Genetic Diversity Of Lepturichthys Fimbriata In The Upper Reaches Of The Yangtze River

Lepturichthys fimbriata is a benthic small fish that feeds on invertebrates and fixed algae.It absorbs on the surface of the bottom rock and adapts to Torrent.It produces drifting eggs,mainly distributed in the middle and upper reaches of the Yangtze River and Its tributary.In recent years,with the construction and operation of cascade hydropower stations in the upper reaches of the Yangtze River,the suitable habitat of L.fimbriata is gradually being compressed.With the pollution of waters and overfishing,its resources are facing a downward trend.Therefore,we uses microsatellite and mitochondrial Cyt b and D-loop sequences as molecular markers to explore the genetic diversity,genetic structure and population historical dynamics of L.fimbriata in the upper reaches of the Yangtze River.1.Microsatellite primer developmentBased on Illumina Novaseq 6000 sequencing method,the microsatellite loci of the polymorphic loach were developed.25180 microsatellite loci which can be used for primer design of L.fimbriata,180 pairs of microsatellite loci were selected for poly-morphism screening,and 23 pairs of polymorphic loci with good amplification results were finally obtained.The number of alleles(NA)of 22 pairs of microsatellite loci ranged from 9 to 30,the observed heterozygosity(Ho)ranged from 0.48 to 1.00,and the expected heterozygosity(HE)ranged from 0.70 to 0.98.The polymorphism information content(PIC)is 0.75 to 0.96,which indicated their applicability for genetic analysis.2.Research on Genetic Diversity Based on Microsatellite Molecular MarkersWe selected 8 pairs of microsatellite loci with stable polymorphism and good polymorphism from 22 pairs of polymorphic microsatellite loci.The genetic diversity and genetic structure of 189 individuals in 7 population including Panzhihua,Qiaojia,Nanxi,Hejiang,Zhuyangxi,Banan and Minjiang in the upper reaches of the Yangtze River.The results showed that the average number of alleles(NA)ranged from 6.88 to20.75,and the average observed heterozygosity(Ho)range is 0.78~0.89,the averageexpected heterozygosity(HE)range is 0.89~0.92,and the average polymorphic information content(PIC)range is 0.77~0.90,indicating that L.fimbriata has a high level of genetic diversity.The analysis of molecular variance(AMOVA)showed that the genetic variation within the population(99.47%)was significantly greater than the genetic variation between the populations(0.53%).The genetic variation mainly originated within the population,and the absolute value of the gene flow among the 7populations was all greater than 4,that means gene communication is frequent among populations,and there is no genetic differentiation among the populations(Fst=0.0053).Structure cluster analysis results further showed that the 7 populations of the upper Yangtze River clustered into one group,indicating that there was no genetic differentiation among the 7 populations of L.fimbriata.3.Analysis of genetic diversity based on mitochondrial DNACyt b gene and D-loop sequence were used to analyze the genetic diversity of 7populations of L.fimbriatais.The results showed that: 84 mutation sites were detected in Cyt b gene,and 90 haplotypes were defined.Haplotype diversity(Hd)and nucleotide diversity(Pi)were 0.95 and 0.0039 respectively;90 mutation sites were detected in the D-loop sequence,defining 124 haplotypes,haplotype diversity(Hd)and nucleotide diversity(Pi)were 0.982 and 0.0076,respectively,indicating that L.fimbriatais has high genetic diversity.Molecular variance analysis(AMOVA)based on Cyt b gene and D-loop sequence shows that most of the variation comes from within the population,and the absolute value of the gene flow among the 7 populations is greater than 4,indicating a high level of gene exchange and a genetic differentiation index(Fst)Both are less than 0.05,indicating that there is no genetic differentiation among the populations of L.fimbriatais;the haplotype network structure diagram and the NJ phylogenetic tree show that the L.fimbriatais haplotypes are not clustered by geographical distribution,there is no lineage differentiation.The neutrality test results showed that the 7 populations of Tajima’s D and Fu’s Fs based on the Cyt b gene and D-loop sequence were negative,the Tajima’s D of Cyt b did not reach a significant level;the single based on the Cyt b gene The haplotype mismatch distribution map is in single-peak curve mode,and the haplotype mismatch distribution map based on D-loop sequence is in double-peak curve mode.The population expansion signals are not uniform,combined with BSP for further analysis,based on Cyt b gene and D-loop the sequences all show that there was a population expansion event about 50,000 years ago,that is,the fourth century ice age.