*4.4. Fine Mapping of the YL Gene*

Three F2 populations generated from reciprocal crosses between the *yl* mutant and two soybean cultivars, Williams 82 and Zhonghuang 13 (*yl* × Williams 82, *yl* × Zhonghuang 13 and Zhonghuang 13 × *yl*), were used for genetic analysis. In the F2 populations, genomic DNA was isolated from selected etiolated seedlings exhibiting mutant phenotype for gene mapping. The cetyl trimethyl ammonium bromide (CTAB) method was used for genomic DNA extraction from the fresh young trifoliate leaf tissue and for all subsequent DNA extractions using this method unless otherwise stated. Briefly, frozen tissue was powdered and dispersed in 2 × CTAB extraction buffer, and incubated at 65 ◦C for about 60 min. Chloroform/Tris-phenol, 1:1 (vol/vol), was added and mixed to form an emulsion that was centrifuged for 10 min. The upper aqueous phase was transferred to a new tube, and 2/3 vol of isopropanol was added for DNA precipitation. The extracted DNA was treated with RNase to remove RNA contamination.

For primary mapping, 92 mutant individuals were selected from two F2 populations between *yl* and Williams 82 or Zhonghuang 13. The markers used for primary mapping were 71 published SSR markers (http://soybase.org/) [47]. A total of 770 mutant individuals selected from the F2 population between the *yl* mutant and Williams 82 were used for fine mapping. New molecular markers were developed for fine mapping (Table S3). To identify the candidate gene, the corresponding DNA fragments within the fine mapping region were amplified from the wild type and *yl* mutant using special primers (Table S3) and sequenced.
