Genome-wide Analysis Of NAC Transcription Factor And Population Evolution In Juglans Regia L.

Common walnut（Juglans regia L.）is a hardwood tree which is economically and ecologically important and produces edible nuts and quality wood,growing in the subtropical zones and warm temprate areas of the Northern hemisphere.In addition to the natural influence of geomorphological barriers and climate,humans have significantly changed their genetic structure.It is commonly believed that J.regia endures and developed in entirely confined stands in Asia after the last glaciation.Antiquated people distributed walnuts into current territories through social extension and trade.The world production since 2012,provided mostly by China,USA,and Iran exceeds three million tons.The breeding began in the 20th century in spite of the very ancient culture of walnut species（Juglans spp.）.Using a range of methodologies,from morphological markers to the most recent advances in genome analysis,many genetic studies of walnut have been conducted during the past 30 years,including diversity examinations,phylogenetic relationship,origin elucidation,and genetic map construction.（i）NAC[no apical meristem（NAM）,Arabidopsis transcription activation factor（ATAF1/2）,and cup-shaped cotyledon（CUC2）]are plant-specific transcription factors that participate in various plant developmental processes such as flowering,plant growth regulation,and development.However,in J.regia,there is no report of the NAC gene family.In this study,we identified 102 NAC genes and clustered into 10 subfamilies based on the phylogenetic tree,protein motif,and gene structure analysis.Importantly,in J.regia,some NAC collinear gene pairs identified in relationship with Populus trichocarpa,Olea europaea,and Quercus robur.The motif composition and conserved domain analysis showed that common walnut contains 5 conserved motifs and 7 conserved protein domains.Most of the NAC genes contain 3 exons,as revealed by structural analysis.All JrNAC genes distributed unevenly on 16 chromosomes with the maximum number found on chromosome 10.Seven pairs of paralogous NAC gene（JrNAC 13-5/Jure＿05048.t1,JrNAC 1-5/Jure＿13612.t1,JrNAC10-5/Jure＿17221.t1,JrNAC 4-2/Jure＿19897.t1,JrNAC 2-11/Jure＿20271.t1,JrNAC9-7/Jure＿20272.t1,JrNAC 10-8/Jure＿28343.t1）were identified in walnut.We identified 60JrNAC WGD（whole-genome duplication）and two tandem duplication that played critical roles in the evolution of the NAC gene family.The ratio of K_a/K_s is less than 1 for 61 gene pairs indicated the negative selection,and only two NAC genes have K_a/K_s values greater than1 indicated positive selection.The JrNAC promoter regions contain 77%of elements that respond to light,7%of the response to environmental stress,3%of the response to plant growth,and 13%of elements associated with site binding.The patterns of cis-acting regions vary among members of the JrNAC.Surprisingly,the JrNAC 1-4 gene promotes the auxin response component,while the gene JrNAC 2-6 has a protein binding site.We detected the interaction relationship between the JrNAC genes family,and the results indicate a strong relationship between the JrNAC 1-4 and the At WRKY12:a gene that regulates the development of flowers.In fruits,three genes（JrNAC 9-8,JrNAC 14-1,and JrNAC 3-4）revealed high expression and have a role in fruit maturation.Furthermore,both the transcript data and qRT-PCR showed that 2 NAC genes（JrNAC 1-4 and JrNAC 2-6）having higher expression levels in female and male flowers compared to leaves.A total of 22 JrNAC were expressed highly in reproductive tissues,while little expressed in vegetative tissues,proposed that the gene family（JrNAC）have a role in flower development.Our results provide valuable evidence for the general characteristics of the common walnut NAC gene family.（ii）We evaluated the spatial genetic structure and genetic diversity of 2,929 individuals from 150 walnut populations sampled using 14 SSR’s markers.STRUCTURE analysis based on 14 loci showed that K=3 was the best representation of the underlying hierarchical structure for the 150 J.regia samples.According to STRUCTURE,most samples generally grouped into predicted populations related to the geographical location.The principal coordinate analysis（PCoA）accounted for 44.0%of the observed variance for the first two axes of coordinates（31.31%and 13.55%）.The results of PCoA indicate three well-separated groups,which are the same as groups identified by STRUCTURE.Our study revealed that Chinese populations had the highest genetic diversity for J.regia in line with Geostatistical IDW technique(H_O,H_E,PPL,N_A,and R_S)interpolation in ArcGIS.Analysis of Molecular Variance（AMOVA）evaluation accounted for 64%of genetic variation distributed mainly within individuals.The main genetic boundary appears only in Central Asia（China,Pakistan,Kyrgyzstan,and Tajikistan）.θand M values were greater than zero as determined by Migrate-n analysis.The size of the immigration rate（M）andθ-value showed a highly asymmetric historical gene flow.Scaled immigration rates（M）revealed asymmetric historical gene flow from population I to population II（M=6.68）,population III to population I（M=5.58）,and population II to the population I（M=4.89）.Meanwhile,the gene flow values（N_M）per locus ranged from 0.23 to 0.79（average=0.52）.The current study thus provides direct evidence of the interspecific historical gene flow.Geneticists,paleoecologists,and biogeographers are of primary interest in the endurance and distribution of trees during the last glacial maximum（LGM）.Ecological Niche Model（ENM）that associate existence and abundance of species with environmental factors are widely used to increase evolutionary and natural insight to anticipate the distribution of species over time and space.Our study mainly focused on walnut history through ecological modeling and genetic analysis of current,last interglacial（LIG）,and LGM.To project the walnut dispersion,LGM,LIG,current,and future were designed using the specie distribution model.We support,based on our genetic data that common walnut originated from central Asian（Uzbekistan,Kyrgyzstan,Tajikistan,and Pakistan）and extends toward Himalayan Mountains～60 Mya.The common walnut has experienced population expansion eastward to western Asian and Europe,and westward to eastern Asian,with the continuous climate changes.These analyses showed that the intraspecific variation in walnut influenced partly by climatic changes.During LIG,the population has been dispersed to eastern China and west to Europe.The present and LIG correlation of the SDMs,northern China is favorable during warmer periods.During the Last Glacial Maximum（LGM）,there were multiple refugia existed in Europe,Central Asian,and China.After the LGM（Last Glacial Maximum）,walnut endured and expand instinctively in almost entirely secluded places in Europe,central Asia,especially the Xinjiang region of western China and to the Caucasus from central Asia.