| Purple-caitai originates from China,it is the special vegetable in the region of Yangtze and plants in Hu Nan, Hu Bei, An Hui et al, tasting crisp and being rich of nutrition; its stem is bright purple, containing high anthocyanin, so it has good health care function and commodity value. To dig the purple gene in purple-caitai and the metabolic pathways of anthocyanin synthesis, we do this experiment. In the experiment, we used the purple-caitai multi-generational inbred lines “Lan1205” as the female parent and the white-caitai multi-generational inbred lines “Lan1206” as the male parent to hybridize to form the F1﹑F2﹑BC1 generations, then observed the color of each generation, making statistics of the separation ratio of color, and analyzed genetic relationships, and used molecular markers to locate the purple traits preliminary. The main results are as follows:1. Investigations of the field character indicate that: F1 generation, the reciprocal cross plants between two parents, is all red, but the red part is much weaker than the purple-caitai P1; BC1(P1) generation is red too; F2 generation appears separation, but it is not simply separated into only two characters: red and white, it presents continuous change of red color, conforming to the normal distribution curve as a whole, but the separation ratio do not conform to the laws of Mendelian inheritance.In conclusion, the purple trait behave incomplete dominance to white trait, and the color trait may be controlled by two or more genes, the purple trait is a quantitative trait.2. Molecular marker screening and map building of the purple gene. Screen 1098 pairs of SSR primers with two parents, preliminary obtaining 222 pairs of SSR markers that the amplification bands are clear, so the effective primers ratio is 20.2%.After two times of repeatedly screening, we obtain 86 pairs of significant and well stability SSR markers. Use the 86 pairs of SSR primers to screen the PCR amplification of F2 population’189 individuals, eventually selecting 37 SSR primers to construct the genetic map. Finally get a molecular genetic map containing 28 markers and composing of two linkage group, the total length is 351.1c M, the averagedistance between markers is 12.54 c M.3. QTL preliminary positioning of purple trait.Take QTL mapping analysis toward purple traits of 189 plants of F2 population with CIM mapping method,getting two QTL loci which locate on c1 and c2 linkage group respectively.The loci on c1 locates between ACMP0242 and ACMP0458, and the LOD value is 43.1, the additive effect value is-2.2, the dominant effect value is-1.7, the rate of phenotypic variation explaining is 72%; The loci on c2 locates between ACMP0561 and ACMP0284, and the LOD value is 61.1, the additive effect value is-0.9, the dominant effect value is 2.6, the rate of phenotypic variation explaining is 57%. The two QTL loci contribute very high, so we deduce that the two QTL loci may be the effective gene loci controlling the purple gene, and they may be locate on 5 or 9 chromosome of Chinese cabbage genome.4. The molecular marker supplement on the linkage group. Developed 110 pairs of In Del primers on 5 and 9 chromosome of Chinese cabbage genome, including 45 pairs on A05 and 65 on A09. Screen of these primers with two parents, preliminary obtaining 32 pairs of In Del markers that the amplification bands are clear, so the effective primers ratio is 29.0%. After two times of repeatedly screening, we obtain 23 pairs of significant and well stability In Del markers. Use the 23 pairs of In Del primers to screen the PCR amplification of F2 population’189 individuals, eventually selecting14 In Del primers to construct the genetic map. Add In Del markers to the the genetic map in composition of the SSR markers, eventually form a map contains 41 markers and one linkage group, the total length is 661.4c M, and the average distance between markers is 16.13.5. The integration of genetic map. The QTL loci changes after adding the In Del markers, and it produces four QTL loci totally. There has one QTL loci between ACMP0094 and ACMP0617, its LOD value is 14.4, the additive effect value is-0.9,the dominant effect value is 1.7; there have two QTL loci between ACMP0255 and ACMP0561, one of the LOD value is 7.6, the additive effect value is 0.3, the dominant effect value is 2.5, the another LOD value is 24.2, the additive effect value is 2.1, the dominant effect value is-0.2; there has one loci between ACMP0561 andBr ID1121, the LOD value is 28.7, the additive effect value is 2.1, the dominant effect value is-2.6. The highest rate of phenotypic variation explaining among these QTL loci is 88%. Through the comparison, we find that the loci between ACMP0561 and Br ID1121 is consistent with the QTL loci and between ACMP0561 and ACMP0284,so we speculate that this loci is an effective loci associated with purple traits, and this loci is located on chromosome 9 of the Chinese cabbage genome.6. The expression analysis of anthocyanin synthesis. Take the white-caitai as comparison, the expression level of gene that related to the anthocyanin synthesis in purple-caitai is as following: the expression level of bra027457 gene on A9 is 0.84 times, and the bra005221 gene on A5 is 2.75 times, the other gene amplify unsuccessfully with a level less than 0.05.Bra027457 and bra005221. So the bra027457 gene and bra005221 gene the minor genes that control the purple trait of purple-caitai.7. Exploit SNP locus related to the purple gene by the second generation sequencing using the two extreme pools of F2. It can be seen from the returned DNA-seq result that the Chr7 has the highest average SNP delta value which is about0.53, suggesting that the main loci that controls purple trait is on chromosome 7. Also there have high average SNP delta value on Chr2, Chr4,,Chr5 and Chr9, they may have loci that control purple traits loci. According to the results of DNA-seq, we develop some markers on chromosome 7 at the location of about 7.5M, and the marker A0716.9 M-4 is preliminary deduce that is linked with the purple gene. |
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