Identification And Genetic Diversity Evaluation Of Casuarina Equisetifolia Clones In Coasta L Shelterbelts Of South Ern China

Casuarina equisetifolia has become an indispensable tree species for shelterbelt construction in coastal areas of southern China because of its excellent characteristics of windbreak,sand-dune stablization,poor soil tolerance,high resistance against adverse environment and high growth rate.At present,casuarina shelterbelt plantations are mainly planted with clones of C.equisetifolia.But in the past decade,coastal shelterbelts have been under serious theats due to confusing naming and unknown number of clone used,couple with unclear genetic background and diversity of planting materials.Therefore,it is necessary to study the identification,genetic diversity assessment and potential origin analysis of C.equisetifolia clones.According to the issues mentioned above,the 109 samples collected from42 locations in three littoral provinces of south China,were used to undertake clone identification,fingerprint construction genetic diversity assessment,genetic relationship analyses,and potential geographical origin inferring using EST-SSR markers.Meanwhile,morphological markers were used to analyze the genetic diversity and relationship of the clonal samples as well.It would be helpful for genetic improvement and development of new clones of C.equisetifolia,and be of great significance in protection of the variety right of casuarina and the legitimate rights and interests of breeders.The main research results of this paper are as follows:(1)Twelve primer pairs with high polymorphism and stable amplification products in C.equisetifolia were screened out and optimized from 71 candidate EST-SSR primer pairs.The 12 primer pairs,along with 9 known reference materials for clone identification were used to identify 109 clonal samples collected from coastal areas of southern China using EST-SSR genotyping technique.As a result,all the samples were determined as 22 clones,indicating only 22 C.equisetifolia clones in the three coastal provinces are currently being used for coastal shelterbelt construction.Among them,in addition to 9 clones were identified to be ramets of the 9 reference clones,the remaining 13 clones are unknown.The results indicatedthat mutual introduction,mislabeling or re-naming of C.equisetifolia clones is no unusual in South China.(2)The fingerprints of 22 C.equisetifolia clones based on EST-SSR genetic genotyping were constructed,which provided a reliable molecular marker technique for the identification and protection of casuarinaclones or varieties in China.Each primers pair can distinguish about2-5 C.equisetifolia clones,but only 7 of primer pairs can distinguish all the 22 C.equisetifolia clones completely when these primer pairs were used through optimizing combination of some primer pairs.Finally,fingerprints of the 22 C.equisetifolia clones were constructed based on 7primer pairs and each clone was coded with a 7-digit fingerprint.(3)The similarities and differences of morphological marker method and EST-SSR marker method in evaluating the genetic diversity of the C.equisetifolia clones were compared.Results showed that the diversity index(H')of the 12 morphological traits ranged from 0.347 to 2.053,among which the qualitative traits presented larger variation than quantitative traits,and the phenotypic similarity of the C.equisetifolia clones was higher.A total of 50 alleles were detected in 12 EST-SSR marker pairs,with an average of 3.42 alleles at each locus.Observed heterozygosity(Ho)and Expected heterozygosity(He)were 0.867 and 0.641,respectively.The average Shannon diversity information index of the population was 1.099.Molecular marker analysis showed that the genetic diversity of C.equisetifolia shelterbelt in coastal areas of southern China was low.Based on the results of clustering analysis of morphological characters and EST-SSR markers,109 C.equisetifolia clones can be clustered into 13 and 22 categories,respectively.The clustering results of morphological and molecular markers are quite different.Mantel consistency test was conducted using the genetic distance matrix of different morphological markers and EST-SSR markers,and the correlation between the two matrices was not significant,indicating that the consistency of the above two markers was low.In conclusion,it is suggested that molecular markers should be used as the main method and morphological markers as the auxiliary method to study the genetic differences of casuarinas.(4)The potential genetic relationship and geographical origins of the tested clones were analyzed.By comparing genetic parametes obtained from the 22 clones and 13 provenances originated from different countries and regions,it was found thatthe 22 clones were clustered into two main groups based on UPGMA clustering analysis,and the Nei's genetic distances of the most cloneswere less than 0.3,but Nei 's genetic distance between the two main groups was0.78,indicating that C.equisetifolia clones in coastal shelterbelts of South China presented close genetic relationship and low genetic diversity.Furthermore,the most likely geographical origins of these clones are from Southeast Asia,including Malaysia,Philippines and Thailand,and the possibility that originated from Oceania is minimal.it is highly unlikely to come from Oceania.