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基因FTi在外来大麦群体HEB-25中的功能，WP. WP 1，QTL克隆-我们将与Andy Flavell教授博士的小组合作，在染色体4 H上的一个新鉴定的位点分离控制HEB-25中晚花的外来基因，邓迪大学。基于1，420个HEB品系的外显子组捕获测序数据，将选择区分重组HEB品系的早开花池和晚开花池的两个顶级候选基因，并进行三种转基因方法。这些涉及过表达、敲除敲低和启动子-报告基因测定。前两种方法将有助于鉴定导致晚开花表型的致病基因，后一种方法将用于表征候选基因的空间和时间表达。WP 2，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处的等位基因效应的变化。为此，FTi基因的12个外来单倍型将从HEB系中克隆，并用于在隐性春大麦Golden Promise基因型的基因组背景中产生过表达转化体。将研究转化对FTi的影响，以鉴定显示改变的FTi表型的新等位基因变体。WP 3，胁迫耐受性-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胁迫耐受性的串扰。为此，我们将选择48个HEB系，其显示开花时间的变化，并且分别在染色体2 H、3 H、5 H和7 H上的四个主要FTi基因Ppd-H1、denso、VrnH 1和Vrn-H3处分离。这些HEB品系将在两年内（2015年和2016年）在两种处理（胁迫与对照）下的田间小区中进行测试，并在全球五个地点进行四次重复。基于随后的建模，我们将量化所研究的FTi基因对产量和胁迫耐受性的影响。由于所测试的环境沿纬度梯度沿着不同，我们也可以选择有利的外来FTi等位基因，以响应日长的变化。澳大利亚阿德莱德大学的Jason埃格林顿教授和博士，干旱胁迫2。沙特阿拉伯阿卜杜拉国王科技大学教授Mark Tester博士，盐胁迫3。迈克尔·鲍姆博士，ICARDA，约旦，热应激4。威廉托马斯博士，詹姆斯赫顿研究所，邓迪，氮缺乏。

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.