2.1 Materials
Dried seeds of A18-1 were treated with 150Gy 60Co-γ radiation to construct a snap bean mutant library. M3 generation plants were grown at the horticulture research base of Hulan campus, Heilongjiang University, Harbin, Heilongjiang, China. The purple phenotype was observed in the pv-pur mutant. In order to locate the PV-PUR mutant gene, we constructed the A18-1ⅹpv-pur F2 hybrid population for preliminary mapping, and the Golden hookⅹpv-pur F2 hybrid population for fine mapping. These F2 isolated populations were grown in the field at the horticulture research base in Hulan campus, Heilongjiang University. Columbia ecotype A. thaliana plants used for transgenic experiments were grown in a light incubator at the horticulture laboratory of Heilongjiang University, provided by BioRun Biotechnology Company (Wuhan, China).
2.2 Bulked-segregant analysis coupled to whole genome sequencing (BSA-seq) analysis
An F2 generation mapping population was constructed by crossing the purple mutant pv-pur with WT A18-1. In the F2 population, 50 plants with the same phenotype as WT plants were selected for DNA extraction, and the 50 DNA extracts were mixed in equal amounts to construct a mixed pool, which was denoted W type. Fifty plants with the same phenotype as the mutant were screened from the F2 population, and the 50 DNA extracts were mixed in equal amounts to construct a mixed pool, which was denoted M type. DNA was extracted from leaves of one mutant and one WT parent, and samples were denoted P_ mutant and P_ wild, respectively.
The library was constructed and sequenced by Hangzhou Lianchuan Biotechnology Co., Ltd (Hangzhou, China). After the sequencing data were acquired, quality control was carried out. After removing low-quality sequences and sequences with connectors, clean data were obtained. Clean data were compared with the reference genome, and SNPs and indels were detected and annotated according to the comparison results. The SNP index and difference value of hybrid pools were calculated, regions with significant differences in SNP index between the two offspring were selected, and target trait regions were located on chromosomes.
2.3 PV-PUR gene fine mapping
Construction of the F2 fine mapping population by hybridisation between ‘Golden Hook’ and purple mutant pv-pur plants. In total, 588 F2 plants segregating for the recessive phenotype were selected for fine mapping. According to the preliminary candidate regions identified by BSA-seq, SSR markers were designed for fine mapping of the PV-PUR gene. We downloaded candidate region sequences from the P. vulgaris v2.1 reference genome database. Primer Premier 6.0 software (Premier Biosoft International, Palo Alto, CA, USA) was used to design primers, which were sent to Genewiz Biotechnology Co., Ltd. for synthesis (Supplement Table 1). Primers were screened against the two parents for polymorphisms, and the selected primers were used to screen the F2 population. The number of recombinant bands was recorded for fine mapping. We performed PCR amplification in a 10 µL reaction comprising 1 µL (50 ng) of template DNA, 0.5 µL of 10 µM forward and reverse SSR primers, 5 µL of 2 × Rapid Taq Master mix, and 3 µL of DEPC (diethypyrocarbonate)-ddH2O. PCR was carried out on an iCycler thermocycler (Bio-Rad, Hercules, CA, USA) using the following reaction conditions: an initial denaturation at 95°C for 3 min, followed by 35 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s, and a final extension at 72°C for 5 min. A 5% denaturing polyacrylamide gel was then used to separate PCR products, followed by silver staining.
Table 1
Prediction of candidate genes within the gene-mapped region on Chr 06
Gene ID | Start | End | Gene annotations |
Phvul.006G018800.3 | 4328039 | 4334826 | K13083 - flavonoid 3',5'-hydroxylase (CYP75A) |
Phvul.006G018911.1 | 4347887 | 4349636 | PTHR23155//PTHR23155:SF560 - LEUCINE-RICH REPEAT-CONTAINING PROTEIN // SUBFAMILY NOT NAMED |
Phvul.006G019022.1 | 4349689 | 4351601 | PTHR23155//PTHR23155:SF560 - LEUCINE-RICH REPEAT-CONTAINING PROTEIN // SUBFAMILY NOT NAMED |
Phvul.006G018600.1 | 4382741 | 4385405 | PTHR11413//PTHR11413:SF50 - CYSTATIN FAMILY MEMBER // SUBFAMILY NOT NAMED |
Phvul.006G018500.1 | 4403804 | 4407407 | PTHR32468:SF17 - CATION/H(+) ANTIPORTER 10-RELATED |
Phvul.006G018400.1 | 4430330 | 4431271 | Unknown protein |
Phvul.006G018300.1 | 4442500 | 4447162 | PTHR32468:SF17 - CATION/H(+) ANTIPORTER 10-RELATED |
Phvul.006G018200.1 | 4476622 | 4478137 | PF00646//PF01344 - F-box domain (F-box) // Kelch motif (Kelch_1) |
Phvul.006G018100.1 | 4517068 | 4520052 | PF03168 - Late embryogenesis abundant protein (LEA_2) |
Linkage analysis data for mutant genes in the F2 population were integrated, and linkage relationships between SSR markers and mutant genes were analysed by MapChart software (Wageningen, Netherlands). Finally, TBtools software (Nanjing, China) was used to construct a genetic linkage map from polymorphic SSR markers and segregation data linked to genes.
2.4 Anthocyanin content determination
Separate 0.5 g samples of A18-1 and pv-pur pod walls were used for extraction of total anthocyanins using a mixture of methanol:hydrochloric acid:purified water (16:1:3), the absorbance was measured at 637 nm and 530 nm using a UV spectrophotometer, and the relative content of total anthocyanins was calculated according to the formula:
Relative content of anthocyanin = (A530–0.25 ⋅ A637) / fresh weight.
Levels of six anthocyanins (delphinidin, cyanidin, petunia, pelargonidin, peonidin and malvidin) in pod walls of A18-1 and pv-pur plants were determined by HPLC using the analytical method referred to in the agricultural industry standard of the People’s Republic of China (ny/t2640-2014).
2.5 Candidate gene cloning
The DNA reference sequences of candidate genes were searched against the database P. vulgaris v2.1 database (http://brassicadb.org/brad/downloadOverview.php), and specific primers were designed by Primer Premier 6.0 software (Supplement Table 2). DNA from A18-1 and pv-pur plants was used as template, and candidate gene-encoding regions were amplified by PCR. After the PCR products were separated by 1.5% agarose gel electrophoresis, target bands were excised using a UV Gel cutter and fragments were recovered with gel recovery reagent (Vazyme Biotech Co., Ltd). The recovered products were sent to Genewiz Biotechnology Co., Ltd. for sequencing. DNAMAN (LynnonBiosoft, USA) was used for sequence alignment.
2.6 Construction of PV-PUR gene expression vectors
Total RNA from tender leaves of pv-pur plants was extracted using a Plant Total RNA Extraction Kit (Tiangen, Beijing, China). First-strand cDNA was synthesised using a reverse transcription kit (Novozan, Nanjing, China). According to the above primer design (Supplement Table 3), PCR amplification was carried out with cDNA as template in 50 µL reactions. Agarose gel electrophoresis was performed, the recovered target band was ligated to rDNAg, the pBWA (V) HS-ccdb GLosgfp vector and rDNAg1 were digested, and their digested products were combined and purified using a PCR Purification Kit (the purified product was named P-rDNAg1) for the next step of the ligation reaction. The vector map is shown in Supplementary Fig. 1, and the vector was named PV-PUR-pBWA-GFP. The vector could be used as an overexpression vector for subsequent subcellular localisation and Arabidopsis genetic transformation.
2.6 Subcellular localisation of candidate genes
The leaf injection method was used for subcellular localisation of candidate genes. A small wound was made on the back of tobacco leaves with a needle. The PV-PUR-pBWA-GFP fusion expression vector and a suspension of pBWA-GFP were injected into the wound. After 3 days of dark culture, the intact epidermis of the injected wound was taken for microscopic observation.
2.7 Candidate gene expression analysis by qRT-PCR
The cDNA samples from stems, leaves, flowers, pods, hypocotyls and cotyledons of A18-1 and pv-pur plants were obtained as described above, and specific primers for candidate genes were designed by Primer Premier 6.0 (Supplement Table 4). A MY17295272 fluorescence quantitative PCR instrument (Agilent Technologies Inc. CA., USA) was used for qPCR experiments. Actin was used as the internal reference gene, the 2− ΔΔ CT method was used to calculate relative expression levels of genes (Hongjian et al. 2009), the least significant difference (LSD) test was used for single factor difference analysis, and GraphPad software (https://www.graphpad.com/scientific-software/prism/) was used for mapping. The reaction system for qPCR was 10 µL of 2×Fast qPCR Mix, 0.4 µL of forward and reverse primer (10 µM), 2 µL of cDNA and 7.2 µL of DEPC-ddH2O. Thermal cycling included an initial denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 15 s. Three technical replicates and three biological replicates were performed.
2.8 Genetic transformation of PV-PUR candidate genes
The PV-PUR-pBWA-GFP overexpression vector was transformed into Agrobacterium tumefaciens GV3101 by electric shock. Colonies were picked and grown to OD600 = 0.8−1.2, silwet-77 was added to a final concentration of 0.02%, Arabidopsis inflorescences were dipped in the suspension for 2−3 s, sealed with film and keep the humidity > 90%, and incubates at 25°C for 24 h. The soaking period was 7 days and three replicates were included. Genetic transformation of WT A. thaliana was performed by dipping flowers using the method of Zhang et al (2006). Hygromycin was used as a screening marker for T1 generation screening, and Arabidopsis genomic DNA was extracted by the CTAB (cetyltriethylammnonium bromide) method for PCR detection. Phenotype identification of homozygous transgenic A. thaliana lines was performed on T3 generation plants.