THE CYTOLOGICAL IDENTIFICATION OF T
CHROMOSOME ASSOCIATED WITH THE R
    LINKAGE GROUP IN ZEA MAYS

BARBARA McCLINTOCK AND HENRY E. HILL
    Cornell University, Ithaca, New York

       Received March 1. 1930

T-ABLE OF CONTENTS

INTRODUCTION ...................
hlethods ..............................
Observations on trisomic inheritance of R. ...
Independence of the R-G linkage group .......
SUmI.~RS ...............................
.iCKNOWLEDGMEXTS ......................
LITERATURE CITED ......................
;\PPEXDIS ............................













. .

PAGE
175
176
176
186
190
190
190
190

INTRODUCTION

Genetic investigations with Zea mays have established ten linkage
groups. Likewise, cytological investigations have revealed the presence
of ten morphologically identifiable chromosomes composing the haploid
complement (MCCLINTOCK 1929b). It is the aim of the present investi-
gation to correlate a particular linkage group with a particular chromo-
some.

The method employed has been to obtain 2nfl plants trisomic for
different members of the haploid complement, and then, by means of
trisomic inheritance, to determine which chromosome carries a particular
group of genes.

The 2nf 1 plants have been obtained from the progeny of one original
triploid (MCCLINTOCK 1929a).  The chromosome-number in the female
gametes of a triploid varies from ten to twenty. Trisomic individuals were
obtained directly from theFr of a cross triploid X diploid and from the 2nf 1
progenies of FI individuals with more than one extra chromosome.

Inheritance data obtained from the 2nfl plants of culture 131 sug-
gested that in this culture the r-g carrying chromosome was present in
triplicate. An effort was therefore made to test the validity of this inter-
pretation and to verify the genetic inference that the nine other linkage
groups are independent of the r-g chromosome.

The evidence indicates that the smallest chromosome of the haploid
complement carries the genes of the r-g linkage group. Thus, 2n + 1 plants
of culture 131 showing trisomic inheritance for r have the smallest chromo-
some in duplicate in their 11-chromosome microspores. Likewise, 2nfl

(hi~~rcs 16: li5JIr 1931


176         BARBARA McCLINTOCK AND HENRY E. HILL

plants of other cultures which show the smallest chromosome in duplicate
in ll-chromosome microspores have given trisomic inheritance for r in
later genetic investigations.

                           METHODS

Root-tips were fixed in a chromic-acetic-formalin mixture and sectioned
in paraffin. The aceto-carmine smear method was used for sporocytes
which previously had been fixed in an acetic-absolute alcohol mixture.
The microspores were fixed in an acetic-absolute alcohol mixture, stained
with carmine and cleared with chloral hydrate.
For morphological studies of the chromosomes, late prophase stages of
the division of the microspore nucleus were found most valuable, since
the chromosomes at this stage are longer, their constrictions are more
obvious and the relative length of their arms is more readily determined
than in the contracted metaphase stage. From comparative studies of the
most easily distinguishable chromosomes of the complement it is clear
that the morphology as shown by the prophase and metaphase microspore
figures is essentially similar to that shown in the root tips. The presence
of only the haploid complement and the ease of observation in the micro-
spore favored the use of this stage for cytological studies.

OBSERVATIONS ON TRISOMIC INHERITANCE FOR K

As has been stated, the 2n+l individuals of culture 131 were trisomic
for the r-g chromosome. This culture arose from a 2n+l plant which had
been selfed. Of 61 individuals examined, 39 (or 63.7 percent) were 2n,
21 (or 34.4 percent) were 2n+l and 1 (or 1.6 percent) was 2n+2. It can
be safely assumed that all the 2n+l plants of this culture were trisomic
for the same chromosome, since the normal rate of non-disjunction in
Zea mays is very low.

As a result of the distribution of the members of the trivalent at
meiosis, 1 l-chromosome carrying and lo-chromosome carrying gametes
are formed. In a normal pollination, the extra chromosome carrying pollen
grains do not function well in competition with the n-carrying pollen grains
(see table 1). For genetic investigations, therefore, it is necessary to
                         TABLE 1

  Percenl of 2n+l individuals resulting from th cross Zn 0 XZn+l cf.


2n? x2n+k?            2n            2n+1          Percent 2n+1

1921x224          261             3            1.13
1928x225s           83             2            2.35


IDENTIFICATIOS OF LINKAGE GROUPS          177

consider the functioning of 11-chromosome carrying gametes only in
the case of the female. On the basis of random distribution of the three
similar chromosomes at meiosis one should expect half of the eggs to
carry the extra chromosome, but actually only about one-third of the
eggs carried it (see above and table 2). This discrepancy can be explained
on the basis of irregularities at meiosis.  Nine bivalents and one tri-
valent are found at metaphase I only in approximately two-thirds of the
sporocytes; in the other sporocytes there are ten bivalents and one uni-
valent.  When the extra chromosome appears thus as a univalent its


                         TABLE 2
      aTumber Zn+l : 2n ittdividuals from the cross Zn+f Q XZnd.

CTx.TCRE

166
168
176
224
225
229
230
231
232

-


_-









-_

I

2n .
-____

11
1
5
2
14
10
14
17
9

-


_-









--

I

2nfl


4
1
2
2
5
10
4
2
11

Totals

83

41 33.06 percent
    2n+l

behavior is very irregular (MCCLINTOCK 1929a). It may not go into the
spindle figure but remain in the cytoplasm.  It may be found in an ab-
normal position in the spindle. Again, the univalent may lag in the central
part of the spindle with or without showing evidence of. a separation of its
split halves. If the halves should be included in the two telophase I
r&lei, they would probably lag in the second meiotic mitosis. In all of
these cases a loss of the univalent will occur in meiosis, with the formation
of all n-carrying nuclei instead of half n-carrying and half nf 1.  This
phenomenon could account for the increased ratio of n to nfl gametes.
The difference in many cases is probably not due to lack of viability of
n+ 1 gametes or 2nf 1 plants, since in many ears of Zea mays the regular-
ity of row and kernel position allows undeveloped kernels to be readily de-
tected. Some 2nfl plants bore almost perfectly filled ears. It is possible,
also, that the lowest megaspore, if it contains the extra chromosome, does
not fuuction to produce the embryosac but, in a certain percent of the

GENETICS 16: XI 1931


178         BARBARA McCLINTOCK ASI, HENRY E. HILL

cases, one of the megaspores that contains the haploid complement func-
tions instead.

The trisomic individuals of culture 131 were crossed so that their prog-
enies were heterozygous for at least one factor of every linkage group.
Backcross and FZ ratios were obtained to determine which factors were
inherited on a trisomic and which on a disomic basis. Abundant evidence
for trisomic inheritance of r in the 2nf 1 progenies of culture 131 (cultures
189, 209, 224, 225, 229, 231) was obtained.
Simple ratios may best be considered first. When the R factor for red
aleurone is duplex (RRr) the gametic ratio expected from random dis-
tribution of the extra chromosome is 2R:2Ry: 1 KR: lr, or a total of 5R: 1~.
Since only the n-carrying pollen grains need be considered, the functioning
male gametic ratio is 2R: 1~. In the 2n+ 1 progenies of culture 131 duplex
for R the backcross ratios through the pollen were 646R:3.5 (table 3),

  ~9 XRRrS

1928X2253
192gX209,g
19210x224~


Totals

COLORED                 COWRLEES


240                  91
198                   141
20s                  123


646                  35.5

a fair approximation to 2: 1. On the same basis, crosses of 2n KY P X2n
+lRRr $ should give 5X: Iv. Table 4 shows a total count of 2102R:135.
A sib cross between two heterozygous 2n individuals gave 290R:88v, or
the expected 3 : 1 ratio (table 7).

RrO XRRr8

22511X2242
225,tX224$
2317x2319
231*4x2319
23116)<231!J
23117x2313

Totals

-









_-


-

TABLE 4

CoLORED

COLORLEsa

26s                  74
235                   49
3s2                   61
419                  82
357                   s2
444                  Si

2102 .   I         435

When R is simplex (Rrr) the expected gametic ratio is 1 R : 2 RY : lrr : 2~.
-With elimination of the n+l carrying pollen grains the functioning male


IDENTIFIChTIO?U' OF LIhKiGE GROUPS          179

gametic ratio is 1 R : 2~. In the progenies of culture 131 only one individual
tested was so constituted and gave a backcross ratio through the pollen
of 110X: 23-b (table 5). Conversely a 2: 1 ratio is expected in crossing a
Sn Rr with this 2nfl of constitution Rrr; actual counts showeh.348R: 182r
(table 5).

TABLE 5

Crosses imohing Ihe simpler (R77) iudividzral, 224~.

Rr 0 X Rrrc?
2244X2241                  lS2                   97
224, X 2243 second ear           166                  85


Totals                     348                 182

Rrr Q XRr$
2243x224,

160                  57

The extra chromosome having thus been shown to carry a factor for
the r-2 linkage group, cytological examinations were made in order to
determine which of the ten chromosomes of the haploid complement it
was. Since the ten chromosomes are all morphologically distinguishable,
it was only necessary to examine the 1 l-chromosome carrying microspores
and see which chromosome was present in duplicate. Observations on
diakinesis had already indicated that the chromosome involved was either
the smallest or the next to the smallest.

The methods devised at the time of the investigation for the observa-
tion of the late prophase chromosome made it somewhat difficult to obtain
ligures with all of the chromosomes lying perpendicular to the optical
axis. Some good figures were obtained, however (figure 1). Many figures
were found with all but one or two chromosomes lying flat. Since the
differences in size between the four smallest and the six largest chromo-
somes arc obvious in the later prophase stage almost regardless of the posi-
tion of the chromosomes in the nucleus, it is comparatively easy to know
when the four smallest chromosomes are lying flat, and hence to obtain
accurate figures (figure 2). In the 1 l-chromosome microspores in which one
chromosome is present in duplicate it is easy to determine whether it
belongs to the group of four small chromosomes or to the group of six
large ones. When it belongs to the former group one observes five small

GENETICS 16: hlr 1931


180         BARBARA McCLINTOCK AND HENRY E. HILL

instead of four small chromosomes, and the frequency of figures with all
five chromosomes lying in the desired plane is sufficiently high to make
accurate comparisons of the chromosomes for the purpose of determining
which of them is present in duplicate. Such a case is shown in figure 3.

FIWRE l.-Late prophase chromosomes in an ll-chromosome (n+l) microspore. The arrows
indicate the duplicated chromosomes of the haploid complement; these are the r-g carrying
chromosomes. At this stage the chromosomes are frequently very angular. X2150.

FIGURE 2.-The four smallest chromosomes from a normal (n) microspore; late prophase
                      X2130.

FIGURE 3.-The five smallest chromosomes from an ll<hromosome (n+l) microspore.
Note that the chromosome in duplicate is the smallest chromosome of the set. Compare with
figure 2. X2150.

It is clear that the extra chromosome which carries the genes for the
r-g linkage group is the smallest chromosome of the haploid complement.
The presence in triplicate of the smallest chromosome does not markedly
affect the growth or appearance of the plant. One is unable to detect
even a moderate difference in comparing 2n+l with 2n individuals. On


IDENTIFIC-ITION OF LINKAGE GROUPS          181

the average, the 2nf 1 individuals are somewhat weaker than the 2n
individuals. Since genetic material of Zea mays is very heterozygous for
growth and morphological characters, it is possible that the morphological
changes produced by the presence of this extra chromosome would be
recognizable only in fairly homozygous material. In consequence of the
inability to recognize 2n+ 1 plants with certainty in the field, every plant
must be examined cytologically or tested genetically to detect the presence
of the extra chromosome.

". ._

FIGURE 4 .-Photomicrograph of microspore showing late prophase chromosomes of first
nuclear division. The smallest chromosome of the complement, that which carries the genes of
the R-G linkage group, is the second chromosome from the Ieft. It is the only chromosome in focus
throughout its entire length. X 1800.

Another 2n+l plant (144J not directly related to culture 131, an FZ
individual from the cross triploid X diploid, was examined cytologically.
The examination showed clearly the presence of the smalIest chromosome
in triplicate. It was therefore necessary to prove that this plant or its
2n + 1 progeny would show trisomic inheritance for r. Colored kernels from
the cross 1441 (2n+l) xAC~ (2n) were grown. The 2n and 2nfl F1 in-
dividuals were backcrossed to I testers (A Cr). Pollen of 2nfl plants
placed on r testers gave a total count of 1282 colored to 2451 colorless
kernels, a simple trisomic ratio of 1: 2 (table 6). Plant 14& probably had
the constitution Rrr, since each of the 2n+l offspring was Rrr. The 2n
sibs (culture 232, table 7) showed, in contrast, a good disomic 1: 1 ratio.

bum 16: hr.r 193~


182      BARBARA McCLINTOCK AND HENRY E. HILL

mQXRnd

COLORED

19412X 2322                 99
194~x232~                 33
1941,X232,                 82
19416x2324                 79
19% X2326                 97
194nX232a                 78
194; X232*                  82
194, X232,1                  69
1946 X23211                104
19212x23232                 89
194, X23212                  98
194,6x 232,9                  52
19419x23233                 99
1932 X232,6             27
1933 X232,6                 79
1936 X23216                 115

Crosses iitvolvirq

Rr BELPED

22510
23b

Totals

Rr9 xRr$

225,7x225,s

RY 0 Xrd

22512X 192,
22513X 1924
232, X 1941;
232s X1926
2321 X1926
2321,X 192s
232s X1926
23210X194,,
23218X 192s

Totals


779 Rr3


192,X189Aso

:i
-


.-




.-


.-


.-


.-


.-









--


--


_-

-

-


.-









_-

178
75
153
184
140
174
167
132
15-l
199
202
100
195
55
143
200

--
         1282      I         2451

Totals

       TABLE 7
1~ Rr individuals of cuhwes 1

       COLORED


       274
       334


       608

290

184
177
172
113
221
182
70
20
22

1161

132

99
105

204

88

219
187
195
104
198
168
73
30
22

1196


IDENTIFICATIOS OF LINKAGE GROUPS           183

Still another unrelated trisomic plant (219Aj) was found which possessed
the smallest chromosome in triplicate. Its genie constitution with respect
to r was unknown, but its pollen was used on r testers with the thought
that it might be heterozygous.  The total backcross ratio from the three
ears obtained (table 8) was 295R: 131r, indicating a duplex (RRr) con-
dition in this plant.

                         TABLE 8

  rrQ XRRrcY

192vjX219rij
1931 X219&
19411X219&


Totals

COLORED                 cotomea


192                   94
15                   11
88                   26
            -

295                 131

Thus far only ratios through the pollen have been considered. The ratios
are simple and agree with the expectations. The trisomic ratios asex-
pressed through the female resulting from the cross 2nfl 9 X2n# are
complicated by the presence of an excess of 2n over 2n+l individuals.
Possible explanations for this have been given on page 177. As was stated
there, if there were random assortment of the three homologous chromo-
somes at meiosis 50 percent of the gametes should be n and 50 percent
n+ 1. Actually, only about one-third of the eggs carry the extra chromo-
some. This condition materially changes the expected ratio.
In a simplex plant (RYY) the gametic ratio, on the basis of random
assortment of the three homologous chromosomes would be 1 R : 2 Rr : lrr : 2r
which would give on backcrossing to or plants, a phenotypic ratio of
1K: lr. If, however, lagging and loss of one of the three chromosomes
occur and at random (or if there is also some selection against nfl
megaspores in favor of n) the gametic ratio produced as a result of these
occurrences would be 1 R: 2v and all the gametes would be n. The total
gametic ratio, therefore should fluctuate between these two extremes de-
pending upon the relative amounts of random distribution of the three
homologous chromosomes as compared to lagging and loss that have
occurred. It is therefore necessary to know what proportion of the female
gametes were produced after random assortment and what proportion
after loss.

If a 2nfl simplex plant (XV) is pollinated with pollen from a 2n rr
plant the proportion of R-carrying and r-carrying gametes may be
directly determined.  The amount of lagging, or of lagging plus mega-

`.~NETICS 16: Sir 1931


184         BARBARA McCLINTOCK .4XD HENRY E. HILL

spore selection, then, may be computed from the distortion of the 1: 1
ratio (expected with no lagging) in the direction of a 1: 2 ratio (expected
with 100 percent lagging). An approximate measure of the total amount of
lagging and loss that has occurred during megasporogenesis in the ovules
of the 2nfl plants of culture 232 (table 9) has been obtained in this man-

TABLE 9

232,X 192,               137                  178
2326X 19416               130                  149
2328X 192s               11.5                  150
2329X 192s               137                 178
23216X 1920               160                 181

Totals     I         679

I         S36

ner. The 2n+l plants of culture 232 were all Rrr (see table 6). They
were pollinated with pollen from 2n r-testers (AC?). Each ear obtained
showed a deviation from a 1: 1 ratio expected with random assortment of
the three homologous chromosomes toward the 1: 2 ratio expected from
iagging and loss, or from lagging and loss plus megaspore selection. Of the
1515 kernels obtained 679 were colored (dominant) and 836 were colorless
(recessive).

1R: lr =gametic ratio due to random assortment of three homologous
chromosomes. 1

1R: lr : 1~ =gametic ratio due to lagging and loss.
When these two gametic ratios are placed one above the other, it is
seen that 679 represents the number of R-carrying gametes produced by
both types of distribution, and similarly, 836 the number of r-carrying
gametes produced. If 679 represents the number of R-carrying gametes pro-
duced, then 679 can be given to a similar proportion of r-carrying gametes.
The total number (679+679) is 13.58. The difference between 1515,
the total number of gametes, and 1358 is 157, which is the number of the
remaining recesives gametes (or kernels). It is easy to see by this method
that this number (157) represents one-third of the total number of gametes
produced as the result of lagging and loss (or lagging and loss plus mega-
spore selection).  The number 157 is also the amount by which the re-
cessives exceed the dominants. Thus, in a population composed of indi-
viduals, some of which, taken as a group, represent a 1: 1 ratio and others


IDENTIFICATION OF LINKAGE GiROUPS          185

of which, similarly, represent a 1: 2 ratio, the number by which the re-
cessives in the total population exceed the dominants should equal one-
third of the individuals representing the 1:2 ratio, or, in the present in-
stance, one-third of the individuals produced as a result of lagging.

The number of colorless kernels (recessives) exceeds the number of
colored kernels (dominants) by 157; hence, the total number of gametes
produced after loss should be three times 157, or 471. This number repre-
sents approximately 30 percent of the total number of kernels. Assuming
that the data represent a sufficiently uniform random sample, it can be
concluded that approximately 30 percent of the gametes were produced
after loss of the extra chromosome through lagging at the first or second
meiotic mitosis, or possibly through lagging and loss plus any loss that
may have occurred through selective viability of megaspores.

If 30 percent of the gametes were produced through such loss, then
loss occurred in 30 percent of the ovules.  From the remaining 70 per-
cent of the ovules half, or 35 percent, should possess nf 1 female gametes
and consequently 35 percent of the plants from the cross 2n+ 19 X2n 3
should carry the extra chromosome. This, in turn, can well explain the
deviation from the expected ratio of 2n+l to 2n individuals in the cross
2n+l X2n (see page 177 and table 2). It is expected that both cytological
and genetical evidence will be obtained which will indicate more definitely
how much of the deviation is actually due to lagging and loss, and how
much may be due to a possible selective viability of nfl gametes or
2n+ 1 embryos.

Assuming this same amount of loss, deviations from the 5R: lr ratio
expected in the cross RRrXrr (table 10) can be interpreted. Each in-

                      TABLE 10


   RRrO Xrro'                COL4OP.m                 coLoBLEs-3

2094,X 1929 (hst ear)           130                  33
20941X 192s (second ear)          207                  56
209dX 1933 (tiller ear)           193                   52
20968X 1926                 289                  72


Totals                   819                  213

dividual cross shows a deviation from the 5: 1 ratio in an increase in the
number of colorless kernels. over expectancy. The total count of 819
colored:213 colorless represents a ratio of 3.84: 1 instead of 5:l. A ratio

GENETICS 16: MI 1931


186         BARBARA McCLIXTOCK AND HENRY E. HILL

of 3.61: 1 would be expected if it were assumed that 30 percent of the
female gametes were produced as the result of loss. The ratio due to loss
is 2R: lr, as contrasted with the ratio of 5X: lr produced through random
assortment of the three homologous chromosomes.

These same assumptions are utilized in explaining the deviations ob-
served in selfing an individual of the constitution RRr.  On the basis of
random assortment and no lagging of the three homologous chromosomes,
the female gametic ratio should be 2X: 2Rr: 1RR: lv, the male, because
of the elimination of the extra chromosome carrying pollen, 2R: lr. One
would expect, therefore, a 17: 1 ratio in F?. On the basis of 30 percent of
the gametes,being produced as the result of loss of the extra chromosome
a 12.8: 1 ratio would be expected instead. Table 11 shows the results
obtained.

`J'ARLC 11

AACCRRr SELFED               COLORED                 COLORLEBS

2319 first ear               396             41  S?=2.47, P=O.lOf
2319 second ear              188              24


Totals                    584             65 X2=7.42, I'= >O.Ol

Determinations of goodness of fit by means of the X? method indicate
a significant deviation on the total counts. A full ear of well developed
kernels of unmistakable classification resulted upon selfing the first ear.
A x2 determination on this ear alone gives a fit well within the probability.
The second ear on this plant produced by selfing was poorly filled, with
many kernels underdeveloped. It is possible that in this ear the color in
some of the kernels did not develop. This possibility is supported by the
fact that some kernels on this ear showed the presence of color by only
a slight degree of mottling. It is possible, also, that some 2n+l embryos
did not develop fully on this particular ear.

The description of trisomic inheritance of Y given above shows the nature
of trisomic inheritance in Zea nzqs with regard to the smallest chromo-
some of the haploid set.

INDEPENDENCE OF THE RG LINKAGE GROUP

The method of trisomic inheritance is a convenient means of determin-
ing with certainty the independence of linkage groups. Evidence obtained
from both cytological and genetical observations indicates that the r-,o
linkage group is independent of all the other nine linkage groups estab-


IDENTIFICATION OF LINKAGE GROUPS          187

lished genetically. At least one factor of each linkage group has been tested
(c and w+, sU, b, J!, gll, p,,f, d and a). 2nfl individuals heterozygous for
these genes have been selfed and backcrossed.

                         TABLE 12
yes& of crosses involving c-x'z, ss, b, y, gl,, p,, d and a among 2nfl individuals lrisonzic for
       the r-g chromosome. For erplanatiolt see page 189.

       I. Disomic inheritance of c and w, (see appendix).
    2nfl Cc [C or c]XZn AcR                  C
                                          -

         131aaXB3461                      78
    2n AcRXZn+l Cc [C or c]
         1042X88,                       194
        B346iX131s                      172
                                 W,
                                          -
    2nfl W,u, [W,] pollen counts              535
    2n W.w, pollen counts                   633
    2n xw, X2n+l IV+, [IV?]
         192sX2253                       152

                II. Disomic inheritance of s,,.
    Znfl Sasu [s,] selfed                   ST`
         2203                          282
         22014                          279
         22014                         174
         22015                           223
         22011                          261
         22013                          221
         2319                            318

          Totals                        1758
    2nS,s,X2nfl SJ,, [sI,]
         22011x1s                        228
        22514x2                        208
        231; X9                        324
         23116X9                         316
         23117x9                        359

          Totals                        1495
    2n s,,s,X2n+ 1 .Sus, [x.]
          188X 189B,8                      190
     2n Susu selfed
         2206                          288
         2208                          269
         22012                         352
         22Slo                         278
         2316                          420
         231i6                        327

          Totals                        1934

km'1cs16: .\Ir 1931

c
-

69


149
184

WT
-
549
594


179




su
110
97
53
68
90
86
102

36


98
73
112
126
139

548


169


99
77
112
95
138
112

633


BARBARA McCLINTOCK AND HENRY E. HILL

          TABLE 12-(conlinued)
     III. Disomic inheritance of b (see page 189).
2n+l Bb[b] selfed                       B
   881                            S4
    ,IV. Disomic inheritance of y (see appendix).
2n+l Yy [Y or y] selfed                    I'
   1766                           94
2n YyXZnSl Yy [y]
   19412x2322                      139
   1943aX232,                      113
   1941~x232,                      136
   1943 X232(                      102
   1941xX232a                      128
   194, X2328                      127
   19419x232~                       143

b
19

3:

37
40
46
38
46
40
S2

-~
    Totals                         588       299
2n yyXZn+l Yy [y]
   194, X232,,                       9S        89
   1945 X2321~                       85        63
   1941~x232~                       77        72
   1933 X232,6                       66        74

   Totals                         323       298

         V. Disomic inheritance of grl.
2nSl Gzm [GlJXZn gngz1               Gt1        611
   2297X2011                       113       105
         VI. Disomic inheritance of p,.
2n+l P& [PT] selfed                   Pr        Pr
   2205                            149        45
   22014                         287        89
   22014                         170        57
   22017                         228        87
   22017                          196        78

    Totals                         1030       356
2n+l PrPT P,IXh PA
   22015 X 2031                      129        120
   22019X2038                      110       108

   Totals                         239       228
2x1 P4%XZn+l P,p, [PA                 p,        PI
   22011X22018                      306        80
2n P,p, selfed
   2205                          271        116
   2208                          249        97
   2201%                          362        102

    Totals
2n P,p, X 2n prpr
   2201~x203

SS2       315


194       207


        IDENTIIkICATION OF LINKAGE GROUPS          189
              TABLE 12-(c0tiinwd)

          VII. Disomic inheritance of d (see appendix).
     2nfl Dd [DJselfed                       D        d
         2319                           129        44
     2n ddX2nfl Dd [D]
         2OSX231g                       119        118
     2nfl Dd [D]XZn dd
        2311~x205                       170       140

          VIII. Disomic inheritance of a (see appendix).
     2nfl Aa [A or a] selfed                    A
         881                           73    1:
    2nfl Au [A or a]XZn aa
         1316XRSlla                      SO        53
     2n aaX2n+l Aa [A or a]
         2066X 189&o                     144       173

It is needless to discuss every cross represented in table 12, for the re-
sults are self explanatory.  The crosses involving the recessive sugary
gene (.r,,) can be used as a single example (see table 12, section II). A 2n+l
plant of culture 131, homozygous for sugary, was crossed with pollen
from a 2n starchy plant (S,S,,). The FL 2n and 2n+l individuals were
selfed and backcrossed to test for trisomic or disomic inheritance. In
each section the type of cross is indicated. The symbol of the gene placed
in brackets indicates how the genie constitution of the 2n+l individual
would have differed had it been trisomic for this gene. On selfing 2n+ 1
plants heterozygous for sugary a total of 17.58 S,, to 586 sU kernels were
obtained, precisely a 3: 1 ratio.  Diploid (2n) sibs upon selfing gave a
total count of 1934 S,,: 633 sU kernels.  The two ratios are similar and
disomic. If these 2nfl plants were trisomic for sugary (SusUsU), an ap-
proach to a 2 : 1 ratio would be expected. Similarly, a 2: 1 ratio would be
expected in the sib crosses 2n (S,L) X2nfl. Here a ratio of 1493 S,:548
sU kernels was obtained, a 3 : 1 instead of a 2 : 1 ratio.  It is therefore con-
cluded that the linkage group including s,, is independent of the r-g
linkage group and must be associated with another chromosome. The
data on factors c, We, y, gll, pr and d are sufficiently numerous to need
no further explanation.
In the case of h, genetic data are hardly necessary, since the b-l, linkage
group has been associated with another chromosome.
The data on the n factor are few but indicate a disomic instead of a
trisomic inheritance. In the case of a 2n+l heterozygous a plant selfed
the results (73A : 19~) indicate neither a duplex (AAa) 17 - : 1 ratio nor a
simplex (AU) 2: 1 ratio, but better, a disomic 3: 1 ratio. Further, the cross
of a heterozygous 2nfl plantx 2n au would have given, if duplex, 54 : la
~~WGXS 16: Mr ,931


190        BARBARA hlcCLINTOCK AND HENRY E. HILL

or if simplex, L4 : l+a, SOA : 53a probably represents a 1: 1 disomic ratio.
In the case of 2n aaX2n+l heterozygous (I, a simplex constitution would
have given a 1: 2 ratio and a duplex constitution a 2: 1 ratio.  144-4 : 173~
approaches neither of these but probably represents a 1: 1 disomic back-
cross ratio.

The factor for fine stripe (`j) did not segregate sharply in the seedling
stage so that backcross counts were not sufficiently reliable. Cytological
evidence from Doctor BRINK'S material indicates that the ~`-5~ linkage
group is carried by a long chromosome.

SUMMARY

1. A 2n+l plant of Zea gnays resulting from the cross diploidx triploid
and its 2n + 1 progenies were found to give trisomic inheritance for r.
2. In these plants the smallest chromosome is present in triplicate.
3. Two unrelated 2n+l individuals were found to be trisomic for the
smallest chromosome of the haploid set. These plants. upon later testing,
gave trisomic inheritance for r.

4. In 2n+l individuals one-third of the eggs carry the extra chromo-
some. In a normal pollination the extra chromosome-carrying pollen
grains function only in a small percentage of the cases.
5. Plants trisomic for the r-g linkage group have given disomic inheri-
tance for c, XI,, sU, b, T, RU, p,, d and a.

ACKNOWLEDGMENTS

To MARCUS M. RHO~DES and GEORGE W. BEADLE the authors wish
to express sincere gratitude for their very generous cooperation in this
investigation. Also to LESTER W. SHARP, ROLLINS A. EMERSON and
MERLE T. JENKINS many thanks are due for their careful reading and re-
vision of the manuscript.

LITERATURE CITED

MCCLIXTOCK, B., 1929a .\ cytological and genetical study of triploid maize. Genetics 14: lSO-
     222.

1929b Chromosome morphology in Zeu rrrays. Science 69: 629.

APPENDIX

During the time this paper was in press the following linkage groups
were found to be associated with chromosomes other than the r-g carrying
chromosome: C-sh-wz, Y-P1, A -&l-c,. By the method of association
of linkage groups with particular chromosomes the independence of six of
the ten linkage groups (C--sIL--wz, R-g, B-l,, Y-PI, P-b,, A -&-c,)
has been definitely established.