Dissection Of A Major QTL Cluster Controlling Flowering Time And Characterization Of "Florigen" Family In Brassica Napus

During the whole growth process, flowering is a significant symbol from the basic vegetative phase to reproductive phase and a determining factor for sexual reproduction of higher plant. Timing of the onset of flowering is an important agronomic trait affecting crop production. To meet the challenges of climate change, it is necessary to coordinate flowering within the context of seasonal variations in order to ensure the greatest possibility of pollination, and thus consistently high seed yield. Flowering time is a quantitative trait with a complex genetic basis and regulated by endogenous genes and environmental factors including photoperiod and temperature. FT protein as "florigen" moves from leaves to the shoot apical meristem (SAM) to promote flowering in Arabidopsis. Rapeseed was a crucial oil crops in China, and Brassica napus was planted widely for agricultural production. B. napus belonged to Brassica and was aloso an important member of Brassicaceae. Two genomes, A and C, were contained in B. napus, which resulted in the complex structure of genome. According to the requirement of vernalization or not, rapeseeds can be divided into two types, the winter type and the spring type annuals. Until now the research of flowering time mainly focus on QTL mapping in whole genome, interacting analysis and predicting candidate genes in Brassica, it is a little dissection in depth about the major QTL and the different mechanism of controlling flowering time in the winter and spring type B. napus.A B. napus doubled haploid (DH) population, designated as TNDH and consisting of 202 lines, was generated from an F1 resulting from a cross between a Tapidor, a European winter cultivar, and Ningyou7, a Chinese semi-winter cultivar. Near isogenic lines were developed with NY7 as the recurrent parent and Tapidor as the donor parent. In the research, two gradations were used to dissect the flowering time of B. napus. Firstly, based on comparative map between Arabidopsis and B. napus, near-isogenic line and photoperiod sensitivity, the major QTL cluster controlling flowering time on C6 (qcFT.C6) was dissected in depth, which explored the regulation of flowering in genetic and genomic levels. Secondly, according to the "florigen" locating in the QTL region, six FT copies were isolated in the whole genome of B. napus. Analysis of structure, identity and phylogenetic relationship showed the law of sequence identity among the FT paralogues and the potential evolutionary process. The genotype and phenotype of NILs confirmed that two FT copies were the key genes of the QTL cluster, which was a good continuation for the dissection of QTL cluster. Main results were described as follows:1. Based on comparative mapping between Arabidopsis and B. napus, two copies FT and API were mapped in the target QTL region. In silico mapping indicated that the QTL region corresponded to block E in chromosome 1 of Arabidopsis, where it forms inverted duplication blocks (IDB) on C6. Based on marker identity, two FT paralogues in the IDBs were close to the junctions. Using the new linkage map, we detected the flowering time QTL on C6 again. The QTL cluster was also detected in winter-cropped environment, and two FT specific markers located close to the peak of the QTL. Owing to the addition of new markers, the QTL shifting was found in different environments:(1) The QTL detecting in most environments shared in the same region from PA28 to BRMS015. (2) The QTL in five environments moved to the left region of the IDB. (3) Only one QTL moved to the right region of the IDB.2.In a BC4F2 population, some plants had earlier budding time than bolting time, however, other plants had earlier bolting time than budding time. Based on genotype and phenotype of NILs, the QTL cluster was identified to contain five sub-QTLs (qcFT.C6-1, qcFT.C6-2, qcFT.C6-3, qcFT.C6-4 and qcFT.C6-5). According to the comparative mapping with Arabidopsis, we presumed the candidate genes of the sub-QTLs, for example the key gene of qcFT.C6-3 was FT. No matter in winter-cropped environment or in spring-cropped environment, the phenotype of NILs was earlier than that of Ningyou 7. The analysis of photoperiod sensitivity showed that Tapidor was more sensitive than Ningyou 7, and the NILs also responded the photoperiod.3. The previous flowering time QTL mapping of B. napus showed qcFT.C6 was only detected in winter-cropped environment and the population constructed with a cross between winter type and spring type B. napus. If the parents of population were winter type and winter type B. napus or spring type and spring type B. napus, qcFT.C6 was not detected in winter-and spring-cropped environment. To test whether qcFT.C6 was the prevalent loci to promote flowering in B. napus, eight PCR markers locating the QTL region were analyzed the distribution in twenty nature winter type cultivars of B. napus. The Tapidor alleles of five markers (Bog46-2, BnC6.FTa, BnC6.FTb, Na12A02 and CCSSR1) were very prevalent in winter type B. napus and these markers all linked the sub-QTL. In contrast, the Tapidor alleles of other three markers (Na12A05, BnAP1a and 028L01_AT3) showed low percentage in winter type B. napus.4. The results of QTL mapping showed qcFT.C6 was not detected in spring-cropped environment, however, the QTL located in A10 was only detected in spring-cropped environment, and BnA10.FLC was the key gene of the QTL. To test whether BnA10.FLC regulated the functions of qcFT.C6, the NILs of qcFT.C6 were crossed with NIL of BnA10.FLC, and the NILs of qcFT.C6 and BnA10.FLC were backcrossed with Ningyou 7 to produce two type F1 plants as control. The flowering time of F1 developed with the NILs of qcFT.C6 and BnAlO.FLC had all significant difference with controls indicating that BnA10.FLC maybe partially repress the functions of qcFT.C6.5. The phenotype of NILs was much earlier than Ningyou 7 in winter-and spring-cropped environments showed the alleles of Tapidor controlling flowering had much stronger functions than those of Ningyou 7, which indicated the alleles of Tapidor in the context of Ningyou 7 genome were not repressed and continued to promote flowering. To test whether the alleles of Tapidor promote flowering universally in the context of spring type B. napus, the NILs of qcFT.C6 crossed with five spring type cultivars (Westar, Monty, Andor, Bronowski and NRS-1). The flowering time of five type F1 plants was all much earlier than the spring type parents indicating that the alleles of Tapidor were not repressed to promote flowering in the context of other spring type B. napus.6. The BAC library (JBnB) was probed with FT fragment amplified from Tapidor genome and six complete sequences of FT copies were isolated from the target BAC clones. The six BnFT paralogues were mapped to four linkage groups of the TN genetic map, two in the A genome and two in the C genome. The BnFT paralogues within B. napus were named as follows:BnA2.FT, BnA7.FT.a, BnA7.FT.b, BnC2.FT, BnC6.FT.a and BnC6.FT.b according to their mapped chromosomes. In silico mapping indicates that all of the genomic regions containing BnFT paralogues corresponded to block E in chromosome 1 of Arabidopsis, although block E was inversely duplicated on linkage groups A7 and C6, where it forms inverted duplication blocks (IDB). Based on marker identity, the BnFT paralogues in the IDBs were close to the junctions of each duplicated block.7. Over the coding sequence the six BnFT paralogues had 85-87% identity with AtFT, 81-83% with AtTSF, and 92-99% with each other. Interestingly, the paralogues within homeologous regions of the B. napus A and C genomes, such as BnA2.FT and BnC2.FT, showed the highest nucleotide identity (99%), whilst the paralogues present within inverse duplicated regions, such as BnC6.FT.a and BnC6.FT.b, showed higher identity (97%) than among other pairs of paralogues, such as BnA2.FT and BnC6.FT.a (92%). The degree of amino acid identity showed the same relationships as the coding sequences.8. The phylogenetic relationships among FT-like genes from B. napus, B. rapa, Arabidopsis, poplar, rice and barley were analyzed, with a phylogenetic tree constructed from the amino acid sequences of these genes. Four groups could be identified:Hd3a and HvFT1, ptFT2, AtFT, and Brassica FT genes. The classification of species was consistent with established divergence of plant taxa. It was clear that the extant Brassica genes derived from two ancestral lineages, Bx.FT-1 and Bx.FT-2. Subsequently, Bx.FT-1 gave rise to formation of Bn.A2.FT, BnC2.FT and BrA2.FT. However, a segmental chromosome duplication event involving Bx.FT-2 appears to have given rise to Bx.FT-2a and Bx.FT-2b, which accounts for the homeologous copies in the A and C genomes, BnA7.FT.a and BnC6.FT.a, BnA7.FT.b and BnC6.FT.b, respectively. A characteristic CArG box, which acts as the FLC protein binding site, was detected in the first intron of four BnFT paralogues. However, it was absent in the other two copies which otherwise appear to be derived from Bx.FT-1. Interestingly, the CArG box was also not detected in the intron 1 regions of PtFT2, Hd3a and HvFT1.9. The Ks values between AtFT and eight Brassica FT genes (from 0.33 to 0.36) suggest a divergence time of 12 to 13 MYA between Brassica and Arabidopsis. The Ks values between Bx.FT-1 and Bx.FT-2 (0.17 to 0.19), Bx.FT-2a and Bx.FT-2b (0.071 to 0.072) suggest that the first and second duplications may have occurred in Brassica～6-7 MYA and 2.5 MYA, respectively. The divergence of A and C genomes (0.041-0.06) may have occurred at 1.5-2 MYA, with natural B. napus (0.0033-0.0058) calculated to have emerged approximately 0.12-0.21 MYA. The order of the evolutionary events calculated by Ks values is consistent with the established phylogenetic relationship of the FT genes.10. Three BnFT paralogues, BnA2.FT, BnC6.FT.a and BnC6.FT.b, were mapped to the major QTL region for flowering time in A2 and C6 chromosomes. To test whether the BnFT paralogues represented candidate genes of QTL for flowering time, two near isogenic lines (NILs) with different combinations of introgressed segments on C6 flowered much earlier than Ningyou 7. NIL-5, which had a small additional introgressed fragment containing two BnC6.FT alleles of Tapidor, significantly flowered earlier than NIL-9 which lacked Tapidor alleles. To further confirm whether the BnFT candidates significantly affected flowering time in B. napus, Bn.C6.FT.a, BnC6.FT.b and BnA2.FT were subjected to association mapping analysis with 35 spring and 20 winter cultivars. The flowering time of the accessions was investigated in vernalization-free conditions, and the genotypes tested with the PCR markers. The alleles of Ningyou 7 and Tapidor were significantly ubiquitous in the spring and winter cultivars.11. Based on marker identity, total ten IDBs containing two IDBs of A7 and C6 chromosomes were detected within the TNDH linkage map associated with eight chromosomes. The IDB regions covered a quarter of the whole linkage groups of B. napus that could align with the genome of Arabidopsis. The majority of IDBs were detected in the A genome and not in the corresponding regions of the C genome, since most markers had been developed from B. rapa BACs. Finally, the result of BAC-FISH showed block E formed the IDBs in the genome of B. napus.