Genetic Analysis, Cloning And Characterization Of The Related Genes For Allogenous White Flower Character In Brassica Napus L.

Rapeseed (Brassica napus L.) is one of the main oil crops widely grown in the word, as well as in China. It is an important source of vegetable oils in China with the largest planting area and the largest production. White flower character in rapeseed is rare and has important research and breeding values. In this paper, the inheritance of white flower character in a Brassica napus line, HW243, was studied by a series of methods, including different ways of observation, quantitative genetic analysis, QTL analysis and real-time quantitative PCR. The main results were summarized as the following:1. The three different methods for observation of rapeseed flower colorsWhite flower line'HW243'was crossed to two yellow flower B.napus lines,'HZ21-1' and'Zhongyou821', i.e.,'HW243×HZ21-1'and'HW243×Zhongyou821'. Six generations (Pi, P2, F1, RF1, F2, B1, B2) for each of the two combinations were prepared to investigate the variations in flower colors in Brassica napus L. The colors of flower in the six generations were observed with three different methods:naked eye observation and classification, spectrophotometry of extracted pigments, and digital scanning of petals. The following importamt results were discovered:(1) With the digital scanning method, rapeseed flower colors could be characterized by the RGB values of the petals, which were obtained with a reading software. The results showed that the parameters R and G varied to a very little extent, but the parameter B showed a large range of variation, among the different generations. The variations in B values were highly consistent with the variation in flower colour observed by naked eyes. In present study, the B values of flower petals were used to analize the genetic characteristics of the flower colors in rapeseed. (2) The flower pigments were extracted with absolute alcohol, and the extracted solution was examinated with a visible/ultra-violet spectrophotometer at variable wavelengths. It was observed that the absorbance value at wavelength 439nm showed the largest difference among P1, P2 and F1, with yellow, white and milky flower colors, respectively. The absorbance values of the extracted pigments at wavelength 439nm were used for the systematic analysis in later experiments. (3) The variations in flower color in the six generations of the two crosses could be evaluated by B values, absorbance values and naked eye grading. It was shown that the three methods generated highly consistent observation results. The digital scanning and the pigment extraction methods, however, could reduce the subjective observation errors, and could be used to replace'naked eye' observation of flower colour in Brassica napus L.2. Genetic analysis of the Allogenous white flower color in Brassica napusThe flower colors in the six generations (P1, P2, F1, B1, B2 and F2) of the two crosses (HW243×HZ21-1, HW243×Zhongyou 821) were analyzed using the B values and the absorbance values. It was found that Allogenous white flower color in Brassica napus L. was controlled by both major genes and polygene genes. The B values and the absorbance values in the segregating generations showed continuously distribution. One single peak was distributed in B1 population, and bimodal distributed in B1 and F2. The genetic modes of flower color were analyzed with a mixed model for major genes plus polygenes. The results from the both detection methods were consistant. It was shown that Allogenous white flower color in Brassica napus L. was a quantitative trait and its inheritance mode fitted to a mixed genetic model of two major genes with additive-dominant-epistatic effects plus polygenes with additive-dominant-epistatic effects (the E-0 model). The effects of major genes were high and polygenes were relatively small. The effects of additive, dominant and epistatic actions of the two major genes were equally important. The inheritability rates of the two major genes were estimated to be 46.55% to 98.14% in B1, B2 and F2, higher than that of the polygenes, estimated to be 0.32% to 46.9% in B1, B2 and F2 respectively.3. Construction of molecular genetic map and QTL identification of Allogenous white flower color in Brassica napusTaking the F2 generation of HW243 (white petal parent)×HZ21-1 (yellow petal parent) as a mapping population, a genetic linkage map of Brassica napus L. was constructed using molecular markers of SSR, RAPD and SRAP. QTL's for petal colors were identified. The molecular map included 217 marker loci, covering 2102cM, with an average distance of 9.69cM. Two additive QTL's were checked out for flower colors in Brassica napus L., i.e., qPE4 (Na12-B06—ME25-EM20-3) and qPE6 (Nal0-H06—ME27-EM26-2). The two QTL's explained 27.45% of the phenotypic variation. Additionally, three pairs of epistatic QTL's were detected, which explained 13.33% of the phenotypic variation.4. Cloning and Characterization of genes associated with carotenoid biosynthesis in Brassica napusA group of genes possibly associated with the carotenoid biosynthesis in Brassica napus were cloned with RACE, including BnPSY (HQ260432, HQ260433), BnPDS (HM989806, HM989807, HM989808, HM989809), BnZDS (HQ260435, HQ260436), BnLYCE (HM212502, HM212503), BnLYCB (HM989810), BnBCH (HM212504, HM212505), BnZEP (GU361616, GU561839), BnNCED3 (HQ260434), BnCCDl (HQ260430, HQ260431). The levels of expression of these genes were detected using real-time quantitative PCR in the different tissues from plants of different flower colors. The results showed that the expression levels of all of these genes were higher in the yellow flower petals than in the white flower petals. The expression level of most of these genes was a little bit lower in flower petals of F1 than in the yellow flower petal. But reverse situations were observed for the genes BnZEP and BnCCD1. The results also showed that the expression levels of these genes were, to some extent, variable during the developmental period of the white flower petals in HW243, which was consistant with the change in flower colour from pale yellow in the earlier stage to full white in the later stage. The changes in the white flower color seemed to be due to the co-regulation of BnPSY, BnPDS, BnZDS, BnLYCB, BnLYCE, BnNCED3 and BnCCD1 gene.