The leaves of ZS11 have low chlorophyll content, and the leaves of QU have high chlorophyll content (Fig. 5). To obtain a relatively simple genetic background and to fine-map cqSPDA2, we constructed an NIL population. An F1 line with the QU genotype in the cqSPDA2 region was selected and backcrossed to ZS11 for three generations. BC3F1 individuals were selfed to generate a BC3F2 mapping population backcrossed to ZS11. The flanking markers Indel1 and Indel87 were used to construct the NIL population with foreground selection (Additional file 2: Table S2).
The BC4F1 individuals with a QU genetic background in the cqSPDA2 region as selected by the flanking markers Indel3 and Indel15 (Additional file 3: Fig. S1) were selfed to generate a BC4F2 population, which was used for fine mapping of the cqSPDA2 locus. The detailed process of population development is illustrated in Additional file 12: Fig. S4. BC4F2:3 individuals with the AA genotype (homozygous for cqSPDA2) and aa genotype (without cqSPDA2) were detected by the flanking markers Indel3 and Indel15 and subjected to qRT-PCR analysis. In addition, each population was grown in the experimental plots with a spacing of 30 cm (between rows) × 15 cm (within rows). The BC3F2 and BC4F2 populations were grown at the same density in fields in Yuanmou, Yunnan (altitude 898 m, 101°52′N, 25°42′E) and Xining, Qinghai (altitude 2225 m, 101°49′N, 36°34′E), respectively. The BC4F2:3 and BC6F1 populations were grown in a greenhouse at the Academy of Agricultural and Forestry Sciences, Qinghai University (Xining, Qinghai, China). Standard crop management practices were followed.
Phenotypic trait and data analysis
The testing targets included every plant of the population that was lacking diseases and insect pests. According to the previous method of chlorophyll determination, we measured three positions in the distal third of the first fully developed leaf from the top of each plant at the six-leaf stage by SPAD (SPAD 502, Japan) during the 9:00-11:00 am interval . Each measurement was repeated three times per leaf, and the veins were avoided. Statistical analysis was performed with Excel. Chi-square tests were performed on the segregation data to determine the genetic regulation of the chlorophyll content.
DNA extraction and development of molecular markers development
Total DNA was extracted from fresh leaves using the CTAB method . PCR was performed in a 20-μL reaction solution containing 2 μL DNA, 2 μL 2 mM dNTPs, 2 μL 10× PCR buffer, 1 μL Taq, 1 μL of 2 μM forward and reverse primers and 12 μL of ddH2O. The PCR program was carried out according to Yang’s method with minor modifications . The PCR products were separated on 6% nondenatured polyacrylamide gels and detected by silver staining . Indel (insertion/deletion) markers were developed from the resequencing data of the parents according to the B. napus ‘Darmor-bzh’ reference genome sequence in the primary mapping interval. SSR markers were developed based on the B. napus ‘Darmor-bzh’ reference genome sequence corresponding to the interval. The sequences of the SSR markers were designed using SSR Hunter 1.3 and Primer Premier 5.0 [43,44].
Mapping of the cqSPDA2 locus
The BC3F2 and BC4F2 family populations were used to fine map the cqSPDA2 locus using indel and SSR markers. First, we designed 87 indel markers in the primer mapping interval (21.87-22.91 Mb on chromosome A02) to map the cqSPDA2 locus. A linkage map for the cqSPDA2 locus was constructed using JoinMap 4.0 . The mapping interval for the cqSPDA2 locus was gradually reduced using the mapping results of the BC3F2 population. Finally, additional indel and SSR markers within the new narrow mapping interval were designed to fine-map the cqSPDA2 locus based on the BC4F2 population with WinQTLCart 2.5. The physical location was obtained by blasting the Brassica napus genome database using the indel and SSR sequences.. The physical linkage map was produced with MapDraw 2.1 .
The specific markers closely linked to cqSPDA2 were sequenced by NIL population scanning. Specific fragments were collected according to Yi et al. . The product was ligated into the pDM18-T vector (Takara), and the transformed clone was detected with M13 primers. Six positive clones were randomly selected and sequenced by Sangon Biotech (Shanghai) Co., Ltd. .
Genes in the mapping interval
All genes within the targeted mapping interval on A02 were identified using annotations from the Brassica napus genomes (http://www.genoscope.cns.fr/brassicanapus/) and annotated according to the BRAD annotations (http://brassicadb.org/brad/blastPage.php). The homologous sequences were aligned using BLASTN (http://blast.ncbi.nlm.nih.gov/).
RNA extraction and qRT-PCR analysis
BC4F2:3 (91 AA-genotype plants homozygous for cqSPDA2 and 104 aa-genotype plants without cqSPDA2) were grown in a greenhouse in September 2019. Total RNA was isolated from the first fully developed leaves counted from the top of each plant (at the 4-leaf stage, 6-leaf stage and squaring stage) of the BC4F2:3 population and the parental lines using TRNzol-A+ Total RNA Reagent (Takara, Dalian, China) according to the manufacturer’s protocol. RNA integrity was monitored using 1% agarose gel electrophoresis. cDNA was obtained via reverse transcription of total RNA using the PrimeScript RT Reagent Kit (Takara, Dalian, China) and following the manufacturer’s instructions.
We performed a qRT-PCR analysis to identify the genes in the mapping interval. Real-time PCR was conducted using LightCycler 480 II 96-Well PCR Plates (Roche, Rotkreuz, Switzerland). The utilized reaction system contained 10 μL of 2×SG Fast qPCR Master Mix (B639271, BBI), 2 μL cDNA, and 10 μM gene-specific primers in a final volume of 20 μL. The thermal cycling conditions used were 95 °C for 3 min, followed by 45 cycles at 95 °C for 5 s and 60 °C for 30 s followed by a final extension stage. The relative expression levels of all the genes in the mapping interval were calculated by the 2−△△Ct method based on the QU samples and three replicates were performed for each sample . The housekeeping gene Actin7 was used as the internal control to calculate the relative expression levels of each gene. Three biological replicates were performed in this experiment and t-test was used for statistical analysis.
Phenotyping for agronomic traits
To evaluate the agronomic efficiency of cqSPDA2, 100 individuals (50 AA- and 50 aa- genotype plants) from the BC4F2 population were sampled using the markers Indel3 and Indel15, and their chlorophyll content was characterized. The agronomic traits investigated were as follows: plant height (cm), total siliques per plant, silique length (cm), seeds per silique, 1000-grain weight (g), and yield per plant (g). The mean values, standard deviations and significance analyses of all the agronomic traits were compared between the AA- and aa-genotype plants by Minitab16 and Excel2010.