Studies On The Genetic Diversity And Phylogeny Of Walnut In Xinjiang

China is one of the origins of walnut (Juglans regia) with more than 2,000 years culture history. Walnut is economically important fruit tree and widely cultivated in Xinjiang with a lot of wild and culture germplasms. In present study, a total 75 walnut collections from Hetian, Yecheng, Kuche, Ruoqiang, Wensu walnut cultivars in timberland, Shenmuyuan, wild walnut in Ili were collected, and other 24 samples, considered as controls, were collected other areas of China for the analysis of genetic diversity by means of EST-SSR markers. Meanwhile, the phylogeny analysis in Juglandaceae based on ITS2 sequence and secondary structure were conducted. The results are as follows:1. Development and evaluation of Walnut EST-SSR primersWalnut EST sequences were downloaded from the NCBI database for subsequent SSR mined. As a result of 398 SSR motifs were obtained. After removing the unsuitable loci with short flanking sequences, a total of 123 primer pairs were designed based on the microsatellites. These primers were then used for detection of the polymorphisms among the samples from Yecheng, Hetian, Wensu counties, Tibet, Shandong, and Shannxi provinces and independent Wingnut DNA pools. Finally,41 out of the 123 primer pairs generated high-quality polymorphisms by PCR amplification using walnut genomic DNA as templates.2. The analysis of genetic diversity in Xinjiang walnut germplasmsThe screened 41 polymorphic primers were used to evaluate 99 samples. The accessions collected from Hetian, Yecheng, cultivar in Wensu and wild walnut in Ili were considered as main germplasm in the study. The effective number of alleles, Nei's genetic diversity index and Shannon's genetic diversity index were 1-1.3492,0-0.2506,0-0.4124, respectively. Average polymorphism information content (PIC) in the Hotan groups up to 100%. The population genetic diversity and intra-population were 0.0884 and 0.0763, respectively. The results illustrated the lower polymorphism within populations and inter-group. Genetic structure analysis revealed that the largest genetic distance existed between populations Ili wild walnut, cultivar in Wensu and Hetian (0.1096), accompanied by the smallest genetic similarity (0.9801); meanwhile, the largest genetic similarity existed between cultivar in Wensu and Ili (1), and smallest genetic distance (0). Threrefor, the higher similarity and lower genetic distance among populations showed the lower polymorphism in the Xinjiang's walnut. Coupled with the higher Nei's (1973) and Shannon index of genetic diversity (1972) and the Hitian's genetic diversity was higher than other regions. Analysis of molecular variance (AMOVA) results showed that the variance within populations was (45.73%), and the variance among populations within groups was 54.27%. The gene flow was 0.85. Analysis of genetic distance and geographical distribution of walnut in Xinjiang, the results showed that the population geographical distance and genetic distance had no significant correlation, so the spead of walnut population in Xinjiang may be related to human activities, rather than natural spread. Hetian was an independent group based on the Nei's genetic diatance clustering; the group of (Ruoqiang+Kuche23) paralleled to the (Kuche24+06-3) group and meanwhile falled into Yecheng population, as a result the three groups composed the Yecheng association complex; Ruoqiang 3 encompassed the (the Yecheng association complex+Wensu cultivar population);Ili wild walnut population was clustered into separated taxa and paralleled with XZF accession; Hetian+Yecheng association complex) and Ili wild walnut population paralleled with XCF accessions;3. The analyses of phylogeny in Juglandaceae familyComparison of ITS2 region of 50 species in Juglandaceae family revealed that 130 sites were variable, of which 73 were parsimony informative. All analyses based on Neighbor-joining phylogeny trees supported the Juglandaceae were divided into two subfamilys, one was PLATYCARYOIDEAE another is ENGELHARDIOIDEAE. The PLATYCARYOIDEAE included the PLATYCARYEAE (Platycarya+(Carya+ Annamocarya)) and JUGLANDEAE (Juglans+(Cyclocarya+Pterocarya)). The ENGELHARDIOIDEAE composed of (Engelhardia+(Oreomunnea+Alfaroa)). The Juglandaceae ITS2 secondary structures (i.e., helices) exhibited significant similarities with the described secondary structures for the flowering plants and eukaryotes. Following the proposed nomenclature of the ITS2 structure model, the Juglandaceae family exhibited 3-helices and 4-helices structures. Helix B was typically short, not branched and included the U-U mismatch; the helix C include the highly conserved 6-bp stretch (5'-GGUGGU-3'). The results of the ITS2 structure arrested to the disjunction of Cardiocaryon Dode (Asian butternuts) and Trachycaryon Dode (American butternut). The clustering and structural features of the butternuts completely supported the closely disjunction relationship between Cardiocaryon and Trachycaryon. The structure of J. hopeiensis supplied the direct evidence of the formal characteristics for taxonomic classification and therefore strengthened the clustering results to the Cardiocaryon section.