Analysis of genes for stigma coloration in rice

The anthocyanic pigmentation of the rice apiculus is controlled by three complementary
genes— C, A, and P —which serve as the basic coloration genes. The
genetic control of stigma color is more complex. To shed more light on genes for
stigma coloration, F 1 and F 2 data for 196 varietal crosses were investigated to
explain the whole pattern of segregation by assuming certain Mendelian genes. In
ordinary cases, the stigma is colored only in plants having C, A, and P. Two
independent genes, Ps-2 and Ps-3, take part in stigma coloration, Ps-2 being
relatively frequent in indicas and Ps-3 in japonicas. For Ps-3, an inhibitor, I-Ps-3,
was found, which seems to have two loci according to variety. In addition, two
complementary inhibitors are assumed to be present in some of the varieties. A
Japanese upland variety, Gaisen-mochi, having a colorless apiculus and colored
stigma, has Ps-1, which expresses stigma color even when P is absent (recessive).
An inhibitor for this gene, I-Ps-1, needs P to function. Four genes— P, Ps-3,
Ph, and Ps-1 —are located in linkage group II in that order. Their recombination
values were estimated.

Most rice varieties and derivatives from varietal crosses express anthocyanin color in
the stigma only when the apiculus is colored. The coloration of the stem node and some
other plant parts also shows such a relation, and the genes for apiculus color can be
considered as basic for anthocyanin formation (Takahashi 1964). Apiculus color at
heading ranges from pink to dark purple, for which several isoalleles have been
described, but when only the presence or absence of color is considered, it is controlled
simply by three complementary genes: C (chromogen production), A (activation), and
P (spreading pigment). These three genes are located in linkage groups I, III, and II,
respectively (cf. Kinoshita 1984, pp. 29–30). Accordingly, all varieties with colored
apiculus have the genotype CAP, while colorless varieties can have varying genotypes
(Oka 1989a). Setty and Misro (1973) reported that the P gene has different loci
according to variety. In the material used in this study, however, all F 2 plants derived
from crosses between apiculus-colored parents had a colored apiculus, and the
different loci of gene P were not confirmed.
97
In contrast to apiculus color, the genetic control of stigma color is more complex,
as reported in this paper, but space is too limited to describe the procedures for
identifying genes and determining parental genotypes for all crosses observed.
Materials and methods
Forty-one strains were used as parents, including 21 indicas and 19 japonicas ( Oryza
sativa ), and an Indian wild annual type of O. rufipogon (Table 1). The F 1 and F 2 plants
of 196 crosses were observed for apiculus and stigma color at heading, pollen and seed
fertilities, phenol reaction, and some other traits. In 20 cases, reciprocal crosses were
made and the F 2 populations were observed more than once, even though no significant
differences were detected between reciprocal crosses and replications. In each cross,
about 80–300 F 2 plants were grown and observed in Taichung, Taiwan, China, with the
assistance of several students of Chung Hsing University.
Purple to dark red (P) and red to pink (R) color tones were distinguished, but it was
difficult to classify them into several grades to identify multiple alleles at the C and A
loci. This was largely because distant crosses were tested under varying conditions
during the winter and summer cropping seasons.
In distant crosses, F 2 ratios are often distorted (Oka 1989b). Yet, the underlying
genes could be deduced in many cases from observed segregation patterns. To estimate
recombination values from distorted ratios, the methods described by Oka (1989c)
were employed. To confirm the parental genotypes presumed from the F 2 data, F 3 lines
from selected F 2 plants were examined in 001/325, 108/532, and 5 other crosses, and
some colorless F 3 lines were intercrossed.
Results
The results of the experiments are presented in two sections: one discussing ordinary
varieties in which stigma coloration occurs only in plants with a colored apiculus, and
the other discussing a special variety, Gaisen-mochi (532), and its crosses, in which
plants with colorless apiculus and colored stigma occur.
Crosses between ordinary varieties
In the F 2 populations of crosses between ordinary varieties, three types of segregants
are found: + + (apiculus and stigma both colored), + - (apiculus colored, stigma
colorless), and - - (apiculus and stigma both colorless). No - + (apiculus colorless,
stigma colored) type occurs. The parental genotype for apiculus coloration (the
combination of C, A, and P ) can be judged by the F 1 phenotypes from crosses with
known genotypes and by observation of F 2 plants when necessary (Oka 1989a). The
F 2 ratios for stigma color observed among segregants with colored apiculus (+ + and
+ - types) are summarized in Table 2, excluding those with variety 532.
Different segregation patterns were found; representative ones are briefly described
in Table 3.
98 H. I. Oka
Table 1. Varieties used as parents and their apiculus and stigma color. a
Code Origin Vernacular name
Color
Apiculus Stigma
001
022
060
101
108 b
124
143
160
414 b
421
435
451
612
619
706
717
719
724
727
761
1091
219
221
236
242
318
325
T65 b
501
521 b
532
535
545 b
552
563 b
571
647 b
701
703
871
W106 b
Vietnam
Vietnam
Vietnam
Taiwan
Taiwan
Taiwan
Taiwan
Taiwan
India
India
India
India
Sulawesi
Sulawesi
North China
China
China
South China
South China
Hainan
South China
Philippines
Philippines
Philippines
Philippines
Indonesia
Indonesia
Taiwan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Sulawesi
North China
North China
Taiwan
India
lndicas
70 a som cau
II dauh
RTS23
U-kuh-tsing-you
Peh-ku
Shuang-chiang
Lui-tou-tzu
Hong-ka-chiu
PTB10
PTB8
Pachchai perumal
Surjamkhi
Padi bali
Padi hotjong
He-nan-tsao
Nan-chang-wan
Chin-sen
Kunming tsieh-huan
Chin-tsao
Siao-chung-kuh
Lui-kung sen
Japonicas
Garumbalay
lnakupa
Olag ayau
Malagkit pirurutong
Boegi inda
Kaniranga
Taichung 65
Urasan
Kisshin
Gaisen-mochi
Hirayama
Shinriki
Aikoku
Kinoshita-mochi
Mansaku
Padi ase-banda
Tatung-tsailai
Tamao tao
Nabeshi
O. rufipogon
Cuttack annual type
P
P
P
P
P
P
P
P
P
P
P
R
R
P
P
P
P
P
P
P
P
P
P
P
P
P
R
P
P
a P = purple to dark red, R = red to pink, (–) = colorless. b Used as maternal parent and
intercrossed.
Analysis of genes for stigma coloration in rice 99
– –
– –
– –
– –
– –
– –
– –
–
– –
–
– –
– –
– –
– –
– –
–
– –
– –
– –
– –
– –
–
– –
–
– –
– –
– –
– –
– –
Table 2. Pooled F 2 ratios for stigma color (++:+–) among segregants with colored apiculus
and genotypes presumed for respective strains. a
P 2
P 1
001 545 647 219 521 T65 W106
Genotype of P 2 strain
Common genes b Extra c
Group A d
O22
Group B e
421
619
Group C f
545
871
647
219
242
325
501
521
571
236
703
535
552
T65
W106
0
0
1:0
49:15
49:15
1:0
3:1
3:1
51:13
0
3:1
0
1:0
0
0
3:1
1:0
1:0
3:1
3:1
3:1
51:13
3:61
51:13
0
0:1
3:1
0
0
201:55 j
0
51:13
1:0
1:3
1:3
1:0
0
7:57 g
7:57 g
15:1
21:43 h
0
0
0
15:1
3:1
13:3
1:15
13:3
0:1
0:1
1:15
51:13
0:1
3:13
0
0
51:13
3:61
(3:61)
51:13
0
0
0:1
3:1
0
0
3:1
0:1
15:1
3:13
15:1
1:1
1:0
(61:3)
1:0
0 1:0
21:1235 i
0
0
0
0
0:1
0:1
(63:1)
(63:1)
1:0
(63:1)
c A P Ps-2 ps-3 I-3
c A P ps-2 ps-3 I-3
C A P ps-2 ps-3 I-3'
C A P ps-2 ps-3 I-3'
C A P Ps-2 Ps-3 i-3
C A P Ps-2 Ps-3 i-3
C a P ps-2 ps-3 I-3
C a P ps-2 ps-3 I-3
C a P ps-2 Ps-3 i-3
C A P ps-2 Ps-3 I-3
c A P ps-2 Ps-3 I-3'
C A P Ps-2 ps-3 i-3
c A P ps-2 ps-3 i-3
c a P ps-2 ps-3 l-3
c a P ps-2 ps-3 i-3
C A p ps-2 ps-3 i-3
C a p Ps-2 ? ?
C Br A P Ps-2 Ps-3 i-3
C Br A P ps-2 ps-3 i-3
C Br a P ps-2 Ps-3 i-3
C A P Ps-2 Ps-3 i-3
(l-a)
(l-a)
(l-b)
(l-b)
(l-b)
(l-a)
(Ps-1)
a Ratios in parentheses = assumed without confirmation. b I-3/i-3 = I-Ps-3/i-ps-3, l-3' = I-Ps-3' supposedly having
a locus different from that of I-Ps-3. l-a and I-b = complementary inhibitors for Ps-3, corresponding to I-Ps-a and
= 001, 060, 101, 108, 124, 143, 414, 612, 717, 719, 724, 727, and 761. e Group B = 160, 435, 451, 706, and 1090.
I-Ps-b in Kinoshita (1984, p. 30). c Strains with no "extra" gene are expected to have i-a, i-b, and ps-i. d Group A
f Group C = 221, 318, and 701. g 7:57 = (1:3) (7:9). (1:3) comes from cosegregation for l-3/i-3 (given in b). (7:9)
comes from complementary inhibitors I-a/i-a and I-b/i-b (given in b). h 21:43 = (3:1) (7:9). (3:1) comes from Ps-
3/ps-3. (7:9) is expected from l-a and I-b as mentioned above. j 21:235 = (3:13) (7:9). (3:13) comes from interaction
between Ps-3/ps-3 (3:1) and I-3/i-3 (1:3). (7:9) is expected from l-a and I-b as mentioned above. j 201:55 = (3:1)
(9:7) (1:3). (3:1) comes from Ps-2/ps-2. (9:7) is due to C/C Br and Ps-3/ps-3, where plants with C and Ps-3
are expected to show stigma coloration. (1:3) is expected from I-3/i-3 as mentioned above (b and g ).
Table 3. Representative segregation patterns, F 2 ratios, and genes for stigma
color.
Case P 1 P 2 F 1
0
1
2
3
4
5
6
7
– –
– –
– –
– –
+–
– –
– –
+–
– –
++
– –
+–
– –
++
– –
– –
– –
++
++
+–
+–
++
++
+–
F 2 ratio
++:+–
"0"
1:0
3:1
1:3
3:13
13:3
51:13
0:1
Example Genes for stigma color
Many (No apiculus-colored segregants)
108/160 Same gene in both parents
108/545 Segregation for a coloration gene
647/421 Segregation for an inhibitor
219/571 A coloration gene and an inhibitor
219/221 A color gene and an inhibitor for
another color gene
108/647 Two color genes and an inhibitor
219/521 Same inhibitor in both parents
100 H. I. Oka
–
–
–
–
–
–
Underlying genes in these different cases are presumed as follows:
• Crosses between colorless indicas produced no colored hybrids, because both
parents have c A P (group A; Table 3, case 0). In their crosses with colored indicas
(group B) or colored japonicas (group C), the F 1 and all F 2 plants with colored
apiculus had colored stigmas (+ +:+ – = 1:0; Table 2; Table 3, case 1). In this case,
both parents are thought to have the same gene for stigma coloration, assigned
the symbol Ps-2 to agree with the description in Kinoshita (1984, p. 30).
• When varieties of group A ( c A P Ps-2 ) were crossed with variety 545 ( C a P ),
the F 1 was colored (+ +) and the F 2 plants with colored apiculus segregated into
3 + +:1 + – type (Table 3, case 2). Variety 871 showed the same pattern. These
varieties seem to have ps-2. Variety 647, whose F 2 ratios in crosses with group
A varieties were near 3:1, may also be considered to have ps-2.
• When 647 was crossed with varieties of groups B and C, all F 2 plants with colored
apiculus had colored stigmas (1:0; Table 2; Table 3, case 1). This cannot be
explained without assuming another gene for stigma coloration, Ps-3, present in
both parents (to agree with Kinoshita 1984, p. 30), since 647 would have ps-2.
Then the crosses of 647 ( ps-2 Ps-3 ) with group A varieties ( Ps-2 ps-3 ) are
expected to show a 15:1 ratio in the F 2 . The occurrence of nearly 3:l ratios in
those crosses suggests that group A varieties have an inhibitor for Ps-3, I-Ps-3
(abbreviated I-3 ); in view of the 1:0 pattern in crosses with groups B and C
( Ps-3 Ps-2 ), 647 cannot have I-3. Accordingly, the nearly 3:1 ratio observed in
crosses between 647 and group A varieties may be considered to be 51:13 (Table
2; Table 3, case 6) resulting from 3:1 for Ps-2 and 3:13 for Ps-3 and I-3. The
observed ratio of 374:105 (pooled for 4 crosses whose ratios were homogeneous)
agrees with this expectation ( c 2 = 0.8, P > 0.5).
• A colored variety 325, showing a 15:1 F 2 ratio in its cross with 647 and the 1:0
pattern with group A varieties, is considered to have Ps-2 ps-3 i-3. Variety 545
is thought to have ps-2 ps-3 I-3, because its crosses with 325 and group A
varieties gave a 3:1 ratio, and its crosses with varieties of groups B and C gave
a nearly 3:1 ratio (51:13; Table 2; Table 3, case 6). The observed F 2 ratios in
crosses of 545 with groups B and C—468:132 (4 crosses pooled, homogeneous)—
agree well with 51:13 ( c 2 = 1.2, P > 0.25).
• When variety 219 was crossed with group A varieties, the F 1 was colored (+ +)
and the F 2 showed a 3 + +:1 + – ratio (568:145, c 2 = 1.2, P > 0.25; 4 homogeneous
crosses pooled). This suggests that 219 has ps-2 Ps-3 I-3. The F 1 and F 2 plants
from 219/545 and 219/871 showed no stigma color, because they had I-3 in
common (Table 3, case 7).
• Varieties 242 and 421 showed nearly 1:15 F 2 ratios for stigma color in their
crosses with 219, suggesting that they have an inhibitor for Ps-3 at a locus
different from that in 219, which is tentatively designated as I-Ps-3' (abbreviated
I-3' ). Variety 619, which showed a segregation pattern similar to that of 421, is
also assumed to have I-3'. When 1:15 is combined with 3:1, a 49:15 ratio is
expected, although it is not distinguishable from 3:1 (e.g., 421/group A,
Table 2).
Analysis of genes for stigma coloration in rice 101
• F 2 ratios lying between 1:3 and 1:15, and between 1:3 and 1:1 were found in some
crosses. These may be regarded as resulting from segregation distortion, but
some of these crosses showed high F 1 fertilities. Hsieh (1960, 1961) pointed out
the presence of a set of complementary inhibitors (I ps and I ps 2; redesignated IPs-
a and I-Ps-b in Kinoshita (1984, p. 30); I-PS-3-a and I-PS-3-b or I-a and I-b
in this paper), based on the finding that T65, which seemed to have no ordinary
inhibitor, showed in its crosses with some colored-stigma strains (7111 and two
others) colorless F 1 plants, and nearly 1:2 F 2 ratios. Such complementary
inhibitors will bring about a 7:9 ratio in the F 2 and will give rise to 21:42, 7:57,
and other ratios when combined with 3:1,1:3, and others. Although the data from
this study give no conclusive evidence for their existence, this assumption
facilitates explanation of some of the F 2 ratios observed. Tentatively, it is
assumed that varieties T65, 545, and 647 have I-a; and that 219, 242, and 501
have I-b (Table 2). Then, for instance, the F 2 ratio found in 647/219 (22:226) can
be regarded as 7:57 ( c 2 = 1.1, P>0.25), and that observed in 647/501 (66:159)
may be regarded as 21:43 ( c 2 = 1.2, P>0.25). Probably, if such complementary
inhibitors exist, they would be distributed more commonly, although their
detection is not always feasible.
• When a colorless japonica, 521, having c a P, was crossed with varieties of
groups B and C, the F 2 ratios (++:+–) observed were 89:5, 86:7, 59:5, etc. Its cross
with 219 produced no colored-stigma F 2 plants (0: 1; Table 3, case 7), while its
cross with 421 ( CAP Ps-2 Ps-3 I-3’ ) gave a nearly 1:15 (3:61) ratio. Variety 521
is then assumed to have ps-2 ps-3 I-3, like 545, and the above ratios with groups
B and C are assumed to be 51:13. The observed ratio in 521/451 (86:17) agrees
with 51:13 ( c 2 = 0.9, P>0.25), but others do not. This is considered due to
segregation distortion, since the crosses with 521 showed low F 1 pollen fertilities
(20–50%). A similar colorless japonica, 571, was assumed to have ps-2 ps-3 i-
3, because its F 2 with 219 gave a 3:13 ratio (13:95. c 2 = 3.2, P>0.05). Another
colorless japonica, 703, having C a p, showed the 1:0 pattern in its F 2 s with group
A varieties, suggesting that it has Ps-2. However. its alleles at loci Ps-3 and I-3
remain unknown. Through similar exercises in symbolic logic, the genotypes for
stigma coloration of several varieties were presumed, e.g., 236 = C A p ps-2 ps-
3 i-3 and 501 = c A P ps-2 ps-3 i-3 I-b (Table 2).
• Varieties showing pink color at the apiculus may be assumed to have C Br , while
plants with C Br/ C Br or C Br/ c and A P are thought to express no stigma color even
if they have a gene for stigma color (Takahashi 1958; 1964, p. 217). T65 is known
to have C Br a P for apicuius color (Hsieh 1960; 1961, p. 85); varieties 535 and
552 with pink apiculus may also be assumed to have C Br. This study suggests,
however, that the suspending effect of C Br on stigma coloration is limited to Ps-
3, leaving Ps-2 unaffected. For instance, the F 2 from 108/T65 segregating for Ps-
2 showed a 3:1 ratio (95:32) for stigma color. On the assumption that the
genotype of T65 is C Br a P ps-2 Ps-3 i-3 I-a, its crosses with groups B and C are
expected to show a 15:1 F 2 ratio for stigma color. This agrees well with the data
102 H. I. Oka
(121:9, 2 homogeneous crosses pooled, c 2 = 0.1, P>0.5). Together with the
complementary inhibitors, the suspending effect of C Br on Ps-3 brings about a
complication in F 2 ratios. For instance, the F 2 of 219/T65 involving C Br / C and Ia,
I-b is expected to give a 21:235 ratio (Table 2, footnote i ), which agrees with
the data (12:108). Variety 022, which showed the 0:1 pattern in its F 2 with T65,
would have ps-2 ps-3 I-3. Variety 535 may be considered to have C Br A P Ps-
2 Ps-3 i-3, because its F 2 with 647 showed a 15:1 ratio (82:3). Its cross with 545
is expected to give an F 2 ratio of 201:55 (Table 2, footnote j ), which agrees with
the data (139:46; c 2 = 1.3, P>0.25).
• Variety 563 shows purple-tawny color spread over the hulls at maturity, which
is expressed by gene Pr (linkage group II, Yen and Hsieh 1968), and pink color
in the stigma. The gene for stigma color of this variety could not be identified,
although more than 20 crosses with different varieties were observed. It seems
that Pr/pr has some effect on stigma color, but the variation in color tone is
continuous from pink to colorless or to purple. Accordingly, the data for 563
crosses are not presented in this paper.
• The crosses of W106 with different cultivars all produced colored F 1 plants, and
when the F 2 segregated for apiculus color, all colored-apiculus segregants had
colored stigmas (1:0) in most crosses. When crossed with varieties having I-3,
there were a few segregants with colorless stigmas (+ – type). W106 is then
considered to have C A P Ps-2 Ps-3 i-3. In addition, it may have Ps-1, because
its cross with varieties having C A p produced a few F 2 segregants of – + type.
Accordingly, the occurrence of many + + and a few + – F 2 plants is assumed to
represent a 63:1 or 61:3 ratio (Table 2). Several other wild strains (e.g., W120,
W134, and W145) also showed the same segregation patterns, suggesting that
their genotypes for stigma coloration genes are similar to that presumed for
W106.
Crosses of Gaisen-mochi (532) with other varieties and linkages
Variety 532, having colorless apiculus and purple stigma (– + type), is thought to have
Ps-1 (called Ps by Takahashi [1958]), which causes stigma pigmentation without P as
long as C and A are present. This variety, having C A p Ps-1, was crossed with several
other varieties. The F 2 patterns observed were complex (Table 4) but could be analyzed
by using the linkage of relevant genes with Ph for phenol reaction (532 had Ph ).
P and Ph are linked with a mean recombination value of 27.2+5.6% (Table 5). Also,
Ps-2 and Ph are linked with a mean recombination value of 21.1 ± 4.0% (Table 6).
Accordingly, P and Ps-2 are linked (9.0 ± 5.4%).
The F 2 data obtained in 545/532 and 219/532 suggest that gene Ps-1 carried by 532
is also linked with Ph, as most segregants of – + type showed positive phenol reaction
(Table 4). In both F 2 populations, + + plants were fewer than + – plants, suggesting that
an inhibitor for Ps-1 ( I-Ps-1 ) is present in 545 and 219. However, – + segregants are
more numerous (19% in 545/532) than expected when Ps-1 is completely suppressed
by I-Ps-1 (about 6%). To account for the observed patterns, an assumption is needed
Analysis of genes for stigma coloration in rice 103
Table 4. F 2 segregation patterns for apiculus (Ap) and stigma (St) color and
phenol reaction (Ph) observed in crosses with Gaisen-mochi (532).
F 1
a Plants (no.) with given segregation pattern
Cross
Ap St F 2 Ap:
St:
Ph:
+
+
+
+
+
+
+
+
+
+
+
+
Plants
(no.)
545/532 P
219/532 P
108/532 P
414/532 P
(pooled)
521/532 P
647/532 P
(R)
(R)
P
P
P
P
26
67
102
67
169
20
47
6
5
14
31
64
77
0
3
3
9
11
31
33
7
3
45
55
12
8
20
16
24
1
2
0
3
48
0
123
94
217
53
41
22
4
9
14
243
243
237
172
409
128
174
a P = purple to dark red, (R) = faint pink
Table 5. Linkage relations estimated between loci P and Ph. a
Cross
(P 1 /P 2 )
Parental
genotype
F 2 phenotype b
Ap:
Ph:
+
+
+
+
Plants
(no.)
Recombination
value (%)
Distortion
considered
for
001/703
108/703
414/703
(pooled)
545/532
P 1 : cAP-Ph
P 2 : Cap-ph
P 1 : CaP-ph
P 2 : CAp-Ph
Obs.
Exp.
Obs.
Exp.
149
150.5
90
97.0
25
23.6
37
39.7
266
274.0
93
85.3
126
117.9
23
21.0
566
c 2 = 0.9,
243
c 2 = 1.4,
22.5 ± 4.5
P > 0.75
35.8 ± 9.9
P > 0.5
219/532
647/532
P 1 : CAP-ph Obs. 144 38 59 243 24.5 ± 6.0
P 2 : CAp-Ph Exp. 136.4 36.5 66.5 c 2 = 2.0, P > 0.5
P 1 : CaP-ph Obs. 58 34 65 174 26.4 ± 15.7
P 2 : CAp-Ph Exp. 67.5 30.4 63.0 c 2 = 3.0, P > 0.25
a Mean = 27.2 ± 5.6%, weighted according to the number of plants observed. Standard deviation for mean =
standing for the variation among estimates. Ap = apiculus color, Ph = phenol reactlon, "-" shows linkage. b Obs.
= observed, Exp. = expected.
2
3.6
17
13.1
C:c
Ph:ph
that I-Ps-1 requires P for its function. The F 2 data for 219/532 are also explained by the
same assumption favorably.
In 545/532, – – segregants would be mostly the aa plants: in 219/532, in which no
aa plant occurs, – – segregants were few (Table 4). This suggests the linkages of p with
Ps-1 as expected from the linkage of Ph with P and Ps-1. Thus, the parental genotypes
are assumed to be: 532= C A p—Ph—Ps-1—ps-2 ps-3 i-3 i-Ps-1 ("—"shows linkage),
545 = C a P—ph—ps-1—ps-2 ps-3 I-3 I-Ps-1, and 219 = C A P—ph—ps-1—ps-
104 H. I. Oka
–
– –
–
–
– –
–
– –
–
–
–
–
–
–
Table 6. Linkage relations estimated between loci Ps-2 and Ph. a
F 2 phenotype
Cross
P 1 /P 2
Parental genotype
St:
Ph:
+
+
+
+
Plants
(no.)
Recombination
value (%)
219/160
219/435
(pooled)
647/108
647/143
(pooled)
545/001
545/124
545/414
(pooled)
219/108
219/124
219/414
(pooled)
647/435
P 1 : ps-2—ph Ps-3 l-3
P 2 : Ps-2—Ph Ps-3 i-3
P 1 : ps-2—ph Ps-3 i-3
P 2 : Ps-2—Ph Ps-3 I-3
P 1 : ps-2—ph
P 2 : Ps-2—Ph
P 1 : ps-2—ph
P 2 : Ps-2—Ph
P 1 : ps-2—ph Ps-3 i-3
P 2 : Ps-2—Ph Ps-3 I-3
Obs.
Exp.
Obs.
Exp.
Obs.
Exp.
Obs.
Exp.
Obs.
Exp.
279
285.9
113
116.5
100
96.9
241
237.4
38
40.6
31
40.2
20
21.3
14
11.9
55
45.4
8
7.3
36
29.1
15
13.2
9
11.9
34
45.4
4
3.6
43
33.8
25
21.9
22
24.4
47
48.8
9
7.5
389
c 2 = 6.4,
P > 0.05
173
c 2 = 0.9,
P > 0.75
145
c 2 = 1.4,
P > 0.5
377
c 2 = 5.0,
P > 0.1
59
c 2 = 0.58
P > 0.9
21 .0 ± 3.8 b
20.7 ± 4.3
18.0 ± 3.6
27.8 ± 2.8
18.0 ± 7.3
a Mean = 21.1 ± 4.0%, weighted according to the number of plants observed. Standard deviation for mean =
observed, Exp. = expected. b Segregation distortion for Ph:ph was considered in computation (cf. Oka 1989c).
standing for the variation among estimates. St = stigma color, Ph = phenol reaction, "–"shows linkage. Obs. =
2 Ps-3 I-3 I-Ps-1. The inhibitors are assumed to be independent of coloration genes so
as to better explain the data.
In 545/532, since both parents have ps-3 (recessive), this gene and its inhibitor can
be neglected in analyzing the segregation pattern. Letting the recombination value
between P and ps-1 (repulsion) be p , the frequencies of genotypes for four color classes
are expected to be as in Table 7.
The maximum likelihood estimate of p is 36.1 ± 14.6%, and the expected values
show no significant deviation from observed ones. Letting the recombination value
between Ps-1 and Ph (coupling) be q , the expected frequencies are as shown in Table
8, in which the genes controlling color phenotype are the same as in Table 7. In
estimating q , the P—ps-1 recombination ( p ) is assumed to be 0.30 to be close to the
mean for different crosses (Table 9).
The q value for Ps-1 and Ph recombination was 14.1 ± 5.7%. Similarly, the
recombination value between P and Ph was 35.8 + 9.9% (Table 5). Thus, the observed
segregation pattern could be explained by analyzing linkage relations.
In 219/532, the computation procedures were more intricate, since I-3 for Ps-3 was
involved in addition to I-Ps-1, and distorted segregation for Ph/ph had to be considered.
It must be determined whether I-Ps-1 is independent of I-3 or whether they are the same
Analysis of genes for stigma coloration in rice 105
–
–
–
–
Table 7. Frequencies of genotypes for 4 color classes in 545/532.
Phenotype Genes Frequency
(Ap) (St) concerned expected
Plants (no.)
Observed Expected
+
+
–
–
+ A P-Ps-1 i-Ps-1
A P-ps-1
A P- Ps- 1 l-Ps-1
+ A p-PS-1
– A p-ps-1 and aa
Total
3
4
3
4
3
4
3
4
3
4
1
4
1
4
3
4
1
4
1
4
1
4
1
4
p 2
(2 + p 2 )
(1 – p 2 )
(2 + p 2 )
(1 – p 2 )
+ 1
4
= 0.0938 + 0.0496 p 2
= 0.4687 – 0.0496 p 2
= 0.1875 – 0.1875 p 2
= 0.25 + 0.1875 p 2
1.0 ( p = 0.361)
32
95
66.7
c 2 = 6.9,
P > 0.05
46
70
243
24.3
112.4
39.6
Table 8. Expected frequencies in 545/532.
Phenotype Expected frequency a Plants (no.)
(Ap) (St) (Ph) ( p = 0.30) Observed Expected
Remarks
+
+
+
+
–
–
–
–
+
+
–
–
+
+
–
–
+
–
+
–
+
–
+
–
0.0980 (3-2 q + q 2 ) • 1/3
0.0980 (2 q + q 2 ) • 1/3
(0.2939 (3–2 q + q 2 ) • 1/3)
(0.1706 (2 q-q 2 )
(0.2939 (2 q–q 2 ) • 1/3
(0.1706 (1–2 q+q 2 )
0.1706 (3–2 q+q 2 ) • 1/3
0.1706 (2 q–q 2 ) • 1/3
0.0169 (1–2 q+q 2 ) + 0.0625
0.0169 (2 q–q 2 ) + 0.1875
26
6
64
31
45
1
22
243
48
2.1
71.9
41.0
3.6
46.6
18.2
Pooled in
computing
c 2 value
Pooled in
computing
c 2 value
Total 1.0 ( q = 0.141) c2 = 7.3, P > 0.1 (df = 5)
a 1/3 is a multiplier necessary for estimating the second recombination value by using the estimate of first
recombination value. To forget this is a pitfall in computing 2 linkage relations consecutively.
gene (or closely linked). The expected numbers for eight phenotypic classes gave a
good fit to the observed numbers when I-Ps-1 and I-3 were assumed to be independent,
but deviated significantly from the observed numbers when they were assumed to be
closely linked, as shown in Table 10.
106 H. I. Oka
21.7
37.9
– { }
}
}
{ }
{ }
Table 9. Recombination values obtained between Ps-1 and 3 other loci. a
Cross Recombination Plants Genes controlling F 2 phenotypes
value (%) (no.) (showing dominant genes only)
545/532
219/532
647/532
521 /532
Mean
219/532
545/532
Mean
108/532
414/532
(pooled)
243
243
172
Ps-1—P (repulsion) Apiculus color : stigma color
36.1 ± 14.6 243 A P—Ps-1 I-PS-1
26.4 ± 6.2 243 P—Ps-1 I-PS-1 Ps-3 I-3
31.3 ± 14.5 174 A P—PS-1 Ps-3
31.4 ± 21.9 128 C A P—Ps-1
31.3 ± 3.9 (standard deviation for variation among estimates)
Ps-1—Ph (coupling) Apiculus/stigma color : phenol reaction
14.9 ± 6.1 P—Ph—Ps-1 I-Ps-1 Ps-3 I-3
14.1 ± 5.7 A P—Ph—Ps-1 I-Ps-1
14.5 ± 4.2 (standard error from two standard deviations)
Ps-1—Ps-2 (repulsion) Apiculus color : stigma color
23.9 ± 6.8 C P–Ps-2–Ps-1
(distortion for Ph:ph considered)
(distortion for C:c considered)
P–Ps-2 (repulsion)
9.0 ± 5.4 172 C P—Ps-2—Ps-1
(distortion for C:c considered)
a "–" shows linkage.
Table 10. Phenotypic classes of 219/532.
Apiculus
Stigma
Phenol
Observed no.
+
+
+
67
+
+
–
5
+
–
+
77
+
–
–
33
–
+
+
55
–
+
–
–
–
+
–
–
–
2 0 4
(pooled)
Total
(no.)
243
Expected no., when I-Ps-1 and I-3 were assumed:
Independent 53.2 8.0 90.5 26.8 58.2 3.2 1.1 2.0 c 2 = 8.3, P > 0.1
Closely linked 35.0 7.0 104.9 27.1 56.5 4.3 1.4 1.8 c 2 = 41.3, P < 0.01
This comparison shows that the two inhibitors are independent. The Ps-1–Ph
recombination value was estimated to be 14.9 ± 6.1%. The P-Ps-a1n d P–Ph values
were also estimated in this cross as shown in Tables 5 and 9. The good fit of expected
to observed numbers may serve as a verification of the assumption of relevant genes.
Similarly, the segregation patterns observed in 108/532, 414/532, 521/532, and
647/532 were analyzed. In these crosses, inhibitor I-Ps-1 does not seem to be involved
(it is recessive in both parents). The data from 108/532 and 414/532 were pooled, since
they were homogeneous. In these two crosses, stigma color is controlled by Ps-1 and
Ps-2, which are linked, Ps-3 being recessive in both parents. The Ps-1–Ps-2
recombination value is estimated to be 23.9 ± 6.8%. The P–Ps-3 recombination is
estimated to be 9.0 ± 5.4%. All these genes belong to linkage group II; their sequence
is mapped in Figure 1.
Analysis of genes for stigma coloration in rice 107
1. Mapping of 5 genes in linkage group II. a Yen and Hsieh (1968). b Takahashi (1964, p. 224). c Standard
deviation standing for variation among several estimates derived from different crosses.
Discussion
This study has shown that there are three genes for stigma coloration: Ps-1, Ps-2, and
Ps-3. This is in agreement with the description in Kinoshita (1984, p. 30), which is
based on a survey of literature by T. Kinoshita. Ps-1, a special gene detected in Gaisenmochi,
confers stigma color even when P is recessive if C and A are dominant, as
reported by Takahashi (1958). Ps-2 and Ps-3 require the dominant combination of C,
A, and P for expressing stigma color. Ps-2 seems to be relatively frequent in indica
varieties (86%; Table 2, group A to 619), while Ps-3 is common in japonicas (50%;
group C to T65), although many colored varieties of indicas and japonicas (groups B
and C) have both genes.
This study has also shown that Ps-1 and Ps-2 belong to linkage group II and are
linked with Ph for phenol reaction. In Kinoshita (1984, p. 30) and Committee on Gene
Symbolization, Nomenclature, and Linkage Groups (1987, p. 15), Ps-2 and Ps-3 are
considered to belong to linkage group II, referring to Hsieh (1961) and Yen and Hsieh
(1968). But Hsieh gives no such information on Ps-3, mentioning, on the contrary, that
“presumably, Ps 2 [corresponding to Ps-3 ] is not linked with Ps 1 , [corresponding to Ps-
2 ] nor with the phenol reaction gene” (Hsieh 1961, p. 128); he reported the linkage of
Ps 1 (= Ps-2 ) with A (corresponding to P ) and Ph. In this study, also, no linkage was
found between Ps-3 and other genes, and the location of Ps-3 remains unknown.
Ps-1 in Gaisen-mochi is thought to belong to linkage group V (Kinoshita 1984, p.
30), based mainly on the linkage of Ps (= Ps-1 ) with I-Bf (inhibitor for brown furrows
on glume, Nagao and Takahashi 1963; Takahashi 1964, p. 225). But the recombination
value of 42.1±3.3% is too high to demonstrate the linkage decisively. Referring to this
linkage, Shastry et al (1975) reported that Ps (= Ps-1 ) is also linked with sd ( sd-1 in
IR20, for semidwarfism), but sd-1 is known to belong to linkage group III (Committee
on Gene Symbolization, Nomenclature and Linkage Groups 1987, p. 17). The present
study has clearly shown that Ps-1 is linked with Ph and several other loci belonging to
linkage group II.
108 H. I. Oka
In this study, inhibitors for stigma color were detected for Ps-1 and Ps-3, but not for
Ps-2. The inhibitors appeared to be independent of each other and of other coloration
genes, although their loci remain unknown. The inhibitor for Ps-3, I-Ps-3 (or I-3 ),
seemed to have a different locus in a few varieties, giving a 1:15 ratio for stigma color
in crosses with varieties having I-3 at the ordinary locus. The dual locations of I-3 may
be regarded as corresponding to 1 ps 2a and 1 ps 2b assumed by Hsieh (1961, p. 129).
Furthermore, Hsieh (1960; 1961, p. 87) has considered that there is a set of complementary
inhibitors, one of which is carried by T65. In the present study, no critical evidence
was obtained for this, but this assumption was adopted for T65 and a few other varieties
to account for F 2 ratios that were otherwise not accountable. It seems certain that there
are two inhibitors, I-Ps-3 and I-Ps-1, but the assumption of inhibitors beyond these two
is still provisional. In this kind of genic analysis, we must elucidate all observed
segregation patterns by assuming a minimum number of genes. The web of inhibitors
for Ps-3 is left for more elaborate analysis in the future. It is also an unanswered
question why the genes for stigma color are more complicated than those for apiculus
color.