Screening And Identification Of Lymphocystis Disease-resistant Related Microsatellite Markers, And Cloning And Preliminary Analysis Of Two Immune-related Genes In Japanese Flounder (Paralichthys Olivaceus)

Japanese flounder （Paralichthys olivaceus） has been widely cultured, with higheconomic value, which is one of the most important mariculture fishes. In recent years,a variety of diseases have appeared with the expansion of farming scale. In order toachieve the healthy and sustainable development, we should combine conventionalbreeding method with molecular marker technology to breed new varieties. In presentstudy,we screened out lymphocystic disease-resistant related microsatellite markers inJapanese flounder. In addition, two immune related genes were cloned, analysed andstudied. The following results were obtained.Ⅰ. Screening and identification of lymphocystic disease-resistant relatedMicrosatellite markers in Paralichthys olivaceusIn order to screen out lymphocystis disease resistance-related SSR markers inChinese flounder population,102individuals were used（56disease susceptible and46disease resistant individuals） in the present study. Firstly, two gene pools wereconstructed using15susceptible individuals and15resistant individuals, respectively.The two gene pools were scanned using178pairs of microsatellite primers. Secondly,the differential bands amplified in gene pools were verified using30individualswhich were used to construct the gene pools for the first time. Lastly, a secondverification for102individuals was performed to verify the difference significance ofthe differential bands identified in the first verification. Results of BSA analysisdemonstrated that some differential bands were amplified using four pairs ofprimers（scaffold440₂2585, scaffold826₅003, scaffold703₄284andscaffold185₅97）. The first verification indicated that the differential bands are significantly different in the SSR markers scaffold826₅003（P=0.023） andscaffold185₅97（P178bp=0.028，P173bp=0.009）. And in the second verification, thedifference was extremely significant in the totally102individuals with the SSRmarker scaffold185₅97, the frequency of differential bands in disease resistantindividuals and disease susceptible individuals was60.9%and14.3%,respectively（P=0.001<0.01）. The differential bands amplified from ten diseaseresistant individuals using primer scaffold185₅97were cloned and sequenced.Sequence alignment by blast confirmed that the bands were fragments ofmicrosatellite marker scaffold185₅97published by lab for aquatic genomics and cellengineering in Yellow Sea Fisheries Research Institute, homology of which was up to96%. The present study has shown that the microsatellite marker scaffold185₅97may be associated with lymphocystis disease resistance in Japanese flounder（Paralichthys olivaceus）, providing some basis for the molecular marker-assistedselection in P. olivaceus.Ⅱ.Cloning and preliminary analysis of immune-related genes of ParalichthysolivaceusIn the study, the complement gene C1q-like3and Cytohesin-1gene were choseas disease-resistant genes for exploration their associations with disease caused byvibrio anguillarum. Firstly, cloning and structural analysis of the two immune geneswere done. Afterwards, the expression in transcriptional level of C1q-like3and SNPscreening examination of Cytohesin-1were carried out, respectively. So therelationship between gene function and disease was studied for the two immunegenes.1. Cloning and expression analysis of C1ql3Complement system is one of the important immune effector systems, and playssignificant roles in innate immune defense. Complement-related molecules existextensively, and participate in the host’s immune defense. In this study, we cloned thecomplement C1q-like protein3gene （designated PoC1ql3） from Japanese flounder（Paralichthys olivaceus）. The full-length PoC1ql3cDNA is1482bp long, including a4-bp5′-UTR, an801-bp coding region, and a677-bp3′-UTR. The801-bp openreading frame encodes266amino acids, with two exons and one intron identified.Homology and phylogenetic analyses revealed that PoC1ql3is most closely related to the C1q-like protein3genes of Maylandia zebra and Oreochromis niloticus.Real-time quantitative PCR analysis of normal tissues demonstrated that PoC1ql3wasabundantly expressed in the spleen and liver, sometimes expressed in blood andmuscle, and minimally expressed in other tested tissues. Challenge with Vibrioanguillarum increased the expression of PoC1ql3in the liver, reaching the highestlevel of expression at6h post-injection, with the expression of PoC1ql3in spleentissue reaching its highest level at48h, levels that were2.89-times and2.64-timeshigher than the control values, respectively （P<0.05）. The fact that ingestion ofbacteria can distinctly increase PoC1ql3expression in immune-related organs maysuggest that C1ql3is associated with the inflammatory response as well as withhomeostatic functions in Japanese flounder.2. Cloning and SNP analysis of Cytohesin-1The study of cytohesins in mouse and human has shown that cytohesins hadsome correlation to immunoregulation, acting as immune factor in organism. The fulllength of Cytohesin-1cDNA was1631bp, including175bp5’UTR,1203bp ORF and253bp3’UTR. The1203bp ORF encoded400amino acids. Japanses flounderCytohesin-1gene had11exons and10introns by spidey software. Alignment analysisdemonstrated Cytohesin-1gene had Sec7domain, and was conservative in all species.Phylogenetic analysis indicated that Japanese flounder Cytohesin-1had closerelationship with weever, clustered into one group with other fishes, and had farrelationship with birds and humans. Basing on30vibrio anguillarum disease-resistantindividuals and30disease-susceptible individuals, we detected SNPs by directsequencing of exons. The findings have shown that there were plenty of overlappingpeaks, presumably insertions or deletions of bases might exist. Also, we foundsuspected SNPs in exon9, exon10and exon11. The suspected SNPs require furthertesting to confirm the correlation between SNP and disease, expecting its guidance inbreeding practice.