Analysis Of Genetic Relationship Among Azadirachta

Neem trees,including species of Azadirachta indica A.Juss.,Azadirachta siamensis Val.and Azadirachta excels(Jack)M.Jacobs,are belonged to Azadirachta,which is a genus of tropical evergreen tree in Meliaceae.A large number of chemical compounds have been isolated from various parts of neem trees and thus being used as fungicide,pesticide,etc.The effectiveness of azadirachtin against crop pests has made Azadirachta being one of the most flourish tree in the research and development of biological pesticide.The Azadirachta has been introduced to dry-hot valleys of Yunnan province for decades,and most individuals of different provenances had normal growth and could blossom and fruit in introducing regions.Especially,two variation types including early maturing variety type and leaflet phenotypic variety type found in pure A.indica plantation are different from the other species of Azadirachta domesticated in Yunnan,China.To understand and reveal the genetic background of two variation types,morphological traits and molecular markers were tested by cluster analysis of Azadirachta with Melia toosendan as outgroupusing UPGMA method.Principal results are shown as follows.The analysis of 21 phenotypic traits of the three Azadirachta species and the two variation types showed that there were wide variations in the morphologic features of Azadirachta.Among all morphologic features,the coefficient variation ranged from 0.00 to 49.74%.And the leaflet area range which was 6.09~21.50cm2 varied maximumly,with highest variance coefficient of 49.74%.While the number of sepals,petals and fertile anthers were unchanged among Azadirachta.Meanwhile there were wide variations of the leflet shape,leaflet margin and leaflet base decline among Azadirachta.Principal component analysis showed that the main variations were resulted from leaflet area,fruit size,leaflet shap,leaflet margin and leaflet deflection.One-way analysis of variance showed there was no difference of the number of leaflet pairs,but signicant differences of other phenotypic traits among Azadirachta.Theresult of UPGMA clustering showed that Azadirachta could be divided into two groups.One is formed by A.indica,A.siamensis and the two variation types,and the other group is formed by A.excelsa.The former could be further divided into two subgroups,A.siamensis and and the two variation types could be clustered into the same subgroup,and A.indica formed the other subgroup.And the early maturing type was most similar to the leaflet variationtype in phenotypic traits.It was found that there was distinct genetic diversity among 26 S rRNA,matK,rbcL,atpF-atpH,psbT-psbN and rpl16 from Azadirachta and Melia toosendan.The sequence length ranged from 377 bp to 598 bp,G+C percentage content ranged from 30.1%~57.2%,the percent conserved loci ranged from 75.86% to 97.86% among six gene loci above.The genetic diversity of Azadirachta and Melia toosendan showed that the haplotypes number of different gene fragments ranged from 5 to 27.The range of haplotype diversity(Hd)was Hd=0.332~0.976,and the range of nucleotide diversity(?)was ?=(0.275~7.676)×10-2.Analyses of Molecular Variance(AMOVA)showed variations of six gene loci were broadly existed among Azadirachta,with 40.97%~98.62% of variance among groups,and1.38%~59.03% of variance within groups,and the Fixation indices(Fst)ranged from 0.410 to0.986.For 26 S rRNA,the haplotypes and number of polymorphic sites was the minimum among six gene loci.For atpF-atpH,the haplotypes and number of polymorphic sites was the maximum among six gene loci.Haplotypes and number of polymorphic sites were moderate in the rest four gene loci,and the results of these four gene loci showed that the early maturing type and leaflet variation type were both similar to A.siamensis in regards to haplotypes and polymorphic sites.UPGMA clustering of different gene loci showed that Azadirachta and outgroup Melia toosendan coundn't be divided into two groups by 26 S rRNA,atpF-atpH and rpl16.UPGMA clustering of matK,rbcL,psbT-psbN respectively,and five chloroplast gene fragments,and combination of 26 S rRNA with five chloroplast gene fragments all showed that Azadirachta and outgroup Melia toosendan cound be divided into two groups.One is formed by Azadirachta,and the other is formed by outgroup Melia toosendan.The former could befurther divided into different groups,but all showed the early maturing type and leaflet variation type were both similar to A.siamensis.Analysis of haplotypes and all UPGMA clustering showed the early maturing type and leaflet variation type were more closely ralated to A.siamensis.Chloroplast of most angiosperms is maternal inheritance,so that A.siamensis is the most likely female parent of these two variation types.With limited known of karyogenes and featuring by outcrossing of species in Azadirachta,the male parent of the early maturing type and the leaflet variation type was still not clear.Further research was required to confirm the male parent of the two variation types.