| Camellia oleifera germplasm resources have some economic traits and the corresponding biological and ecological characteristics.As a cross-pollination plant, a rich variation of groups had been generated by the natural hybridization, it is the base to generate superior germplasm. Camellia species with superior traits had been scattered in different regions, found by the survey with a long and a large number of observations. In order to better compare with the main characteristics and economic traits and in-depth study on the value of their identity and comprehensive assessment, the need of collection and preservation of Camellia germplasm resources and evaluation had been asked. 75 samples of Camellia germplasm resources from main producing provinces of the National as the study material, the varieties of the genetic differences and their genetic relationship had been identified by the morphologic marker and ISSR SRAP molecular marker ,and the national Camellia germplasm molecular identification system had been built up initially in the paper. The results and conclusions were showed as follows:(1) This study has collected and preserved 35 samples of represented Camellia oleifera fine cultivars from Minhou county in Fuzhou, Lingcheng County and Shanghang County in Longyan , Qingliu County,Datian County and Youxi County in Sanming , Pucheng County in Nanping , Xiapu County in Ningde , and other kinds of sources in Fujian Province; 22 samples of excellent series of parents and hybrid F1 generation, introduced species such as Camellia yuhsiensis, Camellia oleifera'Cenxiruanzhi'in Tongkou forestry farm of Fujian Province, as well as the superior clones in the main producing provinces, including 15 fine clones from Jiangxi Province such as Changlin 166 #, 6 fine clones from Hunan Province such as Xianglin 47#, Zhe 53# from Zhejiang Province and so on. The Camellia fruit indicator data of 75 samples showed the rich diversity though variance analysis, correlation analysis, principal component analysis, cluster analysis.(2) A set of modified CTAB method for the Camellia oleifera genomic DNA extraction had been explored, which also had been applied to the extraction of old leaves of Camellia oleifera. By using Purification kit,the OD260/OD280 ratio of the purified genomic DNA samples of Camellia oleifera was between 1.70-1.90, which was in full compliance with ISSR-PCR and SRAP-PCR amplification of the template conditions.(3) Through the univariate analysis and orthogonal analysis,the ISSR and SRAP molecular markers amplification reaction had been optimized: ISSR-PCR system optimization result was Mg2 + concentration of 2.00 mmol/L, dNTP concentration of 0.2mmol/L, Taq polymerase concentration of 1U, Primer concentrations of 0.2 mmol/L, and the amount of DNA template was 30ng; SRAP-PCR system optimization result was Mg2 + concentration of 2.25mmol/L, dNTP concentration of 0.20mmol/L, each Primer concentration of the 1.0umol/L, Taq polymerase concentration of 1.25U, and the amount of DNA template was 30ng.(4) 75 samples of Camellia oleifera had been amplified by the 20 primers (complex) of ISSR markers and SRAP marker.The total site number, polymorphic loci, public loci, the average the proportion of polymorphic loci (PPB), and polymorphic bands range,the parameters of genetic diversity ,and SRAP marker ISSR markers observed number of alleles (Na), effective number of alleles (Ne) number, the group of Shannon diversity index (I), Nei's genetic diversity (H) , the group of total gene diversity (Ht), intra-population genetic diversity (Hs), genetic differentiation coefficient (Gst), gene flow (Nm) values and the total population gene flow (Nm) values of 75 samples of Camellia oleifera revealed that both the total group of the tested samples and six groups existed rich genetic diversity.(5) Based on ISSR-PCR and SRAP-PCR results, according to Nei & Li (Czekanowski 1913) similarity coefficient of 75 samples of Camellia oleifera and UPGMA method for cluster analysis of the tested samples. ISSR-PCR clustering results showed that when the average genetic distance GD values was about 0.40, the 75 samples were divided into four broad categories: Camellia oleifera, C.meiocarpa, soft branches Camellia and C.chekangoleosa; SRAP-PCR clustering showed that when the average genetic distance GD values was about 0.34, the 75 samples were divided into five categories: Camellia oleifera, C.meiocarpa, C.yuhsiensis + Camellia from Lincheng county, Camellia oleifera'Cenxiruanzhi'and C.chekangoleosa.The genetic differences and their relationship of Camellia oleifera had been basically discerned.(6) By 1 / 0 matrix data analysis and DNA fingerprint comparison , 11 samples had been identified by 11 specific marker loci of the 9 ISSR primers, 14 samples had been identified by 18 site-specific markers of 10 pairs of SRAP primer simultaneity, even a sample had been identified by 2-3 pair of SRAP primer. The sample as Minyou 48, Fulin-1 had been simultaneously identified by two kinds of molecular Markers , the correctness by molecules identification had been further improved. The number of samples reached 23 ,which had been identified by the two kinds of molecular markers, and the molecular identification card of Camellia oleifera varieties had been listed.It is valuable to reveale the genetic diversity of Camellia oleifera germplasm resources in China by the morphological classification and ISSR SRAP molecular marker, in order to establish and improve the gene pool of Camellia oleifera germplasm, lay the foundation for further hybridization and selection and genetic improvement. |
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