A strategy for identification and characterization of genic mutations using a temperature-sensitive chlorotic soybean mutant as an example.

Screening a transposon-mutagenized soybean population led to the discovery of a recessively inherited chlorotic phenotype. This "y24" phenotype results in smaller stature, weaker stems, and a smaller root system. Genome sequencing identified 15 candidate genes with mutations likely to result in a loss of function. Amplicon sequencing of a segregating population was then used to narrow the list to a single candidate mutation, a single-base change in Glyma.07G102300 that disrupts splicing of the second intron. Single cell transcriptomic profiling indicates that this gene is expressed primarily in mesophyll cells, and RNA sequencing data indicate that it is upregulated in germinating seedlings by cold stress. Previous studies have shown that mutations to Os05g34040, the rice ortholog of Glyma.07G102300, produced a chlorotic phenotype that was more pronounced in cool temperatures. Growing soybean y24 mutants at lower temperatures also resulted in a more severe phenotype. In addition, transgenic expression of wild-type Glyma.07G102300 in the knockout mutant of the Arabidopsis ortholog At4930720 rescues the chlorotic phenotype, further supporting the hypothesis that the mutation in Glyma.07G102300 is causal of the y24 phenotype. The variant analysis strategy used to identify the genes underlying this phenotype provides a template for the study of other soybean mutants.