Bot. Bull. Acad. Sin. (2003) 44: 285-289

Huang et al. — Fine mapping of the nuclear fertility restorer gene

Fine mapping of the nuclear fertility restorer gene for HL cytoplasmic male sterility in rice

Jingyu Huang1, Jun Hu1, Xin Xu2, Shaoqing Li1, Ping Yi1, Daichang Yang3, Fugang Ren1, Xuequn Liu1,.2, and Yingguo Zhu1,*

1The Key Laboratory of Ministry of Education for Plant Developmental Biology and Institute of Genetics, College of Life Sciences, Wuhan University, Wuhan, 430072, P. R. China

2College of Life Sciences and Chemistry, South-Central University for Ethnic Communities, Wuhan, 430074, P. R. China

3Ventria Bioscience 4110 Freeway, Sacramento, CA 95834, USA

(Received December 2, 2002; Accepted May 16, 2003)

Abstract. Bulked segregant analysis (BSA) of a BC1 population derived from congguang 41A/MiYang 23//congguang 41B was used to map the nuclear fertility restorer gene Rf5 for HongLian (HL) cytoplasmic male sterility. The parents and two bulks representing extremely fertile and sterile plants, respectively, were screened for polymorphism with 20 microsatellite primer pairs on chromosome 10, chosen on the basis of previous research. MRG4456 is linked to the fertility restorer gene Rf5 at a distance of 1.57 cM, and another newly developed microsatellite primer, HL01, was locked at a distance of 0.63 cM to Rf5. Closely linked DNA markers will facilitate not only breeding but also the purity management of hybrid seeds. Concrete steps for developing new microsatellite markers using the rice whole genomic sequence database are also described.

Keywords: Fertility restorer gene; Marker-assisted selection; Microsatellite marker; Oryza sativa L.


The phenomenon of cytoplasmic male sterility (CMS), described as the inability of a plant to produce functional pollens, has been observed in over 150 plant species (Wise et al., 2002). Fertility restorer (Rf) genes in the nuclear genome can counteract this inability (Newton, 1988) and restore fertility to cytoplasmic male-sterile plants. Hybrid breedings based on CMS/Rf systems have achieved great success all over the world. Aside from its commercial exploitation, CMS offers a rare opportunity to examine the regulation of mitochondrial genes by nuclear genes in multicellular organisms.

Three primary types of CMS in rice are now known, and their heritance habits and physiological characteristics have been extensively investigated. They are Wild-rice abortive (WA), BaoTai (BT) and HongLian (HL). WA type CMS belongs to sporophytic abortion, which fails to produce normal pollen and finally forms typical abortive pollen. In contrast, BT (japonica.) and HL type CMS (indica.) belong to gametic abortion, but they are also greatly different in terms of abortive phenotype, relationship of restoration, and maintenance.

CMS systems are usually attributed to chimeric ORFs in the mitochondrial genome (Kempken and Ping, 1998; Schnable et al., 1998; Szklarczyk et al., 2000). These ORFs encode novel proteins, which often interfere with mitochondrial function and pollen development. In many instances, the restorer gene suppression of CMS is directly associated with Rf-gene-dependent mitochondrial RNA modification and concurrent reduction of CMS associated protein (Schnabel et al., 1998). Although many mitochondrial genes associated with CMS have been cloned, only two Rf genes have been isolated until now. One is the maize Rf gene named Rf2, encoding aldehyde dehydrogenase (ALDH) (Cui et al., 1996; Liu et al., 2001); The other is Petunia Rf gene, encoding a mitochondrially targeted protein comprised of pentatricopeptide repeat (PPR) motif (Bentolila et al., 2002).

The inheritance of fertility restoration in WA type CMS has been extensively investigated. Most investigators tended to agree that restoration of WA type CMS is controlled by two nuclear genes (Rf3, Rf4) and that their chromosomal loci have been resolved (Zhang et al., 1997; Yao et al., 1997; Tan and Trangoonrang, 1998; Zhang et al., 2002). BT type CMS is restored by nuclear fertility restorer gene Rf1, which was mapped on chromosome 10 (Fukuta et al., 1992; Akagi et al., 1996;Yokozeki et al., 1996). HL type fertility restoration gene Rf5 was also mapped on chromosome 10, 7.8 cM from RM258 (Huang et al., 2000). The studies reported here were undertaken to finely locate the nuclear fertility restoration gene Rf5 for HL type CMS.

*Corresponding author. Tel: 86-27-87876530; Fax: 86-27-87876530; E-mail:

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

repeated di-to tetra-nucleotide motifs with software. Polymerase chain reaction (PCR) primer pair consists of 17-22 nucleotides, each with a GC content around 50% (Tm Ca. 60°C), which meets the criteria of a low frequency primer-dimer, and it is preferably G-or C-rich at the 3'end. Generally, PCR product ranges between 100-250 bp.

Genetic Mapping by Microsatellite Markers

We used 20 microsatellite primer pairs (Table 1) near microsatellite marker RM258 in this study. PCR followed the procedure described by Huang (Huang et al., 2000). The PCR reaction was carried out on a PTC-100 DNA Thermal Cycler. Amplification products were analyzed with regular 4.0% agarose gel. For polymorphic microsatellite markers, the PCR products were analyzed by running a 4.0% denaturing polyacrylamide gel.

RFLP Analysis

Southern-blotting analysis followed the standard procedure described by Sambrook et al. (1989). RFLP marker probes were kindly provided by Dr. Li Zhikang (International Rice Research Institute). The genetic data was analyzed with Mapmaker software (3.0) following the instructions provided.


Identification of Microsatellite Markers Linked to Rf5

DNA from Congguang 41A, Miyang 23, a DNA bulk of 15 male fertile plants, and a DNA bulk of 15 male sterile plants from the BC1 population were used as the template for PCR analysis. 4 out of 20 primer pairs generated the

Materials and Methods

Mapping Population Development and Fertility Evaluation

A male sterile line of HL type: Congguang 41A and male sterile restorer line responding to HL type: Miyang 23 were used as parents to generate F1. The F1 was then backcrossed to Congguang 41B to generate a BC1. The BC1 population, consisting of 1023 individuals, was used as a mapping population for Rf5 gene. Pollen fertility in BC1 population and seed setting were evaluated at the flowering and maturity stages, respectively. For seed setting evaluation, the panicles were covered before flowering by a paper bag to prevent them from pollinating each other. Sterile plants contained less than 5% stainable pollen and produced no fertile seed. All others were treated as fertile. The segregation ratio of fertile to sterile plants was evaluated in the BC1 generation based on the standard mentioned above. If individual lines contained less than 1% stainable pollen and their seed setting rates of bagged panicles were zero at maturity, they were considered as excessive male sterility lines.

Construction of the Bulks

Genomic DNAs were prepared as described by Huang (Huang et al., 2000) from the parents and from individual plants in the BC1 population. Equal amounts of DNA from male fertile and male sterile individuals were pooled to generate a male fertile and a male sterile bulk, respectively.

Microsatellite Marker Development

Microsatellite DNA markers were produced by searching the rice whole genomic sequence database for simple

Huang et al. — Fine mapping of the nuclear fertility restorer gene

amplicon polymorphism between parents. The frequency polymorphism generation using bulk analysis for microsatellite markers was 20.0%.

Genetic Linkage Analysis of Rf5 Restorer Gene Via Microsatellite Markers Using the BC1 Population

The segregation ratio of fertile to sterile plant was about 1:1 (443:580), which shows only one restorer gene exists in Miyang 23. In order to estimate the genetic distance between microsatellite marker and restorer gene Rf5, 158 excessive male sterility lines were used for genetic mapping analysis via microsatellite markers. Two out of 158 individual lines exhibited a genetic recombination when using a microsatellite marker HL01. For microsatellite markers MRG2510 and RM171, 3 and 7 individual lines showed the genetic recombination respectively; On the other side of Rf5, five individual lines showed a genetic recombination when using a microsatellite marker MRG4456 (Figure 1). The genetic linkage analysis revealed that the two closed microsatellite markers, HL01 and MRG4456, flanked the Rf5 gene locus with a genetic distance of 0.63 cM and 1.57 cM, respectively.

Genetic Fine Mapping of Rf5 Via RFLP Markers Using the BC1 Mapping Population

To further confirm chromosome location of the restorer gene Rf5 in rice genome, RFLP markers from the region surrounding the restorer gene Rf5 on chromosome 10 were selected for a parental polymorphism survey based on previous results (Huang et al., 2000). Two RFLP markers, G2155 and S10019, revealed polymorphism between the parents (data not shown). The results showed that G2155 and S10019 linked to the Rf5 locus with a genetic distance 6.1 cM and 2.7 cM, respectively, and the two RFLP markers are located in the same direction as the Rf5 locus on the long arm of chromosome 10. However, the microsatellite markers HL01 and MRG4456 flanked the Rf5 locus, showing the physical order on chromosome 10 as HL01-Rf5 -MRG4456-S10019_G2155 (Figure 2). These results will accelerate the steps of isolating Rf5 gene by a map-based cloning strategy.

Figure 2. The location of the restorer gene Rf5 identified by SSR and RFLP markers. Markers' names are listed to the right of chromosome, and their positions (in cM) are on the left.


Selection of BC1 Population

HL type CMS belongs to gametic abortion, whether pollen is fertile or not is determined by its own genotype. This is different from sporophytic abortion, in which pollen's fertility is determined by sporophytic genothype. The pollen fertilities in F1 hybrid plants differ in their CMS, 50% have gametophytic and 100% have sporophytic cytoplesmic male sterility. Therefore, a backcross population was used here instead of an F2 population, which is widely used in mapping the genes involved in sporophytic abortion.

Multi-Loci of Fertility Restorer Genes

This study has finely located the nuclear fertility restorer gene for the HL type CMS on Chromosome 10. Interestingly, the nuclear fertility restorer gene Rf1 for BT type CMS has been located on chromosome 10, 3.7 cM from OSR33 (Akagi et al., 1996). Subclone Y3-8 from rice YAC clone Y4892 of RGP (Rice Genome Program) was mapped to the Rf4 gene locus with a genetic distance of 0.9 cM, and YAC clone Y4892 was anchored to the RFLP marker S10019 (Zhang et al., 2002). Comparing various molecular linkage maps suggests that three genes are located in the adjacent region on chromosome 10. Just like the disease resistant genes (Grog et al., 1993; Mahadevappa et al., 1993; Ellis et al., 1995), the restorer genes may be clustered on chromosome 10 (Li et al., 1998).

Analysis of Microsatellite Markers

Microsatellite markers combine the rapidity, straightness, and simplicity of RAPD with the stability and reliability of RFLP. They also often detect more allelic variation than RFLP or RAPD markers (Panaud et al., 1996).Additionally, it has been discovered that simple sequence

Figure 1. Amplication profiles obtained with the microsatellite primer pairs MRG4456 are revealed on 6.4% denaturing polyacylamide gel. M, DL2000 marker; s: Congguang 41A; f:Miyang 23; Bf: fertile bulks; Bs: sterile bulks; 1-7: individuals of BC1 population; 4 is recombinant.

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

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repeat content for rice 93-11 occupys 1.7% of its whole genome (Yu et al., 2002) which proves the microsatellite marker's abundance and wide distribution in the rice genome. Therefore, microsatellite markers are the tool of choice in the genetic mapping of rice. Actually by designing new microsatellite markers in the target region, we have got the co-segrative microsatellite marker Rf5 (unpublished data).

The results presented here indicate that microsatellite marker HL01 and MRG4456 will facilitate marker-assisted selection (MAS) of restorer lines in the CMS-HL system, which will promote the development of hybrid rice. Construction of the physical map encompassing the Rf5 gene locus is underway. Our ultimate goal is isolating the Rf5 gene through a map-based cloning strategy.

Acknowledgements. The author would like to thank Dr. Fu Bingyin for his excellent technical assistance. This work was supported by the National Program on Basic Research. (Grant No. 2001CB108806).

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Huang et al. — Fine mapping of the nuclear fertility restorer gene