Genetic Analysis And Identification Of Cultivated Varieties Of Chestnut (Castanea Mollissima) By RAPD Molecular Markers

Chinese chestnut is of high economic value .It has a long cultivation history and abundant cultivar resources. The nut is rich of nutrients. It is also useful for medical use, landscape architecture and high quality timber production because it has the beautiful crow shape and strong resistance to air pollution. The critical problem for nut production is low nut yield. It's the most important factor to use the improved cultivars for chestnut intensive cultivation.The traditional taxonomy of chestnut variety is based on the morphological and phonological traits, biological characteristics and especially on the economical traits. The major traits are the morphology of anthotaxy, bract and nut. But it's a long time for woody plants to set the seeds. So, it is very difficulty to identify the genuine of chestnut variety based on the vegetative tissue or organ. Because of the low level of domestication, the nomination is confused sometime, such as the same given name, but the different cultivars. Even someone take the sexual production progeny as the cultivar. This makes variety identification more difficulty and it is necessary and urgent to identify the genuine of cultivars. The suitable law for new variety certification, registration, protection and management should be established. It also needs the precise, reliable and standardized procedure to detect the genuine of varieties. This method should not be affected by environmental factors and the development stage of plant. It makes it possible to detect the variety in early stage.The genetic diversity of chestnut was studied by RAPD molecular markers. The RAPD standardized procedure and the fingerprints database of chestnut was established. This is the principal basement of chestnut breeding and supply the precise and reliable method identified the chestnut variety. This research also make the comparison between the traditional taxonomic method and molecular markers.The main results and conclusion are as follows:1. The DNA extraction and purification of chestnut is very difficulty because of the high level of phenols and tannin substances. The improved CTAB method was putforward in this study. It was summarized the satisfacory method of DNA extraction and purification which can amplified DNA segment.2. The reliable RAPD reaction system was established. The annealing temperature and Mg2+,which affect the RAPD reaction effect and polymorphism, were investigated. The reaction volume is 20 μl. RAPD program is: 2min at 940C for pre-denaturation; then 38 cycles of 30s at 940C for denaturation, 30s at 400C for annealing, 90s at 720C for extension; finally extension at 720C for 7min. Sixteen primers were selected from 100 primers tested on the base of the number and frequency of polymorphisms produced among 46 chestnut varieties. With the 16 primers,119 bands were produced. The 69 bands out of the 119 were polymorphic(58%).This standardized procedure established in this experiment research is the foundation for RAPD molecular marker research and also useful for completion of chestnut DNA fingerprints database.3. The genetic diversity of varieties was analyzed. With 16 primers,69 polymorphic loci were detected for 46 chestnut varieties. It was shown that the genetic diversity vary greatly on each locus. The effective number of effective alleles (Ne) range from 1.9990 to 1.0444. The Nei's gene diversity (H) range from 0.4998 to 0.0425.Shannon information index (I) range from 0.6929 to 0.1047. The average value of Ne, H, I are 1.5260, 0.3618, 0.4832 respectively. The corresponding deviation are 0.3096, 0.1418, 0.1748 respectively. This three index shown the great gene diversity among chestnut varieties. The maximum genetic distance (GD) between varieties is 0.8001 and the corresponding genetic identity (GI) is 0.4493. The minimum of GD is 0.0910 and the corresponding genetic identity (GI) is 0.9130. Genetic distance is the good index for evaluating the relationship of varieties.4. The fingerprints database of 46 varieties was established and the optimal combination of primers for these varieties identification was summarized. It was shown that the four primers, out of the 16 primers which can produced the polymorphic bands, could be used to distinguish the 46 varieties tested effectively. It is the main purpose to differentiate these varieties precisely by using optimal combination of least primers, that means the optimal combination of polymorphic loci was detected for variety discrimination. In order to summarize the better combination of primers, the genetic distance matrix was calculated. That indicate the particularity of primers. It was found that the four primers selected can be used for genuine determination of the 46 varieties, if we use primer S28,S87,S11 combined with one of the primer S59,S26,S30. This method using molecular markers detect the plant genomic DNA directly. It's a rapid, reliable and accurate variety identification method. It was not affected by the environmental factors, plant tissues and organ and development stage. This is imperative to detect the woody plants with long seed bearing periodicity. The whole genome could be detected probably by using a large scale of primers. It was resulted that there are greatdifferences among the varieties to be studied. The DNA fingerprints database and its standardization are the foundation of varieties certification, verification, bnreeders' rights protection. This principle and results are useful for genetic analysis of plant germplasm, plant new variety protection and arbitration testing of seed and seedling.5. The taxonomy of chestnut varieties was studied by molecular method and also take the comparison with traditional method. Traditional method based on the plant morphological traits, biological characteristics and economic traits especially on the morphology of inflorescence, bract and nut. Because of the long seed bearing periodicity, it's very difficulty to verify the varieties accurately by the morphological characteristics of vegetative organ in early stage. The variety verification using molecular markers has great perspective. The UPGMA dendrogram of 46 varieties was constructed based on the Nei's genetic distance. It was found that molecular taxonomic method can reveal the relationship of chestnut varieties which have the similar morphological and economic characteristics or the similar provenance. But the molecular method do not agree completely with traditional method, which was divided chestnut into six plantation variety cluster according to the geographic cultivation cluster. One of the reason is that most of the samples come from Yangtes River varieties cluster in this study. Another reason is that the chestnut variety' nomenclature was confused sometimes. Because of widespread introduction, some varieties can not detect the exact origin. So the molecular method should be based on the morphological method. Further study should be carried out to reveal the relationship between morphological, economic characteristics and molecular markers. The 16 primers which can produce 69 polymorphic loci is enough for chestnut variety verification theoretically. Chinese chestnut has a long history of manmade plantation. Artificial selection and domestication makes it different from nature mating population. Nature mating population maintains the original geographic variation and evaluation discipline. In despite of this, molecular method is very sensitive and effective tool for plant variety verification, especially for similar morphological variety.Further study should collected the whole main cultivated varieties and use more polymorphic primers. The DNA fingerprints database should be constructed completely. It is also necessary to standardize the procedure of molecular markers, to make the automation of molecular marker manipulation and communion of variety information. It is possible to verify the chestnut varieties accurately.