Screening Of Taxoids Metabolism-mutations And Linkage Map Constraction Of Taxus

Taxus chinensis var. mairei, as ancient relic perennial woody plants, dioecious, which have important ornamental value, medicinal value, economic value and research value, is belonging to the Taxaceae, Taxus. For its wild resources protection by laws, the main resource utilization is by artificial plantations growing development. There is little genetic and genomic information available in Taxus plant, especially effective DNA genetic markers. In this study, the endosperm of T. chinensis var. mairei were used as raw material for callus induction, and a population of endosperm callus lines (TH) were abtained from the same female parent. The facters affecting on embryo seed plantlets of T. chinensis var. mairei was studied by tissue culture, and a half-sib population of embryonic cell lines were acquired by embryos and plantlets intro culture. The four taxanes content variaton, which were10-deacetyl baccatin Ⅲ, baccatin Ⅲ, cephalomannine and taxel, of the population of TH cell lines and the half-sib cell lines, was detective by LC-ESI-MS. The public EST database, fosmid library sequence and transcriptome database was used to mining of large-scale SSR molecular markers. The genetic diversity of half-sib cell lines of T. chinensis var. mairei were studied based on EST-SSR genetic markers. And based on which of190TH, a linkage map using EST-SSR markers was constructed. On the whole, the studies are facilitated to enhance the breeding technology and selection efficiency of T. chinensis var. mairei. The main results and conclusions are summarized as follows:1. The callus induction ratio was70.89%that separated the endsperm from the seeds, using steriled water soaking the seeds for3days, and cultured on B5medium supplementd with2,4-D2.5mg·L-1,6-BA0.5mg·L-1, sucrose30g·L-1, and agar6g·L-1, at darkness. The callus was haploid tissues identified by flow cytometric analysis. The genome size of female T. chinensis var. mairei was23.28pg(2C).2. The embryos, beening separated from the seeds by soaking in sterile water for3to5days, cultured on BLG mediu supplemented with acticarbon5g·L-1, sucrose30g·L-1, agar6g·L-1, at temperature of24centigrade for40days, afer which embryos were grow seedings. The embryomic callus, with loose and fast-growth, were obtained by separated the embryos and cultured on BLG medium supplemented with2,4-D2.5mg·L-1,6-BA0.5mg·L-1, sucrose30g·L-1, and agar6mg·L-1, and the plantlets supplementd with2,4-D2.0mg·L-1,6-BA0.5mg·L-1, sucrose30g’L"1, and agar6mg·L-1, at temperature of24centigrade in darkness condition.3. The metobilsm of4Taxanes content was dissimilar among different individuals, including of99half-sib cell lines and27TH cell lines. The2012-32cell line and2012-197with higher Taxus content that were53.30mg·100g-1and60.78mg-100g-1was got by LC-ESI-MS detectived. There was only one TH cell line (EH-17) found taxus with0.48mg-100g-1.4. By analysis and assembled of the510EST sequences,1923fosmid library sequences, and4transcriptome databases of T. chinensis var. mairei from NCBI, a unigene databases was abtained. It was found that the redundancy of EST sequences was approximately24.12%, and the fosmid library sequences was68.90%, and the transcriptome databases were range from37.92%to99.30%.5. A total of12SSRs were identified at a frequency of3.10%from EST unigenes by MISA program, and the dominant SSR repeated motifs was hexunucleotid (41.67%).96SSRs were exploited at a frequency of4.32%from fosmid library unigenes by MISA program, and the dominant SSR repeated motifs was Dinucleotid (51.04%).2160SSRs were obtained at a frequency of2.24%from trancriptome unigenes of T. chinensis var. mairei by MISA program, and the dominant SSR repeated motifs were trinucleotid (37.08%) and hexunucleotid (37.08%).6. By compared with the distribution of the repeat motifs of SSRs in the transcriptome of T. baccata, T. cuspidata Sieb. et Zucc, T. cuspidata (Japanese Yew) and T. chinensis var. mairei, the results indicated that the average density of SSRs frequency was higher on454NGS platform than Illumina, and the dominant SSR motifs was AG/CT and AT/AT, and lowest ratio of CCG/CGG.7.196primers were selected from designed SSR primers by primer3, and105effective amplification SSR primers and94polymorphsim SSR were found. The effective amplification ratio and polymorphism ration were53.57%and48.98%, respectively. That9pairs of T. baccata EST-SSRs and3pairs of T. cuspidata (Japanese Yew) EST-SSRs were tranfered to T. chinensis var. mairei. 8. The genetic diversity of96half-sib cell lines of T. chinensis var. mairei was studied by using94pair SSR primers exploited above. The polymorphism information content (PIC index) was among0.06to0.69, and the average PIC was0.56, with55pair SSRs of PIC value higher than0.25. The genetic diversity index (H) was range from0.02to0.05, and the average number was0.39. The expected heterozygosity (He) and unbiased expected heterozygosity (uHe) was among0.18to0.50, and the average number was0.39. The observation F-stastic and mean the F-stastic value were range from0.50to0.98, and0.77to0.84, respectively. And the average number was0.61and0.0.81. The results of clustering analysis and PCoA on the96half-sib cell lines was supported that under certain conditions the natural pollination habit had the king structural characteristics.9. A linkage map, based on190TH cell lines, including49EST-SSR of52primers amplified well which selected from94primers, was constructed. The framework linkage map of T. chinensis var. mairei contained23linkage groups (LGs). The total genetic distance was213.00cM, with the average distance was4.63cM.21EST-SSR showed segregation distortion.12of them were clusters distribution in LG1linkage group, and6of them were distribution in6dissimilar linkage groups.