| Purple-caitai(Brassica campestris L.ssp.chinensis var. Purpurea Hort. 2n = 2x = 20) as a subspecies of Brassica variety, which originated in the central Yangtze River Valley, is native to China and it is an important autumn and winter vegetable crop in some regions such as Hubei, Hunan and Sichuan Provinces. Flower stalk is edible organ, with rich nutrient and high anthocyanin content, covered with color, which is good for health. This project aims to clone the genes controling the colour of the purple-caitai’s. Through analyzing the consequence of the QTL-sequence and designed of molecular markers on the rapa chromosome A07 and A09, F2 population was used to screened linked markers. The individual plant of F2 population were idendfied- by those linked markers associated with pure gene. The F2:3 family lines were established to clone the candidate gene for gene-mapping providing the basis for molecular assisted breeding in cruciferous. The results were as follows:1. Linked marker screening and indentification of purple-related gene: Based on the DNA-bulked segregant analysis sequenceing, which is operated by Li J P(2015), more than 300 pairs CAPS and InDel primers were designed on the rapa chromosome A07 and A09. Subsequently, these primers were first screened by the P1, P2 and two extreme pools to identify polymorphism markers which are high reproducible and stability, then the identify polymorphism markers were further verificated in F2 segregating populations. Finally, seven pairs flanking makers associated with purple-caitai traits were obtained, defined as: 09-16、09-31、07-8、BrID10113、07-58、HA07-12 and 09-3, respectively.2. Construction and selection of the F2:3 populations: The 47 F2 plants selected by molecular markers were self-pollination to get F2:3 populations. Each of the F2:3 populations planted 100 individuals. Then investigated the field traits according to color grades, and selected the F2:3 segregating populations which separation ratio was about 3: 1. In this study, we successfully obtained a F2: 3 segregating populations which is in accordance with single gene isolation ratio, named as 56- 4.3. Genetic analysis of the purple traits in the F2:3 segregating populations: The color grades of the candidate population 56-4 is divided into three grades: 0, 3 and 5. The individuals of grade 3: in the seedling stage, the purple are mainly accumulated in the veins and the vicinity of the growing point. While in vegetative growth period, the purple is significantly less than seedlings. There is only a little portion of red anthocyanin distributed in the flower stalk, seed pods and sepals. The individuals of grade 5: in the seedling stage, the tender, especially the smallest 1-2 true leaves presented to be all purple, the 3-4 true leaves showed purple in the edge and back, while others, purple substance only existed in leaf petioles and part of the veins. when it enters to the vegetative growth period, red anthocyanin distributed all parts of the plants..4. Primary targeting of the single QTL gene on the rapa chromosome A07. Combined field traits investigation with the genotype which determinated by the markers(07-8, 07-23, 07-58, BrID10113 and HA07-12) located on the rapa chromosome A07, we found that the maker 07-8, 07-23 and BrID0113 are located in the left of the target gene, while the markers HA07-12 and 07-58 in the other side. The physical distance between t wo microsatellite markers BrID10113 and HA07-12, is 3M, with genetic distances of 8.0 and 11.0 cM, respectively.5. The expression analysis of anthocyanin biosythesis-related gene of A07: The results indicated that Bra004162 and Bra016164 showed a significant difference between two parents, the purple parent were 10.32 times and 2.87 times compared with the white ones, respectively. So, it was speculated that Bra004162 and Bra016164 might be the candidate minor genes controlled purple-caitai traits. |
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