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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. LITERATURE CITED ABBO,S., and G. 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