The Genetic Diversity And Characteristics Of Mineral Nutrition Of The North China Mountains Walnut

Walnut (Juglans) blongs to the Juglans genus, Juglandaceae Family, which is a fruit-timber tree species, and is ancient tree with a long cultivation history, wide distribution, abundant germplasm resources in China. As a preferred afforestation tree species, Juglans has the extremely important ecological benefits and economic values. Walnut is as one of the four nuts (almonds, cashews, hazelnuts), which kernel have edible value and its trunk, branches, leaves, flowers, green husk have certain medical and medicinal value. Especially the walnut oil is developed and utilized in medical and medicinal values, which made it enjoys its reputation as the "Oriental olive oil" in the international market. Due to the simplification of cultivars and nutrition deficiency of tree body, genetic vulnerability to resistance was exacerbated, and due to the quantity demand for walnuts increasing in domestic and international market, cultivation and breeding of walnut were paid attention. This study started from the optimize of ISSR-PCR (Inter-simple sequence repeats-polymerase chain reaction) which were employed to acess genetic diversity for 8 natural populations of the north China mountain walnut with the complex genetic background, to analyze phylogenetic relationships of Juglans spp. and establish DNA fingerprints and of some cultivars, meantime, mineral nutrition characteristics were studied of the north China mountain walnut. The study provided guidance for scientific protection, reasonable development and utilization of genetic diversity of the north China mountain walnut, and provided reference for scientific and rational fertilization, improving the utilization rate of fertilizer. The main results were as follows:1. Levels of ISSR-PCR factors were optimized by the orthogonal design L16(45) and analysis of variance. The results showed that the dominant factor was dNTPs. PCR was performed in a total volume of 25μl containing 0.8^1 of 10 mM dNTPs,0.3μl of 2.5 U/l TaqE,1.2μl of 10 pM primer,0.8μl of 30 ng/1 DNA. Nine primers with high level of polymorphism and scorable bands were screened out from 100 primers by preliminary screening and rescreening, which annealing temperature was determined by PCR amplifier with temperature gradient, and used for further practical ISSR analysis. The establishment and optimization of the system provided the technical support to the related research of walnut ISSR markers.2. Based on ISSR, genetic diversity and genetic variation among and within 8 natural populations, which were made of a total of 138, from the representative area of the north China mountain walnut were studied by agarose gel electrophoresis and polyacrylamide gel electrophoresis method, and the efficiency of the two electrophoresis was assessed. The results showed that using 9 primers,75 polymorphism loci were observed, the percentage of polymorphic loci(PPL) was 80.44%, the average effective number of alleles (Ne) was 1.4001, the average Nei’s genetic diversity index (h) was 0.2390, the average Shannon diversity index (I) was 0.3612 for agarose gel electrophoresis, and for polyacrylamide gel electrophoresis,135 polymorphism loci were observed, PPL, Ne, h, and I were 83.25%,1.3868,0.2454 and 0.3818 respectively, which genetic diversity parameters of populations had no significant correlation with the two electrophoresis methods, but the genetic diversity of polyacrylamide gel were more higher than agarose gel. At the level of genetic differentiation, the coefficient of gene differentiation (Gst) for polyacrylamide gel electrophoresis (Gst= 0.2979) was bigger than agarose gel electrophoresis (Gst=0.1820). Therefore, polyacrylamide gel was suitable for amplification products separation of ISSR-PCR. Based on ISSR-PAGE Nei’s genetic distance, eight populations were divided into three groups Ⅰ, Ⅱ and Ⅲ. The Shanxi Licheng population (LC), Shanxi Yuxian population (YX), and Hebei Shexian (SX) were clustered firstly, which were clustered into group with Shanxi Zuoquan population (ZQ) and Fengyang population (FY) in turn; Hebei Laiyuan population (LY) and Henan linzhou (HN) were clustered into the group Ⅱ, Shanxi Guxian (GX) were clustered into the group Ⅲ by oneself. Clustering results demonstrated that it was not completely consistent with geographical source, but was conformity with nuts character of local cultivars, which reflected genetic exchange in the North China mountain walnut.3. Genetic relationships of 9 closely related species of Juglans genus, which were comprised of 36 samples, was analyzed with 9 ISSR primers. The results showed that the number of polymorphic loci were 57, the average effective number of alleles (Ne) was 1.3293, the average Nei’s genetic diversity index (h) and Shannon diversity index (Ⅰ) were 0.2875 and 0.4300 respectively. Based on the ISSR data, the clustering of nine species, including 36 individuals, were constructed respectively by Popgene32 software and NTSYS software, which results indicated that two different clustering methods were basically consistent in structure, i.e.9 species were divided into two clusters, Juglans and Carya; Juglans were divided into three subclusters, Sect. Juglans, Sect. Cardiocaryon and Sect. Cardiocaryon, which Sect. Juglans and Sect. Cardiocaryon with close geographical origin grouped at first, and grouped with Sect. Rhysocaryon with the further geographical origin, which were consistent with classical classification method of Juglans. The result provided the theoretical basis for breeding new varieties by distant hybridization of inter-genera and inter-genera.4. ISSR-fluorescent markers were employed to establish the fingerprint of Parts of cultivars in the north China mountain walnut. The results show that, the 60 materials, in addition to Jinbaoxiang 2 and Liaohe 7, the rest of the 58 samples possessed specifical bands, and identification rate could reach 96.72% by combination of any two primers, which made clear that ISSR-fluorescent markers was feasible to identify cultivars. Genetic similarity coefficient among 60 cultivars was between 0.860 and 0.938, which showed that the genetic variation was smaller among cultivars, and genetic relationship was closer, because we had used Xinjiang walnut as core parents in seeding-selectinon or hybrid. For selected 11 late mature cultivars and 49 precocious cultivars, their common banding did not be showed, and most of the bands was owned by the two strains, meanwhile the result of clustering indicated late mature cultivars and precocious cultivars blong to same cluster, which suggested closer genetic relationship between two strains. Based on the specifical bands, DNA fingerprint of 60 cultivars was established, which provided the most direct basis to identification of seeding purity and selective breeding.5. Characteristics of mineral nutrition of main cultivars in North China mountain walnut was studied. The result showed that: ① the average contents of nine mineral elements in leaves of the four cultivars ((Xiangling, Luguang, Zhonglin 1 and Bofeng)) were in order of Ca>N>Mg>P>K, and Fe>Mn>Zn >Cu;② The contents variations of nine mineral elements in leaves at different phenological stages showed different temporal dynamic patterns. The contents of N, P and K were on a declining curve; while the contents of Ca, Mg, Fe and Mn increased volatility; and the contents of Cu and Zn varied slightly. These results could be taken as indication that the nutrient contents of walnut trees were stable from May to July; ③ The correlation analysis among the contents of nine mineral elements in walnut leaves indicates that there are significantly positive correlations among the contents of N, P and K, while the contents of N and P were significantly negative correlations with those of contents of Ca, Mg, Mn and Cu, which suggested there is the synergism action among N, P and K, and antagonism actions between N, P, K and Ca, Mg, Mn and Cu, respectively.④ The correlation analysis between contents of mineral element and yield indicated that there are significantly positive correlations at the shoot rapid-growing stage, whose correlation coefficients were 0.819,0.843 and 0.895 respectively, indicating the shoot rapid-growing stage (around May 20) were the optimal leave sampling stage for mineral nutrient diagnosis. ⑤ trunk smearing liquid fertilizer played a notable role on leaves and fruit development in the fruit expanding stage.