Background
The success in hybrid rice is another great breakthrough following dwarf breeding in rice industry in China. Recently, the percentage of hybrid rice combination in hybrid and inbred regional tests in southern growing areas is increasing progressively. In mid indica rice, late indica rice and Huanan late indica rice groups, this percent has arrived more than 90% or even 100% in 2004. Besides, the percentage of early indica rice and Huanan early indica rice groups is over 80% and exceed 60% in late japonica rice group (Yang et al., 2004). Nowadays, the objective of hybrid rice breeding is far more than high yield. On condition of high production character, much attention has been focus on good quality, desirable resistance to multi-biotic or abiotic stresses and nutriment efficiency. The improvement of parents in hybrid rice is of great importance to achieve all of these goals. Both Minghui86, bred by Sanming Academy of agricultural science in Fujian province in 1994, and Shuhui527, bred by Rice Institute in Sichuan Agricultural University, are elite restorer lines in recent 10 years. Gangyou527 and Dyou527, which use Shuhui 527 as an essential restorer line, are developed and popularized to 715 and 439 million mu, respectively, ranking the sixth and eighth in 2004. In addition, II Minghui86, a combination between Minghui86 and II-32, was planted to 369 million mu, which got the tenth place in 2004 (National Agro-Tech Extension and Service Center (NATESC) 2005; Statistics table of major cultivars’ extention status of National Crop in 2004).

The span of rice growing region in China is very large, therefore hybridization requirements differ a lot due to the diversity of environment and breeding objectives. A desirable hybrid rice for extension should have both excellent characters and broad adaptability. At present, the objective of hybrid rice breeding include: high yield, good quality, permanent resistance to rice blast and all kinds of biotic and abiotic stresses in different places, including insect, drought and cold stresses, high N,P,K use efficiency, lodging resistence in Yangtze river. Conventional methods with limited parents hybridization can not satisfied high requirement of modern agricultural industry due to the long period and low efficiency.

A strategy, integrates germplasms’ efficient utilization, exploration of favorable genes with different target characters and new cultivar development, has been brought forward in the program of global molecular rice breeding by Dr Li. (Li et al., 2005; Ali et al., 2006; Lafitte et al., 2006). Using germplasms from all over the world, hybridizations are conducted to widely planted cultivars in local area. Then screening for target characters is carried out after 2~3 times backcrosses and one self-cross. Consequently, backcross introgression of favorable QTL with target characters from different cultivars will be obtained and exploration of gene/QTL will closely be combined with genetic breeding.

In this research, 95 pairs of SSR primer in rice chromosome were selected for preliminary screening and 53 pairs were chosen in view of good amplifycation and clear banding. Genetic diversity and relationship among different cultivars were investigated, by means of PCR amplification to 55 copies of rice originated from different sources, to make reference for large-scale exploration and efficient utilization of favorable gene.

1 Materials and Methods
1.1 Materials
In our study, 55 accessions of rice, originated from major rice growing regions around the world are used. These materials include 36 accessions from China, 4 from Philippines, 4 from International Rice Research Institute, 3 from India, 2 from Iran, 1 from America, from Japan, from Malaysia, 1 from Nepal, 1 from SriLanka and 1 from Indonesia. (table 1)

table 1 Materials used in present study

1.2 Genomic DNA Preparation
Tender leaves from donor materials were selected and genomic DNA was obtained using CTAB method. (Wang et al., 2003)

1.3 SSR loci selection
SSR markers were from RM series of Cornell University. PCR amplification was conducted to 55 copies of material. 95 pairs of SSR primer from rice chromosome were selected for preliminary screening and 53 pairs of SSR primer were chosen because of good amplification and clear banding. (table 2)

table 2 Analysis on diversity of SSR loci

1.4 PCR amplification and loci detection
Method described by Deng was introduced and adjusted in PCR for SSR analysis. (Deng et al., 2003) PCR instrument is MJ PTC-100. 20μL reaction system was chosen for PCR amplification, which included 25 ng total DNA, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP, 1.0 UTaq DNA polymerase and 0.1 μmol/L primer. PCR amplification procedure is listed as follows: 94℃ 5 min, 94℃ 60 s, 55℃ 30 s, 72℃ 60 s, 35 cycles, 72℃ 10 min, 2/5 proportion of STR loading buffer, degeneration at 95℃ for 5 min, immediate cooling, electrophoresis with 4% PAGE using 2000V for 1~1.5hrs and then detection with silver staining method (Panaud et al., 1996).

1.5 Data analysis
Database was set up with number “1” for the allelic variation detected and “0” for not detected. Formula proposed by Smith(1997) was used to calculate polymorphism index content (PIC): , n means the number of test materials and Pi stands for variation frequency at locus “i”. On the basis of genetic similarity, calculated as Jaccard (J), dendrogram was established with UPGMA. Both J and UPGMA were calculated by NTSYS-PC2.11.

2 Results
2.1 Genetic diversity analysis
Genetic diversity was analyzed to 55 copies of rice lines with 53 pairs of SSR primer on rice chromosome (Table 2). A total of 267 allelic variations have been detected, ranged from 4 to 7, the average allelic variation was 5.04. 7 allelic variations were examined on RM21, RM38, RM72, RM206, RM224, RM250, RM252 and RM528, varing from 0.287 to 0.786 with average of PIC value at 0.624. A total of 8 pairs of primer (RM23, RM475, RM55, RM280, RM38, RM257, RM332 and RM19) belonged to intermediate polymorphism (PIC: 0.25~0.50). While the rest were ranged to high polymorphism (PIC>0.50). PIC of RM50, RM206, RM224, RM248, RM270 and RM331 was more than 0.75. And the values of RM23 and RM280, the lowest, were 0.345 and 0.287, respectively.

2.2 Clustering analysis and genetic relationship
According to data from 53 pairs of SSR marker, clustering analysis and dendrogram were conducted to 55 accessions of rice with UPGMA. (Figure 1) With threshold value setting at 0.68, 55 copies of rice could mainly be divided into japonica rice and indica rice. Ranged from 0.614 to 0.944, the average of genetic similarity coefficient was 0.772. Varied from 0.685 to 0.996, the average coefficient was 0.778 for 14 accessions of japonica rice.

figure 1 Rice genetic similarity UPGMA dendrogram

Similarity coefficient between two recurrent parents and 53 accessions of donor parents was summaried in Table 3. It indicated that the similarity coefficient between Minghui86 and 53 accessions of parents was 0.655~0.850. The coefficient between Minghui86 and 14 copies was less than 0.7 (including 0.7). The coefficient between Minghui86 and either Hangxiang10 or Lemont was 0.655. And the value between Minghui86 and Teqing was 0.850, which was the maximum. Additionally, the similarity coefficient between Shuhui527 and 53 accessions of parent ranged from 0.640 to 0.873. Among them, the coefficient between Shuhui527 and 12 accessions was less than 0.7 (including 0.7). The coefficient that the 12 accessions showed to Hangxiang10 and Han 4 was 0.640. The value of IR65600-27-1-2-2 and Minghui63 was 0.873, which was the maximum. Majority of similarity coefficients between two recurrent parents and japonica rices were relatively low and that of indica rices were high. Similarity coefficients between Shuhui527 and Indica rices were generally higher than that of Minghui86. On the other hand, similarity coefficients between Shuhui527 and Japonica rices was commonly lower than that of Minghui86.

table 3 Similarity coefficient matrix between recurrent and donor parent

3 Discussion
Minghui86, an elite restorer line, was bred through individual selection and testcross for eight generations during six years between P 18 (IR54/Minghui63∥IR60/Gui630) and gk148 (Japonic187/IR30∥Minghui63) (Zhang et al., 1996). Shuhui527 which is a key restorer line, was bred between 1318 and 88-R3360 (Wang et al., 2004). Both of them are Indica restorer lines with good combining ability and strong restoring ability. In present study, an average of 5.04 allelic genes were detected in each pair and the diversity index was 0.624, which were generally higher than others. The similarity coefficients between two recurrent parents and Japonic rices were low and that with Indica rices were relatively high. The similarity coefficients between Shuhui527 and Indica rices were generally higher than that between Minghui86 and Indica rices. This result could be verified with breeding pedigree of two recurrent parents. The similarity coefficients in this study are relatively high, which is consist with recent reports (Duan et al., 2001; Huang et al., 2006).

Using SSR molecular markers, the result obtained from UPGMA clustering indicated test materials could be divided into japonic and indica rices. Pusa, Domsiah, Milagrosa and BJ1 differed a lot from other cultivars. Genetic diversity and relationship could be tested through similarity coefficients at molecular level. SSR polymorphism detection could be used as one of efficient and accurate method for genetic diversity study. It will provide important information for identification of cultivars and construction of core germplasm. In addition, this research will provide important theoretical basis for the future study of rice genetic evaluation and parents’ selection.

Genetic diversity and relationship analysis makes efficient references for large-scale exploration of favorable gene/QTL. The objective of this research comprise of enlarged genetic basis for recurrent parents, efficiently resistance to all kinds of biotic and abiotic stresses and development of newly-bred cultivars with desirable characters. Nevertheless, genetic relationship is not the only criterion for selection of parent plants due to the difference in the types of population and breeding targets. Hybridization, between dwarfing plants with good performance and another elite with the same or similar ecotype but different relationship, usually perform well, which is a basic experience for selection of parents in indica dwarfing breeding in China. (Pang et al., 1994) In general, it can be more fast to develop a promising variety using elite parents with close relationship due to good characters and more favorable genes. However, agriculture productivity demands comprehensive varieties, which push on enlarged genetic basis with more favorable genes. Obviously, combination between cultivars from distant relationship will easily get seperation, thus less individuals with good characters will be obtained, hereby the conventional hybridization can’t work well. Forturately, the conflict could be solved with advanced backcross introgression, proposed in the program of global rice molecular breeding in 1998, by introducing favorable genes into the same genetic background from closely related varieties, distant subspecies or even wild varieties. Subsequently, the promising varieties can be developed by pyramiding sister lines with favorable targeted genes from different donors but similar genetic background (Li et al., 2005). This research demonstrated desirable cultivars with similar relationship could be used for improving recurrent parents, which will contribute to hybridaztion success in a short period. On the other hand, those with bad characters and distant relationship could be used for backcrosses for several times to explore specific favorable genes. Furthermore, the ingrossed lines with different favorable genes can be pyramid to an elite genetic background of recurrent parents to develop novel restorer lines with great improvement in several targets traits.

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