The 18Q2513 female parent (P1) is an ornamental kale inbred line with lobed leaves (Fig. 1a); the 18Q2515 male parent (P2) is an ornamental kale inbred line with unlobed leaves (Fig. 1b). 18Q2513 was crossed with 18Q2515 to generate an F1 population. An F2 population was generated from self-pollination of the F1 plants; BC1P1 and BC1P2 were then generated by BCs of F1 × 18Q2513, F1 ×18Q2515, respectively.
Additionally, 118 different cabbage inbred lines (with unlobed leaves) and another ornamental kale inbred line 2523 (with lobed leaves), were screened for BoLl-1 promoter variations. All of the plant materials used in the present study were grown in a greenhouse under normal management. All the plant materials are from the Institute of Vegetables and Flowers, Chinese Academy of Agriculture Sciences (IVFCAAS, Beijing, China).
Genetic analysis and whole-genome resequencing
Leaf shape was investigated visually. Segregation ratios for the F2 and BC1 populations were analyzed by chi-square (χ2) tests using SAS software.
Fifty lobed-leaf BC1 and fifty unlobed-leaf BC1 individuals were selected to construct two bulks. The two bulks and two parental lines were then used to construct paired-end sequencing libraries, which were subsequently sequenced by the Beijing Genomics Institute (BGI) (Shenzhen, China). SNP-index and sliding-window analyses were performed as previously described .
Marker development and fine mapping of the BoLl-1 gene
InDel and SNP markers were designed based on candidate region resequencing data for the two parents. Markers were designed with amplicon lengths of 100-180 bp, GC contents of 40-50% and Tm values of 52-58°C. The markers that were polymorphic between the parents were then used to analyze unlobed-leaf individuals in the BC1P1 populations.
Genomic DNA was extracted from young leaves of the parents and BC1P1 individuals using a modified cetyltrimethylammonium bromide (CTAB) protocol . The DNA concentration was subsequently determined using a spectrophotometer (BioDrop, UK) and adjusted to 40-50 ng/μL.
The 10-μL PCR reaction mixture consisted of 2 µL DNA template, 1 µL 10× PCR buffer (Mg2+ included), 0.8 µL dNTPs (2.5 mM each), 0.4 µL forward primer (10 µM), 0.4 µL reverse primer (10 µM), 0.2 µL Taq DNA polymerase (5 U/µL), and 5.2 µL ddH2O. The reactions were performed in accordance with the follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 45 s; and then 72°C for 10 min. The amplicons were separated by 8% polyacrylamide gel electrophoresis (160 V for 1.2 h), and the gel was stained with silver nitrate.
For each marker, individuals consistent with the 18Q2513 (lobed-leaf) allele, the 18Q2515 (unlobed-leaf) allele, and the F1 allele were categorized as ‘a’, ‘b’, and ‘h’, respectively. Genetic distances between markers were calculated by the Kosambi map function , and a genetic map was constructed using MapDraw .
Candidate gene analysis
To identify the lobed-leaf gene BoLl-1, genes located within the candidate interval were analyzed based on annotations for the B. oleracea ‘TO1000’ reference genome (http://plants.ensembl.org/Brassica_oleracea/Info/Index) . The expression patterns of candidate genes Bo9g181710 and Bo9g181720 were investigated using quantitative real-time PCR (qRT-PCR). Total RNA was extracted from young leaves of the parents using TRIzol reagent (Invitrogen, United States) according to the manufacturer’s protocol, and PrimeScriptTM RT Reagent Kit (Takara, Japan) was used to reverse transcribe cDNA from the total RNA extracted. qRT-PCR was carried out using a CFX96 Real-Time System (Bio-Rad) with SYBR Premix Ex TaqII Reagent Kit (Takara, Japan). The relative expression level of each gene was calculated using the 2−ΔΔCt method . The qRT-PCR primers used are listed in Table S2, and B. oleraceaactin was employed as a control.
Gene-specific markers GL10 (primers GL10-F and GL10-R) and GL20 (primers GL20-F and GL20-R) (Table S2) were used to amplify the promoter and genomic sequences of Bo9g181710 and Bo9g181720, respectively. The resulting PCR products were analyzed by electrophoresis on 1% agarose gels, followed by sequencing and alignment. The co-dominant marker CMLMI1 (primers CMLMI1-F1, CMLMI1-F2 and CMLMI1-R) and the derived cleaved amplified polymorphic sequence (dCAPS) marker DMLMI1 (primers DMLMI1-F and DMLMI1-R) (Table S2) were used to detect variations in the promoter of BoLMI1 in 118 different cabbage and ornamental kale inbred lines.
BLASTP searches were conducted using the amino acid sequence of BoLMI1 to search for homologues within the protein database of the National Center for Biotechnology Information (NCBI) and the B. oleracea reference genome ‘TO1000’. Protein sequence alignment was performed with MAFFT (v7.037) . FastTree (LG + JTT model) was used to construct phylogenetic trees .