Association mapping of major starch biosynthesis genes in Zea mays ssp. mays.

Abstract

Improving maize yield by utilizing natural allelic diversity is a major objective of today's breeders, and has likely been a goal since maize domestication. Starch is the main component of maize yield, and is an important agronomic trait needed for a wide range of uses from human and animal consumption to ethanol production. The level of starch in the maize kernel is controlled by upwards of 20 different loci, and has been the focus of multiple quantitative trait loci (QTL) studies in order to find regions in the maize genome that affect both starch content and starch quality, like the amylose/amylopectin ratio. The objective of this study was to evaluate the starch biosynthesis pathway using an association mapping approach, by evaluating six starch candidate genes from a diverse set of maize germplasm: Ae1, Bt2, Sh1, Sh2, Su1, and Wx1.

The six starch candidate genes were amplified, sequenced, and aligned from 29 inbred lines and then evaluated for the level of diversity present. Estimates of π (nucleotide diversity) indicated, on average, starch genes contained 2.3- and 4.8-fold lower amounts of diversity at silent and nonsynonymous sites, respectively, than 20 randomly sampled genes from chromosome one of maize. Three of the starch loci (Ae1, Bt2, and Su1) had dramatic drops in diversity compared to Zea mays ssp. parviglumis. Furthermore, Hudson-Kreitman-Aguade (HKA) tests for selection were significant for these same three loci. In addition, another test for selection, Tajima?fs D, was significant at Ae1. These data suggest selection on starch genes has lowered diversity in the starch pathway.

Smaller regions throughout each gene were sampled and aligned in a larger set of 97 maize inbreds for association tests. Phenotypic measurements of kernel composition (starch, protein, oil) and viscoamylographic (viscosity, pasting) profiles of starch were used in separate principle component analyses for the association tests. Significant associations (P< 0.05) with kernel composition traits, while controlling for population structure, were found in Sh1, Sh2, and Bt2. Significant associations for starch pasting traits were found in Sh1, Sh2, and Ae1.

Possible phenotypic effects were examined between alleles with significant associations. For kernel composition traits, Sh1and Sh2 showed a general genotype by environment (G X E) effect. In Bt2, a nonsynonymous change at residue 22 caused lower variance in oil content. For starch pasting traits, an allele in Sh1 caused a 1% increase in pasting temperature. At Ae1, a nonsynonymous change at residue 58 had a 1.6% higher pasting temperature and 4.6% higher amylose content. A nonsynonymous change at residue 318 in Sh2 caused a 6% increase in amylose. This study supports previous findings that the Sh2 locus affects amylose content, but has offered much higher resolution than is possible with traditional linkage mapping, while examining a much broader range of alleles. Therefore, even in a moderately heritable pathway, such as the starch biosynthesis pathway, association methods can be successful in narrowing down regions of effect, most times within 1000 bp.