The pepper inbred line BB3 (C. annuum) and its wild relative Chiltepin (C. annuum var. glabriusculum) were used as the female and male parents, respectively, to construct a backcross population (BC1F1, n = 225) with BB3 as the recurrent parent to verify the UP12.1 interval previously detected based on the BA3 (C. annuum) × YNXML (C. frutescens) F2 population (Cheng et al. 2016a). The BB3, Chiltepin, and their F1 and BC1F1 population were grown at SCAU Main Campus Teaching & Research Base, Guangzhou, China (23°N, 113°E).
A total of 1,827 BC1F2 individuals were generated by artificial self-pollination with the heterozygous individuals from the BC1F1 population to narrow down the up candidate interval. Fruit orientation phenotypes were evaluated with at least five flowers/fruits were recorded from stage S3 to S7 (Fig. 1) for each plant based on the ELV (E: erect, LP: lateral pendant, VP: vertical pendant) classification method as described previously (Cheng et al. 2016a).
Insertion and deletion (InDel) loci were identified based on sequence comparisons between the reference genome sequences of Zunla-1 and Chiltepin (Qin et al. 2014). Primer pairs flanking the InDel loci were designed by using Primer3web (version 4.1.0; http://primer3.ut.ee/). DNA was extracted from young leaves by using the modified CTAB (Murray and Thompson 1980) and then preserved at − 20°C before genotyping. The PCR was performed using a 20 µL reaction mixture, which contains 2.0 µL DNA template (50 ng/µL), 2.0 µL PCR buffer (10×), 2.0 µL Mg2+ (25 mM), 1.5 µL forward and reverse primer (1 µM), 0.2 µL dNTPs (10 mM), and 1U Taq DNA polymerase. PCR reactions were performed as follow: 94°C for 3 min, 32 cycles of 94°C for 30 s, 55°C for 30 s, and 1 min at 72°C; and a final extension at 72°C for 10 min. PCR products were genotyped using 6% polyacrylamide gel electrophoresis.
The primer sequences used for candidate gene cloning were listed in Supplementary Table S1. The PCR amplicons of candidate genes were ligated to the pMD-19T cloning vector (Takara, Tokyo, Japan). At least three randomly selected positive colonies for each amplicon were sequenced and assembled. Nucleotides and amino acid sequences were aligned using DNAMAN (version 9).
For comparative expression analysis, the mixed samples comprised of the flower buds (white arrow 1), main stem (white arrow 2) close to the first branching point and the pedicel (white arrow 3) were excised as a whole at four developmental stages including S1 to S4 (Fig. 1), respectively, for RNA isolation. To characterize the expression profile of Capana12g000958, the flower bud/fruit and pedicel samples at seven developmental stages (S1 to S7, as described in Fig. 5) were excised separately from the BB3, Chiltepin, and BB3×Chiltepin (F1), respectively, for RNA isolation. Every sample was collected in three biological replicates, wrapped in tin foil, frozen directly in liquid nitrogen, and then stored at − 80°C for subsequent experiments.
Total RNA was isolated by using the Eastep® Super Total RNA Extraction Kit (LS1040; Promega, Madison, America) following the manufacturer's instructions. First-strand cDNA was synthesized by using a cDNA synthesis kit (Takara, Tokyo, Japan). qRT-PCR was performed in triplicate by using SYBR Green PCR master mix (Takara, Tokyo, Japan) on a BioRad CFX96 system (Bio-Rad, CA, USA). The primer sequences of the candidate genes and Ca-Actin were listed in Supplementary Table S2. PCR was performed with an initial denaturation step set at 94°C for 3 min, followed by 39 cycles of denaturation and annealing at 94°C for 10 s and 55°C for 30 s, respectively. The relative expression level was analyzed using the 2−∆∆CT method (Livak and Schmittgen 2001). All reactions were performed in triplicate with three biological replicates for each sample.