Study On Drought Adaptability For Psammochloa Villosa(Poaceae),a Desert Grass In The Inner Mongolian Plateau And Adjacent Regions

The Inner Mongolian Plateau is the largest plateau in northern China,with higher terrain in the south than low in the north,and trends toward drier climate,throughout windiness and rainless,regional climate change caused a profound influence on plant’s distribution and adaptation.Thus,it’s the principal pathway for ensuring forage breeding and ecological security by studying on distribution and adaptive evolutionary history of natural population across different climate environment.Psammochloa villosa is a desert herb endemic to the Inner Mongolian Plateau and its adjacent regions,and it exhibits strong adaptation to its arid environment.Thus,this species is an ideal material for studying the evolutionary history of adaptation of plants to the dynamic climatic and geological history of these arid environments,which are relatively geologically young,and to mine genes related to drought resistance.Accordingly,we reconstructed phylogeny,analyzed population structure and dynamic history,inferred gene flow and direction,and evaluated the association of genomic variation with climatic and environmental factors using research methods from phylogeography,population genetics,and bioinformatics to clarify the evolutionary mechanism of drought adaption of P.villosa.Our aim is to reveal the effects of natural selection on environmental adaptive differentiation of population.The main results were as follows:⑴Responses of phenotypic variation to environmental factorsPhenotypic diversity analysis of P.villosa showed that interpopulation index of Shannon-Wiener of leaf sheath hair is the highest,color of spikelet is the lowest.Most phenotype traits were significantly different across populations（P<0.05）except the branch number of panicle,length of glume’s and lemma’s awn.The phenotypic differentiation coefficient（Vst）of panicle length was the largest of interpopulation,length of glume’s awn was differed of intrapopulation.Mean square error from Inter-and intrapopulation were 40.71%and 16.93%respectively,which result provided evidence that differentiation of phenotypic traits was derived from interpopulation.We explored the regression relationship of plant’s phenotypic variations constrained by multi-response environmental variates via distance-based redundancy analysis.The results indicated that the difference of altitude and mean wind speed was direct factor for adaptive differentiation of phenotype,for instance severe climate（mean temperature of driest quarter and precipitation of driest month）promoted phenotypic similarity of interpopulation,but aridity index and vapor tension furthered phenotypic difference.The phenotypic variation of P.villosa were derived from interpopulation,reflecting obvious geoclimatic trends.⑵Responses of transcriptional profiling to simulated drought stressWe utilized PEG-6000 to simulate an arid environment（0,10%,and 20%）,combining the advantages of Pac Bio,and Illumina sequencing methods,obtained184,076 full-length transcripts,annotated 168,650 genes（accounting for 91.6%）and filtered 119,005 unigenes,identified 3669 differential expression genes（DEGs）.Comparing with control group,significantly up-regulated expression genes（FDR<0.01）in drought group were annotated as‘Chlorophyll a-b binding protein’and‘photosystem II light harvesting complex protein’.GO functional enrichment analysis revealed that 11 up-regulated genes were mainly involved in coping oxidative stress processes,and KEGG analysis revealed these DEGs enriched at phenylpropionic acid biosynthetic pathway,which produces phenols eliminating reactive oxygen species（ROS）in plants.The result of protein-protein interaction（PPI）networks showed that a core gene hub was formed by DEGs and their interacting proteins,mainly involved in arginine and proline pathways,playing the roles in scavenging ROS and protection of osmoregulatory.We adopted Real-time quantitative flouorescence PCR method to verify transcriptional expression difference of P.villosa under drought stress.⑶Effects of paleoclimatic and geological historical events on genomic adaption evolution of P.villosaBased on a hybrid sequencing approach from Survey,Hi Fi,and Hi-C,we assembled the high-quality chromosomal reference genome and 371 contigs,found that LAI was 21.76,contigs N50 was 63.67 Mb,identified 54,317 protein-coding genes,and 75.84%of the assembled sequences were transposable elements,with long terminal repeats（LTRs）being the most abundant.The LTR retrotransposons may have been activated and expanded in the genome during the Pleistocene between 0.22and 2.12 million years ago（Mya）.Phylogenetic analysis proved that P.villosa belongs to Poaceae,Pooideae,and tribe of Stipeae,with the closest relationship of Achnatherum splendens,forming a sister group and monophyletic clade,occurring the species divergence about Miocene period（7.18 Mya,HPD:6.28-10.95 Mya）.The divergence time between Brachypodium distachyon and this clade was about 40.63Mya.Genomic synteny analysis indicated that P.villosa underwent a common whole genome duplication（c WGD）event at 98.2 Mya shared by Poaceae species,and an additional independent WGD event dated to Miocene（13～19 Mya）.Meanwhile,We found that 15 conserved chromosome kept massive ancestral gene,avoided recursive chromosomal fusion and fission events after P.villosa divided with ancestral grass karyotype（AGK）.Through WGD and species differentiation events during Miocene,there were 14 gene families experienced rapid evolution,48 and 42 gene families suffered significant expansion and constriction.The functions of those expanded gene families were related to regulation pathways such as‘fatty acid metabolism’,‘jasmonic metabolism’,and‘ion binding’and‘peroxidase activity’.Based on transcriptional expression analysis,we identified 21 gene families involved in drought tolerance of P.villosa,found that the xyloglucan endotransglucosylase/hydrolase（XTH）gene family occurred expansion and expression difference,78 XTHs genes contained complete conserved domains（XET＿C and Glyco＿hydro＿16）.The phylogenetic tree of XTHs illustrated that XTHs gene of P.villosa have grouped into three clades,and closely related to the evolution of A.splendens,a drought-tolerant and salt-resistant plant.Our results indicated that the Asian monsoon-arid environment and tectogenesis caused adaptive evolution history in the P.villosa genome such as WGDs,species diversity,and expansion of drought-tolerant gene families since Miocene.⑷Effects of climatic fluctuation on the demographic history and adaptive differentiationBased on neutral theory,we studied on genetic structure of population combining Neighbour-Joining tree,Principal Component Analysis,ADMIXTURE and LDdecay approaches,and identified 27,378,635 SNPs and 29,639 INDELs.The results demonstrated that the natural population of P.villosa presented obvious phylogeographic structure,including northwest group（located in northern Gansu,Ningxia,and Alashan Right Banner,Inner Mongolia）,central group（Etok Banner,Ulanqab,Inner Mongolia and central Shanxi province）,northeast group（Chifeng,Xilingol League,Hulun Buir,Inner Mongolia）,there were visible gene integration in central group.The plot of LDdecay detected linkage disequilibrium in different subgroup,but non-random associations among allele in central group was higher,the degree of linkage disequilibrium was much intensity.Based on decline of genetic diversity and change of Site Frequency Spectrum（SFS）,we identified selective area of genome,analyzed relatedness between genetic variation and environmental factors searching unusual SNP sites from genetic differentiation(F_ST),polymorphism levels（θπ）,ROD index to detect whether natural selection promotes adaptive differentiation of P.villosa population.There were strong signal among different hierarchic subgroup(0.18<F_ST<0.8 andθπ>0.0019),and 187 environmental-adaptive differentiation sites.We used cross-population composite likelihood ratio test to assess that intrapopulation differences in frequency of multiple alleles were derived from environmental-adaptive differentiation.Demographic history analysis using PSMC model revealed that the effective population size（Ne）of P.villosa have undergone sequentially shrinking and an obvious bottleneck during Pleistocene（about 10～1 Mya）.About 10,000 years ago at Holocene,Ne expanded and reached its peak with faint gene flow（only northwest to central group）,which results suggested that the Quaternary climatic fluctuations and natural selection may affect the long-term adaptation of P.villosa population to geographical environment.Based on LFMM model,we excluded the effect of population structure,detected 302 SNPs associated with six environmental factors.We deem these associated sites to be core adaptive variants of P.villosa populations broadly distributed in the genome.