Study On Screening For Molecular Maker Of Male Sterility Gene And Mapping Of Bolting Gene Of Brassica Campestris L.ssp. Chinensis Var. Utilis Tsen Et Lee

Brassica campestris L. ssp. Chinensis var. utilis Tsen et Lee belongs to Cruciferae Brassica and is native to China, and is one of important vegetables mainly grown in the wide area of South China.Researches on male sterility and bolting gene of Brassica campestris L. ssp. Chinensis var. utilis with molecular makers play an important role in innovation of germplasmsa and breeding for new varieties. It hasn't been reported that ISSR is used to analysis male sterility gene and bolting gene of Brassica campestris L. ssp. Chinensis var. utilis. Orthogonal design was used to optomize the ISSR reaction system in the present research. The genomic DNA of male sterile line and its maintainer line of Brassica campestris L. ssp. Chinensis var. communis was studied with ISSR technique. In order to support the use of the male sterility of Brassica campestris L. ssp. Chinensis var. utilis ISSR technique was used to screen the molecular marker linked to the male sterility and cloning of the male sterility was carried out. This experiment employed ISSR and SRAP technicology with the method of bulked segregant analysis to screen the specific molecular mark linked to bolting gene. Four molecular makers of ISSR and SRAP linked to bolting gene were found. The molecular fingerprint of one hybrid of Brassica campestris L. ssp. Chinensis var.utilis was established by using two molecular markers, ISSR and SRAP and then transformed into digital fingerprints. The main results are as follows:1.In this study,orthogonal design was used to optimize ISSR amplification system on Brassica campestris L. ssp. Chinensis var. utilis in four factors(Taq DNA polymerase, Dntp, Primer, Mg2+) at three levels respectively. A better ISSR reaction system was established, namely 20μL reaction system containing TaqDNA polymerase1U, dNTP 250μmol/L, Primer 0.25μmol/L, 1×PCR buffer, Mg2+ 2.5mmol/L, template DNA 30ng. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. It proved the basis for the analysis of diversity,map construction and gene localization of important traits in Brassica campestris L. ssp. Chinensis var. utilis with ISSR markers.2.The genomic DNA of male sterile line and its maintainer line of Brassica campestris L. ssp. Chinensis var. was studied with ISSR technique. At the same time the homology-based candidate gene method was used to identify the specific PCR markers linked to cytoplasmic male sterility in Brassica campestris L. ssp. Chinensis var. utilis. By searching the DNA database of NCBI, correlative genes or open reading frames were identified. Based on the method of biosoft analysis and the conservative regions, one specific primers were designed to amplify the polymorphism of genomic DNA from sterile line and its maintainer line. The result showed that only primer UBC843 amplified different band between sterile line and its maintainer line. The specific fragment was cloned and DNA sequence was determined. The size of specific fragment was 462bp. Comparison between the specific fragment and other specific sequence derived from Chinese cabbage revealed that the homology generally was below 50%. According to the result sequenced, one pair of primers was designed to develop a more stable SCAR marker from the ISSR maker. CMS primer produced a 675bp specific fragment in sterile line. Analysis of the sequence indicated it was homologous 100% with orf224 of PolCMS. The specific fragment amplified by ISSR primers was likely from nuclear DNA. CMS specific amplification results showed that male sterility of the present experimental material was Pol-type cytoplasm sterile. 3.The F₂ population derived from a cross between CT07(early bolting) and T0601(late bolting) was used to identify bolting trait using ISSR and SRAP makers. 45 ISSR primers and 20 SRAP primers combinations were used to screen polymorphism in parents and DNA pools. PCR pattern in parents and DNA pools showed different fragment appeared in father and early bolting pools. Bolting trait was linked with four makers and the closest marker to bolting gene was E13M4 and the distance between them was 16.8 cM. The four makers in the study could be used to maker assistant breeding.4.One hybrid and its parents of Brassica campestris L. ssp. Chinensis var.utilis were analyzed by using ISSR and SRAP. The results indicated that three ISSR primers and three pairs of SRAP primers could produce parental complementary characteristic bands for the hybrid and its parents, and two ISSR primers could distinguish availably parents and their F1. The molecular fingerprint of one hybrid of Brassica campestris L. ssp. Chinensis var. utilis was established, and then transformed into digital fingerprints. It provided a new technology and an important means for identification of seed purity and was good for the protection of intellectual property of new varieties.