QTL-Seq and Sequence Assembly Rapidly Mapped the Gene BrMYBL2.1 for the Purple Trait in Brassica rapa

Abstract

Anthocyanins have strong antioxidant activity and are believed to be healthy for human beings. The Brassica rapa L. ssp. chinensis var. purpurea "Zicaitai" is rich in anthocyanins. We constructed an F₂ population of Zicaitai and "Caixin" (Brassica rapa ssp. parachinensis) and it shows clear segregation of the purple phenotype (i.e., variation in anthocyanin enrichment). Here, quantitative trait locus (QTL)-Seq was performed with two sample groups from the F₂ population: one exhibiting an intense purple phenotype and the other showed a completely green phenotype. The results showed that the QTL-Seq and linkage analysis located different major loci. This indicates that there are two major genetic factors that plays different roles in regulating anthocyanin enrichment in Zicaitai. This was further supported by the data simulation of an in silico F₂ population that QTL-Seq and linkage analysis can locate different major loci. Furthermore, the draft genomes of the two parents (Zicaitai and Caixin) were assembled and utilized to search for mutations in candidate genes. A ~100-bp insertion was found in the third exon of gene BrMYBL2.1 in Zicaitai. BrMYBL2.1 is a negative regulator of anthocyanin biosynthesis, while BrEGL3.2-previously located by linkage mapping-is a positive regulator. For these populations with multiple genes contributing large effects to a trait, a strategy of low depth re-sequencing of F₂ individuals followed by QTL-Seq analysis with the free combination of sample groups is proposed. Furthermore, draft-sequence assembly of parental genomes together with QTL mapping is suggested as an efficient means for fine-mapping genes rapidly in segregating populations.

Figure 1

Major QTL loci of anthocyanin enrichment determined from an F₂ population of Zicantai/Caixin. (a) QTL-Seq located at the major QTL locus at chromosome A07; the grey points are the Δ(SNP-index) of each SNP, the purple points are the average values of the Δ(SNP-index) in a 200-kb sliding window with a 20-kb increment across all chromosomes, and the red lines show the confidence intervals of the Δ(SNP-index). (b) The LOD scores generated by linkage analysis with the MEM algorithm using software MapQTL6.0. The major QTL locus was located at chromosome A09.

Figure 2

QTL mappings in a simulated population with 200 F₂ individuals. (a) The inheritance of parental genomic fragments in each of the F₂ individuals; green fragments are from parent one, orange fragments are from parent two, while grey fragments are heterozygous genotype from both parents. (b) QTL–Seq analysis located the major QTL locus at chromosome A04. The colors denote similar objects as that in Fig. 1. The six black triangles denote the locations of the simulated loci. The bigger triangle at chromosome A04 is epistatic to the other loci. (c) Linkage analysis mapped the major QTL locus at chromosome A02.

Figure 3

A large sequence insertion was found in gene BrMYBL2 in Zicaitai. (a) The depth the reads covered on each nucleotide around the genomic region of gene BrMYBL2; purple points denote the reads depth from the sequencing data of purple pool in the QTL-Seq analysis, while green points denote reads depth from that of the green pool. (b) The gene model of BrMYBL2 and the positions of a sequence insertion and four non-synonymous mutations in the third coding exon of this gene in Zicaitai.

Figure 4

Experimental verification of a sequence insertion in gene BrMYBL2. (a) The electrophoretogram of the PCR products of the target sequence that contains the insertion variant. Three replicates for both Caixin and Zicaitai were labeled as one to three. (b) The alignment of the target sequences from the Chiifu reference genome (Chiifu_V3.0), draft sequence assembly of Caixin (Caixin-ass) and Zicaitai (Zicaitai-ass), and Sanger sequencing results (Caixin-F and Caixin-R, Zicaitai-F and Zicaitai-R, F denotes forward sequence, R denotes reverse sequence). The red color denotes the inserted sequence.