This project was initiated preceding the incorporation and standardization of KPIs. The progress of objectives are as follows:

Achieving objectives:
• Researchers generated two types of transgenic plants (one expressing CenH3-GFP at normal levels and one overexpressing CenH3-GFP while silencing the native CenH3 gene), but were experiencing difficulty using these lines to cross to wild type plants for generation of the haploid inducer lines(i.e. soybean lines missing chromosomes). In the first round of testing, 473 crosses were made, most of which failed to develop seeds. Those that did develop seeds were not missing chromosomes, thus the experiment was not successful.
• A reassessment of the stable transformed lines (those used for crossing), revealed a line that had a very low level of the transgene (from a single copy transgenic event) and none of the native gene. This line (281-6) was used for crossing with 6 different cultivars to look for loss of chromosomes. Screening over 500 putative F1 plants revealed two plants with green hypocotyls, a visual change that indicates a loss of a specific chromosome. These are being genotyped to detect possible haploid or aneuploid individuals.
• A second crossing experiment, using a mutant that will allow for identification putative haploids before planting, is underway. The mutant has a recessive trait that makes the cotyledons in the seed remain green, rather than turning to the normal yellow.
• KeyGene Inc. is screening EMS mutant populations to identify mutations in the soybean native CenH3 gene. Initial screening of Glyma07g06310 (the main candidate gene) revealed 26 total non-synonymous mutations but zero stop codons or splice variants in M1 plants. Screening of the secondary candidate gene Glyma16g02951 revealed 11 total non-synonymous mutations of which one is a stop codon and one is a predicted splice variant. Researchers are still awaiting seed from KeyGene for testing.
• CRISPR/Cas9 targeted mutagenesis constructs were designed for generating mutations in soybean’s CenH3 and these transgenes are being transformed into soybean.

Not meeting objectives:
• Progress in the prior project (1193) was slower than anticipated primarily due to the fact crosses made in the first round did not produce the desired seed. This roadblock seems to have been overcome. The researchers will proceed with the apparent successful crosses and at the same time work to develop additional transgenic lines and generate more crosses.