水稻航天诱变突变体变异分析及应用研究
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摘要
本研究选用航天诱变籼型水稻材料H9808后代中获得的一株突变体材料,首先利用AFLP分子标记和全基因组重测序的方法,对突变体基因组的变化进行了分析,再通过SSR、 InDe、 CAPS等分子标记方法将橙红色突变性状进行了精细定位,并将突变体材料应用于水稻育种中,创制出带橙红色标记性状的水稻新不育系,组配出了系列杂交水稻新组合。主要研究结果如下：
1.水稻航天诱变突变体性状变化与AFLP标记分析
籼型水稻材料H9808的航天诱变突变体,在拔节后期植株节间、节底隔膜中心以及茎杆下部约1/3处为橙红色,幼穗分化期幼叶的叶脉呈橙红色,抽穗时颖壳也为橙红色,并保持到种子成熟。成熟后突变体实粒数减少,籽粒变窄变长,而其它性状无明显变化。利用132对AFLP标记比较突变体与野生型材料在基因组中的变化,发现突变体基因组出现52个多态性片段,表明了突变体无论是在外观性状和基因组均发生了变异。
2.突变体与野生型材料全基因组重测序研究
以籼型水稻9311序列为基础,利用二代测序技术对突变体和野生型材料基因组重测序,结果发现航天诱变因子对水稻基因组的诱变作用分布均匀,诱变率与各对染色体的大小呈正相关,突变类型主要有SNPs、 InDel、 SV的变化,诱变率分别为29.58%、7.49%、3.75%。在SNPs中碱基A的变异率为24.4%,而A突变为T、C、G的几率分别为12.30%、15.57%、72.13%；碱基T的变异率为24.2%, T突变为A、C、G的几率分别为15.70%、69.42%、14.88%；碱基C的变异率为25.5%, C突变为A、T、G的几率分别为17.26%、72.94%、9.80%；碱基G的变异率为25.9%, G突变为A、T、C的几率分别为75.29%、16.60%、8.11%,表明航天诱变因子对A、T、C、G碱基诱变几率一致,但发生突变的碱基中A、G互变、T、C互变的几率最高。利用生物信息学方法进一步分析,发现突变体基因组的变异引起了近2000个水稻功能基因的编码序列发生了变化,这些编码序列的变化是否会引起水稻性状的改变或对应哪些外观变化还需深入研究。
3.突变体橙红色性状的遗传分析与基因定位
利用突变体分别与川恢907、9311杂交的F2代群体进行遗传分析,发现突变体的橙红色性状受1对隐性基因控制。将突变体与9311杂交的F2代中表现为橙红色性状的植株作为定位群体,利用SSR标记将橙红色基因定位在2号染色体RM5350和RM12601标记之间,遗传距离分别为0.55cM和1.38cM,再利用InDel标记,进一步将该基因定位在S1和S2之间,遗传距离分别为0.41cM和0.25cM。在SSR和InDel标记定位的基础上,物理距离约为300kb的序列中,设计了具有多态性的8对CAPS标记,最后将控制橙红色性状的基因精细定位在9412bp区间内,生物信息学预测表明该区间内仅存在1个基因Osl_06158，编码Myb家族蛋白的R2-R3类型转录因子,在拟南芥和金鱼草的研究中表明R2-R3转录因子参与了花青素生物合成途径的调控。
4.突变体在水稻育种中的应用
田间调查发现突变体材料除带有的橙红色标记外,还有较好的农艺性状和经济性状,因此可作为水稻不育系选育的材料。在与不同遗传背景的不育系杂交时发现与宜香1A的杂交后代高度不育,将其与宜香1A连续多年杂交、回交,成功育成了带橙红色标记性状的杂交水稻新不育系花香A,并与川恢907、川恢4016、川恢1618等恢复系配组,组配出了花香7号、花香4016、花香优1618等杂交水稻新组合并通过了省级审定。
A mutant of Indica rice H9808with orange-red trait was obtained by space mutation technology. The variation of the mutant was analyzed by AFLP molecular markers and genome re-sequencing technology. The gene controls orange-red trait of the mutant was fine mapped by SSR, InDel and CAPS markers. We used the mutant in rice breeding and new CMS lines with orange-red trait were obtained.The main results are as follows:
1. AFLP molecular marker. analysis of mutant of space-flight in rice
Compared with the wild-type material planted in the field, we found that the mutant dispalys young leaves with orange-red veins at the stage of panicle initiation its internode and one third of the down part stem become orange-red by the end of booting stage, and the color of the mutant hull is also orange-red from heading to maturity. The full grains of mutant reduced significantlyand become more narrower and longer, while other traits did not change significantly. Comparing the changes of mutant and wild-type material in genomic DNA by132AFLP molecular markers, we found52polymorphic fragments in the genome of the mutant, which showed that the mutant had changed both in the phenotype and the genome.
2. Re-sequencing of the mutant and wild-type
Taking the indica rice9311as the basic sequence, we re-sequenced genome of mutant and wild-type material and found that the mutagenic effects of space mutation factors for the rice genome were basically distributed evenly and the mutation rate was positively correlated with chromosome size. The mutagenesis types are SNPs, InDel and SV. Their mutation rates are29.58%,7.49%,3.75%respectively. The percent of Base A in SNPs Mutant is24.4%, and the percent of A into T, C and G is12.30%,15.57%,72.13%respectively; the percent of Base T in SNPs Mutant is24.2%, and the percent of T into A, C and G is15.70%,69.42%,14.88%respectively; the percent of Base C in SNPs Mutant is25.5%, and the percent of C into A, T and G is17.26%,72.94%,9.80%respectively; the percent of Base G in SNPs Mutant is25.9%, and the percent of G into A, T and C is75.29%,16.60%,8.11%respectively. These results showed that the space mutation factors had thesame effects on the mutation of the A, T, C and G base. The most found base change types are the interchange between A and G, and the interchange between T and C. After comparing the gene sequences oofmutant and wild-type, we found that the mutagenesis of SNPs, InDel and SV have made more than2000genes' encoding amino acid changed in mutant.
3. Genetic analysis and mapping of the mutated gene conferring orange-re d leaves and hull
We found that phenotype of Orange-red trait was controlled by a single recessive allele by comparing the F2plants of the mutant with Chuan Hui907and9311cross. F2population of the cross between mutant and9311was analyzed, and the gene was located on chromosome2, with the genetic distance of0.55cM and1.38cM from SSR marker RM5350and RM12601. Further study was conducted by InDel markers, and the target gene was narrowed down the locus to0.41cM and0.25cM from S1and S2, respectively. After analyzing the300KB sequence attained by the maping analyasis with above molecular markers, we found that there is no available SSR and InDel markers. Therefore, we designed8CAPS markers in this region based on the sequence of indica rice9311, and located the gene into the region of9412bp which only concluding a gene Osl_06158. Analysis showed that the gene Osl_06158encoding a R2R3-MYB protein is a regulatory factor,. The reported studies in Arabidopsis and Petunia have showed that R2R3-MYB protein, bHLH protein of MYC family and WD40repeat protein interact with each other to regulate the biosynthetic pathway of anthocyanin in plant.
4. Application of mutants in rice breeding
The mutant has good agronomic and economic traits, and specific Orange-red trait that facilitates the selection of rice hybrid plants, so it is a good donor in rice breeding. When hybridization with sterile lines or CMS LINES of different genetic background, we found that the offspring from mutant and Yixiang1A is highly sterile. After hybriding, backcrossing with Yixiang1A, and breeding for many years, we obtained a new hybrid rice CMS line HuaxiangA. Afer hybriding HuaxiangA with different rice restorer lines such as ChuangHui907, ChuangHui4016and ChuangHui1618, we have got new hybrid rice combinations of HuaXiang7, HuaXiang907, HuaXiang4016and HuaXiang1618, which have passed the provincial approval.
1.水稻航天诱变突变体性状变化与AFLP标记分析
籼型水稻材料H9808的航天诱变突变体,在拔节后期植株节间、节底隔膜中心以及茎杆下部约1/3处为橙红色,幼穗分化期幼叶的叶脉呈橙红色,抽穗时颖壳也为橙红色,并保持到种子成熟。成熟后突变体实粒数减少,籽粒变窄变长,而其它性状无明显变化。利用132对AFLP标记比较突变体与野生型材料在基因组中的变化,发现突变体基因组出现52个多态性片段,表明了突变体无论是在外观性状和基因组均发生了变异。
2.突变体与野生型材料全基因组重测序研究
以籼型水稻9311序列为基础,利用二代测序技术对突变体和野生型材料基因组重测序,结果发现航天诱变因子对水稻基因组的诱变作用分布均匀,诱变率与各对染色体的大小呈正相关,突变类型主要有SNPs、 InDel、 SV的变化,诱变率分别为29.58%、7.49%、3.75%。在SNPs中碱基A的变异率为24.4%,而A突变为T、C、G的几率分别为12.30%、15.57%、72.13%；碱基T的变异率为24.2%, T突变为A、C、G的几率分别为15.70%、69.42%、14.88%；碱基C的变异率为25.5%, C突变为A、T、G的几率分别为17.26%、72.94%、9.80%；碱基G的变异率为25.9%, G突变为A、T、C的几率分别为75.29%、16.60%、8.11%,表明航天诱变因子对A、T、C、G碱基诱变几率一致,但发生突变的碱基中A、G互变、T、C互变的几率最高。利用生物信息学方法进一步分析,发现突变体基因组的变异引起了近2000个水稻功能基因的编码序列发生了变化,这些编码序列的变化是否会引起水稻性状的改变或对应哪些外观变化还需深入研究。
3.突变体橙红色性状的遗传分析与基因定位
利用突变体分别与川恢907、9311杂交的F2代群体进行遗传分析,发现突变体的橙红色性状受1对隐性基因控制。将突变体与9311杂交的F2代中表现为橙红色性状的植株作为定位群体,利用SSR标记将橙红色基因定位在2号染色体RM5350和RM12601标记之间,遗传距离分别为0.55cM和1.38cM,再利用InDel标记,进一步将该基因定位在S1和S2之间,遗传距离分别为0.41cM和0.25cM。在SSR和InDel标记定位的基础上,物理距离约为300kb的序列中,设计了具有多态性的8对CAPS标记,最后将控制橙红色性状的基因精细定位在9412bp区间内,生物信息学预测表明该区间内仅存在1个基因Osl_06158，编码Myb家族蛋白的R2-R3类型转录因子,在拟南芥和金鱼草的研究中表明R2-R3转录因子参与了花青素生物合成途径的调控。
4.突变体在水稻育种中的应用
田间调查发现突变体材料除带有的橙红色标记外,还有较好的农艺性状和经济性状,因此可作为水稻不育系选育的材料。在与不同遗传背景的不育系杂交时发现与宜香1A的杂交后代高度不育,将其与宜香1A连续多年杂交、回交,成功育成了带橙红色标记性状的杂交水稻新不育系花香A,并与川恢907、川恢4016、川恢1618等恢复系配组,组配出了花香7号、花香4016、花香优1618等杂交水稻新组合并通过了省级审定。
A mutant of Indica rice H9808with orange-red trait was obtained by space mutation technology. The variation of the mutant was analyzed by AFLP molecular markers and genome re-sequencing technology. The gene controls orange-red trait of the mutant was fine mapped by SSR, InDel and CAPS markers. We used the mutant in rice breeding and new CMS lines with orange-red trait were obtained.The main results are as follows:
1. AFLP molecular marker. analysis of mutant of space-flight in rice
Compared with the wild-type material planted in the field, we found that the mutant dispalys young leaves with orange-red veins at the stage of panicle initiation its internode and one third of the down part stem become orange-red by the end of booting stage, and the color of the mutant hull is also orange-red from heading to maturity. The full grains of mutant reduced significantlyand become more narrower and longer, while other traits did not change significantly. Comparing the changes of mutant and wild-type material in genomic DNA by132AFLP molecular markers, we found52polymorphic fragments in the genome of the mutant, which showed that the mutant had changed both in the phenotype and the genome.
2. Re-sequencing of the mutant and wild-type
Taking the indica rice9311as the basic sequence, we re-sequenced genome of mutant and wild-type material and found that the mutagenic effects of space mutation factors for the rice genome were basically distributed evenly and the mutation rate was positively correlated with chromosome size. The mutagenesis types are SNPs, InDel and SV. Their mutation rates are29.58%,7.49%,3.75%respectively. The percent of Base A in SNPs Mutant is24.4%, and the percent of A into T, C and G is12.30%,15.57%,72.13%respectively; the percent of Base T in SNPs Mutant is24.2%, and the percent of T into A, C and G is15.70%,69.42%,14.88%respectively; the percent of Base C in SNPs Mutant is25.5%, and the percent of C into A, T and G is17.26%,72.94%,9.80%respectively; the percent of Base G in SNPs Mutant is25.9%, and the percent of G into A, T and C is75.29%,16.60%,8.11%respectively. These results showed that the space mutation factors had thesame effects on the mutation of the A, T, C and G base. The most found base change types are the interchange between A and G, and the interchange between T and C. After comparing the gene sequences oofmutant and wild-type, we found that the mutagenesis of SNPs, InDel and SV have made more than2000genes' encoding amino acid changed in mutant.
3. Genetic analysis and mapping of the mutated gene conferring orange-re d leaves and hull
We found that phenotype of Orange-red trait was controlled by a single recessive allele by comparing the F2plants of the mutant with Chuan Hui907and9311cross. F2population of the cross between mutant and9311was analyzed, and the gene was located on chromosome2, with the genetic distance of0.55cM and1.38cM from SSR marker RM5350and RM12601. Further study was conducted by InDel markers, and the target gene was narrowed down the locus to0.41cM and0.25cM from S1and S2, respectively. After analyzing the300KB sequence attained by the maping analyasis with above molecular markers, we found that there is no available SSR and InDel markers. Therefore, we designed8CAPS markers in this region based on the sequence of indica rice9311, and located the gene into the region of9412bp which only concluding a gene Osl_06158. Analysis showed that the gene Osl_06158encoding a R2R3-MYB protein is a regulatory factor,. The reported studies in Arabidopsis and Petunia have showed that R2R3-MYB protein, bHLH protein of MYC family and WD40repeat protein interact with each other to regulate the biosynthetic pathway of anthocyanin in plant.
4. Application of mutants in rice breeding
The mutant has good agronomic and economic traits, and specific Orange-red trait that facilitates the selection of rice hybrid plants, so it is a good donor in rice breeding. When hybridization with sterile lines or CMS LINES of different genetic background, we found that the offspring from mutant and Yixiang1A is highly sterile. After hybriding, backcrossing with Yixiang1A, and breeding for many years, we obtained a new hybrid rice CMS line HuaxiangA. Afer hybriding HuaxiangA with different rice restorer lines such as ChuangHui907, ChuangHui4016and ChuangHui1618, we have got new hybrid rice combinations of HuaXiang7, HuaXiang907, HuaXiang4016and HuaXiang1618, which have passed the provincial approval.
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