Background

Rice is one of the most important food crops in China and worldwide, and serves as the main food staple for nearly half of the world's population. Rice blast disease is a serious disease which could cause 10%~30% loss of total rice yield in the world every year (Talbot, 2003; Dean et al., 2005). Breeding and planting resistant varieties is the most economical, effective and environmental measure to control rice blast disease. To date, more than 100 rice blast resistance genes (resistance gene, R gene) have been identified. And at least 23 of these resistance genes have been cloned, including Pib, Pi-ta, Pi9, Pi2, Piz-t, Pid2, Pi37, Pi36, Pik-m, Pi5, Pit, Pid3, Pid3-A4, Pi54, Pish, Pik-p, Pia, Pik, Pi-CO39, Pi25, Pi1, pi21, Pb1, etc (Liu et al., 2014; Tian et al., 2016). The identification and cloning of resistance genes have provided theoretical basis for molecular breeding of blast disease resistance rice.

Rice line IR65482 has broad-spectrum resistance to Magnaporthe oryzae isolates in Korea and Philippines (Jeung et al., 2007). Jeung et al. (2007) identified a rice blast resistance gene Pi40 (t) in the Pi2/9 allele on chromosome 6 of IR65482 by genetic analysis and gene mapping. When breeding using IR65482 as a parent, we observed that IR65482 had broad-spectrum resistance to rice blast isolates in Fujian. In the process of breeding, we found that in addition to the Pi40 (t) gene on chromosome 6, the IR65482 genome also carried different resistance genes from Pi40. In this study, based on bulked segregant analysis sequencing (BSA-Seq) and molecular marker mapping, we identified another R gene on chromosome 11 of IR65482. Our results provide helpful information for application of IR65482 in breeding rice varieties with blast resistance and basis for further cloning the R gene in IR65482.

1 Results and Analysis

1.1 Genetic analysis of blast disease resistance in IR65482

In a previous study, Jeung et al. (2007) mapped a R gene Pi40 (t) on chromosome 6 of IR65482. In order to evaluate the disease resistance of IR65482, 17 Fujian field isolates of blast fungus were used to artificially inoculate IR65482 and a Pi40 (t) single gene line in Nipponbare background (NPB-Pi40). The results showed that IR65482 had broad-spectrum resistance to M. oryzae isolates in Fujian, but the resistance spectrum of NPB-Pi40 was not as good as that of IR65482 (Table 1). The results also indicated that, in addition to the Pi40 (t) gene, IR65482 may carry another R gene.

Rice blast strain RB22 was used to inoculate 6 F2 populations, including IR65482 × Nipponbare, IR65482 × 02428, IR65482 × 9311, 3301 × IR65482, 02428 × IR65482, and 9311 × IR65482. The results showed that 6 F₂ population had different disease resistance / susceptible segregation patterns. The resistant plants / susceptible plants of the F₂ population derived from a cross between IR65482 and Nipponbare were 373/137. Chi-square test showed x²=2.72＜x² (0.05)=3.84 (Table 2), consistent with the 3:1 theoretical ratio, indicating that the resistance was controlled by a single dominant nuclear gene in this population. The F₂ populations derived from a cross between IR65482 and other parents were not conformed to the 3:1 genetic segregation.

1.2 BSA-Seq analysis for R gene in IR65482

Based on second-generation sequencing, a total of 1, 821, 724 polymorphic and homozygous SNP loci were identified between IR65482 and Nipponbare. Using the identified SNP loci, the SNP-index of disease-resistant and susceptible segregation pools were analyzed, and △ (SNP-index) was calculated. The whole genome was scanned with 95% confidence level as the screening threshold. The results showed that there was a continuous region beyond the threshold at the end of chromosome 11, indicating that this region may harbor a R gene (Figure 1).

1.3 Mapping of R gene in IR65482

Based on the BSA-Seq analysis result, a total of 17 polymorphic InDel markers located around the identified region of chromosome 11 were developed (Table 3). Using these InDel markers, 137 susceptible segregant plants were analyzed and the result confirmed a R gene located at the end of chromosome 11 of IR65482.

To finely map the R gene of IR65482, a larger F₂ population derived from a cross between IR65482 and Nipponbare was further constructed. After inoculation with RB22, 1, 048 F₂ susceptible segregant plants were screened. Eight InDel markers, OSL2-16, OSL3-2, OSL3-4, OSL3-5, OSL4-2, OSL4-13, OSL5-5, and OSL6-2 were used to analyze the population. The results showed that the R gene was located in a 425-kb region between OSL3-2 and OSL3-5 (Figure 2).

2 Discussion

Rice contains two levels of resistance to M. oryzae: basal resistance and R gene-mediated resistance. Basal resistance was commonly controlled by quantitative trait genes and showed moderate resistance level. R gene-mediated resistance was a high-level resistance which is triggered by recognizing of M. oryzae avirulence protein (Avr) by R protein. Identification and application of R genes have been attached great importance in rice breeding because of the resistance effect and strong maneuverability in practical application. On the other hand, the “gene for gene” relationship between R gene and Avr gene, represent the limitation of R gene-mediated resistance as a type of race-specific resistance. R gene-mediated resistance will exert strong selective pressure on M. oryzae and promote the emergence of new dominant isolates without the corresponding Avr gene. Usually, rice varieties with R gene-mediated resistance will lose their resistance when the non Avr M. oryzae isolates developed into dominant ones. Therefore, introducing and rotating new resistant germplasm from different countries or rice planting regions to cope with the possible emergence of new dominant M. oryzae isolates would be of great significance for controlling rice blast disease.

Fine mapping and cloning of R genes and the establishment of molecular markers promoted the development of molecular breeding of resistant rice. Up to now, at least 19 major blast resistance genes have been successfully used in rice blast resistance breeding by molecular marker-assisted selection, including Pi1, Pi2, Pi9, Pi33, Pi34, Pi35, Pi39, Pi40, Pi46, Pi54, Pib, Pid1, Pigm, Pik-h, Pish, Pi-ta, Piz, Piz-5, Piz-t, etc (Srivastavaa et al., 2017). Many materials used in molecular breeding for resistant rice are excellent parents with broad-spectrum resistance. On the other hand, many materials newly developed by marker-assisted selection were found to display not the same broad-spectrum resistance as to their original parents. One possible reason for that is that the original parents carry more than one R gene. In the present study, our results indicated that there is another R gene on chromosome 11 in rice line IR65482, in addition to the previously identified Pi40 (t) on chromosome 6. Identification of R gene composition of these elite parental lines would provide helpful information for the scientific use of disease-resistant parents to develop disease-resistant varieties.

The IR65482 R gene was mapped to the long arm end of chromosome 11. The region contains a big number of R gene alleles (Chen et al., 2016), and many of the R genes confer broad-spectrum resistance to M. oryzae isolates from different rice regions of China (Wang et al., 2009). Several genes have been cloned at the long arm end of chromosome 11, such as Pi-km (Ashikawa et al., 2008), Pi-kp (Yuan et al., 2011), Pi-k (Zhai et al., 2011), Pi1 (Hua et al., 2012), Pi-kh (Zhai et al., 2014). Whether the IR65482 R gene is one of the identified genes or a new one remains to be further studied. Our results provided a basis for further identification of the IR65482 R gene.

3 Materials and Methods

3.1 Parents and genetic population of rice

Rice materials IR65482-4-136-2-2 (Abbreviated IR65482), NPB-Pi40, Nipponbare, 02428, 9311, and 3301 are preserved and provided by the Biotechnology Research Institute, Fujian Academy of Agricultural Sciences. F₂ populations were developed by using IR65482 as a female parent, crossing with Nipponbare, 02428 and 9311, respectively, or 3301, 02428, and 9311 as female parents, crossing with IR65482.

3.2 Rice blast disease inoculation

Rice blast isolates NH1, NH2, NH3, NH4, NH5, NH6, SH1, SH2, SH3, SH4, SH5, SH6, SH7, SH8, SH9, SH10, and RB22 were preserved by the Biotechnology Research Institute, Fujian Academy of Agricultural Sciences. The inoculation was carried out according to the method described by Tian et al. (2016). Rice blast isolates were grown onto oat medium under dark culture at 25°C for 5~7 days, and then transferred to light for 3 days to induce spore growth. Rice was seeded in the cement pools of Shoushan Experimental Base of Biotechnology Research Institute, Fujian Academy of Agricultural Sciences. At the leaf age of 3 ~ 4, rice seedlings were sprayed with rice blast fungus spores. The seedlings were shaded 24 h after inoculation, and cultured under high humidity at 24°C ~ 28°C. Rice disease symptoms were investigated after 5~7 days.

3.3 Genetic analysis of IR65482 disease resistance

Genetic analyses of resistance to blast disease of IR65482 were performed according to the resistant, susceptible segregant plant ratios of F₂ populations (IR65482×Nipponbare, IR65482×02428, IR65482×9311, and 3301×IR65482, 02428×IR65482, 9311×IR65482) inoculated with M. oryzae isolate RB22 using a x² test.

3.4 BSA-seq

The IR65482 × Nipponbare F₂ population was inoculated with the M. oryzae isolate RB22 for screening segregant plants. Twenty extremely resistant segregant plants, 20 extremely susceptible segregant plants, 10 IR65482 plants and 10 Nipponbare plants were selected for extracting genomic DNAs. The mix pools of resistant and susceptible plants and the two parental lines were used subjected to whole genome sequencing.

SNP frequency calculation and linkage analysis were carried out according to Abe et al. (2012). The △ (SNP-index) was calculated based on the difference of SNP-index of two offspring separation. After 1 000 times replacement tests, the 95% confidence level was selected as the screening threshold to determine the linkage interval.

3.5 Mapping of IR65482 R gene

The IR65482 × Nipponbare F₂ population was inoculated with RB22, and the susceptible segregant plants for mapping. Genomic DNA of susceptible individual plant was extracted by CTAB method.

Based on the whole genome sequencing results, InDel loci showed polymorphic in IR65482 and Nipponbare within the prediction interval of linkage to IR65482 R gene were screened. Primer Premier5.0 was used to design primers (Cai et al., 2016) for PCR reactions using DNA of F₂ susceptible plants as template. The whole PCR reaction system was 25 μL, including 1 μL DNA template, 12.5 μL 10×PCR Mixture, 1 μL forward primer, 1 μL reverse primer, and 9.5 μL ddH₂O. The reaction procedure was as follows: 94°C predegeneration 5 min; followed 34 cycles, 94°C denaturation 30 s, 56°C annealing 30 s, 72°C extension 20 s. And 72°C extension 7 min after cycle completion.

PCR products electrophoresis on polyacrylamide gel: 0.8~1 μL PCR products were electrophoresed on 8%~12% polyacrylamide gel; 1.0×TBE was used as electrophoretic buffer, and electrophoresis lasted 50 ~ 120 min under 180 V voltage. After electrophoresis, the gel was dyed in 0.05% AgNO₃ solution for 10 min. After dyeing, the gel was washed with ddH₂O for 3 times, and then colored in 0.75% NaOH and 0.5% formaldehyde. Based on the results of electrophoresis, the linkage and mapping interval of disease resistance genes were further analyzed.

Authors’ contributions

LTM participated in the experimental design, carried out the experimental research, completed the data analysis, and drafted the manuscript. GXR, CZQ, TDG, and CZJ involved in material creation, group building, and experimental execution. WF and CSB guided experiment design, experiment execution, result analysis and draft revision. All authors read and approved the final manuscript.

Acknowledgments

This work was supported by a grant from the National Natural Science Foundation (U1405212), the Natural Science Foundation Project of Fujian Province (2014J-07004), and the Basic Scientific Research Projects of Public Welfare Research Institutes of Fujian Provincial Science and Technology Program (2017R1019-1; 2017R1019-10).

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