Comparative Analysis Of Wild Buckwheat Chloroplast Genomes And Phylogenetic Relationship Between Fagopyrum Species

Buckwheat is a nutritional and medicinal crop belonging to Polygonaceae,Fagopyrum.The Southwest China has abundant wild buckwheat resources and it was also recognized as the global center of buckwheat origin.In order to take the advantage of the great germplasm resources in Southwest China,it is necessary to develop high efficient molecular markers to identify the new species of Fagopyrum during the wild resources investigation.However,the shortage of buckwheat genetic resources has affected the process of phylogenetic relationship research between Fagopyrum species,which also influenced the buckwheat molecular breeding and the utilization of wild buckwheat genetic resources.At the same time,chloroplast genome has become an ideal resource for phylogenetic relationship and the genetic evolution studies,while the wild buckwheat chloroplast genomes data are necessary for the bioinformatics and molecular biotechnology studies.To the best of our knowledge,only several chloroplast genomes of buckwheat were sequenced and reported,which is insufficient for the wild buckwheat resource investigation and phylogenetic relationship research.All in all,it will make great value to start research about the wild buckwheat resources investigation and collection,sequencing the wild buckwheat chloroplast genomes,developing potential molecular markers after comparative analysis the buckwheat chloroplast genome sequences,as well as molecular marker-assisted phylogenetic research and new species of Fagopyrum identification.In this way,to better utilizing the resource of buckwheat in Southwest China,also understand the mutation patterns and evolution trend in the chloroplast genomes of buckwheat,found sufficient number of variable regions to explore the phylogenetic relationships of Fagopyrum,the study about comparative analysis of wild buckwheat chloroplast genomes and phylogenetic relationship between Fagopyrum species was processed.The main results are as follows1)After the investigation and collection of wild buckwheat resources in Southwest China,the results shown that 26 Fagopyrum species were distributed in Southwest China,and the distribution ranges of Fagopyrum species were different.F.cymosum,F.gracilipes and F.gracilipes var.odontopterum were widely distributed in Sichuan and Yunnan,while the distribution of F.crispatifolium,F.qiangcai and F.hailuogouense were narrow compared with other species.The wild buckwheat germplasm resources were mainly distributed in Northern and Southwest Sichuan,also the Northwestern Yunnan areas.These results were also proved the Southwest China is the global origin place of buckwheat.On the other hand,after evaluated the nutrients and secondary metabolic products of wild buckwheat resources,we found that the wild buckwheat were comparable to cultivation buckwheat in protein,amino acids,flavonoids content and so on,which proved that wild buckwheat have the great potential in food and pharmaceutical industry.2)Two complete chloroplast genomes of buckwheat including F.cymosum and F.luojishanense were sequenced,and other two Fagopyrum chloroplast genomes were used for comparative analysis.In this study,we reported the information of the whole chloroplast genome sequence of wild buckwheat firstly.After morphological analysis,the main difference among these buckwheat were height,leaf shape,seeds and flower type.F.luojishanense was distinguishable from the cultivated species easily.The chloroplast genome of F.cymosum was 159320 bp while the F.luojishanense was 159265 bp.The SNP number between F.cymosum and F.tataricum was the smallest compared with other Fagopyrum chloroplast genomes,which was only 317.At the same time,we also found that the difference between the F.luojishanense and other buckwheat chloroplast genomes was the largest,the number of SNP were 5940,6260 and 5992,respectively.In order to detect the selection pressure of different genes in chloroplast,the synonymous(dS),nonsynonymous(dN),and dN/dS values of each coding genes were analyzed.The results shown most dN/dS ratios of chloroplast genes were less than 1,only ndhK、ycf1、rpoC2、rpl20、petL and rpl32 had dN/dS values more than 1,which indicating that most genes in chloroplast were under negative selection.After the border analysis of chloroplast,only rps19 and ndhF genes was detected across the LSC/IRb and IRb/SSC border in four Fagopyrum species.On the other hand,the rps15 gene was across only in F.luojishanense and F.esculentum which caused by the normally single-copy gene ycf1.Meanwhile,after comparative analysis of four Fagopyrum chloroplast genomes,48 and 61 SSRs were found in F.cymosum and F.luojishanense respectively.The total repeat sequences among the four chloroplast genomes were 135,and the number of repeat sequences,repeat sequences type and repeat sequences location in F.cymosum were similar with F.tataricum.After that,10 highly variable regions among these buckwheat species were located precisely,including trnS-trnG、rpoB-rpoC、trnT-psbD、trnT-trnL、rbcL-accD、ycf4-cemA、psbE-petL、ndhF-rpl32、ycf3-trnS and ndhA-intron.The phylogenetic relationships among four Fagopyrum species based on complete chloroplast genomes was showed.All the results suggested that F.cymosum is more closely related to F.tataricum compared with F.esculentum.3)Nowadays,the study about the phylogenetic relationships of buckwheat were only based on several gene fragments,the molecular results were limited caused by the insufficient materials and incomplete information,the phylogenetic relationships between the Fagopyrum species were still not clear.In this study,we combined the morphological characteristics with the molecular evidences,as well as we reconstructed the phylogenetic trees of different Fagopyrum species collected from Sichuan and Yunnan based on the molecular markers developed from the comparative analysis of whole chloroplast genomes.The results shown that the molecular markers psbE-petL and ndhA-intron regions could be used as ideal molecular markers to identify the cultivation and wild species of Fagopyrum.Meanwhile the phylogenetic relationships between the Fagopyrum species could be demonstrate charily by using the multi molecular markers,which shown the combinations of matK+psbE-petL and psbE-petL+ndhA could elaborate the relationships between Fagopyrum species easily.After combined results of multiple phylogenetic trees,we further clarify the relationships among species in Fagopyrum,the Fagopyrum should be divided into two large groups.The groups I including cultivation species and F.cymosum,the groups II should including all the wild buckwheat species such as F.lineare、F.qiangcai、F.pugense、F.wenchuanense and F.crispatifolium and so on.Meanwhile,in groups II the F.callianthum is closely related to F.wenchuanense and F.pugense while F.leptopodum is closely related to F.jinshaense and F.lineare,as well as F.macrocarpum clustering with F.gracilipes、F.gracilipes var.odontopterum and F.crispatifolium,which indicated these species have closely relationship.4)The last but not least,in order to verify the molecular markers for the new Fagopyrum species identification,we demonstrated the phylogenetic status of F.longzhoushanense which was found during the wild buckwheat resources investigation of Southwest China and it could be identified as a new species in Fagopyrum.After research,the results showed F.longzhoushanense was a new species of Fagopyrum based on the morphology characteristics and karyotype analysis.The results of phylogenetic analysis based on the molecular marker also highlight the distinction of this new species.To sum up,based on the collection of wild buckwheat germplasm resources in the Southwest China,combined with the chloroplast genomes sequencing results and comparative analysis,we found the potential molecular markers for the buckwheat phylogenetic research and new Fagopyrum species identification.Our data provided valuable genetic information for Fagopyrum species taxonomy,phylogenetic study and molecular breeding in the future.