Published in Crop Sci 39:1711-1715 (1999)
© 1999 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Crop Science 39:1711-1715 (1999)
© 1999 Crop Science Society of America
CELL BIOLOGY & MOLECULAR GENETICS
Mapping and Genetic Analysis of the Genes for Photoperiod-Sensitive Genic Male Sterility in Rice Using the Original Mutant Nongken 58S
M.H. Meia,
X.K. Daia,
C.G. Xua and
Qifa Zhanga
a National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural Univ., Wuhan 430070, People's Republic of China
qifazh{at}public.wh.hb.cn
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ABSTRACT
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Photoperiod-sensitive genic male sterility (PSGMS) rice has a number of desirable characteristics for hybrid rice production. In this study, we conducted a molecular marker-based mapping and genetic analysis of PSGMS genes using two crosses involving the original mutant Nongken 58S as the PSGMS parent. The genomic locations of the PSGMS loci were determined following the "bulked extreme and recessive class" approach, in which the regions containing the PSGMS loci were identified by assaying the bulks of extreme phenotypes and the distances between the PSGMS loci and molecular markers were calculated from the highly sterile plants of the F2 populations. Two PSGMS loci, located on chromosomes 7 and 12, respectively, were identified in both crosses. The locus on chromosome 7 was the same as pms1, identified previously in a cross between an indica PSGMS line and an indica cultivar. The one on chromosome 12 was a new locus that we designated as pms3. Both loci had major effects on fertility and acted like a pair of classical duplicated genes in controlling male sterility. Comparison of the present finding with previous results indicated a complex genetic basis of PSGMS. The implications of such genetic complexity in breeding for PSGMS lines are also discussed.
Abbreviations: ANOVA, analysis of variance • cM, centimorgan • PSGMS, photoperiod-sensitive genic male sterility • RFLP, restriction fragment length polymorphism • 58N, normal cultivar Nongken 58 • 58S, Nongken 58S, the PSGMS mutant of Nongken 58
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INTRODUCTION
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A PHOTOPERIOD-SENSITIVE
genic male sterility rice was found in 1973 as a spontaneous mutant in japonica (Oryza sativa ssp. japonica) rice cultivar Nongken 58 grown in Hubei Province, China (Shi, 1985). Large numbers of studies conducted to date have established that this novel mutant (Nongken 58S, abbreviated as 58S hereafter) possesses a number of desirable characteristics that are useful in hybrid rice development (Yuan et al., 1993). First, pollen fertility of 58S is regulated by photoperiod length: the pollens are completely sterile when the plant is grown under long-day conditions, and fertile pollens are produced when the plant is grown under short-day conditions (Zhang and Yuan, 1987). Thus, PSGMS rice can be used to propagate itself under short-day conditions and to produce hybrid seeds by interplanting it with normal fertile lines under long-day conditions. PSGMS rice, therefore, provides the opportunity for replacing the widely used "three-line" hybrids with a "two-line" system that promises to reduce greatly costs in labor, time, and resources in hybrid rice production. Second, PSGMS rice has a broad spectrum of restoration; almost all normal rice varieties restore the fertility of the F1 hybrid. Deliberately bred restorer lines are consequently not required. A further advantage is that the performance of PSGMS hybrid does not suffer from the adverse effects of male-sterile cytoplasm such as has commonly been the case with the three-line hybrids. Utilization of PSGMS rice for the development of two-line hybrids has thus become a major goal in many rice breeding programs in China and has led to the release of several hybrids that have started to gain large acreage in rice production.
There has been a large number of inheritance studies on PSGMS (Jin, 1995). The results consistently showed that fertility segregation is controlled by a single locus in crosses of 58S with its wild-type progenitor Nongken 58 (abbreviated as 58N) and a few other japonica varieties, whereas two-locus segregation appears to be the case in crosses of 58S with many other varieties. Allelism tests of 58S with a number of PSGMS lines, developed with 58S as the donor parent, indicated that the lines may differ in the loci for PSGMS (Mei et al., 1994).
Studies have also been carried out to determine the chromosomal locations of the PSGMS genes. Results from a genetic analysis based on morphological markers, in crosses with 58S as the PSGMS parent, suggested that one of the PSGMS loci is located on chromosome 5 (Zhang et al., 1990). Using molecular marker-based genetic analysis, Zhang et al. (1994) identified two PSGMS loci in a cross between `Minghui 63', an indica variety, and `32001S', an indica PSGMS line derived from a cross between `IR8' and 58S. They determined that the two loci were located on chromosomes 3 and 7, respectively, and the effect of the locus on chromosome 7 (pms1) was two to three times greater than the one on chromosome 3 (pms2). These results clearly suggested the need for further genetic analyses, especially with populations derived from 58S as the PSGMS parent, to characterize the genetic basis of the PSGMS and to clarify the loci involved in PSGMS.
In this study, we conducted a molecular marker-based genetic analysis of PSGMS using two different crosses, both of which had 58S as the PSGMS parent. The data obtained thus provided a comparison of the genetic behavior of 58S in different populations. The overall objectives of the study were to gain insights into the genetic basis of PSGMS and to determine the loci controlling PSGMS in the original mutant Nongken 58S.
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Materials and methods
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Lines and Populations
The F2 populations from the crosses of (58S x 1514) and (58S x Lunhui 422) were used as the experimental materials in this study. 58S is the original PSGMS mutant whose pollen fertility is sensitive only to day length and relatively insensitive to temperature fluctuations under natural summer conditions in a wide range of eco-geographical areas (e.g., Yuan et al., 1993). 1514 is a japonica variety developed by the Hybrid Rice Research Unit of Huazhong Agricultural University, and Lunhui 422 is a wide-compatibility variety with a broad germplasm base in its pedigree, including japonica, javanica, and indica varieties (Luo and Yuan, 1992). Large F2 populations (1000–10000 individuals, see the Results section) of the two crosses were planted in the 1994-1996 rice growing seasons in the Experimental Farm of Huazhong Agricultural University, Wuhan, China (latitude 30.5°N), where the long-day/high temperature conditions in the summer are particularly suitable for the expression of PSGMS. Seed setting rates (in some cases also pollen fertility, measured as a percentage of dark-staining pollens with iodine) of 2 to 3 panicles per plant under the natural long-day conditions were examined and used as the fertility scores.
Molecular Markers and Assay
RFLP (restriction fragment length polymorphism) markers were selected at regular intervals from the two high density maps published by the Cornell University group (Causse et al., 1994) and the Japanese Rice Genome Research Program (Kurata et al., 1994). If a selected probe failed to detect polymorphism between the parents, another probe from the same chromosomal region was added to the survey. Also, if a marker was found to be linked to a PSGMS locus, all the available probes from the same genomic region were added to the survey. Consequently, a total of 306 probes from the two maps were used in the study.
Total cellular DNA was isolated from fresh leaf tissues harvested from individual plants grown in the fields. DNA isolation, digestion, Southern blotting, and hybridization followed the methods described previously (Liu et al., 1997).
Determining Linkage between Markers and PSGMS Loci
Linkage between molecular markers and the PSGMS loci were determined following the bulked extreme and recessive class approach (Zhang et al., 1994). Two bulks, bulks F and S, were made from each of the F2 populations. Bulk F was made by mixing equal amounts of DNA from 10 highly fertile plants from the population and bulk S by mixing equal amounts of DNA from 10 highly sterile plants. The bulks and the parents were surveyed to identify possible regions containing the PSGMS loci by RFLP markers covering the entire rice genome. Markers that revealed differences between the two bulks (positive markers) were used to assay individuals in samples of highly sterile plants selected from the F2 populations (see the Results section for the sample sizes). The recombination frequency (c) between a marker locus and a PSGMS locus was calculated by the maximum likelihood estimator (Allard, 1956):
, in which N is the total number of sterile plants surveyed, N1 is the number of individuals homozygous for the RFLP band from the fertile parent (recombinant homozygote), and N2 is the number of plants heterozygous for the bands from the two parents. The variance was given by
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Results
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Fertility Segregation in F2 Populations
As expected, the PSGMS parent 58S expressed complete sterility in all plantings under the long-day conditions in the summers of Wuhan, while F1 plants of both (58S x 1514) and (58S x Lunhui 422) were highly fertile. The distribution of seed setting rates under natural long-day conditions in the F2 population (223 plants) of (58S x Lunhui 422) planted in1994 appeared to be bimodal, with a valley at 15 to 20% seed setting. A 
2 test showed that the fertility distribution in this population fit the 15 (fertile): 1 (sterile) digenic segregation ratio (P > 0.10) (data not shown). In the F2 population (1409 individuals) from the cross of (58S x 1514) planted in 1995, the fertility distribution was also in agreement with a 15:1 segregation ratio (P > 0.25) (data not shown).
Identifying Regions Containing the PSGMS Loci
Of the 306 RFLP probes used in this study, 61 detected polymorphisms between 58S and 1514, covering approximately 65% of the linkage map by assuming that the representation of a marker locus was 20 cM on each side. Ninety-five RFLP probes, covering about 88% of the linkage map, detected polymorphisms between 58S and Lunhui 422. In combination, the polymorphic markers in the two crosses covered up to 91% of the linkage map.
The survey of the bulks by RFLP probes identified 33 positive markers in the two crosses, of which 24 were from chromosome 12 (RG958, RG463, RG901, CDO344, RG543, CDO459, RZ76, RG457, RZ261, RG634, RG9, RG341, C996, C1069, G148, C87, R1709, R2708, R643, C751 G402, C1060, C2, and G2140) and 9 from chromosome 7 (RG477, RG511, RZ272, R1807, C67A, R277, C1023A, R1788, and C451). Thirty-one positive markers were identified in the (58S x Lunhui 422) cross; 22 of the 31 were from chromosome 12 and 9 from chromosome 7. In the (58S x 1514) cross, 16 and 2 positive markers were identified from chromosomes 12 and 7, respectively. These results strongly indicated the existence in both crosses of two major PSGMS loci on chromosomes 7 and 12, respectively.
To assess the possibility of the existence of the pms2 locus on chromosome 3 that was identified previously in the (32001S x Minghui 63) cross (Zhang et al., 1994), we specifically surveyed the bulks using the four markers (RG191, RG117, RG450, and RG266) that were linked to the pms2 locus determined by Zhang et al. (1994). All four makers detected polymorphisms between the parents in both crosses, none of the markers detected difference in fertility between the two bulks, indicating that the pms2 locus did not segregate between 58S and 1514 or Lunhui 422, hence was not responsible for fertility segregation in these populations.
The Identities and Locations of the PSGMS Loci
The distances between the PSGMS loci and the polymorphic markers were estimated from highly sterile individuals from the F2 population of the (58S x 1514) cross (Table 1)
. This included 52 highly sterile plants from the F2 population of 1409 plants grown in 1995 and 221 highly sterile plants from the F2 population of >10000 individuals planted in 1996.
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Table 1 Distances between marker and the PSGMS loci calculated using highly sterile individuals from F2 populations of (Nongken 58S x 1514) planted in 1995 (upper line for each marker) and 1996 (lower line for each marker)
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For the PSGMS locus on chromosome 7, the estimated distance between RG477 and the PSGMS locus is similar to that by Zhang et al. (1994) calculated using the 32001S x Minghui 63 cross. Because of the complexity of the genetic background segregation, sterility for a small fraction (<10%) of the plants in the 58S x Lunhui 422 cross did not seem to be the result of photoperiod induction. Consequently, the distances calculated from plants of this population may not be accurate and hence is not presented. However, the estimates (data not shown) clearly indicated the existence of a PSGMS between RG477 and R1807 (or RG511), a position that agreed very well with the pms1 locus identified previously in the 32001S x Minghui 63 cross (Zhang et al., 1994). We concluded that the locus identified is the same as the pms1 locus.
The locus on chromosome 12 is new and was designated as pms3, following the convention we used previously. The map distances between the pms3 locus and a number of RFLP marker loci estimated from highly sterile plants of the F2 populations of the 58S x 1514 cross grown in 1995 and 1996 agreed well with each other (Table 1). On the basis of the map distances, together with the examination of the recombinational events that occurred in the highly sterile individuals, we determined that marker loci RG543, CDO344, R1709, CDO459, R2708, and R643 were located on one side of pms3 and RZ261, C751, C2, G2140, RG9, and RG341 on the other side of pms3. A local linkage map consisting of 9 RFLP markers from this genomic region was constructed based on 246 random individuals from the F2 population of the 58S x 1514 cross (Fig. 1)
. The distances between markers in this map agreed well with those calculated from highly sterile plants (Table 1). Thus, the pms3 locus is located between R2708 and RZ261/C751 (cosegregating), at distances of 
9.0 cM from R2708 and about 5.5 cM from RZ261/C751 (Fig. 1).
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Fig. 1 The location of pms3 in the molecular linkage map of Chromosome 12. The local linkage map was constructed by Mapmaker/Exp. 3.0 (Lincoln et al., 1992) on the basis of 246 random individuals from the F2 population 58S x 1514
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Analysis of Genetic Effect of the PSGMS Loci
The effects of the two loci were evaluated based on the genotypes of the marker loci in these two regions using the random sample of 246 plants from the F2 population of the 58S x1514 cross. One-way analyses of variance (ANOVA) of pollen fertility and seed setting rates (Table 2)
showed that both pms1 and pms3 loci had highly significant effects on fertility. A two-way ANOVA, using the marker loci RG477 and RZ261 located closest to the pms1 and pms3 loci, respectively, revealed highly significant interactions between pms1 and pms3 (Table 3)
. The two loci acted as a pair of classical duplicated genes, in that it was sterile only when both loci were homozygous for the recessive alleles while all other genotypes were fertile (Table 4)
. The estimates of the genotypic values indicated that both pms1 and pms3 have major effects on fertility, although the effect of allelic substitution at the pms3 locus seemed to be slightly larger than that of the pms1 locus.
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Table 2 One-way ANOVA of the effect on male sterility for each of the two PSGMS loci in the F2 population of (58S x 1514) based on marker genotypes at each locus
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Table 3 Two-way ANOVA of the effects of the two PSGMS loci in fertility in the F2 population of (58S x 1514) based on the genotypes at the two marker loci RG477 (closely linked to pms1) and RZ261 (closely linked to pms3)
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Table 4 Fertility (%) for each of the two-locus genotypes marked by RFLPs of RG477 and RZ261 in the F2 population of (58S x 1514)
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Discussion
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The major finding of this study is the identification, in crosses involving Nongken 58S as the PSGMS parent, of two PSGMS loci located on chromosomes 7 and 12. The results raised some important issues concerning the genetic basis of PSGMS that may warrant discussion.
The first issue concerns the genetic basis of PSGMS in different lines or crosses. The PSGMS loci reported thus far have involved four chromosomal locations including chromosomes 3, 5, 7, and 12 (Zhang et al., 1990; Zhang et al., 1994; this study). This number of loci does not seem to be compatible with the results of inheritance studies that the fertility in the progeny of many crosses is conditioned by one or two major loci (Jin, 1995). Thus, apparently, PSGMS in different crosses and lines have different genetic bases, in terms of the number and locations of PSGMS loci.
Comparison of the results obtained in this study and the 32001S x Minghui 63 cross study is particularly intriguing. Although 32001S was developed with 58S as the donor for PSGMS genes, the PSGMS loci are clearly different in these two lines. The pms1 locus was detected in the crosses involving both 58S and 32001S as the PSGMS parents. However, the pms3 locus on chromosome 12 was not segregating in the 32001S x Minghui 63 cross, instead, a different locus on chromosome 3 (pms2) was involved in the fertility segregation. The results of graphical genotypes (M.H. Mei et al., unpublished data) showed that the RFLP patterns of 32001S in the pms3 genomic region is the same as its indica parent (IR8) and different from that of 58S, indicating that the DNA fragment of the pms3 region was not even transferred from 58S to 32001S. Moreover, there was significant segregation distortion in the pms2 region in the F2 population of 32001S x Minghui 63, suggesting that the sterility may somehow be related to indica-japonica hybridization, which often causes partial sterility in the hybrids (e.g., Liu et al., 1996). The differences between 58S and 32001S in the genomic locations of the PSGMS genes imply that the transfer of the complete set of the PSGMS related genes from the donor parent is not necessary for the development of new PSGMS lines.
A further complication is indicated by the result of Wang et al. (1997) who showed that the fertility segregation in the F2 of a cross between 58S and 58N was not caused by the pms1 locus, or in other words, 58S and 58N had the same allele at the pms1 locus. Thus, pms1 is not the locus of mutation that changed 58N to the PSGMS rice Nongken 58S, although this locus was detected in all the aforementioned crosses as having played a major role in controlling PSGMS.
These results have important implications regarding the nature of the PSGMS genes. It may well be that some of the loci are controlling male fertility while others specifying photoperiod sensitivity; and thus all the PSGMS loci identified so far are putative. Further studies should determine the location of the gene for the original mutation that changed the cultivar Nongken 58 to PSGMS rice 58S, and to characterizing the functions of the loci to distinguish between genes for photoperiod sensitivity and male sterility.
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ACKNOWLEDGMENTS
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We thank the Cornell University group and the Japanese Rice Genome Research Program for kindly providing the probes. This study was supported in part by a grant from the National Program for High Technology Development and a grant from the Rockefeller Foundation.
Received for publication April 10, 1998.
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ABSTRACT
INTRODUCTION
