| Walnut (Juglans regia L.) is a antiquated fruit with old-line planting in china, and it distributes abroad and has abundance resourse in china.But there are many disputed points for where walnut origins from, spore fossil and chared walnut and antiquity forest relic have been reviewed and researched, which testifed that walnut in Xinging might be direct ancestor of walnut cultivars and it will be affirmed by more molecular study .Based on optimized SSR reaction system, Using principles of molecular systematics and SSR marker techniques, the genetic diversity and population genetic structure of three groups for Yili wild walnut,Kashi walnut and cultivars were analyzed first in order to explore its evolutional processes and origin of walnut cultivars, and to provide molecular basis for further cultivar selection and breeding of cultivated walnut.The main results from this study are as follow:1. DNA of Qingxiang, Xiangling and Amigo were distilled by CTAB .Every factor of PCR had five different concentration and its variation changed the result of PCR-SSR. Then the factors affecting the SSR results of walnut were studied.The annealing temperature of primer WGA321 was optimized.The results showed that the optimal annealing temperature is 46℃～54℃, and the best reaction system of SSR-PCR is: 1.0 mmol/L Mg2+, 0.10～0.20 mmol/ L dNTPs,0.5U Taq polymerase,1.0 ng/μL template DNA,and 0.4μmol/ L primer in 15μL reaction system. Using above PCR system, SSR fragments of 12 walnut cultivars were obtained. The clearity and abundant polymorphism indicated this system was suitable for analysis of phylogenetic relationship. 2. Seven black walnut simple sequence repeat (SSR) primer pairs were used to study 80 samples of walnut native to Yili and Kashi, and cultivars in order to analyze genetic diversity. The average percentage of polymorphic bands in all primers was 98.1%, the expected heterozygosity was 0.1589～0.4359,and Shannon information index was 0.2734～0.6225, which indicated that each locus had abundant polymorphism. The expected heterozygosity and Shannon information index and percentage of polymorphic loci of the three populations were analyzed and the results reflected that the genetic diversity of Kashi population was highest (He=03085,I=0.4669,P=95.83%) which were almost equal to that of cultivars (He=0.3050,I=0.4646,P=86.84%) and that of Yili population was lowest (He=0.2325,I=0.3361,P=56.52%)3. The parameters-- Genetic differentiation coefficient and gene flow of population genetic structure in walnut were analyzed in this study.Genetic differentiation coefficient (Gst = 0.1928) for three walnut groups showed that apricot genetic variation was mainly within the groups and accounted for 80.72% of total variations.The genetic variation between groups accounted for 19.28% of total variations. The gene flow (Nm= 2.0934) according to the genetic differentiation coefficient between groups indicated that there are partly gene exchanges among walnut populations. It is suggested that the main way of gene exchanges could be seed spreading and human introduction of occasional seedling and cross breeding.4. 10 accessions and varieties were choosed randomly from three populations to be used to cluster analysis which was analyzed by SPSS version 10.3 according to squared euclidean distance.The results was that all samples ananlyzed crossed one another, which made out that gene infiltration and exchange occured between populations(gene flow Nm=2.0934). Walnut native to Kashi and cultivars immingle more severe than wild walnut from Yili in 4 species of the cluster chart, and it reflected that there may be more complicated heredity background in walnut native to Kashi and cultivars.The varieties from outside -- Italy 1 and Amigo and Qingxiang had closer genetic relationship with walnut native to Kashi and walnut from Yili than with other cultivars. Nei′s genetic distance and UPGMA clustering method ware adopted to analyze genetic relationship among walnut populations. Genetic distance betweeen cultivars and walnut native to Kashi was closest which was 0.0559, secondly, genetic distance betweeen cultivars and wild walnut in Yili was 0.0938. Based on all clustering relation, the relation chart of genetic evolvement was primely brought forward among three population. The results of study offered molecular proof for the judgment that China is one of origin centers of walnut cultivars.
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