Evaluation And Phylogenetic Relationship Of Citrus L. Based On DArT Markers And Chloroplast Genomes Analyses

Citrus L.is one of the most important fruit crops in the world.China is a vital birthplace of Citrus L.The genus Citrus was established by Carl Linneaus in 1753.Since the establishment of the genus,Citrus L.has attracted much research interest,domestic and foreign scholars has launched a series research on the classification of Citrus L.for more than 260 years.However,due to the high frequency of bud mutations,the sexual compatibility between Citrus L.and related genera,the long history of cultivation,and wide dispersion,the classification of cultivars and the origin of wild species are still controversial,the phylogeny framework of Citrus L.is still to be improved.Although researches from morphology to molecular have been done,the citrus interspecific relationship is still lack of sufficient molecular evidence.With the development of bioinformatics and high-throughput sequencing technology,the new method on classification and evolution are gradually applied to more and more plants.DArT(Diversity Arrays Technology)is a high-throughput technique in species or individuals identification on the genome level.It combined genomic library and genechip inspection without the need for traditional gel electrophoresis analysis,so that the detection accuracy and throughput are greatly improved;besides this methods has no need to know the genome sequence information in advance,it will be the ideal technique on the assessment of the overall situation for the nuclear genome of citrus.And because the chloroplast DNA is tiny,simple,and monolepsis,it is an ideal means on maternal inheritance analysis.Our study included two parts.On one hand,a DArT microarray was constructed,24 Citrus and it's closely relatives were analyzed based on this microarray.On the other hand,chloroplast genomes of 6 Citrus L.and it's closely relatives were sequenced,and several DNA barcodes for Citrus L.phylogenetic issues were identified by comparisons of chloroplast genomes.DArT microarrays were generated using DNA from 15 citrus species of Swingle classification system.We analyzed 24 citrus and their close relatives on DArT arrays.Phylogenetic analyses based on DArT markers strongly supported delineation of the Citrus L.as a monophyletic group.Most citrus species analyzed formed different clusters,but Ichang papeda and Mangshan wild mandarin formed separate clusters as two single branches,suggesting that various mandarin accessions have different genetic backgrounds.We sequenced six complete cp genomes,four belonging to the Citrus L.,and two belonging to the genera Fortunella and Poncirus.These newly sequenced genomes together with the two publicly available were used for comparative analyses of the Citrus L.and its close relatives.The cp genome of C.aurantiifolia is the smallest one,which is followed by C.medica,C.grandis,C.sinensis,C.mangshanensis,F.hindsii,P.trifoliata,and C.hongheensis Y.L.D.L.,respectively.All 8 cp genomes share similar basic structure,gene order,introns,and gene content.Except for C.aurantiifolia,the other 7 cp genomes share an identical set of 136 predicted functional genes,including 102 protein-coding genes,30 tRNA genes,and 4 rRNA genes.The comparative analyses revealed 60 intergenic regions could be used as markers for the resolution of the interspecific relationships.