Environment-dependent regulation of flowering time in a barley (<em>Hordeum vulgare</em> L.) MAGIC population

Abstract

Barley (Hordeum vulgare L.) is used as a source for food and feed and is the fourth widely cultivated cereal world-wide. Flowering time is a complex trait controlled by endogenous and environmental factors that marks switching of plant life cycle from vegetative to reproductive development. Flowering time mechanism has important role in crop adaptation to environment and abiotic stressors including heat and drought and has an impact on crop yield. Flowering time in barley, as a model for small grain cereals, is extensively studied. Nevertheless, what is known about its flowering time mechanism in response to environment is under-represented compared to model plants such as Arabidopsis thaliana. Therefore, the present thesis aims to improve understanding of environment-dependent regulation of flowering time in barley and provide insights into novel flowering time regulators. For this purpose a series of approaches were employed aiming to 1) better understand the interconnected dynamics of epistasis and environment using a MAGIC population and look for novel regulators; 2) identify candidate gene(s) underlying novel barley flowering time QTL; 3) investigate effect of flowering time genetic regions on yield-related traits under different environments. Spring barley MAGIC population was used in three detailed studies as plant material. This population can provide sufficient diversity and mapping power to study a complex trait such as flowering time and is constructed of an eight-way cross of barley landraces known as “founders of German barley breeding” and one elite cultivar Barke. The first study focused on QTL, epistasis and environment interaction regulation in flowering time pathway of barley under different environments including field and semi-controlled conditions. A set of 534 spring barley MAGIC DH lines were used for analyzing of quantitative trait loci (QTLs), epistatic interactions, QTL × environment interactions, and epistasis × environment interactions effects with single SNP and haplotype approaches. Results of this study, revealed overall 18 QTLs and 2,420 epistatic interactions which included regions for major genes such as Ppd-H1, Vrn-H1, Vrn-H3, and denso/sdw1. Distinguishable epistatic interactions were detected in field and semi-controlled conditions and findings from analysis of QTL × environment interactions and epistasis × environment interactions suggested the influence of temperature on regulators of flowering time pathway. Additionally, the results revealed a novel QTL harboring a flowering-delaying allele on chromosome 1H, engaged in epistatic and environment interactions, which we named “HvHeading”. The findings showed that this region was involved in epistasis and epistasis × environment interactions with regions harboring Ppd-H1, Vrn-H3, and Vrn-H1 and denso/sdw1 suggesting that it might have an important role in environment-dependent regulation of flowering time in barley. The second study aimed to investigate the newly-detected QTL HvHeading to identify the underlying candidate gene(s) by a targeted background effect elimination approach based on epistasis. The spring barley MAGIC DH lines were screened to select flowering-time-specific-near-isogenic pairs of DH lines that have the same background regarding the genes, Ppd-H1, Vrn-H1 and Vrn-H3 which are involved in major epistasis with HvHeading. One DH line in the pair had the haplotype harboring the flowering-delaying allele underlying HvHeading which originated from parental line Danubia. The apex and inflorescence development was investigated using microscopic phenotyping. Differential gene expression analysis by RNA-sequencing and RT-qPCR in apex and leaf tissue was conducted. Phenotypic effect of HvHeading was detected as early as after vegetative-to-reproductive transition. Analyzing transcripts from RNA-sequencing identified differentially expressed genes in HvHeading region in flowering-time-specific-near-isogenic pair of DH lines. These findings led to refining HvHeading interval to <8.50 Mbp and identifying up-regulation of Spt6 gene in delayed-flowering DH line. Differential gene expression analysis using RT-qPCR validated up-regulation of Spt6 starting before double-ridge stage and showed down-regulation of Ppd-H1, Vrn-H1 for the delayed-flowering DH line. Most of the promoter region of Spt6 gene in flowering-time-specific-near-isogenic pair of DH lines was sequenced and showed many mutations. Additionally, comparing the sequenced transcripts of Spt6 gene with published Spt6 isoforms in barley showed that DH line with Danubia haplotype might produce a novel isoform. The third study was conducted to evaluate effect of flowering time genetic regions on yield-related traits including grain yield components under different environments. This study used the same set of 534 MAGIC DH lines and evaluated seven traits under well-watered and terminal drought treatments. The analysis of QTL for each treatment, maker by treatment interaction (M×T) and QTL for drought tolerance was conducted. Results revealed all traits were affected by treatment apart from days to heading. In total 69, 64 and 29 QTL were found under well-watered and terminal drought treatment and drought tolerance, respectively. The M×T analysis revealed total 25 loci for four traits. The identified QTL included loci that co-located with known genes/QTL as well as novel regions. The results revealed genetic regions for various traits which coincided with flowering time loci under well-watered and terminal drought, hinting to pleiotropic effect of flowering time genes such as Ppd-H1, Vrn-H1, Vrn-H3 and denso/sdw1 on grain yield and plant development. Some detected QTL showed favorable effect on grain number, ear number and grain weight under terminal drought treatment including QTL corresponding to major flowering time gene Ppd-H1 for grain weight. Spring barley MAGIC population is a valuable asset which offered powerful mapping of flowering time QTL and epistasis under different environments including novel regulators; these outcomes can be transferred to more complex crops such as wheat. Validating effect of epistatic QTL HvHeading and identifying Spt6 gene as a candidate gene by a targeted elimination of background effect approach based on epistasis showed that mapping epistasis interactions can help compose strategies to facilitate gene identification. The role of candidate gene Spt6 in flowering time pathway of barley should be validated and further studied. Detecting favorable pleiotropic effect of flowering time loci on grain weight components under terminal drought hinted to possibility of usefulness of flowering time genes for improving these traits under extreme environments through timing of flowering or their contribution to developmental pathways. The findings showed the importance of exploring epistasis and environment interaction in addition to more common approaches such as QTL analysis, to explore adaptation of flowering time to different environment in barley. The series of studies presented in this dissertation gives new insights into environment-dependent regulation of flowering time in barley through a multidisciplinary approach which highlights importance of employing approaches that better explain complex traits in future research and breeding programs.