MYB1 transcription factor is a candidate responsible for red root skin in radish (Raphanus sativus L.)

Abstract

Root skin color is one of the economically important traits in radish (Raphanus sativus), and the pigmentation in red skin varieties is largely attributable to anthocyanin accumulation. Pelargonidin was found as a major anthocyanin pigment accumulated in the sub-epidermal layer of red radish roots. In the 20 F2 population generated from the F1 with red root skins, root skins with red and white colors segregated in a 3:1 ratio. Additionally, a test cross between a red F3 individual and a white skin individual gave rise to 1:1 segregation of red and white, indicating that the root skin color of radish is determined by a single locus and red color is dominant over white. We performed association mapping for root skin color using SNPs obtained from RNA-seq analysis. Segregation analysis on the 152 F3 test-cross population revealed an RsMyb1 transcription factor as a candidate gene to determine root skin color. A PCR marker based on the polymorphism within 2 kb of RsMyb1 was developed and tested on 12 and 152 individuals from F2 and F3 test cross populations, respectively, and red and white root skin colors were completely distinguished corresponding to the genotypes. Expression levels of RsMyb1 in red or purple root cultivars were significantly higher than in white root cultivars. These findings suggest that RsMyb1 is a crucial determinant for anthocyanin biosynthesis in radish roots, and the molecular marker developed in this study will be useful for marker-assisted selection for red skin individuals at early seedling stages.

Fig 1. Phenotypes of red and white root skin radishes.

(a) Root skin color phenotypes of segregating F₂ individuals grown in a field. (b) Hypocotyl skin colors of F₃ plants at 21 days after planting (DAP). Note that in many instances, the hypocotyl and petiole colors are already separated but they are not always identical to the root skin color. (c, d) White and red root skin colors of F₃ plants at 120 DAP, when root and hypocotyl thickening is in progress (e) Stereomicroscopic images of transverse sections of the red (left) and white (right) roots stained with toluidine blue O. Samples are from the plants shown in (c) and (d). (f) Accumulation of anthocyanin pigments in the 2–4 layers of the subepidermal cells of red colored root (from c).

Fig 2. Pelargonidin is a major anthocyanidin in red root skin.

HPLC analysis with standards (a), and root skin extracts of red (b) and white roots (c). Pictures for methanol extracts from the red and white root skins were depicted in inlets in (b) and (c), respectively.

Fig 3. Genome-wide association analysis and genetic linkage analysis of F₃ individuals indicate the red root skin color locus is on chromosome R7.

(a) Manhattan plot showing -1og₁₀ P-values of 208,469 SNPs associated with root skin color relative to their genomic positions on the x-axis. The dotted red line indicates 5% significance with Bonferroni correction. (c) Relative positions of PCR markers (black bars) and candidate genes (red bars) on chromosome R7. Note that the marker R7-9.36 and a strong candidate were proximally located within 10 kb. (d) Twelve F₂ individuals were genotyped with PCR makers, which were designated and named after their chromosomal positions. The R7-9.36 Mb marker was further applied to 152 individuals of F₃ test-cross population. The numbers of recombinants/chromosome tested are presented in parentheses (c). M, size marker; R, red; W, white root skin individuals.

Fig 4. Transcription levels of RsMyb1 and other candidate genes in red and white root skins.

(a) Transcription levels were measured by qRT-PCR and presented relative to RsACTIN. Positions on chromosome R7 and predicted functions of the genes are as follows: R7_9141477_9142640(+) (binding partner of ACD11 1-like), R7_9145761_9147238(-) (heat stress transcription factor A-8), Rs388430 (R7_9348525_9349979(+), transcription factor MYB114-like) Rs386970 (R7_10205078_10206725(-), isoflavone reductase homolog P3), Rs386960 (R7_10214323_10216096(-), isoflavone reductase homolog P3-like), Rs386430 (R7_10493232_10495210(-), myb-related protein 306-like), Rs386290 (R7_10584636_10586808(-), transcription repressor MYB6-like), R7_11797046_11798366(-) (transcription factor MYB114-like). * p < 0.05, ** p < 0.01, ***p < 0.005. Bars indicate means ± standard deviation from three biological replicates. (b) Expression level of Rs388430 (RsMyb1) was analyzed with qRT-PCR in six red/purple and three white root radish cultivars as indicated. Rs388430 expression was consistently higher in red or purple root cultivars than in white root cultivars. Error bars indicate means ± standard deviation from the three biological replicates.