IDENTIFICATION AND CHARACTERIZATION OF GENETIC LOCI FOR CULM MORPHOLOGY TRAITS IN BARLEY (HORDEUM VULGARE L.)

Barley (Hordeum vulgare L.) is one of the most important cereal crops worldwide and it has been widely used as a model species in plant genetics and breeding. Stem lodging is a major constraint to yield potential and grain quality in barley. Since the Green Revolution, height reduction has been the main target in breeding to overcome lodging occurrence. However, negative impact on yield and biomass may result from further reduction of plant height. In cereals, lodging resistance has been addressed also by manipulating traits associated with morphology and the composition of the culm. However, limited knowledge is available on the genetic and molecular bases controlling these traits in barley. In a previous study carried out by our group, a multi-environment genome-wide association study (GWAS) on a panel of European barleys led to the identification of stable Quantitative Trait Loci (QTLs) for culm morphology with negative impact on lodging and no influence on plant height. Based on these results, we developed two crossing populations from two large and two small culm cultivars to further characterize two selected QTLs (QTL17-4H and QTL18-4H) and/or identify new loci associated with culm morphology. For each cross, F1 plants were backcrossed with the small culm parent and BC1F1 plants heterozygous at the target QTL were used to develop two Doubled Haploid (DH) populations: 188 and 189 DHs for AV_POP1 and CG_POP2 populations were obtained, respectively. These and the respective parents have been genotyped with an 15K XT Infinium Illumina SNP array to perform single and multi-locus GWAS for culm architecture traits, flowering date (FD), plant height (PH), fertile tiller number (FTN), total tiller number (TTN), second internode length (IL), and second internode weight (IWt) using phenotypic data from one field experiment conducted in Germany during the summer of 2022. By applying several GWAS methods to the same dataset, we were able to identify robust Quantitative Trait Nucleotides (QTNs) that were detected as significant in multiple GWAS methods. A total of 14 QTNs detected with at least two GWAS methods were found for FD, PH, outer and inner diameter (OD and ID), section modulus (SM), secondary moment of inertia (SMI), stem index (SI), stiffness (ST) and IWt in the AV_POP1 population. Among them, AV_qtnFD_2H_1 located near the HvAP2/Zeo accounted for the highest percentage of phenotypic variation for FD and PH and explained up to 31.01 % of phenotypic variation for ID, SI, and ST, suggesting that the HvAP2/Zeo gene may affect culm morphology, beyond its well-known role in controlling spike density and internode elongation. Furthermore, AV_qtnOD_6H_1 was specifically detected for OD, ID, SM and SMI and had no influence on PH. Regarding CG_POP2, a total of 15 QTNs detected with at least two GWAS methods were found for FD, PH, ID, thickness (TH), SI, ST, IL and IWt. Marker-trait associations for FD, PH, TH, SI and IWt were detected in the vicinity of semi-dwarfing gene Sdw1/Denso. In particular, association of CG_qtnPH_3H_1 with PH, IWt and TH is consistent with the major role of Sdw1/Denso gene in controlling culm length and its pleiotropic effects on culm features and flowering. Finally, we identified two specific QTNs for ID and ST on chromosome 3H. In a parallel approach, CENTRORADIALIS1 (HvCEN1) -a close paralog of the well-known barley flowering time CENTRORADIALIS (HvCEN)- was identified as a candidate gene for the QTL18-4H region based on high expression in internodes. Two TILLING lines from the HorTILLUS population (hvcen1.h and hvcen1.r) were identified as carrying mutations with potential impact on protein function and evaluated for their effects on plant and culm development under greenhouse conditions. Compared to their background cultivar Sebastian, both lines exhibited reduced OD, SM and SMI of second internodes sampled at dough stage, supporting a role of HvCEN1 in culm development. Furthermore, line hvcen1.r showed altered spike morphology, plant height, biomass, and flowering time, suggesting an effect of this gene on flowering time and plant architecture traits. To complement this reverse-genetics approach, we carried out a forward screening on a subset of 57 lines from the TILLMore population. Based on replicated greenhouse experiments, three promising mutant lines for culm morphology traits were identified: compared to their background cultivar Morex, lines TM 2544 and TM 3325 showed larger culms, while TM 349 showed smaller culm, providing a starting point for cloning the underlying genes by bulk-segregant analysis. In conclusion, the present work shows that genetic variation for culm morphology is present in the newly developed DH populations that can be used for further validation and/or fine mapping of QTLs. Finally, the TILLING lines identified in this study are useful resources for functional characterization of candidate genes associated with culm architecture.