欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2020, Vol. 46 ›› Issue (11): 1743-1749.doi: 10.3724/SP.J.1006.2020.01016

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦高效转基因受体品系CB037的抗条锈性分析

郑燕燕(), 黄德华, 李金龙, 张会飞, 鲍印广, 倪飞, 吴佳洁*()   

  1. 山东农业大学农学院 / 作物生物学国家重点实验室, 山东泰安 271018
  • 收稿日期:2020-02-24 接受日期:2020-06-02 出版日期:2020-11-12 网络出版日期:2020-06-15
  • 通讯作者: 吴佳洁
  • 作者简介:E-mail:zyy18854889722@163.com, Tel: 0538-8242279
  • 基金资助:
    本研究由国家转基因生物新品种培育重大专项(2016ZX08009003001);山东省高等学校“青创科技计划”项目(2019KJF026)

Analysis of the stripe rust resistance in a wheat line CB037 with high regeneration and transformation efficiency

ZHENG Yan-Yan(), HUANG De-Hua, LI Ji-Long, ZHANG Hui-Fei, BAO Yin-Guang, NI Fei, WU Jia-Jie*()   

  1. State Key Laboratory of Crop Biology / College of Agronomy, Shandong Agricultural University, Tai'an 271018, Shandong, China
    State Key Laboratory of Crop Biology / College of Agronomy, Shandong Agricultural University, Tai’an 271018, Shandong, China
  • Received:2020-02-24 Accepted:2020-06-02 Published:2020-11-12 Published online:2020-06-15
  • Contact: Jia-Jie WU
  • Supported by:
    This study was supported by the National Major Project for Developing New GM Crops(2016ZX08009003001);the Youth Innovation and Scientific Program of Higher Education of Shandong(2019KJF026)

RichHTML

13

PDF

865

摘要：

小麦遗传转化是开展小麦基因克隆、基因编辑及基因工程等研究的重要基础。获得易于转化及遗传背景纯合的受体材料是高效开展小麦遗传转化的关键。近年来, 小麦品系CB037作为受体材料被广泛利用, 并获得了较高的转化效率, 在小麦研究中发挥了重要作用。虽然野生型CB037农艺性状整齐一致, 但遗传背景并不纯合, 对小麦条锈菌(Puccinia striiformis f. sp. tritici)表现高感和高抗两种不同反应。本研究分离了抗病株系CB037-PstR及感病株系CB037-PstS, 并创建了F2分离群体。遗传分析表明, CB037-PstR携带单个显性抗病基因。利用BSR-Seq测序分析及分子标记检测, 将抗病位点定位于1B染色体短臂。进一步荧光原位杂交表明, CB037-PstR为1BL/1RS易位系, 可能携带抗条锈病基因Yr9。本研究明确了CB037的遗传背景及其抗条锈性, 并分离了抗病及感病株系, 为有效利用CB037作为受体材料开展小麦转基因研究奠定基础。

关键词: 小麦遗传转化, 1BL/1RS易位系, 条锈病抗性, BSR-Seq, Yr9

Abstract:

Genetic transformation is used for efficient gene cloning, gene editing and gene engineering, etc. Obtaining recipient lines amenable to transformation and with pure genetic background is critical for high efficiency transformations. For recent years, the wheat inbreed line CB037 had been widely used as a recipient for transgenes and obtain its high transformation potential. Despite having stable agronomic traits, the CB037 is genetically heterogeneous for resistance to wheat stripe rust (Puccinia striiformis f. sp. tritici, Pst). In this study, the Pst-resistant line CB037-PstR and Pst-susceptible line CB037-PstS were isolated, and their F2 population was created. Genetic analysis showed that the CB037-PstR carried a single dominant resistance gene. The identified resistance gene was mapped on the short arm of chromosome 1B using BSR-seq and molecular marker analysis. GISH results further revealed that CB037-PstR is a 1BL/1RS translocation line and likely carried Yr9. This study segregated genetic heterogeneity in CB037 for stripe rust resistance and isolated its Pst-susceptible and resistant lines. These results will facilitate trait-oriented use of CB037 in genetic engineering of wheat.

Key words: wheat transformation, 1BL/1RS translocation line, stripe rust resistance, BSR-Seq, Yr9

图1

CB037所分离出的两个株系对条锈菌的不同反应 1, 2: 苗期叶片; 3: 成株期叶片。"

图2

抗病株系CB037-PstR与感病株系CB037-PstS及F2感病单株混合池间差异位点的染色体分布图 竖线及绿色圆点分别代表小麦染色体及着丝粒。上方数字及字母代表染色体编号, 下方数字为定位于该染色体的SNP及InDel数量, 短横线代表SNP及InDel所在位置。左侧标尺代表染色体长度, 单位Mb。"

表1

本研究开发的分子标记"

分子标记
Molecular marker		 染色体及位置
Chromosome & position (Mb)		 正向引物
Forward primer
(5′-3′)		 反向引物
Reverse primer
(5′-3′)		 退火温度
Annealing temperature (℃)		 内切酶
Restriction enzyme		 多态性
Polymorphism
(bp)
CBSR-53/54 a 1B, 69.9 GAACGGCTCGGATCTGATC TGTACAACAACAAGAAGGAGGAG 55 Dde I CBS: 79+83
CBR: 163
CBSR-1/2 b 1B, 98.7 CCGGAGTTGAAGATAAGTAGTTG CTTCCCTGTATTCGAACAGACTCA 56 N/A CBS: 242
CBR: 0
CBSR-35/36 a 1B, 115.3 ACACCGAGGTGCGTCTAAT TATCCCATGCGGTAAATGGAC 55 Msp I CBS: 150+57
CBR: 207
InDel-3/4 b 1B, 376.6 TCCATGGCAGTACCGCTTGCTT CCGGAGCAGCAATGAAACGTC 56 N/A CBS: 240
CBR: 207
CBSR-25/26 b 4A, 723.7 GCCGCGTTTTGCATGTTTTCCG TGGGTTGAATTTTGGGTTGTTTGAA 56 N/A CBS: 408
CBR: 378

图3

抗条锈病基因Yr9分子标记(iag95, AF1/4, H20)的检测结果 S、R分别表示F2个体的感病及抗病表型; CK为空白对照。"

图4

CB037两个株系的染色体荧光原位杂交鉴定 A, B: CB037-PstS; C, D: CB037-PstR; A和C: GISH鉴定; B和D: FISH鉴定。标尺长度为10 μm。"

[1] Hensel G. Genetic transformation of Triticeae cereals—summary of almost three-decade’s development. Biotechnol Adv, 2020,40:107484. doi: 10.1016/j.biotechadv.2019.107484.
pmid: 31751606
[2] Harwood W A. Advances and remaining challenges in the transformation of barley and wheat. J Exp Bot, 2012,63:1791-1798.
pmid: 22140237
[3] 叶兴国, 徐惠君, 杜丽璞, 何光源, 王轲, 林志珊. 小麦规模化转基因技术体系构建及其应用. 中国农业科学, 2014,47:4155-4171.
Ye X G, Xu H J, Du L P, He G Y, Wang K, Lin Z S. Establishment and application of large-scale transformation systems in wheat. Sci Agric Sin, 2014,47:4155-4171 (in Chinese with English abstract).
[4] Wang K, Riaz B, Ye X G. Wheat genome editing expedited by efficient transformation techniques: progress and perspectives. Crop J, 2018,6:22-31.
[5] Hayta S, Smedley M A, Demir S U, Blundell R, Hinchliffe A, Atkinson N, Harwood W A. An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat(Triticum aestivum L.). Plant Methods, 2019,15:121.
pmid: 31673278
[6] Demirer G S, Zhang H, Matos J L, Goh N S, Cunningham F J, Sung Y, Chang R, Aditham A J, Chio L, Cho M J. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat Nanotechnol, 2019,14:456-464.
pmid: 30804481
[7] Ishida Y, Tsunashima M, Hiei Y, Komari T. Wheat (Triticum aestivum L.) transformation using immature embryos. Agrobact Protoc, 2015,1223:189-198.
[8] 张伟, 尹米琦, 赵佩, 王轲, 杜丽璞, 叶兴国. 我国部分主推小麦品种组织培养再生能力评价. 作物学报, 2018,44:208-217.
Zhang W, Yin M Q, Zhao P, Wang K, Du L P, Ye X G. Regeneration capacity evaluation of some largely popularized wheat varieties in China. Acta Agron Sin, 2018,44:208-217 (in Chinese with English abstract).
[9] Wang K, Liu H Y, Du L P, Ye X G. Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties. Plant Biotechnol J, 2017,15:614-623.
doi: 10.1111/pbi.12660 pmid: 27862820
[10] 张云龙, 王美蛟, 张悦, 褚翠萍, 林志珊, 徐琼芳, 叶兴国, 陈孝, 张宪省. 不同簇毛麦6VS染色体臂的白粉病抗性特异功能标记的开发及应用. 作物学报, 2012,38:1827-1832.
Zhang Y L, Wang M J, Zhang Y, Chu C P, Lin Z S, Xu Q F, Ye X G, Chen X, Zhang X S. Development and application of functional markers specific to powdery mildew resistance on chromosome arm 6VS from different origins of Haynaldia villosa. Acta Agron Sin, 2012,38:1827-1832 (in Chinese with English abstract).
[11] 陈龙飞, 严以苹, 汪信东, 刘艳, 张怀渝, 张增艳. 兼抗白粉病和黄矮病小麦育种材料的创造与鉴定. 植物遗传资源学报, 2013,14:925-929.
Chen L F, Yan Y P, Wang X D, Liu Y, Zhang H Y, Zhang Z Y. Development and detection of wheat breeding materials resistant to both powdery mildew and barley yellow dwarf virus. J Plant Genet Resour, 2013,14:925-929 (in Chinese with English abstract).
[12] 董鲁浩, 姚富泉, 詹悠, 李兴国, 别晓敏. 春小麦成熟胚愈伤组织诱导及再生体系的优化. 分子植物育种, 2019,18:1244-1249.
Dong L H, Yao F Q, Zhan Y, Li X G, Bie X M. Optimization of callus induction and regeneration system of mature embryo in spring wheats. Mol Plant Breed, 2019,18:1244-1249 (in Chinese with English abstract).
[13] Zhang C Z, Huang L, Zhang H F, Hao Q Q, Lyu B, Wang M N, Lynn E, Liu M, Kou C L, Qi J, Chen F J, Li M K, Gao G, Ni F, Zhang L Q, Hao M, Wang J R, Chen X M, Luo M C, Zheng Y L, Wu J J, Liu D C, Fu D L. An ancestral NB-LRR with duplicated 3'UTRs confers stripe rust resistance in wheat and barley. Nat Commun, 2019,10:4023.
pmid: 31492844
[14] Liu K Y, Cao J, Yu K H, Liu X Y, Gao Y J, Chen Q, Zhang W J, Peng H R, Du J, Xin M M, Hu Z R, Guo W L, Vincenzo R, Ni Z F, Sun Q X, Yao Y Y. Wheat TaSPL8 modulates leaf angle through auxin and brassinosteroid signaling. Plant Physiol, 2019,181:179-194.
pmid: 31209125
[15] 韩德俊, 康振生. 中国小麦品种抗条锈病现状及存在问题与对策. 植物保护, 2018,44:1-12.
Han D J, Kang Z S. Current status and future strategy in breeding wheat for resistance to stripe rust in China. Plant Prot, 2008,44:1-12 (in Chinese with English abstract).
[16] Engelhardt S, Stam R, Hückelhoven R. Good riddance? Breaking disease susceptibility in the era of new breeding technologies. Agronomy, 2018,8:114.
[17] Zhu X, Qi T, Yang Q, He F, Tan C, Ma W, Voegele R T, Kang Z, Guo J. Host-induced gene silencing of the MAPKK gene PsFUZ7 confers stable resistance to wheat stripe rust. Plant Physiol, 2017,175:1853-1863.
pmid: 29070517
[18] Line R F, Qayoum A. Virulence, aggressiveness, evolution, and distribution of races of Puccinia striiformis (the cause of stripe rust of wheat) in North America, 1968-87. Technical Bulletin No. 1788, U.S. Department of Agriculture, National Technical Information Service, Springfield, VA, USA, 1992.
[19] Mago R, Miah H, Lawrence G J, Wellings C R, Spielmeyer W, Bariana H S, McIntosh R A, Pryor A J, Ellis J G. High-resolution mapping and mutation analysis separate the rust resistance genes Sr31, Lr26 and Yr9 on the short arm of rye chromosome 1. Theor Appl Genet, 2005,112:41-50.
pmid: 16283230
[20] Francis H A, Leitch A R, Koebner R. Conversion of a RAPD-generated PCR product, containing a novel dispersed repetitive element, into a fast and robust assay for the presence of rye chromatin in wheat. Theor Appl Genet, 1995,90:636-642.
doi: 10.1007/BF00222127 pmid: 24174021
[21] Liu C, Yang Z J, Li G R, Zeng Z X, Zhang Y, Zhou J P, Liu Z H, Ren Z L. Isolation of a new repetitive DNA sequence from Secale africanum enables targeting of Secale chromatin in wheat background. Euphytica, 2007,159:249-258.
[22] Han F P, Liu B, Fedak G, Liu Z H. Genomic constitution and variation in five partial amphiploids of wheat- Thinopyrum intermedium as revealed by GISH, multicolor GISH and seed storage protein analysis. Theor Appl Genet, 2004,109:1070-1076.
[23] Huang X, Zhu M, Zhuang L, Zhang S, Wang J, Chen X, Wang D, Chen J, Bao Y, Guo J, Zhang J, Feng Y, Chu C, Du P, Qi Z, Wang H, Chen P. Structural chromosome rearrangements and polymorphisms identified in Chinese wheat cultivars by high-resolution multiplex oligonucleotide FISH. Theor Appl Genet, 2018,131:1967-1986.
pmid: 29947816
[24] 高德荣, 张勇, 吴宏亚, 陆成彬, 张伯桥, 程顺和. 系统选择在现代小麦品种改良中的应用. 江苏农业科学, 2000, (1):21-23.
Gao D R, Zhang Y, Wu H Y, Lu C B, Zhang B Q, Cheng S H. Application of systematic selection to improvement of modern wheat varieties. Jiangsu Agric Sci, 2000, (1):21-23 (in Chinese).
[25] 曹廷杰, 陈永兴, 李丹, 张艳, 王西成, 赵虹, 刘志勇. 河南小麦新育成品种(系)白粉病抗性鉴定与分子标记检测. 作物学报, 2015,41:1172-1182.
Cao T J, Chen Y X, Li D, Zhang Y, Wang X C, Zhao H, Liu Z Y. Identification and molecular detection of powdery mildew resistance of new bred wheat varieties (lines) in Henan province, China. Acta Agron Sin, 2015,41:1172-1182 (in Chinese with English abstract).
[26] 刘建军, 何中虎, Pena R J, 赵振东. 1BL/1RS易位对小麦加工品质的影响. 作物学报, 2004,30:149-153.
Liu J J, He Z H, Pena R J, Zhao Z D. Effect of 1BL/1RS translocation on grain quality and noodle quality in bread wheat. Acta Agron Sin, 2004,30:149-153 (in Chinese with English abstract).
[27] 李亚青, 张楠, 张士昌, 何明琦, 李孟军. 黑麦1RS染色体臂Sec-1位点的研究进展. 河北农业科学, 2018,22:37-40.
Li Y Q, Zhang N, Zhang S C, He M Q, Li M J. Advances of Sec-1 locus on rye 1RS chromosome arm. Hebei Agric Sci, 2018,22:37-40 (in Chinese with English abstract).
[28] 柴建芳, 王海波, 马秀英, 张翠绵, 董福双. ω-黑麦碱基因沉默对小麦1B/1R易位系加工品质的影响. 作物学报, 2016,42:627-632.
Chai J F, Wang H B, Ma X Y, Zhang C M, Dong F S. Effect of ω-Secalin gene silencing on processing quality of wheat 1B/1R translocation line. Acta Agron Sin, 2016,42:627-632 (in Chinese with English abstract).
[29] 叶兴国, 佘茂云, 王轲, 杜丽璞, 徐惠君. 植物组织培养再生相关基因鉴定、克隆和应用研究进展. 作物学报, 2012,38:191-201.
Ye X G, She M Y, Wang K, Du L P, Xu H J. Identification, cloning, and potential application of genes related to somatic embryogenesis in plant tissue culture. Acta Agron Sin, 2012,38:191-201 (in Chinese with English abstract).
[1] 张怀志,谢菁忠,陈永兴,刘旭,王勇,闫素红,杨兆生,赵虹,王西成,贾联合,曹廷杰,刘志勇. 利用BSR-Seq定位小麦品种郑麦103抗条锈病基因YrZM103[J]. 作物学报, 2017, 43(11): 1643-1649.
[2] 徐中青, 张书英, 王睿, 王文立, 周新力, 尹军良, 陈洁, 井金学. 小麦品系M8003-5抗条锈病基因遗传分析和分子作图[J]. 作物学报, 2010, 36(12): 2116-2123.
[3] 任强,刘慧娟,陈洋,徐世昌,何名召,辛志勇,张增艳.
人工合成小麦CI191抗条锈病基因的鉴定及分子标记定位
[J]. 作物学报, 2010, 36(05): 721-727.
[4] 何名召;王丽敏;张增艳;徐世昌;王丽丽;辛志勇. 硬粒小麦-粗山羊草人工合成小麦CI108抗条锈病新基因的鉴定、基因推导与分子标记定位[J]. 作物学报, 2007, 33(07): 1045-1050.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2