SNPs Identification Of Immune Related Genes From Litopenaeus Vannamei And Their Association Analyses To WSSV Resistance

The Pacific white shrimp(Litopenaeus vannamei) occupies the biggest part ofshrimp farming in China. Maintaining a sustainable farming of shrimp is veryimportant. However, frequent outbreaks of shrimp disease and lack of excellent breedseriously hinder the healthy development of shrimp farming. With characteristics ofhigh lethality, fast infection rate, wide range of hosts and difficult to cure, the whitespot syndrome becomes one of the most severe disease for shrimp farming.Fundamental solution to these problems is breeding WSSV-resistant varieties. Due tothe complexity of selection for resistance traits, molecular markers are very helpfulfor selective breeding. Single nucleotide polymorphisms (SNPs) are regarded as themost promising one. In the present study, SNPsof the immune related genes wereobtained from the transcriptomesof L. vannamei and further confirmed in the newbred variety Kehai No.1. On one hand, the expressions of differentanti-lipopolysaccharid factor (ALF) genes were analyzed and the association analysesof their SNPs to the WSSV resistance of shrimp were performed; on the other hand,the associations of SNPs from genes,which involved in immune signaling pathwaysand immune reactions,with WSSV resistance of shrimp were analyzed. The SNPsassociated with WSSV resistance will be used as markers for selective breeding ofshrimp. The research progress are as follows:1．According to gene annotations,156immune unigenes were selected fromsolexa sequencing database and86were selected from454sequencingdatabasesAmong them,56were immune signal pathways related genes and the others are genesof immune effectors.1644SNPs were predicted based on transcriptome raw data.Seventy two SNPs were randomly chosen for further verification, and the resultshowed that high quality SNPs (predicted accuracy>85%) could be predicted if thefiltering parameterswere set with Q>20(solexa) and duel min≥9(454). Throughalignments of SNPs from the transcriptome data with genomic sequences data in ourlab, a database containing681SNPs (593from solexa,88from454) was constructed. 2．Six ALFs (nLvALF1、2、4、5、6、7) genes acquired from transcriptomes wereverified by sequencings. The ORFs of these ALF genes encoded123,94,120,124,123,122amino acids separately. All these ORFs contained ALF conserved functionaldomain-LPS binding domain (LBD), and they were electropositive except nLvALF6.Amino acid sequences alignment and phylogenetic analysis showed that nLvALF1、2、5、6had closest relationships with FcALF1、2、5、6from Fenneropenaeus chinensis，while nLvALF4、7were the same with the reported LvALF. Tissue distributionprofiles of nLvALFs gene were differentsignificantly. The transcription levels ofnLvALF1-6were up-regulated significantly at36,72h after WSSV infection (P<0.05),while nLvALF7was down-regulated. We inferred that different isoform of ALFsmight perform different functions during the immune response of shrimp.3．The WSSV oral infection experiment was performed in shrimp from20familiesfrom “Kehai No.1” new variety. This work lasted19days. Forty eight shrimp whichdied at the earliest time of WSSV infection were regarded as susceptive group and48survivors at the19thday after WSSV infection were regarded as resistant group. Asignificant difference of average virus copy numbers existed between resistant andsusceptible group (P<0.01)which indicated an efficient process of virus removing inresistant group.After aligning cDNA sequences with ourgenomic database, roughgenomic sequences were assembled and further confirmed after sequencing. AllnLvALFs showed high polymorphisms with one SNP per21bp. Partial SNPs wereselected for genotyping among resistant and susceptible samples. Allele frequenciesof loci g.1361-T>C, g.1370-T>C, g.1419-T>A of nLvALF1between the two groupswere significantly different (P<0.05), and C, C, A were superior genesof the three lociassociated withWSSV resistance. Loci g.2422A>G, g.2466T>C, g.2529G>A ofnLvALF2and g.2994T>A of nLvALF6were also associated with resistance.Meanwhile, it wasfoundthat the specific haplotype CT consisted of g.1415-C>A andg.1419-T>A in nLvALF1had significant association with WSSV-susceptibilityofKehai No.1. 4．By SNaPshot genotyping platform, we did association analyses between SNPsfrom immune gene sets and WSSV resistance/susceptibility. The gene sets containedtwo parts, one set consisted of genes in the immune signaling pathways (JAK/STAT、Toll、IMD) and another set included immnue effector genes. Loci in unigene30237(STAM,3’ UTR) from JAK/STAT pathway, unigene15411(TLR, sSNP) and unigene16729(TRAF6, nsSNP) from Toll pathway were significantly associated withresistance to WSSV in alleles and/or genotypes (P<0.05). Frequencies of CT genotypeof Unigene26970(Hemocyanin),AA of Unigene34569(Cu/Zn SOD, sSNP), CT andT allele of Unigene34129-1in resistant group were much higher than those insusceptible group (P<0.05).This study will not only provide important information for understanding theanti-virus immune mechanism of ALF in shrimp, but also provide molecular markersfor the selective breeding of WSSV resistant shrimp based on “Kehai No.1”.