Moleculer Identification Of Juglans Regia L. And J. Sigillata And Genetic Diversity Studying In Sichuan Province

Both Juglans regia L.and J.sigillata Dode belong to the genus Juglans and are considered to originate in China.The two species are important economic nut trees and are extensively distributed in Sichuan province.Walnut is one of the oldest cultivated crops and traditional edible nut in the world,with high fat,protein,unsaturated fatty acids, carbohydrate,P,K,Ca,Mg,Zn,Mn,Cu,Fe,B and vitamin B,C,E.At present,Research about walnut mainly focused on morphological description,identification of geographical ecology genotypes,relationship identification among species and verification of objective trait,etc.,but genetic diversity and population structure of J.regia have rarely been studied and genetic relationships between the two species at the genus level are disputed in a long time.Accurate and effective evaluation of genetic diversity,population genetic structure and relationships between the two species is especially essential for effective preservation of germplasm resources,breeding,reclaiming the low yield of productivity.RAPD was applied for germplasm verification of the two species in this study.AFLP was applied to study genetic diversity,genetic differtiation,and genetic structure of natural populations of the two species.The contents of this study and the main results are as followings:(1) AFLP analysis needs high DNA quality.Two methods namely improved high salt and low pH and traditional CTAB were used to extract DNA from walnut young leaves. Using agarose gel electrophoresis,ultraviolet absorbance spectrophotometer and AFLP analysis tested the DNA samples extracted from above methods.The former method namely improved high salt and low pH obtained high quality DNA which has a good structural integrity and high molecular weight,with the OD260/OD280 value 1.8～1.9. AFLP banding patterns produced by this method is better than the other.So this method is more suitable for AFLP analysis.(2) Random Amplified Polymorphic DNA(RAPD) was applied to detect the relationship at the species level between J.regia and J.sigillata.Both J.regia DNA-pool and J.sigillata DNA-pool were constrcted by using 8 J.regia samples and 8 J.sigillata samples,respectively.327 primers were screened,of which 10 polymorphic primers were identified.Two DNA-pools and all individuals were verified by using the 10 polymorphic primers by three times.A 250bp DNA band was amplified only from primer D6 in J.regia DNA-pool and 8 corresponding samples of J.regia,but absent both in J.sigillata DNA-pool and 8 corresponding samples of J.sigillata.The results tentatively showed that D_6-250 was a special molecular marker related to J.regia and J.sigillata at the species level. This marker can be used to identify germplasm resources of the two species.(3) Four J.regia populations distributed in add or semi-arid valleys,Mingjiang river and Daduhe fiver namely,and two J.sigillata populations at Jinshajiang dry-hot valley, total 46 samples were selected and analyzed by AFLP—silver staining protocol using 6 pairs of AFLP primers screened,total 475 bands which include 332 polymorphic bands were obtained,the percentage of polymorphic band(P) was 69.89%.As for J.sigillata populations at the genus level,the effective number of alleles per locus(Ae) was 1.2758, and the Nei's gene diversity index(H) was 0.1616,Shannon's information index(I) was 0.2422.For J.regia populations at species level,the estimates were Ae=1.2972,and H=0.1745,I=0.2629.Genetic differentiation coefficients among J.regia populations and J. sigillata populations(Gst) were 0.3794,0.2252,respectively,which showed small partial genetic diversity present among populations both in two species.This result was approved by AMOVA.Cluster analysis,the dendrogram analysis and the principle component analysis(PCA) for all of the samples and populations were constructed.Results obtained through cluster analysis were consistent with PCA results.The UPGMA cluster based on Nei's distance matrix was analyzed and some further analysis about the relationships among the populations was made.UPGMA cluster analysis based on genetic identity of 6 populations of Juglans and DNA molecular dendrogram of 46 samples reflected relationships of materials.Firstly,four J.regia populations were departed from two J. sigillata populations.Secondly,the population WC was departed from LD,HY,and DB. At last,population LD and population HY show the closest relationships.There was a significant relationship between genetic and geographical distances(Pearson coefficent was 0.847^**) among Juglans populations analyzed by SPSS software.The possible reasons for the genetic diversity and genetic structure of the six populations in this region were mainly related to the geographic isolation,inbreeding and gene flow of the populations.In a word,the marker D_6-250 can be used to identify germplasm resources of the two species and provide a possibility for molecular breeding selective.In the meantime,this study revealed molecular marker technology,such as AFLP could screen relative higher level of polymorphism,and be effective tools to construct genetic maps,to identify germplasm resources and to construct DNA fingerprinting for protecting new varieties of walnnut.At last,based on the results from the present study and in combination with results from other researchers,breeding strategies for walnut were put forward in order that the information on genetic diversity of walnut germplasm could be fully utilited,and walnut germplasm resources could be rationally exploited for creating new excellent germplasms and improvement of walnut varieties.