【作者】 赵良侠；

【导师】 马传喜；

【作者基本信息】 安徽农业大学 ， 作物生物技术， 2017， 博士

【摘要】 收获前穗发芽(Pre-harvest sprouting,PHS)是世界范围内小麦生产上面临的主要问题之一,严重影响小麦产量及品质。穗发芽是由多基因控制的数量性状,同时受环境条件的影响。前人已鉴定的穗发芽抗性相关的基因数目有限,在不同材料中,这些基因等位变异与穗发芽表型的关系尚不清晰。因此,克隆和挖掘更多穗发芽抗性调控基因及其优异等位类型,开发功能标记,聚合不同抗穗发芽优异等位基因将有助于培育穗发芽抗性持续性强的小麦品种。TaMFT是FT(flowering time)家族成员,前人研究表明TaMFT抑制小麦籽粒萌发,是位于3AS上一个控制种子休眠的主效QTL区域的候选基因。本研究在穗发芽抗性极端材料中检测TaMFT-3A的等位变异,开发功能标记,并在275份中国微核心种质和262份小麦育种材料中分析其对穗发芽抗性的效应,同时结合TaMFT-3A已知等位变异类型(SNP-222,SNP646,SNP666)分析聚合多个优异等位类型对穗发芽抗性的效应。PTF1是一个具有bHLH结构域的转录因子,在玉米中被报道与种子发芽相关,但在小麦中TaPTF1与种子休眠的关系及其调控机制尚不清晰。本研究利用已开发的TaPTF1-B1基因标记,分析TaPTF1-B1等位变异与穗发芽抗性的相关性,通过测定激素ABA和GA含量以及相关基因的表达水平,探讨其参与穗发芽抗性的调控机制,主要研究结果如下:1.在中抗穗发芽品种(安农0711)和感穗发芽品种(河农825)中检测到TaMFT-3A基因在启动子-194bp位置(SNP-194)存在一处33个碱基的插入/缺失,以此开发功能标记TaMFT-A2,并鉴定出TaMFT-A2a和TaMFT-A2b两种等位类型,其中,TaMFT-A2a与抗穗发芽相关,分布频率较高;而TaMFT-A2b与感穗发芽相关。在安农0711/河农825构建的重组自交系群体中,TaMFT-A2标记与控制穗发芽抗性的主效QTL共分离,位于TaMFT-3A标记SNP646/SNP666与Xbarc310之间,可解释穗发芽抗性表型变异的13.85%-16.12%。2.在中国微核心种质与小麦育种材料中共检测TaMFT-3A 4处等位变异(SNP-222、SNP-194、SNP+646、SNP+666)与穗发芽表型相关,多重比较结果表明,携带SNP-222优异等位变异的材料的种子发芽指数和田间自然发芽率显著低于携带SNP-194、SNP+646/666优异等位变异的材料的种子发芽指数和田间自然发芽率,即TaMFT-3A基因在启动子-222bp位置的变异对穗发芽抗性的效应最大。同时聚合4个优异等位类型的小麦材料的种子发芽指数较其它携带0-3个优异等位类型的材料的种子发芽指数显著降低,说明不同优异等位基因对穗发芽抗性存在一种聚合效应。3.在中国微核心种质与小麦育种材料中检测到TaPTF1-B1两种等位类型,TaPTF1-B1a和TaPTF1-B1b。携带TaPTF1-B1a等位类型的材料其种子发芽指数显著高于携带TaPTF1-B1b等位类型的材料的种子发芽指数。随着育种年代进程,TaPTF1-B1b的分布频率越来越低,受到负向选择。在京411/红芒春21群体中,TaPTF1-B与控制穗发芽的QTL(Qphs.jhau-7B)紧密连锁,可解释8.39%-11.46%的穗发芽表型变异。4.TaPTF1在休眠/休眠解除的红芒春21籽粒中不同浸润时期的表达变化趋势与ABA/GA的比例变化趋势一致,而与GA合成途径中关键酶基因(TaGA20ox与TaGA3ox2)的表达变化趋势相反,说明TaPTF1作为转录调控因子,通过参与ABA和GA合成/代谢途径,从而调控种子休眠与萌发。更多还原

【Abstract】 Pre-harvest sprouting(PHS)is an important problem in wheat production worldwide.PHS in wheat causes terrible loss in grain quality and yield,particularly in regions with prolonged wet weather during the harvest season.As a quantitative trait,PHS is influenced jointly by genetic and environmental factors.At present,PHS resistance genes in wheat have not been well characterized at the nucleotide sequence level.Additionly,the numbers of genes with rich allelic variations are not enough to meet the needs of improvement of PHS resistance in wheat breeding.Therefore,identification of superior alleles for improving PHS resistance and development of functional marker will contribute to develop wheat varieties with high PHS tolerance.Mother of FT and TFL1(MFT),belongs to phosphatidyl ethanolamine binding protein(PEBP)family,has recently been shown to inhibit seed germination in wheat.PTF1 was a transcription factor with a bHLH domain,which was proved to relate with PHS in maize;however,its homologus genes have not been reported on wheat.In this study,we detected novel variations inTaMFT-3A,developed functional markers for PHS resistance in Chinese micro-core collections of wheat,and validated effects of the novel alleles on PHS resistance using linkage and association analysis.Based on the results,wheat varieties pyramiding favorable alleles of TaMFT-3A were selected for PHS resistance breeding.Simultenously,the association of TaPTF1-B1 allelic variations with PHS resistance was analyzed through gene-specific marker.The regulation mechanisims of TaPTF1-B1 on PHS resistance was also discussed in this study.The main findings of this study are as follows:1.An InDel variation(33 bp)in the-194 of the promoter of TaMFT-3A was detected between Chinese white-grained wheat varieties Annong0711(PHS tolerant)and Henong825(PHS susceptible),and developed a gene-specific marker(TaMFT-A2).Linkage analysis indicated that the TaMFT-A2 co-segregated with a major QTL(designed Qphs.ahau-3A)controlling PHS resistance on 3AS in the population from the cross of Annong0711×Henong825,explaining 13.85-16.12% of phenotypic variations.Associationanalysis showed that the TaMFT-A2 was significantly associated with PHS resistance in262 wheat varieties and 275 Chinese mini-core collections of wheat,suggesting that it can be used for marker-assistant selection of PHS resistance breeding.2.Four mutations in TaMFT-3A,including SNP-222,SNP646,SNP666 and TaMFT-A2,were evaluated the association with PHS.Multiple comparison analysis indicated that varieties carried favorable alleles SNP-222 had averages GI and FS of 0.30 and 0.14,which were significant lower thanthe materials with other mutations across different environments(P < 0.01).Several white-grained varieties pyramiding four favorable alleles of TaMFT-3A,which hadsignificantly lower GI and FS values,could be used for improvement of PHS tolerance in wheat breeding.3.TaPTF1-Bl contained two allelic varieties,which were designated as TaPTF1-B1 a and TaPTF1-B1 b.GI and FS values of wheat varieties carrying TaPTF1-B1 a were significantly higher than that of wheat varieties carrying TaPTF1-B1b(P < 0.01).TaPTF1-B1 b was considered as a superior allele for PHS resistance in wheat.With the breeding process,the distribution frequency of TaPTF1-B1 b is getting lower and lower,which is negatively chosen..In the Jing411×Hongmangchun21 RIL population,a major QTL for seed dormancy on 7B was detected at 5 and 15 days after harvest,explaining 8.39%-11.46% of phenotypic variations.4.In different imbibition of dormant and non-dormant Hongmangchun21 seed,the TaPTF1 gene expression is consistent with the changes of the ratio of ABA/GA,and contrary to the expression of the TaGA20 ox and TaGA3ox2,key genes in GA synthesis pathway.We speculate that the TaPTF1 gene positively controls seed germination through participation in GA and ABA synthesis pathway,however its details still needs to be further verified.更多还原