Allele mining in wild barley: finding new exotic genes which control flowering time in the barley nested association mapping (NAM) population HEB-25.

在PP-9项目的第二阶段，我们计划在以下三个工作包中研究控制开花时间的主要基因FTI在外来大麦种群HEB-25中的功能，WP.WP1，QTL克隆-我们将与邓迪大学的Andy Flavell教授团队合作，在染色体4H上的新发现的座位分离控制Heb-25晚花的外来基因。根据1,420个Heb品系的外显子捕获测序数据，将选择两个区分重组Heb品系早花池和晚花池的候选基因，并进行三种转基因方法。这些方法包括过度表达、敲除-敲除和启动子-报告分析。前两种方法将有助于确定导致晚花表型的原因基因，后一种方法将用于表征候选基因的时空表达。WP2，等位基因挖掘-我们将研究25个外来Heb供体在四个显性主要FTI基因PPD-H1、Denso、VRN-H2和VRN-H3上的等位基因效应的变异。为此，将从Heb系中克隆12个外源单倍型的FTI基因，并用于在隐性春大麦Golden Promise基因的基因组背景下产生高表达的转化子。将研究这些转化对FTI的影响，以确定显示FTI表型改变的新的等位变异。WP3，耐逆性-我们将在全球范围内研究FTI基因对产量的开花效应及其与逆境耐受性的串扰。为此，我们将选择48个表现开花时间变异的Heb系，它们分别位于染色体2H、3H、5H和7H上的四个主要FTI基因PPD-H1、Denso、VrnH1和Vrn-H3上。这些Heb品系将在两年(2015和2016)的田间地块中进行测试，在两种处理下--压力和对照--并在世界各地的五个地点进行四次重复试验。在后续模型的基础上，我们将量化所研究的FTI基因对产量和抗逆性的影响。由于测试环境沿纬度梯度不同，我们也可以选择有利的外来FTI等位基因来响应日长变化。外部伙伴、他们的从属关系和研究的胁迫如下：1.澳大利亚阿德莱德大学的Jason Eglinton教授，干旱胁迫2。沙特阿拉伯阿卜杜拉国王科技大学马克·泰斯特教授，盐压力3。迈克尔·鲍姆博士，ICARDA，乔丹，热应激4。威廉·托马斯博士，詹姆斯·赫顿研究所，邓迪，缺氮。

During the second phase of the PP-9 project, we plan to study the function of major genes controlling flowering time, FTi, in the exotic barley population HEB-25 within the following three work packages, WP.WP1, QTL cloning - We will isolate an exotic gene that controls late flowering in HEB-25 at a newly identified locus on chromosome 4H in collaboration with the group of Prof. Dr. Andy Flavell, University of Dundee. Based on exome capture sequencing data of the 1,420 HEB lines, two top candidate genes, which differentiate between early and late flowering pools of recombinant HEB lines, will be selected and subjected to three transgenic approaches. These involve over-expression, knock-out-knock-down and promoter-reporter assays. Whereas the first two approaches will assist to identify the causal gene that causes the late flowering phenotype, the latter approach will be used to characterize the spatial and temporal expression of the candidate genes.WP2, Allele mining - We will study the variation of allelic effects of the 25 exotic HEB donors at four dominant major FTi genes, Ppd-H1, denso, Vrn-H2 and Vrn-H3. For this, 12 exotic haplotypes of the FTi genes will be cloned from HEB lines and used to generate over-expression transformants in the genomic background of the recessive spring barley Golden Promise genotype. The effects of the transformations on FTi will be studied to identify new allelic variants displaying altered FTi phenotypes.WP3, tress tolerance - We will study the flowering effects of FTi genes on yield and their crosstalk with stress tolerance on a global scale. For this, we will select 48 HEB lines, which reveal variation in flowering time, and, which segregate at four major FTi genes, Ppd-H1, denso, VrnH1 and Vrn-H3 on chromosomes 2H, 3H, 5H and 7H, respectively. Those HEB lines will be tested during two years (2015 and 2016) in field plots under two Treatments - stress versus control - and in four replications at five locations world-wide. Based on the subsequent modelling, we will quantify the impact of the studied FTi genes on yield and stress tolerance. Since the tested environments differ along a latitudal gradient, we may also select favorable exotic FTi alleles in response to day length variation.The external partners, their affiliations and the stresses studied are:1. Prof. Dr. Jason Eglinton, University of Adelaide, Australia, drought stress2. Prof. Dr. Mark Tester, King Abdullah University of Science and Technology, Saudi Arabia, salt stress3. Dr. Michael Baum, ICARDA, Jordan, heat stress4. Dr. William Thomas, James Hutton Institute, Dundee, nitrogen deficiency.