Development Of Molecule Markers And Gene Differential Expression Analysis Of Miichthys Miiuy Based On Transcriptome Sequencing Data

The miiuy croaker(Miichthys miiuy) which belongs to family Sciaenidae, is an important species of marine fish that supports capture fisheries and aquaculture. At present commercial scale aquaculture of this species is limited due to diseases caused by pathogens and parasites which restrict production and limit commercial value. The lack of transcriptomic and genomic information for the miiuy croaker limits the ability of researchers to study the pathogenesis and immune system of this species. In this study we firstly constructed a c DNA library from healthy miiuy croakers liver, spleen and kidney which was sequenced using Illumina paired-end sequencing to enable gene discovery and molecular marker development. In addition, in order to understand the mechanism of miiuy croaker host defense against Vibrio harveyi infection and obtain abundant immune related genes with high expression, we sequenced the transcriptome of healthy miiuy spleen and spleen injected with V. harveyi suspension. In this study, according to the twice miiuy croaker transcriptome sequencing, we obtained some main conclusions:1. We sequenced the transcriptome of miiuy croaker liver, spleen and kidney using Illumina Hi Seq 2000. After strict data cleaning and quality testing 25,760,602 high-quality reads were obtained and assembled 69,071 unigenes. By searching against NT, NR, Swiss-Prot and KEGG databases, 45,676 were successfully annotated. In total, 8,423 unigenes were classified into 51 function categories under the three ontologies of GO, 21,662 unigenes were assigned the COG classification, which could be grouped into 25 functional categories and a total of 23,927 unigenes with significant matches in the database were assigned to 123 KEGG pathways. According to these annotations, we found 498 unigene sequences showing significant homology to immune-relevant genes and divided these genes into fourteen categories.2. We obtained a total of 14,885 SSRs from 11,251 unigenes and the most abundant type of repeat motif is Di-nucleotide repeats. Eighty-seven primer pairs were designed and synthesized, 58 pairs primer were successfully amplified and 25 microsatellite loci were examined showing allelic polymorphism. Allele number of these loci ranged from 2 to 9 with an average of 3.88. The observed heterozygosity(Ho) ranged from 0.100 to 1.00 with an average of 0.433, while the expected heterozygosity(He) ranged from 0.095 to 0.784 on average of 0.489. Polymorphism information content(PIC) values of per locus varied from 0.005 to 0.991 with anaverage of 0.49. Additionally, we also identified a total of 8,510 predicted single nucleotide polymorphisms, including 6,182 transitions and 2,328 transversions.3. A total of 50,339,252 reads and 50,661,542 reads were obtained by sequencing the transcriptome of healthy miiuy spleen and spleen injected with V. harveyi suspension. After alignment with genome and screening, 37,410,818 were assembled into 78,176 unigenes, and bioinformatics analysis was carried out.4. By quantitative expression analysis, 41,845 and 41,989 genes were expressed in Ctrl and Vha sample, respectively. A total of 1,350 significant differentially expressed genes were received, including 782 up-regulation genes and 568 down-regulation genes. The GO function and KEGG pathway enrichment analysis of the differentially expressed genes were researched.5. On the basis of the differentially expressed genes GO and KEGG pathway enrichment analysis, we obtained a large number of immune related genes and their regulatory mechanism after V. harveyi infection. Such as immunoglobulin Ig M, Ig D and Ig A, chemokine and chemokine receptors CCL19, CCL21, CCL25, CXCR3 and CXCR7, and complement C1 Q, C3, C6 and C7.This is the first time for miiuy croaker transcriptome study, provides a large resource to identify new genes involved in many processes including those involved in the response to pathogens and diseases, and the differential expression analysis will help us to understand the mechanisms involved in the immune response to bacterial infection in miiuy croaker. Furthermore, the thousands of potential SSR and SNP markers found in this study are important resources with respect to future development of molecular marker assisted breeding programs for the miiuy croaker.