小米类胡萝卜素积累的分子机制研究

The color of the processed foxtail millets is the key quality index that consumers are interested in, and also one of the key targets of foxtail millet breeding. There have been many varieties of foxtail millets, yet there is not much breakthrough on the millet color. Jingu 21 won several national awards for its excellent quality and one of its key traits is its yellow color of the millets, yet it is still the leading variety in Shanxi Province, though it was bred over 20 years ago, demonstrating how difficult it is to improve the millet color. The reason behind the difficulty is that it is very hard for breeders to select for the millet color during traditional foxtail millet breeding. Therefore, it is very important to study the molecular mechanism of carotenoid accumulation and identify the related genes for molecular breeding on millet color in foxtail millet. We proposed here to carry out genome wide association study (GWAS) on the color and carotenoid content of the whole grain and process grain with two populations, a natural population of 300 germplasm accessions with known genome wide SNPs and a F2 population from a cross between white and yellow millet varieties, whose genome wide SNPs will be analysed using reduced-representation genome sequencing method such as RAD-seq. The expression pattern of candidate genes from identified alleles will be analysed, from which key genes related to the accumulation of carotenoids may be identified. The functions of the key genes will be tested by genome editing using CRISPR system. This study could provide insight into the genetic and molecular mechanism of millet color quality, which would promote molecular breeding on millet quality in foxtail millet.

小米米色是消费者关注的重要表观及营养品质，也是谷子育种的重要方向。虽然谷子品种层出不穷，但在米色上有所突破的甚少。多次获得国家金奖的晋谷21米色很好，但育成20多年来仍然是山西主导品种，难以超越。究其原因，在于传统育种很难在较大群体中对米色进行选择。因此，开展米色形成分子机制研究、发掘米色相关分子标记以促进米色分子育种具有重要意义。本项目拟对300份中国核心种质资源（自然群体）和白色黄色小米品种的杂交F2代（杂交群体）分别进行米色性状（全米、加工后小米表面颜色及类胡萝卜素含量）分析，并分别与自然群体的全基因组SNP（已获得）及F2群体的简化基因组SNP进行关联分析，定位相关基因位点，在不同米色品种的小米成熟过程中对候选基因的表达模式进行分析，确定米色关键基因并利用CRISPR系统对其编辑以验证其功能，从而初步探索小米米色品质形成的遗传与分子机制，为谷子米色品质分子育种奠定基础。

米色是评价小米品质的主要指标，与小米的营养和商品价值密切相关。本项目对300份中国谷子核心种质资源（自然群体）进行了小米米色性状分析，以全基因组重测序数据为基础，对米色及类胡萝卜素含量进行全基因组关联分析（GWAS），小米籽粒总类胡萝卜素含量GWAS结果显示，在Chr3 和 Chr4上检测到包含74个基因的强关联信号。同时，我们选用谷子材料牛毛白（小米颜色为白色）和GBS（小米颜色为黄色）为亲本进行杂交，构建了一套高世代的重组自交系（RIL）群体。在对两个品种杂交所获得的后代群体性状调查中，发现米色分离差异明显，受多基因控制。从F6RIL群体内筛选黄度值极高和极低株系，分别构建黄池和白池，进行BSA测序分析，共关联到2个候选区域，分别位于3号和4号染色体。值得注意的是，前期自然群体小米类胡萝卜素GWAS分析与群体米色BSA测序分析定位的物理位置高度一致。且编码类胡萝卜素合成途径第一个关键酶的基因SiPSY1和SiPSY2位于该区域。利用qRT-PCR对不同米色（黄、白）品种籽粒不同成熟时期SiPSYs基因家族成员进行表达模式分析，结果显示在籽粒成熟的整个过程中黄色小米品种SiPSY1基因表达量均显著高于白色小米品种。再次证明黄小米品种SiPSY1基因的过量表达是促进类胡萝卜素积累，导致其米色形成的关键因素之一。