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

Abstract

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.

Figure 1

Parents and F1 progenies: (a) white sweetcorn parent (‘Tims-white’-derived accession); (b) purple maize parent (‘Costa Rica’); (c) F1 round kernels with white-pericarp, with or without blue-aleurone; (d) reciprocal cross showing F1 round kernels with purple-pericarp. F2 progenies: (e) cobs with purple-pericarp and reddish-purple-pericarp kernels with a 3:1 ratio of round to shrunken kernels; (f) white cob with a 3:1 ratio of round to shrunken kernels.

Figure 2

Purple pigmentation in different tissues of F3 plants at different stages of plant development: (a) sheath-leaf, (b) stem, (c) auricle, (d) anthers, and (e) silks. Developed purple F3 cobs: ‘Tim1’ to ‘Tim5’ (f–j) at milk/eating stage, prior to exhibiting the mature shrunken phenotype.

Figure 3

Linkage break and recombination: (a) length of a1 and sh2 genes on chromosome 3, along with the position of yz1 and x1 genes; (b) linkage and recombination between A1 and sh2 alleles; (c) mapping of the whole genome (WG) of ‘Costa Rica, ‘Tim1’ and ‘Tims-white’ lines against the reference B73 genome, with four genes (a1, yz1, x1  and sh2) within the a1-sh2 interval annotated (blue arrow), red bar indicates variants, with height of bar meaning read coverage; (d) red arrows showing putative linkage break and recombination site between a1 and sh2 genes of the developed F6 line, ‘Tim1’.

Figure 4

One-way factorial analysis of variance (ANOVA) using Fisher’s least significant difference (LSD) test for anthocyanin and sugar concentrations. Multiple comparison showing ns non-significant, *significant at P < 0.05, **significant at P < 0.01, and ***significant at P < 0.001. (a) Anthocyanin profiles of ‘Costa Rica’ and ‘Tim1’ kernels at 23 days after pollination. The anthocyanin-base grouping (e.g. cyanidin) is indicated on the y-axis; (b) sugar profiles of ‘Costa Rica’, ‘Tims-white’ and ‘Tim1’ kernels at 23 DAP.

Figure 5

Parental plants: (a) white shrunken2 super-sweetcorn accession, ‘Tims-white’, (b) purple-pericarp Peruvian maize accession, ‘Costa Rica’. Pollination and crossing: (c) covered ear, (d) covered tassels (and ear), (e) bagging after pollination.