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Map-based Cloning of Flowering Time QTL--qFT10-4-and Expression Regulation of the Candidate Gene BnFLC.A10in Brassica Napus L.

Author: HouJinNa
Tutor: MengJinLing
School: Huazhong Agricultural University
Course: Crop Genetics and Breeding
Keywords: Brassica napus Flowering Vernalization map-based cloning FLOWERING LOCUS C MITE
CLC: S565.4
Type: PhD thesis
Year: 2013
Downloads: 46
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Abstract

There is a network about interaction between various environmental signals and flowering genes in plants. Elucidating the molecular mechanisms of the network underlying flowering is critical to develop strategies for manipulating and optimizing the flowering times to maximize crop yields. Rapeseed (Brassica napus L.) was bred three types-winter-, semi-winter and spring-type for adapting to different growing seasons. The molecular basis underlying the evolutionary transition from spring-to winter-type rapeseed is not known, however, and needs to be elucidated.In this research, backcrossed populations developed from TN-DH population (a doubled haploid (DH) mapping population of rapeseed derived from a cross between winter rapesseed Tapidor and semi-winter rapeseed Ningyou7) were used for fine-mapping the spring environment specific quantitative trait locus (QTL) for flowering time, qFT10-4. The candidate gene BnFLC.A10, an ortholog of FLC in Arabidopsis, was cloned from the QTL. The regulation mechanism of BnFLC.A10expression was elucidated. The significance of BnFLC.A10behind domestication of rapeseed and winter rapeseed formation also be discussed. Here are the main results:1. To construct a high-resolution map of the qFT10-4locus, we analyzed a large BC5F2population (9,000plants) that was derived from a cross between the DH line TN DH043(winter-type) and Ningyou7(semi-winter-type). Four molecular markers developed from the sequence of the BAC clone JBnB75D10, which contains BnFLC.A10, were used for the analysis. Eight recombinants were identified and the QTL qFT10-4was delimited to an80-kb region that showed collinearity with the top of chromosome5of Arabidopsis thaliana. None of the genes in this region except FLC are known to be involved in floral transition.2. To analyze the basis of the vernalization requirement in rapeseed, we cloned and compared BnFLC.A10sequences from Tapidor and Ningyou7. No polymorphism was found in the coding sequence between the two alleles. However, there were two insertion/deletions (indels Ⅰ and Ⅱ) in the upstream region, together with two indels (indels Ⅲ and Ⅳ) and eight single nucleotide polymorphisms (SNPs1-8) in intron1of BnFLC.A10. Expression analysis showed that BnFLC.A10-N was markedly down- regulated upon exposure to cold treatment after1week, whereas expression of BnFLC.A10-T decreased gradually over7weeks of cold treatment.3. We conducted an association analysis using a panel of79diverse rapeseed cultivars representing winter, semi-winter and spring genotypes. All of the cultivars were planted in spring environments. The621-bp insertion showed a highly significant correlation with flowering phenotype. Haplotyping of BnFLC.A10specific markers for indels I-IV and SNPs1-6also suggest that indel I plays a very important role in modulating flowering time in natural rapeseed germplasm and potential development of a winter growth habit.4. The inserted sequence possessed typical characteristics of a Tourist-like MITE, with14-bp TIR sequences flanked by TSDs of TAA. Between the TIR sequences, an AT-rich (67%) core that contained12classes of important motifs was identified. It is the first MITE discovered in B. napus genome and be named BnTrst-1.5. The similar sequences of the BnTrst-1were screened in the published sequence database of B. rapa and B. oleracea genome. Seventy-five and70intact copies were found in B. rapa and B. olerecea, respectively. The intact copies of BnTrst-1were extracted from B. rapa and B. oleracea genome. Analyzed the distribution and characterization of BnTrst-1in A and C genome in Brassicacea. The nucleotide composition and variation of TIR and TSD also be analyzed. Provide experimental evidence to MITE activities caused inter-and intra-diversities. MITE activities also influence genomic structure and size which provide fuel for selection about adaptation.6. To understand the evolutionary process behind the adaptation associated with the insertion of BnTrst-1into the upstream region of BnFLC.A10and to trace its origin and transmission, we investigated an additional154spring cultivars of B. napus and103cultivars (including the genome sequenced cultivar, Chiifu) belonging to nine subspecies of B. rapa (oilseed, swede and fodder types). No BnTrst-1insertion was detected in the upstream region of BnFLC.A10in any of the accessions, even though the empty site of insertion was almost100%identical to the sequences that flanking the BnTrst-1insertion in BnFLC.A10in winter rapeseed. On the other hand, hundreds of copies of BnTrst-1were detected in the whole genome, but not in the BrFLC.A10upstream region of B. rapa ’Chiifu’. This suggests that BnTrst-1may have pre-existed in the B. rapa genome but was inactive, after the generation of B. napus, it was activated and inserted into the upstream region of BnFLC.A10, giving rise to winter rapeseed.7. Primary elucidates the molecular mechnasim of BnTrst-1induced gene regulation. Between the TIR sequences, an AT-rich (67%) core that contained12classes of important motifs was identified. These motifs might function in transcriptional initiation or promotion, or in response to different stimuli and signals. The actual protein binding ability of BnTrst-1insertion was evaluated using EMSAs. Nuclear protein(s) extracted from Tapidor and Ningyou7before vernalization were able to bind to some fragments from the middle of the BnTrst-1that contained TATA box motifs. These results suggest that BnTrst-1can bind to specific transcription factors that may initiate or enhance BnFLC.A10expression in winter rapeseed cultivars, giving rise to their stronger vernalization requirement. DNA methylation level of MITEs located in different position and its flanking region show different profiles. DNA methylation level might also refer to MITE induced gene regulation.

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