Download Hybrid Sterility and Hybrid Breakdown

Copyright 0 1997 by the Genetics Society of America
Genetics of Hybrid Sterility and Hybrid Breakdownin an Intersubspecific
Rice ( O y a sativa L.) Population
Zhikang Li,**+Shannon R. M. Pinson,: Andrew H. Patemon,” William D. Parks and James W. Stanselt
*Plant Genome Mapping Laboratory, Department of Soil and Crop Sciences, ”Department of Biochemistry and Biophysics, Texas A&”
University, College Station, Texas 77843, tTexas A&M University Agricultural System Research and Extension Center, Beaumont,
Texas 77713 and xUSDA-ARS, Beaumont, Texas 77713
Manuscript received September 26, 1996
Accepted for publication December 19, 1996
ABSTRACT
F, hybrid sterility and “hybrid breakdown” of F2 and later generations in rice (Oryza sativa L.) are
common and genetically complicated. We used a restriction fragment length polymorphism linkage
map and F4 progeny testing to investigate hybrid sterility and hybrid breakdown in a cross between
“widely compatible” 0.sativa ssp. japonica cultivar Lemont from the SouthernU.S. and ssp. indica cultivar
Teqing from China. Our results implicate different genetic mechanisms in hybrid sterility and hybrid
breakdown, respectively.Hybridsterility appeared to be due to recombination within a number of
putative differentiated “supergenes” in the rice genome, which may reflect cryptic structural rearrangements. The cytoplasmic genome had a large effect on fertility of both male and female gametes in the
F, hybrids. There appeared to be a pair of complementary genes that behaved like “widecompatibility”
genes. This pair of genes and the “gamete eliminator” ( S I ) or “egg killer” (S-5) may influence the
phenotypic effects of presumed supergenes in hybrids. Hybrid breakdown appeared to be largely due
to incompatibilities between indica and japonica alleles at many unlinked epistatic loci in the genome.
These proposed mechanisms may partly account for thecomplicated nature of postreproductive barriers
in rice.
S
PECIATION, the process by which an ancestral lineage splits into two or more reproductively isolated
lineages, is the core of evolution. A fundamental component of this process is the origin of reproductive isolation. In the modern synthesis, DOBZHANSKY
(1936) and
MULLER(1940) proposed that generalincompatibilities
between differentiated “complementary” (epistatic)
genes often accounted for postzygotic isolation, since
these genes affect viabilityor fertility of hybridprogeny
but not the parentalspecies. However, STEBBINS
(1958)
proposedthatformulation
of reproductive barriers
might differ considerably from one groupof organisms
to another.
In plants, rates of genetic divergence between populations are affected by breeding systems (JAIN 1975).
Asian cultivated rice (Oryza sativa L.) is a predominantly
self-pollinating species. The differentiation of 0. sativa
into two subspecies, ssp, indica and ssp. japonica, has
been well documented (OKA1988). Considerable differences between the two subspecies both in morphology and at themolecular level [isozymesand restriction
fragment length polymorphism (RFLP)] are associated
with their adaptability to different environments
(GLASZMANN
1987; OKA1988; WANG and TANKSLEY
1992; LI and RUTGER1997). Varying degrees of hybrid
sterility (sterility in F1 hybrids) and hybrid breakdown
Corresponding author: Zhikang Li, Department of Soil and Crop Science, Texas A&M University, College Station, TX 77843-2474.
E-mail: [email protected]
Genetics 145 1139-1148 (April, 1997)
(sterility and weakness in F2 or later generations) are
commonly seen in crosses between indica and japonica
varieties (STEBBINS
1958; OKA1988).
Hybrid sterilityin rice has been asubject of extensive
investigation but itsbasis remains controversial. Although hybrid sterilitydue to chromosomal aberrations
has been suggested (HENDERSON
et al. 1958; YAO et al.
1958; SHASTRY
and MISRA1961; DELORES
et al. 1975), it
could not, in the majorityofcases, be attributed to
cytologically detectable abnormalities (CHUet al. 1969).
In addition, several lines of evidence implicate involvement of major genes in which alleles at a single locus
or two complementary loci could either cause sterility
or recover fertilityinhybrids
(KITAMURA 1962; OKA
1974 and 1988; IJSEHASHI
and ARAKI1986).Certain rice
varieties produce fertile F1 hybrids when crossed to either ssp. indica or ssp. japonica and thus are called
“widely compatible varieties” (MORINAGAand KURIYAMA 1958;JENNINGS 1966; IKEHASHI
and ARAKI1986).
Hybrid sterility and hybrid breakdown often coincide
in indica/japonica crosses, but hybrid breakdown is
sometimes found in advanced-generation progenies
from completely fertile F1 hybrids. OKA(1974) found
a pair of complementary recessive sterility genes that
caused hybrid breakdown in indica/japonica crosses.
While OKA(1988) speculated that there may be many
sets of such complementary sterility genes responsible
for hybrid breakdown in rice, this hypothesis remains
untested.
%.Li rt al.
1140
Here, we investigate the genetic basis of postreproductive barriers in rice. UsingDNA markers, we sought
to determine the number and locations of quantitative
trait loci (QTL) and their epistatic interactions, which
were associated with hybrid sterility and hybrid breakdown in rice.
MATERIALSAND METHODS
Plant materials: Two 0. sativa cultivars, Lemon1 and Teqing, were the parentq in the study. Lemont is a semidwarf
commercial variety from Southern U.S., belonging to the japonica group based on data of isozymes and RFLP (XIE 1998;
LI and RUTGER1997). It is also widely compatible since it
produces fertile Fl’s when crossed with typical indica and
ponicn cultivars (XIE:1993; Z.L I , unpublished data). Teqingis
a semidwarf indica cultivar from China that generally produce
highly sterile F,’s when crossed with typical japonica varieties.
Reciprocal F, crosses were made between the parents. Two
hundred sixty-seven F2 plants from the Lemont/Teqing cross
were randomly selected to produce 255 F:I lines (12 F, plants
produced <35 seeds and thuswere not used in both genotyping and phenotyping experiments). Seeds ( > X ) from each
o f the remaining 255 F-, lines were used to reconstruct the Fy
marker genotype and to produce 2418 F., lines (seven to 11
F, lines from each of the F:, lines, which in turn derived from
a single F2 plant) that were used for replicated phenotyping.
RFLF’ markergenotyping: The genotypes of each of the
2.55 F? plants for 119 RFLP marker loci were determined as
described previously ( L I ut nl. 1995). The genotypes of the
2.55 F2 plants for two morphological markers ( C for purple
apiculus and gl-1 for leaf pubescence) were also determined
from progeny testing of the F4 lines. The resulting map with
115 markers and anaverage 19.1 cM between markers covered
a11 12 rice chromosomes.
Phenotypic data: The field experiment to phenotype the
2418 F., lines derived from the 255 F2 plants was conducted
in 1990, as described previously (IJ ut al. 1995). The F, lines
were drill planted into the field at the Texas A&M University
System Agricultural Research and Extension Center in Beaumont, Texas in 1990. Each F., line was planted in a single row
plot 2.4 m long with a spacing of 28 cm between rows. The
F, lines were planted in family groups separated by tworow
check plots of Gulfmont, a sister line of the female parent
Lernont. Gulfmont was used to determine within plot variation and the F, data were adjusted accordingly. Eleven plots
of Teqing were also randomly located in the field. Ten panicles from different plantsin each of the F4 lines were collected
and dried at 50” for 72 hr. Panicles from each of the F, lines
were hand-threshed andassayed for Several grain yield component traits including total floret number per panicle (FN),
sterilefloret(unfilledflorets)
numberper panicle, grain
number (filled spikelets) per panicle, 200 kernel weight, and
grain weight per panicle. Spikelet sterility (SS) was calculated
as the percentage of empty spikelets over total spikelets per
panicle for each of the Fa lines. Plant height (pH) andheading date (HD) were assayed on each of the F, lines as described previously ( L I rt al. 1995). PH was used as atrait
associated with hybrid weakness since weak and/or slowly
growing plants tend to have reduced height (OK\ 1988). The
breeding value of each o f the 255 F2 plants for these traits
was obtained by averaging its seven to 11 F.+lines (70- 110
observations) and used in the data analyses. In 1993, the parents and reciprocal F, plants were planted in the greenhouse
for assay of SS and pollen fertility using I&I staining.
Dataanalyses: Mapping of QTL used interval mapping
(L,ANI)ER and BOTSEIN 1989) and Mapmaker/QTL (L,IN(:oI.N
rt rcl. 1992). A threshold of LOD 2.4 was used to claim the
, l a -
presence of a QTL. Genomic regions with LOD scores between 2.0 and 2.4 were considered as putative QTL. Identified
QTL were further evaluated using a nmltiple regression
model (ZEN(: 1998, 1994; 1.1 rt (11. 1997). To identify epistasis
affecting SS and PH, twz.o-way analyses of variances (ANOVA)
were performed between 95 selected markers (noncodominant markers and those with >9.5 missing data were not used)
based on the model and the methods by L I rt nl. (1997) using
SAS PRO<: GLM (SAS I w r r r L m IN(:. 1987). The threshold
10 claim a significant interaction w a s P < 0.001 and
@,,,<.,.,,,,,,,,> 6%. All significant interactions were ftu-thcr ~ I K ly~edusing multiple regression models with all identified QTL
fixed in the models to control the background genetic effects
of segregating QTI. based on the model and methods de1993,1994; LI ~t n / . 1997). Estimascribed previously (ZEN(;
tion of digenic parameters and testsof interaction effects
using t tests, also followed the methods desrribed i n L I r / (I/.
(1997).
RESULTS
Difference in sterility between reciprocal
Fl plants
and segregation of spikelet sterility in the F2 and F4
generations: In the greenhouse experiment, the reciprocal Fl hybrids differed significantly in fertility of both
male and female gametes even though the hybrid plants
were virtually identical morphologically (Table 1 ) . Teqing/Lemont F, plants had significantly higher SS a n d
pollen sterility than Ixmont/Teqing F, plants. This indicated that gene(s) in the cytoplasmic genome had a
significant impact o n t h e fertility of both male and female gametes of the hybrid
FI plants and that Lemont’s
cytoplasmic gene(s) may play an important role in its
“wide compatibility.” The parents had high pollen fertility (>%%) a n d low SS (9.6% for Lemont and 5.8%
for Teqing).
In the field experiment, the 2418 F4 lines from the
Lemont/Teqing cross had mean SS of 28.3%, significantly ( P < 0.01) higher than the parents (15.7 2 4.2%
f o r L e m o n t a n d19.1 2 3.5% for Teqing). TheF.l distribution of SS was slightly skewed, ranging from
8 to
nearly 70% (Figure 1).T h e b r e e d i n g values of the 255
F2 plants for sterility had an approximately normal distribution, ranging from15 to 50%. Sterility was strongly
correlated with heading date ( r ( ; = 0.447, P < O.OOOl),
weakly correlated with plant height
( q , = 0.258, P =
O.001), and not correlatedwith floret number per panicleorkernelweight
( , r ( ; = 0.148 a n d 0.007,respectively).
Identification of SS QTL FourQTLaffecting
SS
were mapped to chromosomes
3, 6, 8, a n d 10 (Table
2 and Figure 2). T h e s e Q T L h a d I? ranging from 7.2
to 21.1% a n d collectively explained 60.4% of the genotypic variance. T h e p r e s e n c eof two putative QTL ( QL%2
a n d QSs5) on chromosomes 2 a n d 5 W d S suggested by
subthresholdLODscores(LOD
= 2.14 a n d 2.35).
W h e n a full model with the four QTL (LOD
> 2.4)
fixed was performed on the two putative QTL by Mapmaker/QTL and by multiple regression analysis, @.s5
was confirmed (with a LOD of3.25) a n d y S s 2 remained
suggestive (LOD = 2.1). It was particularly interesting
1141
Reproductive Isolation Barriers in Rice
TABLE 1
Comparison of reciprocal FI hybrids between ‘Lemont’ and ‘Teqing’ for spikelet sterility (SS), pollen sterility (PS),
heading date (HD), plant height (PH), panicle length (PL), floret number per panicle (FN), and
kernel weight/1000 grains (KW) in the greenhouse experiment
9.6 ? 4.6
5.8 ? 3.2
16.8 ? 5.6
50.7 f 6.3
-33.9***
Lemont
Teqing
F, (Lemont/Teqing)
R F , (Teqing/Lemont)
F,-RF1“
“ **,
4.2 f 3.2
4.7 f 2.5
38.4 f 5.1
57.0 +- 5.7
- 18.6**
108
117
98
98
0
*** represent the significance levels of P 5 0.01 and P 5 0.001, respectively, based on
to note that these two putative QTL had zero additive
effects but very large underdominance effects of 10.8%
(QSs2) and 13.6% ( Q S 5 ) for fertility.
Identification of epistasis affecting SS and PH: Of
4465 two-way ANOVAs performed, the observed numbers of significant interactions at P < 0.001 were 83
and 74 for SS and PH, of which 18 for SS and 20 for
PH were due to linkage of the markers involved in
highly significant interactions. Results from multiple
regression analyses (with all previously identified QTL
fixed in the model) indicated that
38 additional interactions for SS and 45 for PH were due to background
genetic effects caused by the SS QTL and three plant
height QTL (LI et nl. 1995). Thesewere removed from
further analyses. The remaining interactions could be
divided into twotypes: (1) interactions between unlinked markers (21 for SS and 11 for PH) that were
supposed to be due to epistasis and (2) those between
linked markers (six for SS and seven for PH) , which did
not represent cases of epistasis but cases where novel
phenotypes of SS and PH resulted from rare recombination events within certain genomic regions. The latter
cases will be examined separately in a later section.
Themagnitudesandcharacteristics of additivedigenic epistatic estimates: Table 3 shows the interaction
effects between homozygotes in the 21 and 11 interac-
4O0
21.5
223.0
236.2
-13.2
0.4
t tests. -, data was not available.
tions affecting SS and PH, and the markers involved.
For SS, the mean I? explained by individual interactions was 7.62 % 1.28% and ranged from 6.18to
10.84%, significantly higher than the mean l? (6.05 ?
1.85%, obtained by ANOVA) of the four SS QTL. The
average value of 45significant interaction effects (based
on t-tests) was 5.3 ? 2.9% (rangingfrom 2.7 to 21.2%),
which was slightly larger than the doubled mean additive effect (4.9 +- 0.4%) of the SS QTL. No interactions
were detected between the SS QTL. However, three of
the 32 different markers involved in the interactions
flanked three SS QTL (QSs3, QSs6, and QSsX), which
were involved in five interactions (Tables 4 and 5). The
remaining 27 (84.4%) interactions occurred between
29 unlinked non-QTL markers. On average, each of the
32 markers was involved in 1.31 interactions, suggesting
the presence of higher-order interactions.
Evidence of sterility arising from “incompatibilities”
between indica andjaponicaalleles at epistaticloci:
When the digenic interaction effects between homozygotes in Table 3 (except for the one
between CD0348b
and RZ660 that will be discussed later) were classified
into the parentaltypes and the recombinanttype (interactions between indica alleles and jnponicn alleles), we
found that sterility was usually associated with the recombinant type interaction (Table 4). The sum of11
TABLE 2
Frequency
“c
-
24.6
25.221.1
-0.6
79.2
81.5
110.0
113.2
-3.2
QTL and their estimated effects on percentage SS detected
by MapMaker/QTL in the Lemont X Teqing rice cross
n
n II n
t
Imean of F4lines
0 F2breeding values
QTL
300
Flanking
markers
R“(;
II
d
(%)
LOD
QL%3
100
0
RG348-RG944
3.9 -2.0 11.7 5.40
GRG424
3.1 -1.2
9.6 2.88
QSS8
RG20-RG1034 -4.921.1 1.2
4.23
3.31
QSSlO
RG752-E786
7.2 2.4 2.9
QSS5
RG556-gl-1
-0.2 13.6 6.4 3.25
QSs2
RG598bRG139 -0.1 10.8
2.14 4.1
Multi-QTL model
60.4 17.64
QSsh
200
5
11
17 41
35
29
23
47
Spikelet sterility(SS,in %)
59
53
65 68
FIGURE1.-Frequency distributions of the mean spikelet
sterility of 2418 F4 lines and the breeding values of the 255
F2 plants (10 times the observed frequecies) of the Lemont
X Teqing cross.
” a is the additive effect due to substitution of the Lemont
allele by the corresponding Teqing allele at QTL, and d is
the dominance effect associated with the heterozygote. LOD
scores are based on a “free genetics” model (PA’rERSON et al.
1991), including both additive and dominance effects.
%.1.i
I142
CM
2
I
3
4
5
7
6
111.
9
IO
I1
12
RG403
RGIX?
RG13
DO3XXr
RG4XZa
RG716
CDO177
RG34h
CDOS44
RZ7hX
---RGXl I
I
CDOX7
KG173
RG532
R7.2XRx
RG472
R(iJ47
RG433
RGYlOn
-
RGh53
RG41X
SSQTL
0 Purative“supergenc”regionsaffecting PH
Pur;lrive“super~ene”regions
causing S S
(5.06 to 10.295%(Table 4), which was lower thanthe
~neanI?’ (8.00 f 6.12%) of the four PH QTI. itlcntified
previously (LA NI. 199.5). N o interaction was tletectetl
between the PH QTI. ( h I>/ d . 199.5), but two markers
flanking PH QTI, (Ql’h? antl Ql’lt9) were involved i n
tligenic interactions. Thc remaining nine (81.8%) i n teractions occurred between 18 ranclom markers. On
average, each of the 20 markers was involved i n 1 . I
interactions.The mean o f the 19 significant interxtion
effects hetween homozygotes was 7.0 5 2.0 cm (ranging
from 4.1 to 12.1 c m ) , which w a s smaller than the douI~ledlnean additive effect (7.8 f 2.9 cm) of the four
PH QTI, (LI d d . 199.5).The sum of the parental interaction cffccts resulted i n 32.1 cm increased height. I n
contrast, thesum ofthe recombinant interactioneffects
resulted i n decreased PH by 4.9 cm.
Putative “supergenes” affecting SS and
PH
in
rice: Table .5 shows three (SS) antl t w o (PH) genomic
regions within which significant interactions were detected between linked markers. I n these cases, large
phenotypic emects \vert almost exclusivelyassociated
with the recombinant genotypes. I t was realized that
the recomhinant genotypes homoz)pus a t the flanking
markers may actually be heterozygous somewhere i n
the region since the prohahility that crossing-over
events occurred at thesame point of this region in hoth
n1ale and female gametes is extremely l o w .
SS: Six pairs of linked markers represented three genomic regions i n chromosolnes 5, i, and 11, within
which recombination resulted i n tlrasticAly reduced
fertility. Thc first casewas two interactions representing
a 14c,M genomic region betwren RG207 and $1 on
Reproductive Isolation Barriers in Rice
1143
TABLE 3
Interaction effects ( T ~on
) spikelet sterility (in %) and plant height (incm) between homozygous alleles at unlinked marker
pairs in the Lemont X Teqing rice cross
Chromosome
Marker 1
Marker 2
RG236
RZ390a
RZ288b
RG532
RZ390a
RG447
RZ390a
RG520
RG171
RG418
RZ676
RG9 1Oa
CD087
RG482a
RG944
RG104
CDO109b
CD0348b
CDO109b
RG30
RG757
RG9 1Oa
CDOlO9a
RG 143
RG143
RG424
RZ276Q
RG90 1
RG1034
RG103
RG143
RG182
RG381
RG532
RZ776
RZ390a
CD0388a
RZ776
RG437
RZ761
RG13
RZ2
RG4
RG437
RG944
RG104
RG424
RG716
Chromosome
1L/2L
1L/ 2T
1T/2L
6.24
7.26
6.32
8.46
7.53
7.26
7.93
7.66
6.18
7.25
6.34
6.63
8.59
7.22
7.47
8.73
10.84
8.08
7.88
7.72
8.51
-6.4****
3.3*
-0.2
1.3
4.7***
-1.2
3.1*
-7.7""""
0.1
5.1***
-2.8"
2.7*
-3.8""
-2.5
0.8
2.6
7.7****
-0.0
1.0
-6.7****
-2.4
2.1
-3.9**
-2.6
6.8****
0.5
1.6
-2.1
0.3
3.5*
-1.4
2.8*
-2.2
-1.5
3.1*
1.6
3.7**
-4.7**
-0.9
6.7***
6.4****
9,2****
3.1"
0.9
5.5****
-2.7*
-4.7***
4.1**
-0.2
-1.7
3.0**
-1.6
-0.7
5.4***
3.8**
5.7***
6.1****
1 .0
-7,5****
-5.9***
-2.9
1.o
-1.0
-3.9""
1.7
1.8
0.6
1.0
6.64
6.42
6.06
8.92
7.46
7.66
6.08
7.74
10.26
6.47
10.29
-8.0***
1.1
6.0**
-4.2"
-2.3
-2.8
-0.4
1.6
-4.7*
10.4****
2.8
-7.7***
2.9
4.5"
1.7
-7.4***
-8.9***
0.9
7.0**
-6.5**
-3.5
6.4**
3.1
-5.1*
1.7
5.9**
2.6
3.6*
1.2
3.1
8.3***
- 1.4
Spikelet sterility
1
1
1
1
1
1
1
2
2
3
?
?
?
?
3
3
6
6
6
7
9
Plant height
1
1
1
1
1
1
2
?
5
6
7
c
RG1034
RG1034
CD098
RZ397
RG4
RZ660
CD098
RG463
RZ660
Rz777
RG9 1
RG190a
CD098
RG241b
RG1022
3
3
4
4
6
9
12
8
11
4
5
6
8
8
10
12
7
9
10
9
9
2
3
?
6
6
9
4
4
10
10
11
Digenic genotypes"
R'
(%)
0.0
4.1"
8.6***
6.3**
12.1****
0.1
-0.4
3.0
-3.3
-1.0
1T/2T
8.0****
6.7****
-0.3
-3.9""
3.9**
-3.5**
2.3
6.2****
-3.7**
-0.0
-(jJ****
1.8
21.2****
- 1.6
1.4
4.6**
2.2
-
1.4
*, **, ***, and **** represent the significance levels of P 5 0.05, 0.01, 0.001, and 0.0001 based on t tests. 1L/2L and IT/
2T are the parental genotypes where L and T represent homozygotes of Lemont and Teqing, and 1L/2T and 1T/2L are two
homozygous recombinant genotypes.
"
chromosome 5 where an underdominant QTL (QSs5)
was identified. This QTL hada zero additive effect
(0.1%) and a very large dominance effect for increased
SS by 13.6%. Again, significantly increased SS was associated with the recombinants within this region. The
second case was a 15-cM-longregion flanked by RG711
and RG678b on chromosome 7, which was supported
by the data from three pairs of linked markers flanking
this region. While we did not detect any SS QTL at this
region, one class of the two homozygous recombinants
generated by crossing over within this region significantly ( P < 0.0001) increased SS by 16.1%. This was
unlikely to be caused by mistyping of the marker genotypes because the probability that the same mistyping
error occurred in three markers detected by different
enzyme digestions and scored in different hybridization
blots was very low.The thirdwas a 26cM-long genomic
region flanked by RZ781 and RG1022 on chromosome
11. One of the classesof homozygous recombinants
(the genotype 1L/2T) had 6.2% increased SS (and 49.3
reduced florets per panicle). The other homozygous
recombinant (the genotype 1T/2L) had a reduced SS
by 5.1%, which could be attributed to its effect on reduced floret number (data not shown). Two heterozygous recombinant classesalso resulted in significant
effects for increased SS. In addition, the heterozygotes
at this region increased SS by 4.8% ( P = 0.03).
PH: The seven interactions between linked markers
affecting PH involved two genomic regions. Similar to
the cases for SS, there were two genomic regions on
chromosomes 2 (between RG256 and RG139) and 6
(between RG179 andCD0544) where crossing over
produced
recombinants
with
significantly
reduced
height. It was also noted that theputative "underdomi-
Z. Li et al.
1144
TABLE 4
Characterization of significant additive interaction effects
(T+)on spikelet sterility and plant height between
homozygous alleles at unlinked marker pairs
in the Lemont/Teqing rice cross
Spikelet
Plant height
sterility
Parental types
+"
+
Lemont
-
1
1
Teqing
-
+
Total
-
+
Recombinants
-
No. of
Zr,
T'/
(%)
No. of
rt/
CT,~
(cm)
6
5
4
6
10
11
-0.8
4
22.1
8.3
2
9.1
7.5
15
31.2
16
6
52.5
7
5
6
-4.9
" + and - represent the parametersfor increased SS or PH
and decreased SS or PH, respectively.
nance" QTL for fertility was detected in the putative
supergene region on chromosome 2.
DISCUSSION
Genetic analysis of postreproductive isolation barriers between 0. sativa ssp. indica and ssp. japonica may
shed light on the process of speciation in plants, and
provide important information for rice improvement.
Our results from QTL mapping and interaction analy-
ses suggest several genetic mechanisms that might be
responsible for hybrid sterility and hybrid breakdown
in rice.
Genetic
mechanisms
causing
hybrid
sterility
in
rice: Three genetic mechanisms, cytoplasmic gene(s),
putative differentiated supergenes, and putative complementary R m genes, have been implicated in hybrid
sterility by our results.
Cytoplasmic gene(s): The impact of cytoplasmic
gene(s) on hybrid sterility has been reported in rice
(CHANGet al. 1990; PHAM1990). In the present study,
sterility of the reciprocal Lemont/Teqing F, hybrids
differed as much as 33.9%. Since 43-57% normal pollen of the reciprocal F, plants was sufficient to fertilize
the female gametes, such a big difference in SS was best
accounted forby the cytoplasmic gene(s) onthe female
gametes. The effect of cytoplasmic gene(s) on hybrid
sterility may provide an explanation for the observed
varietal specificity of some wide compatibility varieties
(KUMARand VIRMANI1992; LIN et al. 1992).
Putative supergenes: Supergenes are definedas groups
of tightlylinked genes, within whichrecombination will
cause reduced fitness (DARLJNGTON
and MATHER1949).
The five genomic regions on chromosomes 2, 5, 6, 7,
and 11, revealed both by QTL mapping (underdominant SS QTL) and by interaction analyses in the present
study, appear to behave as such supergenes. A unique
characteristic of these putative supergene regions was
that reducedfitness (sterility, reduced height,and grain
number per panicle)
was exclusively associated with heterozygotes. It is conceivable that a limited number of
differentiated supergenes may cause hybrid sterility in
TABLE 5
Large phenotypic effects of crossing over within certain genomic regions on percentage spikelet sterility andon plant height
revealed putative supergene regionsin the rice genome
~~~~~~
~
Genomic regions
Flanking markers
Interval
(r)
R'
(%)
Digenic genotypes"
1L/2T
1T/2L
1T/2T
1L/2H
1T/2H
1H/2L
1H/2T
1 .o
1.3
-1.2
-0.7
-1.0
2.3
2.3
-1.1
0.2
1.3
3.0*
0.3
1.7
1.5
1.3
0.6
0.4
1.5
3.2""
0.1
1.9
2.7*
3.2*
-0.4
-4.0*
-1.3
1.4
1.9
2.3
2.9
3.3
1.6
1.9
1.2
0.3
2.7
2.1
2.5
3.8
3.1
2.3
-1.0
-0.8
2.3
1.7
Spikelet sterility
5
5
7
7
7
11
RG207
RG556
RG4
RG711
RG711
RZ781
gl-1
gLJ
RG678b
RG678b
RG30
RG1022
0.14
0.13
0.19
0.15
0.26
0.18
4.20
4.43
4.40
4.62
5.64
8.04
-0.9
-1.1
0.0
-0.7
-2.2
-2.6
3.8**
3.4**
11.7****
16.1****
15.1****
6.2****
14.6****
-
-
-3.9""
-0.3
2.0
-5.1***
2.1
4.0**
0.1
1.8
0.8
Plant height
2
RG256
2
6
0.17
RG256
RG598b
-~
6
6
6
6
RG424
RG424
RG179
RG179
RG716
RG139
0.19
CD0544
0.26
E768
0.29
CD0544
-3.8 5.340.24
E768
0.29
RZ768
0.24
5.52
4.51
3.50
3.34
5.43
6.71
4.1*
2.1
-2.6
-2.9
-2.9
-1.2
4.4*
3.1
-2.5
-0.4
-6.1**
-3.8*
-4.3*
-9.4***
-10.9****
-17.1****
-16.3****
-17.4****
-16.5****
-18.2****
-1.1
-0.8
-1.0
-0.9
0.1
-1.0
-0.6
1.1
0.9
1.4
0.3
0.9
1.9
3.2
" L, T, and H represent the Lemont alleles, the Teqing alleles, and the heterozygotes at the flanking markers;*, **, ***, and
0.05, 0.01, 0.001, and 0,0001, respectively. -, cases of
a missing genotype. Underlinedmarkers flanked two regions where underdominance SS QTL were identified.
**** indicate that the interaction effects are different from zero at P 5
-:
RcprodtIcIivc Isolatic ) I I kuricrs in l i i w
I 1-13
gions arc related to fitness traits. Segregation distortion
against
the 0 . s d i r w s.s/l. itlclietr allcle w i t h i n chromoP-RGS20
RG.520
RGZW
some 5 supergene region was also rrportetl i n several
HC.556
cases invol\ing ,j~rpotlic/r/irttlie~r
crosses (/;I
L.ls I>/ cd.
RZ446X
20
RZ3YOh
'-RZM6x
"256
1992). M'c further ohset~ctla 1:s ratio against the ittdicct
gr- I
40
. -RG256
RZ273
allele a t gl-I i n I28 R C I F l [(I,cmont X Tcqing) X 1.c1u2~1
mont] plants, which suggested that the distorted scg~.ca60
RG139
.LRZ260
gation within this rcxgion WIS clue t o gametic sclcction.
RG4Q3
O u r rcsdts indicated that the phcnot!.pic eff'ecr o n k r RG403
RG139
~-CD0718,RZ386
tility o f thissllpe1-genc was rlcpcntlent 0 1 1 the cytoRG182
plasmic genotype. This is much like the case of meiotic
RG I82
RZ213
:I$?$$
drive
i n nlouse i n which sllppresion o f rccorn1)ill;~tion
(RZ476)
RG83
I20
t l w t o inversions within thc I complex appeared t o
occur only i n fcmalrs (FKIS(:I
I A ~ T 198.5).
RG13
RGSSS
RG13
I40
Supergenes ( o r the presumed cryptic Iyhritlit!~) arc
m386
cxpcctrtl t o inflrlcncc hybrid l,re;ddo\w primarily i n
(Caussc cf ol. 1994)
...RG470
160
W718
c * d y segregating gencwtions, and their c f f c ~ t swill tli...RG470
minish ;IS the progeny ;Ipproach complete llomoz!y)sI80
RG437
ity by sclfing.
Rz476
200
7Kr p l r l n l i r w K t n p t w / r t d ils rnotl~jiw:M'hile the preUS99
sumed supergenes may cause vaning tlcgt-ecsof l l ! h k l
220
sterility, they do not adeqrlatcly explain the indcpcntlence of' hybrid sterility and hybrid bre;lktlo\\*ni n t!lc
I,emont/Tcqing cross, o r i n many other crosscs whcrc
RG346
RG346
cytopl;tsmic
c f f c ~ t s\\we not ;I k~ctor.Thc presence of
260
241
( Y u e f d . 199s:
witlc compatibility gcne(s) that c m p r c ~ ~the
1 t abor(Li er ol. unpublished)
Caussc cf ol. 1994) (Li "a'' 199s)
tion of F, hybrid gametes has been suggcstecl i n ricc
(KIT~\\IL.IL\
1962; IKI.II.\SIII ;lnd AIG\KI1986; K ~ . \ I . \ Iand
<
FK;~.IW
:~.-(:oml';lrison
:
o l ' ricr Iinktgr m;qx sr~ggcbsrst w o
\'IK\I:\SI
1992),
tomato
(
KK:K
1
S
(
i
(
197
i
,
I
)
,
wheat
illvcrsions in rhr p t l c t t i v c . supwgvw regions o n ricr chromoS ~ I I I C S2 ; t n d 5 .
(LOIX;I<KIS(;
and SI-;\KS19(i3), tobacco ( C \ J I I - I W S and
h 4 0 ~ \ .l9.57), ;Ind lentil (Alma and I A ) I X I S S K ~ 1994).
~
I n Drosophila, single mutations (1,hr antl Htttv) were
specific crosscs. Each o f these clifferentiatetlsupergenes
reportedly able to restore hybridviability (M':vI..\s.\I~I<
indiviclwlly m;y only cause partial sterility, since only
1979; HL"I-I'EKand ASIIIILXSICK
1987). I n f k t , allelic
thc g;\metcs that arc recombinant within any specific
interactions a t S I and S 5 on ricc cl1romosomc h have
stlpcqcnc rcgion will be affectctd. Further,different
hcen ofrered a s one hypothesis for the genetic basis o f
supergenes m;~!' havc different cffccts, and their phentr
hyhritl sterility ( 1 ~ 1 . L\SI
1 11 and A&\al 198(i; S.\SO 1 9 9 0 ) .
typic cf'fccts on sterility and/or viability i n hybrids are
Although w e did not detect a major SS QTI. on the
rxprctctl t o l,c crlmdativc. In agreement with these
S,/S-5 region o f chromosome 6,we did identify an intcrexpectations. the ovcrall heterozygosity (cstimatcd
action between 21 pair of putative complementary genes
from a l l 11.5 m;llk-s throrlghout the genome) o f intli(mapped near CD0348h on chromosome 6 and R i M i 0
vitltlal F2 plants was not rc+ltctl t o SS o f their derived F I
on chromosome 9 ) , which behaved like wide compatilines ( r = 0 . 0 1 3 ) , b u t the heterozygosity a t the putative
bility genes in the Lemont/Teqing cross. The recessive
supcrgcnc regions (flanking markers) was significantly
nature antl gamete killer behavior of the gene loci arc
associatcttl w i t h SS ( r = 0.43.5, 1'< 0 . 0 0 0 1 ) . These results
suggested by the observation that when all but one o f
suggest that these tlilrercntiatctl supergenes may be an
the FL'plants with the Teqinggenotype (non\videlycomimportant C ; I I I S ~ of' hybrid sterility i n rice.
patible) were eliminated,the homozygorls rccombiAlthough the nature o f ' these putative suprrgenes is
u n k n o w n , some ma!' he c t y p i c sl,rrclrr,nlrc~~rl-angc~nents nants m t l the heterozygotes did not exhibit high SS.
The o n l y remaining Teqing (nonwitlcly compatible in(cytologicallyundctcctahlc minorchromosomal ahcrrations) (S-n-lrslss 19.50, 19.58). A direct comparison hediccr) genotype at the complementm? gene locihad
22.2% reduced fertility, which was equivalent t o that o f
t~vreno u r maps (constructcd from the F2 population
S5 (IKI<II,\SIIIa11dAK;\KI 1986). CDO348h mapped I I C Y I ~
and a set of219 tlcrivctl RILs from the Lcmont/Tcqing
the location (rcportcd on chromosome 6 ) of' the sacross) and the ricc RFLP maps pd~lishctlby CALWI; PI
mete eliminator S I causing hyhritl SS of interspecific
d . (19!)4) suggests the presence of inversion polymorcrosses between 0 . s d i u c r and 0 . gldwritnttto ( S.\SO
phism i n thc putative supergene regions on chromo1990) antl the egg killer S-5 affecting intergroup h!hid
somes 2 ;und 5 (Figure 3 ) . Rcducetl fitness in heteroz\lgous plants :wising from crossing over within the
SS hct\vcc~njcrj)otlirn and inrliccr varieties ( II<I-I I.\SI1 1 and
invcrtctl regions is expected if gcnes w i t h i n these reAKAKI1986).
cM
0
2
+
Ij
I :L
2
5
5
I ',:E:
RGSS6.A
1
1 'S'"'
:
1
~
I
1146
Z. Li et al.
Unlike many reported mutations that cause nuclear
male and/or female sterility in rice (Hu and RUTCER
1992), the recessive alleles at the putative complementary loci do not kill gametes in their original parent
(Teqing) but only in the indica/japonica hybrid background. Then, a pertinent
question is,what kind of
genes are they and how can they cause SS or recover
fertility in hybrids? Our speculation is that if the gene
or gene pair influences recombination frequencies between homologous chromosomes, like the RMl gene
in Petunia hybrida (CORNUet al. 1989), they may behave
like gamete killers or wide compatibility genes. As discussed above, a primary mechanism causing hybrid sterility is inferred to be recombination within differentiated supergenes located in several regions of the rice
genome. Then, any gene(s) thatcan influence homologous recombination may behave like wide-compatibility
(dominant) or gamete killers (recessive) by affecting
recombination frequencies inmany supergene regions.
Thus, themajor gamete killer near the wx and Cloci on
chromosome 6 reported in previous studies (KITAMURA
1962; IKEHASHIand ARAKI1986; SANO 1986, 1990; KUMAR and VIRMANI 1992), including
one of the (putative)
complementary genes identified in the present study,
may be recombination-modulating genes. Large effects
of S-5 and S, leading to two- to fivefold variation in
recombination frequencies along their surrounding
genomic regions were observed in rice (IKEHASHI
and
AKAKI 1986; SANO 1990), which strongly suggested the
true nature (recombination modulating)
of the wide
compatibility gene or the gamete eliminator gene in
rice.
Control of recombination may also be influenced by
a polygenic system, as described by BROOKS
and MARKS
(1986). PFEIFFER
and VOCT (1990), SALL (1990) and
FATMIet al. (1993) reported polygenes that affect local
recombination frequency primarily in a region-specific
manner in maize, barley, and soybeans. Such a polygenic system may alsoinfluence postreproductive isolation of rice and may explain some of our epistatic interactions that resulted inreduced
SS but were not
attributable to the related traits.
Geneticmechanismscausinghybridbreakdownin
rice: SS and hybrid weakness in F2 and later generations from indica/japonica crosses, or hybrid breakdown
as defined by STEBBINS
(1958), are much morecomplicated than F1 sterility since these properties are influenced by large numbers of genes functioning in both
gametophytic and sporophytic stages, as well as by environment. Our results indicated thatthe primary mechanism causing hybrid breakdown in rice is the uncoupling of coadapted indica and/or japonica gene
complexes by recombination.
The presence of such coadapted gene complexes as
a result of natural and/or artificial selection in related
plant populations has long been suggested &LARD
et
al. 1972; WEIRet al. 1974; CLEGG1978; &LARD
1988;
OKA1988). In rice, intersubspecific progeny tend to
quickly revert back to their parental types as generations advance (Om 1988; SATO1990), strongly suggesting the presence of indica and japonica gene complexes. Our results indicated
that
sterility arises
primarily from the incompatible interactions between
indica (Teqing) and japonica (Lemont) alleles at many
unlinked loci in the genome. This implies that epistasis
is an important factor in maintaining the integrity of
these gene complexes, in the indica and japonica gene
pools.
Our results provide direct evidence for thehypothesis
that hybrid breakdown results primarily from disharmonic interactions between unlinked loci, which refers
to the complementary genesystem (DOBZHANSKY
1936;
MULLER 1940;
STEBBINS
1958; OKA1988). Genes in this
complementary system appear to directly (QTL) or indirectly (epistasis) affect fitness correlates such as heading date and floret number perpanicle. The presumed
coadapted gene complexes appear to have large effects
on hybrid breakdown but little impact on hybrid sterility since the F, plants were largely fertile and vigorous.
This suggests that dominance relationships at many of
these epistatic loci afford fertility to F1 plants, as predicted by recent theoretical work (ORR1995; TURELLI
and ORR1995).
Recombination is a key genetic element for origin
of
reproductive isolation: Both hybrid sterility and hybrid
breakdown appear related to recombination. In selfpollinated plants like rice, chromosomal mutations may
evolve quickly since they impose minimal genetic load
on a population of homozygous individuals. Reproductive isolation mechanisms based on cryptic chromosomal rearrangements may be more effective. Genes
that suppress homologous recombinationat meiosis
may enhance the formation of reproductive isolation
(hybrid sterility).
Sterility and weakness can also arise in interspecific
hybrid progenies from random assortment of nonhomologous chromosomes (DOBZHANSKY
1936; MULLER
1940; STEBBINS
1958). Ourresults indicated that hybrid
breakdown mayinvolve large numbers of genes and
complex high order gene interactions, as reported in
Drosophila (CABOT et al. 1994; PALOPOLIand WU 1994)
and predicted by recent theoretical work (ORR1995;
TURELLI
and O m 1995).
Impacts of genetic mechanisms affecting
SS on segregation distortion and gene mapping: Segregation distortion has been commonly observed in wide crosses
between distantly related taxa. In our study, distorted
segregation at 18 marker loci (LI et ai. 1995) could be
attributed to selection at either gametophytic and/or
sporophytic stages as well asto random sampling variation. In the Lemont/Teqing population, 14 of the 18
markers showing segregation distortion were located
on four genomic regions on chromosomes 3, 5, 6, and
9, three of which wereassociated with SS QTL. Severely
distorted segregation due to the differentiation of coadapted genecomplexes may occur insegregating pop-
Reproductive Isolation Barriers in Rice
ulations from divergent parents, as was noted in an
indica/juponicu recombinant inbred population ( W ~ G
et al. 1994). Selection for differentiated coadapted epistatic genes could explain “pseudolinkage” between
some unlinked markers (WANGet al. 1994).
Differentiated supergenes or cryptic chromosomal
variation may have an impact on genomic studies. First,
breakdown of supergenes by crossing over wouldresult
in recombinants with reduced fertility, and thus reduced genetic distances between markers in affected
genomic regions. Second, presence of cryptic structural
variation between parents of a mapping population
may
cause problems in determining marker order within a
supergene region since the parents may have different
orders of the affected markers. Thus, in populations
generated from wide crosses including interspecific or
subspecific crosses, information about the number and
effects of such supergene regions is valuable in designing gene mapping
and map-based cloning experiments.
We are grateful to Dr. TRUDYF. C. MACKAY and two anonymous
reviewers for many critical comments and suggestions in the early
draft of this manuscript. We also thank S. D.TANKSLEY and S. R.
MCCOUCHfor providing us with the DNA probes. This research was
supported by USDA-ARS, Southern Plain Area, The Texas A&M UniversitySystem Agricultural Research and Extension Center,The
Texas Rice Research Foundation, and the Texas Advanced Technology Program grants to W.D.P., Z.L. and J.W.S.
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