Conservation Genetics Of Pteroceltis Tatarinowii Based On Transcriptome And Chloroplast Genome Sequences

Pteroceltis tatarinowii Maxim.is an endangered deciduous tree endemic to China,which is distributed in 19 provinces in China.Due to its high economic value,Pteroceltis tatarinowii has been cut down in large numbers,resulting in the continuous reduction of its germplasm resources and wild population distribution area.In this study,we used Illumina HiSeq^TM 2500 sequencing platform to conduct transcriptome sequencing and chloroplast genome sequencing of two geographically distant individuals of Pteroceltis tatarinowii,Twenty polymorphic EST-SSR loci of Pteroceltis tatarinowii were developed by analyzing the sequence information.Based on EST-SSR molecular marker technology,conservation genetics of five Pteroceltis tatarinowii natural populations（Langyashan,An Hui（LYS）;Jingxian,Anhui（JX）;Tianmushan,Zhejiang（TMS）;Yanziji,Jiangsu（YZJ）and Shidu,Beijing（SD））in China was studied.The main processes and conclusions are as follows:（1）Based on Illumina HiSeq^TM 2500 sequencing platform,two individuals of Pteroceltis tatarinowii from Langyashan（LYS）and Shidu（SD）populations were sequenced.The polymorphic EST-SSR loci were searched and primers were designed by Candi SSR software.369 theoretically polymorphic EST-SSR loci were obtained,with an average length of 18 bp.We randomly selected 30 candidate EST-SSR loci with 3-5 bp repeat units for subsequent primer effectiveness,polymorphism and transferability experiments.Finally,20 polymorphic EST-SSR loci（Gen Bank Accession Number JZ980980-JZ980999）were successfully developed.In addition,the 20 polymorphic EST-SSR loci of Pteroceltis tatarinowii were tested for the universality in Ulmus gaussenii Cheng and Ulmus pumila Cheng.It was found that 17of these polymorphic EST-SSR loci could be successfully amplified in both Ulmus gaussenii Cheng and Ulmus chenmoui Cheng,and showed high polymorphism,with the transferability of 85% and the polymorphism of 100%.Therefore,the selected EST-SSR loci can be used to study the population genetics of other Ulmaceae species.（2）The genetic diversity and genetic structure of five natural populations of Pteroceltis tatarinowii were analyzed by using the data of above 20 EST-SSR polymorphic loci,including LYS,JX,TMS,YZJ and SD.A total of 63 alleles were generated from 20 EST-SSR polymorphisms in Pteroceltis tatarinowii.The number of alleles per locus was 2-6,and the average number of alleles per locus was 3.02.At the same time,the average genetic diversity of Pteroceltis tatarinowii population was in the middle level:the average observed heterozygosity H_o was 0.402,the average expected heterozygosity H_e was 0.405,The overall population genetic differentiation coefficient F_st was 0.075,the inbreeding coefficient F_is was 0.031,and the total inbreeding coefficient F_it was 0.102.the genetic diversity level of the LYS population was the highest（the average number of alleles detected per locus was 3.15,the observed heterozygosity was 0.411;the expected heterozygosity was 0.422）;the genetic diversity level of YZJ population in Nanjing was the lowest（The average number of alleles detected per locus was 2.80;the observed heterozygosity was 0.384;the expected heterozygosity was 0.373）.Based on the results of population structure analysis by STRUCTURE software,five natural populations of Pteroceltis tatarinowii can be divided into three lineages:LYS population lineage;YZJ population lineage,JX population,TMS population and SD population lineage.It was found that there was no obvious relationship between the genetic structure of Pteroceltis tatarinowii’s populations and its geographical distance,and it was speculated that the unique gene pool occupied by the YZJ population is related to the Founder Effect.Combined with the habitat survey results of field investigation,it can be judged that the protection priority of LYS population and YZJ population is higher.（3）Based on HiSeq^TM 2500 sequencing platform,we sequenced the chloroplast genome of two individuals of Pteroceltis tatarinowii from LYS population and TMS population.The total gene length of the individuals was 158635bp and that of TMS was 158619bp.The chloroplast genome of the two individuals was similar to that of other Ulmaceae species in structure,consisting of two reverse repeat regions（Inverted repeats region,IRs）,one large single copy region（Long single copy seguence,LSC）and one small single copy region（Short single copy seguence,SSC）.At the level of chloroplast genome,there are 119 genes in chloroplast genome,including 80 protein coding genes,29 t RNA genes,8 r RNA genes and 2 pseudogenes.Through the sequencing and preliminary analysis of the chloroplast genome of Pteroceltis tatarinowii,it laid a foundation for the development of chloroplast molecular markers and the construction of phylogenetic relationship of Pteroceltis tatarinowii.