Explore gibberellin (GA)‑related genes for barley productivity

Gibberellin (GA) is an important plant hormone that regulates many aspects of plant growth. During the Green Revolution (GR), the semi-dwarf phenotype in plants with high lodging resistance due to mutations in GA-related genes has led to the deficiency in GA. However, the shorter stature caused by GR genes is usually accompanied by adverse impacts on the other agronomic traits, including shorter coleoptile length, decreased seed weight, smaller seed size, and later flowering time. A full understanding of the biochemical and molecular functions of GA-related genes will provide useful information for breeding semi-dwarf varieties with minimum impacts on desirable traits. To effectively overcome the undesirable agronomic traits of shorter plants, identification and functional validation of novel GA synthesis- or regulation-related genes that control plant height without adverse effects on other agronomic traits is of prominent value. In barley, the GR gene sdw1/denso is the first key enzyme involved in the late stage of the GA biosynthesis pathway and it corresponds to Gibberellin 20-oxidase (HvGA20ox2) which is well studied.
Another metabolic enzyme involved in the regulation of GA biosynthesis is gibberellin 3-oxidase (HvGA3ox). To investigate if HvGA3ox can be a potential gene for modifying agronomic traits by altering GA content, I first conducted a genome- wide association study (GWAS) using a natural population consisting of 895 worldwide barley accessions and identified several genetic variants (single polymorphic sites) of HvGA3ox1, which were associated with plant height, days from planting to flowering (days to Z49), and grain yield (GY) (Chapter 3). Using the natural population consisting of 632 barley accessions, a correlation analysis was conducted between the haplotypes of HvGA3ox1 and phenotypic data from six trials in 2015 and 2016. The results revealed that the haplotype “Hap05” in HvGA3ox1 was associated with low plant height but late flowering. The loss-of-function mutation of HvGA3ox1 showed longer coleoptile length and prolonged seed dormancy by reducing the bioactive GA.
Enhancing GA catabolic enzymes and reinforcing the GA deactivation pathway can cause a consequential decrease in GA content, which can lead to shorter stature. Gibberellin 2-oxidase (GA2ox) is the major catabolic enzyme in regulating GA deactivation. Preliminary GWAS results discovered several genetic variants related to plant height and flowering time in HvGA2ox8a, HvGA2ox3 and HvGA2ox4. Using the tool of CRISPR-Cas9 technology for gene editing, I confirmed that HvGA2ox8a is a functional gene controlling plant height, flowering time, and seed width (Chapter 4). Correlation analysis between the identified 34 genetic variants across 632 barley accessions and the existing phenotypic data revealed that Hap05 in HvGA2ox8a is a semi-dwarf haplotype contributing to early flowering, and increased seed weight and size, which could be used for breeding cultivars more adapted to drought environments that potentially occurs during the later growth stage. In addition, 19 genetic variants across 632 barley accessions were commonly found in both HvGA2ox3 and HvGA2ox4 (Chapter 5). A correlation analysis was also performed between phenotypic data collected from 12 environments and the haplotypes based on their insertions and deletions (InDels) and single nucleotide polymorphisms (SNPs). Hap04 in HvGA2ox3 was detected as a functional haplotype possessing a semi-dwarf phenotype with early flowering time and high yield, while Hap02 in HvGA2ox4 showed semi-dwarf phenotypes with increased yield. In terms of the InDel-based haplotypes, both HvGA2ox3 and HvGA2ox4 harbored Hap03, exhibiting low plant height and early flowering by decreasing expression levels in young stem and young leaves compared with other haplotypes. Besides, both HvGA2ox3 and HvGA2ox4 were mainly expressed in the leaf rather than in the young stem. This study provided a deeper understanding of the biochemical and molecular mechanisms of how GA2ox genes regulate plant growth. With the identification of their functions, the four GA2ox-related genes could be used as new alternative semi-dwarfing genes that contribute to minimum influence on other traits. This research not only delivered new strategies for the validation of GA-related genes but also confirmed the high possibility of applying genetic approaches to overcome the negative effects on agronomic traits due to the use of GA-related genes