水稻广谱抗稻瘟病基因PigmR功能标记的开发及应用

【目的】稻瘟病是世界上最严重的水稻病害之一。通过功能标记的开发,为加快广谱持久抗稻瘟病基因PigmR在水稻育种中的应用提供依据。【方法】利用Snapgene 2.3.2软件分析PigmR的碱基变异特征,用Oligo 7设计特异功能标记。为了避免因PCR扩增失败引起的假阴性,设计扩增内参基因Actin1的引物作为参照,对功能标记进行优化。用功能标记对水稻亲本材料、丽江新团黑谷的单基因系材料、育种中间材料和南粳53045/谷梅4号BC₁F₃群体株系进行鉴定。稻瘟病鉴定的供试菌株为江苏省稻瘟病代表菌株（2018-4、2018-65、2018-102、2018-222和2018-241）的混合菌。将供试菌株移植RCA培养基上,25℃培养7 d,用黑光灯照射72 h,待稻瘟病菌产生孢子后,再用无菌水洗下,配成10×10倍显微镜下每视野30—40个孢子的悬浮液。于水稻抽穗前3—4 d注射混合菌株,每穗注射1 mL菌液,做好标记。在水稻灌浆饱满后进行抗性调查。【结果】根据PigmR与PigmS、Pigm-R4序列比对及差异分析,设计了8对分子标记引物。通过分子检测,筛选获得的PigmR功能标记GMR-3,能特异扩增来源于谷梅4号的PigmR,获得98 bp产物,而不携带PigmR扩增不出产物。以不同浓度配比优化GMR-3与内参基因Actin1检测引物Actin1-1,发现以0.4 μmol·L ^-1 GMR-3与0.1 μmol·L ^-1 Actin1-1组合浓度扩增出PigmR和Actin1特征条带的效率相当,效果最优,将该标记命名为GMRA。用该标记扩增水稻样品,携带PigmR的样品能扩增出146和98 bp条带,不携带PigmR的样品仅能扩增出146 bp条带。利用GMRA标记检测229份水稻材料,只有谷梅4号能扩增出146和98 bp条带,其他籼稻和粳稻均只能扩增出146 bp条带。进一步对29份丽江新团黑谷的单基因系材料进行检测发现,GMRA标记能有效区分PigmR与Pi9、Piz、Piz-t等同源性较高的基因,有很好的特异性。利用GMRA标记,从240份育种中间材料中筛选到3份携带PigmR的材料,可作为该基因的供体材料。利用该标记进行分子标记辅助选择,将PigmR通过回交转育到优质食味粳稻南粳53045。对南粳53045/谷梅4号BC₁F₃群体单株进行分子标记检测及稻瘟病人工接种鉴定,发现携带PigmR的单株均表现为抗或中抗,不携带PigmR的单株均表现为高感,表明导入PigmR能显著改良南粳53045的穗颈瘟抗性。【结论】PigmR的功能标记能有效用于抗稻瘟病遗传改良和资源筛选。

Development and Application of the Functional Marker for the Broad-Spectrum Blast Resistance Gene PigmR in Rice

【Objective】Rice blast is one of the most serious rice diseases in the world. The objective of this study was to develop the functional marker for broad-spectrum blast resistance gene PigmR in rice. The marker improved the application of PigmR in blast resistance rice breeding. 【Method】The characteristics of PigmR were analyzed by Snapgene 2.3.2, and the specific function markers were designed with Oligo 7. To avoid mistake results of the PCR amplification failure, the functional marker was optimized by the specific primers of Actin1 as a reference. The parent materials, the monogenic lines of Lijiangxintuanheigu (LTH), the bridge materials, and the BC₁F₃ population lines of Nangeng 53045/Gumei 4 were identified by the functional markers. The blast isolates in this study were the mixed representative strains of rice blast in Jiangsu Province (2018-4, 2018-65, 2018-102, 2018-222, and 2018-241). The tested isolates were transplanted into RCA medium, cultured at 25 ℃ for 7 d, and irradiated for 72 h. After spores were produced, they were washed with sterile water and then formulated 30-40 spores per field in 10×10 microscope. The mixed spores were injected into each panicle with 1 mL of blast isolates solution at 3-4 days before heading. The resistance was investigated after the rice grains were matured.【Result】Eight pairs of molecular markers were designed, according to the sequence difference of PigmR and PigmS, Pigm-R4. By molecular detected, the function marker of PigmR, GMR-3, could specifically amplify PigmR from Gumei 4 with 98 bp fragment, and no fragment was amplified in the samples without PigmR. The functional marker was optimized by different concentrations of GMR-3 and Actin1-1 (a marker for the internal reference gene Actin1), results shown that the marker consist of 0.4 μmol·L ^-1 GMR-3 and 0.1 μmol·L ^-1 Actin1-1 had the best effect. The functional marker was named GMRA. Samples carrying PigmR were amplified the expected size of 146 and 98 bp by GMRA. By contrast, samples without PigmR were only amplified a 146 bp fragment. The 229 rice materials were detected with GMRA, only Gumei 4 amplified the size of 146 and 98 bp, others only amplified a 146 bp fragment. Furthermore, the results detected the monogenic lines of LTH by GMRA suggested that the marker had a strong specificity and could effectively distinguish PigmR from homology genes, such as Pi9, Piz, and Piz-t. Moreover, three donor materials carrying PigmR were obtained from 240 bridge materials by GMRA. By molecular marker-assisted selection (MAS) of GMRA, PigmR was transferred to the good eating quality rice cultivar Nangeng53045 by backcrossing. The BC₁F₃ plants with PigmR showed resistant/middle resistant to the panicle blast, while others showed high susceptible. It was suggested that PigmR observably improved the resistance of Nangeng53045-Pigm lines in the panicle blast.【Conclusion】In conclusion, the functional marker of PigmR can be effectively used for genetic improvement in breeding and germplasm screening.