Fine mapping of the QTL cqSPDA2 for chlorophyll content in Brassica napus L

Abstract

Background: Chlorophyll is the most important factor enabling plants to absorb, transfer and transform light energy and plays an important role in yield formation. Brassica napus is one of the most important oil crops. Breeding Brassica napus for high light efficiency by improving photosynthetic efficiency has considerable social and economic value. In Brassica napus, there have been studies of the initial location of chlorophyll in seed embryos and pericarps, but there are few reports on the fine mapping of chlorophyll QTLs. We constructed near-isogenic lines (NIL), fine-mapped a chlorophyll locus, and evaluated the effect of this dominant locus on agronomic traits.

Results: The cqSPDA2 locus was mapped to an interval of 21.87-22.91 Mb on the chromosome A02 of Brassica napus using doubled haploid (DH) lines. To fine-map cqSPDA2, we built NIL and designed Indel primers covering the mapping interval. The 469 individuals in the BC₃F₂ population were analyzed using these indel primers. Among these indel primers, 15 could narrow the mapping interval to 188 kb between Indel3 and Indel15. Next, 16 indel primers and 19 SSR primers were designed within the new narrower mapping interval, and 5 of the primer-amplified fragments were found to be polymorphic and tightly linked to the cqSPDA2 locus in the BC₄F₂ population. The mapping interval was narrowed to 152 kb on A02 between SSR2 and Indel15. By gene expression analysis, we found three annotated genes in the mapping interval, including BnaA02g30260D, BnaA02g30290D and BnaA02g30310D, which may be responsible for chlorophyll synthesis.

Conclusions: The locus cqSPDA2, a dominant QTL for chlorophyll content in Brassica napus, was fine-mapped to a 21.89-22.04 Mb interval on A02_. Three annotated genes (BnaA02g30260D, BnaA02g30290D and BnaA02g30310D) that may be responsible for chlorophyll synthesis were found.

Keywords: Brassica napus; Chlorophyll content; Fine mapping; Near-isogenic line; qRT-PCR.

Fig. 1

Phenotypic frequency distribution of chlorophyll SPAD in the BC₄F₂ population. The lowest point of the column chart represents the dividing value (SPAD = 43). Leaves were considered to have high chlorophyll content(n = 1514) with a SPAD value≥43 and a low chlorophyll content(n = 547) with a SPAD value < 43. A chi-square test showed that the segregation pattern of the chlorophyll content trait was in keeping with the expected Mendelian segregation ratio of 3:1 (χ2 = 2.53)

Fig. 2

Genetic and physical maps of the cqSPDA2 gene locus and candidate gene analysis. a Genetic linkage map of the cqSPDA2 region on chromosome A02. The numbers between the markers indicate genetic distance in centimorgans. b Fine mapping of the cqSPDA2 locus in the BC₄F₂ population. The cqSPDA2 was narrowed down to a 152-kb interval between the markers SSR2 and Indel15. The numbers between the markers indicate physical distance. c Genetic and physical maps of the candidate genes in the targeted interval and the annotated genes in the Brassica napus genome annotation database (http://www.genoscope.cns.fr/brassicanapus/)

Fig. 3

Codominant marker and closely linked to cqSPDA2. The aa group (low-chlorophyll phenotype): the mean of SPAD = 40.0 ± 0.27; the Aa group (medium-chlorophyll phenotype): the mean of SPAD = 44.8 ± 1.44; the AA group (high chlorophyll phenotype): the mean of SPAD = 53.3 ± 0.80

Fig. 4

Differential expression of 3 genes in the mapping interval in the parents and NILs. The relative expression levels 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. Values shown are means ± SD (n = 3). Student’s t-test was used to compare QU with ZS11 and BC₄F_2:3 (AA) with BC₄F_2:3 (aa). * denotes significance at the probability level of 0.05. ** denotes significance at the probability level of 0.01

Fig. 5

Leaf color phenotypes of the parents QU and ZS11. QU: deep green leaves, high- chlorophyll content, SPAD value = 50.4; ZS11: light green leaves, low-chlorophyll content, SPAD value = 40.6