高抗白粉病小麦-山羊草新种质TA002的创制和遗传研究

doi:10.3864/j.issn.0578-1752.2016.03.002

摘要/Abstract

摘要： 【目的】小麦白粉病是世界范围内对小麦危害最严重的病害之一。培育和推广抗病品种是防治小麦白粉病最经济、有效、安全的途径。偏凸山羊草和柱穗山羊草是普通小麦的近缘物种，蕴藏着丰富的抗病、抗虫、抗逆和优质等优异基因。利用远缘杂交途径，将偏凸山羊草和柱穗山羊草的抗白粉病基因导入普通小麦，培育抗白粉病小麦新种质，为小麦白粉病抗性遗传改良提供新抗源。【方法】通过细胞学分析和结实率调查，明确TA002及其与普通小麦杂种的细胞学稳定性和育性特点。利用十二烷基磺酸钠聚丙烯酰氨凝胶电泳（odium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE）及酸性聚丙烯酰胺凝胶电泳（acid polyacrylamide gel electrophoresis，A-PAGE）方法，分析TA002的高分子量麦谷蛋白亚基（high molecular weight glutenin subunit，HMW-GS）、低分子量麦谷蛋白亚基（low molecular weight glutenin subunit，LMW-GS）及醇溶蛋白亚基组成；通过白粉病菌分小种接种鉴定，明确TA002对小麦白粉病的抗性及其遗传特点；利用基因组原位杂交（genomic in situ hybridization，GISH）、多色原位杂交（multicolor genomic in situ hybridization，mc-GISH）、多色荧光原位杂交（multicolor fluorescence in situ hybridization，mc-FISH）及分子标记技术，分析明确TA002的染色体组成特点。【结果】TA002染色体数目为42条，它及其与普通小麦的杂种F₁减数第一次分裂中期细胞（pollen mother cells at metaphase I，PMCs MI）均含有21个二价体，减数第一次分裂后期细胞（pollen mother cells at anaphase I，PMCs AI）中的染色体分离均衡，结实率正常。在成株期，TA002、SDAU18及其双亲偏凸山羊草和柱穗山羊草对白粉病均具有良好抗性，而烟农15对白粉病表现髙感。TA002/辉县红F₁、TA002/铭贤169F₁对白粉病菌种E09表现免疫，F₂单株对E09的抗感分离比例符合3﹕1。种子贮藏蛋白分析结果表明，在TA002的醇溶蛋白和谷蛋白中均含有SDAU18的特有亚基，其醇溶蛋白还含有双亲没有的新型亚基。分别以单芒山羊草和尾状山羊草的基因组总DNA为探针，烟农15的基因组总DNA为封阻，对TA002的根尖细胞染色体进行基因组原位杂交，均未检测到杂交信号。同时以不同荧光素标记的乌拉尔图小麦和粗山羊草基因组总DNA为探针，拟斯卑尔脱山羊草基因组总DNA为封阻，对TA002的根尖细胞染色体进行mc-GISH，并利用以不同荧光素标记的寡核苷酸pSc119.2和pTa535对TA002的根尖细胞染色体进行mc-FISH，发现TA002具有完整的A、B和D基因组，但其4A、5A、6B、7B和5D染色体在FISH带型上与亲本烟农15的对应染色体具有显著差异。分子标记检测结果表明，TA002含有来源于偏凸山羊草和柱穗山羊草的遗传物质。【结论】TA002是一个细胞学稳定、农艺性状和育性良好的小麦-山羊草渐渗系，它含有偏凸-柱穗山羊草双二倍体特有的贮藏蛋白亚基及其双亲没有的新亚基，且高抗小麦白粉病，其白粉病抗性受显性单基因控制，该基因可能是来自偏凸山羊草或柱穗山羊草的一个新的白粉病抗性基因。

关键词: 小麦, 山羊草, 白粉病, 种质系, 鉴定

Abstract: 【Objective】 Powdery mildew is one of the most devastating diseases of wheat. It is widely accepted that the most economic, efficient and safest way to control powdery mildew is breeding and planting powdery mildew resistant cultivars. Aegilops ventricosa and Aegilops cylindrica, which possess many favorable characters and good qualities such as resistance to diseases, tolerance to environmental stresses, are close-related relatives of wheat. The objective of this study was to develop novel powdery mildew resistant germplasm line via wide hybridization between common wheat and Aegilops ventricosa and/or Aegilops cylindrica for genetic improvement. 【Method】 Improved fuchsin squash and seed set examination were used to determine the cytogenetic stability and fertility of TA002. Acid polyacrylamide gel electrophoresis (A-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to analyze the subunit composition of gliadin, high and low molecular weight glutenin (HMW-GS and LMW-GS) respectively. Genomic in situ hybridization (GISH), multicolor genomic in situ hybridization (mc-GISH), multicolor fluorescence in situ hybridization (mc-FISH) and molecular markers were employed to detect the genetic nature of TA002. Respective inoculation of powdery mildew isolates was performed to detect the reaction of TA002 to powdery mildew.【Result】 TA002 (2n = 42) showed equivalent seed set to that of common wheat. Both TA002 and its hybrid F₁ with common wheat Mingxian169 housed 21 bivalents in the pollen mother cells observed at metaphase I (PMCs MI) and took on chromosomal segregation of 21 to 21 in pollen mother cells examined at anaphase I (PMCs AI). Contrast to wheat parent Yannong15 highly susceptible to powdery mildew, TA002, similar to SDAU18 and its parents Aegilops ventricosa and Aegilops cylindrica, was highly resistant to powdery mildew. Immunization and resistant-susceptible segregation of 3﹕1 was observed respectively in F₁and F₂ of the crosses between TA002 and common wheats susceptible to powdery mildew. The analysis of seed storage proteins of TA002 and its parents revealed that TA002 not only had new glutenin and gliadin subunits specific to SDAU18, but also possessed a novel gliadin subunit derived from its neither parents. When probed respectively by genomic DNA of Aegilops uniaristata and Aegilops caudata in root tip cell chromosomes of TA002, no probe signals were detected. Multicolor genomic in situ hybridization (mc-GISH) and multicolor fluorescence in situ hybridization (mc-FISH) indicated that TA002 had three complete genomes (A, B and D) although five pairs of chromosomes 4A, 5A,6B, 7B and 5D in it showed significantly different FISH pattern from that of corresponding ones in Yannong15. Mc-GISH was carried out simultaneously using genomic DNA of Triticum uratu and that of Aegilops tauschii labeled with different fluoresceins as probes, and with genomic DNA of Aegilops speltoides as blocker. Mc-FISH was performed with two oligonucleotides pSc119.2 and pTa-535 fluorescing differently. Molecular marker analysis revealed TA002 housed alien genetic materials not only from Aegilops ventricosa but also from Aegilops cylindrica. 【Conclusion】TA002, derived from Aegilops ventricosa or Aegilops cylindrica with high powdery mildew resistance controlled by a single dominant gene, was found as a novel wheat-Aegilops introgression line of cytogenetic stability and good fertility. In addition, TA002 possessed novel seed storage protein subunits either specific to Aegilops ventricosa -Aegilops cylindrica amphiploid or in neither parents.

Key words: Triticum aestivum, Aegilops, powdery mildew, germplasm line, introgression line

王玉海，何方，鲍印广，明东风，董磊，韩庆典，李莹莹，王洪刚. 高抗白粉病小麦-山羊草新种质TA002的创制和遗传研究[J]. 中国农业科学, 2016, 49(3): 418-428.

WANG Yu-hai, HE Fang, BAO Yin-guang, MING Dong-feng, DONG Lei, HAN Qing-dian, LI Ying-ying, WANG Hong-gang. Development and Genetic Analysis of a Novel Wheat-Aegilops Germplasm TA002 Resistant to Powdery Mildew[J]. Scientia Agricultura Sinica, 2016, 49(3): 418-428.

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参考文献

[1] Li G Q, Fang T L, Zhang H T, Xie C J, Li H J, Yang T M, Nevo E, Fahima T, Sun Q X, Liu Z Y. Molecular identification of a new powdery mildew resistance gene Pm41 on chromosome 3BL derived from wild emmer (Triticum turgidum var. dicoccoides). Theoretical and Applied Genetics, 2009, 119: 531-539.

[2] Roelfs A P. Foliar fungal diseases of wheat in the People's Republic of China. Plant Disease Reporter, 1977, 61: 836-841.

[3] 王心宇. 分子标记技术在小麦抗白粉病育种及指纹图谱分析中的应用研究[D]. 南京: 南京农业大学, 2000.

Wang X Y. Application of molecular markers in the improvement for powdery mildew resistance and fingerprint mapping of wheat [D]. Nanjing: Nanjing Agricultural University, 2000. (in Chinese)

[4] 刘万才, 邵振润. 我国小麦白粉病大区流行的气候因素分析. 植保技术与推广, 1998, 18(1): 3-5.

Liu W C, Shao Z R. Analysis on climatic factor for epidemiology of wheat powdery mildew in China. Plant Protection Technology and Extension, 1998, 18(1): 3-5. (in Chinese)　

[5] 解超杰, 杨作民, 孙其信. 小麦抗白粉病基因的分子标记. 中国农业大学学报, 2003, 8(1): 1-6.

Xie C J, Yang Z M, Sun Q X. The molecular markers of powdery mildew resistance genes in wheat. Journal of China Agricultural University, 2003, 8(1): 1-6. (in Chinese)

[6] Huang X Q, Hsam S L K, Mohler V, Röder M S, Zeller F J. Genetic mapping of three alleles at the Pm3 locus conferring powdery mildew resistance in common wheat (Triticum aestivum L.). Genome, 2004, 47: 1130-1136.

[7] Qiu Y C, Zhou R H, Kong X Y, Zhang S S, Jia J Z. Microsatellite mapping of a Triticum urartu Tum. derived powdery mildew resistance gene transferred to common wheat (Triticum aestivum L.). Theoretical and Applied Genetics, 2005, 111: 1524-1531.

[8] 郝元峰. 小麦抗白粉病基因的分子标记定位及标记辅助选择[D]. 泰安: 山东农业大学, 2008.

Hao Y F. Location of wheat powdery mildew resistance genes with molecular marker and marker-assisted selection [D]. Tai’an: Shandong Agricultural University, 2008. (in Chinese)

[9] Huang X Q, Röder M S. Molecular mapping of powdery mildew resistance genes in wheat: A review. Euphytica, 2004, 137: 203-223.

[10] Luo P G, Luo H Y, Chang Z J, Zhang H Y, Zhang M, Ren Z L. Characterization and chromosomal location of Pm40 inTheoretical and Applied Genetics, 2009, 118: 1059-1064. common wheat: A new gene for resistance to powdery mildew derived from Elytrigia intermedium.

[11] Hua W, Liu Z J, Zhu J, Xie C J, Yang T M, Zhou Y L, Duan X Y, Sun Q X, Liu Z Y. Identification and genetic mapping of pm42, a new recessive wheat powdery mildew resistance gene derived from wild emmer (Triticum turgidum var. dicoccoides). Theoretical and Applied Genetics, 2009, 119: 223-230.

[12] He R L, Chang Z J, Yang Z J, Yuan Z Y, Zhan H X, Zhang X J, Liu J X. Inheritance and mapping of powdery mildew resistance gene Pm43 introgressed from Thinopyrum intermedium into wheat. Theoretical and Applied Genetics, 2009, 118: 1173-1180.

[13] 何方. 小麦-长穗偃麦草杂种后代的分子细胞遗传学分析及种质材料的筛选鉴定[D]. 泰安: 山东农业大学, 2014.

He F. Molecular cytogenetic analysis of wheat-Elytrigia elongata hybrids and identification of Trititrigia germplasms [D]. Tai’an: Shandong Agricultural University, 2014. (in Chinese)

[14] Mohler V, Bauer C, Schweizer G, Kemf H, Hartl L. Pm50: A new powdery mildew resistance gene in common wheat derived from cultivated emmer. Journal of Applied Genetics, 2013, 54: 259-263.

[15] Petersen S, Lyerly J H, Worthington M L, Parks W R, Cowger C, Marshall D S, Brown G, Murphy J P. Mapping of powdery mildew resistance gene Pm53 introgressed from Aegilops speltoides into soft red winter wheat. Theoretical and Applied Genetics, 2015, 128: 303-312.

[16] Hao Y F, Parks R, Cowger C, Chen Z B, Wang Y Y, Bland D, Murphy J P, Guedira M, Brown G G, Johnson J. Molecular characterization of a new powdery mildew resistance gene Pm54 in soft red winter wheat. Theoretical and Applied Genetics, 2015, 128: 465-476.

[17] Ekiz H, Safi Kiral A, Akçin A, Simsek L. Cytoplasmic effects on quality traits of bread wheat (Triticum aestivum L.). Euphytica, 1998, 100: 189-196.

[18] El B M, Benlhabib O, Nachit M M, Houari A, Bentika A, Nsarellah N, Lhaloui S. Identification in Aegilops species of resistant sources to Hessian fly (Diptera:Cecidomyiidae) in Morocco. Genetic Resources and Crop Evolution, 1998, 45: 343-345.

[19] Zaharieva M, Dimov A, Stankova P, David J, Monneveux P. Morphological diversity and potential interest for wheat improvement of three Aegilops L. species from Bulgaria. Genetic Resources and Crop Evolution, 2003, 50: 507-517.

[20] Spetsov P, Plamenov D, Kiryakova V. Distribution and characterization of Aegilops and Triticum species from the Bulgarian Black Sea Coast. Central European Journal of Biology, 2006, 1(3): 399-411.

[21] Zaharieva M, Prosperi J M, Monneveux P. Ecological distribution and species diversity of Aegilops L. genus in Bulgaria. Biodiversity and Conservation, 2004, 13: 2319-2337.

[22] Farooq S, Azam F. Co-existence of salt and drought tolerance in Triticeae. Hereditas, 2001, 135: 205-210.

[23] Landjeva S, Merakchijska-Nikolova M, Ganeva G. Copper toxicity tolerance in Aegilops and Haynaldia seedlings. Plant Biology, 2003, 46(3): 479-480.

[24] 颜济, 杨俊良. 小麦簇生物系统学: 小麦-山羊草复合群. 北京: 中国农业出版社, 1999: 124-132, 165-166.

Yan J, Yang J L. Triticeae Biosystematics: Triticum-Aegilops Complex. Beijing: China Agriculture Press, 1999: 124-132, 165-166. (in Chinese)

[25] Bao Y G, Wu X, Zhang C, Li X F, He F, Qi X L, Wang H G. Chromosomal constitutions and reactions to powdery mildew and stripe rust of four novel wheat-Thinopyrum intermedium partial amphiploids. Journal of Genetics and Genomics, 2014, 12: 663-666.

[26] Kato A, Jonathan C L, Birchler J A. Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(37): 13554-13559.

[27] Tang Z X, Yang Z J, Fu S L. Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis. Journal of Applied Genetics, 2014, 3: 313-318.

[28] Fu S L, Lv Z L, Qi B, Guo X, Li J, Liu B, Han F P. Molecular cytogenetic characterization of wheat-Thinopyrum elongatum addition, substitution and translocation lines with a novel source of resistance to wheat fusarium head blight. Journal of Genetics and Genomics,2012, 39: 103-110.

[29] Ahmadpoor F, Asghari-Zakaria R, Firoozi B, Shahbazi H. Investigation of diversity in Aegilops biuncialis and Aegilops umbellulata by A-PAGE. Natural Product Research, 2014, 19: 1626-1636.

[30] Yan Y M, Yu J Z, Jiang Y, Hu Y K, Cai M H, Hsam S L K, Zeller F J. Capillary electrophoresis separation of high molecular weight glutenin subunits in bread wheat (Triticum aestivum L.) and related species with phosphate-based buffers. Electrophoresis, 2003, 24: 1429-1436.

[31] Zhao C H, Cui F, Zong H, Wang Y H, Bao Y G, Hao Y F, Du B, Wang, H G. Transmission of the chromosome 1R in winter wheat germplasm Aimengniu and its derivatives revealed by molecular markers. Agricultural Sciences in China, 2009, 6: 652-657.

[32] Cifuentes M, Garcia-Agüero V, Benavente E. A comparative analysis of chromosome pairing at metaphase I in interspecific hybrids between durum wheat (Triticum turgidum L.) and the most widespread Aegilops species. Cytogenetic and Genome Research, 2010, 129: 124-132.

[33] Burt C, Nicholson P. Exploiting co-linearity among grass species to map the Aegilops ventricosa-derived Pch1 eyespot resistance in wheat and establish its relationship to Pch2. Theoretical and Applied Genetics, 2011, 123: 1387-1400.

[34] Arabbeigi M, Arzani A, Majidi M M, Kiani R, Tabatabaei B E S, Habibi F. Salinity tolerance of Aegilops cylindrica genotypes collected from hyper-saline shores of Uremia Salt Lake using physiological traits and SSR markers. Acta Physiologiae Plantarum, 2014, 36(8): 2243-2251.

[35] Kiani R, Arzani A, Habibi F. Physiology of salinity tolerance in Aegilops cylindrica. Acta Physiologiae Plantarum, 2015, 37: 1-10.

[36] 王玉海. 偏凸-柱穗山羊草双二倍体SDAU18的细胞分子遗传学分析[D]. 泰安: 山东农业大学, 2009.

Wang Y H. Molecular cytogenetic analysis of amphiploid SDAU18 from Aegilops ventricosa and Aegilops cylindrica [D]. Tai’an: Shandong Agricultural University, 2009. (in Chinese)

[37] 曹新有, 刘建军, 程敦公, 宋健民, 李豪圣, 刘爱峰, 赵振东. 小麦VPM1后代Yr17-Lr37-Sr38、Pch1基因分子标记检测. 山东农业科学, 2012, 44(8): 1-4.

Cao X Y, Liu J J, Cheng D G, Song J M, Li H S, Liu A F, Zhao Z D. Identification of Yr17-Lr37-Sr38 and Pch1 genes in segregation wheat plants of Jimai22 × VPM1 using molecular markers. Shandong Agricultural Sciences, 2012, 44(8): 1-4. (in Chinese)

[38] 孙喜营, 崔磊, 孙蕾, 孙艳玲, 邱丹, 邹景伟, 武小菲, 王晓鸣, 李洪杰. 抗禾谷孢囊线虫小麦新种质H3714和H4058的培育与鉴定. 作物学报, 2015, 41(6): 872-880.

Sun X Y, Cui L, Sun L, Sun Y L, Qiu D, Zou J W, Wu X F, Wang X M, Li H J. Development and identification of wheat lines H3714 and H4058 resistant to cereal cyst nematode. Acta Agronomica Sinica, 2015, 41(6): 872-880. (in Chinese)

[39] Singh S, Franks C D, Huang L, Brown-Guedira G L, Marshall D S, Gill B S, Fritz A. Lr41, Lr39, and a leaf rust resistance gene from Aegilops cylindrica may be allelic and are located on wheat chromosome 2DS. Theoretical and Applied Genetics, 2004, 108: 586-591.

[40] Babaiants L T, Dubinina L A, Iushchenko G M. The detection of nonallelic to known genes of resistance to Tilletia caries (DC) Tul. in wheat strains from interspecific hybridization (Triticum aestivum× Aegilops cylindrica). Cytology and Genetics, 2000, 34(4): 32-40.

[41] Babaiants O V, Babaiants L T, Horash A F, Vasilev A A, Trackovetskaia V A, Paliasnǐǐ V A. Genetics determination of wheat resistance to Puccinia graminis F. sp. tritici deriving from Aegilops cylindrica, Triticum erebuni and amphidiploid 4. Cytology and Genetics, 2012, 46(1): 9-14.