粳稻保持系日本晴中一个野败型恢复基因的定位与克隆

WA-type cytoplasmic male sterility (CMS) has been used widely in three-line indica hybrids, and study on the fertility-restorer genes (Rf) for WA-type CMS is help for the development of WA-type indica hybrids. In former study, the fertility restoration of WA-type CMS lines are controlled by two major gene, Rf3 and Rf4, and are affected by minor effect restorer genes, which are considered to exist in the restorers. 9311(Yangdao6) is an elite restorer for HongLian type indica CMS lines, but have no capability to restore the fertility to WA-type CMS lines; Nipponbare, a typical japonica variety, is the maintainer for WA-type CMS lines. C119, one chromosome single segment substation line (CSSL), which were developed by using two sequenced varieties, 9311 as recipient parent and Nipponbare as donor parent, has been proved to have the restoring ability for WA-type CMS lines, indicating that there is Rf for WA type CMS in maintainers. In this study, we will reconstruct the CSSLs with the short introgression segments covering the target gene from the cross between 9311 and C119, and the genotypes of each CSSLs will be identified by fertility levels of the corresponding testcrossed F1 plants, by which the fertility-restorer gene in Nipponbare would be mapped and cloned. Additionally, the function of the Rf will be analyzed for uncovering the mechanization under fertility restoration for WA-type CMS, which would provide useful informations for breeding of WA-type indica hybrids.

三系杂交籼稻以利用野败（WA）型不育胞质为主，对WA型育性恢复基因的研究有助于WA型杂交稻的选育。已有的研究表明，WA型不育系育性恢复受主效恢复基因Rf3、Rf4控制，受一些微效基因的影响，恢复基因多认为来自恢复系。9311为红莲（HL）型强恢复系，但不能恢复WA型不育系，日本晴为常规粳稻品种，为WA型保持系；本实验室构建的以日本晴为供体、9311为背景的1套染色体片段代换系中，C119能够正常恢复WA型籼稻不育系的育性，表明WA型粳稻保持系中也存在恢复基因。本研究拟利用C119与9311配组，构建染色体单片段叠代系，进而利用测交后代的表型来鉴定基因型，对日本晴中的恢复基因进行定位与克隆，采用RT-PCR等分子生物学的方法对基因功能进行初步研究。相关结果有助于进一步阐明WA型不育系育性恢复机理，对WA型杂交稻选育具有指导意义。

三系杂交稻是水稻杂种利用的主要形式。在生产上，三系杂交籼稻主要利用野败（WA）型不育胞质，对微效恢复基因的研究不足限制了WA型杂交稻育种效率，是制约杂交稻发展的因素。因此，加强WA型微效恢复基因的研究对于杂交稻的发展及保障我国粮食安全具有重要意义。本课题组前期研究中，利用一套日本晴为供体、9311为背景的染色体片段代换系，发现日本晴中存在WA型微效恢复基因。本项目中，我们利用该染色体片段代换系鉴定到1个新的恢复基因位点Rf21(t)，初步将日本晴中的恢复基因定位于第1染色体长臂上；利用代换系衍生群体构建了一套染色体单片段叠代系，采用叠代作图的方法对Rf21(t)进行精细定位，发现Rf21(t)位点内存在两个连锁的恢复基因Rf21a(t)、Rf21b(t)，分别将两基因定位于570kb与48kb的物理区间内；在Rf21b(t)定位区间内，包含2个具有明确注释的基因LOC_0s01g71310 和LOC_Os01g71320，通过测序及表达分析，确定LOC_Os01g71320为Rf21b(t)的候选基因，编码线粒体定位蛋白己糖激酶；通过对可育测交系与部分不育测交系中WA352表达量的检测，初步揭示Rf21(t)通过转录后调控的机制来恢复WA型不育系的育性；通过可育测交系衍生的F2群体，明确Rf21(t)以孢子体不育模式恢复育性，且其育性恢复作用依赖于9311核背景；创制了9311背景的近等基因系NILrf6及多基因聚合系PPLrf5+rf6，通过测交鉴定明确9311对WA型不育系的恢复力非由Rf5、Rf6所致；创制了代换系C31为供体、WA型不育系天丰A为背景的回交高代同质恢材料，鉴定并定位了9311中的WA型恢复基因Rf24(t)。