The Development Of SSR Markers And Genetic Structure Analysis Of An Endangered Plant Tetracentron Sinense Oliv.

Tetracentron sinense Oliv.is the only species in the family Tetracentraceae.The species has been over-harvested for use in medicine,timber,and ornaments,resulting in poor regeneration of its populations.Therefore,it has been listed as a national second-grade protected endangered plant species in China.In this paper,SSR markers of T.sinense were developed based on simplified genome sequencing(RAD-seq)techniques and applied to the analysis of genetic diversity and population genetic structure.The results are of great significance for the scientific research and conservation countermeasures of T.sienese.The main results are as follows:(1)Development of SSR markers for T.sienese.High-throughput sequencing was used for large amount of sequences.A total of121022 SSRs were identified in the 8322400 sequence,which 6672 were compound and63985 were dominant nucleotide repeats.The dominant nucleotide repeats were single nucleotide repeats,accounting for 59.14%of the total repeat type,followed by dinucleotide repeats accounting for 28.71%.The proportion of AG and AT base repeats were the highest in dinucleotide repeats,and that of AAG/CTT,AGC/GCT were the highest in trinucleotide repeats.Using the minimum repetition rate of 5 as the criterion,we designed 1212 pairs of primers,in which 67 primers were selected for primer synthesis and polymorphic detection.Among 67 pairs of primer,52 pairs of primers had amplification products,and 48 pairs of primers were polymorphic,accounting for 92.31%of the total primers.Based on loci polymorphic information content(PIC)values,14 pairs of primers were screened for subsequent population genetics analysis.(2)Genetic diversity and population structure of T.sienese.Based on the 14 pairs of SSR markers,the genetic diversity and population structure of T.sienese were analyzed.Total 102 alleles were detected from 22 natural populations(193 individuals).At the species level,T.sienese has moderate-level genetic diversity with an expected heterozygosity(0.559)and Shannon information index(1.631).At the population level,the expected heterozygosity was 0.566 and the Shannon information index was 0.937.The results of AMOVA analysis showed that the genetic variation among the population within the groups was 84%,and the genetic variation between the groups was16%,which indicated that the genetic variation of T.sienese mainly occurs between individuals within the population.The genetic differentiation coefficient of Fst was 0.31,indicating that 31%of the genetic variation was among the populations and genetic differentiation in T.sienese populations was high.Structure analysis demonstrated that the models with K=3 provided satisfactory explanations of the observed data.The bar plot for k=3,all populations were separated into three groups,which were correlated with Ice Age Shelter in southwest,Hengduan Mountains and Qinling Mountains.Bottleneck results suggest that some populations experienced recent bottleneck events.The DIYABC software was used to simulated the divergent event and estimate divergent time among three groups,which were radially differentiated.The time of differentiation was 1.115×10~4 years ago,the size of NA was2.34×10~4,and the total of N1+N2+N3 was 9.345×10~3,which indicating that the existing genetic structure of T.sienese was mainly affected by the deterioration of Quaternary climate,the arrival of glaciers and cold climate.(3)conservation strategy for T.sinenseAccording to the genetic structure analysis,the existing natural populations should be divided as 3 evolution management units,which should be managed separately.The BSJ and WFHH populations with relatively high levels of genetic variation should be considered as the priority protection populations.The HBY and GLGM populations with low genetic diversity can be enriched by communicating with individuals from other populations in the same management unit to promote gene exchange among populations.