Fine mapping of the target bud-keeping-green trait gene
Thirty-one sets of KASP primers were designed according to the SNP information in the target region between C9: 58,700,000 - 63,300,000 of the parent lines. Initially, the parental lines and random 20 plants out of 987 BC2F2 individuals were used to screen the primers, and the resultant 30 sets of KASP markers could be used for further testing of the remaining individuals of the segregating population. The 987 individual plants in the BC2F2 population were phenotyped and genotyped with the 4 sets of KASP primers which were at both side of the target region to find the recombined individuals, and finally, 96 individuals with recombined chromosome segment in the target region were used for fine mapping. These recombined individuals were genotyped by the left 26 sets of KASP markers. Phenotyping and genotyping (with 30 KASP markers) results of the recombined individuals are depicted in Figure 3A. The genotypic data were arranged from top to bottom according to ascending order of 30 KASP markers lined from left to right by physical location in the reference genome as described in the materials and methods. The data strongly supported the mapping result that the target area was closely associated with bud color at low temperature.
According to the genotypic and phenotypic data of the recombinants with chromosome segment exchange in the target region, the trait was fine mapped in the region of about 271 Kb between bropK17 (C9:59974705) and bropK19 (C9:60246054).  In the range of C9:59974705-60246054, another 6 groups of KASP primers were developed according to the relative SNP information, and 19 individual plants with recombined chromosome segment in the above range were genotyped with these 6 KASP markers. The phenotyping and genotyping analysis of the 19 recombinants reduced the target area into 241Kb range between bropK41 (C9:59998177) and bropK43 (C9: 60239356) (Figure 3B).  Then, according to the InDel information in region of C9: 59998177-60239356 of the parental lines, another new 6 sets of KASP primers were developed and used to genotype the 17 individuals with the recombined genome in this area (Figure 3C). Finally, the target gene was fine mapped to a region of about 240 kb between bropK47 and bropK43, located between C9:59999594 and 60239356 (Figure 3C).