Molecular Tagging Of Flowering Time-related Genes As Well As Cloning And Expression Analysis Of Vernalization-related Genes In Brassica Rapa

Brassica rapa ssp. chinensis var. communis and B. rapa ssp. chinensis var. parachinensis is the extreme representative of flowering habit, and the former is a winter biennial, and need a prolonged period of cold time, a process named vernalization to induce flowering, the latter, however, is a summer annual dose not need vernalization to induce flowering. In cultivation practice, premature bolting is serious problem that not only cause a great of loss in production, but also reduce the commercial value. So bolting-resistance is an important breeding goal.In the present study, a F₂ segregating population derived a cross between a flowering Chinese cabbage cultivar 'Sijiu' (B. rapa ssp. chinensis var. parachinensis cv. Sijiu) and Chinese cabbage-pak-choi cultivar 'Shanghai-qing' (B. rapa ssp. chinensis var. communis cv. Shanghai-qing) was developed for the the study of genetic control of flowering time.A trait-based analysis methodology, bulked segregant analysis (BSA)was adopted to make up two bulks that were composed of individuals of extreme early flowering and extreme late flowering .Bulked segregant analysis in combination of AFLP techoneques was used to screen the two bulks for differential bands that may be candidate marker associated with early flowering habit or late flowering habit. On the other hand, 'Shanghai-qing' was artificially vernalized for the study on the morphological differentiation of floral bud in Chinese cabbage-pak-choi, meanwhile RNA was extrated from the SAM of 'Shanghai-qing' pak-choi at different time point of vernalization treatment, and from the leaf of non-vernalized 'Shanghai-qing' and 'Sijiu' that was used for the cloning of vernalization-related gene by PCR method and for the expression analysis of them by RT-PCR in the different differentiation stage of bud .The main result was listed as follows:(1) Sevral specific AFLP bands between two bulks were obtained by BSA-AFLP analysis.Among them, E2-AAG/M8-CTT317 and E6- ACG/M6-CTC₂₆₆ is specific to the extreme late flowering bulks and parent 'Shanghai-qing';and E3-ACA/M1-CAA₂₉₅, E8-AGG/M3-CAG₂₇₀, E8-AGG/M4-CAT₁₇₄ is specific to the extreme early flowering bulks and parent 'Sijiu'. Then, three of them was converted to SCAR or CAPS marker, and were named E3MlSCAR₂₇₁, E8M3SCAR₁₅₁ and E6M6CAPS_238/168. The segregant analysis in F₂ segregating population show E3MlSCAR₂₇₁ and E8M3SCAR₁₅₁ is linked to a putative flowering time QTL, flanking it on both side .being away from it 21.3 cM and 28.5 cM.(2) 'Shanghai-qing' was artificially vernalized for the study on the morphological differentiation offloral bud in Chinese cabbage-pak-choi, the results show treatment for 20 d at 4"C is sufficient for the transition from vegetative growth to reproductive development.(3) The nucleotide sequence of intron 1 of 'Shanghai-qing' and 'Sijiu' show no substantial difference. RT-PCR analysis of the expression of flowering time-related genes BrcFLCJircFCA and BrcFT shows that the expression level of BrcFLC is reduced gradually and was at very low level after 20 days of vernalization treatment ,and was undetectable after 30 days of vernalization treatment, which is consistent with the fact that treatment for 20 d at 4*C is sufficient for the transition from vegetative growth to reproductive developmentThe expression of BrcFT was progressively enhanced, which show an inverse expression pattern compared with that of BrcFLC. The expression level of BrcFCA was only slightly elevated with time.