Fine-Mapping Of QFT.A10 And Dissection The Relationship Between DNA Methylation Of BnA10.FLC And Flowering In Brassica Napus

Flowering is a significant symbol from the basic vegetative phase to reproductive phase of higher plants. In many factors that effect flowering, temperature is a key one, because several crops such as wheat、rapeseed are all need low temperature to flower. Until now, lots of researches relating to genetic network of controlling flowering time in Arabidopsis have been done well. During the network, FLC is an important gene in venalization, it can work together with many other genes to control flower in Arabidopsis. While Arabidopsis and Brassica napus have the same ancestor, it’s much more different to study on genetic mechanism of flowering in B. napus owing to its large genome. In recent years, researches on flowering in B. napus mainly focus on QTL analysis, and it’s known a little about the mechanism of controlling flowering time in B. napus.Tapidor, a European winter cultivar, Ningyou7, a Chinese semi-winter cultivar and the near isogenic lines (NIL) which developed with Ningyou7 as the recurrent parent and Tapidor as the donor parent were used as materials. In the research, two layers were used to dissect the key gene BnA10.FLC which controlled flowering time of B. napus. Firstly, as a spring-cropped environment specific QTL (qFT.A10) had been found and the result that BnA10.FLC maybe the candidate gene of the QTL by bioinformation analysis (Long, 2007), higher generation of NIL were builded. In the process, molecular marks which developed from the intervar of the QTL were used to assist selecting. The aim to build higher generation is to fine mapping the QTL and ascertain the gene controlling qFT.A10. Secondly, detected the DNA methylation status of BnA10.FLC and tried to explain the differences expression level of BnA10.FLC between Tapidor and Ningyou7. These results could offer some help on clarifying the genetic mechanism of BnA10.FLC. Main results were described as follows:1. Molecular marks developed from the intervar of qFT.A10 and also BnA10.FLC specific marks were used to select the BC5F2 population.6 recombinated individual plants were found in a population contained 2727 individual plants. The qFT.A10 was fine mapped between two marks, T11 and Niab009. The results of genscan showed that BnA10.FLC, the homologus gene of FLC in Arabidopsis, was the gene controlling qFT.A10.2. It was a key section to design methylation detecting primers during the process of bisulfit-sequencing. Aimed at BnA10.FLC,35 pairs of primers were designed, while 9 of them were suitable for methylation detection. The primers were distributed in the promoter、coding region and 3’end of the gene.3. During DNA methylation detection, no methylated sites were found in the coding region, whereas several methylated sites were found in the promoter region (ATR600) which winter cultivar specific and 3’end of the gene.4. When analysed the methylated sites, it seemed that there was little relationship between methylation status in 3’end and BnA10.FLC expression. But in the ATR600, methylation exsited in different vernalization times of Tapidor and Bakow. In this region, several CpG and CpNpG sites were maintained methylation stuats in the whole vernalization time; while the methylation degree of CNN and other CpNpG sites were decreased during the vernalization time, what’s more, these sites were continuous in physical location.5. Quantitive PCR had done in different vernalization times of Tapidor, Ningyou7, Bakow and Westar. The results showed that before vernalization, the expression level of BnA10.FLC was the highest. While with the vernalization time lengthening, the expression levels of BnA10.FLC were decreased generally. In the winter cultivar Tapidor and Bakow, the tendency of BnA10.FLC expression was much more the same, whereas there were differences in NY7 and Westar owing to different sensitivity to vernalization.6. During analyse, the sites which methylation degree decreased during the vernalization time maybe related to BnA10.FLC differential expression in Tapidor and Ningyou7. According to the motif prediction, some mode about these two factors was speculated.