-20= €<— Eight of the C sh ba kernels were ix, None of these, however, showed any 4x to wx variegation, In order to rapidly visualize the tyoes and fresuencies of chromosomes 9 constitutions that the gametes of this plant could have, a diagram of the usual synaptic association of the two chromosomes 9 in this plant and the resulting tyves of cross-over chromatids, is included with table l4ea, It may be seen from this diagram that the 8 C sh bz Wx kernels could arise from erossing over in the region of the arrow--between the distal #x locus and the point that marks the beginning of the proximal duplicated segment, These chromosomes--oarrying C sh bz ix=-should be normal in morphology and should have no Ds locus, Therefore, no /x « wx varie-= gation should appear in the kernels receiving such a chromosome, As stated above, no 4x « wx variecation appeared in the C sh bz jx kernels, This same .lant was crossed to an ac ac female plant having the rearranrced chromosome 9 with ec 3h Bz wx ds and a normal chromosome 9 with C sh bz wx ds (table 14-b). In this cross, the 65 C + ¢,3h Bz Wx ~ wx kernels arose from fusion of a female nucleus carrying the c 3h Bz wx chromosome with a male nucleus carrying the Dunlication chromosome, in the resulting varlersted kernels, all the oc sreas were wx, No extensive wx sectors regularly appeared in the C areas, Again this shows that the Ds locus in the Duplication chromosome 9 of the male parent -ust be to the right of the Wx loci, dith recard to chromosomes 9, plant 46282 had the same genic and chromosomal constitution as plant Kel. It was, however, ac ac, Nhen crossed to ¢ sh bz wx ds ac female plants (table 15-a) or to ¢ sh 32 wx dg ac female plants (table 15-b), no variecated kernels appeared, “There were only three types of kernels on the ear in each of these two crosses, On the basis of the civen constitution of Table 15 a © sh bz wx ds ac female Table 1l5«d e ¢. sh Bz wx ds ac ac 2@ x 4688K-2 o ec Sh Bz Wx Ds 4435—=] C Sh Wx | C sh ix Cross ln non-varie- | C=e, Sh(+sh }* and gated wx lowx) C sh wx non-varie- geted ' | 4435-1 x 4628K-2 20 79 se * Some kernels are Shesh and Wxewx variegated; others are not, as expected from constitution of 9, Zoe Plant (table 16-a) and to three o sh Bz wx ds ac female plants (table 16-b). The types of kernels appearing on the ears of these test crosses have made it possible to write the scenic constitution of the chromosomes 9 in this plant. Crossing over between the normal chromosome 9 and the Duplication chromosome 9 in the region of the distal duplicated semment occurred frequently and was normal in relative frequencies between the marked loci, ‘The percentages in the marked regions are given in tables lé-a and 16-b, The explanatory supplement accompanying each of these tables will make this evident. No Wx - wx variegation or extensive wx sectors regularly appeared in the C §z areas of the variegated kernels of table lé~a, all of the C bz areas were wx; none showed Wx = wx variegation. In the cross to the ¢ sh 33 wx ds ac female plants, the Ds mutation in the ¢C $h 4x kernels resulted in o sh wx sectors, No #x - wx variesation was present in the © areas and, as stated, all the c areas were wx, Again, large wx sectors were not appearing in the C areas, This type of variegation would be expected if only one Ds locus were present and if its position were to the right of the proximal duplicated segment. It should be noted that none of the © Sh bz wx or C Sh Bz wx kernels in table l6-a was © = c variegatec. None should be variegated for these kernels should have a normal chromosome 9 with no 3s locus. These ¢ Sh bz wx and C Sh Bg wx chromosomes arose from a crossover in regions 1 and 2, resvectively. It should be noted that no ¢ sh bz dx kernels were produced, These would appear only if a rollen grain carried a double crossepver chromatid (regions 2 and 3), Such a double cross-over chromatid should appear relatively infreocuently. No such chromatid was represented on this ear. Table 16-4 C ds Sh Bz 4x Wx Bz Sh Ds Cshbzwxdsac? x §-43-S0 Sa ix Wz bz Shoe, AG Aco d 43563<3 4625L-1 a C Sh Ba Wx /C BaeC ba, Shesh C Sh Bz wx C Sh ba wx C sh bz fx C sh bz wx Wx=wx non-varie- fated p 1st 125 74 10 o 188 * Several have a few wx spots. Supplement to table lé<-a a 3 C ads Sh | oe bug Wx Bz Sh Ds aes, Cds sh bz wx ds > Non-crossovers C da Sh C as sh Region 1 C as ah C€ da Sh Region 2 C da sh C de Sh Region 3 C ds sh C ds 3h Summary: Crossing over Region 1] Region 2 Region 3 * Too high; probably includes some No Da or No Ao gametes due to Ba bz bz bz Bz bz Bz Wx Wx Bz Sh Ds wx ds Wx Wx Bz Sh Ds Wx Wx Wx Ba Sh Ds wx ds wx dix Bz Sh De #x ds 4.4% “32. 517 loss or transposition of Ds or Ao, “ C BaeC bz Sh, Wx-wx - © eh bz wx = ¢ Bec bz Sh, W#xewx = ¢ Sh bs wx = ¢ BaeC bz, Sh, ¥x=wx = ¢ Sh Bz wx = ¢ Bz-C bz 3h, Wx=wx ~ € Sh Be Ax 128 10 74 13* Table 16-b We c sh wx ds ac 2? x 46281L-]1 ¢ Cross GC Sh wx § Cwc, Shesh, cCShwx C sh dx C shwx Odds non-varie- $$$ W#xewx gated : 4347~4 x 46281-1 10 129 — 68 = eB | oa 46 4418F-2 x 4628L-1 8 50 360260 aL SR Colorless dx-wx;3h A416F-2 x 46281-1 4 59 46 0 - 3 Totals 22 218 160 = 3* AB * 1 has defective embryo; probably carries an abnormal chromosome 9. ™ at J Feed Atg ye v3 f ey ; WK ve Fak D Tr Aes necl Ve Cac ~ ——_ | 3 ° ~ , é ol } - A \ocukiad paren Cnbamerchey - Wet T Tee Dh [coh uy ar 4 } 4 Table 17a C ds sh bz Ax Wx Bz Sh Ds” Ac ae C sh bz wx ds ac & x C ds sh bz wx ds 46281L~2 Cross Cc Sh Bs Wx C Ba-C bz, 3shesh, C sh bz ax C sh bz wx non-varie~ Wxowx hon variegated gated (Ac ac ac) (Ac ac ac and (Ae ac ac and {ac ac ao) ac ac ac) ac ac ac) 4361-16 x 4628L-2 59 59 li 255 4366-9 x 4628L-2 37 34 ll 245 Totals 96 93 22 500 Cross~over region C da sh bz axt Wx Bz Sh Dsa& ec ds sh bz wx Fe Cross-overs: C ds sh bz wx dx Wx Bz Sh Ds® Duplication chromosome 9 . C ds sh bz 4x ds Normal chromosome 9 = 22 Normal chromosome class "rossovers 522 4.2% es of Supplement to table 16=b @ sh B2 wx ds ac ¢ x € ds Sh Bz Wx Wx Bz sh De? Ae Ae of C ds sh bz wx 12 ty C.Sh Wx Wx sh Dee C sh wr dg Non-crossoverg C Sh #x 4x sp De® “= Cec, Shesh, dxewx C ah wx da = C bh wx £42 Region 2 C 8h ¥x ¥x Sh Dg2 “ Cea, Shesh, dxawx C Sh wx ag | * C Sh wx 150 Region 2 C sh wx wx Sh De® = C«o, Sheesh, dx=wx C Sh Wx ag - C Sh Wx g2* Regions 1 & p C Sh wx Wx 3h De® = Cee, Shesh, dxewx C gh 4x dg = C¢ sh ax 3 * Teo high; probably includes No Ds, or No Ac ¢ gametes due to loss or transposition of Ds or Aa, Summary of crossing over; Region 1; 36.7% Region 2 ; 6% 0.72% (with no interference Would exnect 2.27%) Doubles =e wD So Plant 4628L=2 was similar to plants 4628Ke] and K-2 in the genie and chromosomal constitutions of ite chromosomes 9, It was heterozygous for Ac (Ac ac). In crosses to © sh bz wx ds ac female plants, half of the © Sh Bg Wx carrying kernels were ° Bz - © ba, Sh = sh, wx + wx variegated (the Ac ac ac kernels) and half were none-variegated (the ac ac ac kernels), table 17a, Again, in this cross, a small percentage of the C sh bz kernels were Wx (4.2%) and again, none of these kernels were Wx « wx variegated. The reason for this has been stated previously in the description of similar crosses involving 4628K-1 and Ke2, dhen plant 46281~2 was crossed to ac sh Bz wx ds ac female plant, the expected types of kernels appeared, table 1?-b. Half of the C Sh Wx kernels were non-variegated and half were C ~ c, Sh sh, ax = wx variegated, In the variegated kernels, all c areas were wx and no large wx sectors were regularly appearing in the C areas, The 13 ¢ sh Wx kernels (3,9% of the sh class) were non-variegated, To repeat, since they carry a normal chromosome 9 with no Ds locus, no variegation should appear, Plant 4628L~2 was crossed to a c sh x, Ac Ac female plant, The kernela resulting from this cross are indicated in table 17-c, No obvious C - o variegation could be observed in approximtely half of the C Sh kernels, These are probably the Ae Ao Ac kernels, The kernels showing C « c variegation had a pattern of Ds mtations characteristic of Ac Ac ac constitutions, «again, it may be noted that no C ~ c variegation was present in the ¢ sh class of kernels, No variegation is expected, Table 17 b © sh wx dsp ac ¢ x 46281-2 ¢ ! Cross C Shix | Cec, Shesh, 4x-wx C sh Wx C sh wx non-varie~ non-varie~ ted __ gated AO AG and ac Ac and ao 4347-24 x 46281-2 39 35 . 13 31? Cross-over recion C ds sh Wx ix Sh Ds _ C de sh wr 48 gw Cross-over: Duplication chromosome 9 C ds eh wx Wx Sh De a Normal chromosome 9 C ds sh ux _ = 13 Total kernels = 404 Normal chromosome class : 330 crossovers : 3.9% Table 17-0 co ah Wx ¢ o 8.1 Wx Ao Ae @ x 46281-2 ¢ Ae ac ‘Duplication chromosome from d | Normal chromosome CO sh Wx | Ceo,Sh-sh, ix | Cc sh dx Not obviously ' nonevariegated variegated (Ac Ac Aco and {Ac Ae Ac) (Ac Ac ac) Ao Ac ac) 4365-20 x 46201<2 65 59" 219 * 86 kernels are speckled with o; late Ds mutation 1 Kernel has an Ao ae ac type of C — co variegation, Yoo, og aif on Table 18 on 2b Plants arising from various kernels frem self-pollinatien,of plant 4306 (see Table 3). Chromosomal and genic constitutions of plants of culture 4628; sub-sultures C to L. Dup. * Duplication chromosome 9. Nor. = Normal chromosome 9 Appearance of Aotivator Table or page Plant Chromosome 9 constitution constitution feference peers from which Dup. I Ds? 3h Bz Wx Wx Bz Sh Ds* 4628C-9 pup. I Del Sh Be Na Wx Be Sh Det Ac Ac Table 7 I Sh 4x 1s ax 3 3 2 m pezo Dupe I Ds” Sh Bz 4x 4x Bz Sh De Ac ac Table 8 I-¢ Bz-C bz,Sh-sh, Nor. C ds sh bz wx ds dxewx ° si s dx Wx Bz S 2 * Dell Dup. I De® Sh Ba tx Wx Be Sb Ds Ae ac Table 8 I-C Ba-C bz,Shesh, Nor. C &s sh bz wx ds 4x=wx 1 sh? "Fe 1 Non . oe = ve a Ao Ae Table 9 I 3h wx *,*., s Fe 2 Nor. I ds Sh Bz wx ds ? Table 10 (see page's) I Sh wx Nor. C ds sh bz wx ds - Nor. I Ds! Sh Bz wx ds - " Ge 1 Nor. C ds sh bz wx ds Ac ac Table 11 IeC Ba-C bz,Sh-sh,wx © eH Dsl sh Bz wx ds ac ac Table 11 I-c Bz-C bz,Sh-sh,wx Nor. oo bet la Ds+ Sh Bz wx ds AG ac Table 11 I-C Ba-C bz,Sh -_— = ae Lo C ak , ca AS geen . Hy guuol chun Such kernels would not appear, however, if a Wx locus were carried by the proximal duplicated segment, Such kernels did not appear in the crosses. It could be concluded, therefore, that a #x locus must be present in this segment. Sh and Bz loci must likewise be present in this serment,. The evidence for this is apparent from several considerations, It is most obvious, of course, in the phenotypes produced by the Duplication chromosome 9 in plants 4628K«1, K-2 and L-2, It is the Sh and Bz loel in this proximal segment that accounts for the Sh and Ba phenotypes that appear in the kernels from which each of these plants arose and in the kernels having the Duplication chromosome 9 in crosses of these plants to sh b2 planta, Neither I nor C can be present in the proximal duplicated segment, The absence of a C locus in this segment was considered on page 4 . The absence of an I locus in this segment is apparent from the phenotynes that result from a Dat mutation in an 1 Dat Sh Bz Wx Wx Sh Bz pe* / c sh bz wx / © sh be wx, Ac ac ac kernel, A Dat mutation deletes the I locus distal to Dat. The resulting vhenotyve is C. Again, if I were present in this segment a Duplication chromosome 9 with the constitu- tions shown to be present in sub-cultures 4628 K and L would not be recovered, These chromosomes have no I locus in the proximal segment. Neither an I locus, nor a C locus, therefore, is present in the proximal s8epment, 27a All of these considerations point towards the exact, composition of the two segments with regard to the loci they carry, The position of breakage that could give rise to such serments is also indicated, The distel segment extends from a position just to the right of I (demarked by the inserted Det locus) to a position 3 or 4 crossover units to the right of the 4x locus in this seement. Crossing over between a normal chromosome 9 and the Duplication chromosome 9 ia normal in kind and relative frequency within this distal segment, as the tables have shown, The crogsover unit distance between this distal ‘x loous ang the junction with the proximal sepment in the plents heterozygous for the Duplication chromosome 9 is the same as the crossover unit distance between Bx and Ds-standurd in plants with two normal chromo- somes 9, A summary of the cross over percentages in this region, that oecurred in plants 4626K-1, e2 and le2 where it could be determined, is given in table 19, The junction of the two segments is marked by the position that Dsestandard oceunies in a normal I Sh Bg wx Os chromosome, aAs shown in tables 1 and 2, the chromosome 9 in plant 4108¢81 that carried I Sh Bz Wx and De, was normal in ite senetic behavior and had Ds at the standard location. The presence of only two recognizable odd gametes was observed in the crosses of this plant. One of these had the duplication that arose from an aberration ocourring within this I Sh Bz #x Ds chromosome, In this Duplication chromosome 9, the »roximal segment has the same genes within it as the distal segment, This indicates that chromosome breakage occurred in a cell of the parent plant (4108C+]) at the Ds locus and also at a position just to the right of the I locus. This was followed by fusions of broken encs that at the same time included a transposition of a Ds locus between two of these ends, X3efore this event may be reconstructed, Table 19 Summary ef table of per_cent crossing over between the distal ix locus and the end of the distal duplicated segment based on the recovered normal chromosomes 9 in crosses of plants 4628K-1, 4628K-2 and 4628L~2 Cds sh bz ¥x wx Be Sh De® * 2 -- ¢ ds sh bz wx} ae “ON . crossing over region Cross GC sh wx Oo sh wx Percent crossing over 4362C<3 x 4626K~) 323 8 2.4 4365-1 x 4628K~) 150 1 0.66 4561-11 x 4628K~2 132 4 2.9 4349-6 x 4628K~2 214 3 1.4 4462C=2 x 4628K=2 215 13 5.7 4361-16 x 4626L<2 255 ll 4,1 4566-9 x 46268L<2 245 ll 4.3 4347-24 x 4626L<2 317 13 3.9 Totals 1849 64 3.5 Total kernels: 1915 ~28< it is necessary to inquire into the order of the genes in the proximal segment. The order of genes in the proximal segment has been determined from two general types of evidence: first, the type of variegation patterns in the kernels having the Duplication chromosome 9 and secondly, the tyoes of chromatids that plants heterozygous for the Duplication have produced, The order could be (1) I pal sh Bz dx Sh Bz ax Da® or (2) I Det Sh Be Wx dx Bz Sh Ds", If (1) were correct, it would be difficult to explain how the large wx areas could arise that frequently appear in the C Ba sectors in the I - ¢ Be ~- C ba, Sh «= sh, ix = wx variecated kernels of tables Yea and 8, If the second of the two genic orders was present, just such wx regions should appear because the vreakage~fusionebridge cycles that are initiated by pat mutations should often result in deletions of the two #x loci from some cells while retaining the proximal 32 locus. In the crosses indicated, the resulting cells would be C Bz wx. If order (1) were present, the C Bz sectors that are variegated should have some C bz areas within them that are Wx = wx variegated, As stated earlier, no such C b2 areas are present. All of the C bz areas are wx, If order (2) were present, all the ¢ bz areas within the C 32 sectors should be wx for the #x loci should be lost from some cells by the breakare-fusion-bridge mechanism before the proximal Bz locus is lost. In other words, 3z will not be lost before the Wx loci are lost, In the crosses of plants 4628 Kel, K-2£ and Le2 to © sh bz wx plants, no large wx areas appeared in the J 32 sectors of the variegated kernels and none are expected as thers is no Ds locus to the left of Ds* that, ny mtation, could initiate a dicentric chromatid having Bz and 4x in the region between the two centromeres, These several observations, then, strongly support the given inverted order of the genes in the proximal segment, Figure 2 Types of chromatids that should be produced by crossing over with order of genes (1) and (2) in Buplication chromosome 9 Type (1) order Type (2) order A. Synapsis of distal segment of Duplication Ae Synapsis of distal segment of Duplication chromosome 9 with homologous segment in chromosome 9 with homologous segment in the normal chromosome 9 normal chromosome 9 tay , Single crossover chromatids Single erossover chromatids Vy Yo : ‘ Ape TpchRg © wet shy wep” TOS By we We . Be Shh a i . seine eeagpene : pete en ve en oe ee . . ate meen maestro nee crt } ~-Or- oe C & ab, Ly by - an - deadn by mt og i e ; ON Region 1 : Region 1 I ds sh bz wx ds Normal chromosome I ds sh bz wx ds Normal chromosome C Del sh Bz ax Sh Bz Wx Ds Duplication c Dsl Sh 32 Wx Wx Bz Sh Ds2 Duplication chromosome chromos ome 2 Det sh bz wx ds ds Sh Be Ax Sh Bz Del Sh bz wx ds ads sh Bz 4x Sh Bz Dst 3h Bz wx ds ds sh bz ix Sh Bz Dsi Sh Bz 4x as ds sh bz wx Sh Bz Figure 2 continued Region 2 Normal chromosome ix De® Duplication chromosone Region 3 Normal chromosome Wx Dae Duvlication chromosome Region 4 Normal chromosome ux Bse Juplication chromosome Region 5 Normal chromosome Wx Ds® Duplication chromosome QO Lo} Region 2 pst sh bz wx ds ds Sh Bz #x Wx Be Sh Ds® Region 3 Dsl Sh bz wx ds ds sh Bz ¥x 4x Bz Sh Ds* Region 4 Del sh Bz wx ds a@s sh bz dx Wx Bz Sh Ds® Region 5 Det Sh Bz dx ds ds sh b2 wx sx Bz Sh Ds” Normal chromosome Duplication chromosome Normal chromosome Mplication ehromo some Normal chromosome Duplication chromosome Normal chromosome Duviteation chromosome Figure £ continued B, Synapsis of proximal segment B. Segment of proximal segment of Duplication chromosome 9 of Duplication chromosome 9 with homolagous segment in with homologous segment in Normal chromosome 9 Normal chromosome 9 aa S om . ’ . vL : y, + ! EWR, PR cafe ee TeisR, Wi we tty ee - ade . 7 : + met carter niet atm ane ES cn enameet Gene me stevatnee on <> dec a ne cee mee ne “St ye . a ee eee nD Glen * ~ OQ Cdn ah bs hy oo de des ee Lo . Lo m we i, . . a Single crossovers All single crossovers would give a dicentric chromosome and an acentric Region 1 fragment. The dicentric chromosoxe 1 would be deficient for the terminal I Ds” Sh Bz Wx sh bz wx ds Duplication third of the short arm. chromosome C ds Sh Bz Wx Da® Normal chromosome Region 2 I Dsl Sh Bz vx Sh bz wx ds Duplication chromosome C ds sh Bz Wx Ds* Normal chromosome Region 3 I Dsl Sh Bz Wx Sh Bz wx ds Duplication chromosome C ds sh bz ax Ds” Normal chromosome region 4 I Dal Sh Bz ix Sh Bz vx ds Duplication chromosome C ds sh bz wx Ds@ Normal chromosome