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. 2022 Mar 4;12(3):402.
doi: 10.3390/biom12030402.

Fine Mapping and Characterization of a Major Gene Responsible for Chlorophyll Biosynthesis in Brassica napus L

Chengke Pang  1   2 , Wei Zhang  2 , Menlu Peng  1   2 , Xiaozhen Zhao  1   2 , Rui Shi  1   2 , Xu Wu  2   3 , Feng Chen  2 , Chengming Sun  2 , Xiaodong Wang  2 , Jiefu Zhang  1   3
Affiliations

Fine Mapping and Characterization of a Major Gene Responsible for Chlorophyll Biosynthesis in Brassica napus L

Chengke Pang et al. Biomolecules. .

Abstract

Rapeseed (Brassica napus L.) is mainly used for oil production and industrial purposes. A high photosynthetic efficiency is the premise of a high yield capable of meeting people's various demands. Chlorophyll-deficient mutants are ideal materials for studying chlorophyll biosynthesis and photosynthesis. In a previous study, we obtained the mutant yl1 for leaf yellowing throughout the growth period by ethyl methanesulfonate mutagenesis of B. napus. A genetic analysis showed that the yl1 chlorophyll-deficient phenotype was controlled by one incompletely dominant gene, which was mapped on chromosome A03 by a quantitative trait loci sequencing analysis and designated as BnA03.Chd in this study. We constructed an F2 population containing 5256 individuals to clone BnA03.Chd. Finally, BnA03.Chd was fine-mapped to a 304.7 kb interval of the B. napus 'ZS11' genome containing 58 annotated genes. Functional annotation, transcriptome, and sequence variation analyses confirmed that BnaA03g0054400ZS, a homolog of AT5G13630, was the most likely candidate gene. BnaA03g0054400ZS encodes the H subunit of Mg-chelatase. A sequence analysis revealed a single-nucleotide polymorphism (SNP), causing an amino-acid substitution from glutamic acid to lysine (Glu1349Lys). In addition, the molecular marker BnaYL1 was developed based on the SNP of BnA03.Chd, which perfectly cosegregated with the chlorophyll-deficient phenotype in two different F2 populations. Our results provide insight into the molecular mechanism underlying chlorophyll synthesis in B. napus.

Keywords: Brassica napus; candidate gene; chlorophyll deficiency; fine mapping; molecular marker; quantitative trait loci sequencing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Phenotypic expression of mutant yl1 over the entire growth period. (A) The phenotype of NY18 and yl1 in seedling stage; (B) leaf color of NY18 and yl1; (C) the phenotype in budding stage; (D) the color of flower buds in NY18 and yl1; (E) the size of petals; (F) the color and size of siliques.
Figure 2
Figure 2
SNP-index and Δ(SNP-index) graphs from the QTL-Seq analysis. SNP-index of (A) Y-pool (homozygous mutant leaves) and (B) G-pool (wild-type leaves); (C) Δ(SNP-index) of Y-pool and G-pool. The BnA03.Chd on chromosome A03 was regarded as the candidate interval, with an α = 0.05 significance level.
Figure 3
Figure 3
Fine mapping of the BnA03.Chd gene in (Holly × yl1) F2 population. (A) The BnA03.Chd locus was fine-mapped in a 304.7 kb interval containing 58 genes using 1524 chlorophyll-deficient individuals. (B) The BnaA03G0054400ZS was the final candidate gene and had an SNP (G to A) of the gene. (C) The SNP caused the change of glutamic acid at 1349th position to lysine.
Figure 4
Figure 4
(A) Differentially expressed genes (DEGs) between yl1 and NY18. (B) The expression level of genes involved in chlorophyll biosynthesis pathway. (C) KEGG pathway categories of differentially expressed genes between NY18 and yl1 at leaves from the seedling stage. The size of the point represents the number of genes, and the color of the point represents the degree of enrichment.
Figure 5
Figure 5
Sequence alignment of the coding sequence of BnaA03G0054400ZS gene between NY18 and yl1. There was a base substitution (G to A) at 4045 bp of the CDS in the fifth exon in yl1.
Figure 6
Figure 6
The expression level of BnaA03G0054400ZS in leaves, stems, pods, and flower buds of NY18 and yl1, as determined by quantitative real-time PCR (qRT-PCR). The values are presented as the mean ± SD (n = 3). The statistical analyses in this study were performed using one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) test.
Figure 7
Figure 7
Genotyping results using marker BnaYL1 in (A) (ZS11 × yl1) F2 and (B) (NY18 × yl1) F2 segregation populations. The blue dot represents homozygous alleles derived from parent lines with normal chlorophyll content, the green dot represents homozygous alleles derived from parent line “yl1” with low chlorophyll content, and the red dot represents heterozygous loci. The grey dot represents negative control.
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