中国两系杂交水稻光温敏核不育基因的鉴定与演化分析  

Identification and Transition Analysis of Photo-/Thermo-Sensitive Genic Male Sterile Genes in Two-Line Hybrid Rice in China

作　　者：张华丽[1] 陈晓阳 黄建中[1] 鄂志国[3] 龚俊义[3] 舒庆尧[1] 

机构地区：[1]浙江大学原子核农业科学研究所/水稻生物学国家重点实验室,杭州310029 [2]金华市农业科学研究院,浙江金华321017 [3]中国水稻研究所,杭州311401

出　　处：《中国农业科学》2015年第1期1-9,共9页Scientia Agricultura Sinica

基　　金：国家公益性行业(农业)科研专项(201103007);浙江省8812项目;IAEA协调研究项目(16818)

摘　　要：【目的】鉴定中国生产上应用的两系不育系所携带的光温敏核不育基因,揭示两系杂交稻光温敏核不育基因的演变过程。【方法】收集光温敏核雄性不育水稻材料共90份,包括农垦58S、安农S-1和株1S等衍生系。采用改良CTAB法从水稻叶片中提取基因组DNA,根据光敏不育基因所含突变设计1个功能性CAPS标记,用引物NK-F(5′-ATCCCACAAATCCTTTAGCA-3′)和NK-R(5′-CCGTTATAGATAGACCCGAGA-3′)扩增包含该突变位点的片段,用内切酶RsaⅠ酶切并检测,可清晰区分纯合光敏不育系(lnc Rm)(329 bp)、纯合野生型(lnc Rwt)材料(414 bp)和杂合型(lnc Rm/lnc Rwt)材料(414和329 bp)。温敏不育基因RNZ的分型则采用功能性d CAPS标记d CAPS-rnz,待测材料分别用引物对RNZ1F(5′-ACCGCGCCGCCACCGGGTCGGCCGGAG-3′)/RNZR(5′-TGAAGAGGAACTCCTGCGAGACGG-3′)和RNZ2F(5′-ACCGCGCCGCCACCGGGTCGGCCCAAG-3′)/RNZR扩增,产物分别用内切酶HinfⅠ和StyⅠ酶切,酶切产物经电泳分离检测,纯合温敏不育系(RNZm)不能被上述2种内切酶所酶切,纯合野生型RNZtc和RNZgc可分别被HinfⅠ和StyⅠ完全酶切,而杂合型RNZm/RNZtc和RNZm/RNZgc可分别被HinfⅠ和StyⅠ不完全酶切。采用上述功能性分子标记鉴定两系不育系所携带的光敏(lnc Rm)和温敏核不育基因(RNZm),并结合不育系的系谱信息和中国水稻数据库的年度推广面积数据分析lnc Rm和RNZm在中国两系杂交稻生产中应用的历史与现状。【结果】以农垦58S为唯一不育基因源的47个两系不育系中,12个携带lnc Rm,29个携带RNZm,2个同时携带lnc Rm和RNZm,4个不携带这两个基因;衍生自安农S-1和株1S的18个不育系则均携带RNZm;由农垦58S衍生系(如培矮64S)与安农S-1复交育成的不育系也全部携带RNZm;由培矮64S与株1S复交育成的2个不育系中1个携带RNZm,而另1个则同时携带lnc Rm和RNZm;另外16个与农垦58S、安农S-1及株1S无血缘关系的独立起源两系不育系中有6个携带lnc Rm,9个�Objective]The objective of this study is to identify and reveal the transition of the male sterile gene（s） in photoperiod- and temperature-sensitive genic male sterile （P/TGMS） lines utilized in the two-line hybrid rice system in China.[Method]A total of 90 environment-conditioned genic male sterile （EGMS） lines including descendents of Nonken 58S, Annong S-1 and Zhu 1S, were used in the present study. Genomic DNAs were extracted from rice leaves by modified CTAB. One functional CAPS marker based on the C to G mutation in the long non-coding RNA （lncR） gene was designed for PGMS genotyping; Namely, a pair of primers NK-F （5′-ATCCCACAAATCCTTTAGCA-3′） and NK-R （5′-CCGTTATAGATAGACCCGAGA-3′） were used to amplify segments harboring the mutation site, followed by digestion overnight at 37 with restriction endonuclease℃ RsaⅠ and separation on 1% agarose gel electrophoresis. Homozygous PGMS allele （lncR^m） （329 bp） can be readily distinguished from homozygous wild type （lncRwt） （414 bp） and heterozygous type （lncR^m/lncRwt） （414 and 329 bp） based on the sizes of digestion products. For TGMS genotyping, functional dCAPS markers were deployed with the following steps： Two pairs of primers RNZ1F （5′-ACCGCGCCGCCACCGGGTCGGCCGGAG-3′）/RNZR （5′-TGAAGAGGAACTCCTGCGAGACGG-3′）, RNZ2F （5′-ACCGC GCCGCCACCGGGTCGGCCCAAG-3′）/RNZR were used to amplify segments harboring the mutation site （SNP-＋70TA/TC/GC）;Amplified products were digested overnight at 37 with re℃ striction endonucleases HinfⅠ and StyⅠ, respectively, and separated on 8% polyacrylamide gels. Homozygous lines with the TGMS allele （RNZ^m） cannot be digested by these two restriction enzymes; on the other hand, homozygous wild type lines with the alleles of RNZtc or RNZgc, can be digested completely by HinfⅠ and StyⅠ, respectively; heterozygous genotypes, RNZ^m/RNZtc and RNZ^m/RNZgc, can be digested incompletely by HinfⅠ and StyⅠ, respectively. By using these functional

关 键 词：杂交水稻 光敏核不育 温敏核不育 品种演化 分子标记 

分 类 号：S511[农业科学—作物学]