Genetic Analysis Of Core Germplasms And Fingerprint Identification Of Main Cultivars Of Juglans Spp

Genetic analysis of core germplasm is necessary for walnut breeding, it can provide scientific support for the innovation, utilization and improvement of the germplasm resource. Meanwhile, germplasm identification is also the crucial step for genetic improvement and intellectual-property protections. Though convenient and intuitive, the traditional morphological identification is now inadequate for the identification and purity analysis of the abundant varieties because, the morphological characters in new cultivars become more and more undistinguishable. Molecular markers are now being widely used for identifying the authenticity and purity of cultivars for their ability to detect differences between individuals in DNA level directly.Based on the walnut bacterial artificial chromosome (BAC) library end sequences of Juglans regia L, we designed SSR primers for genetic analysis and cultivars identification of walnut germplasm resources. The genetic background for selfed progenies and the differences for thdir phenotypes were also studied. Finally, fingerprints of some walnut cultivars were established based on the phenotypes and SSR. The main results are as follows:1. From all NCBI database J. regia cv. Chandler Mbol genomic BAC clones,22740end sequences (BES) were downloaded and analyzed.4732SSRs (microsatellite) were found by using the MISA program. The frequency of SSRs was approximately1/2.8kb. Among them, A/T, AT/AT and ATT/AAT motifs were the most abundant types in the mono-, di-and tri-nucleotide repeat categories respectively. Three hundred and ten pairs of SSR primer with different types and the number of repeats were designed and synthesized, and116pairs of SSR primer with high polymorphic were screened from them.Nineteen of the116pairs of primer were used to amplify SSR for20genotypes that belonged to6species and1hybrid in the genus Juglans. The results showed that2-9polymorphic loci were amplified per pair of primers with an average of5.4. Polymorphism information content was greater than0.5in17SSRs loci and with a mean value of0.662in all loci, which showed high polymorphic. The cluster analysis showed that groups were clustered in accordance with species and origin. After polymorphisms analysis, the number (percentage) of polymorphic primers in J regia. J. sigillata Dode, J. hopeiensis Hu and J. mandshurica Maxim were107(92%),82(71%),57(49%) and47(41%), respectively.2. Morphological and molecular differences in J. sigillata cv. Dapaohetao and9its selfed progenies (SP1～SP9) in1979-1982were investigated for evaluating changes of phenotype and genetic composition in selfed progenies. Nine representative selfed progenies presented obvious variations such as length of juvenile period, tree height and nutshell thickness. Among them, SP1～SP4initiated flower2years after germination, SP5～SP8initiated flower7-8years while SP9had not flowered since1980. The selfed progenies have three types of nuts:Pao walnut (with0.1～0.9mm thickness of shell), Jiamian walnut (with1.0～1.5mm thickness of shell) and Tie walnut (with1.6～2.0mm or over2.0mm thickness of shell). And, some selfed progenies presented lower vigor, dwarfing and inferior resistant to adverse situation with only4.5-5.5m tree height. Fifty-eight SSR markers were used to analyze the genetic composition of selfed progenies, and the results showed that the increased homozygosity of genome and inbreeding depression in the selfed progenies. And, the homozygous effect was most prominent in SP9. The most similar phenotype and genetic composition was showed between the parent and SP5that had82.8%same SSR loci.3. The genetic diversity of62J. regia germplasms was implemented using28microsatellite loci. The results showed2to7alleles were amplified in per primer. The expected hoterozygosity (He) ranged from0.382to821with an average of0.609. The Shannon’s information index (Ⅰ) changed from0.637(WJR291) to1.750(WJR265) with an average of1.122. The genetic similarity ranged from0.47to0.97among62germplasms which showed higher genetic diversity. The cluster analysis suggested that62germplasms could be categorized into three groups inclined to geographical classification. The results provided reference for cultivars classification and parental selection in cross breeding. Genotypes with distant relationship in different groups as cross parents are beneficial to breed abundant types of progenies.4. Fingerprint identification method of walnut cultivars was established by combining nut phenotypes and SSR markers. Molecular identification by12simple sequence repeat (SSR) markers and 22nut shape characters were conducted on35cultivars of J. regia. All35cultivars were identified on the basis of both the circular,(broad) ovate,(broad) elliptic shape in the longitudinal section through the suture and the medium prominence of apical tip, as well WJR031, WJR069, WJR265and WJR281. These cultivars were classified into4broad categories according to the nut phenotype, and then all accessions of each category were differentiated by no more than two SSR primer pairs. Every cultivar had unique fingerprints which jointed phenotype and SSR alleles. Regardless of nut phenotype characters, only the the four loci also allowed the unambiguous separation of35cultivars studied.