水稻芒性基因Awn3-1、Awn4.1、Awn8-1的精细定位及Awn4-2的图位克隆

发布时间：2018-06-07 14:27

本文选题：水稻(Oryza + sativa　；参考：《中国农业大学》2016年博士论文

【摘要】：芒是水稻及其他禾本科作物重要驯化性状之一,同时也是重要的农艺性状。本研究构建了一系列芒性作图群体,利用4个精细作图群体分别定位了4个调控芒性发育基因,并对一个进行了克隆。利用粳稻品种SLG(具芒)作为供体亲本,粳稻品种日本晴(无芒)作为受体亲本构建近等基因系,在前人定位基础上,利用8103株BC4F4群体和定位区间内6个多态性连锁标记,将Awn3-1定位于3号染色体上Indel标记In316和SNP标记S9之间,物理距离为101.13kb,该区间内有9个候选基因。对候选基因在近等基因系显隐性材料间进行序列分析未发现差异。但表达分析显示Os03g0418600在显隐性材料间存在差异,推测其为调控芒性发育的基因。此外,Awn3-1和本实验室已发现的调控芒性发育基因Awn4-2对芒长和芒分布存在积加作用。利用粳稻品种高丽早稻(具芒)作为供体亲本,粳稻品种日本晴(无芒)作为受体亲本构建近等基因系,结合前人定位结果,利用7072株BC6F5群体和3个新开发多态性连锁标记,将Awn4.1在4号染色体上的定位区间缩小至20.02kb,该区间内有2个候选基因。利用三亚抽穗期极晚的粳稻材料(具芒)作为供体亲本,与粳稻品种日本晴(无芒)杂交后构建芒性重组自交系。对F3群体的遗传分析表明,芒性状受一对显性核基因控制,命名为Awn8-1。利用BSA法结合连锁分析,将Awn8-1初步定位在8号染色体长臂上SSR标记RM7556和M3381之间。利用12204株F4群体和6个多态性连锁标记,将Awn8-1定位于SSR标记RM23337和SNP标记S3之间,物理距离为25.66kb,该区间有3个候选基因。序列分析表明,Os08g0485500基因第二外显子上存在4bp插入缺失,3’UTR上存在2个SNPs。利用粳稻品种抚宁小红芒(具芒)为供体亲本,粳稻品种日本晴(无芒)为轮回亲本构建近等基因系,对调控芒性发育的基因Awn4-2进行定位及克隆。利用11206株BC4F6群体和定位区间内7个多态性连锁标记,最终将Awn4-2定位在4号染色体SSR标记M1126和Indel标记In73之间,物理距离为62.4kb。根据RAP-DB的预测,该区段内有3个注释基因。序列分析表明,在日本晴和隐性材料中,预测基因Os04g0350700启动子区存在约4.4kb的转座子插入,基因序列存在7个SNPs。结合半定量表达分析,Os04g0350700可能是Awn4-2。Os04g0350700编码一个具有bHLH保守结构域的转录因子。序列分析表明,Awn4-2第二个外显子处的SNP可能不影响基因功能,启动子区域转座子的插入可能导致基因表达受抑制,导致芒不能正常发育。为验证其功能,我们分别构建了Awn4-2基因的RNA干扰载体和过表达载体。表达特性分析显示,该基因主要在有芒材料幼穗中表达。根据调控表型和序列差异,推测该基因为An-1的等位基因。
[Abstract]:Awn is one of the important domestication traits and agronomic characters of rice and other gramineous crops. In this study, a series of awn mapping populations were constructed, and four genes regulating the development of awn were located using four fine mapping populations, and one of them was cloned. Japonica rice (SLG) was used as donor parent, japonica rice variety Nippon (without awn) as recipient parent to construct near-isogenic lines. On the basis of previous mapping, six polymorphic linkage markers were used in 8103 BC4F4 populations and locational intervals. Awn3-1 was mapped between Indel marker In316 and SNP marker S9 on chromosome 3 with a physical distance of 101.13 kb. There were 9 candidate genes in the region. No difference was found in the sequence analysis of candidate genes between Nisogenic and recessive materials. But the expression analysis showed that there were differences in Os03g0418600 between recessive materials, which suggested that Os03g0418600 was a gene regulating the development of awn sex. In addition, Awn3-1 and the Awn3-1 gene Awn4-2, which has been found in our laboratory, have accumulative effects on Awn3-1 and the distribution of Awn3-1 and Awn3-1. Using japonica rice variety Gaoli early rice (Awo) as donor parent, japonica rice variety Nippon (without awn) as recipient parent to construct near isogenic line, combined with the results of previous mapping, 7072 BC6F5 populations and 3 newly developed polymorphic linkage markers were used. The mapping interval of Awn4.1 on chromosome 4 was reduced to 20.02 kb, in which there were two candidate genes. After crossing with japonica rice cultivar Nippon (without Awns) at very late heading stage in Sanya as donor parent, the Awned recombination inbred line was constructed. Genetic analysis of F3 population showed that the awn traits were controlled by a pair of dominant nuclear genes and named Awn8-1. Using BSA method and linkage analysis, Awn8-1 was preliminarily located between SSR marker RM7556 and M3381 on the long arm of chromosome 8. Using 12204 strains of F4 population and 6 polymorphic linkage markers, Awn8-1 was mapped between SSR marker RM23337 and SNP marker S3 with a physical distance of 25.66 kb. There were three candidate genes in this region. Sequence analysis showed that there were two SNPs on the second exon of Os08g0485500 gene. A near isogenic line was constructed by using Japonica rice cultivar Funing small red awn as donor parent and japonica rice cultivar Nippon clear (without awn) as recurrent parent to map and clone the gene Awn4-2 which regulates the development of awn. Using 7 polymorphic linkage markers in 11206 BC4F6 populations and loci, Awn4-2 was mapped between M1126 and Indel marker In73 on chromosome 4, with a physical distance of 62.4 kb. According to RAP-DB, there are three annotated genes in this region. Sequence analysis showed that there were about 4.4kb transposon insertions in the promoter region of the predicted gene Os04g0350700 and 7 SNPs in the gene sequence in Japanese sunny and recessive materials. Combined with semi-quantitative expression analysis, Os04g0350700 may be a transcription factor encoding a conserved domain of bHLH in Awn4-2.Os04g0350700. Sequence analysis showed that the SNP at the second exon of Awn4-2 might not affect gene function, and the insertion of transposon in promoter region might result in the inhibition of gene expression and the failure of Awn4-2 to develop normally. To verify its function, we constructed RNA interference vector and overexpression vector of Awn4-2 gene respectively. The expression characteristic analysis showed that the gene was mainly expressed in young panicle of Awned material. According to the regulatory phenotypic and sequence differences, we speculated that the gene was the allele of An-1.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

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