Expression quantitative trait loci analysis of BAN, F3H and TT19 genes in Brassica napus Kun Lu, Cunmin Qu, Kai Zhang, Hao Yu, Junxin Lu, Yourong Chai, Jiana Li* Chongqing Rapeseed Engineering & Technology Research Center; Engineering Research Center of South Upland Agriculture, Ministry of Education, P. R. China; College of Agronomy and Biotechnology, Southwest University, Tiansheng Road 2, Beibei, Chongqing 400715, P. R. China ljn1950@swu.edu.cn drlukun@swu.edu.cn Abstract—Flavonone 3-hydroxylase (F3H), anthocyanidin reductase (ANR) encoded by BANYULS (BAN) gene and glutathione S-transferase encoded by TRANSPARENT TESTA 19 (TT19) gene are three key enzymes in flavonoid pathway, playing important roles in the synthesis and transport of anthocyanin and proanthocyanidin. However, little is known about the expression variations and the regulatory network in Brassica. In this study, we examined their expression levels in seeds of 30 days after flowering (DAF) in F 7 recombinant inbred lines (RILs) derived from a cross between Brassica napus cultivars Youyan2 (black-seeded) and GH06 (yellow- seeded), and characterized the locus-level regulatory network related to B. napus BAN, F3H and TT19 genes. Expression quantitative trait loci (eQTL) mapping revealed five, seven and eight eQTLs for BAN, F3H and TT19 genes, respectively. Marker E4M8 was associated with qBAN-8-2 and qTT19-8-2, while marker H022L18-2 was linked with qBAN-16-4 and qF3H-16-6, implying that there might be upstream regulatory genes in E4M8 and H022L18-2 marker flanking regions. In further analysis, we identified four trans-eQTLs (qBAN-16-5, qF3H-16-5, qBAN-16-4 and qF3H-11-2) close to the location of the major QTL controlling yellow-seeded trait of B. napus. The 200-kb flanking sequences of the four eQTL on B. rapa chromosome A09 showed well synteny to B. oleracea genome sequence and partial continuous fragment of chromosome 1 of Arabidopsis genome, suitable for candidate gene predication of eQTLs. Based on gene finding results, seven transcription factors were suggested to be the potential upstream candidate(s) controlling expression variations of BAN, F3H and TT19 genes. These results could provide a new approach for constructing regulatory pathways that contribute to complex traits, such as yellow-seeded trait. Keywords-Brassica napus; transparent testa; expression quantitative trait loci; regulatory network I. INTRODUCTION Oilseed rape (Brassica napus, AACC, 2n=4x=38) is one of main oil crops in the world. It is an important source to the vegetable oil, meal protein and industry material. Compared with black-seeded cultivars, yellow-seeded B. napus shows many good quality traits, such as lower seed coat pigment content, lower meal fiber content, higher seed oil and meal protein content, etc. Selecting of yellow-seeded oilseed rape cultivars with stably inherited yellow seed trait and good agronomic traits has become the most important breeding goals. However, it is very slow advance and difficult improvement to breeding of yellow-seeded B. napus, since the genetic and molecular mechanisms of yellow-seed trait were still unclear. It had been proved that plant seed coat pigments are mainly composed of polymers of proanthocyanidin (PA), which is synthesized via the flavonoid-anthocyanin- proanthocyanidin pathway (simplified as flavonoid pathway here), a core branch pathway of phenylpropanoid pathway. F3H, BAN and TT19 are 3 key enzymes in flavonoid pathway, playing important roles in the synthesis and transport of anthocyanin and proanthocyanidin. In Arabidopsis thaliana, the loss of function mutants of the 3 genes all leads to the transformation of the wild-type dark- brown seed to yellow or pale yellow seed, i.e. transparent testa (TT) trait [1]. At present, the gene families encoding the 3 key enzymes have been cloned by RACE method in our lab, which laid the foundation to further study their molecular and regulatory mechanism. In order to determine the upstream regulatory network of the 3 gene families, analysis of transcript abundance was carried out on RNA from seeds of 30 DAF in RIL population. Regarding as quantitative traits, the transcript levels of the 3 gene families were examined by QTL mapping method for eQTL detection. Using this method, it was possible to construct regulatory pathways that contribute to complex traits, demonstrating that the principles of eQTL mapping could be applied to B. napus. II. MATERIALS AND METHODS A. Plant materials and total RNA extraction The RILs were developed through successive selfing up to six generations from a cross between yellow-seeded female parent ‘GH06’ and black-seeded male parent ‘Youyan2’ by single seed descent (SSD). Parental lines and RILs were sown in field trials at plant breeding station of Chongqing Rapeseed Technology Research Center (CRTRC) in 2009 as previously described [2]. Seeds of 30 DAF in 180 F7 recombinant inbred lines were harvested and used for total RNA isolation. Total RNA was extracted using the Plant RNA Mini Kit (Watson Biotechnologies, Inc., China). To remove contaminated genomic DNA, the total RNA was treated with RNase-free DNase I (TaKaRa). Quality and concentration of total RNA samples were 2012 International Conference on Biomedical Engineering and Biotechnology 978-0-7695-4706-0/12 $26.00 © 2012 IEEE DOI 10.1109/iCBEB.2012.214 272