Pigm,功能標記,分子標記輔助選擇," /> 3d近1000期基本走势图

中國農業科學 ?? 2019, Vol. 52 ?? Issue (6): 955-967.doi: 10.3864/j.issn.0578-1752.2019.06.001

? 作物遺傳育種·種質資源·分子遺傳學 ? 上一篇    下一篇

水稻廣譜抗稻瘟病基因PigmR功能標記的開發及應用

王芳權1,2,陳智慧1,2,許揚1,2,王軍1,2,李文奇1,2,范方軍1,2,陳麗琴1,陶亞軍1,2,仲維功1,楊杰1,2()   

  1. 1 江蘇省農業科學院糧食作物研究所/國家水稻改良中心南京分中心/江蘇省優質水稻工程技術研究中心,南京 210014
    2 揚州大學/江蘇省糧食作物現代產業技術協同創新中心,江蘇揚州 225009
  • 收稿日期:2018-11-18 接受日期:2019-01-18 出版日期:2019-03-16 發布日期:2019-03-22
  • 通訊作者: 楊杰 E-mail:[email protected]
  • 作者簡介:王芳權,Tel:025-84390320;E-mail: [email protected]
  • 基金資助:
    國家重點研發計劃(2017YFD0100400-3);江蘇省農業科技自主創新資金項目(CX182022);江蘇省現代農業重點研發項目(BE2017368);江蘇省現代農業重點研發項目(BE2018388);江蘇省自然科學基金面上項目(BK20171326)

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

WANG FangQuan1,2,CHEN ZhiHui1,2,XU Yang1,2,WANG Jun1,2,LI WenQi1,2,FAN FangJun1,2,CHEN LiQin1,TAO YaJun1,2,ZHONG WeiGong1,YANG Jie1,2()   

  1. 1 Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement/Jiangsu High Quality Rice R & D Center, Nanjing 210014;
    2 Yangzhou University/Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou 225009, Jiangsu
  • Received:2018-11-18 Accepted:2019-01-18 Online:2019-03-16 Published:2019-03-22
  • Contact: Jie YANG E-mail:[email protected]

摘要:

【目的】稻瘟病是世界上最嚴重的水稻病害之一。通過功能標記的開發,為加快廣譜持久抗稻瘟病基因PigmR在水稻育種中的應用提供依據。【方法】利用Snapgene 2.3.2軟件分析PigmR的堿基變異特征,用Oligo 7設計特異功能標記。為了避免因PCR擴增失敗引起的假陰性,設計擴增內參基因Actin1的引物作為參照,對功能標記進行優化。用功能標記對水稻親本材料、麗江新團黑谷的單基因系材料、育種中間材料和南粳53045/谷梅4號BC1F3群體株系進行鑒定。稻瘟病鑒定的供試菌株為江蘇省稻瘟病代表菌株(2018-4、2018-65、2018-102、2018-222和2018-241)的混合菌。將供試菌株移植RCA培養基上,25℃培養7 d,用黑光燈照射72 h,待稻瘟病菌產生孢子后,再用無菌水洗下,配成10×10倍顯微鏡下每視野30—40個孢子的懸浮液。于水稻抽穗前3—4 d注射混合菌株,每穗注射1 mL菌液,做好標記。在水稻灌漿飽滿后進行抗性調查。【結果】根據PigmRPigmSPigm-R4序列比對及差異分析,設計了8對分子標記引物。通過分子檢測,篩選獲得的PigmR功能標記GMR-3,能特異擴增來源于谷梅4號的PigmR,獲得98 bp產物,而不攜帶PigmR擴增不出產物。以不同濃度配比優化GMR-3與內參基因Actin1檢測引物Actin1-1,發現以0.4 μmol·L -1 GMR-3與0.1 μmol·L -1 Actin1-1組合濃度擴增出PigmRActin1特征條帶的效率相當,效果最優,將該標記命名為GMRA。用該標記擴增水稻樣品,攜帶PigmR的樣品能擴增出146和98 bp條帶,不攜帶PigmR的樣品僅能擴增出146 bp條帶。利用GMRA標記檢測229份水稻材料,只有谷梅4號能擴增出146和98 bp條帶,其他秈稻和粳稻均只能擴增出146 bp條帶。進一步對29份麗江新團黑谷的單基因系材料進行檢測發現,GMRA標記能有效區分PigmRPi9PizPiz-t等同源性較高的基因,有很好的特異性。利用GMRA標記,從240份育種中間材料中篩選到3份攜帶PigmR的材料,可作為該基因的供體材料。利用該標記進行分子標記輔助選擇,將PigmR通過回交轉育到優質食味粳稻南粳53045。對南粳53045/谷梅4號BC1F3群體單株進行分子標記檢測及稻瘟病人工接種鑒定,發現攜帶PigmR的單株均表現為抗或中抗,不攜帶PigmR的單株均表現為高感,表明導入PigmR能顯著改良南粳53045的穗頸瘟抗性。【結論】PigmR的功能標記能有效用于抗稻瘟病遺傳改良和資源篩選。

關鍵詞: 水稻, 稻瘟病, Pigm, 功能標記, 分子標記輔助選擇

Abstract:

【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.

Key words: rice (Oryza sativa L.), rice blast, Pigm, functional marker, molecular marker-assisted selection

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