CMS (Cytoplasmic Male Sterility) line is the base of hybrid rice. At present, Japonica hybrid rice cytoplasm sterile is Chinsurah Boro II type (BT-type) planted large area on the production in the north of China. BT-CMS is gametophyte sterility, abortion period late, self fruitfulness at the high temperature; therefore fertility is not stable enough. Using BT-CMS lines in Japonica hybrid rice seed production, once the preservation measures is adverse, it is vulnerable to be biological confounding, mechanically mixed, seriously affected the purity of hybrid rice. Nowadays, most BT-CMS existing the problem is low outcrossing rates, low yield of seed, as well as poorer purity and quality. Reducing the risk of genetic vulnerability caused by single hybrid rice CMS cytoplasmi is also the main direction to be focused on in recent years. Therefore, the diversification requirements of cytoplasmic male sterile types become more and more obvious.

The single set sterile of selfing bag in Japonica YS-CMS type cytoplasmic male sterile breeded by Rice Research Institute of Liaoning Province is 100%, abortion period is single nucleus stage, pollen types is the typical abortion and the fertility is stable. Compared with BT-CMS lines, YS-CMS lines of Japonica hybrid rice have a wide application prospect. But the lack of support with the restoration, so YS-type restorer lines in Japonica rice breeding efficiency has become a serious problem (Chen et al., 2008; 2009).

As a result of the same period in Indica rice YS-CMS lines and f WA-type male sterility abortion, the restoring and maintaining relationship was similar. Research results showed that the fertility restorer of Indica type sterile lines were controlled by two pairs of major genes, one was located in the middle of the long arm on the tenth chromosome (Yao et al., 1997; Tan et al., 1998; Zhang et al., 2002), named as Rf4, and the other restorer gene Rf3 was located on the first chromosome (Zhang et al., 1997). Theoretically, we can screen YS-type Japonica restorer lines from WA-type restorer line in rice, but WA-type restorer line wasn’t in Japonica rice. Furthermore, there was reproductive isolation between YS-type Japonica CMS line and Indica rice restorer lines (Li, 2009), which was restricted the application of YS-type Japonica CMS line.

Molecular marker-assisted selection, a tool for crop improvement, makes up for the traditional selection techniques for low accurate rate in crop breeding. The basic principle is to screen the target gene or the whole genome from the selected individuals by using the molecule markers which was tightly linked or co-separation. Consequently, it can reduce the chain cumbersome to obtain the desired individual and to improve the breeding efficiency. Using the restorer gene linkage the target gene of candidate plant with marker-assisted selection, we chose the plants with these two restorer genes. In the case of existing these two fertility restorer genes, molecular markers detection showed that the offspring seed-setting rate is highest, more than 60%; whereas, when these two genes was absent, the offspring of seed-setting rate was only 20%, the correlation between genotype and phenotype was very high. Studies have shown that the selection result was reliable by using SSR markers RM10353 and RM6100 on Rf3, Rf4 (Qi et al., 2008).

Therefore, we employed the Japonica rice material with wide compatibility and WA-type restorer line crossing using molecular marker-assisted selection of RM10353, RM6100 to breed Japonica rice restorer lines, which can be the restorer lines of restor the Japonica rice YS-CMS. In this paper, we discussed the application of technique f molecular marker-assisted selection for breeding restorer lines.

1 Result and Analysis
1.1 Results of molecular marker-assisted selection
Using molecular markers RM10353, RM6100 for the homozygousp in Japonica rice wide compatibility materials with Wanlun 422 and Indica WA-type restorer line Shuhui 527, Miyang 42, Miyang 46, the amplification product was about 180 bp and 160 bp, respectively. Their hybrid offspring F₂, F₄ restorer genes were detected by these two molecular markers (Table 1; Figure 1). In accordance with the requirements on the selection, the agronomic traits of plants were investigated, when both were containing Rf3, Rf4 gene markers. We selected the plant based on the plant type, seed-setting rate, as well as took the reference condition for the gametophyte generation resistance the premature aging, disease and insect resistance. The selected plants were crossed with the sterile lines to carry out the fertility restoration in the field experiments.

Table 1 Marker test results of restorer genetic in F2, F4 groups

Figure 1 Two pairs of marker primers for PCR amplified electrophoresis of restorer gene

1.2 Detection results of testcross and the fertility of pollen
The number of 113 pairs in testcross was obtained in winter of 2008 in Hainan, and the gametophyte generations F₁ were planted in Shenyang in 2009. The bagged selfing seed-seting rate of was more than 60%, which was 26.6% in the total combinations (Table 2). The pollen fertility was positive correlation with the seed-setting rate and its correlation coefficient was 0.98. The gametophyte generation agronomic trait comprehensive investigation showed that the heterosis was obvious (Figure 2) and the grain quality displayed better appearance (Figure 3) in later autumn.

Table 2 Test results of seed-setting rate and pollen fertility of measurement gamete generation

Figure 2 Performance of plant heterosis in gamete generation

Figure 3 Performance of the appearance quality of rice in gamete generation

Testcross combinations derived from “Wanlun 422×Shuhui 527” as the paternal parent, 40 plants, 4 F₂ lines, among them, the fertility of 10 combinations in 1 lines was stability, whereas, the fertility of 30 combinations the other 3 strains was separated. The results showed that there were 1 homozygous 3 heterozygous in the F₂ molecular marker genotype. On the other hand, from “Wanlun 422×Miyang 42” as the paternal parent, 38 plants, 4 F₂ lines, of them, the fertility of 10 combinations in 1 linens was stable, when the fertility of 28 combinations in the other 3 lines was separated. The results show that there were 1 homozygous 3 heterozygous in the F₂ molecular marker genotype. “Wanlun 422×Milyang 46” as the male paternal, 35 plants, the fertility separated in 35 combined of 4 F₂ lines, which showed that the F₂ molecular marker genotype was heterozygous (Table 3).

Table 3 The fertility results of gamete generation

The results showed that we can quickly, accurately found the restorer gene in plants by using molecular markers, we can select the applied plants under strict agronomic traits, linking with the other breeding methods to verify, which can make the restorer gene expressed more accurately.

2 Discussion
There are some research progresses in rice breeding such as resistant cultivars (Cao et al., 2003; Zhang et al., 2009), quality improvement (Li et al., 2004; Xia et al., 2004), restorer line breeding (Chen et al., 2009; Xue et al., 1998) in china by using marker-assisted selection. With the rapid development of molecular biology technology, more and more important agronomic traits in rice genes were localized, some tightly linked molecular markers of these traits have been screened. Adopting these molecular markers for indirect selection, we will get a multiplier effect. However, so far, there are still little new rice varieties (lines) developed via molecular marker-assisted selection. Considering the reasons, maybe one is that the main effect gene is not much among the important traits in the positioned rice, and the genes for auxiliary selection are limited, the distance between many genes positioned and their linked molecular markers is too large to be used for help. And the other is that the studies of gene mapping and breeding is out of touch, the purpose of many researchers is only to locate the target gene, which has some differentiae in breeding practice (Wang et al., 2009). Therefore, to find the practical combination is the main aspect of this research.

It is a sample for combining the conventional breeding with molecular marker techniques for selecting the restorer lines in Japonica rice in this study. Using the different marker polymorphism for molecular marker-assisted selection, although it wasn’t the marker of the gene itself, the linkage distance is closer, which presented some persuasive, and the selection effect is good. In recent years, with the deepening of the research on different cytoplasmic male sterility type of CMS line, breeding requirements of whose ancillary restorer line is getting higher and higher, especially in the present study, YS-CMS Japonica rice restorer lines has become a key select object, in the course of the experiment, although the combination of generation is low, the degree of separation is larger, it does not affect restorer selection.

In this study, the results showed that YS-type cytoplasmic male sterile line overcame the defects of the existing BT-CMS line, which is a new attempt in Japonica rice CMS research in the north of China. We selected supporting restorer line, which can enhance the heterosis in YS-type cytoplasm Japonica hybrid rice, and it can be applied to production.

3 Material and Methods
3.1 Plants material
We selected the wide compatibility Japonica rice materials Wanlun 422 as maternal parent, and Indica rice WA-type restorer lines Shuhui 527, Miyang 42, Miyang 46 as paternal parent for crossing. They generated F₂-F₄ generations respectively.

3.2 Establishment and cultivation of generations
The tested materials were planted in Rice Research Institute of Liaoning Province experimental field in 2008 and 2009. The seeds were planted on April 13 to 15, transplanted on May 20 to 25 when the seedling was 1 meters long, each strain was 5 to 10 rows, spacing at 30 cm×13.3 cm. The field management was according to conventional method. 10 strains were planted in each parental material of Wanlun 422, Shuhui 527, Miyang 42, Miyang 46, and 100 strains were grown in F₂ generations of Wanlun 422×Shuhui 527, Wanlun 422×Miyang 42, Wanlun 422×Miyang 46, respectively. We detected the restorer genes with agronomic trait selection of single plant by using molecular marker-assisted. F₃ generation was grown continually in winter of 2008 in Hainan, 4 lines of each combination, 50 strains in each line. We chose 10 strains from each line to do the testcrossing with the breeding 8 generation and the stable Japonica rice YS-CMS line, respectively, a total of 120 pairs of combination (Figure 4).

F₁ generations were planted in spring of 2009 in Shenyang, 20 strains in each combination. When heading, they were bagged selfing for calculating the seed-setting rate. At the same time we planted 55 strains of paternal parent (F₄), and were paratactically planted with the gametophyte generation. And then selected 11~12 strains the single row (Figure 4) in F₄ generation. According to the calculation, the homozygous genotype probability in F₄ generation plant is approximately 1/5. The hang tag for molecular detection was carried out based on the order, and elected per plant of selection for agronomic traits. After the gametophyte generation heading, we adopted the pollen staining with 10 lines in each combination, each line with 3 visual fields, 150~200 pollen particles in each visual field, and then calculated the average number for verifying the seed-setting rate. According staining the reaction differentia of the pollen grains to 1% I-KI, the pollen were divided into 4 kinds, the deformity without staining was typical abortive, circular no staining was round defeats, circular shallow staining was stained abortion and circular deep staining was normal fertile, respectively.

Figure 4 Process of the restorer selection by MAS

3.3 DNA extraction
The experimental materials were cultivated in Rice Research Institute of Liaoning Province experimental field, after transplanting for 30 days, DNA from leaves were extracted by using the CTAB method. Taking 10 parents, the F₂ generation for each combination a total of 163 plants in 2008, as well as a total of 1265 plants from the F₄ group of each combination in 2009, we hang tags on each plant sequentially.

3.4 PCR amplification and electrophoresis validation
Two pairs of primers used in SSR analysis, named as RM10353, RM6100 from http://www.gramene.org/, were synthesized from Shanghai Biological Engineering Company. RM10353 used for identification of Rf3 is located on the first chromosome and linkage distance with the target gene is 6.79 kb. RM6100 used for identification of Rf4 is located on the tenth chromosome, and linkage distance with the target gene is 2.4 cM. Primer sequence and their name were shown in Table 4.

Table 4 Primer sequence and their name

The volume of PCR reaction system was 20 μL reaction, including Mg²⁺ (25 mmol/L) 2.0 μL, PCR Buffer (no Mg²⁺) 2.0 μL, dNTP (10 mmol/L) 0.3 μL, each primer (15 ng/μL) 2.0 μL; template DNA (about 15 ng/μL) 1.4 μL, Taq enzyme (10 U/μL) 0.15 μL, double-distilled water sterilization 12.15 μL.

PCR reaction were carried out in the Biometra PCR instrument, and reaction conditions are as follows: 94℃ degeneration for 5 min, and then 94℃ 1 min, 55℃ 1 min, 72℃ 1.5 min, a total of 32 cycle, at last, 72℃ extension for 10 min. the PCR products were stored at 4℃.

Authors’ Contributions
XJW performed the experiment and wrote the manuscript. XLT and GJS conducted experimental design and modified the manuscript. HM, YJC, CZ, and WQS took part in the experiment. All authors have read and approved the final manuscript.

Acknowledgment
This project was supported by Liaoning Province Science and Technology Reserch (2006201002) and Liaoning Province Science and Technology Fund (20052129).

References
Cao L.Y., Zhan X.D., Zhuang J.Y., and Cheng S.H., 2005, Breeding of Indica hybrid rice Guodao 1 with good quality, high yield and resistance to bacterial leaf blight by molecular marker-assisted selection technique, Zajiao Shuidao (Hybrid Rice), 20(3): 16-18

Chen J.M., Fu Z.Y., Quan B.Q., Tian D.G., Li G., Wang F., 2009, Breeding hybrid rice restoring line with double resistance to rice blast and bacterial blight by marker-assisted selection, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 7(3): 465-470

Chen Y.J., Ding F., Wang X.J., Zhang C., Shao G.J., and Qiu F.L., 2008, Selection and study on Yinshui Type Japonica sterile rice(I), Beifang Shuidao (North Rice), 38(1): 15-19

Chen Y.J., Ding F., Wang X.J., Zhang C., Shao G.J., and Qiu F.L., 2009, Selection and study on Yinshui Type Japonica sterile rice(II), Beifang Shuidao (North Rice), 39(1): 6-9

Li G.X., Yao F.Y., Zhuang J.Y., Hou H.J., and Jiang M.S., 2006, Inheritance of fertility restoration and M olecular mapping of restoring genes of CMS in rice, Zajiao Shuidao (Hybrid Rice), 21(3): 1-6

Li H.J., Li P., Gao F.Y., Lu X.J., and Ren G.J., 2004, Improvement of amylose content of G46B by SSR marker-assisted selection, Zuowu Xuebao (Acta Agronomica Sinica), 30(11): 1159-1163

Li J.Y., 2009, Breakthrough progresses on rice hybrid sterility between Indica and Japonica contributed by Chinese scientists, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 7(1): 1-4

Qi F.L., Jiang M.S., Yuan S.J., Yao F.Y., Gao J., and Li G.X., 2008, Mapping of fertility-restoring gene Rf3 for wild-abortive cytoplasmic male sterility in rice, Zhongguo Nongxue Tongbao (Chinese Agricultural Science Bulletin), 24(8): 114-1117

Tan X.L., Vanavichit A., Amornsilpa S., and Trangoonrung S., 1998, Genetic analysis of rice CMS-WA fertility restoration based on QTL mapping, Theor Appl Genet., 97(5-6): 994-999 
http://dx.doi.org/10.1007/s001220050983

Wang C.L., Chen T., Zhang Y.D., Zhu Z., Zhao L., and Lin J., 2009, Breeding of a new rice variety with good eating quality by marker assisted selection, Zhongguo Shuidao Kexue (Chinese J. Rice Sci.), 23(1): 25-30

Xia M.Y., Li J.B., Zhang J.H., Wan B.L., and Qi H.X., 2004, Breeding of early Indica rice with intermediate amylose content by molecular marker-aided selection, Huazhong Nongye Daxue Xuebao (Journal of Huazhong Agricultural University), 23(2): 183-186

Xue Q.Z., Zhang N.Y., Xiong Z.F., Li Y.Z., and Zhu L.H., 1998, The development of the rice restorer lines with the resistance of the bacterial biight disease by the M arker assisted selection, Zhejiang Nongye Daxue Xuebao (Journal of Zhejiang Agricultural University), 24(6): 581-582

Yao F.Y., Xu C.G., Yu S.B., Li J.X., Gao Y.J., Li X.H., Zhang Q.F., and Zhang Q., 1997, Mapping and genetic analysis of two fertility restorer loci in the wild-abortive cytoplasmic male sterility system of rice (Oryza sativa L.), Euphytica., 98(3): 183-187
http://dx.doi.org/10.1023/A:1003165116059

Zhang G., Bharaj T.S., Lu Y., Virmani S.S., and Huang N., 1997, Mapping of the Rf-3 nuclear fertility-restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers, Theor Appl Genet., 94(1): 27-33
http://dx.doi.org/10.1007/s001220050377 PMid:19352741

Zhang H.G., Li B., Zhu Z.B., Cui X.F., Tang S.Z., Liang G.H., and Gu M.H., 2009, Improving resistance of a good-quality Japonica Variety Wuyujing 3 to rice stripe virus via molecular marker-assisted selection, Zhongguo Shuidao Kexue (Chinese J. Rice Sci.), 23(3): 263-270

Zhang Q.Y., Liu R.G., Zhang G.Q., and Mei M.T., 2002, Molecular marker of the fertility restorer gene Rf-4 for WA cytoplasmic male sterility in rice, Yichuan Xuebao (Acta Genetica Sinica), 29(11): 1001-1004