The Conservation Genetics Research Of Parrotia Subaequalis(Hamamelidaceae),An Endangered Palaeoendemic Plant In China

Parrotia subaequalis is an endangered palaeoendemic tree from disjunct montane sites in eastern China and is classified as 'Grade I Key Protected Wild Plant' in China.Due to the lack of effective genomic resources,the genetic diversity and population structure of this endangered species are not clearly understood.In this study,we conducted the low-coverage whole genome paired-end sequencing(2 x 125 bp)for two geographically distant individuals of Parrotia subaequalis on the Illumina HiSeqTM 2500 platform.Based on the resulting sequences,we have successfully assembled the complete chloroplast genome of the two Parrotia subaequalis,as well as identified the mutational hotspots of chloroplast and the polymorphic chloroplast microsatellites(cpSSRs)in a comparative genomic way.Besides,from the two databases excluding the chloroplast and mitochondria genes,we further initiatively developed the polymorphic nuclear microsatellites(nSSRs)by means of the CandiSSR software.Finally,we used the the polymorphic cpSSR loci and the polymorphic nSSR loci to investigate the population genetics of Parrotia subaequalis from six natural populations.The main results were as follows:(1)Based on the two next-generation high-throughput sequencing databases,we successfully assembled the P.subaequalis(TX)and P.subaequalis(SJD)whole chloroplast genomes,respectively.The whole cp genome sequences of two P.subaequalis were 159,280 bp(TX;GenBank:MG252374)and 159,324 bp in length(SJD;GenBank:MG334121).The cp genomes of P.subaequalis exhibited a typical quadripartite structure,consisting of a pair of inverted repeats(IRs)separated by one large single-copy(LSC)region and a small single-copy(SSC)region,which was consistent with previous published plastid genomes of other Saxifragales species.Besides,there were 133 predicted functional genes in both P.subaequalis cp genomes,of which 115 were unique and 18 were duplicated in the IR regions.(2)Using a comparative genomic method,we found 16 chloroplast divergence hotspot regions of the two cp genomes including the seven intergenic spacers(matK-trnK,trnT-psbD,trnS-psbZ,psbZ-trnG,psaJ-rpl33,rps18-rpl20,and ccsA-ndhD)and the nine genes(psaC,rps4,cemA,petD,ycfl,ndhD,ndhG,ndhI,and ndhA).These regions of the cp genome can be further used to evaluate maternally inherited genomic diversity for phylogeographic and population genetic studies of P.subaequalis.Additionally,we identified 10 polymorphic cpSSR loci to conduct the study of population genetics of P.subaequalis.Moreover,all cpSSR loci were successfully amplified(the universal rate was 100%)in all the other five species of Hamamelidaceae(Parrotia persica,Parrotiopsis jacquemontana,Sycopsis sinensis,Distylium racemosum,Hamamelis virginiana).Therefore,we can apply these loci to promote the population genetics of other species of Hamamelidaceae.(3)From the two databases excluding the chloroplast and mitochondria genes,we further initiatively developed the 15 polymorphic nuclear microsatellites(nSSRs)by means of the CandiSSR software to investigate the population genetics of P.subaequalis.Besides,the above loci showed good transferability(the universal rate was 47%)in all the other five species of Hamamelidaceae(Parrotia persica,Parrotiopsis jacquemontana,Sycopsis sinensis,Distylium racemosum,Hamamelis virginiana).Hence,they can also be used for the population genetics of other species of Hamamelidaceae.(4)We selected ten polymorphic cpSSR loci and 15 polymorphic nSSR loci to genotype 96 individuals of P.subaequalis from six populations to estimate genetic diversity.From the results of cpSSR' loci data,a total of 43 alleles were identified,the mean value of haplotype diversity for each loci was 0.548.Based on the results of P.subaequalis' populations data,we calculated the mean value of the effective alleles for each population was 2.207 and the mean value of haplotype diversity was 0.713.Besides,at the species level,P.subaequalis exhibited abundant genetic diversity(e.g.,cpSSRs:Hcp = 0.862).Above all,we can conclude that since the Tertiary,P.subaequalis had maintained high level of chloroplast diversity during its long evolutionary histories.For the nSSR dataset,a total of 64 alleles with the mean value of the polymorphic information content was 0.515,indicating the high polymorphism of these nSSR loci.At the population level,average estimates of genetic diversity were generally low(AR = 2.274,HE = 0.464,HO = 0.520),being lowest in population HBS(1.814,0.395,and 0.495)and highest in population TX(2.558,0.540,and 0.620).Combining the results of field investigation,P.subaequalis maintained more individuals in population TX than HBS.More importantly,TX's habitat was better than HBS's.P.subaequalis in HBS grew in a road which is influenced by the people,thus leading the low level of genetic diversity.(5)We further selected ten polymorphic cpSSR loci and 15 polymorphic nSSR loci to figure out the genetic structure of P.subaequalis from six populations.From the results of cpSSR data,15 haplotypes were identified across the 96 individuals from the six populations based on the 10 polymorphic loci(H1-H15),H1,H2,H3,and H4 were the most frequent haplotypes,and shared by all the populations.While there were more particular cpSSR haplotypes in population ZXC and HBS.The AMOVA partitioned genetic variation into 65.45%among six populations and 34.55%was contributed by differences within populations(P<0.001),indicating the chloroplast genome' differentiation among the populations of P.subaequalis.For the nSSR dataset,STRUCTURE yielded the results that we can separate the above six natural populations as three distinct genetic clusters or lineages.The population ZXC was the southern lineage,the population HBS was the northern lineage and the other four populations(SJD,TX,LWS and WFS)were central lineage.Mantel tests of IBD revealed no significant correlation between geographical and genetic distances(r =0.228,P = 0.564).Additionally,the pollen/seed migration ratio(r)was calculated as 1.78,indicating a slightly higher level of pollen flow as compared to seed flow and the block effect of gene flow resulted of the genetic drift.The hierarchical AMOVA revealed that 16.39%of the total variation was attributed to differences among six populations and that 83.61%was contributed by differences within populations(P<0.001),indicating the nuclear genome' differentiation within the populations of P.subaequalis.(6)Therefore,follow both cpSSRs and nSSRs data analysis results,we should consider the above lineages as three separate evolutionary significant units or conservation units in any program.In addition,population ZXC and HBS should have the highest conservation priority in conservation programs because of the rare alleles and particular cpSSR haplotypes.