An in-depth interrogation of the genetic control of grain size and response to heat stress in barley (Hordeum vulgare L.)

Watt, Calum John (2020) An in-depth interrogation of the genetic control of grain size and response to heat stress in barley (Hordeum vulgare L.). PhD thesis, Murdoch University.

Abstract

Barley (Hordeum vulgare L.) is the fourth most important cereal crop grown globally and represents an important livestock feed and major raw material for the malting, brewing and distilling industries. Despite the importance of barley, it is relatively under researched compared to other cereal crop species such as wheat and rice. However, their exposure to the challenges imposed by climate change are likely to be similar, necessitating the need to improve barley’s productivity. Yield, is a reflection of two key components, number of grains/m2 and individual grain weight. “Yield is king” from a barley improvement point of view thus it is surprising that there has been limited research aimed at increasing productivity through the individual grain weight avenue either directly or indirectly by targeting component traits such as grain size. This thesis fills some of this research void by identifying candidate genes contributing to grain size variation and improves our current understanding surrounding the response of grain size to heat stress events which are predicted to become more detrimental under a future climate.

To achieve these objectives whole genome linkage analyses were performed on a doubled haploid (DH) population derived from two Australian malt barley varieties with distinctly different grain sizes, predominantly grain length. Multiple stable grain size QTL were identified, two of which were fine mapped to identify candidate genes for further interrogation. Two candidate genes underlying major grain length QTL on chromosomes 2H (HORVU2Hr1G089310) and 5H (HvDEP1) were identified and reinforced through comparative gene analysis. Sequence analysis and expression profiling of HvDEP1 between the population’s parents indicated that mutations of multiple cis-regulatory elements and a reported uORF were likely drivers of grain length variation in addition to plant architecture as reported in similar studies in rice and barley. Additionally, a predicted R2R3-MYB transcription factor was identified as a likely candidate gene driving grain length variation on chromosome 2H. To further interrogate these two putative candidate genes through reverse genetics approaches, single guide-RNAs targeting each gene were identified and CRISPR-cas9 binary vector plasmids were constructed using an adapted protocol with improved efficiency and proven to be highly efficient at transforming barley immature embryos. Material developed through these experiments represent important resources to assist in gene functional analysis.

In addition to our genetic understanding of grain size control this research aimed to address the deficiencies in knowledge regarding the grain size response to heat stress events during anthesis and grain development. We were able to conclude that grain length was not significantly affected, conversely grain width and thickness did respond adversely but not until more intense and longer duration heat stress events were experienced. Reflecting research in rice and wheat, grain number was the major driver of yield variation under heat stress conditions rather than grain size indicating future research should improve grain number retention under stressful conditions foremost. Further to this, this thesis suggests that transcription factors, such as that identified in this research represent promising targets for genetic modification to effectively improve the grain size and overall productivity of barley under adverse abiotic conditions.

Item Type:	Thesis (PhD)
Murdoch Affiliation(s):	Agricultural Sciences
United Nations SDGs:	Goal 12: Responsible Consumption and Production
Supervisor(s):	Li, Chengdao, Zhou, Gaofeng and Moody, David
URI:	http://researchrepository.murdoch.edu.au/id/eprint/59276

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