Towards a better molecular and physiological understanding of spikelet fertility in barley (Hordeum vulgare L.)

本研究主要集中在大麦小穗育性的分子研究上。它主要由两个主要部分组成：第一个感兴趣的领域涉及发育突变体，六排穗2（vrs 2）的分子分析;而第二部分则集中在Vrs 4.1的推定下游基因的分子和生理特征上。 通过遗传作图，我们确定了一个vrs 2候选染色体区域包含两个预测的基因模型在大麦物理图谱。然而，对于vrs 2基因座，迄今为止仅发现一个已知的等位基因（vrs2.e），这使得该基因座的分析变得困难。因此，我们建议使用不同的遗传和分子方法来寻找或创建新的vrs 2等位基因。通过（I）中间大麦（H.普通褶曲intermedium），和（II）两个候选基因的TILLING分析。(III)应检测新等位基因是否与vrs2.e等位。(IV)转基因方法应包括使用两个野生型候选基因互补vrs2.e突变体;但类似地通过TALEN技术产生新的等位基因。(V)最后，vrs2.e和其他假定的等位基因将进行详细的表型分析以及代谢和激素分析，以揭示与两个vrs 2候选基因之一的功能联系。 在我们最近的研究（Koppolu et al. 2013）* 中，我们发现Vrs 4调节属于海藻糖生物合成途径的基因的转录物。海藻糖-6-磷酸（T6 P）被认为是一种重要的信号代谢产物，参与植物生长发育对碳源供应的调控。T6 P水平可能受一组海藻糖-6-磷酸合酶（TPS）和海藻糖-6-磷酸磷酸酶（TPP）同源物控制。本文拟对大麦花序中这两类酶的功能进行详细的研究，以阐明它们的系统发育关系、拷贝数、组织特异性、mRNA表达模式和催化活性（即TPS，合成T6 P的能力; TPP，去磷酸化T6 P的能力）。这项研究提案旨在从更深层次的分子，大麦小穗和小花育性的遗传和生理学研究，以提高小粒谷物的未来产量。

This research proposal we will focus on the molecular elucidation of spikelet fertility in barley. It largely consists of two major parts: The first area of interest deals with the molecular analysis of a developmental mutant, six-rowed-spike2 (vrs2); whereas part two focuses on the molecular and physiological characterization of putative down-stream genes of Vrs4.1) Through genetic mapping we identified a vrs2-candidate chromosomal region containing two predicted gene models in the barley physical map. However, for the vrs2 locus only one known allele has been found so far (vrs2.e), which makes analysis of this locus difficult. Hence, we propose to search for, or create new vrs2 alleles using different genetic and molecular approaches. New alleles shall be found through (I) SANGER-sequencing of intermedium-barleys (H. vulgare convar. intermedium), and (II) TILLING analyses of the two candidate genes. (III) Putatively new alleles shall be tested for being allelic to vrs2.e. (IV) Transgenic approaches shall include a complementation of the vrs2.e mutant using the two wild type candidate genes; but similarly to create new alleles through TALEN technology. (V) Finally, vrs2.e and other putative alleles shall undergo a detailed phenotypic analysis as well as metabolic and hormonal profiling to reveal functional links to one of the two vrs2 candidate genes.2) In our very recent study (Koppolu et al. 2013)* we showed that Vrs4 regulates transcripts of genes belonging to the trehalose biosynthesis pathway. Trehalose-6-phosphate (T6P) has been implicated to control meristem determinacy and generally has been considered as an important signaling metabolite that is involved in the regulation of plant growth and development in response to carbon availability. T6P levels are potentially governed by a group of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) homologs. Here we would like to propose a detailed functional characterization of these two enzyme classes in barley inflorescences to elucidate their phylogenetic relationships, copy number, tissue-specificity, mRNA expression pattern and catalytic activities (i.e. TPS, ability to synthesize T6P; TPP, ability to dephosphorylate T6P).This research proposal aims at a deeper molecular, genetic and physiological insight into spikelet and floret fertility of barley for improving future yields of small grain cereals.