| Artemia is not only one of the most important live feed used in larviculture,but also an ideal experimental organism for scientific research.In the study,SLAF-seq method was used to develop SNP markers and construct the first high-density genetic linkage map for Artemia franciscana,and mapping QTL for sex and growth-related trait,which provide a valuable resource for genetic improvement and genome study in Artemia.In order to study the mechanism in the reproductive development of bisexual Artemia,four reproduction related candidate genes,namely Piwi 、 Sox2 、 Fru and Sxl,were obtained from A.franciscana transcriptome,and bioinformatics analysis including sequence analysis and domain prediction was performed.The expression characteristics of these genes in different stages of gonad development of A.franciscana were studied by q PCR,and its function was verified by microinjection RNAi technology.In this study,8709 effective single nucleotide polymorphism markers detected using specific-locus amplified fragment sequencing and were mapped into 21 linkage groups(LGs)by using a full-sib family with 106 progenies.The map spanned 2636.43 c M and 105 loci per LG,with an average inter-marker distance of 1.19 c M,of which the largest linkage group contains 596 markers with 194.17 c M,and the smallest linkage group contains 137 markers with 87.96 c M.In addition,male and female genetic maps were constructed respectively.The female map spanned 2203.04 c M and 4288 makers,with an average inter-marker distance of 1.93 c M.The male map spanned 2885.65 c M and 5619 makers,with an average inter-marker distance of 1.92 c M.Given of the lower female/male recombination rate of 0.77,the WZ-ZZ sex determining system was further confirmed in A.franciscana.Based on the high-density genetic map,10 QTLs were mapped for sex and four growth-related traits.The phenotypic variability explained(PVE)by each QTL ranged from12.5% to 100%.Two QTLs of body width were located on LG3 and contained 131 SNP markers,with a maximum logarithm of odds(LOD)threshold of 3.66 and a maximum PVE of 17.1.The other 7 growth-related QTLs were located on LG6,of which 3 QTLs for body length and 3 QTLs for full length were located on the same region,each containing 118 SNP markers,with the maximum LOD of 8.83 and the maximum PVE of 30.7.One QTL for body weight was distributed on LG6 and contained 17 SNP markers,with a maximum LOD of 3.41 and a PVE of 14.0%.The sex-associated QTL was distributed on LG6 and contained 466 sex-associated SNP markers,with the maximum LOD of 1629 and the highest PVE of 100%,including 15 female-specific SNPs.QTL-association analysis further identified 22 candidate genes that probably related to the biological process of reproduction,growth and immunity.Four reproduction related cand idate genes contain complete open reading frame(ORF),which were widely expressed in gonads and had significant differences in expression levels at different developmental stages.The expression of these genes showed an upward trend at different stages of ovary development and a downward trend of testis development,suggesting that these genes may play a role in female Artemia entering different reproductive pathways and early testis development.RNAi microinjection further confirmed that the knockdown of Piwi and Fru genes resulted in 100% cysts production,suggesting that Piwi and Fru genes not only play an important role in the regulation of A.franciscana reproductive development,but also may play a key role in determining the reproductive modes of A.franciscana. |
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