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, 9 (1), e1003215

Switchgrass Genomic Diversity, Ploidy, and Evolution: Novel Insights From a Network-Based SNP Discovery Protocol

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Switchgrass Genomic Diversity, Ploidy, and Evolution: Novel Insights From a Network-Based SNP Discovery Protocol

Fei Lu et al. PLoS Genet.

Abstract

Switchgrass (Panicum virgatum L.) is a perennial grass that has been designated as an herbaceous model biofuel crop for the United States of America. To facilitate accelerated breeding programs of switchgrass, we developed both an association panel and linkage populations for genome-wide association study (GWAS) and genomic selection (GS). All of the 840 individuals were then genotyped using genotyping by sequencing (GBS), generating 350 GB of sequence in total. As a highly heterozygous polyploid (tetraploid and octoploid) species lacking a reference genome, switchgrass is highly intractable with earlier methodologies of single nucleotide polymorphism (SNP) discovery. To access the genetic diversity of species like switchgrass, we developed a SNP discovery pipeline based on a network approach called the Universal Network-Enabled Analysis Kit (UNEAK). Complexities that hinder single nucleotide polymorphism discovery, such as repeats, paralogs, and sequencing errors, are easily resolved with UNEAK. Here, 1.2 million putative SNPs were discovered in a diverse collection of primarily upland, northern-adapted switchgrass populations. Further analysis of this data set revealed the fundamentally diploid nature of tetraploid switchgrass. Taking advantage of the high conservation of genome structure between switchgrass and foxtail millet (Setaria italica (L.) P. Beauv.), two parent-specific, synteny-based, ultra high-density linkage maps containing a total of 88,217 SNPs were constructed. Also, our results showed clear patterns of isolation-by-distance and isolation-by-ploidy in natural populations of switchgrass. Phylogenetic analysis supported a general south-to-north migration path of switchgrass. In addition, this analysis suggested that upland tetraploid arose from upland octoploid. All together, this study provides unparalleled insights into the diversity, genomic complexity, population structure, phylogeny, phylogeography, ploidy, and evolutionary dynamics of switchgrass.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The analytical framework of UNEAK.
(A) Multiple DNA samples are digested and sequenced using GBS (red arrows represent cut sites). The inputs of UNEAK are Illumina Qseq or Fastq files. All of the reads are computationally trimmed to 64 bp. The solid colored lines represent error-free (“real”) reads, while the dashed lines are reads containing one or more sequencing errors. (B) Identical reads are classified as a tag. The colored bars are real tags, whereas the shaded bar is a rarer error tag. (C) Pairwise alignment is performed to find tag pairs differing by only a single bp mismatch. (D) Topology of tag networks. The colored circles are real tags. The shaded circles are error tags. Lines (“edges”) are drawn only between tags that differ by a single bp mismatch. (E) Only reciprocal, real tag pairs are retained as SNPs.
Figure 2
Figure 2. The networks of 802 representative tags from actual switchgrass data.
The red circles are putative “real” tags. The blue circles are low frequency, putative error tags (see Methods). The size of each circle denotes the count of a tag. Lines connecting the circles (“edges”) join tags that differ by a single bp mismatch. Of the 802 tags, 192 (24%) formed reciprocal tag pairs and thus, were identified as SNPs by the network filter.
Figure 3
Figure 3. Allele frequency of 50,000 SNPs (call rate >0.8) in the full-sib F1 population (n = 130) of upland tetraploid switchgrass, showing the classic signature of a cross between two heterozygous diploids.
Figure 4
Figure 4. Eighteen paternal linkage groups identified in the full-sib tetraploid linkage population.
Three thousand markers are clustered into 18 linkage groups, matching the haploid number of chromosomes in switchgrass. The color scale represents the Spearman's rank correlation between markers.
Figure 5
Figure 5. Sequence alignment of SNPs in switchgrass paternal linkage groups to the foxtail millet genome.
Nearly 10% (299/3000) of the SNPs previously mapped to switchgrass linkage groups were also mapped to unique sites in the foxtail millet genome. For each linkage group, the majority of SNPs that aligned to one chromosome of the foxtail millet genome are labeled red; the few exceptions are in blue. Each foxtail millet chromosome matches two switchgrass linkage groups, clearly indicating a genome duplication.
Figure 6
Figure 6. Geographic distribution and phylogenetic groups of switchgrass in the association panel.
Each population is indicated by a dot on the map in its approximate source location and a branch in the phylogenetic tree of the same color. Clades are labeled with ecotype, ploidy and geographical descriptors.
Figure 7
Figure 7. Upland 4× arose from 8×.
(A) A NJ tree of 29,221 markers. The lowland clade is the outgroup. (B) Multiple dimensional scaling (MDS) plot of the upland ecotypes.

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Publication types

Grant support

This project was funded by the United States Department of Energy and United States Department of Agriculture Plant Feedstock Genomics for Bioenergy Program (Project no. DE-AI02-07ER64454), National Science Foundation awards 0820619 and 0965342, and the United States Department of Agriculture. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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