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.