Molecular Basis of Self-Incompatibility in Petunia

IBN-9982659 PI：高辉矮牵牛具有配子体型自交不亲和性，其自交不亲和性受一个多态位点S-位点控制。花粉和雌蕊所携带的S等位基因的配对抑制了花柱中花粉管的生长。在S-位点的多态性基因，称为S-RNase基因，控制雌蕊功能在SI已被确定。然而，在花粉互作中控制花粉功能的基因，即花粉S基因，还没有被鉴定出来，本项目的目的是利用功能基因组学的方法来鉴定胀果侧耳花粉S基因。目的一是用14个S位点的cDNA标记和S-RNase基因作为探针，从已构建的S2 S2 BAC文库中分离克隆。目的II是构建S1 S1基因型的BAC文库，并使用与目的I相同的探针分离阳性克隆。 目的III是通过脉冲场凝胶电泳和荧光原位杂交来表征目的I和II中分离的所有BAC克隆，以确定它们之间的任何重叠，将它们在S位点排序，并估计它们之间的间隙大小。将从两个BAC文库中分离额外的克隆，以尽可能广泛地覆盖整个S基因座。目的四是将所有BAC克隆分别导入S1 S2基因型的胀果蓝植株中，以确定它们中是否含有花粉S基因。如果一个BAC克隆含有花粉S2-基因，则转基因植株产生的花粉中有四分之一携带花粉S1-等位基因和花粉S2-转基因，它们将由于竞争相互作用而与S1 S2雌蕊亲和。 将研究具有自交亲和性的转基因植物，以鉴定负责表型的转基因。花粉S-基因的功能将通过功能丧失实验进一步确定，这一研究的完成将进一步加深对基于RNA酶的自我/非自我识别机制的理解。分离的BAC克隆可用于研究附加的S位点基因的功能，以及两种不同S单倍型S1和S2的S位点的比较研究。在实践方面，可探索将S1性状恢复到自交亲和栽培种中以利于杂交制种的可能性。 如果成功，这将产生非常重要的农艺影响。

IBN-9982659PI: Teh-hui KaoPetunia inflata possesses gametophytic type self-incompatibility which iscontrolled by a polymorphic locus, the S-locus. Matching of S-allelescarried by the pollen and pistil results in inhibition of pollen tubegrowth in the style. A polymorphic gene at the S-locus, termed the S-RNasegene, that controls pistil function in SI has been identified. However, thegene that controls pollen function in SI interactions, termed the pollenS-gene, has not been identified.The goal of this proposed project is to use a functional genomic approachto identify the pollen S-gene of P. inflata. Obj. I is to use 14 cDNAmarkers for the S-locus and the S-RNase gene as probes to isolate clonesfrom an S2S2 BAC library already contsructed. Obj. II is to construct a BAClibrary of the S1S1 genotype and use the same probes employed in Obj. I toisolate positive clones. Obj. III is to characterize all the BAC clonesisolated in Obj. I and II by pulse-field gel electrophoresis andfluorescence in situ hybridization to determine any overlap between them,to order them at the S-locus, and to estimate the size of the gaps betweenthem. Additional clones will be isolated from both BAC libraries to coveras extensively as possible the entire S-locus. Obj. IV is to introduce allthe BAC clones separately into P. inflata plants of S1S2 genotype todetermine whether any of them contains the pollen S-gene. If a BAC clonecontains the pollen S2-gene, a quarter of the pollen grains produced by atransgenic plant will carry the pollen S1-allele and the pollenS2-transgene, and they will be compatible with S1S2 pistils due tocompetitive interaction. Transgenic plants that have becomeself-compatible will be studied to identify the transgene that isresponsible for the phenotype. The function of the candidate(s) for thepollen S-gene will be further ascertained by loss-of-function experiments.Accomplishment of this proposed research will advance the understanding ofan RNase-based self/non-self recognition mechanism. The BAC clones isolatedcan be used for studying the functions of additional S-locus genes, and forcomparative study of the S-locus of two different S-haplotypes, S1 and S2.On the practical side, one can explore the possibility of restoring the SItrait to self-compatible cultivated species to facilitate hybrid seedproduction. If successful, this will have a very important agronomicimpact.