Leaf is the main photosynthetic organ, and chlorophyll content is an important agronomic trait for plant yield. Ninety to ninety-five percent of plant dry matter is produced by photosynthesis, and crop yield is primarily derived from the photosynthetic products of leaves . Chlorophyll is an important pigment involved in photosynthesis in chloroplasts, which can absorb and transform light energy. Chlorophyll is also an important index to evaluate the photosynthetic capacity of leaves . Increasing crop yield by increasing chlorophyll content is one of the important breeding objectives of high light efficiency breeding . Chlorophyll content is a quantitative characteristic that is primarily controlled by nuclear genes and has high heritability. At present, research on chlorophyll QTLs has been performed on various crops, such as rice [8, 10, 12, 13, 15], wheat [9, 11], beans [24, 25], and cabbage , especially rice . There have been studies on the location of chlorophyll in the embryo  and pericarp  of winter Brassica napus. Under drought and salt stress conditions, QTLs were also detected, and even a candidate gene related to salt tolerance was predicted [18–20]. However, there is no report on the fine location of the chlorophyll QTL locus in Brassica napus. This report is the first to describe fine mapping of cqSPDA2 on Brassica napus. In this study, the NILs of cqSPDA2 regions were constructed using recurrent parent ZS11 with flanked markers. Indel and SSR markers were used to scan the populations of BC3F2 and BC4F2, and the mapping interval was reduced to 152 kb. This strategy for mapping the target genes is reasonable, inexpensive, and highly efficient. According to the BC4F2 phenotype and BC6F1 genotype analysis, the strategy is consistent with the Chi-square test. The results indicated that cqSPDA2 is the main QTL locus. A positive correlation between cqSPDA2 and agronomic characteristics, such as yield, was determined through the analysis of NIL.
BnaA02g30260D, BnaA02g30290D and BnaA02g30310D were identified as good candidate genes of qSPADA2 among the twenty-nine genes annotated from Brassica napus genomes in the mapping interval according to the qRT-PCR analysis. BnaA02g30260D, which is a disease-resistance protein family, has transmembrane receptor activity, nucleoside-triphosphatase activity, nucleotide binding, and ATP binding function and is involved in signal transduction, defense response, apoptosis, and innate immune response according to the annotations. Further study is needed to determine whether BnaA02g30260D affects chlorophyll synthesis. BnaA02g30290D is FK506- and rapamycin-binding protein 15 kD-2 (FKBP15-2) and has peptidyl-prolyl cis-trans isomerase activity related to protein folding. Luan et al.  found that AtFKBP15-1 and AtFKBP15-2 had the highest homology to FKBP13 and encoded functional homologs of FKBP13. AtFKBP13 was reported to be associated with Rieske protein, both before and after the import of the proteins into the chloroplast stroma. AtFKBP13 can play a role in the downregulation of Rieske protein accumulation. Rieske is a subunit of the cytochrome b6f complex, which is one of the four complexes of the photosynthetic electron transport chain . It was also reported that ScFKBP12 was transferred into Arabidopsis, chloroplast formation was inhibited and the expression of genes related to chloroplast formation was inhibited . In this study, the expression levels of BnaA02g30290D (AtFKBP15-2) in NILs (aa) and ZS11 were all higher than those in NILs (AA) and QU at the three stages, which inhibited the formation of chlorophyll and was consistent with the above mentioned results. BnaA02g30310D is homologous to GCH-1 in Arabidopsis thaliana. GCH-1 is the first enzyme for tetrahydrobiopterin (BH4) biosynthesis . BH4 is an essential coenzyme for all three kinds of nitric oxide synthase (NOS) . AtNOA1 (AtNOS1) is located in Arabidopsis chloroplasts, and OsNOA1 (OsNOS1) is also located in rice chloroplasts [31–33]. Yang et al.  found that the chlorophyll content decreased with increasing OsNOA1 at a low temperature (22 °C). He  suggested that OsNOA1 directly regulates the chloroplast self-coding protein by affecting the function of the chloroplast ribosome and then transmits the signal to the nucleus through the chloroplast retrograde signal pathway mediated by Mg protoporphyrin IX, further affecting the expression of chloroplast protein encoded by the nuclear gene. Qinghai Province is located in the Qinghai-Tibet Plateau. The average temperature during crop growth is lower, and the expression level of BnaA02g30310D (GCH-1) in NILs (aa) and ZS11 was more strongly expressed at three stages in the study, especially at the six-leaf stage, which is consistent with the results obtained by He [33, 34].
In addition, more research, such as a transgenic complementary test, CRISPR/Cas9, VIGS and RNAi, is warranted to examine whether BnaA02g30260D, BnaA02g30290D and BnaA02g30310D are the target genes of cqSPDA2. Analysis of the regulatory network for chlorophyll synthesis will facilitate Brassica napus molecular breeding for high yield.