Sequence variations associated with novel purple-pericarp super-sweetcorn compared to its purple-pericarp maize and white super-sweetcorn parents.

Recently, a novel purple-pericarp super-sweetcorn line, 'Tim1' (A1A1.sh2sh2) was derived from the purple-pericarp maize 'Costa Rica' (A1Sh2.A1Sh2) and white shrunken2 (sh2) super-sweetcorn 'Tims-white' (a1sh2.a1sh2), however, information regarding anthocyanin biosynthesis genes controlling purple colour and sweetness gene is lacking. Specific sequence differences in the CDS (coding DNA sequence) and promoter regions of the anthocyanin biosynthesis structural genes, anthocyanin1 (A1), purple aleurone1 (Pr1) and regulatory genes, purple plant1 (Pl1), plant colour1 (B1), coloured1 (R1), and the sweetcorn structural gene, shrunken2 (sh2) were investigated using the publicly available annotated yellow starchy maize, B73 (NAM5.0) as a reference genome. In the CDS region, the A1, Pl1 and R1 gene sequence differences of 'Tim1' and 'Costa Rica' were similar, as they control purple-pericarp pigmentation. However, the B1 gene showed similarity between the 'Tim1' and 'Tims-white' lines, which may indicate that it does not have a role in controlling pericarp colour, unlike the report of a previous study. In the case of the Pr1 gene, in contrast to 'Costa Rica', 6- and 8-bp dinucleotide (TA) repeats were observed in the promoter region of the 'Tims-white' and 'Tim1' lines, respectively, indicating the defective functionality (redder colour in 'Tim1' rather than purple in 'Costa Rica') of the recessive pr1 allele. In sweetcorn, the structural gene (sh2), sequence showed similarity between purple-sweet 'Tim1' and its white-sweet parent 'Tims-white', as both display a shrunken phenotype in their mature kernels. These findings revealed that the developed purple-sweet line is different to the reference yellow-nonsweet line in both the anthocyanin biosynthesis and sweetcorn genes.

Profiling and Quantification of Anthocyanins in Purple-Pericarp Sweetcorn and Purple-Pericarp Maize.

Purple-pericarp sweetcorn accessions, derived from crossing purple-pericarp maize with white shrunken2 sweetcorn, were assessed for differences in anthocyanin profile at both sweetcorn eating stage and at full kernel maturity. The 'Tim1' sweetcorn line developed a similar total anthocyanin concentration to its 'Costa Rica' parent when assessed at sweetcorn-eating stage. At full maturity it surpassed the purple maize parent, but this was mainly due to the presence of starch diluting the anthocyanin concentration of the latter. The anthocyanin/colour relationship was affected by both total anthocyanin concentration and the ratio of cyanidin- to pelargonidin-based anthocyanins. Malonylation of anthocyanins was also found to vary and did not appear to be linked with either cyanidin:pelargonidin ratio or total anthocyanin concentration. In addition, anthocyanin synthesis was affected by kernel maturity at harvest, with colour development increasing in conjunction with a progression of anthocyanin development across the kernel surface. Pigmentation was present in the aleurone, pericarp and vitreous endosperm of kernels of the purple-pericarp maize parent and purple-pericarp sweetcorn accessions when fully mature, but pigmentation was only apparent in the pericarp at sweetcorn-eating stage. Importantly for consumers, anthocyanin pigmentation covered almost the entire kernel surface at sweetcorn-eating stage.

Breaking the tight genetic linkage between the a1 and sh2 genes led to the development of anthocyanin-rich purple-pericarp super-sweetcorn.

The existence of purple-pericarp super-sweetcorn based on the supersweet mutation, shrunken2 (sh2), has not been previously reported, due to its extremely tight genetic linkage to a non-functional anthocyanin biosynthesis gene, anthocyaninless1 (a1). Generally, pericarp-pigmented starchy purple corn contains significantly higher anthocyanin. The development of purple-pericarp super-sweetcorn is dependent on breaking the a1-sh2 tight genetic linkage, which occurs at a very low frequency of < 1 in 1000 meiotic crossovers. Here, to develop purple-pericarp super-sweetcorn, an initial cross between a male purple-pericarp maize, 'Costa Rica' (A1Sh2.A1Sh2) and a female white shrunken2 super-sweetcorn, 'Tims-white' (a1sh2.a1sh2), was conducted. Subsequent self-pollination based on purple-pericarp-shrunken kernels identified a small frequency (0.08%) of initial heterozygous F3 segregants (A1a1.sh2sh2) producing a fully sh2 cob with a purple-pericarp phenotype, enabled by breaking the close genetic linkage between the a1 and sh2 genes. Resulting rounds of self-pollination generated a F6 homozygous purple-pericarp super-sweetcorn (A1A1.sh2sh2) line, 'Tim1'. Genome sequencing revealed a recombination break between the a1 and yz1 genes of the a1-yz1-x1-sh2 multigenic interval. The novel purple-pericarp super-sweetcorn produced a similar concentration of anthocyanin and sugar as in its purple-pericarp maize and white super-sweetcorn parents, respectively, potentially adding a broader range of health benefits than currently exists with standard yellow/white sweetcorn.