Loss of Function of the Carotenoid Isomerase Gene BrCRTISO Confers Orange Color to the Inner Leaves of Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Abstract

Chinese cabbage varieties with orange inner leaves are of interest due to their attractive appearance and high carotenoid content. Previously, a recessive locus, Br-or, that confers this trait was mapped to linkage group A09 in Brassica rapa, and Bra031539 was identified as the candidate gene. We used a backcross population consisting of 1,392 individuals for fine mapping; three linked markers delimited the Br-or locus to a region of 9.47 kb, which contained only one predicted functional gene, Bra031539 (the carotenoid isomerase gene BrCRTISO). We identified two bacterial artificial chromosome (BAC) clones carrying Br-or and confirmed that this allele of BrCRTISO confers the mutant phenotype by sequencing. Many DNA sequence variations are present in Br-or, including a 90-bp DNA deletion in the promoter and a 501-bp insertion at the 3′ end of the gene. GUS staining showed that the spatial expression of BrCRTISO was perfectly coincident with the site of carotenoid synthesis and that the 90-bp deletion in the promoter slightly downregulated expression of the gene. Using an in vitro enzyme assay, we showed that the mutant Br-or protein cannot convert prolycopene to all-trans-lycopene. Light has a positive effect on the expression of carotenoid biosynthesis genes, except for beta-ring carotene hydroxylase (CHXB), and this coincided with the increase in total lycopene and β-carotene in the Br-or mutant. We also demonstrated that loss of BrCRTISO function, upregulation of the upstream genes, and downregulation of downstream genes lead to the accumulation of prolycopene and confer an orange color to the inner head leaves in Chinese cabbage. Keywords Brassica rapa L. ssp. pekinensis . Orange leaf . Br-or . BrCRTISO . Carotenoid biosynthesis Abbreviations CHXB Beta-ring carotene hydroxylase DH Doubled haploid GAPDH Glyceraldehyde-3-phosphate dehydrogenase IPI Isopentenyl pyrophosphate isomerase LCYB Lycopene beta-cyclase LCYE Lycopene epsilon-cyclase PDS Phytoene desaturase PSY Phytoene synthase VDE Violaxanthin de-epoxidase ZDS Zeta-carotene desaturase ZEP Zeaxanthin epoxidase

Transcriptome Analysis of Orange Head Chinese Cabbage (Brassica rapaL. ssp.pekinensis) and Molecular Marker Development

“…This result is inconsistent with some previous studies which show that the mRNA level of a Br-or candidate gene (Bra031539) is lower in the orange inner leaves than in the white inner leaves [10, 15]. To understand the difference, the promoter sequence of Bra031539 was analyzed.…”

Fine mapping and characterization of the or gene in Chinese cabbage (Brassica rapa L. ssp pekinensis)

“…A BAC library was developed to clone BrCRTISO in the orange head material. A 90-bp DNA deletion in the promoter and a 501-bp DNA insertion prior to the stop codon are the distinctive features revealed between the two experimental materials (Su et al, 2014). Li et al (2015) found that an orange-type A21530 mutant also lacks the 3' end sequence.…”

“…Using an F2S4 population, a single recessive gene, Br-or, was previously localized to a 16.7-kb region, with three ORFs (open reading frames) predicted in the target area (Zhang et al, 2013). Br-or was then localized to a 9.47 kb region containing one predicted gene (Su et al, 2014). Using two insertion-deletion (InDel) markers, SB14001 and SB13049, the target gene was subsequently localized to a 65.555 kb region containing 17 candidate genes .…”

“…The use of this marker may enable identification of hybrid orange head materials in the early stage of breeding, which could speed up the breeding process of orange head Chinese cabbage. Compared with the markers developed in fine mapping studies, such as those closely linked to or (Zhang et al, 2008;Feng et al, 2012;Zhang et al, 2013;Su et al, 2014), functional markers are developed in mutated genes, thereby allowing genotypes to be distinguished accurately. At the same time, an allele-specific marker (Br-Pro-Indel) was also developed to identify allelic variations in Chinese cabbage cultivars .…”

Sequence Analysis of the Carotenoid Isomerase Gene in Potato (Solanum tuberosum)

“…It is reported the identification of a CRTISO as the candidate gene for orange head by high-resolution genetic mapping using F2S4 population [15]. Loss of BrCRTISO function, upregulation of the upstream genes, and downregulation of downstream genes lead to the accumulation of prolycopene and confer an orange color to the inner head leaves in Chinese cabbage [16]. Previous studies have shown that the CRTISO protein is relatively conserved in plants.…”