*2.2. Fine Mapping of the YL Gene*

To unravel the molecular mechanism of the *yl* phenotype, we performed genetic mapping to isolate the *YL* gene. The *yl* mutant was crossed to two soybean cultivars with normal green leaves, Williams 82 and Zhonghuang 13, and three F2 mapping populations were produced by reciprocal crosses, *yl* × Williams 82, *yl* × Zhonghuang 13 and Zhonghuang 13 × *yl*. The leaf color of all F1 plants was normal green. The segregation ratio of green to yellow leaves appeared to be 3:1 in F2 populations, indicating that the *yl* phenotype is controlled by a single recessive nuclear gene (Table 1).

**Table 1.** Segregation pattern and chi-square tests for green/yellow leaves of F2 progeny from the crosses between *yl* and Williams 82 or Zhonghuang 13.


The *yl* mutation was primarily mapped on chromosome 20 between microsatellite markers Satt162 and Sat\_155, which were 9.9 centimorgans (cM) and 1.1 cM from *yl*, respectively (Figure 3A). The *YL* locus was ultimately fine mapped into a 28-kb interval between single nucleotide polymorphism (SNP) markers S3 and S7-3 (Figure 3A). According to the soybean gene annotation database (www. phytozome.net) [31], there were three putative open reading frames (ORFs) within this 28-kb region

(Figure 3A). We sequenced the 28-kb sequences between the wild type and *yl* mutant and found a C to A transition at the eighth exon of *Glyma.20G187000* (Figure 3B).

**Figure 3.** Fine mapping of the *YL* gene. (**A**) The *YL* locus was initially mapped to a region between markers Satt162 and Sat\_155 on soybean chromosome 20. The gene was finally delimited to a 28 kb region between markers S3 and S7-3. Three predicted open reading frames (ORFs) were within this region. (**B**) *YL* (*Glyma.20G187000*) structure indicating nine exons (gray boxes), eight introns (line segments between the exons), and 5 and 3 untranslated regions (white boxes with black frame). Start (ATG) and stop (TGA) codons are marked. The *yl* mutation in the *YL* gene is shown.
