Development of mPing-based activation tags for crop insertional mutagenesis.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

Characterization of genetic heterogeneity within accessions in the USDA soybean germplasm collection.

Soybean breeding relies on the use of wild (Glycine soja Sieb. and Zucc.) and domesticated [Glycine max (L.) Merr.] germplasm for trait improvement. Soybeans are self-pollinating and accessions can be maintained as pure lines, however within-accession genetic variation has been observed in previous studies of some landraces and elite cultivars. The objective of this study was to characterize within-line variation in the accessions housed in the USDA Soybean Germplasm Collection. This collection includes over 20,000 accessions, each previously genotyped using the SoySNP50K Chip. Each SoySNP50K genotype was developed by pooling approximately three individuals per accession. Therefore, clusters of SNPs called as heterozygous within an accession can be inferred to represent putative regions of heterogeneity between the three individuals sampled. In this study, we found high-probability intervals of heterogeneity in 4% of the collection, representing 870 accessions. Heterogeneous loci were found on every chromosome and, collectively, covered 98.4% of the soybean genome and 99% of the gene models. Sanger sequencing confirmed regions of genomic heterogeneity among a subset of ten accessions. This dataset provides useful information and considerations for users of crop germplasm seed banks. Furthermore, the heterogeneous accessions and/or loci represent a unique genetic resource that is immediately available for forward and reverse genetics studies.

Similar Seed Composition Phenotypes Are Observed From CRISPR-Generated In-Frame and Knockout Alleles of a Soybean KASI Ortholog.

The ß-ketoacyl-[acyl carrier protein] synthase 1 (KASI) gene has been shown in model plant systems to be critical for the conversion of sucrose to oil. A previous study characterized the morphological and seed composition phenotypes associated with a reciprocal chromosomal translocation that disrupted one of the KASI genes in soybean. The principle findings of this work included a wrinkled seed phenotype, an increase in seed sucrose, a decrease in seed oil, and a low frequency of transmission of the translocation. However, it remained unclear which, if any, of these phenotypes were directly caused by the loss of KASI gene function, as opposed to the chromosomal translocation or other associated factors. In this study, CRISPR/Cas9 mutagenesis was used to generate multiple knockout alleles for this gene, and also one in-frame allele. These soybean plants were evaluated for morphology, seed composition traits, and genetic transmission. Our results indicate that the CRISPR/Cas9 mutants exhibited the same phenotypes as the chromosomal translocation mutant, validating that the observed phenotypes are caused by the loss of gene function. Furthermore, the plants harboring homozygous in-frame mutations exhibited similar phenotypes compared to the plants harboring homozygous knockout mutations. This result indicates that the amino acids lost in the in-frame mutant are essential for proper gene function. In-frame edits for this gene may need to target less essential and/or evolutionarily conserved domains in order to generate novel seed composition phenotypes.

Integration, abundance, and transmission of mutations and transgenes in a series of CRISPR/Cas9 soybean lines.

BACKGROUND: As with many plant species, current genome editing strategies in soybean are initiated by stably transforming a gene that encodes an engineered nuclease into the genome. Expression of the transgene results in a double-stranded break and repair at the targeted locus, oftentimes resulting in mutation(s) at the intended site. As soybean is a self-pollinating species with 20 chromosome pairs, the transgene(s) in the T0 plant are generally expected to be unlinked to the targeted mutation(s), and the transgene(s)/mutation(s) should independently assort into the T1 generation, resulting in Mendellian combinations of transgene presence/absence and allelic states within the segregating family. This prediction, however, is not always consistent with observed results. RESULTS: In this study, we investigated inheritance patterns among three different CRISPR/Cas9 transgenes and their respective induced mutations in segregating soybean families. Next-generation resequencing of four T0 plants and four T1 progeny plants, followed by broader assessments of the segregating families, revealed both expected and unexpected patterns of inheritance among the different lineages. These unexpected patterns included: (1) A family in which T0 transgenes and mutations were not transmitted to progeny; (2) A family with four unlinked transgene insertions, including two respectively located at paralogous CRISPR target break sites; (3) A family in which mutations were observed and transmitted, but without evidence of transgene integration nor transmission. CONCLUSIONS: Genome resequencing provides high-resolution of transgene integration structures and gene editing events. Segregation patterns of these events can be complicated by several potential mechanisms. This includes, but is not limited to, plant chimeras, multiple unlinked transgene integrations, editing of intended and paralogous targets, linkage between the transgene integration and target site, and transient expression of the editing reagents without transgene integration into the host genome.

Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes.

Developments in genomic and genome editing technologies have facilitated the mapping, cloning, and validation of genetic variants underlying trait variation. This study combined bulked-segregant analysis, array comparative genomic hybridization, and CRISPR/Cas9 methodologies to identify a CPR5 ortholog essential for proper trichome growth in soybean (Glycine max). A fast neutron mutant line exhibited short trichomes with smaller trichome nuclei compared to its parent line. A fast neutron-induced deletion was identified within an interval on chromosome 6 that co-segregated with the trichome phenotype. The deletion encompassed six gene models including an ortholog of Arabidopsis thaliana CPR5. CRISPR/Cas9 was used to mutate the CPR5 ortholog, resulting in five plants harboring a total of four different putative knockout alleles and two in-frame alleles. Phenotypic analysis of the mutants validated the candidate gene, and included intermediate phenotypes that co-segregated with the in-frame alleles. These findings demonstrate that the CPR5 ortholog is essential for proper growth and development of soybean trichomes, similar to observations in A. thaliana. Furthermore, this work demonstrates the value of using CRISPR/Cas9 to generate an allelic series and intermediate phenotypes for functional analysis of candidate genes and/or the development of novel traits.

Genomic changes and biochemical alterations of seed protein and oil content in a subset of fast neutron induced soybean mutants.

BACKGROUND: Soybean is subjected to genetic manipulation by breeding, mutation, and transgenic approaches to produce value-added quality traits. Among those genetic approaches, mutagenesis through fast neutrons radiation is intriguing because it yields a variety of mutations, including single/multiple gene deletions and/or duplications. Characterizing the seed composition of the fast neutron mutants and its relationship with gene mutation is useful towards understanding oil and protein traits in soybean. RESULTS: From a large population of fast neutron mutagenized plants, we selected ten mutants based on a screening of total oil and protein content using near infra-red spectroscopy. These ten mutants were regrown, and the seeds were analyzed for oil by GC-MS, protein profiling by SDS-PAGE and gene mapping by comparative genomic hybridization. The mutant 2R29C14Cladecr233cMN15 (nicknamed in this study as L10) showed higher protein and lower oil content compared to the wild type, followed by three other lines (nicknamed in this study as L03, L05, and L06). We characterized the fatty acid methyl esters profile of the trans-esterified oil and found the presence of five major fatty acids (palmitic, stearic, oleic, linoleic, and linolenic acids) at varying proportions among the mutants. Protein profile using SDS-PAGE of the ten mutants did exhibit discernable variation between storage (glycinin and ß-conglycinin) and anti-nutritional factor (trypsin inhibitor) proteins. In addition, we physically mapped the position of the gene deletions or duplications in each mutant using comparative genomic hybridization. CONCLUSION: Characterization of oil and protein profile in soybean fast neutron mutants will assist scientist and breeders to develop new value-added soybeans with improved protein and oil quality traits.

Screening populations for copy number variation using genotyping-by-sequencing: a proof of concept using soybean fast neutron mutants.

BACKGROUND: The effective use of mutant populations for reverse genetic screens relies on the population-wide characterization of the induced mutations. Genome- and population-wide characterization of the mutations found in fast neutron populations has been hindered, however, by the wide range of mutations generated and the lack of affordable technologies to detect DNA sequence changes. In this study, we therefore aimed to test whether genotyping-by-sequencing (GBS) technology could be used to characterize copy number variation (CNV) induced by fast neutrons in a soybean mutant population. RESULTS: We called CNVs from GBS data in 79 soybean mutants and assessed the sensitivity and precision of this approach by validating our results against array comparative genomic hybridization (aCGH) data for 19 of these mutants as well as targeted PCR and ddPCR assays for a representative subset of the smallest events detected by GBS. Our GBS pipeline detected 55 of the 96 events found by aCGH, with approximate detection thresholds of 60 kb, 500 kb and 1 Mb for homozygous deletions, hemizygous deletions and duplications, respectively. Among the whole set of 79 mutants, the GBS data revealed 105 homozygous deletions, 32 hemizygous deletions and 19 duplications. This included several extremely large events, exhibiting maximum sizes of ~ 11.2 Mb for a homozygous deletion, ~ 11.6 Mb for a hemizygous deletion, and ~ 50 Mb for a duplication. CONCLUSIONS: This study provides a proof of concept that GBS can be used as an affordable high-throughput method for assessing CNVs in fast neutron mutants. The modularity of this GBS approach allows combining as many different libraries or sequencing runs as is necessary for reaching the goals of a particular study. This method should enable the low-cost genome-wide characterization of hundreds to thousands of individuals in fast neutron mutant populations or any population with large genomic deletions and duplications.

Whole-genome re-sequencing reveals the impact of the interaction of copy number variants of the rhg1 and Rhg4 genes on broad-based resistance to soybean cyst nematode.

Soybean cyst nematode (SCN) is the most devastating plant-parasitic nematode. Most commercial soybean varieties with SCN resistance are derived from PI88788. Resistance derived from PI88788 is breaking down due to narrow genetic background and SCN population shift. PI88788 requires mainly the rhg1-b locus, while 'Peking' requires rhg1-a and Rhg4 for SCN resistance. In the present study, whole genome re-sequencing of 106 soybean lines was used to define the Rhg haplotypes and investigate their responses to the SCN HG-Types. The analysis showed a comprehensive profile of SNPs and copy number variations (CNV) at these loci. CNV of rhg1 (GmSNAP18) only contributed towards resistance in lines derived from PI88788 and 'Cloud'. At least 5.6 copies of the PI88788-type rhg1 were required to confer SCN resistance, regardless of the Rhg4 (GmSHMT08) haplotype. However, when the GmSNAP18 copies dropped below 5.6, a 'Peking'-type GmSHMT08 haplotype was required to ensure SCN resistance. This points to a novel mechanism of epistasis between GmSNAP18 and GmSHMT08 involving minimum requirements for copy number. The presence of more Rhg4 copies confers resistance to multiple SCN races. Moreover, transcript abundance of the GmSHMT08 in root tissue correlates with more copies of the Rhg4 locus, reinforcing SCN resistance. Finally, haplotype analysis of the GmSHMT08 and GmSNAP18 promoters inferred additional levels of the resistance mechanism. This is the first report revealing the genetic basis of broad-based resistance to SCN and providing new insight into epistasis, haplotype-compatibility, CNV, promoter variation and its impact on broad-based disease resistance in plants.

Genome Editing in Soybean with CRISPR/Cas9.

CRISPR/Cas9 mediated genome editing technology has experienced rapid advances in recent years and has been applied to a wide variety of plant species, including soybean. Several platforms have been developed for designing and cloning of single CRISPR targets or multiple targets in a single destination vector. This chapter provides an updated working protocol for applying CRISPR/Cas9 technology to target a single gene or multiple genes simultaneously in soybean. We describe two platforms for cloning single CRISPR targets and multiplexing targets, respectively, and reagent delivery methodologies. The protocols address crucial limiting steps that can limit CRISPR editing in soybean hairy roots, composite plants, and tissue culture-based regenerated whole plants. To date, transgenic soybean plants with mutagenesis in up to three target genes have been obtained with this procedure.

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

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 ß-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.

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

BACKGROUND: The 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. RESULTS: On 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. CONCLUSIONS: Overall, 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.

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.

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.

Genomic heterogeneity and structural variation in soybean near isogenic lines.

Near isogenic lines (NILs) are a critical genetic resource for the soybean research community. The ability to identify and characterize the genes driving the phenotypic differences between NILs is limited by the degree to which differential genetic introgressions can be resolved. Furthermore, the genetic heterogeneity extant among NIL sub-lines is an unaddressed research topic that might have implications for how genomic and phenotypic data from NILs are utilized. In this study, a recently developed high-resolution comparative genomic hybridization (CGH) platform was used to investigate the structure and diversity of genetic introgressions in two classical soybean NIL populations, respectively varying in protein content and iron deficiency chlorosis (IDC) susceptibility. There were three objectives: assess the capacity for CGH to resolve genomic introgressions, identify introgressions that are heterogeneous among NIL sub-lines, and associate heterogeneous introgressions with susceptibility to IDC. Using the CGH approach, introgression boundaries were refined and previously unknown introgressions were revealed. Furthermore, heterogeneous introgressions were identified within seven sub-lines of the IDC NIL "IsoClark." This included three distinct introgression haplotypes linked to the major iron susceptible locus on chromosome 03. A phenotypic assessment of the seven sub-lines did not reveal any differences in IDC susceptibility, indicating that the genetic heterogeneity among the lines does not have a significant impact on the primary NIL phenotype.

Addition of individual chromosomes of maize inbreds B73 and Mo17 to oat cultivars Starter and Sun II: maize chromosome retention, transmission, and plant phenotype.

Oat-maize addition (OMA) lines with one, or occasionally more, chromosomes of maize (Zea mays L., 2n = 2x = 20) added to an oat (Avena sativa L., 2n = 6x = 42) genomic background can be produced via embryo rescue from sexual crosses of oat x maize. Self-fertile disomic addition lines of different oat genotypes, mainly cultivar Starter, as recipient for maize chromosomes 1, 2, 3, 4, 5, 6, 7, 9, and the short arm of 10 and a monosomic addition line for chromosome 8, have been reported previously in which the sweet corn hybrid Seneca 60 served as the maize chromosome donor. Here we report the production and characterization of a series of new OMA lines with inbreds B73 and Mo17 as maize chromosome donors and with oat cultivars Starter and Sun II as maize chromosome recipients. Fertile disomic OMA lines were recovered for B73 chromosomes 1, 2, 4, 5, 6, 8, 9, and 10 and Mo17 chromosomes 2, 4, 5, 6, 8, and 10. These lines together with non-fertile (oat x maize) F(1) plants with chromosome 3 and chromosome 7 of Mo17 individually added to Starter oat provide DNA of additions to oat of all ten individual maize chromosomes between the two maize inbreds. The Mo17 chromosome 10 OMA line was the first fertile disomic OMA line obtained carrying a complete chromosome 10. The B73 OMA line for chromosome 1 and the B73 and Mo17 OMA lines for chromosome 8 represent disomic OMA lines with improved fertility and transmission of the addition chromosome compared to earlier Seneca 60 versions. Comparisons among the four oat-maize parental genotype combinations revealed varying parental effects and interactions on frequencies of embryo recovery, embryo germination, F(1) plantlets with maize chromosomes, the specific maize chromosomes retained and transmitted to F(2) progeny, and phenotypes of self-fertile disomic addition plants. As opposed to the previous use of a hybrid Seneca 60 maize stock as donor of the added maize chromosomes, the recovered B73 and Mo17 OMA lines provide predictable genotypes for use as tools in physical mapping of maize DNA sequences, including inter-genic sequences, by simple presence/absence assays. The recovered OMA lines represent unique materials for maize genome analysis, genetic, physiological, and morphological studies, and a possible means to transfer maize traits to oat. Descriptions of these materials can be found at http://agronomy.cfans.umn.edu/Maize_Genomics.html .

Maize centromere mapping: a comparison of physical and genetic strategies.

Centromere positions on 7 maize chromosomes were compared on the basis of data from 4 to 6 mapping techniques per chromosome. Centromere positions were first located relative to molecular markers by means of radiation hybrid lines and centric fission lines recovered from oat-maize chromosome addition lines. These centromere positions were then compared with new data from centric fission lines recovered from maize plants, half-tetrad mapping, and fluorescence in situ hybridizations and to data from earlier studies. Surprisingly, the choice of mapping technique was not the critical determining factor. Instead, on 4 chromosomes, results from all techniques were consistent with a single centromere position. On chromosomes 1, 3, and 6, centromere positions were not consistent even in studies using the same technique. The conflicting centromere map positions on chromosomes 1, 3, and 6 could be explained by pericentric inversions or alternative centromere positions on these chromosomes.

Dissecting the maize genome by using chromosome addition and radiation hybrid lines.

We have developed from crosses of oat (Avena sativa L.) and maize (Zea mays L.) 50 fertile lines that are disomic additions of individual maize chromosomes 1-9 and chromosome 10 as a short-arm telosome. The whole chromosome 10 addition is available only in haploid oat background. Most of the maize chromosome disomic addition lines have regular transmission; however, chromosome 5 showed diminished paternal transmission, and chromosome 10 is transmitted to offspring only as a short-arm telosome. To further dissect the maize genome, we irradiated monosomic additions with gamma rays and recovered radiation hybrid (RH) lines providing low- to medium-resolution mapping for most of the maize chromosomes. For maize chromosome 1, mapping 45 simple-sequence repeat markers delineated 10 groups of RH plants reflecting different chromosome breaks. The present chromosome 1 RH panel dissects this chromosome into eight physical segments defined by the 10 groups of RH lines. Genomic in situ hybridization revealed the physical size of a distal region, which is represented by six of the eight physical segments, as being approximately 20% of the length of the short arm, representing approximately one-third of the genetic chromosome 1 map. The distal approximately 20% of the physical length of the long arm of maize chromosome 1 is represented by a single group of RH lines that spans >23% of the total genetic map. These oat-maize RH lines provide valuable tools for physical mapping of the complex highly duplicated maize genome and for unique studies of inter-specific gene interactions.