Blast is one of the most important rice diseases in the world. Identifying and mining the resistance genes might be important foundational work in the breeding program. Chen and research team collected 7l5 rice blast from the South Central China region, and the virulence were determinated by inoculated the rice blast on the donor parent C101LAC of Pil gene and found that for the parent C1O1LAC, the frequency of virulence to the rice variety C101LAC was 10.35%, which indicated that C101LAC (harboring Pi1) were resistance to most of physiologic races of rice blast fungus (Pyricularia grisea (Cooke) Sacc.) in Chinese (Chen et al., 2001). In Yunnan province of China, Utilization value of 20 rice resistance gene have evaluated, and the results shows that seven vertical resistance genes Pi9, Piz-5, Pil, Pita-2, Piz, Pik-h and Piz-t showed higher resistance, and those resistance gene would be employed in indica cropping region of Yunnan; Pi9, Piz-5, Pi1, Pita-2, Piz, Pik-h, Piz-t, Pi12, Pita and Pib would be employed in japonica-growing-region; Pi9, Piz-5, Pil, Pita-2 can be employed in indica and japonica cropping region in Yunnan province of China (Li et al., 2005). The above results showed that Pi1 has broad-spectrum resistance in Yunnan, and can be used widely. The identification and utilization of Pi1 is significance for blast resistance breeding program.

The resistance gene of Pi1 is a main effect dominant blast resistance gene (Hittalmani et al., 2000), and is located on the end of long arm of chromosome 2, the genetic distance from RFLP markers RZ536 to Pi1 was 11 cM (Yu et al., 1991), and the genetic distance from SSR markers RZ536 and NpB18l to Pi1 were 7.9 cM and 3.5 cM, respectively (Mew et al., 1994). Further analysis found that the genetic distance from the RFLP marker RZ536 to Pi1 was 9.7 cM, and between SSR markers RM144 was 6.8 cM. While the genetic distance between the rice SSR markers RM224 and RM144 was 5.7 cM (Liu et al., 2003b). By estimate, the physical distance between Pi1 gene and SSR markers RM144 and RFLP markers RZ536 were 57 kb and 72 kb, respectively. The genetic distance from SSR marker MRG4766 to Pil was 1.3 cM (Chen et al., 2005). Using the above three of closely linked SSR markers of rice blast resistance genes Pi1, the identification of Pi1 had been detected among 49 rice varieties (lines) collected from Heilongjiang Province, and cleared the distribution of Pi1 in the major rice cultivars in Heilongjiang Province (He et al., 2006). SSR markers of Pi1 gene used as a tool in molecular-assisted breeding, and found the results have better accuracy (Yao et al., 2010).

Pi1 gene molecular marker assisted selection (MAS) has been effectively applied in breeding program. Three rice blast resistance genes (Pi1, Pi2, Pi4) has polymerized via MAS technology by Yang et al (1994). Hittalmani et al (2000) successfully polymerizatied three blast resistance genes Pi1, Pi5, Pita by used of MAS technology. Liu et al (2003a) polymerizated the Pi2, Pi3, Pil, Pi3, the resistance was confirmed by inoculation of rice blast fungus, and found that polymerization genes can effectively enhance the rice resistance to rice blast fungus.

There were collected and conserved about 6,000 rice resources in Yunnan Province, among these ancient landraces, some of them were continuous planted near one century in the same area, and still showed resistance to rice blast (Liang et al., 2001). In Yunnan Province, topography, climate and cultivation methods are various, as well as the physiological rice of rice blast fungus is diverse. The rice varieties and the rice diseases were co-evolution in fields, there may be harboring the novel resistance genes and durable resistance to rice blast fungus in landraces (Liang et al., 2001). 

In this paper, we will identify the Pi1 gene by using the SSR markers MRG4766 linked with Pi1 gene of landraces collected in Yunnan, to make clear the distribution of Pi1 gene in Yunnan province of China, and provide the useful information for mining and utilization of Pi1 gene in rice blast resistance breeding and production. 

1 Result and Analysis
1.1 Genotype of Pi1 detected by molecule marker and the confirmation of resistance
The genomic DNA of 173 landraces were PCR amplified by using SSR markers MRG4766, the PCR products were separated by 8% non-denaturing polyacrylamide gel electrophoresis, and developed by silver staining after electrophoresis. PCR validation results showed that bands with different fragment size can be distinguished by polyacrylamide gel electrophoresis (Figure 1), the susceptible variety Lijiangxintuanheigu without Pi1 gene can be amplified a small fragment (Figure 1, lane 1). The results were the same with the previous research by Chen et al (2005), the PCR products of the landraces were the larger fragments containing Pi1 gene, whereas, that of the landraces were the small fragments without Pi1 gene.

Figure 1 Genotype profile of Pi1 generated by primer MRG4766 among 30 rice landraces

In order to confirm the accuracy of the results by SSR markers detected, thirty landraces (17 with small fragment, 13 with larger fragment) were selected for artificial inoculated rice blast isolates 96-2-2b (AVR-Pi1) at seedling stage in greenhouse, Lijiangxintuanheigu as susceptible varieties control. The results showed that Lijiangxintuanheigu and the landraces with a small fragment were all susceptible, whereas, the landraces with large fragment were all resistant to 96-2-2b. The inoculation results were entirely consistent with the results of PCR detection, which demonstrated that it was viable for detection the Pi1 gene by SSR marker, and the landraces with small fragment were not carrying Pi1 gene, while the landraces with large fragment were harboring Pi1 gene. 

Base on PCR detection, there were 64 carrying Pi1 gene (36.99%) among 173 landraces. The distribution of Pi1 in japonica and indic rice were difference, 33 of 102 indica rice were holding Pi1 gene (32.35%), 31 of 71 japonica rice carrying Pi1 gene (43.66%). The results showed that indica rice harboring Pi1 genes were higher than that of japonica rice. 

1.2 The distribution of landraces holding Pi1 gene in rice-growing-region of Yunnan Province
The Pi1 gene is distributed in all rice growing regions (Table 1). The frequency of landrace holding Pi1 gene in paddy-upland rice region in South marginal of Yunnan, single and or double indica cropping region in south of Yunnan, single indica and or japonica cropping region in center of Yunnan, japonica cropping region in northeast of Yunnan and japonica cropping region in northwest highland of Yunnan, was 41.03%, 32.69%, 35.29%, 35.29% and 33.33%, respectively. The frequency of landrace carrying Pi1 gene in paddy-upland rice region in Southen marginal was higher than the remained rice-growing-regions, and the frequency of Pi1 gene were below 36.99% in other regions. The 64 landrace with Pi1 were distributed in 29 counties of 11 prefectures, and distributed widely. There were not detected the Pi1 gene in landraces from Dali, Wenshan, and other 17 counties (cities), which may be due to less materials for test. The frequency of Pi1 in each county (more than four landraces) was quite different. The first main different was the frequency of landraces carrying Pi1 in some of counties were higher, but in some of counties were low in the same prefecture. Example, Lianghe County (6 test materials) of Baoshan City, Wenshan County (4 test materials) of Guangnan prefecture, Yanjin County (4 tested material) of Zhaotong City, all landraces not carrying the Pi1 gene. However, the Pingbian county of Red River state (4 test materials, the frequency was 100%), Yongde county of Lincang city (5 test materials, frequency was 60%) and Gengma county (5 test materials, frequency was 60%), those counties have a higher frequency. The second different was the geographical proximity, but large differences in the frequency of Pi1. Such as Jiangcheng county and Mojiang county were proximity in geographical, but the frequency of Pi1 were 41.67% and 20.83%, respectively. Similar results in Tengchong county (frequency was 50%) and Lianghe county (frequency was 0%), the ecological conditions are similar in relative county, but the frequency of Pi1 gene was large different. The results showed that Pi1 gene were broader distribution in Yunnan, and showed irregular distribution.

Table 1 Distribution of the tested landraces containing Pi1 gene in each county of Yunnan Province

2 Discussion
Ancient landraces and rice blast fungus has a long coexistence history, and the specific interaction between the rice blast fungus and rice were according to the "gene for gene" relationship. Landrace resources are the results of a long-term co-evolution between varieties resources and rice blast fungus, and rice blast fungus played natural selection role in the process of long-term cultivation. The distribution of resistance genes and the rice blast were closely related. This study showed that Pi1 gene distributed in 29 counties and 11 prefectures of Yunnan Province, but we haven’t found the Pi1 gene in landraces collected from remaining 17 counties (cities) which may be due to the small test materials in those regions, if increase large number of test materials, may be can detected it in more counties, those indicated that Pi1 gene was widely distribution in Yunnan Province. It is relative between the distribution of Pi1 gene and rice blast races. Pi1 gene can be employed in indica and japonica cropping region (Li et al., 2005). This result also showed that the analysis of the distribution of resistance genes in ancient populations of landraces can direct the rice blast resistance breeding program and rice blast control by genetic diversity. 

Pi1 resistance gene is a broad-spectrum blast resistance gene. In this study, molecular markers detection results showed that 36.99% of the Yunnan landraces contained the Pi1 gene and those landraces were distributed in different ecosystem, the different ecological types and sub-species provide more choices for resistant material in breeding program, and the blast resistance breeding is a crucial work in China. Our results confirmed that the effectiveness of SSR marker identification for Pi1 gene, which was an effective method for identification the Pi1 gene in a mass of resources.

3 Materials and Methods
3.1 Rice materials
One hundred and seventy-three rice landraces were used for this study, and these landraces were randomly selected from the Yunnan core collection of resources library, which were conserved in Institute of Biotechnology and Genetic Resources, Yunnan Academy of Agricultural Sciences, Kunming, China. The selected 173 landraces were collected from 46 counties of 13 prefectures in Yunnan, China. Among them, 102 are japonica, 71 are indica. All materials were identified by SSR marker (MGR4766) of Pi1 gene. Based on the PCR detected results, 30 landraces were selected for seedling artificial inoculation, Lijiangxintuanheigu variety as susceptible control.

3.2 Identification blast isolate
The identification blast isolate 96-2-2b was selected base on the reaction on the monogenic lines in Plant Pathology Laboratory of Agricultural Environment and Resources Research Institute, Yunnan Academy of Agricultural Sciences, China. 

3.3 Rice genomic DNA extraction
DNAs were isolated from three to four leaf stage of rice, genomic DNA was prepared by CTAB method following instruction of Xu et al (1998). 

3.4 SSR primers
SSR primers MGR4766 were designed according to Chen et al (2005), and the forward primer: 5'- ATTGCTGCAAAGTGGGAGAC-3', the reverse primer: 5'-AAGTGGAGGCAGTTCACCAC-3'. The primers were synthesized by Sangon Biotech. (Shanghai) Co., Ltd.

3.5 PCR amplification and polyacrylamide gel electrophoresis
PCR reaction volume was 25 μL, containing 2.5 μL 10× Buffer, 2.0 μL dNTP, 3.0 μL 4 mol/L forward and reverse primers, 0.2 μL 5 U/μL Taq enzyme, 3.0 μL 20 ng/μL template DNA, ddH₂O 14.3 μL. PCR program follows: 94℃ predenaturation 5 min, followed by 35 cycles of 9℃ for 45 s, 55℃ for 45 s, 72℃ extension of 1 min, then 72℃ extension of 7 min. PCR products were separated on 8% non-denaturing polyacrylamide gel electrophoresis, and then silver staining followed by Li et al., 2008. 

3.6 Rice blast fungus inoculation in the greenhouse
The 173 landraces seeds are sown in a plastic tray (5 cm×15 cm×10 cm) half-filled with sieved garden soil. Each landrace are sown about 16 seeds and placed in greenhouse. At 2.5-leaf stage applied 0.2 g urea each plate, a total of 2~3 times.

Inoculation of blast isolates was carried out following the method. The spore concentration was standardized to 2~5×10⁵ spores per ml. Plants were inoculated at approximately the 3.5^th~4.5^th-leaf stages by spraying spore suspension on each tray using a fine atomizer, and then placed in charmber under 24~26℃ for 16~20 h, and then back to the greehouse. The degree of disease of each seedling was evaluated at 6~7 days after inoculated. The rice varieties resistant evaluation as described by Ahn et al (1994).

Authors’ Contributions
JBL worked on the inoculation, resistance test, confirmation and modified the manuscript. DL worked on PCR detection and finished draft. YDS analyzed the data. MHX performed the experiment designs, analyzed the data, wrote and modified the manuscript. All authors have read and approved the final manuscript.

Acknowledgements
This research was supported by National Natural Science Fund of China (31160355) and Natural Science Fund of Yunnan of China (2010ZC173).

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