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作物学报 ›› 2018, Vol. 44 ›› Issue (03): 324-331.doi: 10.3724/SP.J.1006.2018.00324

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻抗咪唑啉酮类除草剂基因ALS功能标记的开发与应用

王芳权1,2(), 杨杰1,2,*(), 范方军1,2, 李文奇1,2, 王军1,2, 许扬1,2, 朱金燕1,2, 费云燕1, 仲维功1,2   

  1. 1江苏省农业科学院粮食作物研究所 / 国家水稻改良中心南京分中心 / 江苏省优质水稻工程技术研究中心, 江苏南京 210014
    2扬州大学 / 江苏省粮食作物现代产业技术协同创新中心, 江苏扬州 225009
  • 收稿日期:2017-07-19 接受日期:2017-11-21 出版日期:2018-03-12 网络出版日期:2017-12-11
  • 通讯作者: 杨杰
  • 作者简介:

    wfqjaas@163.com, Tel: 025-84390320

  • 基金资助:
    本研究由国家重点研发计划项目(2017YFD0100400-3), 江苏省现代农业重点研发项目(BE2015355), 江苏省农业科学院探索性项目(ZX(17)2014), 江苏省自然科学基金面上项目(BK20171326)和国家公益性行业科研专项项目(201303102)资助

Development and Application of the Functional Marker for Imidazolinone Herbicides Resistant ALS Gene in Rice

Fang-Quan WANG1,2(), Jie YANG1,2,*(), Fang-Jun FAN1,2, Wen-Qi LI1,2, Jun WANG1,2, Yang XU1,2, Jin-Yan ZHU1,2, Yun-Yan FEI1, Wei-Gong ZHONG1,2   

  • Received:2017-07-19 Accepted:2017-11-21 Published:2018-03-12 Published online:2017-12-11
  • Contact: Jie YANG
  • Supported by:
    This study was supported by the National Key Research and Development Program (2017YFD0100400-3), the Jiangsu Province Key Research and Development Program (Modern Agriculture, BE2015355), the Exploratory Project of the Jiangsu Academy of Agricultural Sciences (ZX(17)2014), the Natural Science Foundation of Jiangsu Province of China (BK20171326), and the Special Fund for Scientific Research on Public Causes (201303102).

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摘要：

选育和利用抗除草剂水稻品种具有重要的生产实践意义。通过筛选水稻资源, 发现了抗除草剂材料金粳818, 其ALS基因编码区第1880位碱基存在一个由G到A的碱基变异, 导致丝氨酸突变为天冬酰胺, 从而具有除草剂抗性。本研究基于该位点的碱基变异, 设计了11条等位基因特异PCR (allelic-specific PCR, AS-PCR)引物, 经过优化筛选, 获得两个引物组合F1N (S1/S9)和F1M (S1/S10), 将该标记命名为AS-ALS。利用F2群体及其亲本和杂交种, 结合AS-ALS标记检测和除草剂抗性分析, 结果表明感除草剂ALS-G等位基因型只能被F1N引物对有效扩增, 抗除草剂ALS-A等位基因型只能被F1M引物对有效扩增, 而杂合基因型能同时被两对引物F1N和F1M扩增, ALS-A纯合或杂合等位基因型都表现抗除草剂, ALS-G纯合基因型表现感除草剂。因此本研究开发的标记能有效区分除草剂抗性基因的3种基因型, 基因型与表型完全对应。该标记用于回交育种, 可以选择ALS-A杂合基因型单株, 剔除ALS-G纯合等位基因型, 在自交的F2保留ALS-A纯合基因型单株, 连续自交, 能快速获得除草剂抗性稳定的水稻材料。该除草剂抗性基因的功能标记还可用于咪唑啉酮类除草剂抗性资源筛选。

关键词: 水稻, 除草剂, 乙酰乳酸合成酶, ALS基因, 功能标记

Abstract:

Breeding and utilization of herbicide resistant rice are significant to rice production. By screening the rice germplasm, we found the herbicide resistant material “Jinjing 818”. An SNP mutation G to A was present in Acetolactate synthase (ALS) gene at 1880 bp position, leading to the alteration from serine (S, AGT) to asparagine (N, AAT), which confers herbicide resistance. In this study, 11 allelic-specific PCR (AS-PCR) primers were designed based on the functional mutation. After optimized these primers, we obtained two primer combinations F1N (S1/S9) and F1M (S1/S10), named AS-ALS marker. Using this marker detected the genetic population, its parents, F1 hybrid, F2 and also rice collections, inbred lines, showing that the herbicide susceptibleness allelic ALS-G could be amplified by F1N, the herbicide resistance allelic ALS-A by F1M, and heterozygous genotype by F1N and F1M simultaneously. The genotype of those tested materials perfectly matched with the phenotype of herbicide resistance or susceptibleness. Aided by AS-ALS marker selection, the homozygous ALS-A pedigrees in multi-generation backcross or self-cross showed stable herbicide resistance. Therefore, the allelic-specific PCR functional marker AS-ALS can be used in herbicide breeding efficiently, also screening herbicide resistant rice germplasm. In conclusion, the AS-ALS marker developed in this research is inexpensive and effective in breeding practice.

Key words: rice (Oryza sativa L.), herbicide, acetolactate synthase, ALS gene, functional marker

表1

ALS基因分子标记设计"

编号
No.		 引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
S1 AS-ALS-F1 GAGGCAATCATCGCTACTGG
S2 AS-ALS-R1-1 ACAAACCTAGACAGCAGGAAG
S3 AS-ALS-R1-2 CTCTTTATGGGTCATTCAGGTC
S4 AS-ALSn-F2-1 TGTGCTGCCTATGATCCtgAG
S5 AS-ALSn-F2-2 TGTGCTGCCTATGATCCCtAG
S6 AS-ALSm-F2-1 TGTGCTGCCTATGATCCCgAA
S7 AS-ALSm-F2-2 CATGTGCTGCCTATGATCtCgAA
S8 AS-ALSn-R2-1 TGTCCTTGAATGaGCCCCtAC
S9 AS-ALSn-R2-2 TGTCCTTGAATGCGCCCCtAC
S10 AS-ALSm-R2-1 TGTCCTTGAATGCGCCCCtAT
S11 AS-ALSm-R2-2 TGTCCTTGAATGCGCCCttAT

图1

ALS基因序列比对图日本晴的ALS基因序列GenBank登录号为NC_008395.2, StrawhullS的ALS基因序列GenBank登录号为AY885673.1。"

图2

不同引物组合对日本晴和金粳818的PCR扩增结果 A为日本晴, B为金粳818; M为DL2000标记, 由大到小分别为2000、1000、750、500、250和100 bp; 泳道1~11分别为S4/S2、S4/S3、S5/S2、S5/S3、S6/S2、S6/S3、S7/S2、S7/S3、S1/S8、S1/S9和S1/S10。"

图3

引物组合提高退火温度优化后PCR扩增结果 A为日本晴, B为金粳818; M为DL2000标记, 由大到小分别为2000、1000、750、500、250和100 bp; 泳道1~6分别为S4/S2、S4/S3、S5/S2、S5/S3、S1/S9和S1/S10。"

图4

AS-ALS标记检测部分F2单株及表型对应情况M为DL2000 DNA标记(由大到小分别为2000、1000、750、500、250和100 bp), P1为金粳818, P2为南粳9108, F1为南粳9108/粳9108/金粳818杂交种, 1~21为F2单株; R表示单株表现抗除草剂, S表示单株表现感除草剂。"

图5

喷施除草剂后F2植株表型1为金粳818, 2为南粳9108, 3为南粳9108/金粳818杂交种, 4为ALS-A纯合基因型后代, 5为杂合基因型后代, 6为ALS-G纯合基因型后代。"

图6

用AS-ALS标记检测高代品系及表型对应情况 M为DL2000 DNA标记(由大到小分别为2000、1000、750、500、250和100 bp), P1为金粳818, P2为南粳9108, 1~22为BC3F3群体单株; R表示单株表现抗除草剂, S表示单株表现感除草剂。"

图7

利用AS-ALS功能标记筛选水稻资源 M为DL2000标记, 由大到小分别为2000、1000、750、500、250和100 bp; 泳道1~24分别为金粳818、日本晴、南粳40、南粳41、南粳44、南粳45、南粳46、南粳49、南粳9108、南粳5055、淮稻5号、苏秀867、武运粳21、武运粳24、武运粳27、徐稻3号、徐稻8号、镇稻88、镇稻99、连粳7号、常农粳7号、9311、IR36和南京16。"

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