Revealing Mechanisms Underlying Lineage Divergence Of Ginkgo Biloba Using Plastid Genomic And Comparative Transcriptomic Approaches

Abstract:Ginkgo spp. were widely distributed in the northern hemisphere in the Mesozoic Jurassic. However, they had disappeared in most areas since the Miocene, such as North America, Europe, Eastern Asia and Japan. The minor populations survived in the downstream region of Yangtze River. These populations are divided into two genetic lineages, located in Eastern China (Tianmu Mt., Zhejiang Province) and Central-Western China (Wuchuan, Guizhou Province; Jinfo Mt., Chongqing City; Enshi, Hubei Province etc.). But due to the deficiencies and neutrality of traditional molecular markers, phylogenetic relationship, divergence time and evolutionary forces of Ginkgo biloba have not yet been solved. Therefore, we selected 10 cpDNA haplotype individuals of Ginkgo biloba based on the previous work. In order to obtain a high resolution of DNA mutations, we sequenced the chloroplast genomes via Next Generation Sequencing, which can be used for resolving the differentiation pattern, phylogenetic relationship and divergence time of Ginkgo lineages. At the same time, the ecological niche model (ENM) of Ginkgo biloba in East Asia region and the T test analysis between East (Tianmu Mt.) and West (Jinfo Mt.) geographical lineages were used to identify the main factors in ENM and the differentiated factors in T test analysis, followed by comparative transcriptomes analysis of two representative populations (Tianmu Mt. and Jinfo Mt.) analysis, in order to test the existence of natural selection and identify functional candidate genes under positive selection. Results are as follows,1.Structure evolution of chloroplast genome:The genome size of Ginkgo chloroplast ranged from 156,929-156,999, including 99,202-99,269 long single copy (LSC) region,22,256-22,283 short single copy (SSC) region, and two inverted repeat (IR) regions with 35,464-35,366 bp. The structure variation (SV) among 10 individuals was tiny. All the indels appeared in two single copy regions, while only one SV appeared in IR.152 mutation regions were identified, including 115 SNP and 37 Indel loci. In addition,5 EST-SSR sites were identified specifically in the Western populations, which could be used for the identification of resources origin.2. Phylogenetic relationship and divergence time of genetic lineages:the cp genome data from 10 haplotype individuals were divided into 3 clades, Western refuge clade (first diverged), Eastern refuge clade, and H1 haplotype clade. This result strongly supports the previous hypothesis there are two refugia in China, and further reveals that there are two divergence events happened in lineage divergence of Gingko biloba. Combined 53 shared gene in different species and four fossils calibration of Cycas, we estimated the first divergence event of Ginkgo lineages happened in 1.072 Mya (95% HPD:0.2740-2.312 Mya), and the divergence time of Eastern clade and H1 clade was 0.6920 Mya (95% HPD:0.1730-1.459).3. The main environmental factors and differentiated environmental factors of geographical distribution:ENM results indicated that the main environment factors are related to water and cold temperature. Similarly, results from T test analysis demonstrated that the differentiated environmental factors are relevant to maximum temperature, precipitation and sunshine time (P<0.05), implying that there may be a force of environmental differentiation selection for divergence of Ginkgo lineages.4. Candidate genes for environmental differentiation selection. Totally,59,585 unigenes were obtained, and 46,133 of them were annotated.9114 genes differently expressing between two transcriptomes were found, which are related to biological process is most, followed by membrane component gene, while the least one is molecular function genes. Considering the ortholog genes from Populus, Oryza sativa, Vitis vinifera, and Arabidopsis thaliana as reference, we selected 141 single copy genes with length longer than 600 bp.18 single copy genes were found with 2-fold different expression, with the higher expression amount showed in Tianmu individual.528 genes were found under positive selection by using MA method, with Ka/Ks ratio greater than 1.In conclusion, this thesis revealed there are three lineages in Ginkgo biloba. And the divergent time of them began in Pleistocene (1.072 Mya,95% HPD:0.274-2.312). Beside the driving forces of lineage divergence from genetic drift, they may also include environmental differentiation selection, selection pressure of which temperature and humidity.