| Common walnut (Juglans regia) as a model species in the genus Juglans, widely distributed, has a long history of cultivation and has high edible and medicinal value. For a long time, the genetic background and the important value of walnut is the focus of attention of scholars.. At present, the walnut plants research reports mainly focus on the morphology, anatomy embryology, palynology, cytology and other aspects. The application of molecular markers of walnut plant reported research is limited to a few individual populations or individuals, molecular genetic markers used are mostly generic primers, polymorphism and the genetic information contained are relatively limited. So it is necessary to use molecular markers to study the walnut of different geographical populations and develop more genetic marker. On the one hand, This research, on the one hand, by the DNA sequencing technology, using mitochondrial, chloroplast and nuclear DNA fragments on the geographical distribution of walnut colony phylogeographical, mainly by partheno genetic cytoplasmic molecular markers and biparental inheritance of nuclear DNA fragments walnut spectrum Department of geography study; on the other hand, Illumina HiSeq high throughput sequencing platform based on the leaves of walnut bud, female flowers and male flowers four kinds of plant organs of transcriptome sequencing, obtained data 4.98G, followed by biological information method of data were assembled, annotates and related biological information analysis,, and using the microsatellite sequences of Walnut in different Unigene developeasy amplification, polymorphic EST-SSR primers. Through the study of the two aspects of the analysis, walnut population evolutionary history, provide theoretical foundation and basis for the development and utilization of genetic markers and germplasm resources and protection. The main conclusions are as follows:(1) Using 3-9 mitochondrial gene fragments and TrnL-F chloroplast gene fragment for J. regia and outgroups (Juglans cathayensis, Juglans mandshuric, Juglans hopeiensis) study their genetic diversity and phylogenetic relationship. The results showed that J. regia genetic diversity the highest among 5 species and other species are rare. J. cathayensis, J. mandshurica and J. hopeiensis close relations as a group, called the Sect. Cardiocaryon, and J. regia differences between the Sect. Cardiocaryon was divided into two groups different. Based on chloroplast fragments beast analysis results show that China may be the center of origin of walnut. Two gene fragments can not only distinguish different species of Juglans, but also have significant difference in J. regia.(2) ITS gene fragment of J.regia genetic structure analysisshow that different geographic populations of walnut phylogeographic structure significantly, and different geographic groups formed their unique haplotypes. Population genetic structure analysis shows in Southwest China has its own unique haplotypes and haplotype diversity is rich, speculation southwest may be a genetic diversity of neutral of walnut. AMOVA analysis results show that, walnut genetic variation mainly exists within populations and genetic differentiation was significant.(3) JRD5680,15R-8 are used to analyze the genetic diversity of two gene fragments of walnut and genetic structure. According to the analysis of the two gene fragments of AMOVA, we find that the genetic variation of walnut mainly existed within populations, and genetic differentiation was significant. According to the geographical distribution of haplotypes JRD5680,15R-8 two gene fragments, We derive walnut genetic diversity of higher population groups mainly cso we think that walnut genetic diversity of high may be due to existence of the cultivated populations and wild populations, gene flow caused by the presence of the we obtained a higher genetic diversity in the population of walnut the group mainly cultivated population present in the area,, so we think the walnut high genetic diversity may be due to the presence of gene flow between cultivated populations and wild populations. Two gene fragment neutrality test and mismatch distribution analysis showed that the sequence of the two genes is consistent with the neutral theory, research shows that JRD5680.15R-8 two gene fragments based on the experience we draw the walnut no recent expansion events.(4) Using Illumina sequencing method of walnut leaf, bud, flower, and four kinds of tissues of male flowers the transcriptome information, through the Trinity to assemble to obtain 250222 transcripts,117229 Unigene. with the length of Unigene frequency, the total number is more than 500 bp of the total data of 60%. The 117,229 Unigenes respectively compare with GO, COG, KEGG the three databases,. Total 45,029 (38.4%) Unigene and GO database compared to the success.11,983 Unigene and COG annotation database successfully. 25,269 Unigene and KEGG annotation success. Unigene and GO, COG, KEGG three database comparison results show that transcriptome sequencing received a large amount of diverse information.(5) The use of MISA software on Walnut transcriptome data of SSR microsatellite loci were mining, and has been a lot of specific SSR sequences. By primer3 design primer, and from the design of primers randomly selected 78 pairs of EST-SSR primers were amplified, the amplified results showed 78 pairs of EST-SSR primers in 53 of amplified have bands,39 pairs of polymorphism is better. The results show that the transcriptome data design EST-SSR primers, amplification efficiency highly. The polymorphism of 39 pairs of EST-SSR primers of walnut populations 88 individual genetic diversity research, discovery of walnut genetic diversity is high, different geographical environment populations form a specific genetic structure. |
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