Selecting Proper Restriction Enzymes For Genotyping-by-sequencing Library Construction And Local Adaptation To Climate In Eucalyptus Grandis

The speeding up of global climate change is a major challenge to human,and the response and adaptation of species to climate change will have major impacts on the stability of terrestrial ecosystem structure and function.The organisms adaption to the continuous changes of habitat conditions is of great significance to the long-term preservation of germplasm resources and the formulation of breeding strategies.Recent climate-change research largely confirms the profound impacts on ecosystems and biodiversity.To carry out population adaptation research has become an important part of population genetics and evolutionary biology study since the intensification of global climate change and unclear adaptation mechanisms of natural populations.Eucalyptus is an ideal tree species for adaption research due to its rich germplasm resources,high genetic diversity and strong adaption.With the rapid development of next-generation sequencing technology,GBS technology has been increasingly applied in adaption research with the advantages of cost effective,simplicity,and independent of reference genome.However,the number of developed SNPs is rely on selecting proper restriction enzymes for different speices.Therefore,our study firstly used the published ddGBS protocol to consturct the GBS library in different eucalypts species,develop SNP loci and construct an evolutionary tree,and indentify the applicability and reliability of ddGBS technology in Eucalyptus.Then we optimize the restriction enzymes of ddGBS library construction by using six samples of Eucalyptus grandis and apply it to other nine tree species.Finally,through optimization of ddGBS protocol,the study of local adaptation to environment change was carried out in E.grandis natural populations,which provides molecular evidence for further understanding the adaptive mechanism in eucalypts.The main conclusions are as follows:（1）The ddGBS technology can be widely used for high-quality SNPs development in Eucalyptus,and the result provides theoretical support for the systematics and evolution research in Eucalyptus.By constructing ddGBS library on 14 eucalypts tree species,high-quality SNP loci were developed,SNP annotation and nucleotide diversity analysis were carried out,and the maximum-likelihood phylogeny tree was constructed by using IQ-TREE.In total,30.49 Gb clean sequence data are obtained with average 1.09 Gb for each sample and 42 222 high-quality SNP loci were totally obtained,of which only 428～2 107 unique SNP loci are found in each species.A total of 12,237 SNPs were located in the exon region by functional annotation analysis with Snp Eff.The phylogeny tree of the fourteen eucalypts matched Hill&Johnson’s taxonomic classification of the subgenus Symphyomyrtus,which indicated the high quality SNPs development from ddGBS.（2）It was proved the success of ddGBS optimization in Eucalyptus for the efficiency of enzyme digestion and SNP development.Based on Poland’s protocol（the combination enzyme of Msp Ⅰ-Pst Ⅰ）,a new enzyme digestion combination（Msp Ⅰ-Mse Ⅰ）protocol was screened and optimized.Throug contrast test with 6 samples of E.grandis,the new enzyme combination protocol was showed great improvement on the fragments number,the efficiency of gel electrophoresis,the number of developed SNP markers,and the distribution density of the markers on the E.grandis genome.The successful optimization of the ddGBS protocol indicates that the new enzyme digestion combination has a broad application prospect in the study of genetic diversity,phylogeny and molecular marker-assisted breeding in trees.The optimized protocol was also applied to construct ddGBS library for the other 9 tree species:Aquilaria sinensis,Casuarina equisetifolia,Tectona grandis,Quercus dentata,Hopea hainanensis,Acacia melanoxylon,Archidendron clypearia,Mytilaria laosensis and Dalbergia cochinchinensis,and develop SNP markers and analysis loci polymorphism.The results showed that the sequencing data coverage of 9 trees ranged from 0.39 to 9.29,producing 9 583～131701 SNPs.In addition to Quercus dentata and Acacia melanoxylon,the coverage of other seven trees are close to 1×,or higher.The highest number of developed SNP loci are up to 62,114 in Archidendron clypearia（coverage is 0.91）,indicated that the new protocol can be used to discover high-quality SNPs among the 9 tree species.In addition,polymorphism analysis showed that E.grandis had higher nucleotide polymorphism with the new protocol,which also was successfully carried out on the other 9 species.（3）The optimized ddGBS system was successfully applied to the SNP development,genotyping and adaption analysis in E.grandis,and a set of adaptive candidate genes related to precipitation and temperature were successfully screened.The optimized protocol（Msp Ⅰ-Mse Ⅰ）was utilized to develop SNP loci for 137 samples of 16 natural populations in E.grandis,and genetic diversity,environmental adaption and selective sweep analysis were also carried out.A total of 87,282 high-quality SNP markers were developed with average 2.1 Gb sequence data for each sample.High genetic diversity was detected in E.grandis population withthe He mean is0.191,the averageπvalue is 0.206 and PPL%is 70.576%.Nevertheless,mean F_ST value in 0.054demonstrated the level of genetic differentiation is low and the differentiation coefficient（0.064>0.046）demonstrated the northern population（5 population）was slightly higher than that of the southern population.The results of evolutionary tree categorized the populations of E.grandis into two groups.The Ward clustering results with climate factors were also clustered into north and south groups,identified that climate factors also drive population differentiation.The adaption analysis showed that 390 FST outliers were detected by OA using the eleven low-correlation environmental factors.In GEA analysis,three models of MLM,GLM and Farm CPU were used for environmental factor association analysis,and GLM model showed better results.A total of 53 loci at the intersection of the GEA and OA were taken into the adaption significant associated loci,of which 16 loci were significantly associated with at least2 environmental factors.These 53 locis belong to 23 gene regions and annotation analysis found that 21 genes had functional information,of which 9,7,and 6 genes were significantly associated with temperature,precipitation,and altitude factors respectively.For example,the loci associated with the altitude and temperature factors are found located on the genes Eucgr.D02167（bio2/alt）and Eucgr.K03195（bio2/bio6/alt）,and the annotation functions on nitrate transporter（NRT1.5）and protein Kinase superfamily proteins.It also found that plant protein kinases in E.grandis responded to high salt,drought,low temperature,high temperature,and high osmotic stress.Therefore we estimate that E.grandis has potential adaption to future rapid climate changes since it is widely involved in stress response processes.Our study selected proper restriction enzymes for GBS library construction and assess local adaptation to current climate change in E.grandis by using environmental association analyses to identify genetic variants associated with environmental variables.Discovering valuable candidate functional genes and identifying genetic variants,help to find the potential in locally adaptive evolution or plasticity in tree response.This research potentially sheds light on how forest trees will act on future global warming,and will provide a solid genetic basis for the future tree breeding.