Genome resilience and prevalence of segmental duplications following fast neutron irradiation of soybean

Abstract

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.

Keywords: deletion; duplication; fast neutron; soybean; structural variation.

Figure 1

Fast neutron-irradiated mutants analyzed by aCGH for genomic SV. The soybean fast neutron mutant population resource consists of >27,000 M₂-derived lineages. Three categories (forward screen, no-phenotype, and reverse screen) of mutants were assayed based on distinct selection criteria. aCGHs on 216 forward screen mutants representing 100 unique M₂-derived lineages were chosen from among mutants with documented aberrations in visual above-ground appearance, seed composition, or root and nodule screening. In some cases, multiple sibling plants from segregating families were assayed. No-phenotype plants showing no observed differences from the wild-type background were represented by 35 unique M₂-derived individuals. Ten M₂ plants with large deletions/duplications in specific homeologous intervals were identified from the Sequenom MassARRAY reverse screen. Thirteen plants representing these 10 lineages were analyzed by aCGH.

Figure 2

Genome-wide view of segmental duplication and deletion event coverage detected in fast neutron-irradiated soybean mutants. Each bar represents 1 of 20 soybean chromosomes. Light gray lines indicate pericentromeric regions while euchromatic and centromeric regions are shown in dark gray. Homozygous deletion event coverage is represented by red bars to the left of the chromosome, and segmental duplication event coverage is represented by blue bars to the right of the chromosome. The scale across the vertical chromosomes is represented in kilobase pairs (kbp).

Figure 3

Frequency of SV across chromosome regions in fast neutron-irradiated soybean mutants. Each chromosome was divided into 1000 bins of equal size in numerical order. The number of chromosomes (out of 20) with at least one SV event for each representative chromosome bin was calculated. (A) Segmental duplication events are most prevalent at chromosome ends. (B) Homozygous deletions are most prevalent in the pericentromeric regions. (C) Hemizygous deletions are found across the entire chromosome.

Figure 4

Validation of large segmental deletions and duplications in soybean fast neutron plants. Sequenom MassARRAY analysis was used to provide a relative quantification of DNA copy number between the ∼1-Mb syntenic intervals on chromosomes 8 and 15. SNPs differentiating a series of homeologous genes on chromosomes 8 and 15 were identified and quantified relative to one another. Red spots indicate the proportion of SNPs at each position that match the chromosome 8 homeolog, while blue spots indicate the proportion of SNPs at each position that match the chromosome 15 homeolog. Twenty-seven SNPs located throughout the ∼1-Mb intervals are shown. Plants with wild-type chromosomes would be expected to exhibit approximately equal proportions (e.g., 0.50 and 0.50, respectively) of blue and red spots throughout the interval, while plants harboring large duplications or deletions in these intervals would exhibit biased proportions (e.g., 0.33 blue and 0.67 red proportions when a deletion spans the chromosome 15 interval on one of the homologous chromosomes). (A) Results for two M₂ individuals are shown, representing a wild-type proportion (plant FN0170246) and a biased proportion (plant FN0170255). FN0170255 shows a strong bias favoring the chromosome 8 SNPs throughout the syntenic interval, indicating the presence of either a large duplication in the chromosome 8 interval or a large deletion in the chromosome 15 interval. (B) The plots are tightened to display the data for more individuals. For each segregating family, the M₂ parent is shown on the far left. The M₃ individuals (numbered on the x-axis) are grouped according to whether they maintained the large deletion/duplication (left side) or whether they segregated back to wild-type proportions (right side). The aCGH data (shown in Figure S5) indicated that the FN0170255 M₂ plant harbored a large deletion that included the syntenic interval on chromosome 15. Therefore, the FN0170255 M₃ family showed segregation of this large deletion. Meanwhile, the aCGH data (Figure S5) indicated that the FN0170228, FN0170522, and FN0170712 M₂ plants, respectively, harbored large duplications, each including the syntenic interval on chromosome 15. Therefore, these families showed segregation of the large duplications.

Figure 5

Summary of observed SV number and size within fast neutron mutant selection classes. (A) Bar graph of average number of SVs in forward screen, no-phenotype, and reverse screen mutants for duplications, homozygous deletions, and hemizygous deletions. (B) Bar graph of the median size of SVs in forward screen, no-phenotype, and reverse screen mutants for duplications, homozygous deletions, and hemizygous deletions.

Figure 6

Association of a fast neutron mutant deletion with a quantitative change in seed composition. (A) The aCGH log₂ ratios of mutant FN0172932 to wild-type (WT) signals are shown for chromosome 10. A deletion is visible from ∼34 to 36 Mb. (B) The exact coordinates of the deletion were resolved by high-throughput paired-end sequencing followed by PCR. Deletion junctions are represented by inverted triangles from 34,724,721 to 36,054,429 bp, framing a 1,329,809-bp deletion. (C) PCR across the deletion region with flanking primers (shaded trapezoids in B) confirmed the presence of the deletion allele. (D) PCR within the deletion region using internal primers (shaded parallelograms in B) confirmed the presence of the wild-type allele. (E and F) Bar graphs show that the presence of the deletion allele on chromosome 10 corresponds to a simultaneous decrease in percent protein (E) and additive increase in percent oil (F) composition of the soybean seed.

Figure 7

Association of a copy number gain with the short petiole phenotype. (A) The aCGH normalized log₂ ratios of mutant FN0163764 to the ‘M92-220’ progenitor are shown for the first 10 Mb of chromosome 17. The short petiole segregant in the FN0163764 family showed segmental duplication coverage from the start of the chromosome end to ∼7 Mb and also showed an additional level of genomic copy number gain above this region from ∼2 to 7 Mb. (B) The wild-type petiole segregant of the FN0163764 mutant showed a single level of segmental duplication coverage from the chromosome end to ∼7 Mb. (C) NGS-derived results provided evidence for a tandem duplication on chromosome 17. (D) The presence of the tandem duplication was confirmed by the presence of the 145-bp PCR product (lower band) in the short petiole mutant (mut) but not in the wild-type (WT) ‘M92-220’ background. Evidence for the inheritance of the tandem duplication (designated by the “+” symbol) was also found in 50% of FN0163764 × ‘MN1410’ progeny. (E) NGS results also provided evidence for a translocation between chromosome 17 and chromosome 11. (F) The presence of the translocation was confirmed with the expected 210-bp PCR product in the mutant and not the WT ‘M92-220’ background. No-template control lanes (labeled as “c”) are also shown. (G) Association of the presence of the tandem duplication (PCR confirmation) with a decrease (P-value 2.22E-05) in petiole length in F₃ segregants of the FN0163764 × ‘MN1410’ outcross, shown with standard error bars.