Effect of structural variation in the promoter region of RsMYB1.1 on the skin color of radish taproot

Abstract

Accumulation of anthocyanins in the taproot of radish is an agronomic trait beneficial for human health. Several genetic loci are related to a red skin or flesh color of radish, however, the functional divergence of candidate genes between non-red and red radishes has not been investigated. Here, we report that a novel genetic locus on the R2 chromosome, where RsMYB1.1 is located, is associated with the red color of the skin of radish taproot. A genome-wide association study (GWAS) of 66 non-red-skinned (nR) and 34 red-skinned (R) radish accessions identified three nonsynonymous single nucleotide polymorphisms (SNPs) in the third exon of RsMYB1.1. Although the genotypes of SNP loci differed between the nR and R radishes, no functional difference in the RsMYB1.1 proteins of nR and R radishes in their physical interaction with RsTT8 was detected by yeast-two hybrid assay or in anthocyanin accumulation in tobacco and radish leaves coexpressing RsMYB1.1 and RsTT8. By contrast, insertion- or deletion-based GWAS revealed that one large AT-rich low-complexity sequence of 1.3-2 kb was inserted in the promoter region of RsMYB1.1 in the nR radishes (RsMYB1.1^nR), whereas the R radishes had no such insertion; this represents a presence/absence variation (PAV). This insertion sequence (RsIS) was radish specific and distributed among the nine chromosomes of Raphanus genomes. Despite the extremely low transcription level of RsMYB1.1^nR in the nR radishes, the inactive RsMYB1.1^nR promoter could be functionally restored by deletion of the RsIS. The results of a transient expression assay using radish root sections suggested that the RsIS negatively regulates the expression of RsMYB1.1^nR, resulting in the downregulation of anthocyanin biosynthesis genes, including RsCHS, RsDFR, and RsANS, in the nR radishes. This work provides the first evidence of the involvement of PAV in an agronomic trait of radish.

Keywords: GWAS; RsMYB1.1; anthocyanin; presence/absence variation; promoter; radish; red skin color.

Figure 1

Identification of genomic loci associated with the taproot skin color of radish by GWAS. (A) Photographs of selected red-skinned radish accessions in the core collection. (B) Manhattan and Q-Q plots of SNP (upper panel) and InDel (lower panel) GWAS. Solid and dotted lines in Manhattan plots depict thresholds for significant and suggestive, respectively. (C) Variation in the candidate genome region on the R2 chromosome. Triangles represent SNP (red) and InDel (green) variation.

Figure 2

Genotyping of nonsynonymous SNP alleles in the third exon of R2.009390. The target regions of three SNP alleles showing nonsynonymous changes between the nR radish WK10039 and the R radish WK10024 were amplified by PCR and genotyped using the MassARRAY markers. (A) Nucleotide and amino acid sequences of SNP alleles. Arrows indicate primers for MassAARY genotyping. (B) MassARRAY spectrograms of WK10039 (left) and WK10024 (right). Peaks in the spectrograms represent nucleotides extending from the target alleles.

Figure 3

The PAV in the promoter region of RsMYB1.1. (A) The 1.3 kb RsIS is inserted 1.4 kb upstream from the start codon of RsMYB1.1 of WK10039; the corresponding region of WK10024 has no RsIS. Arrows indicate primers for PCR amplification. Numbers are upstream positions from the start codon (+1). (B) Agarose gel electrophoresis of PCR amplicons from the promoter region of RsMYB1.1 of radish accessions. Only the non-red-skinned (nR) radishes have RsIS insertions with length variation. The positions and sequences of nonsynonymous SNP alleles for each accession are also shown.

Figure 4

Expression levels of the MBW complex (RsMYB1.1, RsTT8, and RsTTG1) and selected structural genes (RsCHS, RsDFR, and RsANS) of the anthocyanin biosynthesis pathway in the radish accessions as determined by qPCR. Root tissues were harvested 42 days after germination. Comparative cycle threshold (2^–ΔΔCt) values represent relative expression calculated using the CUR076 (RsMYB1.1) and WK10024 (other genes) samples as a reference. Error bars depict the standard deviations of three independent biological replicates. Asterisks represent statistical significance (^**p < 0.01, ^***p < 0.001) between nR and R radishes by t-test.

Figure 5

Pairwise interaction between RsMYB1.1 and RsTT8 in the yeast two-hybrid (Y2H) assay. The prey vector pGADT7 (AD) and the bait vector pGBKT7 (BD) are Y2H vectors without an insert. RsMYB1.1 and RsTT8 of WK10039 (nR) and CUR034 (R) were cloned into pGADT7 and pGBKT7. PBN204 yeast was cotransformed with the bait (BD) and prey (AD) vectors. Interactions between the two proteins were identified in both cases using RsMYB1.1 as the bait and RsTT8 as the prey (A) and RsMYB1.1 as the prey and RsTT8 as the bait (B).

Figure 6

Transient expression of RsMYB1.1 and RsTT8 in tobacco leaves. (A) RsMYB1.1 and RsTT8 of WK10039 (nR) and CUR034 (R) under the control of the 35S promoter were expressed individually or coexpressed in N. benthamiana leaves. Photographs were obtained 5 days after Agrobacterium infiltration. (B) Total anthocyanin levels in Agrobacterium-infiltrated tobacco leaves. Error bars depict the standard deviations of three independent biological replicates.

Figure 7

Histochemical analysis of transient GUS expression in radish taproot section. (A) Schematics of RsMYB1.1 promoter::GUS fusion constructs. P1 to P6 are successive deletion constructs for pRsMYB1.1^nR of WK10039. P7 is a construct for pRsMYB1.1^R of CUR034. The white box is the RsIS in pRsMYB1.1 of WK10039 and the gray boxes are the GUS coding regions. ATG is the start codon. Numbers are upstream positions from the start codon. (B) Photographs of GUS-stained WK10039 taproot sections obtained 3 days after Agrobacterium infiltration. GUS staining was performed for 24 h. (C) GUS activity expressed as nmol 4-methyl-umbelliferone min^–1 mg^–1 soluble protein. Error bars depict the standard deviations of three independent biological replicates.