‘泰国小香占’香味基因的鉴定和功能分子标记的开发

Identification of the Fragrance Allele and Development of the Functional Markers for Fragrance in’Taiguoxiaoxiangzhan’

香稻育种已成为当前杂交水稻育种的一个重要方向,选择和利用株型好、米质优、抗性强等综合性状优良且具有香味的水稻亲本,尤其是香型水稻恢复系亲本的利用,对进一步促进杂交水稻香稻的选育具有重要意义。‘泰国小香占’是目前国内水稻香稻育种中应用较广且综合性状优良的恢复系亲本,其米质优、抗性强、香味浓。为了探究‘泰国小香占’香味产生的原因,本研究鉴定了‘泰国小香占’的香味基因及其突变类型,发现其香味来源于编码甜菜碱脱氢酶基因Badh2的功能缺失突变,且通过对该基因完整基因组序列扩增和测序分析发现其等位突变类型为badh2-E7类型,即在第7外显子处存在8 bp缺失和3个单核苷酸位点多态性突变(SNPs)。通过气相色谱-质谱联用技术(GC-MS)测定其籽粒中香味物质2-乙酰-1-吡咯啉(2-AP)的含量为0.108 mg/kg,明显高于非香味品种‘日本晴’和‘9311’的含量(<0.01 mg/kg)。实验还证明了特异性分子标记FMbadh2-E7、InDel-E7以及自主开发设计的特异性功能标记FMbadh2-E7A和FMbadh2-E7B均可高效、准确鉴别出非香纯合(Badh2/Badh2)、非香杂合(Badh2/badh2)和香味纯合(badh2/badh2)3种基因型。同时本研究还进一步通过对‘泰国小香占’和非香味水稻品种杂交F2代单株进行了基因型和遗传规律分析,发现其基因型Badh2/Badh2:Badh2/badh2:badh2/badh2的比例为1∶2∶1,符合孟德尔细胞核单基因控制分离比。本研究通过对‘泰国小香占’香味基因的鉴定、遗传特性分析以及特异性功能分子标记的开发和验证,为进一步通过分子标记辅助选择育种技术,应用‘泰国小香占’香味基因培育优质、高产且抗性强的杂交水稻香稻提供了重要的理论基础。

The fragrant rice breeding has become an important direction for hybrid rice breeding program.The selection and utilization of parent materials with ideal plant type,fine grain quality,strong resistance and other excellent agronomic characteristics with strong fragrance,especially the utilization and cultivation of the fragrant rice restorer lines,is of great significance to further promote the fragrant hybrid rice breeding.’Taiguoxiaoxiangzhan’is a restorer line with excellent grain quality,strong resistance and fragrance,which has been widely used in fragrant rice breeding in China.In order to trace the fragrance in’Taiguoxiaoxiangzhan’,we identified the type of the fragrant gene in’Taiguoxiaoxiangzhan’was badh2-E7 which has an 8 bp deletion and three single nucleotide polymorphisms(SNPs)in exon 7,and this gene is a non-functional badh2 allele and that the functional Badh2 allele encoding betaine aldehyde dehydrogenase(BADH2)could render rice non-fragrant.Furthermore,we found that the’Taiguoxiaoxiangzhan’increased 2-acetyl-1-pyrroline(2-AP)content(0.108 mg/kg)compared with the non-fragrant rice varieties of’Nipponbare’and’9311’(<0.01 mg/kg)by the GC-MS method.We also proved that FMbadh2-E7 and InDel-E7,two molecular markers reported earlier,can be applied in different genotypes,including Badh2/Badh2(Non-fragrance homozygote),Badh2/badh2(Non-fragrance heterozygote)and badh2/badh2(Fragrance homozygote).In addition,based on sequence divergence amongst the functional Badh2 and null badh2-E7 allele,we developed two functional markers,FMbadh2-E7 A and FMbadh2-E7 B,which can be easily used to distinguish non-fragrant from fragrant rice.Genetic analysis shows that the segregation of Badh2/Badh2,Badh2/badh2 and badh2/badh2 in the F2 population derived from crosses between’Taiguoxiaoxiangzhan’and different restorer lines fitted a theoretical ratio of 1∶2∶1,indicating that the fragrance of’Taiguoxiaoxiangzhan’is controlled by a single nuclear recessive gene(badh2).In this study,the identification and genetic analysis of the fragrant gene in’Taiguoxiaoxiangzhan’and the development of functional markers for fragrance have been studied,which provides a theoretical foundation for the further breeding of fragrant hybrid rice with excellent quality,high yield and strong resistance by molecular marker-assisted selection technology.