Identification And Analysis Of QTL Controlling Flowering Time In The Genome Of Brassica Napus L.

Flowering is the symbol of the transition from vegetative to reproductive stage for plants,and it's a determining factor for sexual reproduction of plant.Flowering in time can make plants avoid bad environments,such as harsh winters or hot summers,to get good harvest.Flowering of plants is not only regulated by multiple endogenous genetic factors,also controlled by complex environmental factors.Temperature and light are the most two important environmental factors.Rapeseed is an important oil plants and flowering time is both an important agronomical and a complex quantitative trait. Rapeseeds can be divided into two types,the winter annuals and the spring annuals, according to the requirement of vernalization or not.Until now it's known a little about the mechanism of controlling flowering time and differentiation of the winter- and spring-typed cultivars in Brassica napus.The winter-typed B.napus variety Tapidor and the semi-winter typed variety Ningyou7 have different requirement for vernalization.A DH population named with TN DH population was constructed with these two varieties,and a RC-F2 population was constructed based on TN DH population in our lab.The field trial of one location in two years for spring environments was designed and the flowering time data was investigated. Besides the data,the flowering time data of 9 winter environments of TN DH population and 2 winter environments of RC-F₂ population were collected.About one hundred methylation sensitive AFLP markers were added to the TN basic linkage map which including 352 markers.Besides that,about 230 SSR markers were collected and a high density linkage map including 621 markers was constructed by using the software joinmap3.0.After that,229 markers with known DNA sequence information were used as anchored markers to do comparative mapping between TN linkage map and the genome of Arabidopsis.There were 40 synteny blocks and 84 islands were mapped to TN linkage map by using in silico mapping analysis.Then,QTL mapping analysis and epistatic interaction detection were done by using the softwares WinQTLcart2.5 and QTLmapper2.0.The aim of the study was to construct a genetic network controlling flowering time in B.napus.Transgressive segregation of flowering time was found in all the environments and it showed that flowering time of TN DH population was a quantitative trait.In order to fully discover the characters of the flowering time trait,two types of QTL were collected.One type is the QTL attached to the significant level(P=0.05) in each of the 13 different environments for the two populations,the other type is the MR-QTL(Micro-real QTL) with small effect under low significant level(0.5＜P＜0.05) while could be repeatedly deteced in two or more environments.Totally there were 42 unique flowering time QTL detected,including 6 MR-QTL.Among the QTL,there were 55%of the QTL could only be detected in the winter-cropped environments,and one QTL could only be deteced in the spring-cropped environments,which showed that it was a spring-cropped environment specific QTL.Sixty-three interacting loci pairs were detected.Five hundred and forty-eight homolougous genes representing 120 flowering time genes of Arabidopsis were mapped to TN linkage map by in silico mapping analysis.Among these genes,there were 27%and 9%mapped to the QTL regions and the marker interval of interacting loci pairs.To a large extent,these homologous genes represented the flowering functional genes of the QTL regions in B.napus.Therefore,the genetic network of flowering time composed of hundreds of QTL,interacting loci pairs and the functional genes of the regions in B.napus was constructed.It not only provided the further research basis of the flowering time in B.napus,also provided the research clues for dissecting other complex agronomical traits.In order to certify the accuracy of QTL mapping results and the reliability of the mapped functional genes by in silico mapping analysis,meanwhile provide the important genetic resources for genetic improvement for rapeseed,the spring-cropped environment specific QTL was further analyzed.This QTL was located on N10 linkage group and named as qFT10-4.It could explain the 52%of phenotypic variation in the spring-cropped environments,while it could not be detected in any of the winter-cropped environment.Former researches have discoved the vernalization pathway controlling the flowering time in Arabidopsis.The comparative mapping analysis in this study showed that the position of the key gene FLC in the vernalization pathway was homologous to the confidence interval of qFT10-4.The probe designed according to the homologous sequence of FLC in one of the parent Tapidor was hybrized with the total genomic DNA of TN DH population.As a result,the RFLP maker of the gene was mapped to the peak of the QTL region.So the gene located in N10 linkage group was named as BnFLC10.The genomic sequence of the BnFLC10 alleles of the two parents in TN DH population was isolated and they were about 4.5kb long.There were only three differences existing in the region of intron 1 and the gene specific primers M1 and M2 were designed according to the two differences.The primers were used to analyze the genotype of BC₃F₂ population with the recurrent parent Ningyou7 and harboring the target gene BnFLC10.The flowering time data was also surveyed in spring-cropped environment.The Chi-square analysis showed that the ratio of flowering time was fit to the ratio of 3:1(late flowering or not flowering:early flowering).The genotype of all the early flowering plants was consistent with the early flowering parent Ningyou7.These results showed that BnFLC10 was the major gene controlling the flowering time in TN DH population.Gene expression experiment result showed that vernalization could repress the gene expression of BnFLC10.The gene expression amount was stronger in winter-typed cultivar Tapidor than that in spring-type under the same condition,and the gene expression amount of semi-winter cultivar Ningyou7 was in the middle. Seventy-three different cultivars were used to do association mapping alaysis of the BnFLC10 by using the primers M1 and M2.The polymorphic marker of M2 showed significant relative to the flowering time(P=0.007).All the above results showed that the candidate gene BnFLC10 of the QTL qFT10-4 was the key gene and controlling the differentiation of the winter- and spring-typed rapeseed.Three aspects of research work are listed in the last section of the thesis.They are as follows:1.Certification of MR-QTL and the improvement of QTL detecting system;2. Dissecting the mechanism of BnFLC10 regulates the differentiation of winter- and spring-typed rapeseed;3.Dissecting the winter-cropped environment specific QTL cluster.