Waxy allele diversification in foxtail millet (Setaria italica) landraces of Taiwan

Abstract

Foxtail millet (Setaria italica (L.) P. Beauv.), the second most cultivated millet species, is well adapted to diverse environments and remains an important cereal food and forage crop in arid and semiarid regions worldwide. A symbolic crop for indigenous Austronesian peoples, foxtail millet has been cultivated in Taiwan for more than 5,000 years, and landraces reflect diversifying selection for various food applications. A total of 124 accessions collected within Taiwan were assessed for Wx genotypes. Four identified Wx alleles, I, III, IV, and IX were caused by insertion of various transposable elements (TEs) and resulted in endosperm with non-waxy, low amylose content (AC), and waxy, respectively. A total of 16.9%, 4.0%, 49.2%, and 29.8% of accessions were classified as type I, III, IV, and IX, respectively; approximately half of the accessions belonged to the waxy type, indicating that glutinous grains were favored for making traditional food and wine. The TE insertion affected splicing efficiency rather than accuracy, leading to significantly reduced expression of wx in types III, IV, and IX, although their transcripts were the same as wild-type, type I. Consequently, the granule-bound starch synthase I (GBSSI) contents of the three mutated genotypes were relatively low, leading to waxy or low AC endosperm, and the Wx genotypes could explain 78% of variance in AC. The geographic distribution of Wx genotypes are associated with culinary preferences and migration routes of Taiwanese indigenous peoples-in particular, the genotype of landraces collected from Orchid Island was distinct from those from Taiwan Island. This information on the major gene regulating starch biosynthesis in foxtail millet endosperm can be applied to breeding programs for grain quality, and contributes to knowledge of Austronesian cultures.

Fig 1. Assessment of Wx genotypes and phenotypes.

(A) Schematic of the foxtail millet Wx gene. The positions of primer sets used to analyze genotypes are depicted, and primer sequences are listed in S2 Table. (B) PCR amplicons of foxtail millet Wx genotypes. Primer combinations, ex1 and ex2 (1–4), ex2int2 and ex4r (5–8), ex7 and ex10 (9–12), M7 and R9 (13–16), are indicated at the top of each the figure. M1 and M2 indicates 1 kb DNA ladder (DM3100 ExcelBand) and 100 bp DNA ladder (100 bp DNA ladder plus, GeneTeks BioScience Inc.), respectively. (C-D) The distribution of apparent amylose content (AAC) for each Wx genotype. A total of 114 accessions used for AAC assays were grown in the first crop season of 2015.

Fig 2. Gene expression of Wx and quantity of GBSSI protein for different Wx genotypes.

(A) Expression levels of Wx at different seed development stages. The relative expression levels of four Wx genotypes, each represented by two accessions indicated by solid and dotted lines, were estimated at 5, 10, 15, and 22 DAF. Inserts on the right corners of figures of three mutated genotypes are the original expression patterns drawn without normalized scales. (B) Comparison of expression levels of Wx genotypes at different endosperm development stages. Genotype I, III, IV, and IX are represented by green, violet, orange (could be seen at 10 DAF, but not in other three stages), and blue, respectively. Means with different superscripts are significantly different at p<5%, according to Fisher’s LSD test.

Fig 3. Genetic information for four Wx genotypes.

(A) Transcripts of four Wx alleles in developing seeds at 10 DAF (upper panel). The housekeeping gene, EF1a, was used as internal control (lower panel). (B) SNP sites on different Wx genotypes. (C) Assay of granule-bound proteins of 2 sorghum and 8 foxtail millet accessions separated by SDS-PAGE. Lane 1: BTx623 (non-waxy sorghum); lane 2: V9 (waxy sorghum); Lanes 3 and 4: Type I (non-waxy type); lanes 5 and 6: Type IX (low AAC type); lanes 7 and 8: Type IV (waxy type); lanes 9 and 10: Type III (low AAC type). (D) Immunoblotting of GBSSI for different Wx genotypes. (E) GBSSI contents were reflected by band intensities of immunoblot quantified by ImageJ. Means with the different letter superscripts are significantly different at p<5% level, according to Fisher LSD test.

Fig 4. The geographical distribution of foxtail millet Wx genotypes in Taiwan.

A total of 50 (40.3%), 32 (25.8%), 24 (19.4%), 5 (4%), 5 (4%), 2 (1.6%), 2 (1.6%), and 2 (1.6%) of 124 accessions were collected from Taitung, Nantou, Pingtung, Orchid Island, Kaohsiung, Yilan, Taichung, and Hsinchu, respectively. The map was adopted from https://simplemaps.com/resources/svg-tw.