Mapping of the keeping-green trait at cold temperature (KGLT) of the buds in broccoli
HX-16 and HX-11, whose buds would keep green and turn purple in cold temperature, respectively, were chosen to construct a BC2F2 segregating population to map the trait (Figure 1F, G, H). Bulked segregant analysis (BSA) was used to map the target region of the trait. In the BC2F2 segregating population with 987 individuals, 252 and 735 plants exhibited green buds and turned purple at low temperature, respectively, which confirmed the segregation ratio of 1:3. It indicated that a single locus controls KGLT.
From the BC2F2 segregating population, 30 KGLT and 30 TPLT individuals were utilized to analyze the locus linked to KGLT/TPLT buds by BSA-seq after experiencing low temperature (ranging from 0-50C). Genomic DNA was sequenced by the Illumina NovaSeq platform, and 63.86 G of clean data were obtained (Table S1). Low N rates (0) and high scores of Q20 (96.66-98.13) and Q30 (91.06-94.51) in table S1 showed that the reads obtained were of high quality. High-quality data were produced by filtering raw data, and more than 99.34%-99.59% of the data were mapped to the reference genome HDEM (http://www.genoscope.cns.fr/externe/plants/chromosomes.html ) (Table S2). The average sequencing depths of maternal, paternal, keeping-green, and turning-purple pools were 22.58, 22.87, 27.30, and 27.82, respectively, and the mapping coverage ranged from 86.50%-89.03% (Table S3). In the parental lines, 2334791 homozygous SNPs same as the reference genome were called (ref), and 3120872 homozygous SNPs were different from the reference genome (alt) (Table S4). Similarly, 322714 homozygous InDels, same as the reference genome and 615872 homozygous InDels, different from the reference genome were called in the parental lines (Table S5). The SNP-index distribution in the genome of the G-pool and P-pool was analyzed and shown in Figure 2A and 2B. The subtraction of the two SNP indexes produced Delta (Δ) SNP index (Figure 2C). According to the Δ SNP index value, the peak area above the 99% confidence coefficient was chosen as the candidate region associated with the target trait. Therefore, the 4.6Mb of area between 58,700,000 and 63,300,000 bp on chromosome 9 was identified as the target region for the KGLT/TPLT at cold temperature.