Fast neutron mutagenesis in soybean enriches for small indels and creates frameshift mutations.

The mutagenic effects of ionizing radiation have been used for decades to create novel variants in experimental populations. Fast neutron (FN) bombardment as a mutagen has been especially widespread in plants, with extensive reports describing the induction of large structural variants, i.e., deletions, insertions, inversions, and translocations. However, the full spectrum of FN-induced mutations is poorly understood. We contrast small insertions and deletions (indels) observed in 27 soybean lines subject to FN irradiation with the standing indels identified in 107 diverse soybean lines. We use the same populations to contrast the nature and context (bases flanking a nucleotide change) of single-nucleotide variants. The accumulation of new single-nucleotide changes in FN lines is marginally higher than expected based on spontaneous mutation. In FN-treated lines and in standing variation, C→T transitions and the corresponding reverse complement G→A transitions are the most abundant and occur most frequently in a CpG local context. These data indicate that most SNPs identified in FN lines are likely derived from spontaneous de novo processes in generations following mutagenesis rather than from the FN irradiation mutagen. However, small indels in FN lines differ from standing variants. Short insertions, from 1 to 6 bp, are less abundant than in standing variation. Short deletions are more abundant and prone to induce frameshift mutations that should disrupt the structure and function of encoded proteins. These findings indicate that FN irradiation generates numerous small indels, increasing the abundance of loss-of-function mutations that impact single genes.

Environmental Association Identifies Candidates for Tolerance to Low Temperature and Drought.

Barley (Hordeum vulgare ssp. vulgare) is cultivated from the equator to the Arctic Circle. The wild progenitor species, Hordeum vulgare ssp. spontaneum, occupies a relatively narrow latitudinal range (∼30 - 40° N) primarily at low elevation (< 1,500 m). Adaptation to the range of cultivation has occurred over ∼8,000 years. The genetic basis of adaptation is amenable to study through environmental association. An advantage of environmental association in a well-characterized crop is that many loci that contribute to climatic adaptation and abiotic stress tolerance have already been identified. This provides the opportunity to determine if environmental association approaches effectively identify these loci of large effect. Using published genotyping from 7,864 SNPs in 803 barley landraces, we examined allele frequency differentiation across multiple partitions of the data and mixed model associations relative to bioclimatic variables. Using newly generated resequencing data from a subset of these landraces, we tested for linkage disequilibrium (LD) between SNPs queried in genotyping and SNPs in neighboring loci. Six loci previously reported to contribute to adaptive differences in flowering time and abiotic stress in barley and six loci previously identified in other plant species were identified in our analyses. In many cases, patterns of LD are consistent with the causative variant occurring in the immediate vicinity of the queried SNP. The identification of barley orthologs to well-characterized genes may provide a new understanding of the nature of adaptive variation and could permit a more targeted use of potentially adaptive variants in barley breeding and germplasm improvement.

Development of a Multiparent Population for Genetic Mapping and Allele Discovery in Six-Row Barley.

Germplasm collections hold valuable allelic diversity for crop improvement and genetic mapping of complex traits. To gain access to the genetic diversity within the USDA National Small Grain Collection (NSGC), we developed the Barley Recombinant Inbred Diverse Germplasm Population (BRIDG6), a six-row spring barley multiparent population (MPP) with 88 cultivated accessions crossed to a common parent (Rasmusson). The parents were randomly selected from a core subset of the NSGC that represents the genetic diversity of landrace and breeding accessions. In total, we generated 6160 F5 recombinant inbred lines (RILs), with an average of 69 and a range of 37-168 RILs per family, that were genotyped with 7773 SNPs, with an average of 3889 SNPs segregating per family. We detected 23 quantitative trait loci (QTL) associated with flowering time with five QTL found coincident with previously described flowering time genes. A major QTL was detected near the flowering time gene, HvPpd-H1 which affects photoperiod. Haplotype-based analysis of HvPpd-H1 identified private alleles to families of Asian origin conferring both positive and negative effects, providing the first observation of flowering time-related alleles private to Asian accessions. We evaluated several subsampling strategies to determine the effect of sample size on the power of QTL detection, and found that, for flowering time in barley, a sample size >50 families or 3000 individuals results in the highest power for QTL detection. This MPP will be useful for uncovering large and small effect QTL for traits of interest, and identifying and utilizing valuable alleles from the NSGC for barley improvement.