Molecular Identification And Genetic Diversity Evaluation Of Tea-oil Camellia Resources Of Hainan Island

Tea-oil Camellia is a key development woody oil tree species in China.Hainan Island is the southernmost edge of the tea-oil Camellia distribution,which has obviousfeatures in resources and industrial development.Due to late start of the researching and developing of tea-oil Camellia resources in Hainan Island,a systematic review of this resource at home and abroad has not been carried out.The classification of tea-oil Camellia resources in Hainan Island is still inconclusive,which is extremely unfavorable for the protection,innovative utilization and industrial development.Meanwhile,there are major differences on the classification and merging of Camellia oleifera species.Moreover,evaluation of domestic Camellia vietnamensis including tea-oil Camellia resources to further clarify the resource characteristics,understand the genetic diversity level and genetic structure.It can provide a scientific basis for the protection and innovative utilization of related resources.Therefore,a total of 867 individuals of C.oleifera were collected from 42 populations were analyzed in this study.The classification status and genetic relationship of Camellia oleifera,Camellia vieinamensis,Camellia meiocarpa,Camellia gouchowensis Chang,Camellia Drupifera Loureiro,Camellia osmantha are analyzed using ISSR and SRAP markers.And the tea-oil Camellia germplasm resources in Hainan Island were classified and identified.At the same time,the genetic diversity of tea-oil Camellia resources in Hainan Island and Camellia vieinamensis resources in China was evaluated based on SRAP markers,and the genetic structure and differentiation among populations were analyzed.The main results were as follows:(1)Using tea-oil Camellia in Hainan Island genomic DNA as a template,this study used single factor test and orthogonal test method to optimize SRAP-PCR reaction system in tea-oil Camellia in Hainan Island.The optimum SRAP-PCR reaction system(20 ?L)includes 1.8 ?L 10 x PCR buffer,4 U Taq DNA polymerase,0.20 mmol/L dNTPs,0.6?mol/L each of SRAP forward and reverse primers and 5 ng genomic DNA.After verification,the system is stable and reliable.(2)Based on combined SRAP and ISSR data,489 total bands were obtained from 18 populations,with an average PPB of 96.52%.The results showed that the genetic diversity of the tested Camellia oleifera population was low,and the average Na,Ne,H,I,and PPB values of the population were 1.46,1.28,0.16,0.24,and 46.47%,respectively.The level of genetic variation among populations was quite different,with the CV values of H,I,and PPB being 16.32%,15.66%,and 15.17%,respectively.The genetic diversity of the small fruit camellia population and the common camellia population was relatively high.The genetic diversity of C.meiocarpa and C.oleifera was relatively high.The genetic variation mainly exists in the population,and the genetic differentiation coefficient(Gst)was 0.22.The gene flow(Nm)is 1.7696,which is greater than 1,indicating that there is a certain intensity of gene exchange between different populations.(3)The analysis of the genetic relationship and genetic structure of C.oleifera germplasm resources showed that the UPGMA cluster map clustered the 18 test populations into three categories.The populations of C.meiocarpa and C.oleifera were grouped into group I.The populations of C.osmantha were grouped into group II alone.Group III consists of all the populations of C.gouchowensis Chang,C.vieinamensis,C.Drupifera Loureiro and tea-oil Camellia.The results of PCoA were basically consistent with the 18 phylogenetic trees constructed by UPGMA method.Based on the clustering results and the genetic distance between populations,C.meiocarpa should be distinguished as one variant of C.oleifera at least,and should not simply be classified as C.oleifera.C.gouchowensis Chang,C.vieinamensis and C.Drupifera Loureiro were classified into the same species,and it is not supported to merge C.vieinamensis into ordinary C.oleifera.It supports C.osmantha as an independent species,and its relationship with C.vieinamensis is closer than ordinary C.oleifera.(4)The tea-oil Camellia in Hainan Island belongs to another branch to C.gouchowensis Chang,C.vieinamensis,C.Drupifera Loureiro in groups ?,GD from0.03 to 0.09,with an average of 0.07.Based on the analysis results of UPGMA,PCoA and STRUCTURE,the analysis believes that the tea-oil Camellia in Hainan Island belong to C.vieinamensis.However,tea-oil Camellia in Hainan Island is different from C.vieinamensis outside the island.The tea-oil Camellia in Hainan Island should be an ecological type or a variant of C.vieinamensis.(5)Two hundred and ninety-eight total bands were produced by 22 SRAP primers from 33 populations,and the average amplification of each primer was 14.14 bands,with an average of PPB was 96.71%.Among them,the 24 populations of tea-oil Camellia in Hainan Island could amplify 285 bands,with an average of 12.95 per primer,with an average polymorphism percentage(PPB)of 92.70%.The average and inter-population genetic diversity of tea-oil Camellia in Hainan Island is low.The inter-population levels of H,I and P are 0.17,0.27 and 80.45%,and the average population levels are 0.10,0.15 and30.44%,respectively.The H,I,and PPB coefficients of variation(CV)were 8.97%,8.44%,and 9.22%,respectively.The coefficients of variation were low,and there was no significant difference in genetic variation among populations.The genetic diversity of all the C.vieinamensis were slightly higher than that tea-oil Camellia in Hainan Island.The average of H,I,and Pwere 0.11,0.17,and 32.87%,respectively.The coefficients of variation(CV)of H,I,and PPB were 14.20%,14.79%,and 14.90%,respectively,with significant differences.The average H,I,and P of C.oleifera population outside the island were 0.14,0.21,and 39.35%,which was higher than that of tea-oil Camellia in Hainan Island.(6)The tea-oil Camellia in Hainan Island showed a high level of genetic differentiation among populations,with a genetic differentiation coefficient(Gst)of 0.38.The gene flow was only 0.78,which revealed that gene exchange between populations was restricted.The results of UPGMA,PCoA and STRUCTURE showed that 33 test populations were divided into two groups.All of the population outside the island were grouped one group,and all the population inside the island were grouped another group.The tea-oil Camellia resources in Hainan Island are different from other C.vieinamensis populations outside the island.The origin of tea-oil Camellia in Hainan Island is more complicated than that outside the island.A small number of populations have the same genetic background as C.gouchowensis Chang and C.Drupifera Loureiro.The correlation between genetic distance and geographic distance among tea-oil Camellia populations in Hainan Island was not significant(r = 0.20),which revealed that the existing genetic structure of tea-oil Camellia populations in Hainan Island can't be explained by geographical distance.These analysis suggest that the existing genetic structure of tea-oil Camellia in Hainan Island has a great relationship with the historical culture and cultural exchanges.