| Chinese cabbage(Brassica campestris L.ssp. pekinensis) is originated in China, it's one of the most important vegetable crops of Brassica species. It is thermophilic but can't withstand too high or too low temperature. Chinese Cabbage belongs to the type of plants that the seeds of which can be vernalized, germimated seeds could response to the low-temperature to complete vernalization and bolt after a transition from vegetative growth to reproductive growth under a certain sunlight condition. In spring and summer, the changes of temperature and the increase of day length are conductive for the reproductive growth of cabbage, and it can impact the vegetable's quality and yield badly. It's of great urgency to study the bolting-resistant basic theories and breed species with strong character which is appropriate to grow in low temperature and bolting-resistant.In present study, we designed and synthesized the primers based on the sequences of BrFLC2, AtFRI, AtVIN3 and cloned certain sequences of BrFLC2, BrFRI and BrVIN3 which were then analyzed on InDel and SNP to develop the association study and functional mark between sequences variation and bolting time. We used the CAPS, AS-PCR, SSR, EST-SSR and SRAP markers to find out the bolting time QTL localization with F2 lines constructed on inbred lines C3-1 and B2-2. We took an Multiple Sequence Alignment using the cloned sequences of BrFRI, BrVIN3, AtFRI, BoFRI and BoVIN3, AtVIN3 and studied their homology and difference. Main results are as follows:1 There are 34 mutations which include 32 SNPs, 2 single-base InDels in the sequence exon2 to exon7 of BrFLC2 in 20 Chinese Cabbage inbred lines. Correlation analysis showed that one A-G(BrFLC2-1660) variation in intron 6 has highly significant positive correlation with the bolting time and the flowering time. Its correlation with the bolting time and flowering time is r= 0.77** and r= 0.79** (**p <0.01) respectively.2 Part of the homologous sequence of AtFRI was cloned in two inbred lines of Chinese Cabbage P1 (C3-1, late boltinging), P2 (B2-2, early bolting). By comparison we found that BrFRIP2 was 10 base longer than BrFRIP1 and there were 18 SNPs and 4 InDels between them. According to the differences of restriction site and the specific primers, we designed a CAPS marker: BrFRI-Bglâ… and an AS-PCR marker: Fras1 successfully, and they could be used for gene mapping. There was an amino acid sequence which belongs to a frigida superfamily in both of the infered amino acid sequence of the partial sequence of BrFRIP1 and BrFRIP2. However, compared with P1 the amino sequence of P2 lacks the 7 amino acids at the beginning but had 37 amino acids more in the end. The two inferred amino acid sequence had a similarity of 91.6%.3 Part of the homologous sequence of AtVIN3 was cloned in two inbred lines of Chinese Cabbage P1 (C3-1, late boltinging), P2 (B2-2, early bolting). By comparison we found that they both had 967 bases and there were 4 SNPs between them. The similarity of the infered amino acid sequence of the partial sequence of BrVIN3P1 and VIN3P2 was 99%.4 We cloned part of the homologous sequence of AtFRI and AtVIN3 in a Cabbage variety(8398). They have 1304 bases and 967 bases respectively. Through the analysis of the four partial sequence of BrFRIP1, BrFRIP2, BoFRI and AtFRI, we found that the similarity of the partial sequence of FRI in Chinese Cabbage and Cabbage was more than 93%. Compared with the AtFRI, the similarity of them were both less than 80%. By the evolutionary analysis of the four partial sequence, we found that BoFRI had a smallest difference with the ancestor's gene. Besides, through the analysis of the four partial sequence of BrVIN3P1, BrVIN3P2, BoVIN3 and AtVIN3, we found that the similarity of the partial sequence of VIN3 in Chinese Cabbage and Cabbage was more than 99%. Compared with the AtVIN3, the similarity of them were both less than 86%. The evolutionary analysis of the four partial sequence showed that BrVIN3P1 had a smallest difference with the ancestor's gene, then followed was the BoVIN3. The BrVIN3 of P2 which bolts and flowers early was more similar to BoVIN3.5 Using DNA pooling analysis method, the C3-1 and B2-2 were used as the template. 82 pairs of EST-SSR primers, 23 pairs of anchored SSR primers, 7 pairs of SSR primers and 320 pairs of SRAP primers were screened. 7 pairs of EST-SSR primers, 9 pairs of anchored SSR primers, 3 pairs of SSR primers and 11 pairs of SRAP primers were selected out because they can get clear and stable lines among P1, P2 and F1.6 By the CAPS, AS-PCR, EST-SSR, SSR and SRAP analysis of F2 groups, we constructed 5 linkage maps containing 21 markers by Mapmaker/EXP version 3.0 software. 4 linkage maps could correspond to the international Chinese Cabbage A genome linkage groups. Then, we found 2 QTLs locations related with the bolting time: qbt-1, qbt-2 and 2 QTLs locations related with the flowering time: qft-1,qft-2 by Win QTL Cart 2.0 software using composite interval mapping method. These QTL locations were distributed in two linkage groups, and the contribution rate was 17%, 7%, 16% and 9% separately. |
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