, “hen, in M1 turn, thetp peemtes weve crossed by plants that were homozygous for ay but had no Spm, it was learned that Spm was present in some of them and Wolp phenotype produced by the absent in others, However, in all c&@es, the/mutated locus worthe Ooune in gl AD Cerne, VT ‘whom wade, Wy dello Ws Coco Ute WY QTd W the pounce of 2 foun - plants were also crossed bv plants carrying Spm and from this test it was oho learned that the mutant lecus in those plants that had no Spm would remain A These unaltered in expression when Som was introduced, The most gravhic illustrati;n of stability of mutants in the presence a . . m= 1 2 of »pm is derived frome'rosses of plants carrying the state of a, (sro) hat produces no anthocyanin in the absence of Spm but gives rise to many early occurring mvtations to low alleles of A. in its presence, When 1 this state is present, the frequency of occurrence of germinal mutations is high, and they are revealed xmxxnustixisuakxkerneks by the appearance of kernels exhibiting a uniform distribution of pigment over the aleurone layer. The intensity of this pigment among the different kernels la ving germinal mutants varies from very faint in some to rather dark pale in others, The same range in intensity of vigmentation is expressed in the plants grown from these kernels, the degree correspondine with that shew, A ‘ A - : shown by the kernel from which the plant arose, tiese plants, in turn, t. Cc C Srrwacgyar \or Qi, Baek Reno woSpi) were crossed with a Spm tester stocks +2 order to determine the presence or absence of Spm in them, aged Spm was found to be present in some of them, The stability of the mutant in the presence of Spm was cleirly revealed in this test cross. Some of the kernels on the kek ear resulting from this cross canmied the mutated locus and Spm desived from one parent and the a," locus demixed from the other parent, im Phese exhibited kernels spots of deep pigmentation in a uniformly pale colored background, figure 3. The deeply pigmented spots represent Spm induced mutations at the MeL ay locus contributed by the tester stock, The lightly pigmented background in which these appear reflects the action of the mutated locus Vo Snjaseaion us um ef atact Us contributed by the other parent which tp Obviously stots the presence A of Sorte sm ie The phenotypic expression “Py the a element, considered in the previous sections, was remarkably constant and predictable, notwithstanding butt Doug vv Han tig UPs0 ARK A - thdmany diff that wes homozygous for ay > Sho, y, and had 1 Spm-s element closely Jinked : Abe wo : 3 with y in one " nromosomes 6. Some of the pollen parents were Pr/pr and 3 A aclu HY QM! Lycee Orel ue becer aiTorae others were pr/pr. From these tests it was concluded that an Spm tise A element with much xedumxedt weakened action was-peesent in Qa=heemesnees—< by * the piephs derived from the kernels that were eaither teextry colorless c of deep color snowed only 1 or several small spots/in a colorless beckground, and that this element was absent in the plants derived from the uniformly colored kernels, the reason for tnis conclusion is evident from the types of that anpeared on the test-cross ears that kernels/these plants produced, as shown in table (a), Those entered in A of this table are from ears of vlants derived from the uniformly colored Chu kernels. Those entered in B of -eete-+erte are dexiwenxkeenxEneExkeKrners from ears of plants derived from the totally colorless kernels or those 1 or several . in woich only g small spot deep color appesred. Only two classes of amt carrving kernels appeared on the ears of plants entered in A ofthe tabhe, Half of them were uniformly dark pale in color (no Spm) and half exhibited meny spots of deep pigmentation in a colorless background m=] (Spm-s present). On the other hand, the a, carrving kernels on ears produced by plants entered in B of this table fell into four classes. Naif of them (1323 kernels) exhibited the typical pattern of variegation produced when Spm-s is present. A quater of them (629 kernels) were unifor:ly dark pale in color (no Spm), “he remaimhng quater (693 kernels) showed either a few dots of dem pigmentation in a colorless background (52h kernels) or the were totally colorless (169 kernels). In some of the crosses entered in Kxm& this table, the male parent was pr/pr gyel ANd Cunt burst wR, eo¥a: inen these piers were used aacpothepesert, the distribution of Pr to pr anions the three classes of a, mt Sho ecsrrying kernels indicated khak the presence in the pistiallate parent of a factor, carried in the pr Collere Rewutlo chromosome, that is resncnsible for ve pRenetipe exhnibitadg very few or no A, dots, These ratiog ape entered £, the foot of B of sable (a)e neu Thef alee indicated that kkeaxfauckex the Spm-s eloneny lmerodcuced by the ss alu sy pollen parent, was epistatic to th& Peston, One of the plants belonging A r= to group B of tabie (a) had been crossed by a plant homozygous for ay “Hos Yy and pr and having no Spm, the types of kernels this cross produced. also indicated the presence of the weakened Spm-typne factor that was carried in the pr chromosome, There were 253 uniformly pigmented kernels of wich 70 were Pr and 183 were pr; 16 kernels snowed 1 or several small Ay dots in a colorless background and 112 of these were Pr and 3h. wers pre In addition, there were 69 totally colorless kernels, Another sories of progeny tests were conduced with plants derived from the several classes of kernels on the ear wjust described and also from kernels entered in 8 of table (al, These progeny tests confirmed 60,4 obeny we rb the conclusions derived from the tests just described, Details will not A oe. &iven here but in order to indicate the obviousness of the conclusions, data from test crosses of some of these plants are given in table Cb), It was learned from these studies that the Spm-w elevent belmved as a weakened Spm element both with regard to suppression of pigment formation - “1 “hen it is Mm: ea ‘ . : at ay and witn regard to mutation producing capvacity. * 5 * Me : _ im 2 present, the plants having aq 1 develop pigment but the rate of this is uty much slower than in the a_i plants that heave no Spm element, Also, suppression of vigment formation in the kernel is not complete for a very A Fy oo coloration ma. aopear ah the base of kernels that have Spm-w. In order to a A detcrmine if the Spm-w element has a weakened capacity to induce mutations, - . . . 7 . . . M= it wes incorporated into plants having various different states of as 1 but no *pm-s. These tests indicated that the presence of Spm-w results in a marked reduction of the freauency of occurrence of mutation but does not their alter the time offoccurence,c’ This latter remains a function of the state of the a.™+ jocus, 1 Although Spm-w elements residing in different chromosomes of tne Cor KOA complement have been detected, no evidence of transrosition of t is element has yet been obtained, fests of this are not extensive, nowever, and thus no conelusions regarding this may yot be drawn, The origins of Spm-w elements from modifications of Spmes elements is to be suspected but this tonclusion’ could only be sonsidered as tentative since evidence in sunport of it is limited to,|two cases , wreme Spm-w elements appeared in the same A A wp Ow eewear Fr pdeouueed h wiulow chromosome that had carried Spins Uae ty f) Blue Jaubarp Weed 0 Seu, Loufn. Ulu» Uororcrve.0n Sa fod aloes. Coin Awe Veokis b - Q pu Shy lqQrmSbe ot Qval Shi ide % x QQ, A/G, iy | Sposa of ALA 7 Ply Aly Tele oe len Pimury She ae FE, Feo pole Arte Unley sw Pde rototly | Coledow ere Plowt | Comtitubiow — | Poe | GON, flee bed | Colants Tonk, | br | tétals A-3 | Qe BWr| am She] et | 193 De — ~ 397 Ke) " " 25 | 29% o _ - 494 R-s « fade} 122) 136) 1 » | ase] o SYUb A-uy v " 1o% Od 0 O 139 O 399 f-s M " NZ 21 D 20D & 434 2 " u 1344 44 o 257| 0 53% was: i not jap 4b | oe | I | zoe] Oo | Wady Totst$ lois | 106% 2 ] a eer clarleao | i natuve Re ola oe WT WE fina, C3 am ld sue | sr | dbl | Ga | 64 - 543 O-} " . 137 aAtTy | WY iO) ~ He C-4 \ " q aHF | AS 34 - TY 0-2 | A™ShgSe | S4 | laal 4g 4 Pale] | 44 | co TB ow . qi | WB | 69 3 | 373 aL b33 -* \ 68 | 139) Na 4 faey} od | sun D-b \ i ST | 133] HI | ao | at | Oo | 44S Tole ls 6324 | \323| 524) 164 Tevale ° ne Tote ap a aokten aby lf oT EPO 4S2Pr W33ye | pe Gap eal ut pink ovegf = | Q-u “25 3} oO | Hb " 4q 5 Sn gowl Rahs soo. 193 Le naPai34 a Colne ft ume fen loot vary 6449C-37 gm sh Qua Wy Pe Spe er | pe sly % br ¢2 C Uitte (2629 14Spu]y + a C- ar Ww \\ Nv t\ i Pre [ tulle Try 3.1 + Aly Pre ysual 4. S95 D-2 Dw) As Pa Sem] ee D4 % aM Ge py ey tulle Te 47 no Span) Woe ty 6 mein. - 6298-6" % Potlene) 1040 @ ras3y Qsmishe] ads VL hy 4 Pa | Pe Pot |S n-o 4 Y Cylordors, fer A ol Pry > 66%3 Rey L\as4] 06638 &| % b64A-s O,m She] gabe Q WS Qh 14 eepy hi fr AU Ly Prt] Pa Spo ot Yl Wey Stuy . tf / {ue L885 tlle [193s J Tobo(he 4 LL yt ' I Q.*'xh, | q.™' Shr ; ba Spun-ur | yet K qa mich, | Gym Qh 2 yee KO Spy IC “ue” . “ % Qa, Ayla, dr } rir, 44 Syn 0] 404 vv? mr * ve wK " \ \ Po] pe i YM J YoteG= 15, bo Spm-o3 W Soar N AL Plone farm dod pale Bowie, ue S "EW dur dylan Krwb with natal Ar azo. Pods am Ss yg ye weet Benya Proua yo oY Rowe 1 Cot itu vento aod tnleos wel a | take Pau ys Audyl 2, dd. CrfsrCose : a te a oe _ Tobe a | Boeeln | W831 12.07 7O2 232 bow | 3174 ris adn wl toe T64 BT 390 O15 aw: Frese. w} nr 299 | Ao 150 403 129 {ae 4ST LOTS OS Aas oe 2159 : isya 7 -asg — 4H {| 1 VT” wea Tet Liew | 203, eH AD 323 | 1354. — en bee af cee nt nee vee BED Pelle purrs ast | — ' WH3 aur SbF. TUB - Pere @ - 1263 C- 3 C-7 Pardo ows Rorwwta wird utp, Age ukow® apa v4 Udkardoro lo grat Copur-0) 2 Pa AB Am eo Stee SUN 1a advent AWegbl Oye oy COON UA Ab Ly ohewk en, ig, 3 = spe wo Ia Cyfirdous (rel, cueel | Cptortorg Ind cet ae ” ‘ me - : . __ _ me | Pa Po ip Poy ET 3. Lely spa-? ! : : | To Potwlyer 360 84e \ q |200 9 Iz Lays Do 4 op mi ly Spn-e hot cH Pal wSpenr 30D 287 0 |787 a Hy 5) |e a “Hey, \Span-0 | Poca pe m1. = (fa Speed fe) 4% | 30 No} 5 12210. [22] 31, 7S Yala doses a bo 0 67 — ib - [ost = NY. 3Sym- 9 ~ Hoot a CRetom-cwln+) 7 Th v 3 2 1 14399 Aagstione Pe a Pa WR i) V4 | ~~ 0 N35) ~ 2 [- 436 Wawty il. a Soo Pn. 7 "en h rf ma. eee lye AT | 104K “4S ° aL TN pee — 1%3 21 0 Bt Va | 342 x o> Vpw-o Pala Wo Can | 12] \Ub } ee 20 | 5] §32 os ews ea na | 135 fmt | sn oN \Syn Pel Pa ae a Ap fie G rea 50. 54 HOY 1 rhe aa, ee Wn evita. sw ytetaae. SE se 0 | : uno ax} Modifier element in the Spm system In the course of a test undertaken to examine Spm number and location in the »rogeny of a plant carrving two Spm elements, both located in one we = vio, chromosome 5, a kernel appeared on an ear of a sdueee plant in wich the freauency of mutation at at was greatly augmented, Subsequent tests of the plant derived from this kernel and its vrogeny indicated the presence of an element, belonging to the Spm system, that increases the aie me1 with each of five tested -‘frecuency of occurrence of mutation at a, A states of At, but does not alter the tine during te cevelopment when “ dts presence may be detected only when Spm also is nresente Sy? N Ne these will occur, / Like Spm, it may undergo transposition, In all essential respects, it acts like a commhementary controlling element within the Spm system, Cul. f-Wwo The kernel carrying the modifier element appeared on tee eargof a tuo_of its ang Cohonr plant that was a,™"1 Sh,/a, shj, Pr/Pr, Wx/iWx im-eonstitutien when/ik had been used in a cross with a plant that was homozygous for Bas sh, pr, and Wx and had no Spm, / On thésbeearg,there were 167 uniformly dark pale colored kernels in the Shp class (no Spm), 186 Sh, kernels w th spots of deep color C Sen youd) in a colorless background ,with a pattern of mutant spots similar to that +O OW Car vid uy stow ¢ QW-l . shown in Ror figure 1p There were also 38h, colorless, sho kernels, MG “4 , Ax In addition, on one of these twa ears, a single kernel appeared that A exhibited a very marked increase in the number of pigmented spots, thehs L awe for ef kernel_was-removed~and $he plant g rown from £4 also exribited a very high A me] i ‘ rate of mutation at ay, in its somatic cells, In order to examine the nature of the modification that was responsible for this marked increase in frequency of mutation, one ear of this plant, (number 6889), was self~ pollinated, Another ear received pollen from a plant that was -omozypfous :& for ae (state )y Sho, pr, and wx, The reciprocal cross was also made. In addition, podilgm from plant 6889 was placed on the s#lks of éups—ef two plants that were homozygous for a,m sho, pr, and had not Spm, The kernel types and the ratios of them that appeared on the ears produced by these crosses indicated the presence of one Spm element in plant 6889 but it was not linked with Pr. Among the variegated kernels, there were two classes, In one, the kernels exhibited the expected number of mutant spots, In the other, one the other hand, the number of these spots was variegated greatly incre sed, The ratios of these two types of/kernels suggested the presence in plant 6889 of an independently located modifer element that serves to increase the frequency of mutation at amt and does so suaket fue either with state e or state = . In order to verify this, kernels were selected from the various classes emt these ears and the plants grown from ; them were again tested for presence or absence of th¥@ modifier in accordance with the phenotype of the kernel from which the plant arose, These tests verified the presence of the modifier in those plants that were derived from the kernels sha ing an increased freauency of mutation and its absence in those plants that were derived from the kernels that exhibited the usual frequendy of mutation associated with the state of ay ~ present in the plant. They also showed that Spm wes required for the modifier to be expressed, It was also possible to learn from these tests that the modifier undergo:s transposition in somatic eaiis. This results in removal of it from some cells, and increase in its number in others, In one case, this resulted in its insertion into chromosome 9, and linkage of the modifier with Wx covld be detcrmined in plants having the modifier in this location, R¢moval from this location and insertion elesewhere coul also be followed, Detailed evidence for the statements given above cannot be included in this report, Hoever, in order to indicate some of the methods used in obtainang this evidence, several of the tests will be outlined, sh In the cross of plant 6889 to a plant thet was homozygous for ays 2 and had no Spm, 12 plants derived from the variegated kernels showing the usual number of mutant spots (Spm, no modifier) were crossed by vlants homozygous for ay, sh2 and having no Spm, and also b: plants homozygous for age ai, (state ‘ys Shy and having no Spm, The types of kernels on the ears these crosses produced are entered in A and B of table - bight plants derived from the variegated kernels with an increased number of mutant spots (Spm plus modifier) were also used in crosses of the same type, The tyvnes of kernels avpearing on the ears resulting from thec ross with the plants homozygous for ay and sh, are entered in © of table * M1 Because the kernels on the ears produced by the cross with the a4 wegen = > m- Sh, no pm tester plants have different states of ay 1 in them that would require additional categories of kernel txpes in the table, they heve been excluded from it, 1261 Cudknre oh ‘ Wipe ES ~% wish »| chs “ % oa labs ween, so “eet G7 ile, aa poke sae lav a Dud, (0p - Gagner \ eu eds hee oud *. AvMShe [A te oh wo Wwaebfon 4 x Os ele | Orbs, Wo Spun, wo ys 3 ia Sp, bo Whee tibs Sha Ar fhe Stan , ) Tasdd oy af ee ce | ToTale. a o , f 37? Brv-3 7 { y oy g \\ = : - , 5 |s57 |) 3 692 | ! | , Tmo) tg tg a OF OF re WP a3y — of le7 | | 0 0 Oo | 26 | = Hoy Abin D-S Oo as O 0 O | , ooo yee Hp e a et \) Stee I] Ly LD\3 | 7 14q5 - oO ~ | 0 - U4OolZ LD hue an \ au | bb — | 253 —~ | Oem ! O -_ 32) nee — mf co ee tL eee ee | ing Be 2 b 4 121d 0 165 D | IST 3 74849 iL 146] cp DAS pus 2 Woot, i | own | | . aes 2 HD 30 pl eo , 2.00. 4B ; 7 7 an wep ee bee cee Sear e e f n ee — _. : - | a - _ a —— es ~ ( | | un? wal 20 Bot . . . o m= present in some of its gametes, Following introduction of the a, locus f from the male parent, the presencé of Spm in tnose kernels that received it from the female parent should ye revealed by the anvearance in them of small deeply pigmented spots in a folorless background due to activation of the a & — f 2 2 a," 1 locus by the Spm element. In those kerngls that did not receive 5Spn, the aleurone layer shoudd be uniformly pigmented. Among the 30 plants wat derived from the colorless, sho, Y class Af kernels, it could be determined on this basis that/15 had a single Spy’ element and 15 had no Spm, In 13 of the 15 plants/that had Spm, linkAge of it with Y was evident (A, table @) but in the 2 rémaining plants, ng linkage of Spm with Y was noted (B, tabled (The reason Jor the absence of linkage of Spm with Y in these 2 plants will be consideyed in the next gection, It need cnly be menticned nere that t unexpected.) Among the 2l: plants derived from the colorless, sho, y class of kernels, 6 had a single Spm element (C, table QJ), and 18 had no Spm, ee orien te ah ARRANGE Nene hatha ie a, nee renee FA ce In the above described test, the state of ay present in the tester stock (pollen parent) was either that snown in = or ~ of Bigure 1. All of the kernels having Spm exhibited the pattern of variegation characteristic of the introduced state,-- small spots of deep pigmentation in a colorless background, and these spots were rather uniformly distributed > g e 21 over the aleurone layer, Also, all those that had no Spm were uniformly \ pigmented either darkky pigmented if state - had been introduced by the L male parent, or lightly ovigmented if state ~- had been so introduced, This made: More than one fertile @r was wroduced by some of tvese plants. it possible to place on the silks of some of these additional esrs pollen i of plants in weich Spm was considered to be absendt but in wich other This was done with plants having the states i i t i states of at were present, It was found that if the element - i present / Shown in =, ~,=, and » of figure 1. mM~ 1 ta present in the pistillate parent had activated the state of a er states of | x in the tester stock, it would also activate each of these ot! However, thevattern of variegation that appeared in the kernels \ m=] a7 . that received Spm was not the same as that given by the tester stocks. mej] 1 | Inste:d, it was that which characterized the particular state of a I i that hed been introduced by the pollen parent. That the activating elenenti, } nevertheless, was the same for each state could be s:own in some of these tests by whéans of its linkage with Y and an illusttion of tsis is piven | OV, | in D of table Qe i=armeseeoet testswhich showf the activation of the different states of ay ““ by the same Spm element wef conducted and the In this test, result of one of these teste is illustrated in figure 2, m=1 ; shown in - of figure 1 was a variegated plant having the state of aj" 22 ape was crossed by the Spm tester stock having the state shown in =~ of figure 1, A Without exception, all kernels that exhibited the pattern of variegation me] . characteristic of the state of aj, present in the female parent likewise exhibited the pattern of variegation characteristic of the state of a min] 1 that was present in the male parent, The same element activated both states of at, Another type of test involved use of pethben—*ror «a \ Qua gre or WT plants thet wi hor nozygous for the standard recessive, AL in wh ‘Ach tie Spm le a ’ “4 . Gesrstitittien could e follien ont durables gn (BAAD aT from such g plant wa S S Peeeee BSR the silksot /a-number of non=-variegated A ajmnL Woke WD deouh at plants among wich aepmmbesof different states/vone manmeaantod, Paro ; Atul; wo each stom testy the peemesticn of variegated to non-variegated kernels was the sane, pegerdless of the state of ay that was present in the pistillate parent, heavonhwhi on -of these tua slceseret-hecneks cag A we. Wad wnee Prloou? indtested the number of Spm elements in the pollen parent, Again, if ut, linkage of Spm to a given marker was expressed among the kernels on one A ear, it was also expressed in all ears where this could be detected, as LAT ee bbs, Phau aise dy Sorwls Oppiarny on Table bob. Wrrsd, Laat Lavy covet nun Pouce of Konme we ued whan pli aul w» \o" + WEA om ryt Vaupoung (or arm She, ry Wes pheemt S Pau ch db - doap tom. ‘ V WA iy Se 07. Se iS = Mind =» Cane wl la Sf ~ Roenulfoat » wou veatba 3, 5 ln aU 4 ee ate Hoon ow Adehnn ws Lowe oath. bt . Sew “62 Rolin: Bi fn dork fn pe oda lak. a ‘| MA _ \| YL : a x v / bb696-1 + 44 Rea bt \3D UY | Ly f- bot ebtorr@ bo 140 1224 'G@ t 200 w a Ss wl 7 - ~bb%c-$ Ebtol- vt _ 244 — o oy Az Wear a mE boo R-b6 4 b6%¢R-3 19s yw OA.)O, 4, vehun | C1 pi Dauste) 5$6l-7 __$%b 19% | Ly 335% uv C% 10 Spy LS QD Oo N17 hsy 4 pale Q, 7h, 7 686'-] x oe sl-o (stot - | | 224 Tt be59-\4 x 6 $6)-] 4 Dot be 13D OMe) | 24S” 247 | lQs Sy 74 loy 6499R Colap-) 468 bI-! Cy : wet Ow haga Bes Sb 74 [era R-3 Wb lod 173 R-9 6h 6% sy P=! 1 TT $37 B-4 _b0 b2 IS2 %-] 1o7 64 13> 530 Wu Ss QS | ate bb 44 AE tad, woSpan . WS ly » (PCy w~ 19 lor " BM@t w W ls 4y 160 U BO ye 0 \ L234 25h wv RB -1 t Ww vl 1 Q [332 a1 4 ——— “14\ 195 ag Pale She An Shs 6461-1 $ x» vbasTt-3 Catop-) 139 14S __ 0 361-7 tulle lache 57 19 | 2bb. 28% 0 2 ey 25S 247 217 1 106 “~pTals rr SS Stood 9220 Q™ shila d. % bBbl-7 6399 P- 2 oe. 129 ae 21) es Rew Lov SD o te — "Reb lop 92 O 179 —, w QM \20 UW 0 125 = \ B-2 q7 $3 o 69 | \ Bs 100 lid QD 176 “ w Rb 17 MI D Hy "B-¥ ZY 21 0 0 B94 13 6D 0 Iso u Bevo bd 49 O uy ™ Totels $86 19% ] (671 a4. Wo >wa 63% + cue Sh, cite, , ——TeteIs Rw os & ? Jj2y R4 1S 4 Ios 30% Re \ 43 lo 2937 A 43 lo7 200 R-2. 255_ 229 HSY¥ - \Le )2¢9 2557 B-b 21D 202 HI (3-10 \2) 13y 255 Totals L157 Wu 2334 + Phowp Tyo) Asuud- Cont t vor 3 3 Opt ulin 4 oT | 4B Joh. Spun On thi Sha]q™ cha Wp sw | | Ci StOb 5713. ea | yi Gon Toleale Paw 6% 61- | 2249 I74 | +03 Plouit 04 61-7 1349 I43 27 RB. Qywrshe/ QM che Cabed S717 p-3) wound Q,Ar]o,dr Suu 57» gluil 6 36! -I Que) 6361-7 H % plouta. 27° 6961-1 Ss Q ‘. Cc. Q\w Sho Caboly 5720) | Q) oe Woyw? yx __ totals 0461-7, 4%} _ 1567 \Q16 GM | Ae, No S{eu.o7 wd 0; bathe | [yan 37 he Oa a cholo She wee tea AUB yur Ot 1 Oh Alrvlode | Sin - _ Ply 646 Ie 1 520 T oa sey Sie mM Aa a Ofeg, A Th: i SUG th en ee Wo wa ++ 1 ee D0 _ 54 S| _1Grb | . =f uo Pm 14S 1836 ee oa Spy A plow, (661- 7 ! 4b ner ie eae : — AA aloe soa ‘ jaa a sy oe em [3356 ! ATT | 2334