稻瘟病抗性基因Pi25特异性CAPS标记的开发与验证

[19]
This work aims at developing and validating the Pi25 functional markers based on the coding sequences of the locus using three categories of rice materials. Two sets of markers, CAP1/Hinc II and CAP3/Hpy 99I, are able to specifically digest the dominant allele Pi25 and recommended for MAS in rice breeding programs.
稻瘟病抗性基因 Pi25 特异性 CAPS 标记的开发与验证
王惠梅 1,**
1
陈
洁 1,**
施勇烽 1
潘
刚2
沈海超 1
吴建利 1,*
中国水稻研究所 / 水稻生物学国家重点实验室, 浙江杭州 310006; 2 浙江大学农业与生物技术学院, 浙江杭州 310058
摘
要: 为在水稻育种中快速与高效利用稻瘟病抗性基因 Pi25, 本文利用该基因不同等位基因编码区序列差异开发了 4 套
Development and Validation of CAPS Markers for Marker-Assisted Selection of Rice Blast Resistance Gene Pi25
WANG Hui-Mei1,**, CHEN Jie1,**, SHI Yong-Feng1, PAN Gang2, SHEN Hai-Chao1, and WU Jian-Li1,*
CAPS 标记(CAP1/Hinc II、CAP3/Bgl II、CAP3/Nde I 和 CAP3/Hpy 99I), 并利用 169 份稻种资源、98 个重组自交系(RIL)以及 217 个水稻转基因后代, 对 4 套标记的准确性和选择效果进行了验证。结果表明, 4 套标记均能准确地检测 Pi25/pi25 座位。 其中, 标记 CAP1/Hinc II 和 CAP3/Hpy 99I 特异性识别并酶切显性等位基因, 而标记 CAP3/Bgl II 和 CAP3/Nde I 特异性识别并 酶切隐性等位基因。利用稻瘟病菌株 JS001-20 接种 RIL 与转基因材料, 抗性表现与标记检测的结果完全一致, 表明该 CAPS 标记准确可靠。分析稻种资源后发现, Pi25 基因频率较低(1.2%), 说明该基因在我国水稻稻瘟病抗性育种中还没有被充分利 用。 本文的研究结果特别是开发的 2 对识别并酶切显性等位基因的 CAPS 标记可用于分子标记辅助选择, 改良我国早籼稻的 稻瘟病抗性。 关键词: 水稻; 稻瘟病抗性; 酶切扩增多态性序列; 标记辅助选择; 单核苷酸多态性
*
Correspondence author: WU Jian-Li; E-mail: beishangd@; Tel: +86-571-63370326
**
These authors contributed equally to this work.
Received(收稿日期): 2012-04-17; Accepted(接受日期): 2012-07-05; Published online(网络出版日期): 2012-09-10. URL: /kcms/detail/11.1809.S.20120910.1404.023.html
1
State Key Laboratory of Rice Biology / China National Rice Research Institute, Hangzhou 310006, China; 2 College of Agriculture and Biotechnology,
Zhejiang University, Hangzhou 310058, China
[4-12]
. Although a serious of effort has been attempted in elucidating the structure and
function of the genes while there is no fundamental breakthrough in application of the genes to improve rice blast resistance in rice breeding programs. With the development of molecular markers, the selection for targeted blast resistance genes using specific molecular markers is commonly practiced and some intermediate breeding materials
1
carrying the target genes have been bred [13]. One of the quick and effective ways for improving blast resistance in new varieties is to backcross the intermediate breeding materials with commercial elite lines. However, the accuracy of marker-assisted selection (MAS) for the target genes largely depends on the strength of linkage between the markers and the target genes. Fortunately, functional markers for those cloned genes have been developed and could provide the accurate and efficient selection [14-15]. Gumei 2 is the only early-season semi-dwarf indica rice cultivar with stable and broad-spectrum resistance to Magnaporthe oryzae, the causal pathogen of blast disease
More than 50 major rice blast resistance genes have been indentified since late 1980s because of the development and utilization of molecular markers [1-3]. Among them, 15 blast resistance genes have been successively cloned through the map-based cloning strategy
This work was supported by the National High Technology Research and Development Program of China (Grant No. 2011AA10A101 and 2012AA101102) and the Ministry of Finance, China (Grant No. 2012RG002-4).
[16]
. It is ed as a valuable donor for rice blast
[17-18]
improvement in China because of its elite agronomic traits. Previous studies have identified and located at least four blast resistance genes and a number of partial resistance QTLs in Gumei 2 . Among them, Pi25 is a single copy . intronless CC-NBS-LRR type of blast resistance gene located in chromosome 6
Abstract: To promote the application of rice blast resistance gene Pi25 in rice breeding programs, we developed four sets of gene-specific CAPS markers (CAP1/Hinc II, CAP3/Bgl II, CAP3/Nde I, and CAP3/Hpy 99I) based on the coding sequences of the locus. One hundred and sixty-nine rice accessions, 98 recombinant inbred lines (RILs) and 217 transgenic plants were used for the validation of the markers. The results showed that all the four sets of markers were able to accurately and efficiently detect the Pi25/pi25 locus, CAP1/Hinc II and CAP3/Hpy 99I could digest specifically the dominant allele Pi25 while CAP3/Bgl I and CAP3/Nde I were able to digest specifically the recessive allele pi25. RILs and transgenic lines carrying Pi25 allele were resistant to the blast isolate JS001-20 while the lines carrying pi25 allele were susceptible, indicating a perfect detection of the target locus by the CAPS markers. In addition, a low frequency (1.2%) of the dominant allele was detected in the germplasm collections, indicating this gene has not been fully utilized in rice breeding programs in China. Markers CAP1/Hinc II and CAP3/Hpy 99I are recommended and will be useful for the improvement of blast resistance, especially for the early-season indica rice. Keywords: Oryza sativa; Blast resistance; Cleaved amplified polymorphic sequence (CAPS); Marker-assisted selection (MAS); Single nucleotide polymorphism (SNP)