| Paspalum vaginatum is a perennial warm-season lawn grass belongs to family Gramineae,genus Paspalum.This grass is largely diploid(2n=2x=20)with genome size～600Mb。As a kind of halophyte,it has the characteristics of drought resistance,salt resistance and enduring infertility P.vaginatum is able to survive exposure to even ocean-strength levels of salt,and as such has become an important turf grass in coastal and salt-afected areas of the world.P.vaginatum is closely related to some of the world’s most important grain crops such as maize,sorghum and millet,and clarification for molecular mechanisms of stress resistance in P.vaginatum will not only provide theoretical foundations for improving the breeds but possibly may provide a gateway to improving these cereal crops for salt tolerance.Therefore,in this study,we conducted the high quality assembly and annotation of “Sea Isle2000” genome which is an important resource for promoting genomics studies related to the superior traits of paspalum and molecular design breeding.The main results are as follows:1.High quality chromosome-scale genome of P.vaginatum and annotationThis study combines Pac Bio,10 x Genomics,Illumina and Hi-C chromatin interaction mapping based on 167.15 G sequencing data to assemble and anchor the chromosome of P.vaginatum.Finally,the“Sea Isle2000” chromosome-level reference genome was obtained,including 10 chromosomes and 169 scaffolds,the total length of contig is 517.61 Mbp,and the length of contig N50 is 5.46 Mbp;the total length of scaffold was 517.97 Mbp,the scaffold N50 was 9.06 Mbp.Illumina reads,CEGMA,BUSCO,and Tag makers comprehensively evaluated the results of the guillemot genome assembly,showing high completeness and accuracy.Based on de novo,homology prediction and gene annotation of transcriptome data,a total of 28,712 codable protein genes were obtained;2756 non-coding RNAs(555 mi RNAs,331 t RNAs,1236 RNAs and 634 sn RNAs).2.Comparative genomics of P.vaginatum and its relative speciesWe applied the predicted proteomes of S.paspalum and 10 other sequenced species to identify putative orthologous gene clusters.A total of 31,619 orthologous gene families composed of 338,128 genes were identified from 10 plant species of which6,949 clusters of genes were shared by the 10 species,1800(25.9%)of the shared gene clusters were single-copy family.Seashore paspalum genome contained 436 specific gene families containing 849 genes.Gene Ontology(GO)term enrichment analyses of P.vaginatum specific genes showed that the molecular functional categories termed“protein dimerization activity” and “carbonate dehydratase activity” were enriched,“protein dimerization activity” and “response-related activity” were enriched in the biological process categories.which may contribute to the special characteristic of saltresistant in P.vaginatum.A set of single-copy ortholog genes of the 10 plant species were applied to construct phylogenetic relationships tree,revealed S.italica,S.bicolor,Zea mays and P.vaginatum were clustered in the other branch.Based on the phylogenetic tree and calibration points estimate the divergence time of these species.The tree indicated that P.vaginatum,S.bicolor and Z.mays might be share a common ancestor,their divergence time were estimated to be about 23.7 Ma(the interval was 21.1-26.8 Ma).A total of 2,123 gene pairs were identified as segmental duplication in ten chromosomes of P.vaginatum genome.Among these pair genes,318 pair genes(14.98%)were located on the same chromosome.The widespread gene duplications suggested that the WGD might have occurred during P.vaginatum genome evolution.Here,more than half of the predicted P.vaginatum genes were identified as syntenic genes with S.bicolor genes,resulting in total 20,849 pair genes.The synteny analysis between the two genomes provided a clear structural evidence for they might be have a common ancestor.Further,we investigated the collinearity between P.vaginatum and Z.mays,the results showed that 16,046 P.vaginatum genes had good collinearity with Z.mays genes,what interesting is lots of P.vaginatum genes have one-versus-four syntenic genes to Z.mays.The above results indicated that P.vaginatum,S.bicolor and Z.mays might share the common ancestor,and Z.mays might had occurred two WGD events when compared with P.vaginatum.3.N6-methyladenine(6mA)DNA modificationUsing SMRT sequencing data,969,960 N6-methyladenine(6m A)DNA modification were detected and distributed evenly across 10 chromosomes.The coding gene 6m A had the highest methylation rate,but the lowest methylation density overall.The methylation rate of LTR,SINE transposon gene and non-coding gene was higher the closer to TSS site,while the methylation rate of LINE transposon gene and coding gene was contrary,and the coding gene had a special 6m A enrichment peak near TSS.GO analysis of genes encoding different 6m A methylation densities suggested that a series of physiological processes of P.vaginatum adaptation to salt stress may have different 6m A methylation regulation modes.Genes with moderate methylation density showed great functional preference in response to biological stress.4.Resequencing,population structure and genetic diversityA total of 4,994,498 SNP and 2,458,362 InDel were obtained by resequencing 193 P.vaginatum germplasms,the average density was 10.09 SNP/KB,4.97 In Del/KB.By analyzing the genetic structure of P.vaginatum by population structure,principal component analysis,phylogenetic tree and population differentiation coefficient,we found that our natural population could be divided into two subgroups,domestic groups and foreign groups,the population differentiation coefficient,between them was 0.23,probably due to the geographical distribution of planting and ecological adaptability of different leading to greater differentiation.Tajima’s statistics of population gene test showed that there were a large number of medium-frequency alleles in the domestic(3.00)population,and the population was selected for low frequency,while there were a large number of low-frequency alleles in the foreign(-0.32),and it was likely that there was gene expansion.The genetic diversity index(π)showed that the genetic diversity of domestic population(2.94)was higher than that of foreign population(1.21).The LD decay distance of paspalum population was 20.5 KB.5.Comprehensive evaluation of resistance to stress and Genome-wide association analysisThe drought resistance of 181 P.vaginatum was evaluated comprehensively by three treatments of mild,moderate and severe drought stress.The data of 13 characters were analyzed by principal component analysis and 3 principal components were obtained with a cumulative contribution rate of 74.0%.Cluster analysis was performed on the calculated comprehensive scores,and the materials were divided into three populations with different drought tolerance degrees : Drought resistance(71),moderate drought resistance(83),drought-tolerant(27).The morphology observation and the molecular bilology methods indicated that the shrain of tar spot belongs to Phyllachora sp.Pathogenicity test was carried out according to Koch’s rule and confirmed that the strain was pathogenic pathogen of Tar spot in P.vaginatum.We analyzed 12 months of meteorological data and Phyllachora sp incidence data of Tar spot,concluded the regularity of the disease.Genome-wide association analysises were conducted basing on 13 droughtresistant characters and 12 disease-resistant characters of P.vaginatum by using EMMAX.53 Peak SNP and 127 candidate genes were located for drought-resistant traits,75 Peak SNP and 136 candidate genes were located for disease resistance traits. |
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