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Setaria (L.) P. Beauv is a genus of grasses that belongs to the Poaceae (grass) family, subfamily Panicoideae. Two members of the Setaria genus, Setaria italica (foxtail millet) and S. viridis (green foxtail), have been studied extensively over the past few years as model species for C4-photosynthesis and to facilitate genome studies in complex Panicoid bioenergy grasses. We exploited the available genetic and genomic resources for S. italica and its wild progenitor, S. viridis, to study the genetic basis of seed shattering. Reduced shattering is a key trait that underwent positive selection during domestication. Phenotyping of F2:3 and recombinant inbred line (RIL) populations generated from a cross between S. italica accession B100 and S. viridis accession A10 identified the presence of additive main effect quantitative trait loci (QTL) on chromosomes V and IX. As expected, enhanced seed shattering was contributed by the wild S. viridis. Comparative analyses pinpointed Sh1 and qSH1, two shattering genes previously identified in sorghum and rice, as potentially underlying the QTL on Setaria chromosomes IX and V, respectively. The Sh1 allele in S. italica was shown to carry a PIF/Harbinger MITE in exon 2, which gave rise to an alternatively spliced transcript that lacked exon 2. This MITE was universally present in S. italica accessions around the world and absent from the S. viridis germplasm tested, strongly suggesting a single origin of foxtail millet domestication. The qSH1 gene carried two MITEs in the 5'UTR. Presence of one or both MITEs was strongly associated with cultivated germplasm. If the MITE insertion(s) in qSH1 played a role in reducing shattering in S. italica accessions, selection for the variants likely occurred after the domestication of foxtail millet.

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BACKGROUND: Research on orphan crops is often hindered by a lack of genomic resources. With the advent of affordable sequencing technologies, genotyping an entire genome or, for large-genome species, a representative fraction of the genome has become feasible for any crop. Nevertheless, most genotyping-by-sequencing (GBS) methods are geared towards obtaining large numbers of markers at low sequence depth, which excludes their application in heterozygous individuals. Furthermore, bioinformatics pipelines often lack the flexibility to deal with paired-end reads or to be applied in polyploid species. RESULTS: UGbS-Flex combines publicly available software with in-house python and perl scripts to efficiently call SNPs from genotyping-by-sequencing reads irrespective of the species' ploidy level, breeding system and availability of a reference genome. Noteworthy features of the UGbS-Flex pipeline are an ability to use paired-end reads as input, an effective approach to cluster reads across samples with enhanced outputs, and maximization of SNP calling. We demonstrate use of the pipeline for the identification of several thousand high-confidence SNPs with high representation across samples in an F3-derived F2 population in the allotetraploid finger millet. Robust high-density genetic maps were constructed using the time-tested mapping program MAPMAKER which we upgraded to run efficiently and in a semi-automated manner in a Windows Command Prompt Environment. We exploited comparative GBS with one of the diploid ancestors of finger millet to assign linkage groups to subgenomes and demonstrate the presence of chromosomal rearrangements. CONCLUSIONS: The paper combines GBS protocol modifications, a novel flexible GBS analysis pipeline, UGbS-Flex, recommendations to maximize SNP identification, updated genetic mapping software, and the first high-density maps of finger millet. The modules used in the UGbS-Flex pipeline and for genetic mapping were applied to finger millet, an allotetraploid selfing species without a reference genome, as a case study. The UGbS-Flex modules, which can be run independently, are easily transferable to species with other breeding systems or ploidy levels.

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BACKGROUND: The presence of non-coding introns is a characteristic feature of most eukaryotic genes. While the size of the introns, number of introns per gene and the number of intron-containing genes can vary greatly between sequenced eukaryotic genomes, the structure of a gene with reference to intron presence and positions is typically conserved in closely related species. Unexpectedly, the ABCB1 (ATP-Binding Cassette Subfamily B Member 1) gene which encodes a P-glycoprotein and underlies dwarfing traits in maize (br2), sorghum (dw3) and pearl millet (d2) displayed considerable variation in intron composition. RESULTS: An analysis of the ABCB1 gene structure in 80 angiosperms revealed that the number of introns ranged from one to nine. All introns in ABCB1 underwent either a one-time loss (single loss in one lineage/species) or multiple independent losses (parallel loss in two or more lineages/species) with the majority of losses occurring within the grass family. In contrast, the structure of the closest homolog to ABCB1, ABCB19, remained constant in the majority of angiosperms analyzed. Using known phylogenetic relationships within the grasses, we determined the ancestral branch-points where the losses occurred. Intron 7, the longest intron, was lost in only a single species, Mimulus guttatus, following duplication of ABCB1. Semiquantitative PCR showed that the M. guttatus ABCB1 gene copy without intron 7 had significantly lower transcript levels than the gene copy with intron 7. We further demonstrated that intron 7 carried two motifs that were highly conserved across the monocot-dicot divide. CONCLUSIONS: The ABCB1 gene structure is highly dynamic, while the structure of ABCB19 remained largely conserved through evolution. Precise removal of introns, preferential removal of smaller introns and presence of at least 2 bp of microhomology flanking most introns indicated that intron loss may have predominantly occurred through non-homologous end-joining (NHEJ) repair of double strand breaks. Lack of microhomology in the exon upstream of lost phase I introns was likely due to release of the selective constraint on the penultimate base (3rd base in codon) of the terminal codon by the splicing machinery. In addition to size, the presence of regulatory motifs will make introns recalcitrant to loss.

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Loops in proteins that connect secondary structures such as alpha-helix and beta-sheet, are often on the surface and may play a critical role in some functions of a protein. The mobility of loops is central for the motional freedom and flexibility requirements of active-site loops and may play a critical role for some functions. The structures and behaviors of loops have not been studied much in the context of the whole structure and its overall motions, especially how these might be coupled. Here we investigate loop motions by using coarse-grained structures (C(α) atoms only) to solve the motions of the system by applying Lagrange equations with elastic network models to learn about which loops move in an independent fashion and which move in coordination with domain motions, faster and slower, respectively. The normal modes of the system are calculated using eigen-decomposition of the stiffness matrix. The contribution of individual modes and groups of modes is investigated for their effects on all residues in each loop by using Fourier analyses. Our results indicate overall that the motions of functional sets of loops behave in similar ways as the whole structure. But overall only a relatively few loops move in coordination with the dominant slow modes of motion, and these are often closely related to function.