Development of Molecular Markers for Predicting Radish (Raphanus sativus) Flesh Color Based on Polymorphisms in the RsTT8 Gene

Abstract

Red radish (Raphanus sativus L.) cultivars are a rich source of health-promoting anthocyanins and are considered a potential source of natural colorants used in the cosmetic industry. However, the development of red radish cultivars via conventional breeding is very difficult, given the unusual inheritance of the anthocyanin accumulation trait in radishes. Therefore, molecular markers linked with radish color are needed to facilitate radish breeding. Here, we characterized the RsTT8 gene isolated from four radish genotypes with different skin and flesh colors. Sequence analysis of RsTT8 revealed a large number of polymorphisms, including insertion/deletions (InDels), single nucleotide polymorphisms (SNPs), and simple sequence repeats (SSRs), between the red-fleshed and white-fleshed radish cultivars. To develop molecular markers on the basis of these polymorphisms for discriminating between radish genotypes with different colored flesh tissues, we designed four primer sets specific to the RsTT8 promoter, InDel, SSR, and WD40/acidic domain (WD/AD), and tested these primers on a diverse collection of radish lines. Except for the SSR-specific primer set, all primer sets successfully discriminated between red-fleshed and white-fleshed radish lines. Thus, we developed three molecular markers that can be efficiently used for breeding red-fleshed radish cultivars.

Keywords: InDels; RsTT8; SNPs; flesh color; molecular marker.

Figure 1

Four radish samples used in this study: green skin/red flesh (GsRf), red skin/red flesh (RsRf), white skin/white flesh (WsWf), and red skin/white flesh (RsWf) with inbred number (bottom). (A) Anthocyanin contents measured with 35-day-old samples. Real photos are on top. (B) Photographs taken at the harvest period.

Figure 2

Presence (A) and expression (B) of genes related to anthocyanin accumulation in four radishes. (A) All genes were amplified by genomic DNA PCR. (B) Expression levels of genes. L, leaves, H, hypocotyls, R, roots, s, skin, and f, flesh. Asterisks indicated that expression levels were tightly related to anthocyanin accumulation.

Figure 3

Schematic representation of genomic DNA of RsTT8 genes with primer position for cloning (A) and genomic organization of cloned RsTT8 genes (B). (A) Exon and intron are represented by box and solid line, respectively. I and E indicate intron and exon, respectively. Arrows indicated primer position and direction with forward (F) and reverse (R). (B) Genomic organization of cloned RsTT8 genes with promoter regions and 7 exons (box) and 6 introns (solid line). Numbers indicated nucleotide sequence (bp), and InDel and SSR regions were also marked. MRE, MYB-recognition motif; BRE. bHLH-recognition motif. Arrows named pBamHI and pNcoI indicated primers for promoter::GUS construction. Four boxes of dotted red lines indicate regions used in marker validation: PS-P, region amplified by promoter-specific primer set (−979~−818) (Rf-P/Wf-P in Table S1); ID-P, region amplified by 14 bp InDel-discriminating primer set; SSR-P, region amplified by TC SSR including primer set; WD/AD-P, region amplified by primer set that discriminated one CAT InDel and three SNPs (Rf/Wf-P in Table S1).

Figure 4

Phylogenetic relationship among RsTT8 alleles. Numbers in parenthesis indicate nucleotides of each allele.

Figure 5

Alignment of amino acids of RsTT8 from four radishes used in this study. MIR (MYB-interacting domain), WD/AD (WDR interacting region through the acidic domain), bHLH region, and ACT-like domain were well conserved among RsTT8s, indicating that resulting proteins keep their activity. Regulation may depend on the expression level. Numbers on the right denoted the number of amino acid residues.

Figure 6

Promoter activity assay by GUS stain and RT-PCR.

Figure 7

Development of PCR-markers derived from polymorphisms (SSR, InDel, and SNPs). (TC)-SSRs were present in the 2nd intron ranging from (TC)₁₃ to (TC)₆₈. InDel was 14 nucleotide insertion in Wf gene and present at the 35th nucleotide of ATG upstream. Rf-Sp and Wf-Sp indicated that Rf-specific and Wf-specific amplification, respectively, which was designed using CAT InDel in 5th intron and SNP in exon 6. Rf-P/Wf-P indicated SNPs presented in the promoter regions between −979 to −818 nucleotide from ATG start codon. Primer sequences were listed in Table S1. Numbers on the right denoted size of band (bp).

Figure 8

Validation of PCR-markers using F₁ cultivars in Korea. H and R indicate inbred and recombinant lines, respectively, from Hankook Seed Co. Numbers on the right, denote size of band (bp). (A) Morphology of taproots used in this study. (B) Results of PCR amplification.

Figure 9

Validation of PCR-markers using newly developing radish lines in Sejong University. -P indicates the pink color and that lines were still segregated in terms of taproot color. Numbers on the right denoted size of band (bp). 2219 and 2221, F₄ generation; 2220, F₅ generation; 2217 and 2218, F₆ generation; 2223–2226, F₁₀ generation; 2222, F₁₁ generation.