PP-9: Pillen, Kumlehn
Allele mining in wild barley: finding new exotic genes which control flowering time in the barley nested association mapping (NAM) population HEB-25
Enhancing crop diversity is a key issue to cope with current challenges in agricultural production, facing increased incidences of abiotic (e.g. drought and mineral deficiency) and biotic stresses (e.g. pathogen attacks). Many studies have shown that un-adapted germplasm contains a wealth of novel gene variants (i.e. exotic alleles), which are useful to raise crop performance and extend biodiversity of our modern cultivars. In principle, the biodiversity available in a germplasm collection can be exploited by nested association mapping (NAM). The NAM design was originally developed in maize (Yu et al. 2008). Instead of interrogating a single accession for useful biodiversity, numerous diverse genotypes are explored in parallel in NAM sub-families. This concept proved to foster the identification of candidate genes at or near the gene level. In maize the usefulness of the NAM design has been confirmed, for example by dissecting the genetic architecture of flowering time (FTi, Buckler et al. 2009) and other traits.
During the first phase of the priority program SPP1530, we have developed HEB-25 (Halle exotic barley), the first barley nested association mapping (NAM) population world-wide (Schnaithmann et al. 2014, Maurer et al. submitted). HEB-25 is ideally suited to study, both, biodiversity present in wild barley and to serve as a source of exotic alleles for barley breeding. So far, HEB-25 was genetically characterized with a 9k Infinium iSELECT chip and used to map novel as well as previously known QTLs/genes with high precision, which regulate FTi and other agronomic traits. By the start of the second SPP phase, the Pillen lab will have access to exome capture data of HEB-25, which will allow to align the allelic sequences of the 26 HEB parents for more than 20,000 high confidence barley gene models and to study their inheritance in HEB lines. The exome capture sequence data is also useful to define exotic haplotypes and to study their gene function in HEB-25 with a, so far, unmatched genetic resolution in genome-wide association studies (GWAS).
To facilitate the generation of transformation vectors for over-expression and RNAi-mediated knock-down, the Kumlehn lab has developed a set of versatile generic, GATEWAY-compatible binary vectors comprising derivatives that harbor ubiquitous or tissue-specific promoters for the use in monocotyledonous plants (Himmelbach et al. 2007). Since then, these vectors have been successfully employed in dozens of laboratories worldwide. Research approaches of the Kumlehn lab included over-expression, knock-down, mutant complementation and promoter studies in a number of cereal species and in a wide range of research fields, such as domestication and plant architecture (Sakuma et al. 2013), plant-pathogen interaction (e.g. Nowara et al. 2010), response to abiotic stress (Seiler et al. 2014) and grain yield (Weichert et al. 2010). More recently, the group has focused on the regulation of FTi (Gawronski et al. 2014). The Kumlehn group has also a strong focus on site-specific genome engineering, which will greatly facilitate the functional validation of genes and offers versatile novel possibilities of crop improvement. The group has been among the very first to generate mutant plants using customized transcription activator-like effector nucleases (TALENs) in barley. It is anticipated that the method may not only be utilized for the generation of knock-out mutants but also for plants carrying novel alleles with modified functionality.
During the second phase of the SPP, we aim to dig deeper into the wealth of functional diversity we previously identified in HEB-25. In this regard, we have set up the following three work packages (WP), which are jointly coordinated by Dr. Kumlehn and Prof. Pillen.
WP 1: Cloning and characterizing exotic alleles of a novel FTi QTL.
In WP 1, a novel HEB-25 QTL on chromosome 4H will be isolated and characterized, where the exotic barley donor alleles cause late flowering phenotypes across and within the 25 HEB families compared to the recipient parent Barke. By cloning newly identified exotic FTi QTL alleles, we will raise the understanding of FTi regulation to improve the genetic architecture of crop plants via knowledge based breeding.
WP 2: Allele mining for exotic haplotypes of known FTi genes.
In WP 2, barley transformants, stably over-expressing a set of 12 wild barley alleles of known functional FTi genes will be generated, which caused extreme early or late flowering phenotypes in HEB-25. Subsequently, FTi effects and additional pleiotropic effects of the selected transformants will be characterized in greenhouse and field experiments. By transformation of an elite barley genotype with functional wild barley alleles of approved FTi regulating genes, we will study modification of FTi towards crop improvement by altering the expression or function of individual genes either by genetic modification or by mutation.
WP 3: HEB-YIELD: A crosstalk between FTi and abiotic stress tolerance in HEB-25.
In WP 3, a set of 48 HEB lines will be selected, segregating at four important FTi genes (Ppd-H1, denso, Vrn-H1 and Vrn-H3). By means of the selected HEB lines the crosstalk between flowering regulation and tolerance to four important abiotic stresses (drought, heat, salt and nitrogen deficiency) will be studied in a global field trial across a wide range of photoperiod field conditions located in UK, Germany, Jordan, Saudi Arabia and Australia. By testing the exotic NAM population HEB-25 through a global series of environments and under various stress conditions, we will improve the understanding of pleiotropic effects of FTi regulators and how they account for yield components and stress tolerance. In addition, we will contribute to the development of algorithms and databases for integrated analysis of sequence, expression, phenotypic and image data with the goal of deciphering regulatory FTi networks and their evolution.
Schnaithmann F, Kopahnke D, Pillen K (2014). A first step toward the development of a barley NAM population and its utilization to detect QTLs conferring leaf rust seedling resistance. Theor Appl Genet, doi: 10.1007/s00122-014-2315-x
Hoffmann A, Maurer A, Pillen K (2012). Detection of nitrogen deficiency QTLs in wild barley introgression lines growing in a hydroponic system. BMC Genetics 13:88, doi:10.1186/1471-2156-13-88.