Targeted Sequencing Reveals Large-Scale Sequence Polymorphism in Maize Candidate Genes for Biomass Production and Composition

<div><p>A major goal of maize genomic research is to identify sequence polymorphisms responsible for phenotypic variation in traits of economic importance. Large-scale detection of sequence variation is critical for linking genes, or genomic regions, to phenotypes. However, due to its size and complexity, it remains expensive to generate whole genome sequences of sufficient coverage for divergent maize lines, even with access to next generation sequencing (NGS) technology. Because methods involving reduction of genome complexity, such as genotyping-by-sequencing (GBS), assess only a limited fraction of sequence variation, targeted sequencing of selected genomic loci offers an attractive alternative. We therefore designed a sequence capture assay to target 29 Mb genomic regions and surveyed a total of 4,648 genes possibly affecting biomass production in 21 diverse inbred maize lines (7 flints, 14 dents). Captured and enriched genomic DNA was sequenced using the 454 NGS platform to 19.6-fold average depth coverage, and a broad evaluation of read alignment and variant calling methods was performed to select optimal procedures for variant discovery. Sequence alignment with the B73 reference and <i>de novo</i> assembly identified 383,145 putative single nucleotide polymorphisms (SNPs), of which 42,685 were non-synonymous alterations and 7,139 caused frameshifts. Presence/absence variation (PAV) of genes was also detected. We found that substantial sequence variation exists among genomic regions targeted in this study, which was particularly evident within coding regions. This diversification has the potential to broaden functional diversity and generate phenotypic variation that may lead to new adaptations and the modification of important agronomic traits. Further, annotated SNPs identified here will serve as useful genetic tools and as candidates in searches for phenotype-altering DNA variation. In summary, we demonstrated that sequencing of captured DNA is a powerful approach for variant discovery in maize genes.</p></div

SNP functional class membership.

<p>^a SNP is located 1–3 bases into an exon or 3–8 bases into an intron</p><p>^b SNP is located in the first two or the last two bases of an intron</p><p>^c transistion (A <-> G, C <->T)</p><p>^d transversion (C <-> G, A <-> C, G <-> T, A <-> T)</p><p>^∩ intersection using the complete SNP data set of 21 genotypes</p><p>^∪ union using the complete SNP data set of 21 genotypes</p><p>Re-sequencing and variant calling within 21 maize inbred lines discovered 383,145 high quality candidate SNPs (complete <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132120#pone.0132120.s020" target="_blank">S12 Table</a>). Table displays the five least diverse (green) and five most diverse (blue) inbred lines, also differentiating between the dent [‘D’] and flint [‘F’] inbred lines. The intersection (‘<b>∩</b>’) was analyzed in four settings (5, 10, 15, and complete), indicating that the variant site is present in at least the respective number of genotypes.</p

Global diversity classification per genotype.

<p>Variant positions in captured genes (on target) identified after basic filtering (at least 5-fold coverage of read depth at SNP position) and off-target regions for different maize inbred lines.</p

Statistics of raw and preprocessed sequence data.

<p>A total volume of >6.4 Gbp of raw data was sequenced. Each of the 21 maize genotypes is designated by ‘D’ (Dent) or ‘F’ (Flint), according to which gene pool the line belongs.</p

Variant detection performance.

<p>*'CornFed Target Diversity' (CTD) is the final set of VPs in chromosomes.</p><p>Comprehensive overview of eight evaluated variant detection tools. Predicted VPs of each variant caller are compared to the four control data sets (50k, GBS, RNAseq, and HapMap2) in terms of sensitivity (‘S_e’), specificity (‘S_p’), and the F₁-score (‘F₁’). In addition the final set of variants detected in this study (CTD) is showing the proportion each variant caller is capturing.</p

Captured target gene diversity in 21 maize inbred lines for biomass production and composition.

<p><b>(A)</b> The outer circle illustrates, with dark connecting lines, the location of the 4,372 target genes in the 10 maize chromosomes. The inner circles (heat maps 1–21) represent the 21 (ordered in dent and flint inbred lines and subsequently with increasing diversity) inbred lines and their corresponding SNP density, scaled according to the physical position of the target genes in the B73 genome reference <b>(B)</b>. In total, 1 million of the detected high quality SNPs are integrated. Putative regions of high diversity are represented in blue, and regions with low accumulations of mutations are colored red. Centromere positions are indicated with light blue bars adjacent to the outer circle.</p

Evaluation of seven read alignment methods.

<p>*Number of total reads is 10,409,726</p><p>In this broad evaluation 21 inbred maize lines were included. Seven independent read alignment methods were utilized in three different parameter settings. For each alignment methods the best parameter setting is shown with respect to the highest number of reads mapped on target.</p

Bar plots of the STRUCTURE analysis.

<p>Each of the 21 maize inbred lines is represented by a vertical bar, partitioned into K = 8 colored segments that designate the fraction of each population estimated to belong to the inferred subgroups (Population ID corresponds to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132120#pone.0132120.s009" target="_blank">S1 Table</a>).</p

Distribution of 4,785 candidate genes over 10 maize chromosomes.

<p>Selected genes are predicted to control various aspects of plant growth, biomass production, and composition; bar graph depicts their distribution in the maize genome.</p

Venn diagram of marker/variants in maize.

<p>Illustration of the overlapping intersections between four diversity data sets (50K, GBS, RNAseq, and HapMap2) and the ‘CornFed Target Diversity’ (CTD).</p