Thomas J. Y. Kono

A multi-purpose toolkit to enable advanced genome engineering in plants

An integrated reagent toolkit and streamlined protocols work across diverse plant species to enable sophisticated genome edits. We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

A Roadmap for Functional Structural Variants in the Soybean Genome

Gene structural variation (SV) has recently emerged as a key genetic mechanism underlying several important phenotypic traits in crop species. We screened a panel of 41 soybean (Glycine max) accessions serving as parents in a soybean nested association mapping population for deletions and duplications in more than 53,000 gene models. Array hybridization and whole genome resequencing methods were used as complementary technologies to identify SV in 1528 genes, or approximately 2.8%, of the soybean gene models. Although SV occurs throughout the genome, SV enrichment was noted in families of biotic defense response genes. Among accessions, SV was nearly eightfold less frequent for gene models that have retained paralogs since the last whole genome duplication event, compared with genes that have not retained paralogs. Increases in gene copy number, similar to that described at the Rhg1 resistance locus, account for approximately one-fourth of the genic SV events. This assessment of soybean SV occurrence presents a target list of genes potentially responsible for rapidly evolving and/or adaptive traits.

Development and Genetic Characterization of an Advanced Backcross-Nested Association Mapping (AB-NAM) Population of Wild × Cultivated Barley.

The ability to access alleles from unadapted germplasm collections is a long-standing problem for geneticists and breeders. Here we developed, characterized, and demonstrated the utility of a wild barley advanced backcross-nested association mapping (AB-NAM) population. We developed this population by backcrossing 25 wild barley accessions to the six-rowed malting barley cultivar Rasmusson. The 25 wild barley parents were selected from the 318 accession Wild Barley Diversity Collection (WBDC) to maximize allelic diversity. The resulting 796 BC2F4:6 lines were genotyped with 384 SNP markers, and an additional 4022 SNPs and 263,531 sequence variants were imputed onto the population using 9K iSelect SNP genotypes and exome capture sequence of the parents, respectively. On average, 96% of each wild parent was introgressed into the Rasmusson background, and the population exhibited low population structure. While linkage disequilibrium (LD) decay (r2 = 0.2) was lowest in the WBDC (0.36 cM), the AB-NAM (9.2 cM) exhibited more rapid LD decay than comparable advanced backcross (28.6 cM) and recombinant inbred line (32.3 cM) populations. Three qualitative traits: glossy spike, glossy sheath, and black hull color were mapped with high resolution to loci corresponding to known barley mutants for these traits. Additionally, a total of 10 QTL were identified for grain protein content. The combination of low LD, negligible population structure, and high diversity in an adapted background make the AB-NAM an important tool for high-resolution gene mapping and discovery of novel allelic variation using wild barley germplasm.

The Role of Deleterious Substitutions in Crop Genomes

Populations continually incur new mutations with fitness effects ranging from lethal to adaptive. While the distribution of fitness effects of new mutations is not directly observable, many mutations likely either have no effect on organismal fitness or are deleterious. Historically, it has been hypothesized that a population may carry many mildly deleterious variants as segregating variation, which reduces the mean absolute fitness of the population. Recent advances in sequencing technology and sequence conservation-based metrics for inferring the functional effect of a variant permit examination of the persistence of deleterious variants in populations. The issue of segregating deleterious variation is particularly important for crop improvement, because the demographic history of domestication and breeding allows deleterious variants to persist and reach moderate frequency, potentially reducing crop productivity. In this study, we use exome resequencing of 15 barley accessions and genome resequencing of 8 soybean accessions to investigate the prevalence of deleterious single nucleotide polymorphisms (SNPs) in the protein-coding regions of the genomes of two crops. We conclude that individual cultivars carry hundreds of deleterious SNPs on average, and that nonsense variants make up a minority of deleterious SNPs. Our approach identifies known phenotype-altering variants as deleterious more frequently than the genome-wide average, suggesting that putatively deleterious variants are likely to affect phenotypic variation. We also report the implementation of a SNP annotation tool BAD_Mutations that makes use of a likelihood ratio test based on alignment of all currently publicly available Angiosperm genomes.

Genomic variation and DNA repair associated with soybean transgenesis: a comparison to cultivars and mutagenized plants

BackgroundThe safety of mutagenized and genetically transformed plants remains a subject of scrutiny. Data gathered and communicated on the phenotypic and molecular variation induced by gene transfer technologies will provide a scientific-based means to rationally address such concerns. In this study, genomic structural variation (e.g. large deletions and duplications) and single nucleotide polymorphism rates were assessed among a sample of soybean cultivars, fast neutron-derived mutants, and five genetically transformed plants developed through Agrobacterium based transformation methods.ResultsOn average, the number of genes affected by structural variations in transgenic plants was one order of magnitude less than that of fast neutron mutants and two orders of magnitude less than the rates observed between cultivars. Structural variants in transgenic plants, while rare, occurred adjacent to the transgenes, and at unlinked loci on different chromosomes. DNA repair junctions at both transgenic and unlinked sites were consistent with sequence microhomology across breakpoints. The single nucleotide substitution rates were modest in both fast neutron and transformed plants, exhibiting fewer than 100 substitutions genome-wide, while inter-cultivar comparisons identified over one-million single nucleotide polymorphisms.ConclusionsOverall, these patterns provide a fresh perspective on the genomic variation associated with high-energy induced mutagenesis and genetically transformed plants. The genetic transformation process infrequently results in novel genetic variation and these rare events are analogous to genetic variants occurring spontaneously, already present in the existing germplasm, or induced through other types of mutagenesis. It remains unclear how broadly these results can be applied to other crops or transformation methods.

SNPMeta: SNP annotation and SNP metadata collection without a reference genome

The increase in availability of resequencing data is greatly accelerating SNP discovery and has facilitated the development of SNP genotyping assays. This, in turn, is increasing interest in annotation of individual SNPs. Currently, these data are only available through curation, or comparison to a reference genome. Many species lack a reference genome, but are still important genetic models or are significant species in agricultural production or natural ecosystems. For these species, it is possible to annotate SNPs through comparison with cDNA, or data from well‐annotated genes in public repositories. We present SNPMeta, a tool which gathers information about SNPs by comparison with sequences present in GenBank databases. SNPMeta is able to annotate SNPs from contextual sequence in SNP assay designs, and SNPs discovered through genotyping by sequencing (GBS) approaches. However, SNPs discovered through GBS occur throughout the genome, rather than only in gene space, and therefore do not annotate at high rates. SNPMeta can therefore be used to annotate SNPs in nonmodel species or species that lack a reference genome. Annotations generated by SNPMeta are highly concordant with annotations that would be obtained from a reference genome.

Comparative Analyses Identify the Contributions of Exotic Donors to Disease Resistance in a Barley Experimental Population

Introgression of novel genetic variation into breeding populations is frequently required to facilitate response to new abiotic or biotic pressure. This is particularly true for the introduction of host pathogen resistance in plant breeding. However, the number and genomic location of loci contributed by donor parents are often unknown, complicating efforts to recover desired agronomic phenotypes. We examined allele frequency differentiation in an experimental barley breeding population subject to introgression and subsequent selection for Fusarium head blight resistance. Allele frequency differentiation between the experimental population and the base population identified three primary genomic regions putatively subject to selection for resistance. All three genomic regions have been previously identified by quantitative trait locus (QTL) and association mapping. Based on the degree of identity-by-state relative to donor parents, putative donors of resistance alleles were also identified. The successful application of comparative population genetic approaches in this barley breeding experiment suggests that the approach could be applied to other breeding populations that have undergone defined breeding and selection histories, with the potential to provide valuable information for genetic improvement.

Environmental Association Analyses Identify Candidates for Abiotic Stress Tolerance in Glycine soja, the Wild Progenitor of Cultivated Soybeans

Natural populations across a species range demonstrate population structure owing to neutral processes such as localized origins of mutations and migration limitations. Selection also acts on a subset of loci, contributing to local adaptation. An understanding of the genetic basis of adaptation to local environmental conditions is a fundamental goal in basic biological research. When applied to crop wild relatives, this same research provides the opportunity to identify adaptive genetic variation that may be used to breed for crops better adapted to novel or changing environments. The present study explores an ex situ conservation collection, the USDA germplasm collection, genotyped at 32,416 SNPs to identify population structure and test for associations with bioclimatic and biophysical variables in Glycine soja, the wild progenitor of Glycine max (soybean). Candidate loci were detected that putatively contribute to adaptation to abiotic stresses. The identification of potentially adaptive variants in this ex situ collection may permit a more targeted use of germplasm collections.

Natural variation in teosinte at the domestication locus teosinte branched1 (tb1)

The teosinte branched1(tb1) gene is a major QTL controlling branching differences between maize and its wild progenitor, teosinte. The insertion of a transposable element (Hopscotch) upstream of tb1 is known to enhance the gene’s expression, causing reduced tillering in maize. Observations of the maize tb1 allele in teosinte and estimates of an insertion age of the Hopscotch that predates domestication led us to investigate its prevalence and potential role in teosinte. We assessed the prevalence of the Hopscotch element across an Americas-wide sample of 837 maize and teosinte individuals using a co-dominant PCR assay. Additionally, we calculated population genetic summaries using sequence data from a subset of individuals from four teosinte populations and collected phenotypic data using seed from a single teosinte population where Hopscotch was found segregating at high frequency. Genotyping results indicate the Hopscotch element is found in a number of teosinte populations and linkage disequilibrium near tb1 does not support recent introgression from maize. Population genetic signatures are consistent with selection on the tb1 locus, revealing a potential ecological role, but a greenhouse experiment does not detect a strong association between the Hopscotch and tillering in teosinte. Our findings suggest the role of Hopscotch differs between maize and teosinte. Future work should assess tb1 expression levels in teosinte with and without the Hopscotch and more comprehensively phenotype teosinte to assess the ecological significance of the Hopscotch insertion and, more broadly, the tb1 locus in teosinte.

The Effects of Both Recent and Long-Term Selection and Genetic Drift Are Readily Evident in North American Barley Breeding Populations

Barley was introduced to North America ∼400 yr ago but adaptation to modern production environments is more recent. Comparisons of allele frequencies among growth habits and spike (inflorescence) types in North America indicate that significant genetic differentiation has accumulated in a relatively short evolutionary time span. Allele frequency differentiation is greatest among barley with two-row vs. six-row spikes, followed by spring vs. winter growth habit. Large changes in allele frequency among breeding programs suggest a major contribution of genetic drift and linked selection on genetic variation. Despite this, comparisons of 3613 modern North American cultivated barley breeding lines that differ for spike-type and growth habit permit the discovery of 142 single nucleotide polymorphism (SNP) outliers putatively linked to targets of selection. For example, SNPs within the Cbf4, Ppd-H1, and Vrn-H1 loci, which have previously been associated with agronomically adaptive phenotypes, are identified as outliers. Analysis of extended haplotype sharing identifies genomic regions shared within and among breeding populations, suggestive of a number of genomic regions subject to recent selection. Finally, we are able to identify recent bouts of gene flow between breeding populations that could point to the sharing of agronomically adaptive variation. These results are supported by pedigrees and breeders’ understanding of germplasm sharing.