Establishment and Application of a Multiplex PCR System for the Detection of Blast Resistance Genes Pi-ta and Pi-b in Rice

Shu YAO, Yanqing LIU, Yadong ZHANG, Zhen ZHU, Tao CHEN, Qingyong ZHAO, Lihui ZHOU,Chunfang ZHAO, Xin YU, Cailin WANG

Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Jiangsu High Quality Rice R&D Center/Nanjing Branch of China National Center for Rice Improvement, Nanjing 210014, China

Rice blast is one of the most serious diseases of rice that is caused by Magnaporthe grisea, which has become a serious impediment to high and stable yields of rice due to large hazard area and great damage. According to statistics,annual global rice yield loss caused by rice blast accounts for approxi mately 10%-15% of the total yield,resulting in billions of dollars of economic losses[1]. Practice has proved that breeding blast resistant varieties is the most economical and effective method for prevention and control of rice blast. However, due to strong variability of Magnaporthe grisea, resistant varieties may lose resistance after years of cultivation[2]. Therefore,transferring multiple blast resistance genes with different resistance spectra into the same variety by using molecular marker-assisted selection (MAS)technique has become one of the effective measures to breed rice varieties with durable blast resistance[3].

With the rapid development of molecular biology techniques, so far,more than 50 major blast resistance genes have been fine mapped,among which 24 genes are cloned[4-8]. Pi-ta and Pi-b are two earliest cloned major blast resistance genes[9-10]. In recent years, functional markers of these two resistance genes and their allelic susceptibility loci have been developed,which can rapidly and accurately identify blast resistance genes Pi-ta and Pi-b from rice germplasms[11-12]. Li et al.[13]analyzed the distribution of blast resistance genes Pi-ta and Pi-b in rice germplasms from Yunnan Province using specific molecular markers of these two genes.Liu et al.[14]and Shi et al.[15]identified and analyzed blast resistance genes Pi-ta and Pi-b in main popularized rice varieties in Heilongjiang Province and major cultivars in China using these two specific molecular markers. Previous studies are mostly focused on the distribution of blast resistance genes Pi-ta and Pi-b in different rice varieties by molecular biology techniques, but little information is available on the optimization of mark detection methods.Especially, no studies have been reported on multiplex PCR systems for simultaneous detection of blast resistance genes Pi-ta and Pi-b.Compared with conventional single mark detection, multiplex PCR can detect two or more target genes in one PCR reaction with significantly improved efficiency and remarkably reduced costs[16-17]. Therefore, developing simple,rapid and efficient multiplex PCR systems for specific targets has a great significance for promoting the development of molecular breeding of disease-resistant rice.

In this study, by investigating the effects of PCR reaction components and cycle parameters on multiplex PCR results, two multiple PCR systems for blast resistance and susceptibility genes were established. There was no mutual inhibition or mismatch between the primers in each system.Rice varieties with known genotypes were detected repeatedly to verify the accuracy and stability of the established multiplex PCR systems.The results showed that these two multiplex PCR systems could be used to simultaneously screen and identify two rice blast resistance (susceptibility)genes in the same PCR reaction. In addition,blast resistance genes (Pi-ta and Pi-b)and blast susceptibility genes (pi-ta and pi-b) in 336 high generation japonica varieties (lines) were detected with these two multiple PCR systems, aiming at providing rapid and efficient marker-assisted selection methods for screening Pi-ta and Pi-b genes,thereby improving the breeding efficiency of blast resistant rice.

Materials and Methods

Materials

Nanjing 44, Nanjing 45, Wuyunjing 7, Wuyunjing 8, Wuyunjing 21,Wujing 15, Wuxiangjing 14, Changnongjing 5, Yandao 9 and Yangjing 805 were used to establish the multiplex PCR system for the detection of blast resistance genes Pi-ta and Pi-b(referred to as system I).Xudao 3,Xudao 4,Xudao 6, Zhendao 88, Huaidao 5, Huaidao 9, Lianjing 4, Lianjing 5,Lianjing 6 and Lianjing 7 were used to establish the multiplex PCR system for the detection of blast susceptibility genes pi-ta and pi-b (referred to as system II). Jia 33 was used as the resistant control,which has been recognized as a blast resistant variety; Lijiangxintuanheigu was used as the susceptible control[13,18]. By using the developed multiplex PCR systems,blast resistance genes Pi-ta and Pi-b in 336 high generation japonica lines were detected and compared with conventional single mark detection to verify the reliability of the multiplex PCR systems. The above materials were planted in the experimental field of Institute of Food Crops, Jiangsu Academy of Agricultural Sciences,which were sown on May 10 and transplanted on June 10. Each material was planted in eight rows, 40 seedlings per row. The spacing in the rows and spacing between rows were 13.2 cm × 26.4 cm. All the seedlings were plants under conventional field management.

DNA extraction

Fresh tender leaves were collected from rice seedlings at maximum tillering stage.Genomic DNA of control and experimental varieties was extracted by CTAB method[19]with slight modifications[19].

Primer synthesis

Based on the principle of allelespecific PCR, Wang et al.[20]designed specific primers YL155/YL87 and YL183/YL87 according to the sequences of blast resistance and susceptibility genes. The former primers could specifically amplify DNA fragment of blast resistance gene Pi-ta,which was about 1 042 bp; the latter primers could specifically amplify DNA fragment of blast susceptibility gene pi-ta, which was about 1 042 bp.Primers Pi-bdomF/Pi-bdomR were designed according to Fjellstrom et al.[21]to specifically amplify DNA fragment of blast resistance gene Pi-b,which was about 365 bp; primers Lys145F/Lys145R were designed according to Liu et al.[12]to specifically amplify DNA fragment of blast susceptibility gene pi-b, which was about 803 bp. All the primers were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd. The names, sequences and expected amplified fragment sizes of these primers were shown in Table 1.

Table 1 Names,sequences and expected amplified fragment sizes of primers used for multiplex PCR

Multiplex PCR and electrophoresis

The total multiplex PCR reaction volume was 20 μl, containing l × PCR buffer (1.5 mmol/L MgCl2), 200 μmol/L dNTPs, 1.25 U of Taq DNA polymerase [GenScript (Nanjing) Co.,Ltd.], 100-150 ng of template DNA and proper amounts of primers.Multiplex PCR was performed using Tprofessional thermal cycler. The PCR amplification was started with initial denaturation at 95 ℃for 3 min, followed by 6 cycles of denaturation at 94 ℃for 1 min, annealing at 55 ℃for 1 min and extension at 72 ℃for 1 min,and 32 cycles of denaturation at 94 ℃for 1 min, annealing at 55 ℃for 50 s and extension at 72 ℃for 30 s; the amplification was completed by holding the reaction mixture at 72 ℃for 6 min to allow complete extension of PCR products. PCR products were added with 2 μl of loading buffer; 10 μl of the mixture was separated by electrophoresis on 1.5% agarose gel containing ethidium bromide in 1× TAE buffer under 120 V for 45 min. The electrophoresis results were observed and photographed under an ultraviolet light. In order to ensure the stability and reliability of the results, each material was amplified for more than three times.

Results and Analysis

Establishment of multiplex PCR systems

Genomic DNA of 22 rice varieties harboring known blast resistance and susceptibility genes was extracted as the template for gradient PCR using specific primers of four genes, to identify the appropriate annealing temperature range of each pair of primers.Primers with the same annealing temperature and great differences in amplified fragments were selected for multiplex PCR to further detect experimental materials harboring known genes. After identifying the consistent annealing temperature of four primers for system establishment, the original primers, dNTPs and DNA polymerase were prepared into multiplex PCR systems for PCR amplification and electrophoresis assay. Subsequently,combined with the amplification results of control materials, the amount of primers,extension time and number of cycles were optimized. Equal concentration of primers was added into the system firstly, and the amount was adjusted according to the amplification results: the amount of primers with weak amplification was increased,while that with strong amplification was reduced. The extension time of weak amplification was extended appropriately. Finally, in two multiplex PCR systems, each primer could amplify clear specific fragments from control materials harboring target genes(loci),but no specific bands were amplified from control materials harboring no target genes (loci). The established multiplex PCR systems were described as below.

Multiplex PCR system for the detection of blast resistance genes Pi-ta and Pi-b The multiplex PCR system for detection of blast resistance genes Pi-ta and Pi-b involves primers YL155/YL87 and Pi-bdomF/Pi-bdomR. Using genomic DNA of 12 rice varieties (lines) harboring known genes as the template, PCR amplification and electrophoresis of corresponding loci were performed. The concentration ratio of primers, extension time and number of cycles in multiplex PCR system I were adjusted repeatedly to constantly optimize the system. Ultimately, the total multiplex PCR reaction volume was 20 μl, containing 2.0 μl of DNA template (approximately 20 ng/μl), 2.0 μl of 10 ×PCR buffer (25 mmol/L), 2.0 μl of dNTPs(2.5 mmol/L),and 0.8-1.2 μl of each of primers (10 μmol/L); ddH2O was added to a final volume of 20 μl.

As shown in Fig.1, among 12 rice varieties detected with multiplex PCR system I, the same fragments were amplified from ten materials such as Nanjing 44 and Nanjing 45 as blast resistant control Jia 33,which were 1042 bp and 365 bp, respectively; however,no target bands were amplified from blast susceptible control Lijiangxintuanheigu, which was exactly the same as the results of conventional single mark detection by Shi et al.[15]and Yang et al.[22], indicating that these materials all harbor blast resistance genes Pi-ta and Pi-b.

Multiplex PCR system for the detection of blast susceptibility genes pi-ta and pi-b Similarly, the multiplex PCR system for detection of blast susceptibility genes pi-ta and pi-b involves primers YL183/YL87 and Lys145F/Lys145R.Based on repeated system optimization, multiplex PCR system II was established. The total multiplex PCR reaction volume was 20 μl, containing 2.0 μl of DNA template(approximately 20 ng/μl),2.0 μl of 10×PCR buffer (25 mmol/L), 2.0 μl of dNTPs(2.5 mmol/L),and 0.8-1.2 μl of each of primers (10 μmol/L); ddH2O was added to a final volume of 20 μl.

As shown in Fig.2, among 12 rice varieties detected with multiplex PCR system II, the same fragments were amplified from ten materials such as Xudao 3 and Xudao 4 as blast susceptible control Lijiangxintuanheigu,which were 1042 bp and 803 bp, respectively; however, no target bands were amplified from blast resistant control Jia 33, which was exactly thesame as the results of conventional single mark detection by Yang et al.[22]and He et al.(to be published),indicating that these materials all harbor blast susceptibility genes pi-ta and pi-b.

Table 2 Distribution of blast resistance genes(Pi-ta and Pi-b)and susceptibility genes(pi-ta and pi-b)in 336 japonica rice lines

Detection of blast resistance genes in high generation rice lines using multiplex PCR systems

By using two established multiplex PCR systems, blast resistance genes (Pi-ta and Pi-b)and blast susceptibility genes (pi-ta and pi-b) were detected in 336 high generation breeding materials bred independently by Jiangsu Academy of Agricultural Sciences. According to the results,among 336 experimental materials,119 rice varieties harbor only blast resistance gene Pi-ta, accounting for 35.42%of the total number of experimental materials; 319 rice varieties harbor only blast resistance gene Pi-b, accounting for 94.94% of the total number of experimental materials; 1 042 bp and 365 bp specific fragments of blast resistance genes were amplified from 112 rice varieties(33.33%), which were consistent with that amplified from blast resistant control Jia 33, indicating that these varieties all harbor blast resistance genes Pi-ta and Pi-b; 1 042 bp and 803 bp specific fragments of blast susceptibility genes were amplified from 10 materials (3.0%), which were consistent with that amplified from blast susceptible control Lijiangxintuanheigu, indicating that these varieties all harbor blast susceptibility genes pi-ta and pi-b; 1 042 bp specific fragment of blast resistance gene and 803 bp specific fragment of blast susceptibility gene were amplified from seven materials (2.1%),indicating that these varieties harbor blast resistance gene Pi-ta and blast susceptibility gene pi-b;1 042 bp specific fragment of blast susceptibility gene and 365 bp specific fragment of blast resistance gene were amplified from 207 materials(61.61%),indicating that these varieties harbor blast susceptibility gene pi-ta and blast resistance gene Pi-b (Table 2). The above detection results were exactly the same as that of conventional single mark detection by He et al.(to be published). The detection results of blast resistance and susceptibility genes in different rice varieties were shown in Fig.3 and Fig.4.

According to the above analysis,rice varieties harboring two blast resistance genes account for a small proportion of high generation breeding materials; the distribution frequency of blast resistance gene Pi-b is significantly higher than that of blast resistance gene Pi-ta.

Discussion

Advantages of multiplex PCR systems in detection of blast resistance genes Pi-ta and Pi-b

The development of molecular biology greatly promotes the process of genetic research of blast resistance. A large number of mapped rice blast resistance genes lay a solid foundation for breeding blast resistant rice with MAS technologies.Developing an efficient method that is not subject to restrictions of seasons or sample types to detect resistance genes in rice at early growth stage is conducive to accelerating the progress of genetic improvement of blast resistant rice.Conventional blast resistance genotyping depends on disease resistance identification and phenotypic selection, with long breeding cycle,low selection efficiency, heavy workload, complicated operation and other shortcomings.Molecular marker techniques can detect any tissues of plants,and the sampling is not subject to restrictions of growing seasons or sample types,with relatively simple operation. Compared with single mark detection method,multiplex PCR can detect several target genes simultaneously with low cost, simple operation, high efficiency and other advantages, which is suitable for molecular-assisted selection and breeding. Compared with single PCR detection systems established previously,the multiplex PCR systems established in the present study involve more sites with better specificity and sensitivity, higher detection efficiency, which is more suitable for screening and molecular breeding of rice resources harboring blast resistance genes Pi-ta and Pi-b.

Distribution of blast resistance genes Pi-ta and Pi-b in high generation rice varieties (lines) and suggestions for breeding blast resistant rice

Previous studies have shown that Pi-ta and Pi-b genes exhibited high resistance to Magnaporthe grisea strains in Jiangsu, Yunnan, Jilin and other regions[23-26]. In this study, molecular detection results show that the distribution frequency of Pi-ta and Pi-b genes in different rice varieties varies significantly; the distribution frequency of Pi-b is remarkably higher than that of Pi-ta. Analysis results of the blast resistance of rice varieties harboring only Pi-ta or Pi-b indicate that the incidence of rice varieties harboring only Pi-b is significantly higher than that of rice varieties harboring only Pi-ta;Magnaporthe grisea strains demonstrate high race specificity. In subsequent breeding of blast resistant rice,the constitution of resistance genes in existing rice varieties should be improved to polymerize blast resistance genes at various loci into the same variety, thereby breeding new varieties with excellent blast resistance.

Suggestions for establishment of multiplex PCR systems

The experimental design for multiple PCR is more complex than single PCR with great technical difficulties.Therefore, in the establishment of multiplex PCR systems, the main components and reaction conditions should be optimized repeatedly[27]. According to practical experience,the efficiency of multiplex PCR systems may be improved with the following approaches. Firstly, multiplex PCR systems can be combined using genetic markers with large differences in the amplified products,thus ensuring easy and accurate distinguishing of the results by agarose gel electrophoresis.Secondly, in multiplex PCR, annealing temperature is one of the most important factors to be adjusted.Commonly,the annealing temperature is selected based on the melting temperature of the primers, but the results are not necessarily consistent with the expectations. The simplest way is to amplify single gene by gradient PCR with each primer, thereby identifying the optimal annealing temperature that is appropriate for multiple PCR amplification of each target gene. In multiplex PCR systems with no consistent annealing temperature, touch-down PCR can be employed. Thirdly, in the establishment of multiple PCR systems, the amount of primers with weak amplification should be increased, while that with strong amplification should be reduced. By continuously adjusting the relative amounts of primers, sufficient PCR products can be amplified with each primer ultimately. Although primer selection is the most critical factor determining the success of multiplex PCR, DNA template, chemical reagents (PCR buffer, Mg2+, Taq DNA polymerase and dNTPs) and instruments (PCR amplifier and electrophoresis apparatus) can also affect the results of multiple PCR.Therefore,in the initial period, reagents produced by different companies and PCR amplifiers with different types can be used to determine the optimal multiplex PCR system and procedures; after that, consistent instruments and reagents should be used in subsequent research. Moreover, each primer should be diluted and preserved in aliquots without mixing. Other reagents such as dNTPs should be preserved at 4 ℃ without repeated freezing and thawing. Compared with conventional single mark detection,multiplex PCR exhibits significantly reduced reagent costs and remarkably shortened detection time. The multiplex PCR systems for detection of blast resistance genes can be combined with field resistance identification for breeding blast resistant rice,thus effectively enhancing the efficiency of blast resistance improvement of rice.

Conclusion

In this study, two multiplex PCR systems were established for detection of blast resistance genes (Pi-ta and Pi-b) and blast susceptibility genes (pi-ta and pi-b), which led to stable and reliable identification results with low costs and could be applied in rice parent evaluation and marker-assisted selection to polymerize multiple genes for blast resistance in hybrid breeding. Among 336 high generation breeding materials detected with these two multiplex PCR systems, 112 rice varieties harbor blast resistance genes Pi-ta and Pi-b; seven rice varieties harbor only blast resistance gene Pi-ta; 207 varieties harbor only blast resistance gene Pi-b; 10 varieties harbor blast susceptibility genes pi-ta and pi-b.

[1]ZHENG Z (郑钊),CHEN YQ (陈由强),ZHANG JF (张建福), et al. Mapping cloning of rice blast resistance genes and their application(水稻稻瘟病抗性基因的定位、 克隆及应用)[J]. Mol Plant Breed (分子植物育种), 2009, 7: 385-392.

[2]SHEN Y(沈瑛),ZHU PL(朱培良),YUAN XP (袁筱萍), et al. Genetic diversity of Magnaporthe grisea in China(中国稻瘟病菌的遗传多样性)[J].Acta Phytopathol Sin(植物病理学报),1993,23:309-313.

[3]HITTALMANI S, PARCO A, MEW TV,et al. Fine mapping and DNA markerassisted pyramiding of the three major genes for blast resistance in rice [J].Theor Appl Genet, 2000, 100: 1121-1128.

[4]ZHAI C, LIN F, DONG ZQ, et al. The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication[J]. New Phytologist,2011,189:321-334.

[5]HAYASHI N, INOUE H, KATO T, et al.Durable panicle blast resistance gene Pb1 encodes an atypical CC-NBS-LRR protein and was generated by acquiring a promoter through local genome duplication[J].Plant J,2010,64:498-510.

[6]OKUYAMA Y, KANZAKI H, ABE A, et al. A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes [J]. Plant J,2011,66:467-479.

[7]YUAN B,ZHAI C,WANG WJ,et al.The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC-NBS-LRR genes [J]. Theor Appl Genet,2011,122:1017-1028.

[8]TAKAHASHI A,HAYASHI N,MIYAO A,et al. Unique features of the rice blast resistance Pish locus revealed by large scale ret retrotransposon-tagging [J].BMC Plant Biol,2010,10:175.

[9]WANG ZX, YANO M, YAMANOUCHI U,et al.The Pi-b gene for rice blast resistance belongs to the nucleotide binding and leucine rich repeat class of plant resistance genes [J].Plant J, 1999, 19:55-64.

[10]BRYAN GT, WU KS, FARRALL L, et al. A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta [J]. Plant Cell, 2000, 12: 2033-2045.

[11]WANG Z, JIA Y, RUTGER JN, et al.Rapid survey for presence of a blast resistance gene Pi-ta in rice cultivars using the dominant DNA markers derived from portions of the Pi-ta gene[J].Plant Breed,2007,126:36-42.

[12]LIU Y(刘洋), XU PZ(徐培洲), ZHANG HY(张红宇),et al.Marker-assisted selection and application of blast resistant gene Pi-b in rice(水稻抗稻瘟病Pi-b基因的分子标记辅助选择与应用) [J].Sci Agric Sin(中国农业科学),2008,41:9-14.

[13]LI JB(李进斌),WANG T(王甜),XU MH(许明辉).Identification of Pi-ta and Pib genes for rice blast resistance of rice landraces from Yunnan Province(云南地方稻种抗稻瘟病基因Pi-ta 和Pi-b 的鉴定)[J].Chin J Rice Sci(中国水稻科学),2012,26:593-599.

[14]LIU HZ(刘华招), LIU Y(刘延), LIU HL(刘化龙),et al.Distribution of two blast resistance genes Pi-b and Pi-ta in major rice cultivars in Heilongjiang Province in China (黑龙江省种植品种中稻瘟病抗性基因Pi-b 和Pi-ta 的分布)[J].J.Northeast Agric Univ (东北农业大学学报),2011,42(1):27-31.

[15]SHI K(时克),LEI CL(雷财林),CHENG ZJ (程治军), et al. Distribution of two blast resistance genes Pi-ta and Pi-b in major rice cultivars in China (稻瘟病抗性基因Pi-ta 和Pi-b 在我国水稻主栽品种中的分布)[J]. J Plant Genet Resour (植物遗传资源学报), 2009, 10:21-26.

[16]MA W, ZHANG W, GALE KR. Multiplex-PCR typing of high molecular weight glutenin alleles in wheat [J]. Euphytica,2003,134:51-60.

[17]NAKAMURA T, VRINTEN P, SAITO M, et al. Rapid classification of partial waxy wheat using PCR-based markers[J].Genome,2002,45:1150-1156.

[18]DAI XJ (戴小军),YANG YZ (杨远柱),ZHOU L (周亮), et al. Distribution research of blast resistance genes Pi-ta,Pi-b, Pi-9 and Pikm in blast-resistant rice resources(抗稻瘟病水稻资源抗性基因Pi-ta、Pi-b、Pi-9 以及Pikm 的分布研究)[J].Life Sci Res(生命科学研究),2012,16:340-344.

[19]LI W,LEI CL,CHENG ZJ,et al.Identification of SSR markers for a broadspectrum blast resistance gene Pi20(t)for marker-assisted breeding [J]. Mol Breed,2008,22:141-149.

[20]WANG ZH (王忠华),JIA YL (贾育林),WU DX ( 吴殿星), et al, Molecular marker-assisted selection of the rice blast resistance gene Pi-ta (水稻抗稻瘟病基因Pi-ta 的分子标记辅助选择)[J]. Acta Agron Sin (作物学报), 2004,30:1259-1265.

[21]FJELLSTROM R, CONAWAY-BORMANS CA, MCCLUNG AM, et al. Development of DNA markers suitable for marker assisted selection of three Pi genes conferring resistance to multiple Pyricularia grlsea pathotypes [J]. Crop Sci,2004,44:1790-1798.

[22]YANG J (杨杰), YANG JH (杨金欢),WANG J (王军), et al. Distribution of two blast resistant genes Pi-ta and Pib in landrace rice in China(稻瘟病抗病基因Pi-ta 和Pi-b 在中国水稻地方品种中的分布)[J].Acta Agric Boreali-Sin(华北农学报),2011,26(3):1-6.

[23]LI JB(李进斌),LI CY(李成云),CHEN Y(陈艳), et al. Utilization value of twenty-two blast resistance genes in Yunnan(二十二个抗稻瘟病基因在云南的利用价值评价) [J].Acta Phytophy Sin(植物保护学报),2005,6:113-119.

[24]WANG GZ(王国珍), RU QH(茹庆华),GAO LY (高立原), et al. Studies on genes for resistance blast fungus in Ningxia region(日本水稻抗瘟基因对宁夏稻瘟病菌的抗性研究)[J].Acta Agric Bori-Occide Sin(西北农业学报),1997,6(3):1-4.

[25]LU F(陆凡),CHEN ZY(陈志谊),LIU YF(刘永锋), et al. Analysis of population virulences of Magnaporthe grisea in Jiangsu Province (江苏省稻瘟病菌毒性的群体结构分析)[J].Acta Phytophy Sin (植物保护学报), 2002, 29: 289-294.

[26]REN JP (任金平),GUO XL (郭晓莉),ZHENG M (郑民), et al. Studies on pathogenic spectrum of rice varieties with Magnaporth grisea in Jilin Province (吉林省稻瘟病菌对水稻品种的致病谱研究) [J]. J Jilin Agric Sci(吉林农业科学),2006,31(6):35-37.

[27]LIU ZB, GAO QR, WANG RX, et al.Application of multiplex PCR to studies on plant biology [J]. Mol Plant Breed,2005,3:261-268.