The application of the KASP markers
The fine mapping results showed 6 KASP markers (bropsK49, bropsK18, bropsK51, bropsK52, bropsK42, bropsK43) were closely associated with the target gene and, possibly any one of them could be in the target gene region. These 6 markers were used to screen 92 individuals of the F2 segregation population derived from the mutant GS and the wild line PS. The genotyping results of 5 KASP markers (bropsK18, bropsK51, bropsK52, bropsK42 and bropsK43) were in agreement with the phenotypic data by more than 98%, while the genotyping result of bropsK49 was in 100% accordance with the phenotypic data (Figure 3D). It showed that the six KASP markers based on C9: 59999594 to 60239356 could perform well in genotyping for each plant in the new population (Figure 3D).  Based on the genotypic and phenotypic data of the F2 individuals, the range of genomic location of bud-color at low temperature was further narrowed down to 229.649Kb, located between bropK47 and bropK18, i.e., C9:59999594 ~ 60229243, and the gene where bropsK49 locates could probably be the target gene.
Also, the 6 KASP markers were used to screen forty-two germplasm materials in Brassica oleracea L. (Figure 3E). Out of the 6 KASP markers, the genotyping data of two KASP markers (bropsK18 and bropsK49) matched more than 95% with the phenotyping data and were effective in predicting whether the buds would turn purple or keep green in cold temperature in Brassica oleraceaL. Within the 42 germplasm lines, 40 lines showed consistent results for both bropsK18 and bropsK49 with the genotypic data in conformity with the phenotyping, whereas 2 lines (1947 and K2145), which would keep green buds at low temperature, lack a 43-bp deletion in bropsK49 where there was a 43-bp InDel, and exhibited inconsistent results between genotyping and phenotyping.