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

Hindawi International Journal of Genomics Volume 2017, Article ID 6835810, 8 pages https://doi.org/10.1155/2017/6835810 Research Article Transcriptome Analysis of Orange Head Chinese Cabbage (Brassica rapa L. ssp. pekinensis) and Molecular Marker Development Jingjuan Li, Yihui Zhang, Qian Ding, Huayin Li, Lifeng Liu, Fengde Wang, and Jianwei Gao Institute of Vegetables and Flowers, Shandong Academy of Agricultural Sciences and Shandong Key Laboratory of Greenhouse Vegetable Biology and Shandong Branch of National Vegetable Improvement Center, Jinan 250100, China Correspondence should be addressed to Fengde Wang; and Jianwei Gao; Received 14 September 2016; Accepted 16 February 2017; Published 2 April 2017 Academic Editor: Giuliana Napolitano Copyright © 2017 Jingjuan Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Due to the visual appearance and high carotenoid content, orange inner leaves are a desirable trait for the Chinese cabbage. To understand the molecular mechanism underlying the formation of orange inner leaves, the BrCRTISO (Bra031539) gene, as the Br-or candidate gene, was analyzed among the white and orange varieties, and 7 single nucleotide polymorphisms (SNPs) were identified. However, only one SNP (C952 to T952) altered the amino acid sequence, resulting in a mutation from Leu318 to Phe318 in the orange varieties. Additionally, we analyzed differentially expressed genes (DEGs) between the orange and white F2 individuals (14-401 × 14-490) and found four downregulated genes were involved in the carotenoid biosynthesis pathway, which may lead to the accumulation of prolycopene and other carotenoid pigments in the orange inner leaves. In addition, we developed a novel InDel marker in the first intron, which cosegregates with the phenotypes of orange color inner leaves. In conclusion, these findings enhance our understanding of the underlying mechanism of pigment accumulation in the inner leaves of the Chinese cabbage. Additionally, the SNP (C952 to T952) and the InDel marker will facilitate the marker-assisted selection during Chinese cabbage breeding. 1. Introduction Carotenoids, as a group of colorful pigments, are responsible for the yellow, orange, or red color of fruits and flowers [1]. These pigments play an essential role in plant growth and development, such as harvesting lighting energy and preventing photo-oxidation during photosynthesis [2, 3]. Plant carotenoids also play important roles in human nutrition and health, either functioning as precursors for vitamin A synthesis or offering protection to consumers against cardiovascular diseases, cancer, and age-related eye diseases [4–8]. Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an important vegetable crop planted worldwide, especially in Asia. The color of the inner head leaves is usually white, yellow, or orange. The orange color of the inner leaves is a result of the accumulation of prolycopene and other carotenoid pigments [9–11]. In recent years, the orange head Chinese cabbage varieties have gradually captured the attention of breeders and consumers, since they have an attractive color and higher carotenoids compared with the white and yellow head cabbage. Previous studies have shown that the accumulation of carotenoids in the inner leaves of Chinese cabbage is controlled by a single recessive gene, Br-or (Feng et al. [12]; Matsumoto et al. [13]; Zhang et al. [14]). In recent years, the BrCRTISO (Bra031539) gene has been identified as the Br-or candidate by several research groups. Many insertions/deletions were identified in this gene [9–11, 15], resulting in mutant BrCRTISO proteins, which cannot catalyze the conversion from prolycopene to all-trans-lycopene [15]. Furthermore, studies have also shown mutations of the BrCRTISO gene dramatically alter the expressions of many other genes [11, 15]. However, controversial results were reported. For example, Su et al. [15] found the enzymes are upregulated in the upstream pathway of the all-trans-lycopene biosynthesis and downregulated in the downstream pathway in the orange 2 inner leaves using a RT-qPCR method, while Zhang et al. [11] did not observe any expression differentiations of these genes using a RNA-seq technology. In the present study, we employed a high-throughput sequencing method to identify the differentially expressed genes (DEGs) between the orange and white F2 individuals. This study will provide new insights into the underlying mechanism of the pigment accumulation in the inner leaves of the Chinese cabbage. In addition, we analyzed the promoter regions and the coding sequence of the BrCRTISO gene in the white and orange head Chinese cabbage varieties and developed one SNP and one InDel marker, which cosegregate with the orange color phenotype of the inner leaves. These markers will provide a valuable tool in the process of breeding of orange-colored cultivars. International Journal of Genomics vector (Takara) and sequenced using the Sanger method on an ABI3730XL sequence platform (Applied Biosystems, Foster City, CA). 2.3. Validation of the SNP (C952 to T952) in Different White and Orange Cultivars and F2 Populations. To validate the SNP (C952 to T952), the DNA sequence that contains this site was PCR amplified in different white and orange cultivars and F2 populations using the specific primer (OR-SNP952, Supplementary Material: Table S1). PCR reaction was performed in a 50 μl reaction system containing 20 ng template DNA, 5.0 μl 10 × LA PCR buffer, 3.0 μl 2.5 mM dNTPs, 2 U LA Taq polymerase (Takara), and 1 μl 10 μM of primers. The amplified products were inserted into the pMD18-T vector (Takara) and sequenced using the Sanger method on an ABI3730XL sequence platform (Applied Biosystems). 2. Materials and Methods 2.1. Plant Materials. In this study, a total of 25 cultivars were used. Cultivars with white or yellow inner leaves include 14-401, 663, 1466, 1469, 1492, 1505, 1510, 1720, Hanxiu, Jindianchunwang, Kaichun, and Ribenxiayang. Cultivars with orange inner leaves include 14-490, 1480, 14-102, 14-245, 14-253, 14-257, 14-277, 14-426, 14-662, 14-669, Changyanjubao, Shenmengjuhongxin, and Shenshijuhongxin. The F2 individuals (14-401 × 14-490) were grown in the farm station at the Vegetable and Flower Research Institute, Shandong Academy of Agricultural Sciences, Jinan, Shandong Province, China. The inner leaves were harvested and stored in liquid nitrogen for DNA and RNA extraction. 2.2. cDNA and gDNA Analysis for the BrCRTISO Gene. Total RNA was isolated from Chinese cabbage leaves using the TRNzol-A+ reagent (Tiangen, Beijing, China) and treated with RNase-free DNase I (Takara, Dalian, China) for 45 min according to the manufacturer’s protocol. First-strand cDNA was synthesized from 1 μg of total RNA using a PrimeScript 1st Strand cDNA Synthes (...truncated)