| Placocheilus cryptonemus belongs to Placocheilus of Labeoninae,Cyprinidae.The individual is small,generally 60-100 mm in length.It is nearly cylindrical in shape and has a blunt snout.The snout is round and blunt.P.cryptonemus preferring to inhabit the lower reches layers of t he Nujiang River and a few tributaries.It prefers to live in t he lower layer of t he river wit h rocky bottoms,or dwell in t he int erst ices of rocks.They feed on t he periphyton,algae and organic debris by scraping.The Nujiang River basin is one of t he hot spots of biodiversit y in t he world because of it s rich species di versit y,changeable climat e and unique geographical characterist ics.Due to the variable climate and unique geographical features,Nujiang River basin is one of the biodiversity hotspots in the word.With a rich diversity of species P.cryptonemus is one of the many endemic species in the Nujiang River and has been listed as vulnerable in the Red List of Chinese Species in 2004.The research study of the population genetic structure and genetic diversity of P.cryptonemus provides some basis for its phylogeny and adaptive evolution,as well as theoretical guidance for the formulation of its effective conservation and management measures,and also serves as an inspiration for the study of other endemic species in the Nujiang River basin.In this study,two mitochondrial gene markers Cytb and COI,combined with microsatellite molecular markers developed based on transcriptome data were used to study the genetic structure and genetic diversity of nine populations of P.cryptonemus in Nujiang River.The main results are as follows:1.Genetic structure and genetic diversity analysis of P.cryptonemus based on Cytb gene.The spliced sequences were cut by BioEdit 7.0 to obtain Cytb sequences of 889 bp in length.A total of 14 base variation sites were detected,accounting for 1.57 % of the total.A total of 13 haplotypes were distinguished among the nine sampled populations.From the distribution of haplotypes,eight haplotypes belonged to were unique haplotypes.The highest haplotype diversity of the nine populations was 0.76190(Hd>0.5),while the nucleotide diversity ranged from 0.00090 to 0.00271,none of which exceeded 0.005.In the analysis of molecular variance ANOVA 90.48 %of the genetic variation occurred within populations,and only 9.52 % of the genetic variation occurred between the populations.The total overall genetic differentiation index was 0.09515(P<0.001),indicating that there was significant genetic variation among the nine populations.The results of nucleotide mismatch distribution showed that the haplotype mismatch distribution map of P.cryptonemus population showed a unimodal distribution,indicating that the population expansion may occur during the evolutionary process.2.Genetic structure and genetic diversity analysis of P.cryptonemus based on COI gene.The spliced sequences were compared and cut with the software BioEdit 7.0,by comparison with the software BioEdit 7.0,and the sequences were retained to for 1104 bp,and the base composition was counted,and the average contents of A,T,C and G were 27.2 %,29.3 %,26.3 %and 17.2 %,respectively.A total of 9 haplotypes were defined at the nine sampling sites.The distribution of haplotypes showed that 6 haplotypes were unique haplotypes.The Denggeng Estuary populations had higher haplotype diversity and nucleotide diversity,indicating that had higher genetic diversity.The Cytb and COI sequences,which had been matched one by one,were spliced together with a total sequence length of 1993 bp.The results showed that the haplotype diversity of the nine populations ranged from 0.50000 to 1.00000.The haplotype diversity of most populations was at a high level,and the nucleotide diversity of 9 populations ranged from 0.00088 to 0.00671.The genetic differentiation coefficients between populations ranged from-0.5385 to 1.0000,with most populations showing a moderately high degree of genetic differentiation,and reaching significance levels.From the structure of the phylogenetic tree,it can be see that Hap＿4 and Hap＿19 are far from other haplotypes in the branches of the tree,representing populations Denggeng Estuary and Manbang River,respectively,shows that Hap＿4 and Hap＿19 are distant from other haplotypes inside the branches of the tree,and the populations they represent are Dengdeng Estuary and Manbang River,respectively.The genetic structure here is similar to the results of the previous COI and Cytb gene analysis,with the two populations Dengdeng Estuary and Manbang River are genetically distant and genetically differentiated from other populations.The values of Tajima’s test were mostly negative,the mean value of Fu’s Fs test was positive.Nucleotide unpaired analysis showed multiple peaks,indicating that the population has not expanded recently.3.Analysis of the genetic structure and genetic diversity of P.cryptonemus based on transcriptomic data microsatellite markers.Using the MISA microsatellite recogniton tool to perform SSR analysis on unigenes over 1kb,SSR analysis of unigenes above 1 kb was performed using the MISA microsatellite identification tool,and 30,500 SSR markers were identified among 36,764 unigene sequences,and the number of sequences containing SSR was 18,078.For different nucleotide types of repeats,the total number of SSR was significantly different,the repeats showed a decreasing pattern.Among the different types of base repeats(considering sequence complementarity),the most number of A/T repeats were found in the single base repeat sequences,which was 19,266 times.SSR length varied widely among different types of SSR sequences,and the length of 10 bp SSR accounted for the largest proportion,up to 17.8 %.The different types of SSR sequences of P.cryptonemus showed a wide range of variation in SSR length,with the largest proportion of SSRs of 10 bp in length,reaching17.8 %.As the number of repetitions increases,the abundance of this type of SSR shows a gradually decreasing trend,that is,the longer the microsatellite length,the lower the frequency of microsatellite occurrence.Microsatellite sequences have been obtained from transcriptome data,which can be directly screened for microsatellite marker primers.120 pairs of microsatellite primers were designed in this study,and 58 pairs of microsatellite primers with clear bands were obtained,among which 16 pairs of microsatellite primers were highly polymorphic.A total of 61 alleles were obtained in this study,with an average of 3.8125 alleles.The average observed heterozygosity of nine populations was0.9382 and the average expected heterozygosity was 0.6321 for the nine populations of P.cryptonemus.Among the 16 loci of the nine populations,only three loci of low information content appeared,and the rest were all of medium and high information content with high genetic diversity.The whole population was moderately genetically differentiated,and the average value of genetic differentiation index was 0.1371.The highest genetic distance was 0.3943,while there were six populations with genetic distances greater than 0.3.The phylogenetic tree constructed based on Nei’s genetic distance showed that Manbang River clustered with Xiwaluo River Estuary,Liuku damsite and Denggeng Estuary,indicating that these two populations were closely related,while Lichai Dam and Daxingdi were more distantly related to the other seven sampled populations.Two mitochondrial genes and transcriptome-based microsatellite molecular markers were combined to study the genetic structure and genetic diversity of P.cryptonemus.Although there were some differences in the results of various molecular markers,and the results obtained from the nine populations were not completely consistent.However,the overall results indicated that the level of genetic diversity was relatively rich and genetic differentiation was not obvious among the populations of P.cryptonemus. |
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