稻瘟病抗性基因Pi25特异性CAPS标记的开发与验证

作物学报 ACTA AGRONOMICA SINICA 2012, 38(11): 1960−1968 /zwxb/ISSN 0496-3490; CODEN TSHPA9E-mail: xbzw@This work was supported by the National High Technology Research and Development Program of China (Grant No. 2011AA10A101 and 2012AA101102) and the Ministry of Finance, China (Grant No. 2012RG002-4).*Correspondence author: WU Jian-Li, E-mail: beishangd@, Tel: +86-571-63370326 **These authors contributed equally to this work.Received(收稿日期): 2012-04-17; Accepted(接受日期): 2012-07-05; Published online(网络出版日期): 2012-09-10. URL: /kcms/detail/11.1809.S.20120910.1404.023.htmlDOI: 10.3724/SP.J.1006.2012.01960Development and Validation of CAPS Markers for Marker-Assisted Selection of Rice Blast Resistance Gene Pi25WANG Hui-Mei 1,**, CHEN Jie 1,**, SHI Yong-Feng 1, PAN Gang 2, SHEN Hai-Chao 1, and WU Jian-Li 1,*1State Key Laboratory of Rice Biology / China National Rice Research Institute, Hangzhou 310006, China; 2 College of Agriculture and Biotechno-logy, Zhejiang University, Hangzhou 310058, ChinaAbstract: To promote the application of rice blast resistance gene Pi25 in rice breeding programs, we developed four sets of gene-specific CAPS markers (CAP1/Hinc II, CAP3/Bgl II, CAP3/Nde I, and CAP3/Hpy 99I) based on the coding sequences of the locus. One hundred and sixty-nine rice accessions, 98 recombinant inbred lines (RILs) and 217 transgenic plants were used for the validation of the markers. The results showed that all the four sets of markers were able to accurately and efficiently detect the Pi25/pi25 locus, CAP1/Hinc II and CAP3/Hpy 99I could digest specifically the dominant allele Pi25 while CAP3/Bgl II and CAP3/Nde I were able to digest specifically the recessive allele pi25. RILs and transgenic lines carrying Pi25 allele were resistant to the blast isolate JS001-20 while the lines carrying pi25 allele were susceptible, indicating a perfect detection of the target locus by the CAPS markers. In addition, a low frequency (1.2%) of the dominant allele was detected in the germplasm collections, in-dicating this gene has not been fully utilized in rice breeding programs in China. Markers CAP1/Hinc II and CAP3/Hpy 99I are recommended and will be useful for the improvement of blast resistance, especially for the early-season indica rice.Keywords: Oryza sativa ; Blast resistance; Cleaved amplified polymorphic sequence (CAPS); Marker-assisted selection (MAS); Single nucleotide polymorphism (SNP)稻瘟病抗性基因Pi25特异性CAPS 标记的开发与验证王惠梅1,** 陈 洁1,** 施勇烽1 潘 刚2 沈海超1 吴建利1,*1中国水稻研究所 / 水稻生物学国家重点实验室, 浙江杭州 310006; 2浙江大学农业与生物技术学院, 浙江杭州 310058摘 要: 为在水稻育种中快速与高效利用稻瘟病抗性基因Pi25, 本文利用该基因不同等位基因编码区序列差异开发了4套CAPS 标记(CAP1/Hinc II 、CAP3/Bgl II 、CAP3/Nde I 和CAP3/Hpy 99I), 并利用169份稻种资源、98个重组自交系(RIL)以及217个水稻转基因后代, 对4套标记的准确性和选择效果进行了验证。

结果表明, 4套标记均能准确地检测Pi25/pi25座位。

其中, 标记CAP1/Hinc II 和CAP3/Hpy 99I 特异性识别并酶切显性等位基因, 而标记CAP3/Bgl II 和CAP3/Nde I 特异性识别并酶切隐性等位基因。

利用稻瘟病菌株JS001-20接种RIL 与转基因材料, 抗性表现与标记检测的结果完全一致, 表明该CAPS 标记准确可靠。

分析稻种资源后发现, Pi25基因频率较低(1.2%), 说明该基因在我国水稻稻瘟病抗性育种中还没有被充分利用。

本文的研究结果特别是开发的2对识别并酶切显性等位基因的CAPS 标记可用于分子标记辅助选择, 改良我国早籼稻的稻瘟病抗性。

关键词: 水稻; 稻瘟病抗性; 酶切扩增多态性序列; 标记辅助选择; 单核苷酸多态性More than 50 major rice blast resistance genes have been indentified since late 1980s because of the deve- lopment and utilization of molecular markers [1-3]. Among them, 15 blast resistance genes have been successively cloned through the map-based cloning strategy [4-12]. Al-though a serious of effort has been attempted in elucidat-ing the structure and function of the genes while there isno fundamental breakthrough in application of the genes to improve rice blast resistance in rice breeding programs. With the development of molecular markers, the selec-tion for targeted blast resistance genes using specific molecular markers is commonly practiced and some in-termediate breeding materials carrying the target genes have been bred [13]. One of the quick and effective ways第11期王惠梅等: 稻瘟病抗性基因Pi25特异性CAPS 标记的开发与验证 1961for improving blast resistance in new varieties is to backcross the intermediate breeding materials with commercial elite lines. However, the accuracy of marker- assisted selection (MAS) for the target genes largely de-pends on the strength of linkage between the markers and the target genes. Fortunately, functional markers for those cloned genes have been developed and could provide the accurate and efficient selection [14-15].Gumei 2 is the only early-season semi-dwarf indica rice cultivar with stable and broad-spectrum resistance to Magnaporthe oryzae , the causal pathogen of blast dis-ease [16]. It is considered as a valuable donor for rice blast improvement in China because of its elite agronomic traits. Previous studies have identified and located at least four blast resistance genes and a number of partial resistance QTLs in Gumei 2 [17-18]. Among them, Pi25 is a single copy intronless CC-NBS-LRR type of blast re-sistance gene located in chromosome 6 [19].This work aims at developing and validating the Pi25 functional markers based on the coding sequences of the locus using three categories of rice materials. Two sets of markers, CAP1/Hinc II and CAP3/Hpy 99I, are able to specifically digest the dominant allele Pi25 and recom-mended for MAS in rice breeding programs.1 Materials and Methods1.1 Plant materialsA total of 169 rice germplasm accessions including 129 indica rice and 40 japonica rice, 98 recombinant inbred lines (RILs, F 9) derived from the cross Zhongjian 100/Gumei 2 (donor of Pi25) were grown in the paddy field at the Fuyang Experimental Station, China National Rice Research Institute (CNRRI) and 217 transgenic plants derived from Zhongjian 100 transformed with Pi25 were grown in the greenhouse at CNRRI.1.2 Development of CAPS markersThere are six single nucleotide substitutions (g775a, t1197c, t2444a, c2566g, g2680a, and g2687a) in the cod-ing sequences between Pi25, the resistant allele from Gumei 2, and pi25, the susceptible allele from Zhongjian 100, respectively, leading to the substitution of five amino acids (V259I, F815Y , H856D, V894I, and R896Q). One of the single nucleotide polymorphisms (SNPs), t1197c, is a synonymous mutation in both alleles (Fig. 3). Among the other five SNPs, four SNPs (g775a, t2444a, c2566g, and g2687a) corresponding to four restriction endonuclease (RE) recognition sites were chosen for the development of gene specific markers. Hinc II (g775a) and Hpy 99I (g2687a) detect and digest specifically for the resistant allele Pi25 while Nde I (t2444a) and Bgl II (c2566g) detect and digest specifically for the susceptible allele pi25 (Fig. 1). On the base of these SNPs, we deve- loped four pairs of CAPS markers (CAP1/Hinc II, CAP3/Bgl II, CAP3/Nde I, and CAP3/Hpy 99I) for the detection of the Pi25/pi25 locus. The PRIMER3 online program (http:// /cgi-bin/primer3/primer3_www.cgi) was used for primer design and the primers were synthesizedby Invitrogen (Shanghai) (Table 1).Fig. 1 Four SNPs and the corresponding RE recognition sites atthe Pi25/pi25 locusSNPs are underlined; RE recognition sites are boxed. R: resistant allele;S: susceptible allele.Table 1 Pi25 specific primers and the corresponding restriction endonucleasesPrimer Sequence (5′–3′) Product size (bp)Enzyme Pi25 pi25 CAP1F TGAAATGGGTGAAAGATGAG CAP1R GCCACATCATAATTCCTTGA 406Hinc II+ –Nde I – + CAP3F CCTCACGTTTCTACGTCTTG Bgl II – + CAP3R CACACCATTTCTGATGAACC 409Hpy 99I+ –+: Digestion; –: Non-digestion.1.3 PCR and RE digestionThe PCR was carried out as previously described [20]. The PCR products were digested with Hinc II (Fermentas, ER0491), Nde I (Fermentas, ER0585), Bgl II (Fermentas, ER0082) and Hpy 99I (NEB, R0615L) independently. For Hinc II and Hpy 99I, 4 hours incubation at 37°C, 20 min at 65°C for inactivation in 20 µL reaction mixture containing 5 µL of PCR products (0.25 µg), 5 U RE and 1×Tango buffer (33 mmol L –1 Tris-acetate, pH 7.9, 10mmol L –1 magnesium acetate, 66 mmol L –1 potassium acetate, 0.1 mg L –1 BSA) for Hinc II, and 1×NEB buffer 4 (20 mmol L –1 Tris-acetate, 50 mmol L –1 potassium acetate, 10 mmol L –1 magnesium acetate, 1 mmol L –1 dithiothreitol, 0.1 mg L –1 bovine serum albumin) for Hpy 99I. For Nde I and Bgl II, incubation at 37°C overnight in 30 µL reaction mixture containing 8 µL of PCR products (0.4 µg), 10 U RE and 1× buffer O (50 mmol L –1 Tris-HCl pH 7.5, 10 mmol L –1 MgCl 2, 100 mmol L –11962作物学报第38卷NaCl, 0.1 mg mL–1 BSA). Inactivation of Nde I was achieved at 65°C for 20 min while inactivation of Bgl II was achieved at a final concentration of 20 mmol L–1 EDTA, respectively. The digested products were frac-tionated on 2% agarose gel, and stained with 2×GelRed (Biotium, USA) for visualization.1.4 Cloning of Pi25allelesFor the development and validation of the CAPS markers, we isolated the coding sequences of Pi25/pi25 alleles from Gumei 2, Zhongjian 100, G19, C101A51, Gumei 4, and Tetep as previously reported [19]. The frag-ments were purified and inserted into the pGEM-T Easy vector (Promega, USA) and sequenced by Invitrogen (Shanghai). The coding sequences of the alleles from Gumei 2 (accession number HM448480), Zhongjian 100 (accession number JQ838019), and Tetep (accession number JQ838018) have been deposited in the GenBank.1.5 Blast evaluationThe blast isolate JS001-20 was used for inoculation of 98 RILs, 217 T3 transgenic plants and the recipient Zhongjian 100, the donor Gumei 2 and the susceptible con-trol Lijiangxintuanheigu (LTH) as previously reported [19]. Disease evaluation was carried out following the method described by Bonman et al. [21]2 Results2.1 CAPS analysis of germplasm accessionsTo identify the allelic presence/absence of Pi25/pi25 gene in the germplasm collection, we tested all four pairs of CAPS markers CAP1/Hinc II, CAP3/Nde I, CAP3/Bgl II and CAP3/Hpy 99I. The results showed that three out of 169 rice accessions including C101A51, G19 (Gumei 2/Zhong 156) and Gumei 4 were specifically digested by CAP1/Hinc II and CAP3/Hpy 99I and showed the same band pattern as Gumei 2 (Fig. 2 and Table 2). Further-more, the coding sequences of them were identical. Therefore, C101A51, G19 and Gumei 4 possessed the dominant Pi25 allele as Gumei 2. The other accessions except Tetep were detected and digested by the CAPS markers CAP3/Nde I and CAP3/Bgl II and showed the same band pattern as Zhongjian 100 (Table 2). From the comparison of the coding sequences among Gumei 2, Zhongjian 100, and Tetep, we found that Tetep was con-sistent with Zhongjian 100 at two SNP sites (a775a and a2687a) while consistent with Gumei 2 at the other two SNP sites (t2444t, c2566c) (Fig. 1 and Fig. 3). Because the nucleotide substitutions at positions 2 444 and 2 566 between Zhongjian 100 and Tetep did not result in amino- acid substitutions, therefore, Tetep was predicted to have the identical amino-acid residues at the four SNP sites the same as Zhongjian 100, but different from the donor Gumei 2 at positions 775 and 2687, where a valine and an arginine in the Pi25 were substituted by an isoleucine and a glutamine in pi25, respectively. Although Tetep showed the same amino-acid residues at these four sites as Zhongjian 100, it possessed a new allele because its full coding sequence was 312 nucleotides shorter than that of Gumei 2 and Zhongjian 100 (Fig. 3). Nevertheless, our results indicated that the two markers, CAP1/Hinc II and CAP3/Hpy 99I, could specifically recognize and di-gest the dominant allele Pi25.In addition, the Pi25 allele presented in G19 was originated from Gumei 2 since G19 was derived from the cross Zhongjian 100/Gumei 2. Gumei 4 is a sister line of Gumei 2 with unknown progenitor. The Pi25 allele pre-sented in C101A51 is derived from 5173 [22]. Thus, ex-cept G19 and Gumei 4, only Gumei 2 and C101A51 pos-sessed the dominant Pi25 allele in 167 rice germplasm accessions tested (Table 2). The results indicated that the frequency of Pi25 was low at 1.2% (2/167) and it has not been fully utilized in rice breeding programs in China.2.2 CAPS analysis of RILsTo validate the accuracy of the four CAPS markers, we genotyped 98 RILs derived from the cross Zhongjian 100/Gumei 2. The results showed that Zhongjian 100 possessed the pi25 allele while Gumei 2 contained the Pi25 allele as expected. Among 98 RILs, 56 RILs showed the same band pattern as Gumei 2 while 42 RILs exhibited the same band pattern as Zhongjian 100 (Fig.4). The ratio of Pi25 to pi25 in the RILs was consistent with the expected 1:1 (χ2=2.00<χ20.05=3.84), indicating a free segregation of the locus and could be easily appliedFig. 2 CAPS marker analysis of rice germplasm accessions1: Gumei 2; 2: Zhongjian 100; 3: LTH; 4: C101A51; 5: Tetep; 6: CDR22; 7: G19; 8: Gumei 4; 9–21: Other accessions showing the same band pattern.第11期王惠梅等: 稻瘟病抗性基因Pi25特异性CAPS标记的开发与验证1963Table 2 Distribution of Pi25/pi25 locus in the germplasm based on the CAPS marker analysisPi25/pi25Pi25/pi25) Variety Type Pi25/pi25 Variety Type Variety Type1IZhonghan– II-32B I–Lucaihao I–Guangzhan 63S I – Mianhui 725 I – T97 I –9311 I – Minkezao 1 I – IRBB 14 I –––Baxiludao IMinghuiAUS373 I–63 IBasmati 370 I – Nantehao I – H19 I –Vandana I––Minghui–CDR22 I77 I–+IG19 IMinghuiCO39 I–86Zhongyoudao 1 I – Minghui 70 I – T657 I –C101LAC I – Pei’ai 64 I – C Bao J –H161 I––Han9 J–NipponbareJC101A51 I + Shouguangsimiao I – IR68 I ––Wazushandao–IJ517H333 I–ShuhuiI–IRBB13––Shuhui85 IH593 IC101PKT I – Shuhui 881 I – Zhongjian 2 I ––C57 J–I1IR24 I–ShuangguiI–IRBB3–IR30 IShuangqizhan–IJava14J––IR36 IITaizhong–65I–IRBB5––Teqing IIR64 IMorobereken J – Texianzhan 25 I – Nanjing 42 J ––IRBB8 I–JH811 I–Tianjiqing776R402 I – Chunjiang 06 J – Wuyujing 20 J –IRBB11I––R752 IJWuyoudao–1haoJ300–+/–Tetep IWufujing J–To974 I – Xiushui 11 J – Gumei 4 I +IRBB21I– V20B I–JWuyujing–14IR26 I–––ZDZ057 IWuyujing3 JAimeizao 3 I – Wuyujing 7 J – IRBB4 I –Aijiaonante I – Reyan 1 J – Nanjing 15 J –Aizaizhan I – Xianfeng 1 I – Jingang 30 I ––IRBB7I–IBoB I31–XiangzaoxianTHZ I –B I –Suifuruanzhan I – Xieqingzao–JeffersonJ–ICe46 I04–XiushuiDaonuzhong 58 J – Xiushui 110 J – Zhongguang B I –Duoxi 1 I – Yanhui 559 I – R9308 I –Enhui 58 I – Yujing 6 J – CPSLO17 J –Gang 46B I – Yuexiangzhan I – Lunhui 422 I –Fuhui 838 I – H1 I IRBB10 I –Gumei 2 I + Zhangyouzhan I – MXZ2 I –Guanghui 128 I – Zhenlong 13 I – Zhejing 29 J –Guangluai 4 I – Zhenhui 084 I – Quanzhen 10 I –Gui99 I – Zhenzhan 97B I – Donglian 5 I –Guichao 2 I – Zhong 156 I – Jiayu 253 I –Youzhan I–– Jiahezhan I–R8006 IJiayu 948 J – Zhou 903 I – Zhongzao 39 I –Jianghui 151 I – Zhonghan 4 I – Guixiaozhan I –Jin23B I – Zhongjian 100 I – Zaoxian 276 I –9B I–Hefengzhan I–Kendao10 J–ZhongYueguang J – Zuke 2 I – Yongxian 15 I –Kongyu 131 J – Milyang 46 I – Shanxiaozhan I –213I–ZaoxianPKTX ILTH J––I –7Liantangzao I –XiangzaoxianZachaodao I –Liaojing 294 J – IRAT13X J – Longjing 21 J –Daohuaxiang2 J – Longtepu B I – Zhonghua 11 I –Luhui 17 I – Hongjiaozhan I – Songjing 11 J –Lujiangzao 1 I – 02428 J – Changlixiang J –Mianhui 501 I – Zhonghan 3 J – H17 I –Han 2 J – –+: present for Pi25; –: present for pi25; +/–: Tetep shows the same amino acid residues at the 4 SNPs sites as “Zhongjian 100” but a shortercoding sequence and is considered as a new allele; I: indica rice; J: japonica rice.1964作物学报第38卷(to be continued)第11期王惠梅等: 稻瘟病抗性基因Pi25特异性CAPS标记的开发与验证1965Fig. 3 Comparison of the coding sequences in three rice accessions1966作 物 学 报 第38卷Fig. 4 CAPS marker analysis of RILs1: Gumei 2; 2: Zhongjian 100; 3: LTH; 4–21: 18 RILs derived from the cross Zhongjian 100/Gumei 2. R: resistant; S: susceptible.in breeding practice. The results indicated that the CAPS markers were able to detect accurately the Pi25/pi25 lo-cus in a breeding population, and the markers CAP1/ Hinc II and CAP3/Hpy 99I specifically digesting for the dominant allele were more practical with relatively shorter digestion time.2.3 CAPSanalysis of transgenic lines To further validate the accuracy of the CAPS markers, we genotyped 217 T 3 transgenic progenies (balk seed from 10 independent T 2 lines) originally derived fromZhongjian 100 transformed with Pi25 using the four CAPS markers. The results indicated that four out of 217 individual plants possessed the same band pattern as the donor Gumei 2 while the remaining 213 plants showed the same band pattern as the recipient Zhongjian 100 (Fig. 5) showing that the CAPS markers were also able to ac-curately detect the Pi25/pi25 gene in the transgenic progenies. Again, markers CAP1/Hinc II and CAP3/Hpy 99I specifically detecting and digesting for the dominantallele were much easier to use.Fig. 5 CAPS marker analysis of transgenic plants1: Gumei 2; 2: Zhongjian 100; 3: LTH; 4–21: 18 T 3 transgenic plants; R: resistant; S: susceptible.2.4 Blast resistance evaluationTo confirm the RILs and transgenic plants containing the Pi25 allele were truly resistant to M. oryzae , we chose a blast isolate JS001-20 previously identified compatible to pi25 but incompatible to Pi25 for inocu-lating the 98 RILs and 217 transgenic plants. Our results showed that the 56 RILs and four transgenic plants har-boring Pi25 allele were resistant to JS001-20, the same as the donor Gumei 2 while the remaining RILs and trans-genic plants harboring pi25 allele were susceptible to the isolate, the same as the recipient Zhongjian 100 and the susceptible control LTH (Fig. 4 and Fig. 5). Thus, the results from marker-assisted selection were consistent with those of disease evaluation, indicating the four CAPS markers could accurately and efficiently detect the Pi25/pi25 locus. In addition, Tetep was likely to harbor a new allele at the locus, and was also resistant to the isolate JS001-20. However, it is unknown whether the Tetep allele is incompatible to the isolate because Tetep possesses a number of other blast resistance genes [23]. In practice, we recommend two markers CAP1/Hinc II and CAP3/Hpy 99I that specifically detect and digest the Pi25 allele in order to reduce the workload and the cost.第11期王惠梅等: 稻瘟病抗性基因Pi25特异性CAPS标记的开发与验证19673 DiscussionAccuracy and low cost are two key factors affecting marker-assisted selection. The accuracy of selection de-pends on the strength of linkage between the markers and the target genes. Previous marker-assisted selection usu-ally results in the unsatisfactory outcome because the markers are located far from the target genes [24-25]. In addition, it requires a large sample size and consequently increases the workload and the cost. One of the solutions is to employ flanking markers closely linked to the target gene. This approach is able to largely improve the accu-racy of the selection but also increases the workload and the cost because an additional marker is required [26]. With the progress of fine mapping and cloning of blast resistance genes, the accuracy of MAS reaches a new higher level. First, functional markers theoretically im-prove the selection accuracy to 100%. Second, instead of flanking markers, a single marker is generally enough and the workload and the cost are reduced.At present, a number of PCR-based markers for blast resistance genes have been developed. These new gen-eration/functional markers show a reliable accuracy and efficiency in the selection of the target genes [14-15]. In this study, the CAPS markers were developed based on the nucleotide difference in the coding region between Pi25 and pi25 alleles. Using an extensively-used germplasm collection, a breeding population and a balk of transgenic rice lines we verified the accuracy and effectiveness of the markers. The percent of accuracy reaches 100% and the effectiveness of selection for the locus than for the markers previously reported is hugely improved [18]. Breeding for improved blast resistant varieties has been always an important criterion in Chinese rice breeding programs. However, limited information is available on the usage of known blast resistance genes and on the gene frequency and distribution in Chinese rice germplasm [27]. The blast resistance of Gumei 2 is controlled by multiple genes, one of them Pi25 confer-ring both leaf and neck resistance is considered valuable for improving the resistant level especially for early- season indica rice since the donor Gumei 2 belongs to the early-season indica type of cultivars in China. Unex-pectedly, the frequency of this gene is low for unknown reasons in the extensively used germplasm resources. In addition, at least a new allele of Pi25, with a shorter length of coding sequence as well as base substitutions, was identified in the traditional donor Tetep. Further screening and sequencing of the coding sequence would further clarify the number of alleles at the locus in Chi-nese rice germplasm. This study not only provides four sets of reliable markers for the selection of Pi25/pi25, but also would facilitate the further application of the Pi25 gene to the improvement of blast resistance for the early-season indica rice. In practice, we recommend two markers CAP1/Hinc II and CAP3/Hpy 99I for the speci-ficity to the dominant allele and for the simplicity and cost effective as a whole.4 ConclusionsOn the base of the coding sequences of the blast resis-tance gene Pi25 and its susceptible allele pi25, we de-veloped four sets of CAPS markers in the present study. Using various materials including germplasm accessions, recombinant inbred lines and transgenic plants, the accu-racy and efficiency of the CAPS markers are validated. To make full utilization of the dominant allele, we rec-ommend two sets of the markers CAP1/Hinc II and CAP3/Hpy 99I for marker-assisted selection, especially for the early-indica rice improvement of blast resistance in rice breeding programs.References[1] Jeung J U, Kim B R, Cho Y C, Han S S, Moon H P, Lee Y T, JenaK K. A novel gene, Pi40(t), linked to the DNA markers derived from NBS-LRR motifs confers broad spectrum of blast resistance in rice. 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