Regulation of candidate genes in black point formation in barley.

Abstract

Black point of barley refers to discolouration of the embryo end of the grain. Downgrading of malting barley to feed grade due to black point results in significant economic loss to the Australian barley industry. Given that black point normally occurs in regions of Australia that experience high humidity during grain fill, humidity most probably contributes to the severity of black point in susceptible varieties. Previous studies have excluded fungal infection as a cause but enzymatic browning reaction has been recently hypothesised as responsible for black point. More specifically, a role for peroxidases has been proposed. The first major focus of this study was to confirm under what environmental conditions black point formation was likely to occur and whether there was genetic variation contributing to the phenotype. The occurrence of high humidity and low temperatures was associated with the formation of black point in susceptible varieties, with early maturing varieties being more susceptible to black point. These environmental conditions probably create a moist environment during grain development in which the developing grain cannot dry out, enabling stress or wounding to the embryo that subsequently results in black point formation. Analysis combining two South Australian sites (Hatherleigh and Port Wakefield, SA) identified QTL for black point formation on chromosomes 2H (QBpt.AlSl-2H) and 3H QBpt.AlSl-3H) at positions 83.4 cM and 102.6 cM respectively. Additive by environment effects were substantial at both QTL. Linkage of the QTL on chromosome 2H with the earliness per se (eps2) locus and the observation that early maturing varieties were usually more susceptible to black point established a probable association between earliness and black point susceptibility. When an early maturing(susceptible) variety was planted later so that it matured at the same time as a later maturing (tolerant) variety there was no significant difference in black point scores. The second focus of this study was to characterise a number of candidate genes more than likely linked to black point by investigating expression levels during grain fill and subsequently mapping the genomic regions responsible for those changes in expression. Candidate genes chosen were Quinone Reductase (HvQR), Phenylalanine Ammonia Lyase (HvPAL), Barley Peroxidase 1 (HvBP1), stress-related Peroxidase (HvPrx7) and Lipoxygenase A (HvLoxA). Differential expression as detected using northern analysis, between susceptible and tolerant varieties, was only observed for HvBP1, HvPrx7 and HvQR. Quantitative PCR (qPCR)confirmed that HvBP1 and HvPrx7 expression was up to two times higher in black point susceptible varieties during all stages of grain development, while HvQR expression was significantly higher in the hard dough and mature stages of grain fill in susceptible varieties. Increased expression for HvBP1 and HvPrx7 (approximately two-fold) was also apparent in the tolerant variety Alexis between symptomatic and asymptomatic grains. The qPCR data was then used as a quantitative trait, to score the expression of these candidate genes in an Alexis/Sloop double haploid (DH) mapping population. Areas of the genome potentially involved in the regulation of these candidates (expression QTL or eQTL) were mapped on chromosomes 2H (for HvPrx7 and HvBP1) and 5H (for HvQR and HvBP1). The eQTL for HvPrx7 and HvQR were located in the same regions as the corresponding genes, suggesting their expression is regulated via cis-acting factors. In contrast, while HvBP1 is located on 3H, eQTL were located on 2H and 5H suggesting trans-acting factors were involved. The use of comparative mapping studies between barley and rice identified a number of transcription factor genes within these eQTL. The final component of this study was to investigate how HvBP1 and HvPrx7 expression might be affected by examining their promoters and potential interactors with those promoters. Promoter regions for the susceptible variety Sloop and tolerant variety Alexis were isolated, compared and analysed for known motifs. Particular emphasis was placed on those elements that were associated with embryo and endosperm specific expression or responses to environmental stresses. Several regions containing single nucleotide polymorphisms (SNPs) between the promoters from the tolerant and susceptible varieties were identified. A 160 bp region for HvBP1 and 380 bp region for HvPrx7 were used in Yeast One Hybrid (Y1H) screening to identify potential regulatory proteins. In particular, a potential bZIP-containing factor which interacted with the promoter of HvPrx7 was further characterised. Interaction was confirmed by a gel shift assay and gene expression by northern analysis showed expression at the milk, soft dough and hard dough stages of grain development. Increased expression was apparent in the susceptible variety Sloop. The eQTL, Y1H and environmental studies have furthered our understanding of genes that could be involved in the regulation of black point formation under conditions of low temperature and high humidity. This information will contribute to assessing the roles these genes play in black point formation under certain environmental conditions, and more broadly, will assist in improving breeding for resistant barley varieties.