Genetic Diversity Of Calotropis Gigantea And Chloroplast Genome Of Genus Calotropis

The genus Calotropis comprises of two species viz.C.procera and C.gigantea,which made a great attention due to its aesthetic,medicinal,pharmacological,fodder,fiber,and biodiesel production potential.Calotropis is still undomesticated and the genetic and genomic information available for crop improvement is limited.Genetic diversity and population structure of C.gigantea in its native countries(Bangladesh and China)with special interest is still lacking.Till date,there is no complete chloroplast genome published from genus Calotropis.The genomic comparison of Calotropis with other Apocynaceae members is crucial for understanding the evolution of this genus.1.Genetic diversity and population structure of C.giganteaThree hunderds seventy nine individuals from 21 natural populations of C.gigantea from two key areas of its natural distribution range(Bangladesh and China)were genotyped using nine nuclear microsatellite markers.The microsatellite markers exhibited high polymorphism information content(PIC=0.695)and a total of 151 alleles were detected in 379 individuals.Population genetic diversity was higher in Bangladesh than that observed in Chinese populations.There was no significant variation of genetic diversity between mainland and island populations of C.gigantea.Overall,a moderate level of genetic diversity was found(Na=3.73,HE=0.466),with most of the genetic variation detected within populations(65.49%)and substantial genetic differentiation(FST=0.345)between the study regions.A significant correlation between genetic and geographic distances was observed(r=0.287,P=0.001).The Bayesian clustering,UPGMA(Unweighted pair group method with arithmetic mean)tree and PCoA(Principal coordinate analysis)yielded three distinct genetic pools.The number of migrants per generation was stable and high(NM=1.44)among the genetic pools.The analyses also revealed that some populations may have experienced recent demographic bottlenecks.2.Comparison of chloroplast genome of Calotropis with Apocynaceae membersThe whole chloroplast genomes of both C.procera and C.gigantea species were obtained using next-generation sequencing.Comparative chloroplast analyses conducted using two Calotropis species and another four Apocynaceae species(Asclepias syriaca,Cynanchum auriculatum,Nerium oleander,and Carissa macrocarpa).Calotropis procera possessed the largest chloroplast genome(166,010 bp)followed by C.gigantea(165,928 bp)as compared to other studied genomes(varied 154,903-160,840 bp).Both species of Calotropis had 114 genes including eighty protein coding,thirty transfer RNAs,and four ribosomal RNAs.The six investigated genomes exhibited dominant mononucleotide microsatellites and 50-59 predicted RNA editing sites.A negative selection was found in most of the protein coding genes,while three ribosomal small subunit genes such as rps3(ribosomal protein S3),rps7(ribosomal protein S7),and rpsl9(ribosomal protein S19)were showed positive selection in Calotropis species.The shortest and longest accD(acetyl-CoA carboxylase carboxyltransferase beta subunit)gene was observed in Plumeria cubensis(Rauvolfioideae,1,458 bp)and C.gigantea(Asclepiadoideae,4,332 bp),respectively.The expansion of accD gene length in the three subfamilies of Apocynaceae remarked as Asclepiadoideae>Apocynoideae>Rauvolfioideae.A total of 37 types(R1-R37)repetitive elements(tandem repeats)identified in Apocynaceae lineage.Overall,the genetic diversity provides a baseline for exploitation of the genetic resources of C.gigantea in domestication and breeding programs as well as some insights into the germplasm conservation of this valuable plant.The genomic comparison is helpful for understanding the systematics and evolution,phylogenetic studies,diversification,and ancestral state of the genus Calotropis in Apocynaceae.