Phylogenetics Of Musa Spp.and The Origin Of Cultivated Bananas Based On Cytoplasmic Genome Analyses

Bananas belong to Musa L.Musaceae and are important food and fruit crops worldwide.Although domestic and foreign scholars have carried out a series of studies on the classification,origin and evolution of Musa according to morphological analysis and molecular biology,its origin and evolution are still speculative and inconclusive.The genetic origins of many main cultivars remain unclear.To find out the molecular evolutionary relationship between commercial cultivars of banana and their relative Musa spp.and the genetic origin of banana cultivars is of important theoretical and practical significance for the classification,evolution and genetic improvement of bananas.In this study,direct sequencing of gene fragments was used to screen the cytoplasmic molecular barcodes for different taxonomic categories of Musa L.,the cytoplasmic genes of banana cultivars and their wild relatives were also used to construct the maternal and paternal phylogenetic relationships.Furthermore,high-throughput sequencing was applied to carry out the genome sequencing,genome mapping,comparative genomics and phylogenetic analysis on the chloroplast genome of bananas.The main findings are as follows:1.Screening of cytoplasmic molecular barcodes of Musa species: 70 pairs of chloroplast gene fragment primers and 60 pairs of mitochondrial gene fragment primers have been reported used from literatures of plant taxonomic research.These primers were synthesized and screened,resulting 32 pairs of chloroplast primers and 16 pairs of mitochondrial primers which were suitable for the samples from different groups,species and geographic populations of Musa L..The results showed that 25 pairs of chloroplast primers and 12 pairs of mitochondrial primers have genetic diversity in Musa L.In these polymorphic primers,17 pairs of chloroplast primers and 11 pairs of mitochondrial primers were first used in the genus Musa.2.Phylogenetic analysis of Musa species based on multiple chloroplast genes: The maternal phylogenetic relationships of Musa species were analyzed using 25 chloroplast gene sequences.The results showed that the alignment length of 25 chloroplast gene sequences was 22306 bp,generating 617 variable sites,among which 265 were parsimony-infarmative sites,and also generating 1,663 Indel sites.Based on the 25 chloroplast gene sequences,the MP,ML and Bayesian trees showed that all the experimental materials were divided into three distinct clade:,M.beccarii as the outgroup at the root;M.acuminata subspecies,M.laterita,M.yunnanensis,M.chunii,M.balbisiana(ITC0545),a natural wild hybrid M.balbisiana×M.textilis and other cultivated bananavarieties are cluster together;M.itinerans from different populations,M.nagensium,M.balbisiana from China,three different M.balbisiana from ITC,and two cultivated bananas containing the B genome cluster together.M.balbisiana exhibits different maternal origins,and its intraspecific classification deserves further investigation;3.Phylogenetic analysis of Musa genus based on multiple mitochondrial genes:Analysis of the paternal phylogenetic relationships of Musa species using 12 mitochondrial gene sequences.The results showed that the alignment length of 12 mitochondrial gene sequences was 6802 bp,generating 553 variable sites,among which 279 were parsimony-informative sites,and 328 indels sites were also generated;The MP,ML and Bayesian trees constructed based on 12 mitochondrial gene sequences indicated that the tested materials were divided into four distinct clades.The first one contained M.itinerans from different populations,M.yunnanensis and AAB genotype cultivars Cluoi mat.The second clade contains M.acuminata subspecies,M.laterita,M.chunii,and most of the tested cultivated banana varieties.The third clade include all M.balbisiana,M.nagensium,M.balbisiana × M.textilis natural wild hybrids and Fenjiao(AB),Maduranga(ABB),and Namwa Khom(ABB).The fourth clade was the outgroup M.beccarii.From the paternal origin,both M.acuminata and M.balbisiana have consistent paternal origin,but M.itinerans has separate paternal origin.4.The origin and evolution of banana cultivars: Based on 25 chloroplast gene sequences,M.acuminata revealed 5 genotypes and M.balbisiana revealed 3 genetypes.Based on 12 mitochondrial gene sequences,M.acuminata revealed 3 genotypes and M.balbisiana also revealed 3 genotypes.The combination of chloroplast and mitochondrial genotypes analyses,19 cytotypes were identified.Cytotype IV,consisted by Ca2(burmannica/malaccensis/burmannicoides)and Ma3(microcarpa)is primary origin of cultivated bananas with genetype A(AA,AAA)for test,while the Cytotype V composed of Ca3(M.acuminata ssp516)and Ma1(chinensis/siamea/malaccensis/burmannicoides)and Cytotype VII composed of Ca3(M.acuminata ssp516)and Ma3(microcarpa)were the main origin of cultivated bananas of genotype A-B(AB,AAB,AAAB).The results also showed that M.itinerans also participated in the formation of cultivated bananas,of which Chuoi mat(AAB)and M.itinerans have the same paternal origin.5.Structural and phylogenetic analysis of the chloroplast complete genome of Musa species: By the next generation sequencing technology,the whole genome sequencing of the chloroplast genomes of M.acuminata var.chinensis and M.beccarii and Musella lasiocarpa were performed and the genome map was drawn,the first genomic map of the chloroplast of the genus Musella lasiocarpa was obtained.Compared with the published chloroplast genomes of four Musa species,the full length of the chloroplast genome of thesix Musa species ranged from 161347 bp to 170402 bp,which have a typical quartile structure: LSC,SSC and two inverted repeat regions,IRa and IRb.The six Musa spceis encode 79 protein genes,29-31 t RNAs and 4 r RNAs.According to m VISTA analysis,the most differentiated gene in 6 Musa spceis is ycf1.In addition,there are also differences in genes such as mat K,rpo C2,rpo B,atp A,acc D,ycf2,and some intergenic regions,such as trn K-trn Q,trn S-trn G,Atp H-atp I,trn C-trn D,trn T-trn F and psb E-pet G.The ML phylogenetic tree constructed with the genus Musella lasiocarpa as an outgroup indicates that based on the chloroplast genome-wide sequence,6 species of Musa can be divided into three clades,of which M.beccarii is a separate clade,M.acuminata subspecies cluster together,while M.balbisiana,M.itinerans and M.textilis are cluster together.In the previous studies,M.textilis was belong to Australimusa,which was distinct from M.balbisiana and M.itinerans,but in this study,M.textilis has the same maternal origin with M.balbisiana and M.itinerans.