Evaluation And Molecular Marker Of The Germplasm Resources Of Chaenomeles By SRAP

Chaenomeles were originated in China, as famous ornamental plants and traditionalChinese medicinal materials, they have been widely used in landscaping and the produce ofmedicine, food and cosmetics. The Chaenomeles have been cultivated for more than3000years. Because of their easy interspecific hybridization and strong plasticity of form, theyhave formed a lot of cultivars and variations, which is value in commercial horticulture but isdifficult in classification.Based on the survey of germplasm resources in China,98germplasms were analysed bySRAP molecular technique in this research. According to SRAP markers, the germplasmswere clustered, the molecular identification key of Chaenomeles was compiled, and the coregermplasm of Chaenomeles was constructed. This research will lay the foundations for theidentification of cultivars, the selection of cross parent in breeding and the protection ofgermplasm resources in Chaenomeles.The main results were as follows:1. The quadratic orthogonal rotatable combinatorial design was conducted to establishthe reaction system for SRAP-PCR of Chaenomeles. The total25μl reaction volume incluing1.5mmol L^-1of Mg²⁺,0.4mmol L^-1of dNTPs,0.379μmol L^-1of each primer,2U of TaqDNA polymerse,67.15ng of DNA template. The influence of each factor on the PCR reactionsystem is Taq DNA polymerse> Mg²⁺> dNTPs> DNA template> primers.2. The single factor affected the PCR result was discussed, and the interactive effectswithin the five PCR factors were analyzed by SAS software. Some regularities were summedup and they could give references for the other optimization of PCR system.3.17primer pairs were selected from157pairs, which were applied to study the geneticdiversity and relationships of the materials.17primer pairs produced525polymorphic loci,the percentage of polymorphic loci was100%, and each primer combination produced30.9polymorphic loci; the Effective number of allele （Ne） was1.0471¹.2274with an average of1.1289; the Nei’s genetic diversity （H） was0.0428⁰.1652with an average of0.1007; theShannon’s information index （I） was0.0986⁰.2863with an average of0.1911.4. The Nei’s genetic similarity coefficient ranged from0to0.6596with an average of0.2192, showed that the genetic relationship of the materials was far. By UPGMA clusteranalysis, the98materials can be divided into9groups. The species origin should be considered firstly in the classification of Chaenomeles, then the flower type and originatedplace. The genetic diversity analysis of groups showed that, the genetic diversity existedamong groups was low （Gst=0.2317）, the genetic distances among groups were short and thegene flow was high （Nm=1.6580）, they all conformed the present situation that theChaenomeles are easy to hybridize and there are plenty of cultivars and variations.5.6core primer pairs were selected to establish the fingerprint of Chaenomeles, thematerials can be distinguished by the combination of primer pair ME3-EM3and one of theother5primer pairs. Reference to the plant key, the molecular identification key of98germplasms of Chaenomeles was compiled, according to the19polymorphic loci selectedfrom the amplification results of primer pair ME3-EM3and ME6-EM1.6. Based on the SRAP amplification results, the two methods of core germplasmconstruction, no-grouping gradual clustering method and grouping gradual clustering method,were compared at different sampling ratios. Furthermore, combined with C programminglanguage, an appropritate method was invented, called the most-NPL method. Compared withthe others, the new method was more simple and efficient, which can reach the coregermplasm standard with low sampling ratio.7. The genomic DNA of C.sinensis was successfully extracted for the first time by theimproved CTAB method.