胡萝卜肉质根表皮颜色基因(Rc)的精细定位及候选基因筛选

Root color is an important quality trait for carrot. However, there are different views on inheritance of root color in carrot and few studies on related genes mapping and screening were conducted, genetic mechanism of it is unclear yet. Therefore, it is difficult to improve varieties effectively. Our previous studies on inheritance of root color in carrot showed that a single dominant gene controls the orange root color. Based on these studies, we construct two F2 populations derived from two crosses between one orange root color inbred line with two purple-red root color inbred lines and preliminarily map the root color (Rc) gene using the combination of high-throughput sequencing technology and bulked segregation analysis. Furthermore, a high-resolution linkage map within the target region is constructed with markers developed by re-sequencing of parents and two larger F2 populations are used to screen recombinant plants with their own flanking markers linked to Rc gene. Fine mapping of Rc gene is achieved through the comparison of two mapping results in two populations. Finally, the target gene would be found based on sequence alignment of genes within fine mapping region and real-time PCR analysis of differential genes. The results of this study will not only lay the foundation for molecular breeding of root color but also have important significance on appearance and nutritional quality improvement in carrot.

肉质根表皮颜色是胡萝卜重要的品质性状。然而对胡萝卜肉质根表皮颜色遗传规律的研究存在不同的观点，且相关基因的定位和筛选研究也比较少，还不清楚其遗传机制，因此难以有效地进行品种改良。本课题组前期研究结果表明，胡萝卜橙色肉质根表皮颜色对紫红色为单基因显性。本项目计划在此基础上，利用橙色肉质根自交系与2个紫红色肉质根材料杂交构建的2个F2群体，采用高通量测序与集团分离分析法相结合的方法进行肉质根表皮颜色基因（Root color, Rc）的初步定位；进一步通过亲本重测序数据开发多态标记构建目标区段高密度连锁图谱，利用Rc两侧翼标记分别在两个F2大群体中筛选重组单株，比较分析两个群体的定位结果，实现Rc基因的精细定位；通过定位区间的序列比对，寻找差异基因并进行荧光定量PCR分析，筛选Rc目的基因。本研究结果将为胡萝卜肉质根表皮颜色的分子育种奠定重要基础，对胡萝卜外观和营养品质的改良具有重要意义。

红色胡萝卜中由于富含番茄红素而使胡萝卜呈现红色，而番茄红素是重要的功能物质，但控制红色胡萝卜中番茄红素含量遗传规律和分子机理还未知。本项目以两个红色胡萝卜材料（‘PI432906’‘PI319858’）与两个橙色胡萝卜自交系（‘FN2-9’‘9304’）杂交构建的F2，F3，F4群体及254个品种组成的自然群体为材料，利用视觉观察和高效液相色谱（HPLC）结合的方法对胡萝卜肉质根的表型进行鉴定，采用基因分型技术（Genotyping by Sequencing，GBS）进行基因型鉴定，进行基于分离群体和自然群体的全基因组关联分析，并在显著关联区域附近利用基因注释和转录组分析筛选候选基因，并进行qRT-PCR验证，结果表明：1）利用96个F2群体单株，在1号和3号染色体上检测到两个显著相关的区域，最显著的SNP位点分别为S1_3115583（P=4.76E-06）和S3_4807080（P=2.20E-07）；2）利用285个F2，F3，F4代群体单株，在1号和3号染色体上也检测到两个显著相关的区域，最显著的SNP位点分别为S1_30649901（P=4.76E-06）和S3_4857798（P=2.20E-07）；3）利用254个胡萝卜品种组成的自然群体，分别在1，3，6，7号染色体上检测到显著相关的SNP位点，其中1号和3号染色体上最显著的SNP位点分别为S1_32524116和S3_4798053，且以上3种群体检测到的显著位点物理位置较为接近；4）在以上两个区域的显著位点附近筛选到2个与类胡萝卜素代谢相关的基因，DCAR_003216编码番茄红素代谢途径中的关键酶，DCAR_009172可以促进有色体的合成和转录后调控类胡萝卜代谢途径中的八氢番茄红素合成酶（PSY），其中DCAR_009172经转录组分析和qRT-PCR验证在红色胡萝卜肉质根中显著差异表达，推测这2个基因为控制红色胡萝卜中番茄红素含量的候选基因。