Genetic control of Apigenin di-C-glycoside biosynthesis in bread wheat grain and their role as yellow pigments of Asian alkaline noodles.

Abstract

Colour is an important determinant of quality and customer appeal for Asian noodles that are made from bread wheat (Triticum aestivum L.). The Asian noodle market represents approximately one third of wheat exports from Australia and as a consequence maintaining and improving colour for noodles is an important research and breeding objective. The focus of this project is yellow alkaline noodles (YAN) prepared using wheat flour and alkaline salts, sodium and potassium carbonate, and for which a bright yellow colour is desired. Xanthophylls, primarily lutein, and apigenin di-Cglycosides (ACGs) have been shown to be important components of this yellow colour. ACGs were of particular interest since, in contrast to lutein, the content in flour could be increased without adverse effects on colour of other end-products. There was little information either on the genetic variation for ACG content or the mechanism and genetic control of biosynthesis which was surprising in view of their putative role in a wide range of plant processes, food colour and flavour, and possibly human health. The aims of this project were to provide new information on the role of ACGs in YAN colour and genetic regulation of their biosynthesis. To achieve this aims: genetic variation in grain ACG traits in bread wheat and related species was surveyed, the quantitative contribution of ACG to the yellow colour of YAN was determined and compared to lutein, QTL for ACG content and composition were located, and candidate genes associated with variation in ACG composition identified. Substantial variation in both grain ACG content and the ratio, ACG1/ACG2, were identified within bread wheat cultivars and related species. Genotype controlled the major portion of the variation. ACG content appeared to be a multigenic trait whereas variation in ACG1/ACG2 was associated with a limited number of chromosomes, in particular chromosomes 1B, 7B and 7D. In the absence of chromosome 7B (Chinese Spring 7B nullisomics) there was a substantial increase in ACG1/ACG2, i.e. a relative increase in the glucose-containing isomer, possibly indicating the presence of a Cglycosyltransferase on 7B with specificity for UDP-galactose. A similar phenotype observed in some wheat cultivars could be explained by a deletion or mutation of a gene controlling this enzyme. The results suggest that it should be possible to manipulate both ACG content and composition through breeding. Only 30% of ACG (means 19.3 µg/g) is recovered in flour, which contributed to 1 to 3 CIE b* units to the part of the yellow colour of yellow alkaline noodles (YAN) that develops specifically in the presence of alkali. The relatively low recovery of ACG in flour contrasts with the high recovery of lutein (90%, with means 1.011 µg/g). Since the difference between white salted noodles (WSN) and YAN is approximately 6 b* units, this would indicate that another unidentified compound(s) is responsible for the difference. Potential for ACG0-based improvement of bread wheat cultivars for YAN yellowness is likely to be limited by the range of genetic variation, the location of ACG in grain tissues that are largely discarded during milling and the lack of correlation between grain and flour ACG content. Moreover, the observed variation in ACG recovery in small scale milling was not reflected in larger scale milling anticipated to better represent commercial practice. The improvement in flour recovery and the amount of ACG recovered in the flour were not significant and not enough to achieve the yellowness of commercial noodles. Selection that requires larger scale milling is costly, time consuming and not applicable to early generation screening. In this context, further work on QTL associated with variation in ACG content and development of marker-assisted-selection would be very useful. Addition of thirteen new markers to the QTL region for ACG trait on chromosome 7BS in a Sunco/Tasman doubled haploid population reduced the size of the QTL interval from 28.8cM to approximately 5.5cM. In this revised 7BS map, the major QTL for ACG1 and ACG2 content as well as ACG1/ACG2 ratio were detected within 4.7cM of SSR marker Xwmc76. The QTL region linked to Xwmc76 was shown to be syntenic with a region in rice chromosome 6S between AP005387 and AP005761 and a region on Brachypodium chromosome 1. Based on these comparisons, the most likely candidate gene associated with variation in ACG composition appeared to be a glycosyltransferase. Alternate alleles at the 7BS QTL may be associated with amino acid changes within the C glycosyltransferase that shift the substrate specificity from galactose (ACG2, Tasman) to glucose (ACG1, Sunco). Alternatively, based on a comparison of Chinese Spring nullisomic-tetrasomic lines where nullisomic 7B was associated with a phenotype similar to Sunco, it is possible that Sunco contains a null allele. Other candidate genes located on the same chromosome that could potentially be involved in ACG biosynthesis were identified and included a sugar transporter, which could determine the relative sizes of the available pools of UDP-glucose and UDPgalactose, an epimerase required for inter-conversion of these sugars, other glycosyltransferases and a flavone-2-hydroxylase (F2H) involved in the first committed step in the pathway to ACG. Research approaches that could be used to validate the role of the candidate gene are discussed along with other options for improving the colour of wheat cultivars for the YAN market. Options for utilizing ACG as yellow pigment of noodles might include incorporating the embryo or seed coat materials (pollard and bran) into the flour after milling and genetic modification of bread wheat to achieve ACG expression in the starchy endosperm.