中国两系杂交水稻光温敏核不育基因的鉴定与演化分析

doi:10.3864/j.issn.0578-1752.2015.01.001

摘要/Abstract

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（lncR^m）（329 bp）、纯合野生型（lncR^wt）材料（414 bp）和杂合型（lncR^m/lncR^wt）材料（414和329 bp）。温敏不育基因RNZ的分型则采用功能性dCAPS标记dCAPS-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（RNZ^m）不能被上述2种内切酶所酶切，纯合野生型RNZ^tc和RNZ^gc可分别被HinfⅠ和StyⅠ完全酶切，而杂合型RNZ^m/RNZ^tc和RNZ^m/RNZ^gc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（lncR^m）和温敏核不育基因（RNZ^m），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lncR^m和RNZ^m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lncR^m，29个携带RNZ^m，2个同时携带lncR^m和RNZ^m，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RNZ^m；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RNZ^m；由培矮64S与株1S复交育成的2个不育系中1个携带RNZ^m，而另1个则同时携带lncR^m和RNZ^m；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lncR^m，9个携带RNZ^m等位基因，1个（衡农S-1）则既不携带lncR^m也不带RNZ^m。综合这些不育系携带光温敏基因的鉴定结果，结合不育系选育的系谱资料，绘制了包含92个两系不育系所携带的光温敏基因图。另外，根据这些两系不育系所配制组合的种植面积数据（分品种），发现1993—2012年中国两系杂交稻中不育基因的利用经历了从光敏到温敏的演化过程，携带RNZ^m的不育系所配制组合及推广面积在生产中的应用迅速增加，2012年温敏不育系配制的杂交稻已占两系杂交稻种植面积的95%以上，形成了以携带RNZ^m的不育系为主的局面。【结论】系统明晰了中国两系水稻不育系携带的光温敏不育基因及其与品种系谱之间存在的不一致性，从光敏不育系杂交后代中选育出了温敏不育水稻，且携带RNZ^m的不育系已在目前两系杂交稻生产中占绝对主导地位。

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

Abstract: 【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 (lncR^wt) (414 bp) and heterozygous type (lncR^m/lncR^wt) (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 restriction endonucleases HinfⅠand 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 RNZ^tcor RNZ^gc, can be digested completely by HinfⅠ and StyⅠ, respectively; heterozygous genotypes, RNZ^m/RNZ^tc and RNZ^m/RNZ^gc, can be digested incompletely by HinfⅠand StyⅠ, respectively. By using these functional molecular markers, the PGMS (lncR^m) and TGMS (RNZ^m) genes were identified in commercial EGMS lines that had been utilized in the two-line hybrid rice system. Meanwhile, the transition of P/TGMS genes utilized in two-line hybrid rice production in China (1993-2012) was analyzed according to the information of pedigree and growing area. 【Result】Out of the 47 EGMS lines derived from the PGMS line Nongken 58S, 12 lines carry the PGMS gene lncR^m, 29 lines have the TGMS gene RNZ^m, two lines carry both genes, while the remaining four lines contain none of them. All 18 lines derived from Annong S-1 and Zhu 1S carry RNZ^m gene. All P/TGMS descendants from crosses between a Nongken 58S derivative (Pei’ai 64S) and Annong S-1 carry RNZ^m gene. In the two lines derived from Pei’ai 64S and Zhu 1S, one carries both genes, the other has RNZ^m gene. In addition, in 16 EGMS lines with EGMS progenitors independent from Nongken 58S, Annong S-1 and Zhu 1S, six of them have lncR^m, nine of them have RNZ^m gene, while one of them contains neither lncR^m nor RNZ^m gene. A pedigree map with P/TGMS genes was drawn for 92 EGMS lines, including derivatives from Nongken 58S, Annong S-1, Zhu 1S and others. Furthermore, the transition from lncR^m-based to RNZ^m-based two-line hybrid rice production was shown after examination of statistics data of the two-line hybrid rice planting area during 1993-2012, with the RNZ^m-based hybrids occupying ＞95% planting area in the two-line hybrid rice production in 2012. 【Conclusion】The study systematically revealed the P/TGMS genes in commercial EGMS rice and the presence of discrepancy between pedigree of EGMS lines and their P/TGMS gene. The spontaneous emergence of RNZ^m can be the reason for the transition from PGMS to TGMS in some EGMS lines derived from Nongken 58S. EGMS lines with RNZ^m currently dominate the two-line hybrid rice production in China.

Key words: hybrid rice, PGMS, TGMS, cultivar transition, molecular marker

张华丽，陈晓阳，黄建中，鄂志国，龚俊义，舒庆尧. 中国两系杂交水稻光温敏核不育基因的鉴定与演化分析[J]. 中国农业科学, 2015, 48(1): 1-9.

ZHANG Hua-li, CHEN Xiao-yang, HUANG Jian-zhong, E Zhi-guo, GONG Jun-yi, SHU Qing-yao. Identification and Transition Analysis of Photo- /Thermo-Sensitive Genic Male Sterile Genes in Two-Line Hybrid Rice in China[J]. Scientia Agricultura Sinica, 2015, 48(1): 1-9.

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