| Siniperca chuatsi is an important freshwater economic fish in China,mainly distributed in the rivers of East Asian countries such as China,Vietnam and South Korea.It has been used as a valuable food fish by people in these areas for a long time,and has a history of large-scale breeding in China for more than 20 years.In recent years,with the continuous expansion of the scale of S.chuatsi in China,its germplasm degradation is serious,which is reflects in biological characteristics such as slow growth rate,low disease resistance,precocious puberty,especially the prevalence of ISKNV(infectious spleen and kidney necrosis virus),which has increased the breeding risk of S.chuatsi farming and brought huge economic losses to the farming industry every year.In this study,SLAF-seq(Specific-Locus Amplied Fragments Sequencing)technology was used to develop SLAFs in batches,and a high-density genetic linkage map of S.chuatsi was constructed;QTL(quantitative trait locus)positioning of economic traits was performed;Related traits screening and identification of SNP(Single nucleotide polymorphism)molecular markers and comparing the genetic map QTL interval with the full-length transcriptome DEG library.The results of this study will provide some theoretical guidance for the maker-assisted selection of S.chuatsi1.Construction and evaluation of genetic map of S.chuatsi based on slaf-seq technologyISKNV disease-resistant mature S.chuatsi selected from previous groups were used as male parents and wild mature S.chuatsi were used as female parents for artificial insemination,and 160 F1 offspring were randomly selected for breeding Based on SLAF-seq technology to construct the genetic map of S.chuatsi.A total of 744.24 M reads(182.92G)were obtained by SLAF sequencing.A total of 208,841 SLAFs were developed,including 18,418 polymorphic SLAFs,with a polymorphism ratio of 8.82%;12,365 markers were successfully genotyped,and their isolation types were abxcd,efxeg,abxcc,ccxab,hkxhk,lmxll and nnxnp Screen out markers of low quality,lack of parental information,severe partial separation,etc.that are not suitable for constructing the map,and obtain 6,150 SLAFs that can be used for mapping.Filter out the markers with other SLAFs whose LOD value is less than 4,and finally a total of 2344 SLAFs in the genetic linkage map,the average sequencing depth of the parent markers on the genetic linkage map is 189.38X,the average sequencing depth of the offspring is 34.94X,and the completeness of the markers on the genetic linkage map is 99.88%.The genetic map of S.chuatsi consists of 24 LGs,with a total map distance of 3,268.00 cM and an average map distance of 2.34 cM.The largest linkage group on the linkage map is LG22,with a total of 184 markers and a length of 226.23 cM.The smallest linkage group is LG14,with a total of 40 markers and a length of 39.12 cM.The female map contains 1269 SLAFs with a total map span of 2905.55cM;The male map contains 1500 SLAFs with a total map span of 3,196.49 cM.The genetic map constructed this time will provide a certain theoretical basis for the future molecular marker-assisted breeding of S.chuatsi.2.Phenotypic data statistics and economic QTL mapping of S.chuatsiBased on the genetic map constructed by the SLAF technology,the immune,growth and sex traits of the mapped population were analyzed.A total of 11 QTLs related to growth traits were detected.Among them,one QTL regulating body weight,total length,withers height and slaughter weigh traits was all located in LG14;three QTL regulating body thickness traits were located in LG11 and LG18;4 QTLs regulating body length traits were located at LG2,LG 14 and LG24;2 QTLs related to anti-ISKNV traits were detected at LG 18 and LG21;2 QTLs involved in sex determination were detected at LG23.Among all 15 QTLs,the one with the most markers is the QTL interval that controls the body thickness.and there are 7 SLAFs in total;while the QTL interval related to body length has the fewest markers,with only 1 SLAF;The QTLs related to the withers height,body weight,body length,total length and slaughter weight are mainly concentrated on LG 14,and these 5 QTLs have a overlap area(5.255-5.255 cM);Among all the 15 QTLs,the higher contribution rates are related to body weight and body weight traits,which are 31.6 and 32.2 respectively.3.QTL interval immunization and growth-related molecular markers in the genetic map of S.chuatsiPrimers design was performed on 34 SNP in the QTL interval of S.chuatsi and PCR amplification was performed on the parental genome of the mapping population.Finally,16 SNP successfully amplified the target band,and showed polymorphism in the parent.KASP(Kompetitive Allele Specific PCR)genotyping technology was used to test,and 13 SNP were successfully typed,11 of which were in 300 of S.chuatsi population(150 resistant,150 Susceptibility)showed polymorphism;9 SNP showed polymorphism in the 96 S.chuatsi population.After statistical analysis,it was found that Marker23012-2,Marker23657-1 and Marker72680 were extreme significantly correlated with anti-ISKNV traits(p<0.01),allele frequencies of Marker28932 were significantly correlated with anti-ISKNV traits(p<0.05);Marker23657-1 were with body weight extreme significant correlation(p<0.01),Marker23012-2 locus was extreme significantly correlated with body weight(p<0.01),and was significantly correlated with body length(p<0.05).These SNP markers can provide a certain basis for the molecular marker-assisted breeding of S.chuatsi.In addition,in order to further mine candidate genes related to economic traits on the linkage map of S.chuatsi under the condition of no reference genome,The sequence information of the differentially expressed genes in a large number of full-length transcriptomes screened under the stimulation of ISKNV and the markers sequence in the QTL interval of the genetic linkage map were analyzed by blast.The results showed that 273 differentially expressed gene transcripts were located on 10 Markers in the QTL interval,of which 13 differentially expressed genes were in Marker65999 and Marker52563 in the QTL interval related to anti-ISKNV.In the above,it provides valuable information for the development of anti-ISKNV molecular markers and the selection of anti-ISKNV breeding. |
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