The Development And Application Of Microsatellite DNA Markers Of Paramisgurnus Dabryanus Based On RNA-seq

Paramisgurnus dabryanus is an economic fish species belonging to Cobitidae andalso one of the important freshwater fish species in China, which have being exported toKorea largely. In this study, Paramisgurnus dabryanus transcriptome analysis wasperformed and large amounts of Paramisgurnus dabryanus microsatellite markers weredeveloped by the second-generation high-throughput sequencing platform and thenvalidated. With the markers developed, we analyzed the genetic structures of wildParamisgurnus dabryanus populations from four big lakes in China; then analyzed thegenetic structure of a Paramisgurnus dabryanus full-sib family and made a correlationanalysis between the microsatellite markers and its growth traits. Our research will bebenefitial for Paramisgurnus dabryanus germplasm conservation and provide somereference and theoretical basis for Paramisgurnus dabryanus breeding programs.1Transcriptome analysis and development of microsatellite markers ofParamisgurnus dabryanus by RNA-seqIn this study, Transcriptome sequencing were performed using Illuminahigh-throughput plateform and de novo assembly yielded71,887isogenes with anaverage length of1464.58bp. Out of these sequences,38,733and17,434isogenes hadsimilarity to proteins in the Nr database and string database. Then, gene ontologyfunction classification and KEGG function analysis were performed, and the resultindicated that22,102(31.14%) isogenes were mapped to302KEGG pathways. Tofurther evaluate the transcriptome sequencing depth, we compared our database withzebrafish cDNAdatabase and found43,807Paramisgurnus dabryanus cDNAsequenceshad BLASTx homologs with19,064different zebrafish cDNA sequences. In addition,15106(SSRs≥12bp) cDNA SSRs (cSSRs) were identified as potential molecularmarkers in our database. Among those loci, four hundred pairs of SSR primers wererandomly selected for validation with364(91%) pairs of primers successfully amplified in the initial screening test.43of those perfectly-amplified primer pairs were used forfurther assessment of polymorphism in20Paramisgurnus dabryanus individuals fromfour large lakes in China. Our result revealed that38(88.4%) loci were polymorphic.2Genetic diversity of Paramisgurnus dabryanus from four big lakes in ChinaSeventeen polymorphic EST-SSR markers developed by our lab were selected forgenetic diversity analysis in four Paramisgurnus dabryanus populations from Taihulake, Hongze lake, Dongting lake and Chaohu lake in China. Totally,161alleles werefound in all EST-SSR markers and the average number per locus was9.47. The meanFst value was0.2194（>0.15）, mean Nm0.8894, which indicated that highdifferentiation and low genetic exchange existed in these four populations．The allelenumber of all the four populations ranged from5.4to6.2(mean5.8), the observed andexpected heterozygosities ranged from0.2732to0.3864and0.5629to0.5893,respectively. The polymorphism information content were0.5503-0.5754, indicating ahigh genetic diversity and every pupulation should be protected as independent geneticunit. Among them, the Hongze and Taihu populantions had higher genetic diversity andcan be used as the basic populations for further genetic breeding. In addition, the geneticdistance and identity were calculated in four populations, ranging from0.5283to0.9677.The genetic identity of Taihu and Hongze was the highest and the genetic distance wasthe lowest (0.5283), while those of Taihu and Chaohu were reversed. The phylogenetictree based on genetic distance demonstrated that there were two different groups: onebeing composed of Taihu and Hongze population, and the other of Dongting andChaohu.3Correlation anaIysis of microsatellite DNA markers with growth trait ofParamisgurnus dabryanusForty-seven polymorphic EST-SSR markers were randomly selected for analysis ofgenetic diversity of a full-sib Paramisgurnus dabryanus family. Totally97alleles weredetected and the number of observed alleles and effective number per locus were2-3and1.5945-2.8644respectively. The observed and expected heterozygosity were0.1111-1.0000and0.1054-0.6537. The mean polymorphism information content in allloci was0.3207, and the average value for allele number, observed alleles and effectivenumber were2.06,0.4728and0.3968, respectively. Correlation anaIysis of58polymorphic loci with the growth traits of3Paramisgurnus dabryanus samples（3 samples: AB2011-F6a、AB2011-F6b、AB2011-F6c）. The numbers of loci associatedwith full-length, body length, body height, body weight and gut weight significantlywere17,16,16,15and17, respentively. Among them,22loci were correlated withmore than two growth traits and9makers associated with specific growth trait appearedmore than one sample. Our results indicated that, the AB genotype in locus Pda174andthe NN genotype in Pda247C can be used as full-length dominant genotype in markerassistant selection of Paramisgurnus dabryanus marker assistant selection, the ABgenotype in locus Pda310as dominant genotype of body height and the AB genotype inPda301as weight dominant genotype. All the trait-related markers found in this studycan be used in further validation of marker-trait association analysis and Paramisgurnusdabryanus breeding work.