Benjamin W. Campbell

Identical substitutions in magnesium chelatase paralogs result in chlorophyll-deficient soybean mutants.

The soybean [Glycine max (L.) Merr.] chlorophyll-deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a nonsynonymous nucleotide substitution in the third exon of a Mg-chelatase subunit gene (ChlI1a) on chromosome 13. This gene was selected as a candidate for a different yellow foliage mutant, T219H (Y11y11), that had been previously mapped to chromosome 13. Although the phenotypes of MinnGold and T219H are clearly distinct, sequencing of ChlI1a in T219H identified a different nonsynonymous mutation in the third exon, only six base pairs from the MinnGold mutation. This information, along with previously published allelic tests, were used to identify and clone a third yellow foliage mutation, CD-5, which was previously mapped to chromosome 15. This mutation was identified in the ChlI1b gene, a paralog of ChlI1a. Sequencing of the ChlI1b allele in CD-5 identified a nonsynonymous substitution in the third exon that confers an identical amino acid change as the T219H substitution at ChlI1a. Protein sequence alignments of the two Mg-chelatase subunits indicated that the sites of amino acid modification in MinnGold, T219H, and CD-5 are highly conserved among photosynthetic species. These results suggest that amino acid alterations in this critical domain may create competitive inhibitory interactions between the mutant and wild-type ChlI1a and ChlI1b proteins.

Genome Resilience and Prevalence of Segmental Duplications Following Fast Neutron Irradiation of Soybean

Fast neutron radiation has been used as a mutagen to develop extensive mutant collections. However, the genome-wide structural consequences of fast neutron radiation are not well understood. Here, we examine the genome-wide structural variants observed among 264 soybean [Glycine max (L.) Merrill] plants sampled from a large fast neutron-mutagenized population. While deletion rates were similar to previous reports, surprisingly high rates of segmental duplication were also found throughout the genome. Duplication coverage extended across entire chromosomes and often prevailed at chromosome ends. High-throughput resequencing analysis of selected mutants resolved specific chromosomal events, including the rearrangement junctions for a large deletion, a tandem duplication, and a translocation. Genetic mapping associated a large deletion on chromosome 10 with a quantitative change in seed composition for one mutant. A tandem duplication event, located on chromosome 17 in a second mutant, was found to cosegregate with a short petiole mutant phenotype, and thus may serve as an example of a morphological change attributable to a DNA copy number gain. Overall, this study provides insight into the resilience of the soybean genome, the patterns of structural variation resulting from fast neutron mutagenesis, and the utility of fast neutron-irradiated mutants as a source of novel genetic losses and gains.

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.

MicroRNA Maturation and MicroRNA Target Gene Expression Regulation Are Severely Disrupted in Soybean dicer-like1 Double Mutants.

Small nonprotein-coding microRNAs (miRNAs) are present in most eukaryotes and are central effectors of RNA silencing-mediated mechanisms for gene expression regulation. In plants, DICER-LIKE1 (DCL1) is the founding member of a highly conserved family of RNase III-like endonucleases that function as core machinery proteins to process hairpin-like precursor transcripts into mature miRNAs, small regulatory RNAs, 21–22 nucleotides in length. Zinc finger nucleases (ZFNs) were used to generate single and double-mutants of putative soybean DCL1 homologs, DCL1a and DCL1b, to confirm their functional role(s) in the soybean miRNA pathway. Neither DCL1 single mutant, dcl1a or dcl1b plants, exhibited a pronounced morphological or molecular phenotype. However, the dcl1a/dcl1b double mutant expressed a strong morphological phenotype, characterized by reduced seed size and aborted seedling development, in addition to defective miRNA precursor transcript processing efficiency and deregulated miRNA target gene expression. Together, these findings indicate that the two soybean DCL1 paralogs, DCL1a and DCL1b, largely play functionally redundant roles in the miRNA pathway and are essential for normal plant development.

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Summary Processing of double‐stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL‐effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi‐allelic double mutant for the two soya bean paralogous Double‐stranded RNA‐binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9‐generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ‐line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer‐like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer‐like3 gene and the GmHen1a gene was observed in the T0 generation, but these mutations failed to transmit to the T1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole‐genome sequencing to reveal a spectrum of non‐germ‐line‐targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.

An induced chromosomal translocation in soybean disrupts a KASI ortholog and is associated with a high-sucrose and low-oil seed phenotype

Mutagenesis is a useful tool in many crop species to induce heritable genetic variability for trait improvement and gene discovery. In this study, forward screening of a soybean fast neutron (FN) mutant population identified an individual that produced seed with nearly twice the amount of sucrose (8.1% on dry matter basis) and less than half the amount of oil (8.5% on dry matter basis) as compared to wild type. Bulked segregant analysis (BSA), comparative genomic hybridization, and genome resequencing were used to associate the seed composition phenotype with a reciprocal translocation between chromosomes 8 and 13. In a backcross population, the translocation perfectly cosegregated with the seed composition phenotype and exhibited non-Mendelian segregation patterns. We hypothesize that the translocation is responsible for the altered seed composition by disrupting a β-ketoacyl-[acyl carrier protein] synthase 1 (KASI) ortholog. KASI is a core fatty acid synthesis enzyme that is involved in the conversion of sucrose into oil in developing seeds. This finding may lead to new research directions for developing soybean cultivars with modified carbohydrate and oil seed composition.

Soybean (Glycine max) Mutant and Germplasm Resources: Current Status and Future Prospects

Genetic bottlenecks during domestication and modern breeding limited the genetic diversity of soybean (Glycine max (L.) Merr.). Therefore, expanding and diversifying soybean genetic resources is a major priority for the research community. These resources, consisting of natural and induced genetic variants, are valuable tools for improving soybean and furthering soybean biological knowledge. During the twentieth century, researchers gathered a wealth of genetic variation in the forms of landraces, Glycine soja accessions, Glycine tertiary germplasm, and the U.S. Department of Agriculture (USDA) Type and Isoline Collections. During the twenty-first century, soybean researchers have added several new genetic and genomic resources. These include the reference genome sequence, genotype data for the USDA soybean germplasm collection, next-generation mapping populations, new irradiation and transposon-based mutagenesis populations, and designer nuclease platforms for genome engineering. This paper briefly surveys the publicly accessible soybean genetic resources currently available or in development and provides recommendations for developing such genetic resources in the future.