| Foxtail millet (Setaria italica) belongs to the family of gramineous crops with abundant germplasm resources. Since foxtail millet has a small genome, of C4 photosynthesis characteristics, drought resistance and a series of advantages, it is attracting more and more attention recently. Hulled foxtail millet is rich in nutrients, such as carotenoids, one of which is β-carotenoid, the precursor of vitamin A, lacking it could lead to the risk of eye disease or blindness. Carotenoidsare indispensable in the human diet nutrition. Consumers prefer to choose the millets with dark yellow colour, which is mainly due to the presence of carotenoids. There are many different colours of millets such as dark yellow, light yellow, white, and green millets in the bank of germplasm resources. This is the foundation for studying and breeding to provide and produce finer quality. Eight varieties of foxtail millets (2 varieties for each colour)were choosen as research materials. The relationship between the carotenoid and β-carotenoid contents and the colour difference of millet were analysed; The full-length of SiLCYB gene coding for β-lycopene cyclase (LCYB) were cloned and analysed from these varieties; The SiLCYB expression patterns in different tissues and 5 different grouting periods of eight varieties (the beginning of filling, the middle of filling, the late of filling, the end of filling, and the mature stage), were analyzed. The expression patterns of the P-carotenoid biosynthesis and catabolism relevant genes (SiLCYE, SiCCDl, SiHYD) were also analysed, and then the relationship between the β-carotenoid and the different colours of millets were explored from the molecular perspective. It could lay an important theoretical foundation, which is helpful for the breeding of high-carotenoid content millet variety. The main results are as the followings;1. The millet colour differences of eight varieties were tested by using the X-Rite (American) VS450 colorimeter. It suggested that the CCI index of dark yellow millet ’JG21’(4.082) was the highest, however, the CCI index (2.717) was the lowest in the white millet ’ZSG’ among the 8 different varieties. Chromatic meter results were consistent with the visual observation in general.2. The content of total carotenoid and β-carotenoid of 8 varieties were tested by spectrophotometer method and HPLC method. The results showed that, the highest contents of total carotenoid and β-carotenoid was’JG21’,25.946 μg/g and 2.579 μg/g, respectively, and the lowest was ’NMB’,1.445 μg/g and 0.0133 μg/g, respectively.3. The SiLCYB gene was cloned and analysed from eight different foxtail millet varieties. In these varieties, comparing with’YG1’(which the reference genome sequence) and other varieties, there were two mutations in the SiLCYB gene of ’JG21’:one is located at the 234 bp of CDS, G being replaced by C, whereas the other one is at the 1466 bp of the 3’end, A being replaced by G There was one amino acid difference in the protein sequence, with Q replaced by R, corresponding to the 3’end base difference.4. Using ’JG21’ as experimental material, we analyzed the SiLCYB expression pattern in different tissues including roots, stems, leaves and panicles using real-time quantitative PCR (RT-qPCR) method. The results showed that the gene expression level of SiLCYB in the four tissues were not tissue-specific, with the highest gene expression in the leaves, and the least in the roots.5. During the filling stage, the millets of eight varieties were collected, and then RNA were extracted. Using RT-qPCR method, the expression patterns of β-carotenoid metabolism related genes SiLCYB and SiLCYE, SiCCD1, SiHYD were analysed. The result showed that at the S2 stage, the SiLCYB expression level in’LMQG’was about 5 times of that in ’DQG’, which was the lowest. At S5, the expression level of SiLCYB/SiLCYE in’GS4’was approximately 15 times of those in’JG21’. The expression level of SiCCD1 in two varieties of white millet was higher than that in the other varieties. The SiHYD expression level in those white millets was the highest as well, and about 13 times of that in ’DQG’ at the S4 stage. The results showed that:the colour difference of millets was result of the regulation by multiple genes.6. There were correlations between carotenoid gene expression, the content of carotenoid and millet colour. The results showed that there existed a negative correlation (p<0.05) between the index of CCI and b*, between the content of total carotenoid and beta carotenoid and the gene expression level of SiCCDl and SiHYD during the whole filling stages. Therefore, SiCCD1 and SiHYD played an important role in β-carotene metabolism. The decomposition and hydroxylation rate were far higher than the synthesis rate of SiLCYB, which reduced the color of foxtail millet. |
