A selection of phenotypic variations observed in plants obtained from fast neutron-irradiated seed.
Fast neutron irradiation has been known to be a very effective mutagen for decades and has gained widespread recognition as a gene knockout approach for plants. Deletions from fast neutron irradiation range from 1 bp to >30K bp, with most deletions at 1 to 4 kb in size. Fast neutron mutagenesis has been successfully reported in many plants, including Arabidopsis, rice, Glycine soja, M. truncatula, barley, and L. japonicus. The renewed interest in fast neutron irradiation as a mutagen source is due to the wide range of gene knockouts produced and the development of high-throughput screening processes. Detection of deletion mutations involves pooling DNA from multiple M2 plants to use as template in PCR reactions to select for the presence of shorter-than-wild-type gene fragments.
- Continue to generate and characterize FN mutants
- Obtain 50% deletion coverage of the soybean genome (~475MB) as quantified by comparative genomic hybridization and high throughput sequencing
- PCR-verify mutations in genes likely to impact agriculturally relevant traits
- Assess the heritability of attractive mutant phenotypes
- Use target gene editing (CRISPR-Cas9) to establish genotype to phenotype relationships for the most interesting identified mutant phenotypes
Currently 30,000 M3 seed stocks are being maintained by the Stupar group in Minnesota. These seeds are inventoried and tested for viability to ensure continual availability of mutant lines. A well characterized core set of 500 lines has been phenotyped for a variety of above and below ground traits including seed traits. Furthermore, a subset of the 500 lines has been genotyped by either comparative genomic hybridization or high throughput sequencing. Lines are in the process of becoming available both through Soybase.org as well as the USDA Soybean Germplasm Collection.
Fast neutrons have led to the creation of a trichome mutant. CGH identified a region on chromosome 20 as having a deletion in the mutant individual. Interestingly, when the region was resequenced there were multiple deletions, a rearrangement as well as an insertion. The candidate gene affected by these mutations is Glyma.20G019300 whose Arabidopsis homolog is Nck-Associated Protein 1 (NAP1). By inserting the wild-type version of the soybean NAP1 into A. thaliana NAP1 mutants, normal trichome formation was restored.
The group in Missouri has had similar success identifying and curating the fast neutron deletion populations. In particular, a focus has been placed on seed and nitrogen-fixation traits, as these are two aspects of soybean that are especially unique and are relevant to agriculture. A NIR was used to quantify seed oil, protein, and sugar content in FN populations. Fast neutrons have created a line with a reduced level of phytic acid. Notably, follow up phenotypic work has shown that reduced phytic acid does not affect early developmental traits as has been seen in other phytic acid mutants.
Campbell BW, Hofstad AN, Sreekanta S, Fu F, Kono TJ, O'Rourke JA, Vance CP, Muehlbauer GJ, Stupar RM. (2016) Fast neutron-induced structural rearrangements at a soybean NAP1 locus result in gnarled trichomes. Theor Appl Genet (in press). doi: 10.1007/s00122-016-2735-x.
Anderson JE, Michno JM, Kono TJ, Stec AO, Campbell BW, Curtin SJ, Stupar RM. (2016) Genomic variation and DNA repair associated with soybean transgenesis: a comparison to cultivars and mutagenized plants. BMC Biotechnol 16:41. doi: 10.1186/s12896-016-0271-z.
Bolon YT, Stec AO, Michno JM, Roessler J, Bhaskar PB, Ries L, Dobbels AA, Campbell BW, Young NP, Anderson JE, Grant DM, Orf JH, Naeve SL, Muehlbauer GJ, Vance CP, Stupar RM. (2014) Genome resilience and prevalence of segmental duplications following fast neutron irradiation of soybean. Genetics 198:967-981. doi: 10.1534/genetics.114.170340.
Bolon YT, Haun WJ, Xu WW, Grant D, Stacey MG, Nelson RT, Gerhardt DJ, Jeddeloh JA, Stacey G, Muehlbauer GJ, Orf JH, Naeve SL, Stupar RM, Vance CP. (2011) Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean. Plant Physiol 156:240-253. doi: 10.1104/pp.110.170811.
A database for lines generated from Fast Neutron mutagenesis as well as a general repository for other mutant Soybean lines has been created at Soybase.